The impact of sequence variation on Acid

Transcription

The impact of sequence variation on
Acid Sphingomyelinase activity in the context of
Major Depressive Disorder
Der Einfluss von Sequenzvariationen auf die
Aktivität der Sauren Sphingomyelinase
im Kontext der Majoren Depression
Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades Dr. rer. nat.
vorgelegt von
Cosima Rhein
aus Fürth
Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der
Friedrich-Alexander-Universität Erlangen-Nürnberg
Tag der mündlichen Prüfung: 23. Mai 2011
Vorsitzender der Promotionskommission: Prof. Dr. Rainer Fink
Erstberichterstatter: Prof. Dr. Uwe Sonnewald
Zweitberichterstatter: Prof. Dr. Johannes Kornhuber
ἔνθ᾽ αὖτ᾽ ἄλλ᾽ ἐνόησ᾽ Ἑλένη ∆ιὸς ἐκγεγαυῖα
αὐτίκ᾽ ἄρ᾽ εἰς οἶνον βάλε φάρµακον, ἔνθεν ἔπινον,
νηπενθές τ᾽ ἄχολόν τε, κακῶν ἐπίληθον ἁπάντων.
Da entsann die zeusentsprossene Helena andres.
Und sie warf in den Wein, von welchem sie tranken, ein Mittel
gegen Kummer und Groll und aller Übel Gedächtnis.
Homer, Odyssee
Danksagung
Herrn Professor Kornhuber danke ich dafür, dass ich meine Doktorarbeit an der
Psychiatrischen und Psychotherapeutischen Klinik durchführen und die ausgezeichnete
Infrastruktur des Labors für molekulare Neurobiologie nutzen durfte. Das interdisziplinäre
Arbeiten an der Klinik hat mir interessante Einblicke in das Feld der klinischen
Neurowissenschaften ermöglicht. Eine bereichernde Erfahrung war es auch, an verschiedenen
internationalen Kongressen teilnehmen zu dürfen – herzlichen Dank dafür.
Großer Dank gilt Herrn Professor Sonnewald für die Übernahme der Begutachtung dieser
Arbeit.
Bei Herrn Dr. Martin Reichel bedanke ich mich für die inhaltliche Betreuung dieser Arbeit,
seine Diskussionsbereitschaft und sein Vertrauen in mein experimentelles Arbeiten.
Besonders danke ich ihm für die verlässliche Zusammenarbeit.
Herrn Dr. Philipp Tripal danke ich für die praktische Betreuung dieser Arbeit, seine große
Hilfsbereitschaft und die wertvolle Unterstützung. Ich danke ihm sehr, dass er mir viele
Methoden nahe gebracht und mich für das experimentelle Arbeiten begeistert hat.
Ein großes Dankeschön geht an Frau Alice Konrad für die exzellente experimentelle
Unterstützung im letzten Jahr meiner Doktorarbeit. Ihre perfekte und weitsichtige
Arbeitsweise in sämtlichen Methoden des Labors machte es zum Vergnügen, mit ihr
zusammen zu arbeiten.
Frau Michaela Schäfer danke ich sehr für die hervorragende Unterstützung im Bereich der
Zellkultur. Der hohe Standard ihrer Arbeitsweise ermöglichte erst eine Vielzahl an
Experimenten mit verschiedensten Zelllinien.
Bei Frau Sabine Müller bedanke ich mich für die ausgezeichnete Unterstützung bei den
unzähligen Schritten des Klonierens und ihre äußerst zuverlässige Arbeitsweise.
Frau Dr. Christiane Mühle danke ich für TLC-Messungen, die stets interessanten Ideen
bezüglich des Laboralltags und die Versorgung mit Werbematerial und Sprossen.
Ein Dankeschön gilt Herrn Stefan Hofmann für TLC-Messungen und seine unterhaltsamen
Gedankenexperimente über verschiedenste Themen.
Bei Frau Dr. Angela Seebahn bedanke ich mich für die unkomplizierte und erfolgreiche
Kooperation bezüglich der Massenspektrometrie.
Frau Dr. Rotter-Neubert und Herrn Dr. Bernd Lenz danke ich für die gute Zusammenarbeit
und ihre Bereitschaft, meine Experimente jederzeit durch Blutentnahmen zu unterstützen.
Bei den Mitarbeitern der Abteilung für molekulare Neurologie bedanke ich mich für die
fruchtbare Zusammenarbeit und die kollegiale Nachbarschaft.
Allen aktuellen und bereits ausgeschiedenen Kollegen der molekularen Neurobiologie sage
ich Dankeschön dafür, dass sie die Zeit während meiner Doktorarbeit mitgestaltet und meist
bereichert haben.
Meinem Molmädels-Stammtisch danke ich sehr für die fachliche und mentale Unterstützung.
Herrn ORR Bernd W. Göppner danke ich für die umfassende Unterstützung, sein Verständnis
für alle Widrigkeiten des Forscherlebens und seine Fähigkeit, mich stets zu motivieren.
Von Herzen danke ich meinen Eltern und Schwestern für den unerschütterlichen Beistand in
allen Lebenslagen.
Contents
Contents
List of figures .................................................................................VIII
List of tables ...................................................................................... IX
List of abbreviations...........................................................................X
1. Zusammenfassung ........................................................................ 12
Summary ........................................................................................... 13
2. Introduction .................................................................................. 14
2.1 Lysosomal sphingolipid disorders of the brain .......................................................14
2.1.1 Sphingolipids play diverse roles in cellular processes........................................15
2.1.1.1 Sphingolipids: Functional diversity by structural diversity ....................15
2.1.1.2 Roles of sphingolipids for the CNS ........................................................16
2.1.1.3 The lipid messenger ceramide ................................................................16
2.1.2 Molecular defects of lysosomal enzymes ...........................................................18
2.1.2.1 Primary defects .......................................................................................19
2.1.2.2 Posttranslational defects..........................................................................20
2.1.2.3 Defective trafficking ...............................................................................20
2.2 Acid sphingomyelinase in health and disease..........................................................21
2.2.1 Acid Sphingomyelinase – the lysosomal enzyme for sphingomyelin breakdown
.............................................................................................................................21
2.2.1.1 Genomic organization and transcription .................................................22
2.2.1.2 Sequence variations of Acid Sphingomyelinase.....................................23
2.2.1.3 Properties of the Acid Sphingomyelinase protein ..................................23
2.2.2 Regulation of Acid Sphingomyelinase activity ..................................................25
2.2.2.1 Transcriptional level ...............................................................................25
2.2.2.2 Posttranscriptional level..........................................................................26
2.2.2.3 Posttranslational modifications...............................................................26
2.2.2.4 Regulation via degradation .....................................................................26
2.2.3 Acid Sphingomyelinase and pathologic context.................................................27
2.2.3.1 Decreased activity of Acid Sphingomyelinase .......................................27
2.2.3.2 Increased activity of Acid Sphingomyelinase.........................................28
2.3 The role of Acid Sphingomyelinase in Major Depressive Disorder ......................28
2.3.1 Results of the pilot study.....................................................................................29
2.3.2 Sequence variations of Acid Sphingomyelinase in depressed patients ..............30
2.4 Aims of the study........................................................................................................30
3. Material and Methods.................................................................. 32
3.1 Material.......................................................................................................................32
3.1.1 Enzymes..............................................................................................................32
3.1.2 Antibodies ...........................................................................................................32
3.1.2.1 Primary antibodies ..................................................................................32
3.1.2.2 Secondary antibodies ..............................................................................33
3.1.3 Vectors and plasmids ..........................................................................................33
3.1.3.1 Vectors ....................................................................................................33
V
Contents
3.1.3.2 Plasmids ..................................................................................................33
3.1.4 Biological material..............................................................................................35
3.1.4.1 Bacteria ...................................................................................................35
3.1.4.2 Eucaryotic cells.......................................................................................35
3.1.5 Buffers and solutions ..........................................................................................35
3.2 Methods.......................................................................................................................39
3.2.1 Cell-biological methods ......................................................................................39
3.2.1.1 Cell culture..............................................................................................39
3.2.1.2 Transient expression of ASM .................................................................40
3.2.1.3 Immunocytochemistry ............................................................................40
3.2.1.4 Preparation of whole cell extracts...........................................................41
3.2.2 DNA technology .................................................................................................41
3.2.2.1 PCR for cloning approach.......................................................................41
3.2.2.2 Sub-cloning of ASM into pmCherry-N1 expression vector ...................42
3.2.2.3 Site-directed mutagenesis .......................................................................43
3.2.2.4 PCR for detection of splice variants .......................................................43
3.2.2.5 Agarose gel electrophoresis ....................................................................44
3.2.2.6 Cloning of ASM into FLAG-N2 expression vector................................45
3.2.2.7 Transformation of bacteria with DNA....................................................46
3.2.2.8 Isolation of plasmid DNA.......................................................................47
3.2.2.9 Sequencing of cDNA ..............................................................................47
3.2.3 RNA isolation and cDNA synthesis ...................................................................48
3.2.3.1 RNA isolation .........................................................................................48
3.2.3.2 Synthesis of cDNA .................................................................................49
3.2.4 Protein-biochemical analyses..............................................................................49
3.2.4.1 Determination of protein concentration ..................................................49
3.2.4.2 SDS-PAGE and Western blot analysis ...................................................49
3.2.4.3 In vitro determination of ASM activity ..................................................51
3.2.4.4 MALDI-TOF MS analysis of cellular ceramide and sphingomyelin
levels .......................................................................................................52
3.2.5 In silico analyses and statistics ...........................................................................52
4. Results............................................................................................ 53
4.1 Impact of DNA sequence variations on Acid Sphingomyelinase activity .............53
4.1.1 Expression of recombinant ASM variants ..........................................................55
4.1.2 Characterization of ASM variants used for model system .................................56
4.1.2.1 Catalytic activity of ASM model system variants ..................................57
4.1.2.2 Localization of ASM model system variants..........................................58
4.1.3 Characterization of ASM variant p.P325A.........................................................60
4.1.3.1 ASM variant p.P325A is catalytically inactive.......................................61
4.1.3.2 ASM variant p.P325A localizes in lysosomes........................................61
4.1.4 Characterization of ASM variant p.A487V ........................................................63
4.1.4.1 Catalytic activity of ASM variant p.A487V is equivalent to wild type..63
4.1.4.2 ASM variant p.A487V localizes in lysosomes .......................................64
4.1.5 Characterization of ASM variant p.G506R ........................................................65
4.1.5.1 ASM variation p.G506R impacts catalytic activity ................................65
4.1.5.2 ASM variation p.G506R impacts localization ........................................66
4.1.6 Summary: Characterization of ASM variants.....................................................69
4.2 Impact of alternative splicing on Acid Sphingomyelinase activity........................70
4.2.1 Structure of new ASM splice variants ................................................................73
4.2.2 New splicing motifs are conserved in primates ..................................................74
VI
Contents
4.2.3 Biochemical characterization of new ASM splice variants ................................74
4.2.3.1 New ASM splice variants are expressed in a cell culture model............77
4.2.3.2 New ASM splice variants are catalytically inactive in vitro...................77
4.2.3.3 New ASM splice variants are catalytically inactive in vivo ...................78
4.2.4 New ASM splice variants localize within different subcellular compartments..79
4.2.5 New ASM splice variants show differential expression patterns in human blood
cells .....................................................................................................................82
4.2.5.1 New ASM splice variants are detected specifically by RT-PCR............82
4.2.5.2 New ASM splice variants are differentially expressed in healthy
volunteers................................................................................................83
4.2.5.3 New ASM splice variants are differentially expressed in depressed
patients before and after antidepressant therapy.....................................84
4.2.6 New splice variant ASM-5 exerts a dominant-negative effect on full-length Acid
Sphingomyelinase ...............................................................................................86
4.2.6.1 Upon serum starvation ASM-5 localizes within lysosomes ...................86
4.2.6.2 ASM-5 is a dominant-negative ASM isoform ........................................88
5. Discussion ...................................................................................... 90
5.1 DNA sequence variations impact Acid Sphingomyelinase activity .......................90
5.1.1 The C-terminal domain harbours essential parts for activation and secretion of
Acid Sphingomyelinase ......................................................................................91
5.1.2 Processing of Acid Sphingomyelinase is essential for its regulation in pathologic
context.................................................................................................................92
5.2 Alternative splicing impacts Acid Sphingomyelinase activity ...............................93
5.2.1 Alternative splicing of Acid Sphingomyelinase could predominate in brain and
blood cells ...........................................................................................................94
5.2.2 Acid Sphingomyelinase displays a defined set of conserved splicing motifs ....94
5.2.3 Acid Sphingomyelinase activity is regulated by alternative splicing .................96
6. Bibliography.................................................................................. 98
7. Appendix ..................................................................................... 107
7.1 Chemicals..................................................................................................................107
7.2 Technical device .......................................................................................................108
7.3 Auxiliary material....................................................................................................108
VII
List of figures
List of figures
Fig. 2.1
Fig. 2.2
Fig. 2.3
Fig. 4.1
Fig. 4.2
Fig. 4.3
Fig. 4.4
Fig. 4.5
Fig. 4.6
Fig. 4.7
Fig. 4.8
Fig. 4.9
Fig. 4.10
Fig. 4.11
Fig. 4.12
Fig. 4.13
Fig. 4.14
Fig. 4.15
Fig. 4.16
Fig. 4.17
Fig. 4.18
Fig. 4.19
Fig. 4.20
Fig. 4.21
Fig. 4.22
Fig. 4.23
Fig. 4.24
Fig. 4.25
Fig. 4.26
Fig. 4.27
Fig. 4.28
Fig. 4.29
Fig. 4.30
Fig. 4.31
The ‘ceramide / sphingosine-1-phosphate rheostat’ concept.................................17
ASM is subject to alternative splicing events ........................................................23
ASM is composed of several domains...................................................................24
Position of ASM variations investigated in this study...........................................53
Cloning approach for the generation of recombinant ASM constructs containing
different ASM variations and FLAG- or Cherry-epitope ......................................54
All investigated ASM variants are translated into protein and display
predicted sizes........................................................................................................55
ASM variants were transiently expressed to a different extent .............................56
ASM model system variants display predicted enzymatic activity levels. ............58
Subcellular localization patterns of ASM model system variants ASM wild type
and ASM variants p.S508A and p.M381I..............................................................59
Co-localization of ASM model system variants with subcellular organelles. .......60
ASM variant p.P325A does not exert catalytic activity compared to ASM
wild type.................................................................................................................61
ASM variant p.P325A localizes in a dotted subcellular pattern ............................62
ASM variant p.P325A is localized within lysosomes............................................62
Enzymatic activities of ASM variant p.A487V and ASM wild type reach similar
levels ......................................................................................................................63
ASM variant p.A487V localizes in a dotted subcellular pattern ...........................64
ASM variant p.A487V localizes within lysosomes ...............................................64
ASM variation p.G506R causes a decrease in enzymatic activity.........................66
Subcellular localization patterns of ASM variants containing the variation
p.G506R .................................................................................................................67
Co-localization of ASM variants containing variation p.G506R with subcellular
organelles. ..............................................................................................................68
Coding sequences of full-length ASM and new alternatively-spliced transcripts
differ locally...........................................................................................................72
Schematic structure of new splice variants ASM-5, -6 and -7 ..............................73
Splicing patterns of novel transcripts ASM-6 and -7 are conserved in primates...74
Novel variants ASM-5 to -7 differ regarding their putative protein sequence and
protein domain structure ........................................................................................76
Expression of ASM-FLAG constructs in HeLa cells ............................................77
Novel variants ASM -5 to -7 are catalytically inactive..........................................78
New ASM splice variants modulate ceramide to sphingomyelin ratios ................79
Subcellular localization patterns of ASM-1 and new ASM splice variants...........80
Co-localization of new ASM splice variants with subcellular organelles. ............81
Designed oligonucleotides serve for specific amplification of new ASM splice
variants...................................................................................................................83
Expression patterns of ASM splice variants vary in human blood cells................84
Expression patterns of ASM splice variants vary in blood cells of depressed
patients before and after administering of antidepressants ....................................85
Subcellular localization pattern of ASM-5 upon serum starvation........................87
ASM-5 localizes within lysosomes upon serum starvation ...................................87
ASM-5 exerts a dominant-negative effect on ASM-1 upon serum starvation.......89
VIII
List of tables
List of tables
Tab. 3.1
Tab. 3.2
Tab. 3.3
Tab. 3.4
Tab. 3.5
Tab. 3.6
Tab. 3.7
Tab. 3.8
Tab. 3.9
Tab. 4.1
Primary antibodies in cotext of their application...................................................32
Secondary antibodies in cotext of their application...............................................33
Vectors used in this study ......................................................................................33
Plasmids used in this study ....................................................................................34
Oligonucleotides used for cloning of ASM into pSC-B vector and FLAG-N2
expression vector ...................................................................................................42
Oligonucleotides used for cloning of ASM from FLAG-N2 vector into
pmCherry-N1 vector. .............................................................................................42
Oligonucleotides used for site-directed mutagenesis of ASM wild type-FLAG
construct to generate different ASM variants ........................................................43
Oligonucleotides used for amplification of ASM splice variants ..........................44
Oligonucleotides used for sequencing of ASM plasmids ......................................47
ASM variants display differential properties .........................................................69
IX
List of abbreviations
AA
AC
APS
ASM
BES
bp
BSA
c
°C
C-terminal
CNS
cDNA
DMEM
DMSO
DMF
DNA
dNTP
DTT
EC
ECL
E. coli
EDTA
ER
FCS
Fig.
fw
g
g
GAPDH
GFP
h
H4
H2Obidest
HPRT
kb
kDa
l
LAMP1
LB
LPS
M
m
µ
MDD
mRNA
min
MALDI-TOF MS
MCS
amino acid
Acid Ceramidase
ammonium persulfate
Acid Sphingomyelinase
2-hydroxyethyl-2-aminoethanesulfonic acid
base pairs
bovine serum albumine
centi (10-2)
degree Celsius
carboxy-terminal
central nervous system
coding DNA
Dulbecco’s Modified Eagle’s Medium
dimethylsulfoxide
2,5-dimethylfurane
deoxyribonucleic acid
deoxyribonucleoside-5’-triphosphate
dithiotreitol
enzyme classification number
enhanced chemiluminescence
Escherichia coli
ethylenediaminetetraacetate
endoplasmic reticulum
fetal calf serum
figure
forward
grams
gravitation constant
glycerinaldehyd-3-phosphat-dehydrogenase
green fluorescent protein
hour(s)
neuroglioma cell line
aqua bidestillata
hypoxanthine-phosphoribosyl transferase
kilo base pairs
kilo daltons
litre
lysosomal-associated membrane protein 1
Luria Bertani Medium
lipopolysaccharide
molar
milli (10-3)
micro (10-6)
major depressive disorder
messenger ribonucleic acid
minute(s)
matrix assisted laser desorption/ionisation time of flight
mass spectrometry
multiple cloning site
X
ng
nmol
NPD
N-terminal
PAGE
PBMC
PBS
PCR
PDIA4
pH
PVDF
RNA
rev
rpm
RT
RT-PCR
SAP
SDS
sec
SMPD1
SNP
SOC medium
Tab.
TEMED
Tris
Tween-20
U
UV
V
Vmax
YT
nanogram
nanomole
Niemann-Pick disease
amino-terminal
polyacrylamide gel electrophoresis
peripheral mononuclear cell
phosphate buffered saline
polymerase chain reaction
protein disulfide isomerase family A, member 4
-log H+ concentration
polyvinyldifluoride
ribonucleic acid
reverse
rounds per minute
room temperature
reverse transcription-PCR
saposin domain
sodium dodecyl sulphate
second(s)
sphingomyelin phosphodiesterase
single nucleotide polymorphism
super optimal broth medium containing glucose
table
N,N,N’,N’-tetramethylethylenediamine
Tris-hydroxymethyl-aminomethane
polysorbate 20
Unit
ultraviolett
Volt
maximum velocity of enzymes
yeast extract and tryptone
XI
1. Zusammenfassung
1. Zusammenfassung
In einer Pilotstudie, die im Jahr 2005 von Kornhuber und Kollegen veröffentlicht wurde, war
gezeigt worden, dass die Aktivität der sauren Sphingomyelinase (ASM) bei Patienten mit
schwerer Depression im Vergleich zu einer gesunden Kontrollgruppe deutlich erhöht war. In
der vorliegenden Arbeit wurde der mögliche Einfluss von ASM-Sequenzvariationen auf die
Enzymaktivität als molekulare Ursache dieser Assoziation untersucht. Es wurde sowohl die
Bedeutung von allgemein auftretenden DNA-Sequenzvariationen, die auch in einem
Kollektiv depressiver Patienten identifiziert worden waren geprüft, als auch von RNASequenzvariationen, um den Einfluss von alternativem Spleißen auf die zelluläre ASMAktivität zu beleuchten.
Drei nicht-synonyme Sequenzvariationen im Bereich des maturen Proteins wurden im
Hinblick auf katalytische Aktivität und zelluläre Lokalisation untersucht. Dazu wurden die
ASM-Varianten
in
Expressionsvektoren
kloniert
und
in
einem
Zellkulturmodell
überexprimiert. Zwei der Varianten – p.P325A und p.G506R – zeigten im Vergleich zum
ASM Wildtyp eine signifikant erniedrigte Enzymaktivität, während die Aktivität von
p.A487V nicht verändert war. Alle drei untersuchten Varianten kolokalisierten mit LAMP1
in Lysosomen, so dass kein Unterschied im Vergleich zum Wildtyp beobachtet wurde.
