dms4 pro se

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dms4 pro se
© 2006 Nature Publishing Group http://www.nature.com/naturegenetics
ARTICLES
Positional cloning uncovers mutations in PLCE1
responsible for a nephrotic syndrome variant that
may be reversible
Bernward Hinkes1,22, Roger C Wiggins2,22, Rasheed Gbadegesin1, Christopher N Vlangos1, Dominik Seelow3,4,
Gudrun Nürnberg3,4, Puneet Garg2, Rakesh Verma2, Hassan Chaib1, Bethan E Hoskins1, Shazia Ashraf1,
Christian Becker3,4, Hans Christian Hennies3,5, Meera Goyal2, Bryan L Wharram2, Asher D Schachter6,
Sudha Mudumana6, Iain Drummond6, Dontscho Kerjaschki7, Rüdiger Waldherr8, Alexander Dietrich9,
Fatih Ozaltin10, Aysin Bakkaloglu10, Roxana Cleper11, Lina Basel-Vanagaite11, Martin Pohl12, Martin Griebel13,
Alexey N Tsygin14, Alper Soylu15, Dominik Müller16, Caroline S Sorli17, Tom D Bunney17, Matilda Katan17,
Jinhong Liu1, Massimo Attanasio1, John F O’Toole1, Katrin Hasselbacher1, Bettina Mucha1, Edgar A Otto1,
Rannar Airik18, Andreas Kispert18, Grant G Kelley19, Alan V Smrcka20, Thomas Gudermann9,
Lawrence B Holzman2, Peter Nürnberg3,5 & Friedhelm Hildebrandt1,21
Nephrotic syndrome, a malfunction of the kidney glomerular filter, leads to proteinuria, edema and, in steroid-resistant nephrotic
syndrome, end-stage kidney disease. Using positional cloning, we identified mutations in the phospholipase C epsilon gene
(PLCE1) as causing early-onset nephrotic syndrome with end-stage kidney disease. Kidney histology of affected individuals showed
diffuse mesangial sclerosis (DMS). Using immunofluorescence, we found PLCe1 expression in developing and mature glomerular
podocytes and showed that DMS represents an arrest of normal glomerular development. We identified IQ motif–containing
GTPase-activating protein 1 as a new interaction partner of PLCe1. Two siblings with a missense mutation in an exon encoding
the PLCe1 catalytic domain showed histology characteristic of focal segmental glomerulosclerosis. Notably, two other affected
individuals responded to therapy, making this the first report of a molecular cause of nephrotic syndrome that may resolve after
therapy. These findings, together with the zebrafish model of human nephrotic syndrome generated by plce1 knockdown, open
new inroads into pathophysiology and treatment mechanisms of nephrotic syndrome.
A major component of vertebrate fluid homeostasis is the glomerular
filter of the kidney, which in humans comprises 1 million filtering
units (glomeruli) per kidney that allow passage of water and small
molecules but retain most proteins, including albumin. Nephrotic
syndrome is a common kidney disease characterized by leakage of
the glomerular filter, leading to albumin loss (proteinuria). The
resulting low–serum albumin state (hypoalbuminemia) lowers the
protein-driven capillary pressure gradient, leading to failure of fluid
reabsorption with consequent accumulation of fluid in tissues resulting in body swelling (edema). Nephrotic syndrome (the triad of
proteinuria, hypoalbuminemia and edema) is classified as steroidsensitive or steroid-resistant. Steroid-resistant nephrotic syndrome
(SRNS) is frequently associated with a patchy scarring process in
the glomerulus (focal segmental glomerulosclerosis, or FSGS) and
1Department of Pediatrics and 2Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109, USA. 3Cologne Center for Genomics, University
of Cologne, Cologne, Germany. 4RZPD Deutsches Ressourcenzentrum für Genomforschung GmbH, Berlin, Germany. 5Institute for Genetics, University of Cologne,
Cologne, Germany. 6Children’s Hospital Boston and Renal Unit, Harvard Medical School, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.
7Clinical Institute of Pathology, Medical University of Vienna, Vienna, A-1090, Austria. 8Gemeinschaftspraxis Pathologie, D-69115 Heidelberg, Germany. 9Department
of Pharmacology and Toxicology, Philipps-University, Marburg, Germany. 10Department of Pediatric Nephrology, Hacettepe University Faculty of Medicine, Ankara,
Turkey. 11Department of Medical Genetics, Schneider Children’s Medical Center of Israel and Rabin Medical Center, Petah Tiqva, Israel and Sackler School of Medicine,
Tel Aviv University, Tel Aviv, Israel. 12Department of Pediatrics, Freiburg University, D-79106 Freiburg, Germany. 13Children’s Hospital, Technical University,
München-Schwabing, Germany. 14The Scientific Center of Children’s Health, Moscow, Russia. 15Department of Pediatrics, Dokuz Eylul University, Izmir, Turkey.
16Department of Pediatric Nephrology, Charite Children’s Hospital, Berlin, Germany. 17Cancer Research UK Centre for Cell and Molecular Biology, Chester Beatty
Laboratories, The Institute of Cancer Research, Fulham Road, London SW3 6JB, UK. 18Institute for Molecular Biology, Medizinische Hochschule Hannover, D-30625
Hannover, Germany. 19Department of Medicine, State University of New York Upstate Medical University, Syracuse, New York 13210, USA. 20Department of
Pharmacology and Physiology, University of Rochester School of Medicine, Rochester, New York 14642, USA. 21Department of Human Genetics, University of
Michigan, Ann Arbor, Michigan 48109, USA. 22These authors contributed equally to this work. Correspondence should be addressed to F.H. (fhilde@umich.edu).
Received 23 June; accepted 6 October; published online 5 November 2006; doi:10.1038/ng1918
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well as the dominant genes WT1 (Wilms tumor suppressor gene 1)4,
ACTN4 (actinin alpha-4)5 and TRPC6 (canonical transient receptor
potential 6 ion channel)6,7. The results from gene identification
together with data from animal models have placed glomerular
epithelial cells (podocytes) at the center of the disease mechanisms
of SRNS8–10. Podocytes are neuron-like terminally differentiated
a
LOD score
Figure 1 Positional cloning of PLCE1 as mutated
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 2122
6
in NPHS type 3. (a) Parametric multipoint LOD
5
score profile across the human genome,
4
calculated in four consanguineous kindred with
3
nephrotic syndrome (NS). Parametric LOD scores
2
are on the y-axis in relation to genetic position
1
on the x-axis. Human chromosomes are
0
concatenated from p-terminal (left) to q-terminal
(right) on the x-axis. Note the significant
–1
maximum LOD score of 5.1 on human
–2
chromosome 10 (arrowhead), defining a new
–3
0
1,000
500
3,500
1,500
3,000
2,000
2,500
gene locus (NPHS3) for NS on chromosome
Genetic distance (cM)
10q23.32–q24.1. (b) Haplotype analysis refining
Marker
Position
A601
F389
A38
F331
F1063
the NPHS3 locus by homozygosity mapping in
II-1
II-2
II-1
II-2
II-1
II-4
II-1
II-1
[Mb]
five consanguineous kindred with NS. Left:
a
b
a
a
a
a
a
a
a
a
a
a
a
a
a
a
SNP_A1680351
54,387,029
microsatellite and SNP markers on human
D10S1735
90,642,030
191 191
191 191
193 193
193 187
189 189
189 191
190 190
189 189
D10S1242
92,354,658
167 167
167 167
158 158
158 174
149 149
149 158
145 145
163 163
chromosome 10q, with physical map positions
D10S1753
92,402,953
280 280
280 278
278 280
278 280
294 294
294 280
278 280
286 288
D10S564
92,589,720
272 272
272 272
272 272
272 226
272 272
272 272
272 272
270 270
(http://genome.ucsc.edu; May 2004 freeze),
D10S1171
92,684,341
224 224
224 224
232 232
232 220
228 228
228 224
224 224
228 228
showing positions of PLCE1 and two additional
D10S536
92,873,315
147 147
147 147
147 147
147 149
151 149
151 145
147 147
145 145
T
T
T
T
T
T
93,308,518
SNP A1717632
G
G
G
G
G
T
T
T
T
T
candidate genes. Numbers of kindred and
TNKS2-ab TNKS2 93,586,341
b
b
b
b
a
a
a
a
b
b
b
b
a
a
b
b
D10S1173
94,097,176
214 214
214 214
210 210
210 210
206 206
206 206
207 207
223 223
affected individuals (Table 1) are given above
D10S583
94,358,908
219 219
219 219
224 224
224 224
223 223
223 223
222 222
231 231
haplotypes. Alternative haplotypes are shown on
D10S185
95,178,273
167 167
165 165
159 159
159 159
165 165
165 165
167 167
171 171
D10S200
95,303,677
311 311
311 311
311 311
311 311
311 311
311 311
314 314
313 313
yellow and turquoise background, and observed
D10S1680
95,591,364
224 224
224 224
224 224
224 224
230 230
230 230
222 222
222 222
95776014
95,776,014
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
recombinants on gray background. Segments of
D10S677
95,954,428
219 219
219 219
215 215
215 215
227 227
227 227
215 215
197 197
PLCE1 95,984,772
homozygosity are boxed. Under the hypothesis
D10S1690
163 163
163 163
170 170
170 170
170 170
170 170
171 171
nd nd
b
b
b
b
b
b
a
a
a
a
96057937
96,057,937
a
a
a
a
b
b
of homozygosity by descent, marker
D10S520
96,414,616
186 186
186 186
202 202
202 202
194 194
194 194
186 186
198 198
D10S571 PDLIM1 97,128,430
221 221
221 221
217 217
217 217
207 207
207 207
227 227
223 223
SNP_A1717632 (underlined) delimits the
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
97,343,416
SNP A1715598
a
NPHS3 locus on the centromeric side by showing
D10S1758
98,933,672
nd
nd
201 207
207 207
207 207
203 203
203 203
201 201
197 197
D10S1726
100,701,942
nd
nd
211 215
209 209
209 209
209 209
209 209
211 211
209 209
heterozygosity in individual F389 II-2, whereas
D10S192
102,426,246
211 215
211 215
205 205
205 205
205 205
205 205
nd
nd
211 211
SNP_A-1650393
104,766,465
b
b
a
a
a
a
a
b
a
b
a
a
a
a
a
a
marker SNP_A1715598 (underlined) delimits
b
b
b
b
a
a
a
a
a
b
a
b
a
a
a
a
SNP_A-1711839
112,448,484
a
b
a
b
a
a
a
a
a
b
a
b
a
b
b
b
SNP_A-1678711
119,523,246
the NPHS3 locus on the telomeric side by
a
a
a
a
a
a
a
a
a
b
a
b
a
a
a
b
SNP_A-1670332
128,147,200
heterozygosity in individual A601 II-1, refining
the NPHS3 locus to an interval of 4.0 Mb.
(c) The NPHS3 critical genetic region extends
SNP_A1717632
SNP_A1715598
over a 4.0-Mb interval between flanking markers
PDLIM
TNKS2
PLCE
SNP_A1717632 and SNP_A1715598. Arrows
indicate location and transcriptional direction of
three positional candidate genes that showed
334.4 kb
ATG
TAA
increased expression in rat renal glomerulus.
1
2
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
Forty additional candidate genes that localized
500 bp
within the interval are not shown. Mutations were
detected in the PLCE1 gene. (d) PLCE1 extends
over 334.4 kb and contains 34 exons (vertical
bars). (e) Exon structure of human PLCE1 cDNA
2302 amino acids
showing positions of start codon (ATG) at nt +1
F331
A942 (P382fsX)
A38 (R493X)
A1274 (Q1616X)
F1063 (Q1854X)
and stop codon (TAA). Exon size (62–676 bp) is
F389 (R1116X)
(L1281fsX)
A601 (S1484L)
G
X
M
C
X
S
P
Q
Q
Y
X
K
L
X
F
averaged graphically except for exon 2 (1,570
I
L
I
CT T TAGT T T
G G A T G A AT G
T GT TG A AGC
A TAT TGAT T
T A T TA G A A G
C C |T C A A C A G
bp). Arrows indicate relative positions of
mutations (see g). (f) Positions of putative protein
domains, in relation to the encoding exon
position in e. For protein domains, see
Supplementary Figure 1. (g) Seven different
Y
Q
K
G
R
M
C
R
S
P
S
T
L
Q
F
I
S
I
homozygous PLCE1 mutations (six truncating and
G G A CG A AT G
C CG T CA A C A
C T T CA G T T T
T A T CA G A A G
T G T CG A A G C
A T A T CG A T T
one missense) were detected in seven NPHS3
families. Family number and mutations (Table 1)
are given above sequence traces. Nucleotide
sequence and resulting amino acid sequence are
shown for mutated (top) and wild-type (bottom)
sequences. Mutated nucleotides and amino acid codons are underlined and highlighted in gray. Vertical hatches denote single-nucleotide deletions. Lines
and arrows indicate positions of mutations in relation to exons (see e) and putative protein motifs (see f and Supplementary Fig. 1).
b
97.3 Mb
c
93.3 Mb
d
RA2
RA1
C2
PLC_X
PH
f
PLC_Y
e
Ras
GEF
© 2006 Nature Publishing Group http://www.nature.com/naturegenetics
commonly progresses to end-stage kidney disease (ESKD), requiring
renal replacement therapy in the form of dialysis or kidney transplantation for survival.
Mutations in several genes have been identified by positional cloning as causing SRNS in humans. These include the recessive genes
NPHS1 (nephrin)1, NPHS2 (podocin)2 and LAMB2 (laminin-b2)3 as
g
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Table 1 Six different homozygous truncating mutations and one missense mutation of PLCE1 detected in seven families with early-onset
nephrotic syndrome type 3
Alteration(s)
© 2006 Nature Publishing Group http://www.nature.com/naturegenetics
Family Individual
Country
of origin
Parental
Nucleotide
consanguinity alteration(s)
in coding
sequencea
Exon
(segregation)
Age at
onset
Age at
ESKD
Treatment
Histology
(at age)
A942
II-3
Turkey and
Russia
Unknown
1146delG
P382fsX387
2 (hom, M, P)
4 mo
5 mo
No treatment
ND
A38
II-1
II-4
Israel
Y
1477C-T
R493X
3 (hom, M, P)
4 mo
2 mo
10 mo
No NS at 13 yr
SRNSb
SRNS, CSA-Sc
DMS (7 mo)
DMS (5 mo)
F389
II-1
II-2
Turkey
Y
3346C-T
R1116X
10 (hom, M, P)
4 yr
2 yr
5 yr
2 yr
SRNS
No treatment
ESKD/DMS (4.5 yr)
DMS (2 yr and 3.5 yr)
F331
II-3
II-1
Turkey
Y
3843delG L1281fsX1308
14 (hom, M, P)
3 yr
6 mo
3 yr
6 mo
No treatment
No treatment
DMS (2.9 yr)
ND
A601
II-1
II-2
Turkey
Y
4451C-T
S1484Ld
18 (hom, M, P)
8.8 yr
2 yr
12 yr
4 yr
SRNS and CP-R
SRNS and CP-R
FSGS (8.9 yr)
FSGS (4.6 yr)
A1274
II-1
II-3
Turkey
Y
4846C-T
Q1616X
21 (hom, ND, ND) 8 mo
o 3 yr
1 yr
8 mo
SRNS
SRNS
DMS (8 mo)
DMS/FSGS (7 mo)
II-1
Turkey
Y
5560C-T
Q1854X
F1063
25 (hom, M, P)
II-2
I-3
12 mo
None at 6 yr
7 mo
None at 14 mo
8 mo Died of ESKD at 11 mo
SSNSc
ND
No treatment
ND
ND
DMS (11 mo)
hom, homozygous in affected individual; M, heterozygous mutation identified in mother; P, heterozygous mutation identified in father; ND, no data or DNA available; CSA-S,
cyclosporin A–sensitive; SRNS, steroid-resistant nephrotic syndrome; SSNS, steroid-sensitive nephrotic syndrome; DMS, diffuse mesangial sclerosis; FSGS, focal segmental
glomerulosclerosis; ESKD, end-stage kidney disease; CP-R, cyclophosphamide-resistant; mo, months; NS, nephrotic syndrome; yr, years.
aAll
mutations were absent from 478 healthy control subjects from Central Europe and from 60 healthy control subjects from Turkey. bCyclosporin A treatment was attempted for only 10 d.
cases. dAltered amino acid residue positioned in the PLC_X domain and conserved in many species, including a C. elegans ortholog.
cTreatment-sensitive
epithelial cells that function to support and maintain the glomerular
basement membrane (GBM). They have a cell body with projecting
octopus-like major processes branching to form intermediate processes and tertiary (‘foot’) processes. Each foot process links to the foot
process of a neighboring podocyte through specialized intercellular
junctions (the ‘slit diaphragm’) and abuts the GBM through an
integrin-linked adhesion mechanism. This structure serves to create
maximal filtration space between cells while at the same time
supporting and maintaining the GBM10.
As the molecular cause of over 70% of all SRNS is unknown, and
because treatment options have yet to be identified, we performed a
whole-genome search for linkage to identify further causative genes.
We identified recessive mutations in PLCE1 as the cause of a nephrotic
syndrome variant, making this the first report of a molecular cause of
nephrotic syndrome that resolved after therapy in some individuals.
RESULTS
Positional cloning of PLCE1 mutations in nephrotic syndrome
We generated whole-genome haplotype data for 22 consanguineous
SRNS families with one affected child and for four consanguineous
SRNS families with two affected children using an Affymetrix 50K
SNP array. All subjects were negative for mutations in NPHS1,
NPHS2, WT1 and LAMB2. As single affected individuals pose a
high risk of representing phenocopies, we calculated whole-genome
parametric multipoint LOD score analysis for only the four multiplex
families (A601, F389, A38 and F310). This yielded a significant LOD
score of LODmax ¼ 5.1 on chromosome 10q23.32–q24.1, thereby
defining a new gene locus (NPHS3) for nephrotic syndrome type 3
(NPHS3) (Fig. 1a). When we evaluated whole-genome haplotype
analysis for all 26 families at the NPHS3 locus, we found that only
three of the four multiplex families (A601, F389 and A38) and two of
the 22 simplex families (F331 and F1063) showed a continuous
segment of homozygosity suggesting homozygosity by descent11
(Fig. 1b). We confirmed the locus by typing 18 microsatellite markers
in the three multiplex families and two simplex families that had
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shown homozygosity (Fig. 1b). Detection of heterozygosity delimited
the putative critical genetic region to an interval of 4.0 Mb between
markers SNP_A1717632 and SNP_A1715598 (Fig. 1b,c). This region
contained 43 predicted genes. We based candidate gene selection for
mutational analysis on the hypothesis that human nephrotic syndrome is caused by mutations in genes expressed by podocytes1–7,12,13.