Im Rahmen der Arbeit wurden drei bislang nicht beschriebene ASM-Spleißvarianten
identifiziert und kloniert. Die Spleißvarianten führten nach Überexpression nicht zu einem
Anstieg der zellulären ASM-Aktivität. Es wurde allerdings beobachtet, dass sich der relative
Ceramid-Gehalt der Zellen signifikant verringerte, was für eine dominant-negative Wirkung
der Spleißvarianten spricht. Für die Spleißvariante ASM-5, die nur eine unvollständige
katalytische Domäne aufweist, konnte gezeigt werden, dass es unter Serum-Entzug zu einer
Translokation des Proteins ins Lysosom und zu einer Hemmung der zellulären ASM Aktivität
kommt. Unter basalen Bedingungen hingegen lokalisierte das Protein im endoplasmatischen
Retikulum und hatte keinen Einfluss auf die ASM-Aktivität. In RT-PCR Untersuchungen
konnten die neuen Spleißvarianten bei gesunden Probanden nachgewiesen und eine
Veränderung der Expression bei depressiven Patienten während der Therapie gezeigt werden.
Die
vorliegende
Arbeit
liefert
erste
Hinweise
darauf,
wie
verbreitete
ASM-
Sequenzvariationen die ASM-Aktivität beeinflussen können. Weitere Arbeiten sollten zeigen,
ob diese Mechanismen zur Entstehung der Majoren Depression beitragen.
12
Summary
Summary
In a pilot study conducted in 2005 by Kornhuber et al., we detected increased activity of acid
sphingomyelinase (ASM) in patients suffering from major depressive disorder compared to
controls. In this study, we investigated the potential influence of ASM sequence variations on
ASM activity to get insight into the causative mechanism of this association on the molecular
level. On the one hand, we characterized the impact of generally occurring DNA sequence
variations, which were also detected in depressed patients. On the other hand, we investigated
‘RNA sequence variations’, i.e. the role of alternative splicing for cellular ASM activity
levels. Despite just slightly altered protein sequences, the resulting ASM variants could be
prone to pathologic functioning.
At the genomic level, we investigated the impact of three missense variations located in the
coding region of the mature protein on enzymatic activity and cellular localization. Therefore,
different ASM variations were cloned and over-expressed in a cell culture model. Compared
to ASM wild type, two ASM variants – p.P325A and p.G506R – displayed significantly
decreased ASM activity, whereas variation p.A487V did not change enzymatic activity.
Localization patterns were unchanged, since all variants revealed co-localization with
LAMP1 in lysosomes.
In this study, three to date not described ASM splice variants were identified and cloned.
Compared to ASM wild type, new splice variants did not increase cellular ASM activity
levels upon over-expression. However, we detected a significant decrease in relative cellular
ceramide levels, indicating a dominant-negative effect exerted by new ASM splice variants.
For isoform ASM-5, which displays a disrupted catalytic domain, we could demonstrate
translocation into lysosomes and the inhibition of cellular ASM activity upon serum
starvation. In contrast, ASM-5 was located inside the endoplasmic reticulum and did not
impact cellular ASM activity under basal conditions. By RT-PCR differential expression
patterns of ASM splice variants could be detected in blood cells of healthy volunteers. In
patients suffering from major depression expression patterns differed before and after therapy
with antidepressant drugs.
This study offers first evidence how common ASM sequence variations can impact ASM
activity. Further studies should investigate if these mechanisms could account for the
pathogenesis of major depression.
13
2. Introduction
2. Introduction
2.1 Lysosomal sphingolipid disorders of the brain
Groups of diseases, which we today call ‘lysosomal disorders’, had been clinically
recognized around the turn of the nineteenth to the twentieth century (Tay, 1881; Sachs,
1887). To get insight into the molecular basis of these diseases, it still took to the 1960s
(Hers, 1963) – upon the discovery of the lysosome (De Duve et al., 1955). Lysosomal
diseases are defined as a group of inherited metabolic diseases, which are characterized by
the accumulation of non-metabolized macromolecules like lipids, glycoproteins or
oligosaccharides inside the lysosomes. Notably, most of lysosomal disorders show pathology
in the brain, leading to severe clinical symptoms (Meikle et al., 1999). According to the
stored material, a classification system was established, with lipid storage material and
especially the class of sphingolipids being the most complex group. The so called
‘sphingolipidoses’ cover glycosphingolipid storage diseases and not further specified
diseases, including deficiencies of acid sphingomyelinase (ASM) and acid ceramidase, which
lead to Niemann-Pick (NPD) and Farber disease, respectively (Platt and Walkley, 2004,
p.35).
After the biochemical identification of the storage material, also the genetic background of
these diseases could be identified. Most of the to date described 41 disorders are autosomal
recessive diseases (Meikle et al., 1999). Interestingly, not only lysosomal hydrolases are
affected, but also a variety of proteins exerting influence on their cellular processing, e.g.
endoplasmic reticulum (ER)- and Golgi-associated processing enzymes, protective proteins
or transmembrane proteins (Walkley, 2001). Due to the discovery of the molecular
mechanisms of the lysosomal disorders, a classification system on the basis of molecular
defects evolved, which harboured chances for better understanding and potential treatment of
lysosomal disorders (Platt and Walkley, 2004, p.41).
14
2. Introduction
2.1.1 Sphingolipids play diverse roles in cellular processes
The term ‘sphingolipids’ is derived from the enigmatic and therefore sphinx-like properties of
complex lipids, which were firstly described in the 1870s after their discovery within brain
extracts. Sphingolipids play diverse roles in cellular processes: On one hand as structural
membrane components, on the other hand as signalling molecules that mediate cellular stress
responses (Zeidan and Hannun, 2007b).
2.1.1.1 Sphingolipids: Functional diversity by structural diversity
Sphingolipids are a large class of polar and amphipatic lipids, which show structural
diversity. Their common feature is the core component sphingosine, which results from the
condensation of serine with fatty acid-Coenzyme A conjugates (Loeffler, 2003). An N-linked
fatty acid which can be quite heterogenous gives rise to ceramide, which is the source for
more complex sphingolipids: The head group can be derivatized with different moieties,
whereas condensed glycans result in glycosphingolipids, condensed phosphorylcholine in
sphingomyelin. The chemical core structure in combination with the variety of head groups
makes sphingolipids a unique class of lipids with essential functions for cellular processes
(Breslow and Weissman, 2010).
Sphingolipids significantly affect both the structural and functional properties of cell
membranes. For example, sphingomyelin is an important component of the outer cell
membrane (Emmelot and Van Hoeven, 1975). In addition, many sphingolipids are bioactive
signalling molecules which impact diverse cellular functions. As an example, ceramide and
sphingosine-1-phosphate exert signalling properties in intracellular and extracellular context,
respectively. Therefore, nowadays sphingolipids are not seen as merely structural and
‘passive’ molecules anymore, but take an important position as bioactive molecules (Breslow
and Weissman, 2010).
15
2. Introduction
2.1.1.2 Roles of sphingolipids for the CNS
Though in most cell types sphingolipids constitute the minor component of cell membranes
besides glycerophospholipids and cholesterol, they resemble the vast majority in nervous
cells. For example, glycosphingolipids and sphingomyelin are abundant components of
membranes in the nervous system with the latter’s name being derived from the frequent
prevalence within myelin sheaths (Zheng et al., 2006). Besides the structural functions,
sphingolipids also exert important cell-biological functions in the CNS. They are important
for maintaining the functional integrity of neurons concerning survival, cell death, migration
and differentiation, synaptic functioning and also neuron-glia interactions (Piccinini et al.,
2010). These essential roles in signal transduction are exerted via two different paths:
Concerning structural influence, they modulate properties of membrane-associated receptors
due to alterations of membrane composition. Concerning functional influence, they act as
precursors of bioactive lipid mediators. These differential actions lead to a structural and
functional modification of neuronal plasticity (Haughey, 2010; Swartz, 2008; Day and
Kenworthy, 2009; Owen et al., 2009; Stahelin, 2009). For example, synaptic activity both at
pre- and post-synapses is highly influenced by sphingolipids. Vesicle trafficking, -fusion and
transmitter release are regulated by the bioactive sphingolipids ceramide, sphingosin and
sphingosin-1-phosphate (Kajimoto et al., 2007). Also, synapse formation and the location of
receptors are affected by the action of ceramide (Rohrbough et al., 2004). In total, a
dysfunction of sphingolipid metabolism can severely influence synaptic integrity and
neuronal functioning.
2.1.1.3 The lipid messenger ceramide
The most extensively studied sphingolipid ceramide acts as a lipid messenger, exerting
diverse effects on cellular processes, negatively on proliferation (Boucher et al., 1995;
Coroneos et al., 1995) and differentiation (Sallusto et al., 1996; Zhang et al., 1997), blocking
of cell cycle events (Dobrowsky et al., 1993) and apoptosis (Obeid et al., 1993). Due to the
activity of acid sphingomyelinase, ceramide is generated by the breakdown of
sphingomyelin. According to the ‘ceramide / sphingosine-1-phosphate rheostat’ concept
increased ceramide levels promote apoptosis whereas an increase of the phosphorylated
metabolite of ceramide, sphingosine-1-phosphate, counters this effect by inducing
16
2. Introduction
proliferation, which regulates the balance between cell death and cell growth (Spiegel and
Merrill, 1996; Spiegel et al., 1998). The dynamic balance between these bioactive lipids is
influenced by the activity of acid ceramidase, sphingosine kinase, sphingosine 1-phosphate
phosphatase, sphingosine-acetyltransferase and ASM. Among these enzymes, ASM plays an
important role, since the first bioactive molecule within the rheostat is generated upon its
enzymatic activity (Fig. 2.1).
Fig. 2.1 The ‘ceramide / sphingosine-1-phosphate rheostat’ concept.
The dynamic balance between cell death and growth is highly influenced by acid sphingomyelinase (ASM) and
acid ceramidase (AC). Ceramide is generated by ASM-induced hydrolysis of sphingomyelin and exerts proapoptotic effects (red). The opposite effect is mediated by sphingosine-1-phopohate (blue) (modified from
Kornhuber et al. (2010)).
Ceramide impacts the signal transduction of the cell via the modulation of the cell membrane
and via its function as lipid messenger. Alterations affecting the cell membrane can induce
dramatic changes concerning the biophysical properties of membrane microdomains, which
are often built by sphingomyelin and cholesterol (Simons and Ikonen, 1997; Kolesnick et al.,
2000). Ceramide molecules interact spontaneously and fuse to large macrodomains
(Holopainen et al., 1998; Kolesnick et al., 2000; Nurminen et al., 2002). These
macrodomains play an important role in signal transduction processes and exert a general
effect on diverse signal cascades. For example, the generation of ceramide-rich platforms
leads to the reorganization and an increased density of receptors, e.g. of the death receptor
CD95 and accessory signalling molecules, which means an amplification of this signal
(Grassmé et al., 2001a; Grassmé et al., 2001b; Grassmé et al., 2003).
The generation of ceramide is probably an important cellular reaction of the cell on stressful
events (Quintans et al., 1994; Herr et al., 1997). Therefore, ceramide as lipid messenger
17
2. Introduction
exerts direct influence on different kinases like ceramide-activated proteinkinase,
proteinkinase Cζ und ceramide-activated proteinphosphatase, which in turn impact signalling
molecules like c-Raf and NFκb (Zhang et al., 1997). Thus, ceramide exerts the function of an
effector molecule, regulating many important downstream targets.
In total, ASM generating increased levels of ceramide by hydrolizing sphingomyelin plays a
key role for cellular sphingolipid metabolism and impacts diverse cellular processes.
2.1.2 Molecular defects of lysosomal enzymes
The lysosome is the only cellular organelle with an acidic milieu of pH 5 and is covered by a
membrane bilayer. Since the lysosome contains a variety of hydrolases, it was seen as an
‘organelle for garbage disposal’, carrying out the catabolism of internalized material (Alberts,
2002; Loeffler, 2003): After the uptake of extracellular material, for example by receptormediated endocytosis, internalized macromolecules enter the endosomal-lysosomal pathway.
On their way to the lysosome these molecules are partly degraded until they get their final
destination. Inside the lysosome, final breakdown by diverse hydrolases takes place,
completed with the storage of undigestable material (Platt and Walkley, 2004, chapter 1).
But nowadays, the perception of the lysosome takes a turn: Instead of being there for the
mere breakdown of molecules, it is supposed to be a source for many important regulative
processes. For example, the lysosomal form of ASM generates ceramide, which impacts
important signal cascades (Kolesnick, 1991).
Therefore, molecular defects of lysosomal enzymes can be deleterious for proper cellular
functioning. For neurons, the effectiveness of these processes is even more important, since
they have special requirements for endocytic recycling and have to function during their
whole longevity (Slepnev and De Camilli, 2000; Platt and Walkley, 2004, chapter 1).
Molecular defects of lysosomal enzymes can occur at diverse processing levels, via mutations
in the gene itself, abnormal processing of the protein in the ER and in the Golgi, missing
protective or activating proteins or altered transport proteins (Platt and Walkley, 2004,
chapter 4).
18
2. Introduction
2.1.2.1 Primary defects
Mutations in genes coding for lysosomal enzymes are the most common causes of lysosomal
storage diseases. Among sphingolipidoses, many of the glycosphingolipid storage diseases
are caused by primary enzymatic defects, e.g. Fabry disease resulting from mutations within
α-galactosidase. To give examples for the not further distinguished sphingolipidoses,
Niemann-Pick disease type A and B is caused by mutations in the sphingomyelin
phosphodiesterase gene (SMPD1) coding for ASM, or Farber disease, which harbours
mutations in the gene coding for acid ceramidase. In most of these disorders, the catalytic
activity of the respective enzyme is abolished. Therefore, the catalytic function of an essential
enzyme comprising the endosomal-lysosomal system is lacking (Platt and Walkley, 2004,
chapter 4).
The extent of the derived deleterious effect depends on the normal load of the respective
substrate presented to the lysosome and the residual activity of the enzyme. The worst
pathologic effects of primary defects are located in those tissues, where a certain substrate is
subject to extensive catabolism, e.g. gangliosides in neurons. Also, the respective threshold of
storage is of importance (Conzelmann and Sandhoff, 1983). But even if the main problems of
storage arise in tissues outside the brain, also in neuronal and glial cells there probably is a
steady build up of storage material, leading to clinical manifestation at a later stage of the
process. Since these problems may be worse for long living neurons, all lysosomal enzyme
defects could be seen as causes for brain diseases. Lysosomal hydrolases pass many stations
from their synthesis in the rough ER to their destination, the lysosomes. Many posttranslational modifications take place, the removal of the N-terminal signal peptide,
acquisition of the mannose-6-phosphate recognition marker and glycosylation- and
processing steps to reach their final conformation. Any of these steps can be affected by
mutations at a certain position in the gene giving rise to the enzyme (Platt and Walkley, 2004,
chapter 4). Different types of mutations have been linked to lysosomal enzyme deficiencies,
for example SNPs leading to missense, nonsense or splice-site mutations, deletions, insertions
or chromosomal rearrangements. Usually null alleles evolve, which result in the expression of
non-functional enzymes. In some cases, a residual activity of the resulting enzymes is left.
But a clear prediction of the effects of DNA variation on protein properties remains difficult
(Terp et al., 2002). As an exception, substitution of an amino acid which is directly involved
in catalytic action or interaction with activator proteins probably leads to a lack of catalytic
19
2. Introduction
activity. An important tool for detecting effects of DNA variation is the cloning of gene
variants coding for altered proteins. In vitro expression analyses allow insights into the
effects on enzymatic activity caused by mutations. However, if a missense mutation does not
result in the expression of a catalytic inactive enzyme, the processing steps should be
controlled before negating pathologic effects. Also, mutations are able to enhance or lessen
the effects of another sequence variation (Islam et al., 1996). Additionally, the clinical
phenotype can give a hint for null alleles caused by mutations. Severely affected patients
being homozygous for a mutation probably produce null allele enzymes, whereas
heterozygous patients should be less affected then. Consequences which result from certain
mutations, especially how they affect the structure, the intracellular transport and the activity
of the resulting enzyme, are of high importance for enzyme properties and the following
treatment of pathologic effects (Platt and Walkley, 2004, chapter 4).
2.1.2.2 Posttranslational defects
The lysosomal system can be disturbed also on the posttranslational level. For example,
lysosomal processing of the enzymes in the ER can be involved. This is seen in the so far
unique known molecular mechanism of a lysosomal storage disease, the multiple sulphatase
deficiency (Hopwood and Ballabio, 2001). In this defect, the activating step of the
posttranslational generation of a Cα-formylglycine residue in the catalytic centre of sulfatases
is lacking (von Figura et al., 1998).
2.1.2.3 Defective trafficking
Defective trafficking can lead to lysosomal diseases, if the trafficking from ER to the
lysosomal system via the Golgi apparatus is perturbed. For example, the I-cell disease, also
called mucolipidosis type II, is characterized by a defective trafficking due to the lack of
mannose-6-phosphate phosphorylation. Affected is the protein responsible for synthesis of
the
mannose-6-phosphate-recognition
marker,
the
N-acetylglucosaminyl-1-phospho-
transferase. Instead of trafficking to lysosomes, the enzymes are secreted (Kornfeld and Sly,
2001). Alternative routes for lysosomal targeting in addition to the mannose-6-phosphate
receptor system are barely understood. Taken together, defects affecting the trafficking of
20
2. Introduction
lysosomal enzymes can cause two different effects: The lysosome is deprived of an important
hydrolase, and a lysosomal enzyme precursor is secreted, potentially exerting effects outside
the cell (Platt and Walkley, 2004, chapter 6).
2.2 Acid sphingomyelinase in health and disease
Acid sphingomyelinase (EC 3.1.4.12) is a glycoprotein located mainly in the lysosome and
catalyses the breakdown of sphingomyelin into ceramide and phosphorylcholine at a pH
optimum of pH 5 (Quintern et al., 1987). As one important function, ASM activity changes
the lipid composition of membranes. A second important function is the generation of the
bioactive lipid ceramide. ASM exerts biomedical significance, since both increase and
decrease in ASM activity leads to severe pathologic effects. Several sequence variations of
ASM cause the loss of enzymatic activity, resulting in Niemann-Pick disease type A and B.
Since ASM plays a strategic role in cellular stress response, its tight regulation is crucial for
cellular functioning.
2.2.1 Acid Sphingomyelinase – the lysosomal enzyme for sphingomyelin
breakdown
ASM was firstly described in 1966 by Kanfer et al. (1966), who reported a sphingomyelincleaving enzyme in rat liver tissue. The subcellular localization of ASM inside the lysosome
was identified two years later (Weinreb et al., 1968). But it still took until 1991 to clone ASM
(Schuchman et al., 1991b), which was the starting point to investigate biochemical properties
of the enzyme and the molecular mechanisms of ASM regulation.
21
2. Introduction
2.2.1.1 Genomic organization and transcription
The gene coding for ASM, SMPD1, is located on chromosome 11p15.1-p15.4, covering
about 5000 base pairs (da Veiga Pereira, 1991). Interestingly, it gives rise to two different
enzymes, the lysosomal and the secreted sphingomyelinase, which probably play different
metabolic roles (Schissel et al., 1996).
Transcription of ASM is regulated by different upstream promotor elements, including a
TATA-box and binding sites for transcription factors SP1 and AP-2 (Langmann et al., 1999).
The cDNA synthesized from human mRNA displays an untranslated region of 174 and 388
base pairs at the 5’ and the 3’ end, respectively. The SMPD1 open reading frame covers 1896
base pairs coding for 631 amino acids.
On the posttranscriptional level, ASM is subject to alternative splicing events. For the
lysosomal form of ASM, four different transcripts are identified so far (Quintern et al., 1989;
Schuchman et al., 1991b; Schuchman et al., 1992). The ‘regularly’ spliced transcript, termed
ASM type 1 (GenBank Accession Number NM_000543.4; here referred to as ASM-1) is
composed of exons 1 to 6, with the corresponding five introns spliced out. Compared to this
reference, alternatively-spliced transcript ASM type 2 (GenBank Accession Number
NM_001007593.1; here ASM-2) displays a specific insertion of 40 bp derived from intron 2
and lacks exon 3. Alternatively-spliced transcript ASM type 3 (GenBank Accession Number
NR_027400; here ASM-3) is lacking the complete exon 3 which creates a frameshift and
subsequently leads to a premature stop codon. An additional ASM transcript, which lacks 652
bp of exon 2, was isolated from human brain tissue (GenBank Accession Number
AY649987.1; here typed ASM-4) (Fig. 2.2). According to Schuchman and colleagues
(Quintern et al., 1989; Schuchman et al., 1991b), ASM-1 occurs at a frequency of about 90%,
ASM-2 and -3 of about 10%.
22
2. Introduction
Fig. 2.2 ASM is subject to alternative splicing events.
Model of genomic alignment. Compared to ASM-1, ASM-2 displays a specific insertion of 40 bp and lacks
exon 3. ASM-3 is lacking exon 3, whereas ASM-4 lacks 652 bp of exon 2, both creating a frameshift and a
premature stop codon. In the depicted models of ASM exon-intron structure, the line represents the genomic
sequence while black boxes stand for exons translated in ASM-1, grey boxes for exonic sequence translated into
different amino acids due to a frameshift or sequence corresponding to introns in ASM-1, striped boxes for
lacking exonic sequence and white boxes for exonic sequence following a premature termination codon
(indicated by asterisk). Scheme is drawn to scale.
2.2.1.2 Sequence variations of Acid Sphingomyelinase
According to GenBank database SNP, 32 SNPs within the ASM coding sequence are
identified. The most frequent SNPs are c.107T>C / p.V36A with a frequency of 0.32 and
c.1522G>A / p.G506R with a frequency of 0.251. Besides the different SNPs there also exists
a polymorphic part located in the region coding for the signal peptide of ASM, which is
characterized by a three- to eight-fold repeat of the hexanucleotide GCTGGC
(c.108GCTGGC[6] / p.37(LA)3-8) (Wan and Schuchman, 1995).
2.2.1.3 Properties of the Acid Sphingomyelinase protein
The ASM protein displays several domains. The enzyme is composed of the putative signal
peptide (aa 1-48), a saposin-B-domain for proper substrate interaction (aa 91-167), a prolinerich domain (aa 168-200), a calcineurine-like phosphoesterase domain considered as the
catalytic centre (aa 201-463) and a carboxy-terminal domain (aa 464-631) (Marchler-Bauer
A, 2007; Jenkins et al., 2009) (Fig. 2.3).