We used DNA microarrays from rat glomeruli to prioritize and
directly sequence the exons of three candidate genes within the 43
proposed genes in the region of interest (Fig. 1b–d). Expression of the
major candidate gene, Plce1, was 10.7-fold higher in glomeruli
compared with renal cortex and 11.8-fold higher in glomeruli compared with medulla. In addition, expression of Plce1 was 5.9-fold
higher in podocyte-containing glomeruli compared with podocytedepleted glomeruli (data not shown).
Mutational analysis of the PLCE1 gene in the five kindred examined
by haplotype analysis (Fig. 1b) and in two additional individuals
(A942 and A1274) homozygous for microsatellites at the NPHS3 locus
yielded seven different homozygous mutations (Fig. 1e–g and
Table 1). Six of these were truncating mutations (nonsense and
frameshift) in exons 2, 3, 10, 14, 21 and 25. One was a missense
mutation in exon 18 (leading to S1484L) (Fig. 1e–g and Table 1). This
serine residue is positioned in the catalytic domain (PLC_X) of PLCe1
(Fig. 1f) and is fully conserved in evolution, including in Caenorhabditis elegans (Supplementary Fig. 4 online). All mutations were absent
in 478 healthy control subjects from Central Europe and 60 healthy
control subjects from Turkey. Segregation of mutations was consistent
with recessive inheritance when parental DNA was available (Table 1).
We thereby identified mutations in PLCE1 (also known as NPHS3) as
a new cause of recessive nephrotic syndrome type 3 (NPHS3).
The PLCE1 gene extends over 334.4 kb and contains 34 exons
(Fig. 1d,e). PLCe1 belongs to the phospholipase family of proteins
that catalyzes the hydrolysis of polyphosphoinositides such as phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) to generate the second messengers Ins(1,4,5)P3 and diacylglycerol14. These products
initiate a cascade of intracellular responses that result in cell growth
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Figure 2 Glomerular expression of PLCe1 and identification of interaction
PLCε1-FLAG + – + + + + +
Antibody
IQGAP1-GFP (2-210) – – – – + – –
preabsorbed
partner IQGAP1. (a) Protein blot of rat kidney and cultured mouse podocyte
IQGAP1-GFP (2-522) – – – – – + –
IQGAP1-GFP FL – – – – – – +
lysates. We used anti-PLCe1-RA1 (ref. 20) preabsorbed with either GST
Nephrin – + + + – – –
alone (lanes 1–4) or the PLCe1-RA1-GST fusion protein (lanes 5–8). In
250 kDa
lanes 1–4, bands at the expected sizes of approximately 258 kDa and
180 kDa
250 kDaIP: Nephrin IP: GFP
224 kDa for the two PLCe1 isoforms were detected in extracts from cultured
PB: PLCε1 PB: PLCε1
150 kDamouse podocytes and isolated glomeruli but not in extracts from whole renal
PB: PLCε1 250 kDa
cortex or medulla. Lower–molecular weight bands are present in cortex and
100 kDa180 kDa
75 kDamedulla. In lanes 5–8, all bands were absorbed out by preincubation with
180 kDa
PB: Nephrin
the PLCe1-RA1-GST fusion protein. In lanes 9 and 10, the two glomerular
50 kDalanes 2 and 6 have been stripped and reprobed with monoclonal anti1 2 3 4
5 6 7 8
9 10
180 kDa
110 kDa
podocalyxin39 as a podocyte marker to confirm similar loading and transfer
64 kDa
PB:
IQGAP1
+
–
–
IP:
Rabbit
IgG
of the glomerular extract. (b) PLCe1 coimmunoprecipitates with IQGAP1 but
–
+
IP: PLCε1 –
48 kDa
not with nephrin. HEK293T cells were cotransfected with the indicated
+
+
Lysate +
expression plasmids (+/–) and protein blot analysis was performed with the
250 kDa180 kDaindicated antibodies (PB). CoIP in HEK293T cell lysates was performed with
PB: IQGAP1
polyclonal anti-nephrin (for nephrin) and anti-GFP (for IQGAP1). After coIP,
PLCe1 was detected by protein blot (PB) using anti-PLCe1-RA1. Additional protein blots controlling for presence of proteins in the lysates are shown in the
lower three panels. (c) Immunoprecipitation of endogenous IQGAP1 from cultured mouse podocytes. Cells were grown to confluence and then lysed in RIPA
buffer. Immunoprecipitation was performed with affinity-purified polyclonal antibody raised against the RA1-GST fusion protein (Plce1) and blotted with
mouse monoclonal IQGAP1 antibody (BD Bioscience). Rabbit immunoglobulin G (IgG) (Sigma) was used as a control.
© 2006 Nature Publishing Group http://www.nature.com/naturegenetics
GST-RA1
GST alone
Medulla
Cortex
Glomeruli
Podocytes
Medulla
Cortex
Glomeruli
b
Podocytes
a
c
and differentiation and gene expression. PLCe1 isoform A (2,302
amino acid residues) has a relative mobility of 258 kDa. Isoform B
(1994 amino acid residues) has a relative mobility of 224 kDa. PLCe1
contains the following putative protein domains (Fig. 1f)14:
RasGEF_CDC25 (guanine nucleotide exchange factor for Ras-like
small GTPases domain), PH domain (pleckstrin homology domain),
EF hand, phospholipase catalytic domains (PLC_X and PLC_Y), C2
motif (protein kinase C conserved region 2, subgroup 2) and RA1 and
RA2 domains (RasGTP binding domain from guanine nucleotide
exchange factors) (Supplementary Fig. 1 online). Most of the predicted domains and motifs of human PLCe1 are highly conserved in
plce1 orthologs of evolutionarily distant organisms such as Danio rerio
(zebrafish) (65% amino acid sequence identity) and C. elegans (30%
amino acid identity) (Supplementary Fig. 4), suggesting a conserved
function of the domain assembly within PLCe1.
PLCE1 mutations cause severe nephrotic syndrome
We did not observe any extrarenal manifestations in any of the
individuals with PLCE1 mutations. In the six kindred with homozygous truncating mutations of PLCE1, all 12 affected individuals
manifested with proteinuria by 4 years of age (median 0.8 years, range:
0.2–4.0 years) (Table 1). All 12 developed gross proteinuria and
edema. Nine of the twelve individuals with truncating mutations
progressed to end-stage kidney disease (ESKD) by 5 years of age
(median 0.9 years, range 0.5–5.0 years) (Table 1). Notably, of the
individuals with truncating mutations, two children responded to
treatment with corticosteroids or cyclosporin A (underlined in
Table 1), although infantile nephrotic syndrome is traditionally
regarded as treatment resistant15. One child (A38 II-4) responded to
an initial 4-month course of cyclosporin A treatment, which was
extended to 2.5 years. He remains free of proteinuria at his current age
of 13 years under treatment with an angiotensin-converting enzyme
(ACE) inhibitor for hypertension. Another child (A1063 II-1) presented with nephrotic-range proteinuria at 12 months with gross
proteinuria (protein/creatinine ratio (P/Cr) of 13.5; normal is o0.2).
He responded to an 8-month course of steroid therapy and has
been virtually free of symptoms since then. Presently, at the age of
6 years, he shows normal serum albumin, normal serum creatinine
(0.2 mg/dl) and a near-normal P/Cr of 0.37 (Table 1). All other forms
of nonsyndromic childhood nephrotic syndrome, for which the
mutated gene is known, are characterized by a complete lack of
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response to therapy. This has been shown for mutations in NPHS1
(ref. 12), NPHS2 (ref. 16), WT1 (ref. 4) and LAMB2 (ref. 3). Therefore,
identification of mutations in PLCE1 causing NPHS3 represents the
first report of a molecular cause of nephrotic syndrome that is
responsive to therapy in some individuals.
Renal histopathology shows DMS or FSGS
In all eight individuals with homozygous truncating mutations in
whom histopathology of the kidney was available, we found DMS
(Table 1). DMS is a distinct clinicopathologic entity of severe
nephrotic syndrome17. It is characterized clinically by early-onset
nephrotic syndrome within the first 4 years of life and by rapid
progression to ESKD before 5 years of age18,19. In contrast, renal
histology in both siblings of the only kindred (A601) with a homozygous nontruncating missense mutation (leading to S1484L) showed
focal segmental glomerulosclerosis (FSGS) (data not shown, Table 1).