23
2. Introduction
Fig. 2.3 ASM is composed of several domains.
Schematic model of ASM protein structure. Shown are different domains with respect to amino acid numbers.
Mannose-6-phosphate sites are indicated by symbols. Scheme is drawn to scale.
ASM is subject to several steps of processing before the mature enzyme is obtained. In a first
step, ASM is translated into a premature pre-pro-polypeptide with an apparent molecular
mass of 75 kDa (64 kDa in deglycosylated state), which is found in the ER and does not exert
catalytic activity (Ferlinz et al., 1994; Hurwitz et al., 1994b). As usual for proteins located
within subcellular organelles, also ASM displays an N-terminal signal peptide. In comparison
to ‘regular’ signal peptides of about 20 amino acids, the ASM signal peptide seems to be
special due to its length of 48 amino acids (Garnier et al., 1978; Schuchman et al., 1991b;
Wan and Schuchman, 1995). According to the polymorphism on the genomic level, the
motive composed of leucine and alanine (‘LA’) occurs to a different extent, being repeated
three, four, five, six or eight times (Wan and Schuchman, 1995) and probably influences the
hydrophobicity of the ASM N-terminal region. The signal peptide plays an important role for
the functioning of ASM. Is the signal peptide lacking, ASM does not reach the lysosomal
compartment: Neither is it synthesized into the lumen of the ER, nor is it modified properly
and trafficked by vesicular transport. Instead, ASM is localized inside the cytoplasm (Ferlinz
et al., 1994). Besides proper localization, the signal peptide is also essential for generating
catalytic active ASM enzymes. Recombinant ASM constructs without the sequence coding
for the signal peptide are catalytically inactive in vitro, probably due to the lack of
glycosylation and zinc acquisition. Also, no ASM activity can be detected if sorting and
trafficking is altered (Ferlinz et al., 1994; Ni, 2006). In total, ASM targeting to lysosomes is
essential for proper enzymatic functioning, which stresses the importance of the ASM signal
peptide. Therefore, the signal peptide can influence ASM activity, even though it is not
present in the mature ASM protein.
Within the ER and Golgi further processing of ASM takes place. After cleavage of the signal
peptide inside the ER, a 72 kDa precursor form of ASM occurs (61 kDa in deglycosylated
24
2. Introduction
state) – yet without being catalytically active. This precursor protein gives rise to two
distinctly processed ASM forms. The major part is processed to a 70 kDa form and associated
with a zinc ion. This mature ASM protein is localized inside the lysosome and exerts catalytic
activity. In a further processing step inside the lysosome, the 70 kDa form is cleaved to a 52
kDa form. The minor part of the 72 kDa precursor is cleaved already inside the ER and Golgi
to a 57 kDa form, which is not glycosylated and rapidly degraded (Ferlinz et al., 1994). In
total, processing of lysosomal ASM is essential for generating the properly localized and
catalytically active enzyme. In contrast, secreted sphingomyelinase is not further processed
and is detected as 75 and 57 kDa forms when secreted into extracellular space.
2.2.2 Regulation of Acid Sphingomyelinase activity
ASM plays a role for diverse physiological processes. A variety of external stimuli cause an
activation of ASM, e. g. CD95 ligand (Lin et al., 2000), lipopolysaccharide (HaimovitzFriedman et al., 1997), ionizing radiation (Paris et al., 2001), UV-radiation (Charruyer et al.,
2005), cisplatin (Rebillard et al., 2008) or tumor necrosis factor-α (Garcia-Ruiz et al., 2003)
to name a few.
2.2.2.1 Transcriptional level
Different settings lead to an up-regulation of ASM at the transcriptional level (Lecka-Czernik
et al., 1996; Langmann et al., 1999; Murate et al., 2002; Wu et al., 2005; Shah et al., 2008),
representing a slow regulative response to external stress stimuli. For example, differentiation
of macrophages is correlated with an up-regulated expression of ASM, since transcription
factors AP-2 and Sp1 are essential for both mechanisms (Langmann et al., 1999).
25
2. Introduction
2.2.2.2 Posttranscriptional level
Since four different ASM transcripts have been identified to date (see 2.2.1.1), a further
aspect of ASM regulation could be seen in alternative splicing. Thus far, only the full-length
variant ASM-1 has been proven to encode for an active enzyme, whereas ASM-2 and ASM-3
were catalytically inactive, since both encoded proteins show a disrupted catalytic domain
and the latter one additionally lacks the carboxy-terminal domain due to truncation
(Schuchman et al., 1991b). ASM-4 is lacking parts of the saposin-B-, the catalytic and the
carboxy-terminal domain, but its biochemical properties have not been determined so far.
Since only a single ASM splice variant is functional, cellular regulation of ASM at the RNA
level is highly assumed (Zeidan and Hannun, 2010). Mechanisms underlying alternative
splicing of ASM remain unknown and have to be elucidated.
2.2.2.3 Posttranslational modifications
In contrast to the slow transcriptional response to external stress stimuli, posttranslational
modifications serve as a fast regulative response of ASM. Activation of ASM in vitro results
from interaction with zinc (Schissel et al., 1998) and copper ions, the latter causing
dimerisation of two ASM molecules via the most carboxy-terminal located cysteine at
position 631 (Qiu et al., 2003). Also, an activating effect is exerted by protein kinase δ, which
phosphorylates the serine residue at position p.508 in the carboxy-terminal domain of ASM
(Zeidan and Hannun, 2007a).
2.2.2.4 Regulation via degradation
Degradation of ASM is mediated by antidepressant drugs. Different classes of antidepressant
drugs inhibit ASM activity (Kornhuber et al., 2008). These weak organic bases like
desipramine and imipramine act as functional inhibitors of ASM, since no direct inhibition is
exerted (Sakuragawa et al., 1977; Yoshida et al., 1985; Albouz et al., 1986; Kornhuber et al.,
2005). Rather, these antidepressants are supposed to impact the attachment of ASM to the
intra-lysosomal membrane. Thus, ASM is detached from the membrane and probably
26
2. Introduction
subjected to proteolytic cleavage inside the lysosome (Hurwitz et al., 1994a; Kölzer et al.,
2004).
Therefore, it is supposed that ASM activity is regulated via proteolytic degradation. Evidence
was offered by experiments using protease inhibitors, which were able to abolish effects
exerted by antidepressants (Hurwitz et al., 1994a).
2.2.3 Acid Sphingomyelinase and pathologic context
Imbalanced ASM activity is involved in different pathologic contexts. Either way of ASM
dysregulation induces pathologic effects – decreased as well as increased ASM activity.
2.2.3.1 Decreased activity of Acid Sphingomyelinase
Decreased ASM activity is the cause of Niemann-Pick disease type A (OMIM: 257200) and
B (OMIM: 607616), which is an autosomal recessive sphingolipidosis. Deficient ASM
activity occurs due to inherited sequence variations in the SMPD1 gene coding for ASM
(Brady et al., 1966). Type A is a severe infantile neurodegenerative disease, leading to death
within three years of life. It is characterized by massive hepatosplenomegaly and
psychomotor deterioration. In contrast, type B does not display neurological involvement, but
is a visceral form featuring hepatosplenomegaly and affecting the reticuloendothelial and
respiratory system. Patients survive into adulthood. As detected within cultured fibroblasts,
the ASM activity of patients is about 1-5% of normal activity levels (Levran et al., 1991).
On the genetic level, a variety of ASM sequence variations causing NPD type A and B are
known to date. Different genotypes lead to missense mutations, for example p.M381I,
p.R496L, p.G577S and p.L302P, which cause the loss of enzymatic activity, resulting in NPD
type A. Missense mutations p.R608del, p.S436R, p.G242R, p.N383S retain at least partial
catalytic activity and cause NPD type B, to name a few. Notably, mutations affect especially
the integrity of the catalytic and the C-terminal domain (Ferlinz et al., 1991; Levran et al.,
1991; Levran et al., 1992; Takahashi et al., 1992a; Takahashi et al., 1992b).
In total, genetic variation of ASM causing NPD makes clear that SNPs located within the
SMPD1 gene can severely affect ASM activity.
27
2. Introduction
2.2.3.2 Increased activity of Acid Sphingomyelinase
An increase in ASM activity – the opposite effect as seen in NPD – is associated with
different diseases. A common feature of these diseases is the involvement of cellular stress,
which in turn leads to elevated ceramide levels via the activation of ASM. Ceramide is
supposed to be the cause for apoptosis of hepatocytes as described for Morbus Wilson (Lang
et al., 2007) and for the onset of lung odems occurring during acute lung injuries (Goggel et
al., 2004). Interestingly, ASM knock out mice survive much longer after LPS induced
endotoxic shock syndrome compared to wild type mice (Haimovitz-Friedman et al., 1997).
Other diseases displaying elevated ASM activity levels are cystic fibrosis (Teichgräber et al.,
2008) and arteriosclerosis (Tabas, 2004). Also patients suffering from different neuropsychiatric disorders like Alzheimer’s dementia (He et al., 2008), status epilepticus (Mikati et
al., 2008) and major depressive disorder (Kornhuber et al., 2005) show increased ASM
activity levels.
The underlying mechanisms causing increased ASM activity in so many different pathologic
contexts remain unclear. But considering the correlation between unbalanced ASM activity
and pathologic consequences, a tight regulation of ASM is essential for intact cellular
functioning.
2.3 The role of Acid Sphingomyelinase in Major Depressive Disorder
Major depressive disorder (MDD) is a severe psychiatric disorder characterized by pathologic
changes concerning mood and motivation. According to ICD-10, following symptoms serve
for the diagnosis of MDD: Episodes of sadness, loss of interest, pessimism, negative beliefs
about the self, decreased motivation, behavioural passivity, changes in sleep, appetite and
sexual interest, and suicidal thoughts and impulses (DeRubeis et al., 2008). Patients suffering
from MDD show divergent patterns of symptoms, varying from somatic, psychomotor,
emotional, motivational, cognitive to social components (Davison, 1998; Hautzinger, 2003).
Prevalence for the onset of MDD is about 5% with women being affected much more
frequently than men (8% versus 3%) at averaged age between thirty and forty (Davison,
1998).
28
2. Introduction
The etiopathology of MDD is not clear to date. Several correlations of MDD with biological
changes within patients are identified, but do not serve as causative factors. The current
etiopathic model is the diathesis-stress-model: On the genetic level, certain dispositions are
inherited, leading to a potential vulnerability. Environmental factors like stress in
combination with this vulnerability trigger the manifestation of disease pattern. In depression,
altered genetic disposition concerns the neurotransmitter systems, certain hormonal systems
and circadian rhythms, which is affirmed by experimental data on the morphological,
functional and biochemical level (Köhler, 2005).
2.3.1 Results of the pilot study
In our pilot study, Kornhuber and colleagues compared the level of ASM activity in
peripheral mononuclear cells (PBMCs) between patients suffering from MDD (n=17) and
healthy volunteers (n=8). It was shown that depressed patients displayed a significantly
higher level of ASM activity compared to controls. Also, the severity of depression, indicated
by the Hamilton Scale of Depression (17-HDRS), and ASM activity levels were positively
correlated (Kornhuber et al., 2005).
The relation between major depression and ASM activity is strengthened by findings
concerning antidepressant drugs, since different antidepressant drugs like desipramine and
imipramine inhibit ASM activity (Albouz et al., 1986). This effect was also observed during
our pilot study. Cultured PBMCs from healthy volunteers were treated with imipramine or
amitriptyline, which resulted in a clear decrease of ASM activity. After removal of drugs,
ASM activity raised to normal levels again (Kornhuber et al., 2005).
According to our pilot study, there is a clear link between the psychiatric disease MDD and
elevated ASM activity levels. The type of interrelation however remains to be elucidated.
29
2. Introduction
2.3.2 Sequence variations of Acid Sphingomyelinase in depressed patients
Due to the results of the pilot study, we started to investigate the molecular mechanisms of
increased ASM activity levels in depressed patients. A possible reason for elevated ASM
activity levels could be seen in the enzyme itself. Analogous to the primary defect of ASM
causing NPD, also sequence variations leading to an increased intrinsic activity could exist,
as was reported for other enzymes, e. g. thermolysin (Kusano et al., 2010) or tripeptidyl
peptidase I (Walus et al., 2010). DNA sequence variations could lead to ASM proteins which
are prone to dysregulated activation and might predispose to psychiatric and neurological
diseases. Therefore, we investigated a collective of 58 patients suffering from major
depression for ASM sequence variations. We mapped the genetic variation of SMPD1 by
sequencing the complete gene locus including the adjacent 5’ area.
Several variations were discovered: 15 common SNPs with a minor allele frequency of >0.1;
seven rare SNPs with a minor allele frequency of <0.05; a common 5-bp deletion and a
common 6-bp repeat; a GC-rich region preceding exon 1 and two poly-T stretches in intron 2.
Among these variations were three missense variations leading to an altered mature protein
sequence. To get insight into the potential effect of those DNA sequence variations on the
properties of ASM enzymes, it would be inevitable to investigate ASM variants with respect
to their functionality. Due to amino acid exchange, different properties of the enzyme could
be altered, for example enzymatic activity and protein trafficking.
2.4 Aims of the study
As detected in the pilot study, depressed patients display enhanced activity of ASM within
their blood cells. To gain insight into the causative mechanism of elevated ASM activity
levels, different ASM sequence variations should be investigated. Despite the just slight
differences, a change of protein sequence could make these ASM proteins prone to
pathologic functioning.
ASM variants based on three different DNA sequence variations detected within depressed
patients should be characterized concerning their catalytic activity and cellular localization.
According to their alterations in protein sequence, differential characteristics of these variants
were supposed.
30
2. Introduction
To test if alternative splicing is involved in the regulation of ASM activity, also ASM
isoforms resulting from alternative splicing events should be characterized. Besides their
catalytic activity and cellular localization, expression patterns should be investigated. It was
supposed that alternatively spliced ASM variants could contribute to the regulation of cellular
ASM activity levels.
For these purposes, different ASM variants should be cloned and expressed as recombinant
fusion proteins. Using a cell culture model, their catalytic activity should be assayed and their
cellular localization determined. For expression analyses a RT-PCR system should be
established.
The aim of the study was to get a specific characteristic for each of the different ASM
variants. This would mean a deeper insight into the mechanism of dysregulated ASM
activity.
31
3. Material and Methods
3.1 Material
Technical equipment, auxiliary material and chemicals can be found in the appendix.
3.1.1 Enzymes
Restriction enzymes
NEB, Frankfurt, Germany
Shrimp alkaline phosphatase
Roche, Mannheim, Germany
T4-DNA-ligase
Invitrogen, Darmstadt, Germany
KAPA HiFi DNA polymerase
Peqlab, Erlangen, Germany
Tfi DNA polymerase
PfuUltra II Fusion HS DNA Polymerase
Stratagene, La Jolla, CA, USA
In-Fusion Advantage PCR Cloning Kit
Clontech, Mountain View, CA, USA
VILO cDNA reaction kit
3.1.2 Antibodies
3.1.2.1 Primary antibodies
Tab. 3.1 Primary antibodies in cotext of their application.
WB means application within Western Blot analyses, ICC within immunocytochemistry analyses.
Antibody
Species
Application
Source
Diluted in
FLAG M2
mouse
WB
Sigma
0.5 % Milk /PBS/T
1:1000
F1804
GAPDH
mouse
WB
Millpore
0.5 % Milk /PBS/T
1:200.000
MAB1374
LAMP1
mouse
ICC
DSHB Iowa
0.5 % fish skin
1:100
H4A3
gelatine buffer /PBS
PDIA4
rabbit
ICC
Proteintech
5% normal goat
1:100
14712-1-AP
serum /PBS
32
3.1.2.2 Secondary antibodies
Tab. 3.2 Secondary antibodies in cotext of their application.
WB means application within Western Blot analyses, ICC within Immunocytochemistry analyses.
Antibody
Species
Application
Source
Diluted in
anti-mouse IgG
goat
WB
Calbiochem
0.5 % Milk /PBS/T
1:20.000
401253
FITC anti-rabbit
goat
ICC
Calbiochem
0.5 % Milk /PBS/T
IgG
1:100
401214
3.1.3 Vectors and plasmids
3.1.3.1 Vectors
Vectors used in this study are shown in tab. 3.3.
Tab. 3.3 Vectors used in this study.
Vector
Size
pSC-B
3466 bp
Resistance
Ampicillin
Reference
Stratagene, La Jolla, USA
Flag-N2
4016 bp
Kanamycin
M. Reichel, Erlangen
pmCherry-N1
4699 bp
Kanamycin
Clontech, Mountain View, USA
3.1.3.2 Plasmids
Plasmids used for expression studies are shown in tab. 3.4.
33
Tab. 3.4 Plasmids used in this study.
Name
Insert
Size
Reference
Description
pASM_1522A_FLAGN2
ASM G506R
5.9 kb
this study
ASM wild type C-terminal FLAG epitope
pASM_1522G_FLAGN2
ASM wild type
5.9 kb
this study
ASM G506R C-terminal FLAG epitope
pASM_1522A_M381I_FLAGN2
ASM
5.9 kb
this study
ASM G506R/M381I C-terminal FLAG
G506R/M381I
epitope
pASM_1522G_M381I_FLAGN2
ASM M381I
5.9 kb
this study
ASM M381I C-terminal FLAG epitope
pASM_1522A_S508A_FLAGN2
ASM
5.9 kb
this study
ASM S508A/G506R C-terminal FLAG
S508A/G506R
epitope
pASM_1522G_S508A_FLAGN2
ASM S508A
5.9 kb
this study
ASM S508A C-terminal FLAG epitope
pASM_1522A_P325A_FLAGN2
ASM
5.9 kb
this study
ASM P325A/G506R C-terminal FLAG
P325A/G506R
epitope
pASM_1522G_P325A_FLAGN2
ASM P325A
5.9 kb
this study
ASM P325A C-terminal FLAG epitope
pASM_1522A_A487V_FLAGN2
ASM
5.9 kb
this study
ASM A487V/G506R C-terminal FLAG
A487V/G506R
epitope
pASM_1522G_A487V_FLAGN2
ASM A487V
5.9 kb
this study
ASM A487V C-terminal FLAG epitope
pASM_1522A_splvarIV_FLAGN2
ASM-5
5.8 kb
this study
ASM-5 C-terminal FLAG epitope
ASM_1522A_splvarV_FLAGN2
ASM-6
5.5 kb
this study
pASM_1522A_splvarVI_FLAGN2
ASM-7
5.2 kb
this study
pASM_1522A_cherryN1
ASM G506R
6.6 kb
this study
ASM G506R C-terminal cherry epitope
pASM_1522G_cherryN1
ASM wild type
6.6 kb
this study
ASM wild type C-terminal cherry epitope
pASM_1522A_M381I_cherryN1
ASM
6.6 kb
this study
ASM G506R/M381I C-terminal cherry
G506R/M381I
epitope
pASM_1522G_M381I_cherryN1
ASM M381I
6.6 kb
this study
ASM M381I C-terminal cherry epitope
pASM_1522A_S508A_cherryN1
ASM
6.6 kb
this study
ASM S508A/G506R C-terminal cherry
S508A/G506R
epitope
pASM_1522G_S508A_cherryN1
ASM S508A
6.6 kb
this study
ASM S508A C-terminal cherry epitope
pASM_1522G_P325A_cherryN1
ASM P325A
6.6 kb
this study
ASM P325A C-terminal cherry epitope
pASM_1522G_A487V_cherryN1
ASM A487V
6.6 kb
this study
ASM A487V C-terminal cherry epitope
pASM_1522A_splvarIV_cherryN1
ASM-5
6.5 kb
this study
ASM-5 C-terminal cherry epitope
pASM_1522A_splvarV_cherryN1
ASM-6
6.2 kb
this study
pASM_1522A_splvarVI_cherryN1
ASM-7
5.9 kb
this study
34
3.1.4 Biological material
3.1.4.1 Bacteria
For transformation XL1-Blue bacteria from Stratagene (La Jolla, CA, USA) were used in this
work.
3.1.4.2 Eucaryotic cells
The neuroglioma cell line H4, derived from brain of a caucasian male and purchased from
Promochem (Wesel, Germany) and the adenocarcinoma cell line HeLa, derived from cervix
of a black female and purchased from the American type culture collection (ATCC, Wesel,
Germany) were used in this work.
3.1.5 Buffers and solutions
Reagent-grade water from a MilliQ deionizator (Millipore, Eschborn, Germany) with a
specific resistance set to 18.2MW/cm3 was used for buffers, solutions and dilutions.