The missense mutation was positioned in an exon encoding the PLCe1
catalytic domain PLC_X. In this sibling pair, age of onset of proteinuria was comparatively late (8.8 years and 2.0 years), as was the age of
onset for ESKD (12.0 years and 4.0 years) (Table 1). This finding may
indicate that nontruncating PLCE1 mutations might be associated
with the histological finding of FSGS rather than DMS and with
slower progression into ESKD.
PLCe1 is expressed in podocytes of mature renal glomeruli
To study PLCe1 protein expression, we characterized two different
polyclonal antibodies to domain RA1 (ref. 20) and antibody CS117
(ref. 21) of PLCe1, both of which have been described in the literature
(Supplementary Fig. 2 online). We examined the distribution of the
PLCe1 protein in the kidney, as earlier analysis suggested enrichment
of Plce1 mRNA expression in glomeruli and possibly in podocytes,
and because most genes thus far identified as mutated in nephrotic
syndrome are highly expressed in podocytes22. Immunoblotting using
the immunopurified RA1-domain polyclonal antibody (hereafter
‘anti-PLCe1-RA1’)20 showed the presence of PLCe1 in protein extracts
from isolated rat glomeruli and cultured mouse podocytes (Fig. 2).
PLCe1 was not detectable in extracts from whole renal cortical or
medullary extracts (Fig. 2). This result confirms enrichment in
glomeruli, as seen in the mRNA data on Plce1 that we used for
candidate gene selection. To further refine renal glomerular PLCe1
expression, we performed immunofluorescence studies of rat kidney
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Figure 3 PLCe1 localizes to glomerular podocytes in adult rat. (a) AntiPLCe1-RA1 (green) labels specific cells (exemplified by arrowheads)
in the glomerulus. (b) The podocyte apical membrane marker GLEPP1
(red) identifies podocyte cell bodies (arrowheads) and foot processes
along the glomerular capillary outlining glomerular structure. DAPI labels
nuclei (blue). (c) In the merged image, arrowheads demonstrate that
PLCε1
GLEPP1
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PLCe1 is predominantly in podocyte cell bodies. (d,e) Absorption of
PLCe1-RA1 antibody with GST-RA1 blocks fluorescence. Podocyte
marker GLEPP1 outlining glomerular structure is shown (green) together
with PLCe1 (red) and DAPI nuclear staining (blue). (d) Preabsorption of
anti-PLCe1-RA1 (ref. 20) (red) with GST alone did not prevent binding to
podocytes (arrowheads). (e) Preabsorption of the antibody with the cognate
PLCε1
GLEPP1
PLCε1 GLEPP1 preabsorbed
WT1
RA1-GST fusion protein eliminated binding to podocytes, demonstrating
antibody specificity. Tubular staining was not blocked by preincubation
with the antigen, indicating nonspecific staining of tubules. (f) Podocyte
cell nuclei are identified by antibody to WT1 (green). Arrowheads point to
podocytes with WT1-positive nuclei whose cell bodies contain PLCe1 (red).
(g–i) Higher-power views of a portion of a glomerulus. (g) PLCe1 is localized
PLCε1
GLEPP1
Merge
using anti-PLCe1-RA1 (red). (h) GLEPP1 identifies podocyte foot processes
along the outer aspect of the glomerular capillary wall. (i) Merged image
demonstrates that PLCe1 localizes primarily to the podocyte cell bodies (arrowheads) and the neurite-like major processes projecting from the cell
body towards the foot processes abutting the GBM. Cell nuclei are stained with DAPI (blue). The white bar represents 10 mm.
sections. By immunolocalization using anti-PLCe1-RA1 (ref. 20), we
detected PLCe1 localization to podocytes as demonstrated by colocalization with the podocyte apical marker GLEPP1 (protein tyrosine
phosphatase receptor type O) (Fig. 3a–c). This labeling was specific, as
established by preabsorption of anti-PLCe1-RA1 with the antigen
(Fig. 3d,e). Counterstaining with an antibody to WT1, which marks
podocyte nuclei, demonstrated that PLCe1 localization was cytoplasmic (Fig. 3f). PLCe1 was present predominantly in podocyte cell
bodies and major and intermediate processes (Fig. 3g–i).
PLCE1 mutations halt glomerular development
Mutations in two other genes, WT1 and LAMB2, have been described
as causing DMS4,23. Dominant mutations in the WT1 gene are
associated with Wilms tumor, Denys-Drash syndrome (male pseudohermaphroditism and/or Wilms tumor)17 and Frasier syndrome
(female gonadal dysgenesis with nephrotic syndrome)24 but also
with isolated nephrotic syndrome4. Truncating mutations in the
LAMB2 (laminin-beta 2) gene cause Pierson syndrome (microcoria
and congenital nephrotic syndrome)23, whereas missense mutations
may cause isolated early-onset nephrotic syndrome3. The histopathologic changes of DMS on the basis of WT1 (ref. 25) mutations are
known to follow a corticomedullary gradient, with most severe
involvement in the subcapsular zone19, which shows small, simplified,
immature glomeruli with no more than four capillary loops18. We
observed similar features in individuals with PLCE1 mutations (Supplementary Fig. 3 online).
As coronal kidney sections allow evaluation of glomerular development along a corticomedullary gradient, we examined neonatal
(2-day-old) rat kidney for developmental expression of PLCe1 using
podocalyxin as a marker of glomerular development26 (Fig. 4a–i).
Normal nephron development progresses from comma-shaped bodies
through S-shaped bodies to the capillary loop stage and then to mature
glomeruli. In the capillary loop stage, major, intermediate and minor
(‘foot’) processes of podocytes develop in association with a massive
increase in the surface area for filtration due to increased glomerular
basement membrane synthesis. PLCe1 appeared in the developing glomerulus at the S-shaped stage of glomerular development
(Fig. 4a–f) and was highly expressed during the early capillary loop
stage (Fig. 4g–i). In relation to podocalyxin staining, which marks the
apical podocyte domain as it migrates down toward the glomerular
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basement membrane (GBM), PLCe1 was particularly prominent on
the basal aspect of the cell between the cell nucleus and the expanding
GBM (Fig. 4g–i). This finding would be compatible with the notion
that PLCe1 is required for the normal capillary loop stage of glomerular development and that its malfunction leads to an arrest at this
stage and thereby to the morphologic phenotype of DMS25.
Renal glomeruli from individuals with DMS morphologically
resemble the capillary loop stage of developing glomeruli by light
microscopy and are quite different from normal mature glomeruli that
contain multiple capillary loops (Supplementary Fig. 3). To assess this
concept further, we performed immunofluorescence using podocalyxin as a marker of podocyte development (Supplementary Fig. 3).
The pattern of podocalyxin staining showed that podocyte development in the kidney of an 11-month-old with DMS was similar to that
in human kidney in the early capillary loop stage of glomerular
development at week 28 of gestation (Supplementary Fig. 3). Nephrin
is the product of the NPHS1 gene, mutations of which cause
congenital nephrotic syndrome. It is an essential component of the
slit diaphragm, the modified intercellular junction of mature podocytes. The expression of nephrin was extremely reduced in the DMS
glomeruli at a time point at which it was present in normally
developing glomeruli at the early capillary loop stage (Supplementary
Fig. 3). A similarly strong reduction was seen for the expression of
podocin (Supplementary Fig. 3). These results are compatible with
the concept that the absence of PLCe1 owing to truncating mutations
results in failure of the developing glomerulus to progress past the
capillary loop stage.
PLCe1 interacts with IQGAP1
The observation that nephrin expression was very low in glomeruli of
individuals with DMS owing to PLCe1 mutations led us to perform
coimmunoprecipitation (coIP) studies to determine whether PLCe1
could be shown to interact directly or indirectly with nephrin
(Fig. 2b). We were not able to demonstrate a direct interaction between PLCe1 and nephrin by coIP (Fig. 2b). However, IQ
motif–containing GTPase-activating protein 1 (IQGAP1) is known to
directly interact and colocalize with nephrin27,28. It is expressed in the
S-shaped and capillary loop stages of glomerular development27,28.
IQGAP1 is a regulator of cell morphology and adhesion29.
To determine whether PLCe1 might interact with IQGAP1, we
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performed coIP of PLCe1 with green fluorescent protein (GFP)-tagged
IQGAP1 or nephrin after cotransfection into HEK293T cells. IQGAP1
coimmunoprecipitated with PLCe1 and vice versa, showing that
IQGAP1 can form a protein complex with PLCe1. We mapped
this interaction to the C-terminal half of IQGAP1 (amino acid
residues 523–943) (Fig. 2b). We confirmed the interaction of
PLCe1 with IQGAP1 endogenously by coimmunoprecipitation in
cultured podocytes, the most relevant in vivo system (Fig. 2c).
IQGAP1 and podocalyxin colocalized in developing glomeruli at
the capillary loop stage of development along the basal aspect of
developing podocytes, where process formation is known to
occur27 (Fig. 4j–l). As mutations in WT1 also give rise to DMS,
we examined the subcellular localization of WT1 in relation to
PLCe1 in rat glomerular development. During the S-shaped stage
of nephron development, WT1 appeared in the nuclei of developing podocytes (Fig. 4m), by which time PLCe1 was already
expressed (Fig. 4n–o).