BES-Buffer
BES
2.14 g
NaCl
3.28 g
Na2HPO4x2H2O
0.054 g
adjust to pH 6.98 using NaOH
ad 200 ml using H20bidest
35
0.2 % Fish skin gelatine buffer
BSA Fraktion V
2.5 g
Fish Skin Gelatine
0.5 g
Triton X-100
0.25 g
ad 250 ml using 1xPBS
Glycerol solution
Glycerol
65 ml / 81.99 g
1 M MgSO4
10 ml
1 M Tris / HCl pH 8.0
2.5 ml
Ad 100 ml using H20bidest
10 x Laemmli buffer
Tris
Glycin
SDS
30 g
144 g
10 g
LB medium
Trypton
10 g
Yeast extract
5g
NaCl
5g
adjust to pH 7.0 using HCl
4x Lower gel buffer (1.5 M Tris-HCl, pH 8.8)
Tris
18.15 g
H20bidest
50 ml
adjust to pH 8.8 using 6 N HCl
36
Lysis buffer
1 M NaAc pH 5.0
2.5 ml
0.5 M EDTA
26 µl
2% NP40
500 µl
1 Tab. Complete w/o EDTA
PBS / T
10x PBS
500 ml
100x Tween
2050 ml
RIPA buffer
1.5 M Tris / HCl (pH7.5)
3333 µl
5 M NaCl
3003 µl
NP40 (Igepal CA-630)
1 ml
10% Na-Desoxycholat
5 ml
1% SDS
10 ml
10 Tab. Complete w/o EDTA
4 x SDS running buffer
Upper gel buffer (0.5 M Tris-HCl, pH 6.8)
5.0 ml
Glycerol
5.04 g
SDS
0.8 g
DTT (MW 154.3 g/mol)
0.62 g
Bromphenol blue
200 µl
37
SOC medium
Yeast extract
0.5 g
Trypton
2g
MgCl2
0.2 g
MgSO4
0.25 g
Glucose
0.36 g
ad 100 ml using H2Obidest
10 x TBS
Tris
60.55 g
NaCl
87.66 g
adjust to pH 7.5 - 7.6 using HCl
20 x TBE
Tris
432 g
Boric acid
220 g
0.5 M EDTA pH 8.0
160 ml
10 x Towbin-Puffer
Tris
Glycin
30 g
144 g
adjust to pH 8.6
1 x Towbin-Puffer / 20% MetOH
10x Towbin Puffer
100 ml
Methanol
200 ml
38
4x Upper gel buffer (0.5 M Tris-HCl, pH 6.8)
Tris
6.05 g
H20bidest
60 ml
adjust to auf pH 6.8 using 6 N HCl
2 x YT-Medium
Trypton
64 g
Yeast extract
40 g
NaCl
20 g
adjust to pH 7.0 using 5 N NaOH
3.2 Methods
3.2.1 Cell-biological methods
3.2.1.1 Cell culture
Human neuroglioma cells (H4) were cultivated in DMEM medium supplemented with 10%
(v/v) FCS and 4 mM L-glutamine. HeLa cells were cultivated in DMEM medium
supplemented with 10% (v/v) FCS and 2 mM L-glutamine. Starving medium was lacking
FCS and consisted of DMEM medium with 4 mM or 8 mM L-glutamine, respectively. Cells
were kept at 37 °C in a humidified atmosphere at 8.5% CO2 and were routinely split at a ratio
of 1:4. Regular tests monitored by the MycoAlert Mycoplasma Detection Kit (Lonza,
Cologne, Germany) for mycoplasma infection were always negative. All reagents used for
cell culture were purchased from Biochrom (Berlin, Germany).
39
3.2.1.2 Transient expression of ASM
For transfection, 1.5x10e5 H4 cells / 9.6 cm2 and 1x10e5 HeLa cells / 9.6 cm2 were seeded in
6-well plates with 2 ml culture medium. After 24 hours cells were grown to a confluence of
about 60%. Cells were transfected using calcium phosphate precipitation. 7.5 µg plasmid
were diluted in 90 µl H2O and mixed with 10 µl of 2.5 M calcium chloride, then added to 100
µl of 2x N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffer [50 mM BES,
280 mM NaCl, 1.5 mM Na2HPO4, pH 6.98; all obtained from Sigma-Aldrich (Munich,
Germany)] during softly vortexing, incubated for 20 min at room temperature and added to
the cells. The culture medium was changed 17 h after transfection including a washing step
using TBS buffer. For experiments under serum-starving conditions 48 h upon transfection
cells were shifted to serum-free medium for 24h. Transfection efficiency was about 60-70%
as determined from the number of GFP-positive cells using the expression vector pmaxFPgreen-C (Lonza, Cologne, Germany) in a parallel control transfection.
3.2.1.3 Immunocytochemistry
3x10e4 H4 cells / 2 cm2 were grown on glass slides in a 24-well dish (Nunc, Wiesbaden,
Germany) with 0.5 ml culture medium. 48 h upon transfection with ASM constructs cells
were washed with PBS (Gibco, Darmstadt, Germany) and fixed with 10% formalin for 15
min at room temperature. The slides were washed three times with PBS and permeabilized
with 0.1% Triton-X in PBS for 10 min. For LAMP1 staining, cells were blocked with 0.2%
Fish Skin Gelatine Buffer for 10 min and stained with anti-LAMP1 antibody (1:100; mouse)
for 2 h at room temperature. For PDIA4 staining, cells were blocked with 5 % goat normal
serum for 10 min and stained with anti-PDIA4 antibody (1:100; rabbit) for 2 h at room
temperature. For FLAG staining, cells were blocked with 10 % FCS for 10 min at room
temperature and stained with anti-FLAG antibody (1:500; mouse) for 3-5 h at room
temperature. After three washes with PBS, cells were incubated with goat-anti-mouse (1:100)
and goat-anti-rabbit (1:100) secondary antibodies coupled to FITC, respectively, for 2 h at
room temperature in indicated solutions. After three final washes with distilled water slides
were mounted in DAPI containing Fluorescent Mounting Medium (Dako, Hamburg,
Germany) and analyzed by fluorescence microscopy (Axiovert 135, Zeiss, Jena, Germany)
and laser scanning confocal microscopy (DM IRE2 / TCS SL, Leica, Solms, Germany).
40
3.2.1.4 Preparation of whole cell extracts
48 h or 72 h after transfection cells were washed with PBS (Gibco, Darmstadt, Germany). For
assaying ASM activity cells were lysed in 100 µl lysis buffer / 9.6 cm2 [250 mM sodium
acetate, pH 5.0 (Merck, Darmstadt, Germany), 0.1% NP-40 (Sigma-Aldrich, Munich,
Germany), 1.3 mM EDTA (Sigma-Aldrich, Munich, Germany), 1 tablet of complete mini
protease inhibitor mix/10 ml (Roche, Mannheim, Germany)]. For western blot analysis cells
were lysed in 100 µl RIPA buffer / 9.6 cm2 [50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1%
IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, all obtained from Sigma-Aldrich
(Munich, Germany), and complete protease inhibitor tablets from Roche (Mannheim,
Germany)] for 20 min at 4 °C. Cell debris was removed by centrifugation at 10.000 g for 15
min at 4 °C.
For MALDI-TOF MS analyses cells were washed in PBS (Gibco, Darmstadt, Germany) 24 h
or 48 h after transfection, lysed in 100 µl of MALDI- lysis buffer / 9.6 cm2 [20 mM Tris/HCl
pH 7.4, 100 µM butylated hydroxytoluene (spectrometric grade, Sigma-Aldrich, Munich,
Germany)], immediately frozen in liquid nitrogen and stored at -80 °C.
3.2.2 DNA technology
3.2.2.1 PCR for cloning approach
For cloning into pSC-B vector full-length ASM transcripts were amplified by PCR in a total
reaction volume of 50 µl with 1 µl of undiluted cDNA, 1 U KAPA HiFi DNA polymerase
with GC-buffer, 1.7 mM MgCl2, 0.3 mM dNTPs (Peqlab, Erlangen, Germany) and 0.24
pmol/µl oligonucleotide primers (Operon, Ebersberg, Germany). For sub-cloning into FLAGN2 expression vector 500 ng plasmid DNA were used as template. Primers flanking the ATG
initiation codon and the stop codon according to the reference sequence NM_000543.4
carried BspE1 and BglII restriction sites (Tab. 3.5, indicated in bold). For truncated variants
specific reverse primers were used (Tab. 3.5). Cycle parameters were as follows: initial
41
denaturation at 95 °C for 2 min, 35 cycles at 98 °C for 20 sec, 73 °C for 15 sec, 68 °C for 1
min, and a final extension at 68 °C for 5 min.
PCR products were purified using the QIAquick PCR purification kit or QIAquick Gel
Extraction Kit (Qiagen, Hilden, Germany) according to manufacturer’s recommendations.
Tab. 3.5 Oligonucleotides used for cloning of ASM into pSC-B vector and FLAG-N2 expression vector.
ASM variant
Primer
ASM
Sequence 5’ -> 3’
Forward
TCCCTCCGGAATGCCCCGCTACGGAGC
variants
Reverse
GCGAGATCTGCAAAACAGTGGCCTTGG
ASM-6
Forward
Reverse
GGAAGATCTACGGGAACAAAAATTCATATGAAGAGAGAGG
full
length
ASM-7
Forward
Reverse
GGAAGATCTCGGGAACAAAAATTCATATTGAGAGAGATG
3.2.2.2 Sub-cloning of ASM into pmCherry-N1 expression vector
Cloning ASM variants from FLAG-N2 expression vector into pmCherry-N1 expression
vector for better tracking due to the fluorescent epitope was carried out using In-Fusion
Advantage PCR Cloning Kit according to the manufacturer’s instructions (Clontech,
Mountain View, CA, USA). Primers for this procedure are shown in table 3.6. Cycle
parameters were as follows: initial denaturation at 95 °C for 2 min, 35 cycles at 98 °C for 20
sec, 68 °C for 15 sec, 68 °C for 1 min, and a final extension at 68 °C for 5 min. Constructs
were sequenced completely to exclude any mutations.
Tab. 3.6 Oligonucleotides used for cloning of ASM from FLAG-N2 vector into pmCherry-N1 vector.
ASM variant
Primer
ASM
Forward
TCAGATCTCGAGCTCAAGCTTGCCGCCATGCCCCGCTACGGAGC
full-length
Reverse
CTCACCATGGTGGCGACCGGTACGCAAAACAGTGGCCTTGG
variants
ASM-6
ASM-7
Forward
Reverse
CTCACCATGGTGGCGACCGGTACTTTGGTACACACGGTAACCTGC
Forward
Reverse
CTCACCATGGTGGCGACCGGTACCGGGAACAAAAATTCATATTGAGAGAG
42
3.2.2.3 Site-directed mutagenesis
The QuickChange XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) was
used to mutate ASM wild type sequence by PCR. Mutagenesis was done on ASM wild typeFLAG construct according to the manufacturer’s instructions with primers for different ASM
mutations (tab. 3.7). Cycle parameters were as follows: initial denaturation at 95 °C for 1
min, 18 cycles at 95 °C for 50 sec, 60 °C for 50 sec, 68 °C for 6 min, and a final extension at
68 °C for 7 min. Constructs were sequenced completely to exclude any spontaneous further
mutations.
Tab. 3.7 Oligonucleotides used for site-directed mutagenesis of ASM wild type-FLAG construct to
generate different ASM variants.
Construct
Primer
pASM_1522A_M381I_FLAGN2
Forward
CCGCCTCATCTCTCTCAATATTAATTTTTGTTCCCGTGAGAAC
Reverse
GTTCTCACGGGAACAAAAATTAATATTGAGAGAGATGAGGCGG
Forward
CCGCCTCATCTCTCTCAATATTAATTTTTGTTCCCGTGAGAAC
Reverse
GTTCTCACGGGAACAAAAATTAATATTGAGAGAGATGAGGCGG
Forward
GAAACTACTCCAGGAGCGCTCACGTGGTCCTGGAC
Reverse
GTCCAGGACCACGTGAGCGCTCCTGGAGTAGTTTC
Forward
GAAACTACTCCAGGAGCGCTCACGTGGTCCTGGAC
Reverse
GTCCAGGACCACGTGAGCGCTCCTGGAGTAGTTTC
Forward
GGGTAACCATGAAAGCACAGCTGTCAATAGCTTCCCTCC
Reverse
GGAGGGAAGCTATTGACAGCTGTGCTTTCATGGTTACCC
Forward
GGGTAACCATGAAAGCACAGCTGTCAATAGCTTCCCTCC
Reverse
GGAGGGAAGCTATTGACAGCTGTGCTTTCATGGTTACCC
Forward
CTTCCTGGCACCCAGTGTAACTACCTACATCGGCC
Reverse
GGCCGATGTAGGTAGTTACACTGGGTGCCAGGAAG
Forward
CTTCCTGGCACCCAGTGTAACTACCTACATCGGCC
Reverse
GGCCGATGTAGGTAGTTACACTGGGTGCCAGGAAG
pASM_1522G_M381I_FLAGN2
pASM_1522A_S508A_FLAGN2
pASM_1522G_S508A_FLAGN2
pASM_1522A_P325A_FLAGN2
pASM_1522G_P325A_FLAGN2
pASM_1522A_A487V_FLAGN2
pASM_1522G_A487V_FLAGN2
3.2.2.4 PCR for detection of splice variants
For specific detection of different ASM splice variants, RT-PCR was carried out in a total
reaction volume of 30 µl with 3 µl of undiluted cDNA, using 0.1 U/µl Tfi DNA polymerase
with respective Tfi-buffer, 1.7 mM MgCl2, 0.3 mM dNTPs (Invitrogen, Darmstadt, Germany)
and
0.24
pmol/µl
oligonucleotide
primers
(Operon,
Ebersberg,
Germany).
The
oligonucleotides and annealing temperatures used for ASM amplification are shown in table
3.8. Hypoxanthine-phosphoribosyl transferase (HPRT) encoding transcripts were amplified
43
as control (forward, 5’-TCCGCCTCCTCCTCTGCTC-3’; reverse, 5’-GAATAAACAC
CCTTTCCAAATCCTCA-3’).
Cycle parameters for all ASM-products were as follows: initial denaturation at 94 °C for 2
min, 35 cycles at 94 °C for 30 sec, ASM variant specific primer annealing temperature for 1
min, 72 °C for 1 min, and a final extension at 72 °C for 3 min.
Amplification products were analysed by agarose gel electrophoresis followed by ethidium
bromide staining (Roth, Karlsruhe, Germany). A molecular weight standard (Generuler 100
bp plus DNA ladder; Fermentas, St. Leon-Rot, Germany) was used for size determination.
Tab. 3.8 Oligonucleotides used for amplification of ASM splice variants.
ASM variant
ASM-1
ASM-5
ASM-6
ASM-7
Primer
Annealing temperature
62.5 °C
Forward
CCTCAGAATTGGGGGGTTCTATGC
Reverse
CACACGGTAACCAGGATTAAGG
Forward
TGCAGACCCACTGTGCTGC
Reverse
GCCAGAAGTTCTCACG GGAACTG
Forward
CGTGAGAACTTCTGGCTCTTGATC
Reverse
CTGCAAGGATGTGGGGCTGA
Forward
TGCAGACCCACTGTGCTGC
Reverse
ACCCCCCAATTCTTTCTTTTCCC
66.5 °C
64.5 °C
62.5 °C
3.2.2.5 Agarose gel electrophoresis
Using agarose gel electrophoresis DNA and RNA molecules can be identified and purified
efficiently. Driven by voltage negatively charged molecules migrate to the positively charged
anode, thereby being subject to separation by size. Ethidium bromide is incorporated into
nucleic acids and fluoresces under UV light of 302 nm. For molecules larger than 1 kb a 1%
agarose gel was used, for those smaller than 1 kb a 2% gel. Agarose was boiled and
completely dissolved in 1x TBE buffer, cooled down to 50°C, and ethidium bromide (final
concentration 0.5 µg/ml) was added. The dried gel was covered with TBE buffer and loaded
with DNA or RNA mixed with 1x loading buffer (Fermentas, St. Leon-Rot, Germany). For
size determination a molecular weight marker was loaded (Generuler 100 bp plus DNA
ladder; Fermentas, St. Leon-Rot, Germany). Voltage of 5-10 V / cm was applied for 120 min,
then nucleic acids within the gel were detected by UV light (302 nm).
44
3.2.2.6 Cloning of ASM into FLAG-N2 expression vector
Purified PCR products of full-length ASM transcripts were cloned for analysis into p-SCB
vector using StrataClone Blunt PCR cloning kit (Agilent, Waldbronn, Germany) according to
manufacturer’s recommendations. For expression analysis, open reading frames were
amplified by PCR from pSC-B-ASM plasmids. Purified PCR products were digested with
restriction endonucleases BspE1 (recognition site T/CCGGA) and BglII (recognition site
A/GATCT) (NEB, Frankfurt, Germany) and inserted between XmaI (recognition site
C/CCGGG) and BamHI (recognition site G/GATCC) sites within the multiple cloning site of
the FLAG-N2 expression vector in frame with the coding sequence for the 16 amino acid
FLAG-epitope (GTGDYKDDDDKRSLYK) (Fig. 3.1). In detail, vector DNA was digested
with respective restriction endonucleases and purified. 100 ng linearized vector DNA was
dephosphorylated using 1 U Shrimp Alkaline Phosphatase (Roche, Mannheim, Germany) at
37°C for 1 h, followed by an inactivation step at 65°C for 20 min. Ligation of DNA
fragments, 100 ng vector DNA and 6-fold molar excess of insert DNA, was done using T4Ligase (Invitrogen, Darmstadt, Germany) at 16°C for 24 h. After transformation of E. coli,
positive clones were selected and DNA was isolated using QIAprep spin Miniprep kit
(Qiagen, Hilden, Germany). Constructs were sequenced completely to exclude any mutations.
45
Fig. 3.1 Plasmid construction for expression of ASM-FLAG constructs.
ASM cDNA was amplified by PCR and inserted into FLAG-N2 expression vector via restriction endonuclease
cleavage sites BspE1 and BglII in frame with the coding sequence for C-terminal FLAG-epitope.
3.2.2.7 Transformation of bacteria with DNA
For calcium chloride transformation 50 µl competent E. coli XL-1 Blue cells (Stratagene, La
Jolla, CA, USA) were thawed and incubated with 10-50 ng plasmid DNA on ice. After heat
shock of 45 sec at 42 °C cells were placed on ice for 2 min. Upon adding 450 µl SOC
medium (37 °C) transformed bacteria were incubated on a shaker at 37 °C and 200 U/min for
60 min. 150 µl were plated on LB plates containing ampicillin (50 µg/ml) or kanamycin (100
µg/ml). Plates were incubated over night at 37 °C.
46
3.2.2.8 Isolation of plasmid DNA
For isolating plasmid DNA 300 ml 2xYT medium containing kanamycin were incubated with
a single colony of bacteria containing the respective plasmid. Bacteria were incubated over
night at 37°C and shaken at 150U/min. By centrifugation at 4500g and 4°C for 20 min
bacteria were pelleted. Preparation of plasmid DNA was carried out using Nucleobond xtra
maxi EF (Macherey Nagel, Düren, Germany) according to manufacturer’s recommendations.
Upon alkaline lysis of bacteria plasmid DNA is prepared by chromatography with a yield of
500-2000 µg DNA. Endotoxins were removed since plasmid DNA was used for cell culture
experiments.
3.2.2.9 Sequencing of cDNA
Sequencing of cDNA was carried out according to the chain-termination method by Sanger
(Sanger et al., 1977) using Big Dye Mix (Perkin Elmer, Waltham, MA, USA) according to
manufacturer’s recommendations. Following amplification programme was used: 96 °C for
30 sec; 50 °C for 5 sec; 60 °C for 4 min repeated in 30 cycles and final cycle 15 °C for 30
min. Primer are indicated in table 3.9. Analysis of sequencing was done by Lehrstuhl für
Humangenetik, University Hospital Erlangen.
Tab. 3.9 Oligonucleotides used for sequencing of ASM plasmids.
Primer
Sequence (5`→3`)
ASM binding site
ASM_repeat_fw
GGACTCCTTTGGATGGGCCT
85-104
ASM_repeat_rev
CTTTGCAGATTGGGCAGG
269-286
huASM#2_fw
AAGGAACCCAATGTGGCTCG
319-338
ASM_Exon2-1_fw
TGCAGACCCACTGTGCTGC
669-687
huA-SMase_fw
ATTGAGGGCAACCACTCCTC
1000-1030
NM_000543_BamH1_fw
CACGGATCCCGCAGGAC
1194-1210
47
3.2.3 RNA isolation and cDNA synthesis
3.2.3.1 RNA isolation
Total RNA was isolated from peripheral blood mononuclear cells, whole blood and from cell
lines. PBMCs drawn from healthy volunteers were isolated from 30 ml of blood using Ficolldensity gradient centrifugation (Biocoll Separation Solution, Biochrom, Berlin, Germany).
RNA from PBMCs and cell lines was extracted using Qiazol reagent (Qiagen, Hilden,
Germany). 1 ml Qiazol was added to the cell pellet, which was sheared using a 20-gauge
needle. After 5 min incubation at room temperature 200 µl chloroform were added and
mixed. To separate RNA from DNA a centrifugation step was done at 13.000 rpm and 4°C
for 15 min. The upper phase contains total RNA which was collected. To precipitate RNA
500 µl isopropanol were added and mixed. After 15 min incubation at room temperature
RNA was pelleted by centrifugation at 13.000 rpm and 4°C for 10 min. Two washing steps
were done using 75% ethanol, and the dried RNA pellet was resuspended in H2O. For
complete resolving RNA was incubated at 55°C for 10 min.
RNA of depressed patients was isolated from whole blood within the ADT-study conducted
at the Department of Psychiatry and Psychotherapy. RNA isolation of whole blood was done
using PAXgene blood RNA kit (Qiagen, Hilden, Germany) following the manufacturer’s
recommendations.
The concentration of RNA was determined photometrically (Nanodrop, Peqlab, Erlangen,
Germany) at 260 nm, and RNA integrity was assessed by non-denaturing agarose gel
electrophoresis and capillar gel electrophoresis (Experion, BioRad, Munich, Germany).
The collection of blood samples was approved by the local Ethics Committee and conducted
in accordance with the Declaration of Helsinki. Written informed consent was obtained from
all participants.
48
3.2.3.2 Synthesis of cDNA
Synthesis of cDNA was carried out in a total reaction volume of 20 µl with 1 µg total RNA
using the VILO cDNA reaction kit according to manufacturer’s recommendations
(Invitrogen, Darmstadt, Germany).
3.2.4 Protein-biochemical analyses
3.2.4.1 Determination of protein concentration
The protein concentration of cell extracts was determined using the BCA assay (BioRad,
Munich, Germany) according to manufacturer’s recommendations.