Zebrafish plce1 knockdown causes to nephrotic syndrome
To confirm functional conservation of plce1 in the maintenance of the
podocyte filtration barrier, we targeted the zebrafish plce1 ortholog
using antisense morpholino oligonucleotides and assayed barrier
function by vascular retention of a large tracer molecule injected in
the blood. We designed an oligonucleotide targeting the plce1 exon 14
donor sequence to disrupt expression of the highly conserved PLC-X
domain of plce1. We injected embryos with 4 ng morpholino, and then
at day 4 of development, we perfused the vasculature with 500-kDa
fluorescein isothiocyanate–conjugated dextran (FITC-dextran). At day
4, we fixed and stained embryos to assess glomerular passage of tracer
by uptake of FITC-dextran in endocytic vesicles of pronephric tubule
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Figure 4 Colocalization studies of PLCe1 with podocalyxin, IQGAP1 and
WT1 in the developing glomerulus of a 2-d-old rat kidney. (a–i) Expression
of PLCe1 (red) in relation to podocalyxin (green) in glomerular development.
High-power views are shown for S-shaped body (d–f) and capillary loop stage
(g–i). Nuclei appear blue where stained with DAPI. (a–c) Glomerular
development is known to advance from the cortical surface of the kidney
(asterisk, bottom) toward the medulla (top) through the stages of commashaped body, S-shaped body (arrowhead) and capillary loop stage (arrow).
Podocalyxin (green), a marker of podocyte developmental stage39, is initially
expressed on the apical surface of developing podocytes of late S-shaped
bodies (arrowheads in b,c). (d–f) Podocalyxin migrates down the lateral
surface of the developing podocyte as the intercellular junction migrates
toward the basal aspect (arrowhead in e). (g–i) As podocyte foot processes
and slit diaphragms form between the podocyte cell body and the GBM,
podocalyxin extends down to this site (arrowhead and deeper inverted
U-shaped signal in h). PLCe1 (red) appears in the developing glomerulus at
the S-shaped stage (arrowheads in a,c,d,f) and is highly expressed during
the capillary loop stage (g,i), particularly on the basal aspect between the
cell nucleus and the GBM at the site of developing foot processes. Arrow
denotes podocalyxin (green) in endothelial cells invading the glomerular cleft
(e,f) and into the developing capillary loops (h,i). (j–l) Expression of IQGAP1
(red) in relation to podocalyxin (green) at the late capillary loop stage of
glomerular development. (j) IQGAP1 is expressed at the basal aspect of
podocytes (arrow), where it partially colocalizes with podocalyxin (green)
(k,l). (m–o) Expression of PLCe1 in relation to WT1. (m) WT1 (green) is
present in podocyte nuclei at the capillary loop stage (arrow). WT1 is not
detectable in podocyte nuclei at the S-shaped stage (arrowhead). The
merged image (n) of WT1 (m) and PLCe1 (o) confirms that PLCe1 is present
as WT1 appears in developing podocyte nuclei. (o) Cytoplasmic staining by
PLCe1 and nuclear staining by DAPI for comparison to n.
cells, distal to the glomerulus. Embryos injected with 4 ng control
antisense morpholino showed a normal morphology (Fig. 5) and an
absence of tracer in pronephric tubule cells (Fig. 5c), indicating
selective retention of the large dextran in the vasculature. In contrast,
embryos injected with plce1 exon 14 donor morpholino showed
edema at day 4 of development (Fig. 5b), similar to zebrafish nephrin
and podocin loss-of-function morphants30. Sections of the pronephric
kidney in plce1 morphants invariantly showed abundant FITC-positive
vesicles in the pronephric tubule (Fig. 5d), indicating a breakdown of
barrier function in the pronephric glomerulus owing to plce1 loss of
function. Overall, 100% (7/7) of plce1 morphants demonstrated a
failure in barrier function, whereas 0% (0/3) of control-injected
embryos showed glomerular passage of tracer. RT-PCR on mRNA
from embryos injected with plce1 exon 14 donor morpholino showed
a failure to splice intron 14, resulting in a predicted protein truncated
in the PLC-X domain of plce1 but increased in size by 59 nonsense
amino acids at its C terminus (Fig. 5e). Electron microscopy of wildtype samples (Fig. 5f) compared with samples from morphant
zebrafish (Fig. 5g) showed characteristic pathological features of
nephrotic syndrome in the morphants, with foot process effacement
and severe disorganization of slit diaphragms.
We also examined the recently published mouse model of Plce1
targeted deletion31 but did not detect any nephrosis-like phenotype.
Specifically, at 3 months of age, Plce1–/– mice showed a urine protein/
creatinine ratio (30.7 ± 3 mg/g) that was not different from that of
wild-type mice (25.3 ± 3.1 mg/g; P ¼ 0.25; n ¼ 5 per group). The
histological appearance of the Plce1–/– mouse glomeruli, the immunologic distribution of nephrin and glomerular epithelial protein 1
(GLEPP1) and the appearance of podocyte foot processes upon
transmission electron microscopy did not differ between Plce1–/–
mice and wild-type mice. Evaluation of this mouse model under
nephrosis-promoting conditions will be an important next step to
evaluate this model for a renal phenotype.
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a
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plce ex14MO
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DISCUSSION
Here, we have used positional cloning to identify mutations in PLCE1
as causing early-onset nephrotic syndrome. In all individuals with
homozygous truncating mutations, their kidney histology showed
DMS. As two siblings with a missense mutation in the gene encoding
the PLCe1 catalytic domain showed histology of FSGS, PLCE1 mutations may comprise a spectrum of histologic phenotypes ranging from
severe, early-onset DMS to FSGS. The occurrence of DMS in sibling
pairs with healthy consanguineous parents has prompted others to
postulate the existence of an autosomal recessive variant of DMS
without extrarenal involvement18, which we may have identified here.
In individuals with PLCE1 mutations, the presence of a DMS
phenotype with the appearance of immature glomeruli, together
with our finding of reduced nephrin and podocin expression,
indicates that PLCe1 is necessary for proper progression of glomerular
development at the capillary loop stage. This is consistent with the
fact that DMS is also seen in mutations of WT1, which may have a role
in glomerular development. We identified IQGAP1 as an interaction
partner of PLCe1. The distribution of IQGAP1 in the vicinity of
foot processes indicates that PLCe1 might serve as an assembly
scaffold for the organization of a multimolecular complex involved
in morphogenetic processes of glomerular development at the
capillary loop stage.
The full and sustained treatment responses in two individuals with
PLCE1 truncating mutations is notable. We speculate that there may
be a critical time window in glomerular development during which
treatment with glucocorticoids or cyclosporin A may overcome a
putative developmental defect imposed by PLCE1 loss of function.
This may occur, for instance, through induction of a redundant
mechanism such as the activity of another phospholipase C. In
this context, it is of interest that the only two individuals (from
family F601) who had homozygous missense mutations rather than
truncating mutations in PLCE1 showed late-onset nephrotic syndrome and histology of FSGS rather than DMS. This may signify
that a hypomorphic allele allows for proper glomerular development
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Figure 5 Functional analysis of plce1 in the zebrafish pronephros. (a) Fourday-old larva injected at the one-cell stage with control, antisense plce1
morpholino. (b) Four-day-old larva injected with plce1 exon 14 donor
antisense morpholino (ex14MO) showing edema of the pericardium and
yolk sac. (c) Confocal/differential interference contrast (DIC) section
through the pronephric kidney of a control morpholino–injected embryo
at 4 d perfused with 500-kDa FITC-dextran shows fluorescence in the
vasculature but exclusion from pronephric tubule epithelial cells (white
dotted circumference) distal to the glomerulus (gl). Intestine is marked
(‘gut’). Asterisk (*) highlights the lumen of a pronephric tubule.
(d) Confocal/DIC section through a similar region of a plce1 exon 14 donor
morpholino–injected embryo shows abundant FITC fluorescence in endocytic
vesicles of pronephric tubule cells (white dotted circumference) indicating
passage of 500-kDa FITC dextran past the morphant glomerulus (gl).
Asterisk (*) highlights the lumen of a morphant pronephric tubule.
(e) Altered splicing of plce1 mRNA caused by plce1 exon 14 donor
morpholino results in failure to remove intron 14, creating an mRNA
(MO) predicted to encode a PLCE1 protein truncated in the middle
of the PLC-X domain with 59 nonsense amino acids at its C terminus,
thereby yielding a longer band upon RT-PCR and agarose gel electrophoresis
compared to wild-type (WT), as confirmed by sequencing. (f,g) Electron
microscopic ultrastructure of GBM and podocyte foot processes in
wild-type and 4-d-old morphant zebrafish. (f) In the wild-type, the
foot processes are regularly arranged along the GBM with consistent
spacing between foot processes spanned by slit diaphragms (arrows).
(g) In contrast, the morphant foot processes are effaced and
disorganized, with only occasional intercellular junctions (arrows).