3.2.4.2 SDS-PAGE and Western blot analysis
Proteins were separated by polyacrylamide gel electrophoresis under denaturing conditions in
a minigel chamber (Mini Protein 3 cell, BioRad, Munich, Germany). Sample aliquots were
denatured in SDS-running buffer for 5 min. Total protein (10 µg) was separated by 10%
sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which was
composed of a 4% acrylamide stacking gel (upper SDS gel) and a 10% acrylamide separating
gel (lower SDS gel):
Lower SDS gel (10 % T)
4x Lower gel buffer (1.5 M Tris-HCl, pH 8.8)
2.5 ml
30%T/2.6%C Acrylamide
3.3 ml
10% SDS
0.1 ml
H20bidest
4.02 ml
10% APS
40 µl
TEMED
10 µl
49
Upper SDS gel (4 % T)
4x Upper gel buffer (0.5 M Tris-HCl, pH 6.8)
1.25 ml
30%T/2.6%C Acrylamide
0,667 ml
10% SDS
50 µl
H20bidest
2,973 ml
10% APS
40 µl
TEMED
10 µl
For controlling gel electrophoresis a pre-stained molecular weight standard was loaded (Page
Ruler Prestained Protein Ladder, Fermentas, St. Leon-Rot, Germany), for determining the
size of proteins an unstained molecular weight standard (Magic Mark, Invitrogen, Darmstadt,
Germany). Gel electrophoresis was performed in Laemmli buffer (Laemmli, 1970) at 120 V
for about 2 h.
Separated proteins were transferred to a PVDF- membrane (Millipore, Schwalbach/Ts,
Germany) by semi-dry blotting (SemiPhor Transfer Unit TE77, Amersham, Munich,
Germany) at 100 mA for 1 h using Towbin buffer.
Blots were washed with PBS/ T and incubated in 2.5% milk powder (Roth, Karlsruhe,
Germany) and 0.1% Tween 20 (Sigma-Aldrich, Munich, Germany) in PBS overnight at 4 °C
to reduce unspecific binding. After washing with PBS/0.1% Tween 20 for 15 minutes, blots
were incubated for 4 h at room temperature with primary monoclonal FLAG-antibody
derived from mouse (1:1000; Sigma-Aldrich, Munich, Germany), and for 1 hour with
primary Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody from mouse
(1:200.000; Millipore, Schwalbach/Ts, Germany). After washing in PBS/0.1% Tween 20
three times, blots were incubated for 1 hour at room temperature with secondary goat antimouse IgG antibody, coupled to horseradish peroxidase (1:10.000; Dianova, Hamburg,
Germany). All antibodies were diluted in 0.5% milk powder solution (Roth, Karlsruhe,
Germany) and 0.1% Tween 20 in PBS (Sigma-Aldrich, Munich, Germany). Detection was
performed using freshly mixed enhanced chemiluminescence developer reagent (ECL,
Amersham, Munich, Germany). After incubation of 5 min signals were visualized on a highsensitivity camera device (FluorSMax, BioRad, Munich, Germany).
50
3.2.4.3 In vitro determination of ASM activity
Quantitative analysis of ASM activity was performed on 10 µg of total cellular protein
extract, diluted in 250 µl of lysis buffer; 440 pmol choline methyl-14C sphingomyelin
(Perkin-Elmer, Waltham, MA, USA) was suspended in 30 µl of enzyme buffer [250 mM
sodium acetate pH 5.0 (Merck, Darmstadt, Germany), 0.1% NP-40 (Sigma-Aldrich, Munich,
Germany), 1.3 mM EDTA (Sigma-Aldrich, Munich, Germany)] and added to the protein
extract. The enzymatic reaction was incubated at 37 °C for 20 min. The reaction was stopped
by addition of 800 µl of chloroform/methanol (2:1), and phases were separated by
centrifugation. Radioactivity of the
14
C-labelled product phosphorylcholine in the aqueous
phase was determined by liquid scintillation counting and allowed quantification of ASM
activity. Results are given as ASM activity in nmol/mg/h and refer to the mean values
resulting from three independent experiments.
Alternatively, the activity of ASM was determined using the fluorescent substrate BODIPYC12-sphingomyelin (Invitrogen, Darmstadt, Germany). The reaction consisted of 116 pmol
substrate in 200 mM sodium acetate buffer (pH 5.0) with 500 mM NaCl and 0.2% NP-40 in a
total volume of 100 µl and was started by the addition of 2 µl cell lysate typically
corresponding to 0.5-2 µg protein. After incubation for 0.5 to four hours at 37°C, the
fluorescent product ceramide and un-cleaved substrate were extracted by addition of 250 µl
chloroform/methanol (2:1). Following vortexing and centrifugation, the lower phase was
concentrated in a SpeedVac vacuum concentrator and spotted on silica gel 60 plates (Merck,
Darmstadt, Germany). Ceramide and sphingomyelin were separated by thin layer
chromatography using chloroform/methanol (80:20) as a solvent and quantified on a
Typhoon Trio scanner (GE Healthcare, 488 nm excitation, 520 nm emission, 280 V, 100 µm
resolution) with the QuantityOne software (BioRad, Munich, Germany). Enzymatic activity
of ASM was calculated as hydrolysis rate of sphingomyelin to ceramide per time and per
protein of the cell lysate sample (pmol/µg/h).
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3.2.4.4 MALDI-TOF MS analysis of cellular ceramide and sphingomyelin levels
After thawing, cell samples were homogenized and sonicated. Lipids were subjected to
classical chloroform/methanol extraction (Folch et al., 1957), transferred to a clean tube,
dried, and resolved (2% w/v) in chloroform. For MALDI-TOF MS, samples were mixed with
matrix consisting of 0.5 M 2.5-dihydroxybenzoic acid in methanol with 0.1% trifluoroacetic
acid (spectrometric grade, Sigma-Aldrich, Munich, Germany) (Schiller et al., 2004). Aliquots
were spotted on a steel target and subjected to mass spectrometric analysis. Mass spectra
were obtained using an Autoflex MALDI-TOF MS (Bruker Daltonics, Bremen, Germany)
equipped with a nitrogen laser (λ= 337 nm). Mass spectra were acquired in reflectron and
positive ion mode. Each spectrum consists of the average of 100 laser shots.
Obtained mass-to-charge ratio (m/z) values were compared to the lipid database LIPID
MAPS (http://www.lipidmaps.org), according to which relevant corresponding ceramide and
sphingomyelin species were defined for the semi-quantitative analysis (Löhmann et al.). The
relative intensities of both mass signals [M+H]+ and [M+Na]+ or [M+K]+, respectively, were
determined and summarized. The ratio of ceramide to sphingomyelin intensities was
calculated for each individual spectrum and averaged over three individual samples.
3.2.5 In silico analyses and statistics
In silico analyses of ASM orthologous transcripts were performed using the NCBI database
GenBank. Alignments were generated with the ClustalW alignment of the European
Bioinformatics Institute (www. ebi.ac.uk/clustalw).
The molecular weight of the discussed proteins was calculated using the molecular weight
calculator at www.sciencegateway.org/ tools/proteinmw.htm.
Images were edited with Adobe Photoshop CS5.
For statistical analysis GraphPad-Prism 4.0 software (GraphPad Software Inc., La Jolla) was
used.
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4. Results
4.1 Impact of DNA sequence variations on Acid Sphingomyelinase activity
For investigating effects of DNA sequence variations on properties of the ASM enzyme, we
cloned five different ASM missense variations. To establish a model system for validating the
cell-biological methods, we used ASM variants with already published characteristics. For
this purpose we controlled properties of ASM wild type (WT), the ASM variant c.1528T>G /
p.S508A, which was recently investigated (Jenkins et al., 2010a; Jenkins et al., 2010b), and
the Niemann-Pick disease type A variant c.1146G>T / p.M381I, described by Takahashi et
al. (1992b).
ASM sequencing of 58 patients suffering from major depression revealed three ASM
missense variations: Variation c.973C>G / p.P325A is located within the region coding for
the catalytic domain, and variations c.1460C>T / p.A487V and c.1522G>A / p.G506R are
located in the C-terminal coding region of ASM (Fig. 4.1). These ASM variants should be
characterized.
Fig. 4.1 Position of ASM variations investigated in this study.
Five different ASM variants were characterized. ASM wild type and ASM variants p.S508A and p.M381I
served as model system. ASM variations located within the catalytic domain (p.P325A) and the C-terminal
domain (p. A487A, p.G508R) were under investigation. Scheme is drawn to scale.
In our approach, we cloned these different ASM variations and transiently expressed the
resulting ASM variants in a cell culture system. Due to the putative N-terminal signal
peptide, we expressed ASM variants as C-terminal epitope-tagged fusion proteins. First of
all, we cloned cDNA of ASM wild type derived from RNA of a healthy volunteer into the
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4. Results
expression vector FLAG-N2 for protein-biochemical analyses. For a better tracking of ASM
variants in localization studies, we sub-cloned ASM wild type into expression vector
pmCherry-N1, which contains the sequence for a red fluorescent protein (Fig. 4.2). The
resulting ASM-FLAG and ASM-Cherry constructs were sequenced to confirm their integrity.
Fig. 4.2 Cloning approach for the generation of recombinant ASM constructs containing different ASM
variations and FLAG- or Cherry-epitope.
Starting from direct cloning of donor cDNA into expression vector FLAG-N2, all further ASM constructs were
obtained by mutagenesis or infusion cloning, respectively.
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4. Results
4.1.1 Expression of recombinant ASM variants
To characterize the different ASM variants biochemically, ASM-FLAG variants were
transiently expressed in HeLa cells. Expression of ASM-FLAG variants was confirmed by
Western blot analyses 48 h upon transfection using FLAG antibody. Detection of the
housekeeping protein GAPDH served as loading control. Analyses showed that all cloned
ASM variants were translated into protein and displayed predicted sizes. Lysates derived
from vector-transfected cells served as negative control and did not result in detectable
signals (Fig. 4.3).
Fig. 4.3 All investigated ASM variants are translated into protein and display predicted sizes.
ASM-FLAG constructs were transiently expressed in HeLa cells. Lysates were analysed by Western blotting 48
h after transfection. Expression was detected using FLAG antibody. Lysates of vector-transfected cells served as
negative control, GAPDH was detected for loading control. Indicated are representative Western blot analyses
of three independent experiments. M indicates size marker.
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4. Results
Of note, protein expression levels differed highly between ASM variants with weakest
expression of p.G506R/S508A (27% of WT expression) and strongest expression of
p.G506R/M381I and p.M381I (309% and 290% of WT expression) (Fig. 4.4). To account for
expression differences, all further biochemical analyses were corrected for respective
expression levels.
Fig. 4.4 ASM variants were transiently expressed to a different extent.
Protein expression of ASM p.G506R/S508A was reduced to 27% of WT expression, whereas ASM
p.G506R/M381I and p.M381I were three-fold stronger expressed than ASM WT. Other variants reached values
between these extremes. ASM-FLAG constructs were transiently expressed in HeLa cells. Expression of each
ASM variant was verified by Western blot analyses. Signals derived from immunoblot band intensities were
quantified using QuantityOne software (BioRad, Munich, Germany). Expression of ASM variants is given in
percentage expression relative to transient expression of ASM WT (100%). Band intensities of transiently
expressed ASM variants were normalized to the respective GAPDH signal. Displayed data represent mean
values of three independent experiments, error bars represent SD.
4.1.2 Characterization of ASM variants used for model system
For validating the methodical approach, a model system composed of three different ASM
variants was analysed. The ASM wild type is described as the catalytic active enzyme located
inside the lysosome. The artificial ASM variant p.S508A was characterized as an ASM
variant exerting about 30-60% of enzymatic wild type activity with lysosomal localization.
Changing the phosphorylation site p.508 by the substitution of serine by alanine is shown to
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4. Results
affect activation of ASM and seems to be crucial for secretion of ASM (Jenkins et al., 2010a;
Jenkins et al., 2010b). The Niemann-Pick type A variant p.M381I was described by
Takahashi et al. and is characterized by an exchange of methionin by isoleucin, both being
apolar amino acids. This variant did not exert any catalytic activity (Takahashi et al., 1992b)
and should be trapped inside the ER like Niemann-Pick variants according to Jenkins et al.
(2010b).
4.1.2.1 Catalytic activity of ASM model system variants
In our approach, the intrinsic activity of different ASM variants was determined upon in vitro
over-expression. ASM-FLAG constructs were transiently expressed in HeLa cells. Cells were
lysed 48 h upon transfection and ASM activity of crude cell lysates was analysed using a
radioactive enzyme-activity-assay (Gulbins and Kolesnick, 2000). Over-expression of ASM
wild type elevated ASM activity averaged 35-fold with respect to endogenous ASM activity
levels which were controlled by vector control conditions (223 nmol/mg/h vs. 6.4
nmol/mg/h). For evaluating ASM variants, ASM wild type activity was set on 100%. Due to
differential protein expression levels, exogenous enzymatic activity was normalized
according to quantified Western blot signals and corrected for loading variations. Therefore,
enzymatic activity of each investigated ASM variant was given in exogenous ASM activity
per expressed protein with respect to ASM wild type activity.
Compared to ASM wild type, ASM variant p.S508A exerted an enzymatic activity of 33%
(SD ± 2.3), whereas Niemann-Pick variant p.M381I was catalytically inactive. The difference
between ASM wild type and model systems variants was statistically significant (Fig. 4.5).
Enzymatic activities of our ASM model system variants therefore reveal similar levels of
ASM activity as described earlier. The ASM variants wild type, p.S508A and p.M381I thus
qualify this established method for biochemical analyses of further ASM variants.
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4. Results
Fig. 4.5 ASM model system variants display predicted enzymatic activity levels.
Compared to ASM wild type, ASM variants p.S508A and p.M381I exerted 33% (SD ± 2.3) and 0% catalytic
activity, respectively. ASM-FLAG constructs were transiently expressed in HeLa cells. Lysates were subjected
to an in vitro enzyme-activity-assay 48 h upon transfection. Catalytic activity of ASM variants is given in
exogenous ASM activity levels per expressed protein in percentage relative to activity of ASM WT (100%).
Displayed data represent mean values of three independent experiments, error bars represent SD. Statistical
significance was calculated with respect to WT (* p<0.05).
4.1.2.2 Localization of ASM model system variants
With the aim to track the localization of ASM variants we investigated the subcellular
localization of ASM-Cherry constructs, since the red fluorescent tag allows easy detection.
H4 cells were transiently transfected with ASM-Cherry constructs of ASM wild type and
ASM variants p.S508A and p.M381I. After 48 h cells were fixed and immunostained for the
lysosomal marker protein LAMP1 and the endoplasmic reticulum marker protein PDIA4 for
determining localization of lysosomes and endoplasmic reticulum, respectively. Cells were
analysed for their subcellular localization pattern using fluorescence microscopy and for
potential co-localization with lysosomes or endoplasmic reticulum using confocal
microscopy.
ASM wild type localized within a dotted subcellular pattern. Also ASM variant p.S508A was
found to occur within dotted structures, in sharp contrast to p.M381I (Fig. 4.6).
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4. Results
Fig. 4.6 Subcellular localization patterns of ASM model system variants ASM wild type and ASM
variants p.S508A and p.M381I.
ASM wild type (A) and p.S508A (B) localized in dotted subcellular patterns, whereas p.M381I (C) did not.
ASM-Cherry constructs were detected by fluorescence (Excitation: 587 nm, Emission: 610 nm). Shown are
representative cells investigated by fluorescence microscopy. The indicated scale covers 10 µm. Localization
experiments were reproduced three times.
Confocal microscopy revealed that signals of ASM-Cherry wild type construct and LAMP1
were co-localized. Therefore, ASM wild type was found to be localized within lysosomal
compartments. Also p.S508A showed co-localization with lysosomal marker protein LAMP1
(Fig. 4.7 A). In contrast, p.M381I showed co-localization with endoplasmic reticulum marker
PDIA4 (Fig. 4.7 B).
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4. Results
Fig. 4.7 Co-localization of ASM model system variants with subcellular organelles.
ASM wild type and ASM variant p.S508A were localized within lysosomes, ASM variant p.M381I within the
endoplasmic reticulum. Confocal microscopy revealed co-localization of ASM wild type and p.S508A with
lysosomal marker protein LAMP1 (A) and of ASM variant p.M381I with endoplasmic reticulum marker PDIA4
(B). ASM-Cherry constructs were detected by fluorescence (Excitation: 587 nm, Emission: 610 nm). Marker
proteins were detected by immunofluorescence with anti-LAMP1 and anti-PDIA4 primary antibody derived
from mouse and FITC coupled anti-mouse-IgG secondary antibody derived from goat (Excitation: 488 nm,
Emission: 505 – 530 nm). Shown are representative cells detected by confocal microscopy, displaying ASMCherry constructs in red, marker proteins in green and merged signals. The indicated scale covers 10 µm. Colocalization experiments were reproduced three times.
Compared to already published localization studies, ASM-Cherry wild type construct
displayed identical localization patterns as did endogenous ASM wild type (Jones et al.,
2008) and as described for ASM in different over-expression studies (Ni and Morales, 2006;
Jin et al., 2008). This indicates that the C-terminal Cherry-epitope does not influence proper
localization of ASM inside the lysosome. Also published data about localization of ASM
variant p.S508A and predicted localization of ASM variant p.M381I match our results
concerning localization within lysosomes or endoplasmic reticulum, respectively (Jenkins et
al., 2010a; Jenkins et al., 2010b).
4.1.3 Characterization of ASM variant p.P325A
The rare ASM variant p.P325A shows an amino acid exchange within the catalytic domain of
ASM. The apolar amino acid proline is substituted by the apolar amino acid alanine. This
variant was also detected in patients suffering from Niemann-Pick disease type B (Simonaro
et al., 2002).
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4. Results
4.1.3.1 ASM variant p.P325A is catalytically inactive
Catalytic activity of ASM variant p.P325A was determined analogous to the approach of
determining enzymatic activity of ASM variants used for our model system. Compared to
ASM wild type, ASM variant p.P325A did not exert catalytic activity (Fig. 4.8).
Fig. 4.8 ASM variant p.P325A does not exert catalytic activity compared to ASM wild type.
ASM-FLAG constructs were transiently expressed in HeLa cells. Lysates were subjected to an in vitro enzymeactivity-assay 48 h upon transfection. Catalytic activity of ASM variants is given in exogenous ASM activity
levels per expressed protein in percentage relative to activity of ASM WT (100%). Displayed data represent
mean values of three independent experiments, error bars represent SD.
4.1.3.2 ASM variant p.P325A localizes in lysosomes
With the purpose to determine the subcellular organelles in which ASM variant p.P325A
localizes, we conducted localization analyses as described in 4.1.2.2.
ASM variant p.P325A localized within a dotted subcellular pattern when investigated by
fluorescence microscopy. Compared to ASM wild type, patterns were highly similar (Fig.
4.9).
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4. Results
Fig. 4.9 ASM variant p.P325A localizes in a dotted subcellular pattern.
ASM-Cherry construct was detected by fluorescence (Excitation: 587 nm, Emission: 610 nm). Shown are
Signals of ASM-Cherry variant p.P325A and LAMP1 were co-localized as revealed by
confocal microscopy. Thus, ASM variant p.P325A was localized within the lysosome (Fig.
4.10).
Fig. 4.10 ASM variant p.P325A is localized within lysosomes.
Confocal microscopy revealed co-localization of ASM-Cherry variant p.P325A with lysosomal marker protein
LAMP1. The ASM-Cherry construct was detected by fluorescence (Excitation: 587 nm, Emission: 610 nm).
Lysosomal marker protein was detected by immunofluorescence with anti-LAMP1 primary antibody derived
Emission: 505 – 530 nm). Shown are representative cells detected by confocal microscopy, displaying ASMCherry construct in red, marker protein in green and merged signals. The indicated scale covers 10 µm. Colocalization experiments were reproduced three times.
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4. Results
4.1.4 Characterization of ASM variant p.A487V
ASM variant p.A487V shows an amino acid exchange within the C-terminal domain of
ASM. Alanine is substituted by valine, both being apolar amino acids. Also patients suffering
from Niemann-Pick disease type B displayed this ASM variation (Simonaro et al., 2002).
4.1.4.1 Catalytic activity of ASM variant p.A487V is equivalent to wild type
Enzymatic activity of ASM variant p.A487V was determined as described in 4.1.2.1. ASM
variant p.A487V displayed 108 % (SD ± 63.1) of ASM wild type activity, indicating similar
ASM activity levels of both ASM enzymes (Fig. 4.11).
Fig. 4.11 Enzymatic activities of ASM variant p.A487V and ASM wild type reach similar levels.
ASM-FLAG constructs were transiently expressed in HeLa cells. Lysates were subjected to an in vitro enzymeactivity-assay 48 h upon transfection. Catalytic activity of ASM variants is given in exogenous ASM activity
levels per expressed protein in percentage relative to activity of ASM WT (100%). Displayed data represent
mean values of three independent experiments, error bars represent SD.
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4. Results
4.1.4.2 ASM variant p.A487V localizes in lysosomes
As described for the other variants, localization of ASM variant p.A487V was investigated
using the ASM-Cherry construct, which localized in a dotted subcellular pattern (Fig. 4.12).
Fig. 4.12 ASM variant p.A487V localizes in a dotted subcellular pattern.
ASM-Cherry construct was detected by fluorescence (Excitation: 587 nm, Emission: 610 nm). Shown are
Signals of ASM-Cherry variant p.A487V and LAMP1 were co-localized as revealed by
confocal microscopy, which means a lysosomal localization of ASM variant p.A487V (Fig.
4.13).
Fig. 4.13 ASM variant p.A487V localizes within lysosomes.
Confocal microscopy revealed co-localization of ASM variant p.A487A with the lysosomal marker protein
LAMP1. The ASM-Cherry construct was detected by fluorescence (Excitation: 587 nm, Emission: 610 nm).
Lysosomal marker protein was detected by immunofluorescence with anti-LAMP1 primary antibody derived
Emission: 505 – 530 nm). Shown are representative cells detected by confocal microscopy, displaying ASMCherry construct in red, marker protein in green and merged signals. The indicated scale covers 10 µm. Colocalization experiments were reproduced three times.