The GBM is disorganized.
but later in childhood may interfere with glomerular repair processes,
thereby leading to protracted scarring in the form of FSGS. This
prolonged course might exclude the window of opportunity for
treatment that a developmental defect might offer. Evaluation of
further individuals with PLCE1 mutations and their treatment
response will be required to test these hypotheses. The identification
of PLCE1 mutations represents the first molecular cause of a nephrotic
syndrome variant that resolved after therapy in some individuals. We
speculate that the arrest of glomerular development through PLCE1
mutations may be reversible by treatment with glucocorticoids or
cyclosporin A via an unknown mechanism. The zebrafish model of
human nephrotic syndrome that we generated by plce1 knockdown
will provide a useful tool for investing this hypothesis.
METHODS
Subjects. We obtained blood, tissue samples and pedigrees after obtaining
informed consent from individuals with nephrotic syndrome and/or their
parents. Human subject research was approved by the University of Michigan
Institutional Review Board. The diagnosis of nephrotic syndrome was made by
a pediatric nephrologist based on either chronic or recurrent high-grade
proteinuria (440 mg m–2 h–1) or persistent low-grade proteinuria (44 mg
m–2 h–1) (ref. 32). Steroid-sensitive nephrotic syndrome and SRNS were
defined according to standard criteria32,33. Renal biopsies were evaluated by a
renal pathologist (R.W.). Age of onset of ESKD was defined as age at first renal
replacement therapy; that is, dialysis or renal transplantation. Clinical data were
obtained using a standardized questionnaire (http://www.renalgenes.org).
Linkage analysis. We performed a whole-genome search for linkage in
consanguineous families with nephrotic syndrome using a 50K SNP array
(GeneChip Human Mapping 50K Hind Array from Affymetrix). Data were
evaluated by calculating nonparametric LOD scores and scoring for homozygosity (Zhom) across the whole genome in order to identify regions of
homozygosity. Areas of homozygosity were confirmed by high-resolution
haplotype analysis genotyping using published and newly designed
microsatellite markers within the NPHS3 locus. Additional SNPs were typed
by direct sequencing. The GENEHUNTER-MODSCORE program34 was used
to calculate multipoint LOD scores assuming recessive inheritance with
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complete penetrance, a disease allele frequency of 0.001 and the marker allele
frequencies for individuals of European ancestry specified by Affymetrix.
Parametric and nonparametric LOD scores were plotted over genetic distance
across the entire human genome using gnuplot software (http://www.gnuplot.
info/). For exon sequencing primers, see Supplementary Table 1 online.
Glomerular and podocyte gene expression data for candidate gene selection.
The strategy is based on the assumption that mutations that cause a congenital
nephrotic syndrome or FSGS phenotype will be found in genes coding for
proteins preferentially expressed in the glomerulus versus the renal cortex or
medulla and/or preferentially expressed in the podocyte versus the whole
glomerulus2–7,10,12,35. For the glomerular gene expression profile, RNA was
isolated from whole renal cortex, from medulla and from isolated glomeruli
purified 490% by sieving from 2-month-old Fischer 344 rat renal cortex
(n ¼ 4 per group). DNA microarrays were developed from these RNA
preparations using rat Affymetrix microarrays36. For each primer set, we
calculated the change in DNA microarray signal from the glomerulus as a
multiple of the signal from either whole renal cortex or whole renal medulla.
For the podocyte gene expression profile, we intraperitoneally injected
diphtheria toxin receptor–transgenic Fischer 344 rats, which specifically express
the human diphtheria toxin receptor on their podocytes, with diphtheria toxin
(50 mg/kg) in order to deplete podocytes; 3 and 6 d later, we harvested
glomeruli by sieving8. RNA prepared from these glomeruli and from noninjected transgenic rat glomeruli (n ¼ 4 per group) was used to develop an
Affymetrix DNA microarray database36. The change in signal for each primer
set as a multiple of control was calculated at 3 and 6 d after diphtheria toxin
injection to induce podocyte cell death and is expressed as a reciprocal to
describe the degree of preferential podocyte gene expression versus wholeglomerulus gene expression at the two time points (R.C.W. et al., unpublished
data). Of the 43 positional candidate genes in the critical genetic region,
only Plce1 was preferentially expressed (42 s.d. above range) for both
glomerulus versus cortex and medulla and glomerulus versus podocytedepleted glomerulus.
Production of bacterial recombinant glutathione S-transferase (GST) fusion
proteins, immunoprecipitation, immunoblotting and pull-down assay.
Immunoprecipitation, immunoblotting and pull-down experiments were performed using the procedures described previously37.
CoIP of PLCe1 with IQGAP1 and nephrin. HEK293T cells were transfected
with full-length human PLCe1 and nephrin and three different constructs of
GFP-tagged IQGAP1 (constructs comprising residues 2–210, residues 2–522
and the full-length protein). Cells were lysed after 48 h in RIPA buffer,
immunoprecipitation was performed with a monoclonal antibody to GFP
(Sigma) and lysate was resolved by SDS-PAGE. Immunoblotting was performed with affinity-purified polyclonal anti-PLCe1-RA1 (made against RA1GST fusion protein) and polyclonal anti-nephrin37.
Immunofluorescence. Rat kidneys were perfusion fixed with periodate-lysineparaformaldehyde (PLP) as previously described8. Cryostat-cut kidney sections
were treated with Retrieve-All target unmasking reagent (Signet Laboratories)
for 2 h at 90 1C. Two-day-old rat kidney sections were used for developmental
studies. For human studies, we used archived autopsy formalin-fixed paraffinembedded tissue that had been in paraffin for 10 years to ensure comparable
aging effects on antigen retrieval. Sections were blocked with 10% goat serum
in PBS. Double immunofluorescence staining was performed with anti-PLCe1RA1 (immunopurified polyclonal antibody to the RA1 domain of rabbit
PLCe1) and GLEPP1 mouse monoclonal 1B4 antibody38. Cy3-conjugated goat
anti-rabbit and FITC-conjugated goat anti-mouse were used as secondary
antibodies. For absorption experiments, the immunopurified anti-PLCe1RA1 was absorbed as described above. We used the following antibodies:
2A4 monoclonal anti-podocalyxin39,40; WT-1 mouse monoclonal antibody
(SC-7385) (Santa Cruz) and immunoaffinity-purified nephrin polyclonal
antibody (from ref. 37). Nephrin sections were costained with DAPI for
nuclear identification.
Zebrafish studies. Wild-type TL or TÜAB zebrafish were maintained and
raised as described previously41. Embryos were staged and kept as previously
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described30 and observed with a Nikon SMZ 645 or Leica MZ12 dissecting
stereomicroscope equipped with a Spot digital camera (Spot Insight QE). For
morpholino antisense oligonucleotides (GeneTools) targeting the plce1 exon
14 donor site, see Supplementary Table 1. We injected 4.6 nl of a 0.1-mM
morpholino stock solution as previously described30 into one-cell stage
embryos (approximately 4 ng/embryo) using a Nanoliter2000 injector (World
Precision Instruments). RT-PCR from single-embryo total RNA with nested
primers in flanking plce1 exons 12–17 yielded a final 630-bp amplicon from
wild-type and a 782-bp product from morphants (for primers, see Supplementary Table 1). The inside product was sequenced. Fluorescent dye injection
was performed using lysine-fixable FITC-dextran (500 kDa, Molecular Probes)
and injected as previously described30. Uptake of filtered fluorescent dextran by
pronephric tubule cells was evaluated in histological sections using a Zeiss
Pascal LSM5 confocal microscope.
URLs. C. elegans gene interaction predictor database: http://tenaya.caltech.
edu:8000/predict.
Accession numbers. Accession numbers of PLCE1 orthologs and detailed
sequence alignments are given in the legend to Supplementary Figure 4.
Note: Supplementary information is available on the Nature Genetics website.
ACKNOWLEDGMENTS
We thank the affected individuals and their families for participation. We
acknowledge R.H. Lyons for large-scale sequencing. We thank S.J. Allen and
M. Petry for technical assistance and M. McKee for electron microscopy in
zebrafish. GFP-tagged IQGAP1 constructs were provided by G. Bloom (University
of Virginia). This research was supported by grants from the US National
Institutes of Health to F.H., R.C.W. and L.B.H. (P50-DK039255), to R.C.W.
(DK46073), to A.V.S. (R01-GM053536) to I.D. (R01-DK53093) and to G.G.K.
(R01-DK56294) and by a grant from the KMD Foundation and the Thrasher
Research Fund to F.H.; F.H. is the Frederick G.L. Huetwell Professor and a Doris
Duke Distinguished Clinical Scientist. The work was further supported by the
German Federal Ministry of Science and Education through the National Genome
Research Network (C.B., H.C.H., G.N., P.N. and D.S.), by a EuReGene grant to
D.M. (E.U., FP6005085) and by grants from the German Research Foundation
(A.K., A.D. and T.G.).
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Published online at http://www.nature.com/naturegenetics
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ARTICLES
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1994).
1405
SUPPLEMENTARY FIGURES
1
Ex2
MTSEEMTASVLIPVTQRKVVSAQSAADESSEKVSDINISKAHTVRRSGET
50
51
SHTISQLNKLKEEPSGSNLPKILSIAREKIVSDENSNEKCWEKIMPDSAK
100
101
NLNINCNNILRNHQHGLPQRQFYEMYNSVAEEDLCLETGIPSPLERKVFP
150
151
GIQLELDRPSMGISPLGNQSVIIETGRAHPDSRRAVFHFHYEVDRRMSDT
200
201
FCTLSENLILDDCGNCVPLPGGEEKQKKNYVAYTCKLMELAKNCDNKNEQ
250
251
LQCDHCDTLNDKYFCFEGSCEKVDMVYSGDSFCRKDFTDSQAAKTFLSHF
300
301
EDFPDNCDDVEEDAFKSKKERSTLLVRRFCKNDREVKKSVYTGTRAIVRT
350
351
LPSGHIGLTAWSYIDQKRNGPLLPCGRVMEPPSTVEIRQDGSQRLSEAQW
Ex3
YPIYNAVRREETENTVGSLLHFLTKLPASETAHGRISVGPCLKQCVRDTV
Ex4
CEYRATLQRTSISQYITGSLLEATTSLGARSGLLSTFGGSTGRMMLKERQ
400
401
451
501
551
601
651
701
751
PGPSVANSNALPSSSAGISKELIDLQPLIQFPEEVASILMEQEQTIYRRV
RasGEF_CDC25
LPVDYLCFLTRDLGTPECQSSLPCLKASISASILTTQNGEHNALEDLVMR
Ex5
FNEVSSWVTWLILTAGSMEEKREVFSYLVHVAKCCWNMGNYNAVMEFLAG
Ex6
LRSRKVLKMWQFMDQSDIETMRSLKDAMAQHESSCEYRKVVTRALHIPGC
Ex7
KVVPFCGVFLKELCEVLDGASGLMKLCPRYNSQEETLEFVADYSGQDNFL
450
500
550
600
650
700
750
800
851
QRVGQNGLKNSEKESTVNSIFQVIRSCNRSLETDEEDSPSEGNSSRKSSL
Ex8
KDKSRWQFIIGDLLDSDNDIFEQSKEYDSHGSEDSQKAFDHGTELIPWYV
PH
LSIQADVHQFLLQGATVIHYDQDTHLSARCFLQLQPDNSTLTWVKPTTAS
901
PASSKAKLGVLNNTAEPGKFPLLGNAGLSSLTEGVLDLFAVKAVYMGHPG
950
951
IDIHTVCVQNKLGSMFLSETGVTLLYGLQTTDNRLLHFVAPKHTAKMLFS
1000
Ex9
GLLELTRAVRKMRKFPDQRQQWLRKQYVSLYQEDGRYEGPTLAHAVELFG
1050
Ex10
GRRWSARNPSPGTSAKNAEKPNMQRNNTLGISTTKKKKKILMRGESGEVT
1100
Ex11
DDEMATRKAKMHKECRSRSGSDPQDINEQEESEVNAIANPPNPLPSRRAH
1150
Ex12
1200
SLTTAGSPNLAAGTSSPIRPVSSPVLSSSNKSPSSAWSSSSWHGRIKGGM
Ex13
KGFQSFMVSDSNMSFVEFVELFKSFSVRSRKDLKDLFDVYAVPCNRSGSE
1250
Ex14
EF-hand
SAPLYTNLTIDENTSDLQPDLDLLTRNVSDLGLFIKSKQQLSDNQRQISD
1300
801
1001
1051
1101
1151
1201
1251
1301
1351
AIAAASIVTNGTGIESTSLGIFGVGILQLNDFLVNCQGEHCTYDEILSII
Ex15
Ex16
PLC_X
QKFEPSISMCHQGLMSFEGFARFLMDKENFASKNDESQENIKELQLPLSY
850
900
1350
1400
1401
1451
1501
1551
1601
1651
1701
1751
1801
1851
1901
1951
2001
2051
Ex17
YYIESSHNTYLTGHQLKGESSVELYSQVLLQGCRSVELDCWDGDDGMPII
Ex18
YHGHTLTTKIPFKEVVEAIDRSAFINSDLPIIISIENHCSLPQQRKMAEI
Ex19
FKTVFGEKLVTKFLFETDFSDDPMLPSPDQLRKKVLLKNKKLKAHQTPVD
Ex20
ILKQKAHQLASMQVQAYNGGNANPRPANNEEEEDEEDEYDYDYESLSDDN
Ex21
Ex22
ILEDRPENKSCNDKLQFEYNEEIPKRIKKADNSACNKGKVYDMELGEEFY
Ex23
LDQNKKESRQIAPELSDLVIYCQAVKFPGLSTLNASGSSRGKERKSRKSI
Ex24
PLC_Y
FGNNPGRMSPGETASFNKTSGKSSCEGIRQTWEESSSPLNPTTSLSAIIR
TPKCYHISSLNENAAKRLCRRYSQKLTQHTACQLLRTYPAATRIDSSNPN
Ex25
PLMFWLHGIQLVALNYQTDDLPLHLNAAMFEANGGCGYVLKPPVLWDKNC
C2 Ex26
PMYQKFSPLERDLDSMDPAVYSLTIVSGQNVCPSNSMGSPCIEVDVLGMP
LDSCHFRTKPIHRNTLNPMWNEQFLFHVHFEDLVFLRFAVVENNSSAVTA
Ex27
QRIIPLKALKRGYRHLQLRNLHNEVLEISSLFINSRRMEENSSGNTMSAS
Ex28
RA1
Ex29
SMFNTEERKCLQTHRVTVHGVPGPEPFTVFTINGGTKAKQLLQQILTNEQ
1450
1500
1550
1600
1650
1700
1750
1800
1850
1900
1950
2000
2050
2100
2201
DIKPVTTDYFLMEEKYFISKEKNECRKQPFQRAIGPEEEIMQILSSWFPE
Ex30
RA2
EGYMGRIVLKTQQENLEEKNIVQDDKEVILSSEEESFFVQVHDVSPEQPR
Ex31
TVIKAPRVSTAQDVIQQTLCKAKYSYSILSNPNPSDYVLLEEVVKDTTNK
Ex32
KTTTPKSSQRVLLDQECVFQAQSKWKGAGKFILKLKEQVQASREDKKKGI
2251
SFASELKKLTKSTKQPRGLTSPSQLLTSESIQTKEEKPVGGLSSSDTMDY
2300
2301
RQ 2302
2101
2151
2150
2200
2250
Supplementary Figure 1. Protein domains of human SRN3 (AB040949) as predicted by
PFAM (http://www.ensembl.org/Homo_sapiens/protview?db=core;peptide=ENSP00000260766).
Extent of putative domains is highlighted as follows: white on red, RasGEF_CDC25 domain
(guanine nucleotide exchange factor for Ras-like small GTPases) (predicted by PFAM PF00617,
aa 551-725); yellow, PH domain (pleckstrin homology) (aa 852-933; Winn et al. J Biol Chem
276:48257, 2001); white on dark blue, EF-hand (aa 1299-1375; Winn et al. J Biol Chem
276:48257, 2001); light blue, PLC_X domain (Phospholipase C, catalytic domain, part X)
(predicted by PFAM PF00388, aa 1393-1541); white on dark blue, PLC_Y domain (phospholipase
C, catalytic domain, part Y) (predicted by PFAM PF00387, aa 1744-1846); green, C2 motif
(protein kinase C conserved region 2, subgroup 2) (predicted by PFAM PF00168, aa 1871-1953);
white on dark green, RA1 domain (RasGTP binding domain from guanine nucleotide exchange
factors) (predicted by Kelley et al. EMBO J 20:743, 2001, aa 2011-2015); black on red, RA2
domain (RasGTP binding domain from guanine nucleotide exchange factors) (predicted by PFAM
PF00788, aa 2135-2238). Encoding exons are shown on grey background at their start positions.
Amino acids encoded by even numbered exons are underlined.
2
GFP-PLCε1
A
1
2
3
4
5
RA1
B
1
2
3
4
250 150 100 75 -
50 WB:
GFP
WB:
CS117
Supplementary Figure 2. Characterization of two different anti-PLCε1 polyclonal
antibodies. (A) The anti-PLCε1-RA1 domain antibody detects PLCε1 in cultured
podocytes and isolated glomeruli from mouse and rat. Western blot shows relative
abundance of PLCε1 in differentiated (lane 1) and undifferentiated (lane 2) cultured
mouse podocytes, mouse and rat glomerular lysates (lanes 3 and 4, respectively).
Expression of GFP-PLCε1 in HEK293T cells is used as positive control for PLCε1
antibody (lane 5). (B) Following transfection of HEK293T cells with human full length
fusion protein GFP-PLCε1 (lanes 1 and 3) or untransfected control (lanes 2 and 4) SDSPAGE was performed with equal amounts of cell lysates. An anti-GFP antibody as
positive control another anti-PLCε1 antibody (CS117) was tested for specificity by
western blotting (WB) as indicated below lanes. The full length human GFP-PLCε1
fusion protein was detected at 287 kDa (arrow head).
1
Supplementary Figure 3. PLCE1 mutation leads to renal histology of diffuse mesangial
sclerosis (DMS) and is associated with interrupted glomerular development.