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4. Results
4.1.5 Characterization of ASM variant p.G506R
The common ASM variation p.G506R is characterized by an amino acid exchange within the
C-terminal domain of ASM, located two amino acids next to the phosphorylation site p.508.
Due to this close proximity, biochemical effects were supposed. The apolar amino acid
glycine is replaced by the polar and positively charged amino acid arginine. To get insight
into the potential effect of this variation, we cloned the variant p.G506R not only as single
substitution, but also in combination with the so far presented variations p.S508A, p.M381I,
p.P325A and p.A487V.
4.1.5.1 ASM variation p.G506R impacts catalytic activity
For determining the effect of ASM variation p.G506R on catalytic activity, we considered
eight different variants, resulting in four pairs: G506R - WT; G506R/S508A - S508A;
G506R/M381I - M381I; G506R/P325A - P325A; G506R/A487V - A487V. Determination of
enzymatic activity of ASM variants was described in 4.1.2.1. The singular variation p.G506R
showed a statistically significant decrease in ASM activity of 25% compared to wild type
(80% ± 12.7 vs. 100% of wild type activity; p<0.05). Also regarding variant p.A487V, the
combination with variation p.G506R resulted in a decrease in ASM activity of 37% (71% ±
20.1 vs. 108% ± 63.1 of wild type activity). In contrast, nearly no change in ASM activity
was detected between combined variant p.G506R/S508A in comparison with p.S508A (43%
± 18.1 and 33% ± 2.3 of wild type activity). No enzymatic activity was detected for
combined variants p.G506R/M381I and p.G506R/P325A as described for p. M381I and
p.P325A (Fig. 4.14).
The variation p.S508A not only made a statistically significant decrease in ASM activity
when occurring singularly (p.S508A compared to wild type, see also 4.1.2.1), but also when
combined with variation p.G506R (p.G506R - 80% ± 12.7 vs. p.G506R/S508A - 44% ± 18 of
wild type activity; p<0.05) (Fig. 4.14).
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4. Results
Fig. 4.14 ASM variation p.G506R causes a decrease in enzymatic activity.
Compared to wild type, variation p.G506R lead to a 25%-decrease in ASM activity. The combination of
p.G506R with a second variation as described above made differential effects on ASM activity observable: For
variant p.A487V a 37%-decrease, for p.S508A only a slight effect of 10% and for catalytically inactive variants
p.M381I and p.P325A no effect on enzymatic activity was detected. ASM-FLAG constructs were transiently
expressed in HeLa cells. Lysates were subjected to an in vitro enzyme-activity-assay 48 h upon transfection.
Catalytic activity of ASM variants is given in exogenous ASM activity levels per expressed protein in
percentage relative to activity of ASM WT (100%). Displayed data represent mean values of three independent
experiments, error bars represent SD. Statistical significance was calculated by one way ANOVA (* p<0.05).
4.1.5.2 ASM variation p.G506R impacts localization
Analogous to ASM variants described above, subcellular localization of the ASM variants
containing ASM variation p.G506R was investigated.
ASM variants p.G506R, p.G506R/P325A and p.G506R/A487V localized in dotted
subcellular patterns, in contrast to p.G506R/S508A and p.G506R/M381I (Fig. 4.15).
Compared to the singular occurrence of these variations (see above), the combination with
variation p.G506R only makes a difference in localization patterns for ASM variation S508A
(p.S508A - dotted structure, p.G506R/S508A - non-dotted structure).
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4. Results
Fig. 4.15 Subcellular localization patterns of ASM variants containing the variation p.G506R.
ASM variants p.G506R (A), p.G506R/P325A (D) and p.G506R/A487V (E) localized in dotted subcellular
patterns, in contrast to p.G506R/S508A (B) and p.G506R/M381I (C). ASM-Cherry constructs (p.G506R,
p.G506R/S508A and p.G506R/M381I) were detected by fluorescence (Excitation: 587 nm, Emission: 610 nm).
ASM-FLAG constructs (p.G506R/P325A and p.G506R/A487V) were detected by immunofluorescence with
anti-FLAG primary antibody derived from mouse and FITC coupled anti-mouse-IgG secondary antibody
derived from goat (Excitation: 488 nm, Emission: 505 – 530 nm). Shown are representative cells detected by
fluorescence microscopy. The indicated scale covers 10 µm. Localization experiments were reproduced three
times.
Using confocal microscopy we detected that signals of ASM-Cherry construct p.G506R and
the lysosomal marker protein LAMP1 were co-localized, indicating lysosomal localization
(Fig. 4.16 A). Signals of ASM variants p.G506R/S508A and p.G506R/M381I showed colocalization with signals derived from endoplasmic reticulum marker PDIA4, which indicates
that these variants were located within the endoplasmic reticulum (Fig. 4.16 B). For technical
reasons we did not investigate co-localization of ASM variants p.G506R/P325A and
p.G506R/A487V.
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Fig. 4.16 Co-localization of ASM variants containing variation p.G506R with subcellular organelles.
ASM variant p.G506R was localized within lysosomes, ASM variants p.G506R/S508A and p.G506R/M381I
within the endoplasmic reticulum. Confocal microscopy revealed co-localization of ASM variant p.G506R with
lysosomal marker protein LAMP1 (A) and of ASM variant p.G506R/S508A and p.G506R/M381I with
endoplasmic reticulum marker PDIA4 (B). ASM-Cherry constructs were detected by fluorescence (Excitation:
587 nm, Emission: 610 nm). Marker proteins were detected by immunofluorescence with anti-LAMP1 and antiPDIA4 primary antibody derived from mouse and FITC coupled anti-mouse-IgG secondary antibody derived
from goat (Excitation: 488 nm, Emission: 505 – 530 nm). Shown are representative cells detected by confocal
microscopy, displaying ASM-Cherry constructs in red, marker proteins in green and merged signals. The
indicated scale covers 10 µm. Co-localization experiments were reproduced three times.
68
4. Results
4.1.6 Summary: Characterization of ASM variants
Investigating catalytic activity and subcellular localization reveals differential properties of
ASM variants. Five different ASM variations were cloned and transiently expressed in a cell
culture system.
Concerning catalytic activity, ASM variants can be divided in three different categories: full
(p.A487V), reduced (p.S508A, p.G506R) or no catalytic activity (p.M381I, p.P325A).
Concerning subcellular localization, ASM variants build two different groups: lysosomal
(p.S508A, p.P325A, p.A487V, p.G506R) or non-lysosomal (p.M381I, p.G506R/S508A)
localization. A short overview of differential properties of ASM variants is given in table 4.1.
To sum up, most of ASM variations detected also within depressed patients showed
differential characteristics, but they did not cause excessive ASM activity.
Tab. 4.1 ASM variants display differential properties.
ASM variant
Enzymatic
Protein
Nucleotide
activity
expression
change
[% wild type]
[% wild type]
± SD
±SD
Localization
ASM wild type
-
100
100
Lysosome
p.S508A
c.1528T>G
34±2
144±26
Lysosome
p.M381I
c.1146G>A
0±1
290±115
ER
p.P325A
c.973C>G
0±1
181±64
Lysosome
p.A487V
c.1460C>T
109±63
165±67
Lysosome
p.G506R
c.1522G>A
76±13
99±13
Lysosome
p.G506R/
c.1522G>A
S508A
c.1528T>G
44±18
27±6
ER
p.G506R/
c.1522G>A
M381I
c.1146G>A
0±1
309±119
ER
p.G506R/
c.1522G>A
P325A
c. c.973C>G
0±0
228±81
n.d.
p.G506R/
c.1522G>A
A487V
c.1460C>T
72±20
170±61
n.d.
Summarized characteristics of ASM variants. Enzymatic activity indicates mean values ± SD (n=3) of
exogenous ASM activity per expressed protein in % of WT activity. Protein expression indicates mean values ±
SD (n=3) of ASM band intensities per µg protein controlled by GAPDH band intensities in % of WT values.
For p.G506R/P325A and p.G506R/A487V no co-localization studies were conducted (n.d.).
69
4. Results
4.2 Impact of alternative splicing on Acid Sphingomyelinase activity
In the attempt to clone variant full-length ASM transcripts for expression studies, we isolated
total human RNA from different sources and synthesized the corresponding cDNAs. The
complete ASM coding sequence was amplified by RT-PCR using primers flanking the ATG
initiation codon and the stop codon, inserted into a vector and sequenced.
In total, 17 transcripts from two different sources were isolated and analysed, of which 13
transcripts were identical to the reference sequence of ASM with the exception of known
polymorphisms (Schuchman et al., 1991a; Wan and Schuchman, 1995). Sequence alignment
of four transcripts, however, revealed unique local differences that most probably arose from
alternative splicing (Fig. 4.17).
ASM-1
ASM-5
ASM-6
ASM-7
ATGCCCCGCTACGGAGCGTCACTCCGCCAGAGCTGCCCCAGGTCCGGCCGGGAGCAGGGA
************************************************************
60
60
60
60
ASM-1
ASM-5
ASM-6
ASM-7
CAAGACGGGACCGCCGGAGCCCCCGGACTCCTTTGGATGGGCCTGGTGCTGGCGCTGGCG
CAAGACGGGACCGCCGGAGCCCCCGGACTCCTTTGGATGGGCCTGGCGCTGGCGCTGGCG
********************************************** *************
120
120
120
120
ASM-1
ASM-5
ASM-6
ASM-7
CTGGCGCTGGCGCTGGCGCTGGCTCTGTCTGACTCTCGGGTTCTCTGGGCTCCGGCAGAG
CTGGCGCTGGCGCTGGC------TCTGTCTGACTCTCGGGTTCTCTGGGCTCCGGCAGAG
CTGGCGCTGGCGCTGGC------TCTGTCTGACTCTCGGGTTCTCTGGGCTCCGGCAGAG
CTGGCGCTGGC------------TCTGTCTGACTCTCGGGTTCTCTGGGCTCCGGCAGAG
***********
*************************************
180
174
174
168
ASM-1
ASM-5
ASM-6
ASM-7
GCTCACCCTCTTTCTCCCCAAGGCCATCCTGCCAGGTTACATCGCATAGTGCCCCGGCTC
************************************************************
240
234
234
228
ASM-1
ASM-5
ASM-6
ASM-7
CGAGATGTCTTTGGGTGGGGGAACCTCACCTGCCCAATCTGCAAAGGTCTATTCACCGCC
************************************************************
300
294
294
288
ASM-1
ASM-5
ASM-6
ASM-7
ATCAACCTCGGGCTGAAGAAGGAACCCAATGTGGCTCGCGTGGGCTCCGTGGCCATCAAG
************************************************************
360
354
354
348
ASM-1
ASM-5
ASM-6
ASM-7
CTGTGCAATCTGCTGAAGATAGCACCACCTGCCGTGTGCCAATCCATTGTCCACCTCTTT
************************************************************
420
414
414
408
ASM-1
ASM-5
ASM-6
ASM-7
GAGGATGACATGGTGGAGGTGTGGAGACGCTCAGTGCTGAGCCCATCTGAGGCCTGTGGC
************************************************************
480
474
474
468
70
4. Results
ASM-1
ASM-5
ASM-6
ASM-7
CTGCTCCTGGGCTCCACCTGTGGGCACTGGGACATTTTCTCATCTTGGAACATCTCTTTG
************************************************************
540
534
534
528
ASM-1
ASM-5
ASM-6
ASM-7
CCTACTGTGCCGAAGCCGCCCCCCAAACCCCCTAGCCCCCCAGCCCCAGGTGCCCCTGTC
************************************************************
600
594
594
588
ASM-1
ASM-5
ASM-6
ASM-7
AGCCGCATCCTCTTCCTCACTGACCTGCACTGGGATCATGACTACCTGGAGGGCACGGAC
************************************************************
660
654
654
648
ASM-1
ASM-5
ASM-6
ASM-7
CCTGACTGTGCAGACCCACTGTGCTGCCGCCGGGGTTCTGGCCTGCCGCCCGCATCCCGG
************************************************************
720
714
714
708
ASM-1
ASM-5
ASM-6
ASM-7
CCAGGTGCCGGATACTGGGGCGAATACAGCAAGTGTGACCTGCCCCTGAGGACCCTGGAG
************************************************************
780
774
774
768
ASM-1
ASM-5
ASM-6
ASM-7
AGCCTGTTGAGTGGGCTGGGCCCAGCCGGCCCTTTTGATATGGTGTACTGGACAGGAGAC
************************************************************
840
834
834
828
ASM-1
ASM-5
ASM-6
ASM-7
ATCCCCGCACATGATGTCTGGCACCAGACTCGTCAGGACCAACTGCGGGCCCTGACCACC
************************************************************
900
894
894
888
ASM-1
ASM-5
ASM-6
ASM-7
GTCACAGCACTTGTGAGGAAGTTCCTGGGGCCAGTGCCAGTGTACCCTGCTGTGGGTAAC
************************************************************
960
954
954
948
ASM-1
ASM-5
ASM-6
ASM-7
CATGAAAGCACACCTGTCAATAGCTTCCCTCCCCCCTTCATTGAGGGCAACCACTCCTCC
************************************************************
1020
1014
1014
1008
ASM-1
ASM-5
ASM-6
ASM-7
CGCTGGCTCTATGAAGCGATGGCCAAGGCTTGGGAGCCCTGGCTGCCTGCCGAAGCCCTG
************************************************************
1080
1074
1074
1068
ASM-1
ASM-5
ASM-6
ASM-7
CGCACCCTCAG----------------------------------------AATTGGGGG
CGCACCCTCAG------------------------------------------------CGCACCCTCAG----------------------------------------AATTGGGGG
CGCACCCTCAGGTACTTATCGTCCGTGGAAACCCAGGAAGGGAAAAGAAAGAATTGGGGG
***********
1100
1085
1094
1128
ASM-1
ASM-5
ASM-6
ASM-7
GTTCTATGCTCTTTCCCCATACCCCGGTCTCCGCCTCATCTCTCTCAATATGAATTTTTG 1160
-----------------------------------------------------------GTTCTATGCTCTTTCCCCATACCCCGGTCTCCGCCTCATCTCTCTCAATATGAATTTTTG 1154
GTTCTATGCTCTTTCCCCATACCCCGGTCTCCGCCTCATCTCTCTCAATATGAATTTTTG 1188
ASM-1
ASM-5
ASM-6
ASM-7
TTCCCGTGAGAACTTCTGGCTCTTGATCAACTCCACGGATCCCGCAGGACAGCTCCAGTG
************************************************************
1220
1145
1214
1248
71
4. Results
ASM-1
ASM-5
ASM-6
ASM-7
GCTGGTGGGGGAGCTTCAGGCTGCTGAGGATCGAGGAGACAAAGTGCATATAATTGGCCA
GCTAGTGGGGGAGCTTCAGGCTGCTGAGGATCGAGGAGACAAAGTGCATATAATTGGCCA
*** ********************************************************
1280
1205
1274
1308
ASM-1
ASM-5
ASM-6
ASM-7
CATTCCCCCAGGGCACTGTCTGAAGAGCTGGAGCTGGAATTATTACCGAATTGTAGCCAG
************************************************************
1340
1265
1334
1368
ASM-1
ASM-5
ASM-6
ASM-7
GTATGAGAACACCCTGGCTGCTCAGTTCTTTGGCCACACTCATGTGGATGAATTTGAGGT
************************************************************
1400
1325
1394
1428
ASM-1
ASM-5
ASM-6
ASM-7
CTTCTATGATGAAGAGACTCTGAGCCGGCCGCTGGCTGTAGCCTTCCTGGCACCCAGTGC
************************************************************
1460
1385
1454
1488
ASM-1
ASM-5
ASM-6
ASM-7
AACTACCTACATCGGCCTTAATCCTG--------------------GTTACCGTGTGTAC
AACTACCTACATCGGCCTTAATCCTGTCAGCCCCACATCCTTGCAGGTTACCGTGTGTAC
**************************
**************
1500
1425
1514
1528
ASM-1
ASM-5
ASM-6
ASM-7
CAAATAGATGGAAACTACTCCGGGAGCTCTCACGTGGTCCTGGACCATGAGACCTACATC
CAAATAGATGGAAACTACTCCAGGAGCTCTCACGTGGTCCTGGACCATGAGACCTACATC
CAAATAGATGGAAACTACTCCAGGAGCTCTCACGTGGTCCTGGACCATGAGACCTACATC
CAAATAGATGGAAACTACTCCGGGAGCTCTCACGTGGTCCTGGACCATGAGACCTACATC
********************* **************************************
1560
1485
1574
1588
ASM-1
ASM-5
ASM-6
ASM-7
CTGAATCTGACCCAGGCAAACATACCGGGAGCCATACCGCACTGGCAGCTTCTCTACAGG
************************************************************
1620
1545
1634
1648
ASM-1
ASM-5
ASM-6
ASM-7
GCTCGAGAAACCTATGGGCTGCCCAACACACTGCCTACCGCCTGGCACAACCTGGTATAT
************************************************************
1680
1605
1694
1708
ASM-1
ASM-5
ASM-6
ASM-7
CGCATGCGGGGCGACATGCAACTTTTCCAGACCTTCTGGTTTCTCTACCATAAGGGCCAC
************************************************************
1740
1665
1754
1768
ASM-1
ASM-5
ASM-6
ASM-7
CCACCCTCGGAGCCCTGTGGCACGCCCTGCCGTCTGGCTACTCTTTGTGCCCAGCTCTCT
************************************************************
1800
1725
1814
1828
ASM-1
ASM-5
ASM-6
ASM-7
GCCCGTGCTGACAGCCCTGCTCTGTGCCGCCACCTGATGCCAGATGGGAGCCTCCCAGAG
************************************************************
1860
1785
1874
1888
ASM-1
ASM-5
ASM-6
ASM-7
GCCCAGAGCCTGTGGCCAAGGCCACTGTTTTGCTAG
************************************
1896
1821
1910
1924
Fig. 4.17 Coding sequences of full-length ASM and new alternatively-spliced transcripts differ locally.
Since all identified transcripts show sequence identity with full-length ASM except for described local
differences, these transcripts are results of alternative splicing events. Grey parts indicate specific sequences
differing from full-length ASM coding sequence. Stop codons in bold. Alignment was done using ClustalW.
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4. Results
4.2.1 Structure of new ASM splice variants
One of the identified transcripts lacked the first 69 bp of exon 3, which resulted in a transcript
length of 1.818 bp. The second transcript was characterized by a 20 bp intronic insertion
derived from the end of intron 5 adjacent to exon 6 (exon 5a), leading to a transcript length of
1.907 bp. Two transcripts stood out due to a 40 bp intronic sequence derived from the
beginning of intron 2 adjacent to exon 2 (exon 2a), which resulted in a 1.921 bp transcript
(Fig. 4.18). Since Schuchman and colleagues (Schuchman et al., 1991b) identified two ASM
splice variants, termed ASM-2 and -3, and an additional splice variant, termed ASM-4, was
deposited in the NCBI database GenBank by Reichwald et al., the new transcripts were
termed ASM-5, ASM-6 and ASM-7. Of note, the 40 bp intronic sequence from intron 2
found in ASM-7 is the same occurring in transcript ASM-2 and the 20 bp intronic sequence
from intron 5 in ASM-6 is the same used in transcript ASM-4. However, the newly-identified
splice variants were not identical to these transcripts (Fig. 4.18; Fig. 2.2). Both ASM-5 and
ASM-6 were identified in human neuroglioma cells, whereas ASM-7 was found in human
PBMCs. In total, 24% of the analysed ASM transcripts were alternatively-spliced.
Fig. 4.18 Schematic structure of new splice variants ASM-5, -6 and -7.
Model of genomic alignment. Compared to ASM-1, in ASM-5 a part of exon 3 is excluded, which results in a
1.818 bp coding sequence. ASM-6 and ASM-7 display different intronic insertions, which generate transcript
lengths of 1.907 bp and 1.921 bp, respectively, and create frameshifts including premature stop codons
(indicated by asterisk) after 1.521 bp and 1.197 bp, respectively. In the depicted models of ASM exon-intron
structure, the line represents the genomic sequence while black boxes stand for exons translated in ASM-1, grey
boxes for exonic sequence translated into different amino acids due to a frameshift or sequence corresponding to
introns in ASM-1, striped box for lacking exonic sequence and white boxes for exonic sequence following a
premature termination codon. Scheme is drawn to scale.
73
4. Results
4.2.2 New splicing motifs are conserved in primates
We performed an in silico analysis concerning orthologous conservation of new ASM splice
variants. Since the coding sequence of full-length ASM-1 is highly conserved among species,
the comparison of ASM splice variants with orthologous transcripts is also reasonable.
Newly-identified transcript ASM-6 shows exactly the same splicing patterns as the
orthologous ASM transcripts identified in Pan troglodytes (GenBank Accession Number
XM_001164317.1) and Macaca mulatta (Gen-Bank Accession Number XM_001110020.1).
Newly-identified transcript ASM-7 harbours splicing patterns identical to orthologous ASM
transcript expressed in Pongo abelii (GenBank Accession Number NM_001132129.1) (Fig.
4.19). Therefore, the utilization of splice sites at intronic sequences 2a and 5a which are used
within ASM-6 and -7 is highly conserved. No orthologous transcripts were found for the
newly-identified splice site in exon 3 used in ASM-5.
Fig. 4.19 Splicing patterns of novel transcripts ASM-6 and -7 are conserved in primates.
GenBank search revealed orthologous ASM transcripts which show exactly the same splicing patterns as novel
transcripts ASM-6 and -7. In the depicted models of ASM exon-intron structure, the line represents the genomic
sequence while black boxes stand for exons translated in ASM-1, grey boxes for exonic sequence translated into
different amino acids due to a frameshift or sequence corresponding to introns in ASM-1 and white boxes for
exonic sequence following a premature termination codon. Scheme is drawn to scale.
4.2.3 Biochemical characterization of new ASM splice variants
On the predicted protein level, novel ASM splice variants display specific features in
comparison with ASM-1 (631 amino acids, calculated molecular weight of 70 kDa). The
protein derived from the new transcript ASM-5 covers 606 amino acids with a calculated
molecular weight of 67 kDa. The catalytic domain is disrupted, since amino acids 362 to 384
are missing (Fig. 4.20). The protein derived from the new transcript ASM-6 is composed of
506 amino acids with a calculated molecular weight of 56 kDa, since the intronic insertion
74
4. Results
creates a frameshift and subsequently introduces a premature stop codon TAG resulting in a
truncated reading frame with 1.521 bp. Therefore, ASM-6 lacks 136 amino acids at its
carboxyl terminus and carries a unique carboxyl-terminal peptide of 13 amino acids
(VSPTSLQVTVCTK) instead, displaying only a fragmentary carboxy-terminal domain (Fig.
4.20 A). The protein derived from the new transcript ASM-7 consists of 398 amino acids and
has a calculated molecular weight of 45 kDa, since a frameshift introduced by the intronic
insertion creates a premature stop codon TGA, resulting in an open reading frame of 1.197
bp. Thus, ASM-7 lacks 267 amino acids at its carboxyl terminus and carries a unique
carboxyl-terminal
peptide
of
38
amino
acids
(YLSSVETQEGKRKNWGVLC
SFPIPRSPPHLSQYEFLFP) (Fig. 4.20 A). ASM-7 hence displays only a fragmentary
catalytic domain, whereas the carboxy-terminal domain is completely missing. Taken
together, alternative splicing events generating new variants ASM-5 to -7 affect the catalytic
domain and the carboxyl-terminal domain.
A
ASM-1
ASM-5
ASM-6
ASM-7
MPRYGASLRQSCPRSGREQGQDGTAGAPGLLWMGLVLALALALALALALSDSRVLWAPAE
MPRYGASLRQSCPRSGREQGQDGTAGAPGLLWMGLVLALALALALA--LSDSRVLWAPAE
MPRYGASLRQSCPRSGREQGQDGTAGAPGLLWMGLVLALALALALA--LSDSRVLWAPAE
MPRYGASLRQSCPRSGREQGQDGTAGAPGLLWMG--LALALALALA--LSDSRVLWAPAE
********************************** ********** ************
60
58
58
56
ASM-1
ASM-5
ASM-6
ASM-7
AHPLSPQGHPARLHRIVPRLRDVFGWGNLTCPICKGLFTAINLGLKKEPNVARVGSVAIK
************************************************************
120
118
118
116
ASM-1
ASM-5
ASM-6
ASM-7
LCNLLKIAPPAVCQSIVHLFEDDMVEVWRRSVLSPSEACGLLLGSTCGHWDIFSSWNISL
************************************************************
180
178
178
176
ASM-1
ASM-5
ASM-6
ASM-7
PTVPKPPPKPPSPPAPGAPVSRILFLTDLHWDHDYLEGTDPDCADPLCCRRGSGLPPASR
************************************************************
240
238
238
236
ASM-1
ASM-5
ASM-6
ASM-7
PGAGYWGEYSKCDLPLRTLESLLSGLGPAGPFDMVYWTGDIPAHDVWHQTRQDQLRALTT
************************************************************
300
298
298
296
ASM-1
ASM-5
ASM-6
ASM-7
VTALVRKFLGPVPVYPAVGNHESTPVNSFPPPFIEGNHSSRWLYEAMAKAWEPWLPAEAL
************************************************************
360
358
358
356
75
4. Results
ASM-1
ASM-5
ASM-6
ASM-7
RTLRIGGFYALSPYPGLRLISLNMNFCSRENFWLLINSTDPAGQLQWLVGELQAAEDRGD
RTL-----------------------SSRENFWLLINSTDPAGQLQWLVGELQAAEDRGD
RTLRIGGFYALSPYPGLRLISLNMNFCSRENFWLLINSTDPAGQLQWLVGELQAAEDRGD
RTLRYLSSVETQEGKRKNWGVLCSFPIPRSPPHLSQYEFLFP-----------------***
420
395
418
398
ASM-1
ASM-5
ASM-6
ASM-7
KVHIIGHIPPGHCLKSWSWNYYRIVARYENTLAAQFFGHTHVDEFEVFYDEETLSRPLAV 480
------------------------------------------------------------
ASM-1
ASM-5
ASM-6
ASM-7
AFLAPSATTYIGLNPGYRVYQIDGNYSGSSHVVLDHETYILNLTQANIPGAIPHWQLLYR 540
AFLAPSATTYIGLNPGYRVYQIDGNYSRSSHVVLDHETYILNLTQANIPGAIPHWQLLYR 515
AFLAPSATTYIGLNPVSPTSLQVTVCTK-------------------------------- 506
------------------------------------------------------------
ASM-1
ASM-5
ASM-6
ASM-7
ARETYGLPNTLPTAWHNLVYRMRGDMQLFQTFWFLYHKGHPPSEPCGTPCRLATLCAQLS 600
ARETYGLPNTLPTAWHNLVYRMRGDMQLFQTFWFLYHKGHPPSEPCGTPCRLATLCAQLS 575
--------- -------------------------------------------------------------------------------------------------------------
ASM-1
ASM-5
ASM-6
ASM-7
ARADSPALCRHLMPDGSLPEAQSLWPRPLFC 631
ARADSPALCRHLMPDGSLPEAQSLWPRPLFC 606
-------------------------------------------------------------
B
Fig. 4.20 Novel variants ASM-5 to -7 differ regarding their putative protein sequence and protein domain
structure.
In comparison with ASM-1, ASM-5 lacks part of the catalytic domain, ASM-6 part of the carboxyl-terminal
domain and ASM-7 part of the catalytic and the whole carboxy-terminal domain. ASM-6 and -7 carry unique
carboxy-terminal peptides, indicated in grey. A. Putative protein sequences. Different protein domains are
coloured: yellow - signal peptide (aa 1-48), light blue - saposin-B-domain (aa 91-167), purple - proline-richdomain (aa 168-200), red - catalytic domain (aa 201-463), green - carboxy-terminal domain (aa 464-631).
Mannose-6-phosphate sites (Asn 88, 177, 337, 397, 505, 522) are indicated in light green. Alignment was done
using ClustalW. B. Schematic model of putative new ASM proteins. Mannose-6-phosphate sites are indicated
by symbols. Scheme is drawn to scale.
76
4. Results
4.2.3.1 New ASM splice variants are expressed in a cell culture model
In order to analyse protein properties of the identified new ASM isoforms, the corresponding
cDNAs were cloned into the expression vector FLAG-N2. Because of the putative N-terminal
signal peptide, we expressed the ASM variants as C-terminal-tagged fusion proteins. HeLa
cells were transiently transfected with ASM-FLAG constructs and harvested 48 h later.
Western blot analysis showed that ASM-1 and each of the new splice variants ASM-5 to -7
were translated into protein and displayed the predicted sizes (Fig. 4.21).
Fig. 4.21 Expression of ASM-FLAG constructs in HeLa cells.
Lysates were analysed by Western blotting 48 h after transfection. ASM-1 and each of the new isoforms ASM-5
to -7 displayed the predicted sizes. Expression of ASM-FLAG variants was detected using FLAG antibody.
Lysates of vector-transfected cells served as negative control, GAPDH was detected for loading control. M
indicates size marker.
4.2.3.2 New ASM splice variants are catalytically inactive in vitro
To determine the catalytic activity of the new ASM isoforms, H4 cells were transiently
transfected with ASM-FLAG constructs. 48 h upon transfection cells were harvested and
subjected to an in vitro biochemical enzyme-activity-assay (Gulbins and Kolesnick, 2000).
ASM-1 over-expression selectively increased the ASM activity of cell lysates about 9-fold
over endogenous levels (48.9 ± 1.3 nmol/mg/h, in comparison with control 5.4 ± 0.2
nmol/mg/h). In contrast to ASM-1, none of the alternatively-spliced variants exerted any
77
4. Results
catalytic activity, despite well detectable expression of the proteins (ASM-5: 5.2 ± 1.1
nmol/mg/h; ASM-6: 5.2 ± 1.0 nmol/mg/h; ASM-7: 4.6 ± 1.6 nmol/mg/h; Fig. 4.22). To
validate the impact of the FLAG-tag, we also expressed the variants without the C-terminal
modification, which did not alter results (data not shown).
Fig. 4.22 Novel variants ASM -5 to -7 are catalytically inactive.
Selectively ASM-1 over-expression increased ASM activity in comparison with cloning vector control. ASMFLAG constructs were transiently expressed in H4 cells. Lysates were subjected to an in vitro enzyme-activityassay 48 h upon transfection. Data indicate mean values of three independent experiments, error bars represent
SD.
4.2.3.3 New ASM splice variants are catalytically inactive in vivo
Additionally, we investigated in vivo whether the new ASM isoforms modulate cellular
ceramide and sphingomyelin levels. H4 cells were transiently transfected with ASM-FLAG
constructs. A MALDI-TOF MS analysis showed that the ratio of ceramide to sphingomyelin
24 h upon ASM-1 over-expression was clearly elevated by 1.8-fold compared to control
(p<0.01). In contrast, transfection of new ASM splice variants had the opposite effect and
decreased ceramide to sphingomyelin ratio to between 26% and 38% of control (p<0.01 for
ASM-6; p<0.05 for ASM-5 and -7) (Fig. 4.23).
In total, we concluded that new ASM splice variants were catalytically inactive irrespective
of whether they retained the catalytic domain or not and that they even exert an inhibitory
effect on sphingomyelin breakdown.
78
4. Results
Fig. 4.23 New ASM splice variants modulate ceramide to sphingomyelin ratios.
The ratio of ceramide (Cer) to sphingomyelin (SM) levels upon ASM-1 over-expression was significantly
increased (p<0.01), upon ASM-5 to –7 over-expression significantly decreased (p<0.01 for ASM-6; p<0.05 for
ASM-5 and -7) compared to cloning vector control. Lysates were subjected to MALDI-TOF MS for
sphingolipid analysis 24 h after transfection of H4 cells. Data indicate mean values of three independent
experiments, error bars represent SD. Statistical significance was calculated with respect to control (* p<0.05,
** p<0.01).
4.2.4 New ASM splice variants localize within different subcellular
compartments
As described before, ASM wild type localizes in a dotted subcellular pattern and is located
inside the lysosomes (see 4.1.2.2). To identify potential differences of ASM splice variants
we investigated their subcellular localization. Due to the lack of specific antibodies we
expressed splice variants as recombinant ASM-Cherry constructs with the fluorescent
Cherry-tag on their C-termini. ASM-Cherry constructs were derived from ASM-FLAG
constructs using infusion cloning.
H4 cells were transiently transfected with ASM-Cherry constructs of ASM wild type and new
ASM splice variants and fixed 48 h later. Cells were immunostained for the lysosomal
marker protein LAMP1 and the endoplasmic reticulum marker protein PDIA4 to determine
potential co-localization within lysosomes or endoplasmic reticulum, respectively.
Subcellular localization patterns were investigated using fluorescence microscopy and colocalization with lysosomes or endoplasmic reticulum using confocal microscopy.
79
4. Results
As described above ASM wild type localized in a dotted subcellular pattern. Also ASM-7
was found to occur within dotted structures. In contrast, cells expressing ASM-5 and ASM-6
did not exhibit dotted structures (Fig. 4.24).
Fig. 4.24 Subcellular localization patterns of ASM-1 and new ASM splice variants.
ASM-1 (A) and ASM-7 (D) localized in dotted subcellular patterns, whereas ASM-5 (B) and ASM-6 (C) did
not. ASM-Cherry constructs were detected by fluorescence (Excitation: 587 nm, Emission: 610 nm). Shown are
Confocal microscopy analyses revealed that signals of ASM-Cherry wild type and ASM-7
constructs and signals of LAMP1 were co-localized (Fig. 4.25 A). In contrast, ASM-5 and
ASM-6 constructs showed co-localization with endoplasmic reticulum marker PDIA4 (Fig.
4.25 B).
80
4. Results
Fig. 4.25 Co-localization of new ASM splice variants with subcellular organelles.
ASM-1 and ASM-7 were localized within lysosomes, ASM-5 and ASM-6 within the endoplasmic reticulum.
Confocal microscopy revealed co-localization of ASM-1 and ASM-7 with lysosomal marker protein LAMP1
(A) and of ASM-5 and ASM-6 with endoplasmic reticulum marker PDIA4 (B). ASM-Cherry constructs were
detected by fluorescence (Excitation: 587 nm, Emission: 610 nm). Marker proteins were detected by
immunofluorescence with anti-LAMP1 and anti-PDIA4 primary antibody derived from mouse and FITC
coupled anti-mouse-IgG secondary antibody derived from goat (Excitation: 488 nm, Emission: 505 – 530 nm).
Shown are representative cells detected by confocal microscopy, displaying ASM-Cherry constructs in red,
marker proteins in green and merged signals. The indicated scale covers 10 µm. Co-localization experiments
were reproduced three times.
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4. Results
4.2.5 New ASM splice variants show differential expression patterns in
human blood cells
In order to test if the newly-identified ASM transcripts are generally expressed, the cDNA of
PBMCs from different donors was analysed by RT-PCR.
4.2.5.1 New ASM splice variants are detected specifically by RT-PCR
We designed distinct primer pairs for the exclusive amplification of each ASM transcript
(Fig. 4.26 A), which were evaluated for target-specificity. PCR products were detected
selectively if corresponding cDNA and primer pair were combined (Fig. 4.26 B). Each of the
PCR products displayed the predicted size. Thus, designed oligonucleotides served for
specific amplification of new ASM variants.
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4. Results
Fig. 4.26 Designed oligonucleotides serve for specific amplification of new ASM splice variants.
A. Distinct primer pairs were designed for specific amplification of each novel ASM splice variant. Exons are
indicated by light grey boxes, transcript specific regions by symbol and dark grey boxes, primers by arrows.
B. Primer specificity. Amplification of each novel ASM transcript using the respective primer pair resulted in a
specific PCR-product of the expected size separated on a 1% agarose gel. Cloned transcripts were used as
templates, water served as negative control. M refers to 100 bp size marker; indicated are DNA bands of 300 bp
and 500 bp.
4.2.5.2 New ASM splice variants are differentially expressed in healthy volunteers
Using the established RT-PCR system, each of the newly-identified ASM splice variants
could be detected in the cDNA from eight different donors. Notably, the expression level of
each novel ASM splice variant varied in intra-individual comparison, and also the relative
abundance of the splice variants differed between subjects (Fig. 4.27). PCR products were
confirmed by sequencing (data not shown).
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4. Results
Fig. 4.27 Expression patterns of ASM splice variants vary in human blood cells.
cDNA of healthy volunteers (n=8) was analysed for the occurrence of each novel ASM transcript by RT-PCR
and products were separated on a 1% agarose gel. ASM-1 and the new splice variants ASM-5, -6 and -7
exhibited both inter-individual as well as intra-individual differences in their expression. Water served as
negative control. The second band of ASM-6 RT-PCR results from gDNA amplifications. M refers to 100 bp
size marker; indicated are DNA bands of 300 bp and 500 bp.
4.2.5.3 New ASM splice variants are differentially expressed in depressed patients
before and after antidepressant therapy
In order to investigate if expression patterns of new ASM splice variants were changed in
depressed patients compared to healthy volunteers, we conducted additional RT-PCR
experiments. We isolated RNA of whole blood derived from patients diagnosed for major
depressive disorder at two different time points, before administering antidepressant drugs,
i.e. in a complete untreated state, and after treatment for 28 days. The antidepressant therapy
had been chosen according to the patients’ symptoms; therefore amitriptyline, mirtazapine
and fluoxetine, respectively, were administered.
The comparison of pre- and post treatment revealed an up-regulation of all ASM variants
compared to reference gene HPRT, which was shown to be regulated independently from
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4. Results
ASM (see thesis written by Jens Bode, 2010). Notably, strong effects were detected for the
expression of ASM-5. Without antidepressant treatment, expression of ASM-5 was not
detectable. In contrast, after administering of drugs ASM-5 was expressed to a high extent
(Fig. 4.28). Compared to healthy volunteers (see 4.2.5.2) no obvious differences in
expression patterns were observable. Depressed patients also displayed variations regarding
their intra-individual expression patterns and in the relative abundance in inter-individual
comparison.
Fig. 4.28 Expression patterns of ASM splice variants vary in blood cells of depressed patients before and
after administering of antidepressants.
cDNA of depressed patients was analysed for the occurrence of each novel ASM transcript by RT-PCR and
products were separated on a 1% agarose gel. All ASM variants were up-regulated upon antidepressant therapy
when compared to reference gene HPRT. New splice variant ASM-5 was not expressed without antidepressant
treatment, but showed strong expression after administering drugs. Water served as negative control. Due to
limited material obtained from patients control RT-PCR could not be repeated to avoid unspecific bands. M
refers to 100 bp size marker; indicated are DNA bands of 300 bp and 500 bp.
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4. Results
4.2.6 New splice variant ASM-5 exerts a dominant-negative effect on fulllength Acid Sphingomyelinase
Based on expression differences of new splice variant ASM-5 detected in pre- and posttreatment conditions (see 4.2.5.3), we investigated functional effects of ASM-5.
4.2.6.1 Upon serum starvation ASM-5 localizes within lysosomes
As described above (see 4.2.4), ASM-5 localizes within the endoplasmic reticulum under
basal conditions. Since cells of depressed patients are supposed to be in a stressed state, we
conducted experiments under stress conditions, induced by serum starvation. For this purpose
we transiently transfected H4 cells with ASM-5-Cherry construct. 48 h upon transfection
cells were shifted to serum-free medium. 24 h later cells were fixed and immunostained for
LAMP1 and PDIA4 to determine potential co-localization within lysosomes or endoplasmic
reticulum, respectively. Subcellular localization patterns were investigated using fluorescence
microscopy and co-localization with lysosomes or endoplasmic reticulum using confocal
microscopy.
ASM-5 was found to occur within dotted structures under serum-starving conditions (Fig.
4.29), in contrast to basal conditions (data equate to fig. 4.24B).
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4. Results
Fig. 4.29 Subcellular localization pattern of ASM-5 upon serum starvation.
ASM-5 localized in a dotted subcellular pattern under serum-starving conditions, in sharp contrast to basal
conditions (see 4.2.4). ASM-Cherry constructs were detected by fluorescence (Excitation: 543 nm, Emission:
560 – 615 nm). Shown are representative cells investigated by fluorescence microscopy. The indicated scale
covers 10 µm. Localization experiments were reproduced three times.
To evaluate co-localization of ASM-5 with marker proteins under starving conditions,
confocal microscopy analyses were conducted. Signals of ASM-5-Cherry and signals of
LAMP1 were co-localized, indicating that ASM-5 was localized inside the lysosomes (Fig.
4.30).
Fig. 4.30 ASM-5 localizes within lysosomes upon serum starvation.
Confocal microscopy revealed co-localization of ASM-5-Cherry construct with lysosomal marker protein
LAMP1. ASM-Cherry construct was detected by fluorescence (Excitation: 543 nm, Emission: 560 – 615 nm).
LAMP1 was detected by immunofluorescence with anti-LAMP1 primary antibody derived from mouse and
FITC coupled anti-mouse-IgG secondary antibody derived from goat (Excitation: 488 nm, Emission: 505 – 530
nm). Shown are representative cells detected by confocal microscopy, displaying ASM-Cherry constructs in red,
LAMP1 in green and merged signals. The indicated scale covers 10 µm. Co-localization experiments were
reproduced three times.
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4. Results
4.2.6.2 ASM-5 is a dominant-negative ASM isoform
Since ASM-1 and ASM-5 both show lysosomal localization under serum-starving conditions,
interactive effects on ASM activity levels are possible. Therefore, we transiently cotransfected HeLa cells with ASM-1 and ASM-5-FLAG constructs under starving and basal
conditions. Starving protocol was applied as described for localization studies in 4.2.6.1. Cell
lysates were subjected to an in vitro biochemical enzyme-activity-assay.
Exogenous catalytic activity of ASM-1 over-expression was set on 100% for each condition.
Compared to the respective reference, ASM-5 did not exert catalytic activity (starving
condition: 0.2% ± 1.3 of ASM-1 activity; basal condition: 1.3% ± 1.3 of ASM-1 activity).
Upon serum starvation over-expression of a mixture of ASM-1 and ASM-5 lead to a
significant reduction of ASM activity. Adding 25% of ASM-5 to the transfection mixture
reduced ASM activity to 60.2% compared to over-expression of ASM-1 (SD ± 20.0; p<0.05),
while the addition of 50% of ASM-5 reduced ASM activity to 52.5% of ASM-1 activity (SD
± 12.7; p<0.05). In contrast, no effect of varying amounts of ASM-5 over-expression on
catalytic activity of ASM-1 was observed under basal conditions. Compared to ASM-1, overexpression of a mixture of ASM-1 and ASM-5 did not change ASM activity levels (102% ±
12.2 of ASM-1 activity and 97.8 ± 4.1 of ASM-1 activity) (Fig. 4.31).
These results imply a dominant-negative effect of ASM-5 on ASM-1 selectively upon serum
starvation.
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4. Results
Fig. 4.31 ASM-5 exerts a dominant-negative effect on ASM-1 upon serum starvation.
Under starving and basal conditions ASM-1 over-expression increased ASM activity, in contrast to ASM-5 and
vector control over-expression. Over-expression of a mixture of ASM-1 and ASM-5 lead to a significant
reduction of ASM activity under starving conditions, which was not observed under basal conditions. ASMFLAG constructs were transiently expressed in HeLa cells. Lysates were subjected to an in vitro enzymeactivity-assay 72 h upon transfection. Results are given in exogenous ASM activity levels in percentage relative
to activity of full-length ASM-1 (100%). Displayed data represent mean values of three independent
experiments, error bars represent SD. Statistical significance was calculated with respect to ASM-1 (* p<0.05).
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5. Discussion
5. Discussion
In this study, we investigated ASM sequence variations on DNA and RNA levels. The aim
was to gain insight into the molecular mechanisms of increased ASM activity levels which
were detected within depressed patients.
Regarding the DNA level we did not detect sequence variations which harboured the
potential to increase the intrinsic activity of ASM enzymes under experimental conditions.
Nevertheless, we identified differential characteristics of known common and rare ASM
sequence variations which were detected within our patient collective. The data concerning
catalytic activity and subcellular localization give further hints for the importance of proper
processing for the regulation of ASM.
Regarding the RNA level we were able to identify to date undescribed ASM splice variants
and to characterize the biochemical properties of their corresponding proteins. We could
show that alternatively-spliced ASM variants are able to exert regulative functions on cellular
ASM levels and that their RNA expression is up-regulated by antidepressant drugs.
In total, this study presents a further piece of the enigma of ASM regulation in health and
disease.
5.1 DNA sequence variations impact Acid Sphingomyelinase activity
We investigated the impact of ASM DNA sequence variations resulting in missense
mutations of the ASM protein sequence on enzymatic activity and subcellular localization
patterns. Compared to ASM wild type, most of the ASM variants displayed specific
characteristics. The opposite effect of NPD mutations which lack full enzymatic activity,
namely an intrinsic increase in ASM activity, was not detected. To summarize results,
increased ASM activity levels detected in depressed patients are probably not caused by the
mere variation of ASM sequence. However, ASM sequence variations might result in ASM
enzymes which could be prone to differential or dysfunctional processing or regulation.
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5. Discussion
5.1.1 The C-terminal domain harbours essential parts for activation and
secretion of Acid Sphingomyelinase
Several findings indicate that alterations in the C-terminal region impact catalytic activity and
subcellular localization of ASM. The C-terminal domain of ASM was shown to be highly
important for ASM activation and cellular trafficking, mostly due to several posttranslational
modifications. So are the essential glycosylation sites which ensure proper lysosomal
localization positioned at the C-terminus (Ferlinz et al., 1997). Also, it is the disulfide bonds
at the C-terminus, which are highly necessary for correct lysosomal localization and catalytic
activity of ASM (Lee et al., 2007). The C-terminal phosphorylation site p.S508 was reported
to be essential for activation of ASM by protein kinase δ (Zeidan and Hannun, 2007a). The
drastic amino acid substitution of serine by alanine at this position (p.S508A) has several
implications for ASM activity and localization: On one side it alters a phosphorylation site
and therefore the potential of being fully activated, on the other side it prevents its ability of
being secreted (Jenkins et al., 2010a). In this study, we investigated the effect of the common
ASM variation p.G506R. Since this constitutes an amino acid exchange in such a close
proximity to the important ASM phosphorylation site p.S508, it could exert influence on
ASM properties. Concerning catalytic activity we detected a statistically significant
difference of 25% with ASM variant p.G506R being less catalytically active than ASM wild
type (Fig. 4.14). Possibly, the phosphorylation of serine 508 by protein kinase δ could be
affected by the exchange of glycine for arginine on ASM position 506, which constitutes a
dramatic alteration of protein sequence. This would mean an inability of ASM p.G506R to
reach its full activity. Equivalently, the same trend was detected for ASM variation p.G506R
and ASM wild type in combination with ASM variation p.A487V. Only a slight difference in
catalytic activity was measured for ASM variation p.G506R and ASM wild type in
combination with ASM variation p.S508A (Fig. 4.14). Since the essential phosphorylation
site on position 508 is altered in both variants, no full catalytic activity is exerted, regardless
of an exchange at position 506. In contrast, these two variants display differential cellular
localization patterns. The ASM variant covering a combination of variations p.G506R and
p.S508A displays very low protein expression in our Western blot analyses (Fig. 4.3) and is
trapped inside the endoplasmic reticulum instead of trafficking to lysosomes (Fig. 4.16) – in
contrast to ASM variants featuring only the singular variations, which show lysosomal
localization (Fig. 4.7; 4.16). Two mechanisms could be responsible for this effect: ASM
variant p.G506R/S508A may be rapidly degraded upon being trapped inside the ER or could
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5. Discussion
be secreted into extracellular space to a major part. To get insight into the cellular fate, it
should be investigated if ASM variant p.G506R/S508A is detected inside the Golgi which
serves as sorting point for the two ASM forms, the lysosomal one and the secreted one
(Jenkins et al., 2010a). In summary, these results imply a regulatory effect of the amino acid
at position 506 and stress the importance of the C-terminal region around position 506/508
for trafficking and secretion of ASM. Further investigations should be conducted to get
insight into the exact mechanisms leading to differential trafficking of ASM enzymes.
5.1.2 Processing of Acid Sphingomyelinase is essential for its regulation in
pathologic context
Several studies about Niemann-Pick disease type A and B give insight into the relation
between ASM genotype and corresponding ASM activity (Simonaro et al., 2002; Sikora et
al., 2003). However, the residual ASM activity detected within patients did not always
correlate with the severity of clinical symptoms, especially for NPD type B (Chan et al.,
1977; Sogawa et al., 1978; Dawson and Dawson, 1982). Also, studies focussing on ASM
variations differed regarding their results about catalytic activity and localization patterns,
depending on the experimental system – using patients’ material or over-expression strategies
of recombinant constructs. For example, ASM variant p.G242R exerted a catalytic activity of
15% wild type activity in a patient’s lymphoblasts, whereas the recombinant ASM variant
which was over-expressed in COS-1 cells reached a level of about 40% wild type activity
(Takahashi et al., 1992b). Another example gives ASM variant delR608: Whereas Jones et
al. (2008) detect this variant inside the lysosomes in patients’ fibroblasts, Jenkins et al.
(2010b) find the recombinant construct expressed in HEK 293 cells trapped within the
endoplasmic reticulum as did Lee et al. in COS-1 cells (2007). Therefore, over-expression
studies are indeed useful for evaluating intrinsic properties of ASM variants, but are at the
same time somewhat limited, since they do not resemble the processing and regulation of a
pathologic cellular context. In our study, we detected the two rare ASM sequence variations
p.P325A and p.A487V within a collective of patients suffering from depression. These
variants were previously defined as Niemann-Pick type B mutations according to their
reduced ASM activity levels in patients’ fibroblasts and blood cells (Simonaro et al., 2002).
This finding seems to constitute a sharp contrast to the assumed underlying genetic defects,
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5. Discussion
where depressed patients showed increased, patients suffering from NPD decreased ASM
activity. Whereas ASM p.P325A was indeed catalytic inactive (Fig. 4.8) but was located
inside the lysosomes (Fig. 4.10), ASM p.A487V displayed the same properties as did ASM
wild type (Fig. 4.11 and 4.13). Obviously, ASM trafficking is of highest importance for ASM
processing, since only proper lysosomal localization allows full catalytic activity in vivo
(Ferlinz et al., 1994; Ni, 2006). So in principle for both variants the possibility is provided to
be processed and activated in vivo due to lysosomal localization under our experimental
conditions. However, processing of p.P325A can probably not compensate the alteration
within the catalytic domain. In contrast, variant p. A487V displays an intact catalytic domain
and therefore could be processed and activated – probably depending on cellular context. As
a third category, Niemann-Pick disease type A variant ASM p.M381I has an altered catalytic
domain and is trapped inside the ER (Fig. 4.7), which prevents any further processing.
Therefore, variants located inside the lysosome and featuring an intact catalytic domain could
exert varying catalytic activities in dependence of their cellular context. In total, cellular
processing of ASM is probably highly influenced by cell type and pathologic context,
resulting in differential modifications of intrinsic properties in vivo. Thus, experimental
conditions could account for restraints in detecting differences between ASM variants
regarding differential regulation. Further studies should concentrate on cell specific ASM
processing and regulation to get insights into disease related mechanisms.
5.2 Alternative splicing impacts Acid Sphingomyelinase activity
In our study we identified the three additional human alternatively-spliced ASM transcripts
ASM-5, ASM-6 and ASM-7, which are catalytically inactive and distinctly expressed within
individuals. Since alternatively-spliced variant ASM-5 exerts a dominant-negative effect on
full-length ASM, cellular ASM activity levels could be regulated by alternative splicing. This
mechanism seems to be induced by chronic administering of antidepressant drugs. ASM
alternative splicing therefore could be a potential mechanism for ASM regulation.
Investigating ASM alternative splicing especially within brain tissue and blood cells could
offer insights into stress and disease related mechanisms.
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5. Discussion
5.2.1 Alternative splicing of Acid Sphingomyelinase could predominate in
brain and blood cells
In this study, we identified three new human alternatively-spliced ASM transcripts. In our
approach, the cloning of PCR amplified full-length transcripts, alternatively-spliced ASM
transcripts occurred to a higher extent than expected (Quintern et al., 1989; Schuchman et al.,
1991b). From the analysis by Schuchman and colleagues, the rate of alternatively-spliced
transcripts was estimated to be about 10% while in our hands, every fourth transcript arose
from alternative splicing. The diverging splicing rates might be due to the different tissue
under investigation. Splice variants ASM-2 and ASM-3 were identified within cDNA
libraries of fibroblast, placental or testis tissue. However, alternative splicing is particularly
important for neuronal functions (Grabowski and Black, 2001) and the brain exhibits the
highest number of tissue-specific splice forms (Xu et al., 2002). Two of our new ASM splice
variants were isolated from a human neuroglioma cell line, and previously described ASM-4
was also derived from human brain tissue. The third new ASM splice variant was identified
within human primary blood cells. This could be of interest, since the main features of
mammalian stress response are changes within the peripheral and central nervous system,
modification of blood cell composition, and alterations in many gene products. For example,
alternative splicing of the acetylcholinesterase was reported as a stress response in brain and
blood cells (Pick et al., 2004). Similarly, alternative splicing of ASM may be particularly
abundant in brain and blood cells as part of the cellular stress response.
5.2.2 Acid Sphingomyelinase displays a defined set of conserved splicing
motifs
In general, the modification of protein properties by alternative splicing is an oftendiscovered phenomenon (Okazaki et al., 2002; Zavolan et al., 2003). In particular,
concerning enzymes, alternative splicing is able to affect all enzymatic properties, such as
substrate affinity, substrate binding, Vmax, activity regulation and catalytic properties (Stamm
et al., 2005). However, alternative splicing of ASM generally results in catalytically inactive
isoforms, as judged by biochemical in vitro assays. The only functional transcript encoding
an active enzyme thus far is transcript ASM-1 encompassing exon 1 to 6 (Fig. 4.22). The
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5. Discussion
ASM enzyme is composed of a putative signal peptide, a saposin-B-domain, a proline-rich
domain, a calcineurine-like phosphoesterase domain and a carboxy-terminal domain.
Compared to this, alternatively-spliced forms lack essential parts. A common motif is the
differential splicing of exon 3, which leads to the generation of ASM isoforms with a
disrupted catalytic domain (e.g. ASM-2, ASM-3). As a variation of this theme, we discovered
a splice form in which exon 3 was only partly missing (ASM-5). A second common motif is
the inclusion of 40 bp intronic sequence adjacent to the 3’ end of exon 2 (herein referred to as
exon 2a) into the mature transcript (found in ASM-2, ASM-7). If not compensated by the outsplicing of exon 3 (as in ASM-2), the inclusion of exon 2a ultimately provokes a shift in the
reading frame (Fig. 2.2, 4.18 and 4.20). Either way leads to the disruption of the catalytic
domain. The last common motif is the inclusion of 20 bp intronic sequence adjacent to the 5’
end of exon 6 (herein referred to as exon 5a) into the mature transcript, which also provokes a
frameshift and the generation of truncated proteins. The exon 5a is present in the transcripts
ASM-4 and ASM-6. However, since a second alternative splicing event affecting exon 2 and
thus the catalytic domain is encountered in ASM-4, ASM-6 is the only splice form retaining
the complete catalytic domain except for functional ASM-1. Nevertheless, just like the other
splice forms, ASM-6 was found to be inactive in our biochemical assays. In total, alternative
splicing selectively affects the integrity of the catalytic and C-terminal domain. Interestingly,
the ASM sequence variations which lead to catalytically-inactive enzymes and cause
Niemann-Pick disease type A and B, are also selectively located within the ASM sequence
coding for the catalytic domain (75%) or the C-terminal part (25%) (Ferlinz et al., 1991;
Levran et al., 1991; Levran et al., 1992; Takahashi et al., 1992a; Takahashi et al., 1992b;
Levran et al., 1993; Ferlinz et al., 1995; Simonaro et al., 2002; Rodriguez-Pascau et al.,
2009). Therefore, changes solely within the C-terminal part can also abolish enzymatic
activity of ASM. Of note, the usage of the motifs described above is conserved among
different species. GenBank search revealed orthologous ASM splice forms identical to
variants ASM-2, -6 and -7 expressed in different primates. Therefore, ASM splicing covers a
defined set of motifs which are evolutionary conserved.
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5. Discussion
5.2.3 Acid Sphingomyelinase activity is regulated by alternative splicing
Since ASM plays an essential role in the fragile balance of the rheostat, alternative splicing
occurs within a highly-regulated context. ASM enzymatic activity exerts strong consequences
for cellular functioning, deciding between apoptosis and proliferation, and is increased upon
stress signalling (Carpinteiro et al., 2008). External stimuli like cellular stress and receptor
signalling as well are an important source of changes in cellular processes and also affect
splice site selection to a great extent. The switch from enzymatic activity to inactivity by
alternative splicing, triggered by stress signals, is a well-known phenomenon (Kaufer et al.,
1998). According to this, dysbalanced ASM activity upon occurrence of stress stimuli could
be rebalanced by alternative splicing events. Under basal conditions, the rheostat is balanced,
with ASM exerting basal enzymatic activity. Under stress conditions, ASM is fully activated,
exerting increased enzymatic activity, which results in an accumulation of ceramide. The
dysbalanced rheostat could be rebalanced by a switch in splice site selection via the
spliceosome, leading to an increase in inactive ASM enzymes, which would counter the
effects of the dysbalanced rheostat. Experimental evidence for this assumption is offered by
our in vivo results. Ratios of cellular ceramide to sphingomyelin contents upon new ASM
splice variants expression are significantly lower than those of the control condition (Fig.
4.23) and equate to untransfected cells (data not shown). Since transfection of cells always
means cellular stress, the control condition is also subjected to elevated endogenous ASM
activity levels. Expression of catalytic inactive ASM splice variants clearly decreases the
ceramide to sphingomyelin ratio, modulating the stress-induced increase in ASM activity.
The result is a normalized cellular ceramide to sphingomyelin ratio, which corresponds to the
ratio within cells in an unstressed state.
The mechanisms of how primarily inactive ASM isoforms could contribute to total cellular
ASM activity probably depend on the different structural and functional categories of ASM
splicing. It was reported that activated ASM forms dimers in particular via the most carboxyterminal located cysteine (Qiu et al., 2003). Thus, inactive ASM isoforms which retain the Cterminus could act as dominant-negative modulators of ASM activity by binding to active
ASM isoforms (Stasiv et al., 2001; Zhang et al., 2010). This mechanism could take effect on
ASM-5, which was shown to inhibit ASM activity upon co-expression with full-length ASM
and serum starvation (Fig. 4.31). Since ASM-5 displays an intact C-terminus, it should be
capable of dimerizing – despite its disrupted catalytic domain. As shown in figure 4.30,
ASM-5 translocates into the lysosome upon serum starvation, where it could interact with
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5. Discussion
full-length ASM, leading to a dominant-negative effect on active ASM in this stress
condition.
For ASM-6 and -7, different mechanisms should take effect due to their truncation. Splicing
events which result in truncated variants are often subjected to nonsense-mediated decay
(Stamm et al., 2005). Three-quarters of alternatively-spliced transcripts featuring a frameshift
leading to a premature stop codon are supposed to undergo nonsense-mediated decay and are
switched off, probably because of their instability (Lewis et al., 2003; Wollerton et al., 2004).
Therefore, the cellular ASM activity level could be decreased by switching off ASM
transcripts -6 and -7 after a short life within the cell.
Additional hints for a regulative role of alternative spliced isoforms are given by our results
about expression patterns of new ASM splice variants within individuals. Full-length ASM-1
and each of the new ASM splice variants were expressed to a different extent upon interindividual comparison. Also, regarding the intra-individual level, highly-differential
expression patterns of ASM splice variants were observed (Fig. 4.27). These findings clearly
show that alternative splicing of ASM is regulated and therefore bears relevance for cellular
processes. Expression patterns of patients suffering from depression show that ASM splice
variants are differentially regulated by antidepressant drugs. Despite the small case number
and the complexity of influencing factors it is obvious that especially ASM-5 is dramatically
up-regulated after chronic administering of different antidepressants (Fig. 4.28). Effects of
antidepressants on the selective up-regulation of a special splice variant were already shown
for further stress- and depression related molecules (D'Sa et al., 2005; Funato et al., 2006).
Antidepressant drugs therefore could be able to inhibit ASM activity by two different
mechanisms: Functional inhibitors like desipramine and imipramine inhibit ASM activity via
the detachment of ASM from the lysosomal membrane and subsequent proteolytic cleavage
inside the lysosome (Kölzer et al., 2004). Since all ASM transcripts showed increased
expression levels upon administering of antidepressants, this degradation in turn could lead to
enhanced ASM transcription to ensure supply. Potent ASM inhibitors like fluoxetine and
amitriptyline could also inhibit ASM activity via the induction of the dominant-negative
splice variant ASM-5. Accordingly, the effect exerted by ASM-5 due to its unique properties
probably is of strong importance for cellular ASM regulation. Further studies should
elucidate the exact relation between splicing patterns and respective cellular ASM activities.
In total, elevated cellular ASM activity could be regulated by splicing events, generating
inactive variants for acting as modulators of wild type ASM. In pathologic context, this
mechanism could be initiated by the effect of antidepressants.
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6. Bibliography
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106
7. Appendix
7.1 Chemicals
Agarose
APS
Ampicillin
β-Mercaptoethanol
Bacto-Agar
Bromphenol blue
Calciumchloride (CaCl2)
Chloroform
Desoxyribonucleictriphosphat-Set (dNTP)
D, L-Dithiothreitol (DTT)
Dimethylsulfoxide (DMSO)
DMEM-Medium
ECL-Solution
Ethanol
Ethidiumbromide
FCS
Glacial acetic acid
GFP
L-Glutamine
Glycin
Isopropanol
Yeast extract
Kaliumchloride (KCl)
Kaliumdihydrogenphosphate (KH2HPO4)
Kanamycin
Magnesiumchloride (MgCl2)
Magnesiumsulfate (MgSO4)
Milk powder
Natriumchloride (NaCl)
Natriumdodecylsulfate (SDS)
Natriumhydrogenphosphate (Na2HPO4)
Nonidet P40
Oligonucleotides
Protease-Inhibitor Mix (CompleteMini)
Qiazol
Sphingomyelin 14C
TEMED
Tris(hydroxymethyl)-aminomethane (Tris)
Triton-X-100
Trypsin
Trypton
Tween20
Biozym, Oldendorf
Merck, Darmstadt
Sigma-Aldrich, Munich
Roth, Karlsruhe
Roth, Karlsruhe
Merck, Darmstadt
Merck, Darmstadt
Roth, Karlsruhe
Peqlab, Erlangen
Amersham, Freiburg
Roth, Karlsruhe
Biochrom, Berlin
Amersham, Freiburg
Merck, Darmstadt
Roth, Karlsruhe
Biochrom, Berlin
Merck, Darmstadt
Amaxa, Cologne
Roth, Karlsruhe
Merck, Darmstadt
Roth, Karlsruhe
Roth, Karlsruhe
Merck, Darmstadt
Merck, Darmstadt
Roth, Karlsruhe
Roth, Karlsruhe
Amersham, Freiburg
Operon, Huntsville
Roche, Mannheim
Qiagen, Hilden
Perkin Elmer, Waltham, USA
Roth, Karlsruhe
AppliChem, Darmstadt
Biochrom, Berlin
Roth, Karlsruhe
Roth, Karlsruhe
107
7.2 Technical device
Biofuge Centrifuge pico
Block heater
Casy cell counter
Cooling centrifuge
Experion System
Fluoreszenzmicroscope Axiovert 135
Freezer
Gel system for western blotting
Hicclave HV-85 Autoklav
Ice machine
Incubator Hera cell
Laser scanning microscope DM IRE2
Light microscope DM IL HC Bio
Microplate Reader
Nitrogen tank
Oven
pH-Meter
Photometer
Pipettes
Pipetboy
Power Supply Electrophoresis EPS 301
QuantityOne Fluor-S ™ MultiImager
Sterile hood
Thermomixer comfort
Thermocycler
Typhoon
Vortex Mixer
Heraeus, Hanau
Grant Boekel, UK
Innovatis, Reutlingen
Eppendorf, Hamburg
BioRad, Munich
Zeiss, Jena
Liebherr Bulle, Switzerland
BioRad, Munich
Schubert-Weiss, Munich
Scotsman, Herborn
Heraeus, Hanau
Leica, Solms
Leica, Solms
BioRad, Munich
air liquide, Düsseldorf
Memmert, Schwabach
WTW, Weilheim
Eppendorf, Hamburg
Eppendorf, Hamburg/
Gilson, Frankreich
VWR, Darmstadt
Schubert&Weiss, Munich
Amersham, Freiburg
TKA-Lab, Niederelbert
Heraeus, Hanau
Eppendorf, Hamburg
Sensoquest, Göttingen
Amersham, Freiburg
NeoLab, Heidelberg
7.3 Auxiliary material
Cell culture flasks
Cell culture plates
Eppendorf tubes
Medical gloves
Parafilm
Pasteurpipettes
Pipette Tips
PVDF Membrane
Serologic Pipettes
Tissues
TPP, Switzerland
TPP, Switzerland
Eppendorf, Hamburg
Kimberly-Clark, UK
Pechiney, Chicago, USA
Roth, Karlsruhe
Eppendorf, Hamburg
Millipore, Schwabach
GreinerBio-One, Frickenhausen
Roth, Karlsruhe
108

The impact of sequence variation on Acid

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