(A-C) PLCE1 mutation leads to DMS. (A) For comparison histology of a human normal mature
glomerulus is shown. (B) Renal histopathology from NPHS3 patient A38 II-1 (7 months) with a
truncating PLCE1 mutation (PLCE1-/-) reveals the characteristic pattern of DMS of a “primitive”appearing glomerulus with a reduced number of capillary loops. The hypertrophied and vacuolized
podocytes surround the glomerular tuft like a crown (arrowhead). (C) Lower magnification shows the
presence of typical tubular changes of DMS with interstitial infiltrations with fibrosis, tubular atrophy,
and dilated tubules that contain hyaline casts. A glomerulus is indicted by a frame.
(D-O) “Immature” glomeruli of DMS at 7-11 months are reminiscent of fetal developing kidney. The
first column depicts images from renal histology of an 11-months old patient (F1063 I-3) (A38 II-1 in
D) with homozygous truncating mutations (PLCE1-/-), the second column depicts early capillary loop
stage from a normal fetal human kidney (28 weeks gestation), and the third column normal fetal
human kidney at late capillary loop stage.
(D) Trichrome-Masson staining in patient A38 II-1 reveals typical features of DMS with “immature”
glomerulus, circumferential capillary loop, and absence of normal glomerular capillaries in the center
of the glomerulus, which is filled with blue matrix material. This renders the glomerular structure
similar to normal developing glomeruli at the early (E) and late (F) capillary loop stage of normal
glomerular development as shown in a normal fetal human kidney of 28 weeks gestation showing a
similar “crown” of podocytes surrounding the center of the glomerulus. The center of the patient’s
glomerulus is rarefied from capillary loops (D) in comparison to a normal mature glomerulus (A).
(G-I) Podocalyxin is present on the apical surface of developing podocytes and is used to mark
podocyte development. Podocalyxin distribution on the PLC1-/- glomerulus (G) is scant on the basal
aspect (arrowhead) and is thereby similar to that of an early capillary loop stage glomerulus (H)
(arrowhead). It is clearly less developed than a late capillary loop stage glomerulus (I) where
podocalyxin is accumulating along the basal aspect of the podocyte (arrow).
(J-L) (J) Nephrin is barely detectable in PLCE1-/- (F1063 I-3) glomeruli. (K) Nephrin is present
during thse early capillary loop stage of glomerular development prior to foot process appearance
and mostly basal, (L) but easily detectable along the developing infolding GBM at which time foot
processes and slit diaphragms are formed.
(M-O) Merged images including Dapi to mark nuclei show the relationship between distribution of
podocalyxin as a podocyte developmental marker and the appearance of nephrin. (M) The low level
expression of nephrin in PLCe1-/- glomerulus is similar to or less than the early capillary loop stage of
glomerular development (N), and much less than that seen in late capillary loop stage of glomerular
development (O). All non-scarred glomeruli in PLCE1-/- kidney showed the same level of glomerular
development (n=13). Therefore at 11 months of age PLCE1-/- glomeruli appear to be at a level of
glomerular development equivalent to the early capillary loop stage from a 28 weeks of gestation
fetus as assessed by criteria of histologic appearance, podocalyxin distribution, and nephrin
expression.
(P-X) Colocalization study of podocalyxin and podocin using the same specimens and procedures
described in (G-O) for podocalyxin and nephrin. Note the markedly reduced podocin staining in the
specimen of individual A38 II-1, who bears a homozygous PLCE1 truncating mutation.
2
SUPPLEMENTARY FIGURE 4
CLUSTAL W (1.83) multiple sequence alignment
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
MTSEEMTASVLIPVTQRKVVSAQSAADESSEKVSDINISKAHTVRRSGETSHTISQLNKL
MTSEEITASVLIPVTQRKVVSAQSAADESSEKVSDINISKAHTVRRSGETSHTISRLNKL
MTSEGMAASVLTPVTQRKVTFAPSAVDESSEKVSDISVPKAHSVKQS-EQTSTIPWMNKL
MTSEEMAASVLIPVTQRKVASAQSVAEERSVKVSDAGIPRARAGRQGALIPPTISQWNKH
MTSEEMAASFLIPVPQRKVASAQSVAEERGEKVSEAGIPKTRAGRQGGLTPRTISQRNEP
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
60
60
59
60
60
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
KEEPSGSNLPKILSIAREKIVSDENSNEKCWEKIMPDSAKNLNINCNNILRNHQHGLPQR
KEEPSGSNLPKILSIAREKIVSDENSNEKCWEKIMPDSAKNLNINCNNILRNHQHGLPQR
KEESSGSNLPKILSIAREKIASDENSNEECWTESTPVSVKNLNINHNNILTNRQCVLPQS
KEESSRSDLSKVFSIARGELVCDENSNEEGWEENAPDSPENHAMNGNSLVQSHQHQFPRS
EEESPRTDFSQVFSIARGELDSDENHNERCWEENVPGSTKNHAVNCNSLLQSHQHALPPS
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
120
120
119
120
120
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
QFYEMYNSVAEEDLCLETGIPSPLERKVFPGIQLELDRPSMGISP--------LGNQSVI
QFYEMYNSVAEEDLCLETGIPSPLERKVFPGIQLELDRPSMGISP--------LGNQSAI
QSYDTCNSVMEEDPCLETGISSSLERKVFPGIQLEVNRPPMDFRPPGLMDFSTLGSQSAI
QLCEARDSVTEDP-CLQPGIPSPLERKVLPGIQLEMEDSPMDVSP--------AGSQPRI
QLCEVCDSVTEEHLCLQPGIPSPLERKVFPGIELEMEDSPMDVSP--------LGNQPGI
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
172
172
179
171
172
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
IETGRAHPDS-RRAVFHFHYEVDRRMSDTFCTLSENLILDDCGNCVPLPGGEE-KQKKNY
IETGRAHPDS-RRAVFHFHYEVDRRMSDTFCTLSENLILDDCGNCVPLPGGEE-KQKKNY
VDTGQAHPDSNKAAFQIFNYKVDRRMSDTFCTLSGDLILDDCGNCVPLSSGFGGEQKKNY
MESSGPHSDR-NTAVFHFHYEADRTMSDAFHTLSENLILDDCANCVTLPGGQQ---NKNC
MESSGPHSDR-NMAVFHFHYAGDRTMPGAFHTLSEKFILDDCANCVTLPGGQQ---NKNY
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
230
230
239
227
228
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
VAYTCKLMELAKNCDNKNEQLQCDHCDTLNDKYFCFEGSCEKVDMVYSGDSFCRKDFTDS
VAYTCKLMELAKNCDNKNEQLQCDHCDTLNDKYFCFEGSCEKVDMVYSGDSFCRKDFTDS
VAYTCKLMELAENCDNENGQLQCDDYDALDDKYLCFEDSCQRDSVVCSSDSFRREDLTNS
MAYACKLVELTRTCGSKNGQVQCEHCTSLRDEYLCFESSCSKADEVCSGGGFCEDGFAHG
MAYTCKLVELTRTCGSKNGQLKCDHCTSLRDEYLCFESSCRKAEALSSGGGFCEDGFTHG
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
290
290
299
287
288
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
QAAKTFLSHFEDFPDNCDDVEEDAFKSKKERSTLLVRRFCKNDREVKKSVYTGTRAIVRT
QAAKTFLSHFEDFPDNCDDVEEDAFKNKKERSTLLVRRFCKNDREVKKSVYTGTRAIVRT
PPAKTFLSHFEDFPDNGEDVEDFLK-NKKERSTLLVRRFCKNDREVKKSVYTGTRAIMRT
PAAKTFLSPLEDFSDNCEDVDDFFK-SKKERSTLLVRRFCKNDREVKKSVYTGTRAIMRT
PSAKTFLNPLEEFSDNCEDVDDIFK-GKKERSTLLVRRFCKNDREVKKSVYTGTRAIVRT
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
350
350
358
346
347
1
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
LPSGHIGLTAWSYIDQKRNGPLLPCGRVMEPPSTVEIRQDGSQRLSEAQWYPIYNAVRRE
LPSGHIGLTAWSYIDQKRNGALLPCGRVMEPPSTVEIRQDGSQHLSEAQWYPIYNAVRRE
LPSGHIGLEAYSYIDQKRSGPLLPRGRVLEQLPVVAIRQDGSQCLSEAQWYRIYNAVRRE
LPSGCIGPAAWNYVDQKKAGLLWPCGNVMGTLSAMDIRQSGSQRLSEAQWCLIYSAVRRG
LPSGHIGLAAWSYVDQKKAGLMWPCGNGMRPLSTVDVRQSGRQRLSEAQWCLIYSAVRR---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
410
410
418
406
406
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
E-TENTVGSLLHFLTKLPASETAHGRISVGPCLKQCVRDTVCEYRATLQRTSISQYITGS
E-TENTVGSLLHFLTKLPASETAHGRISVGPCLKQCVRDTVCEYRATLQRTSISQYITGS
EEIENTIGSLLHYFTKLPASKTAHERISVGPCLKQCVRDTICEYRATLQRTSISQYITGS
EEIEDTVGSLLHCSTQLPNSETAHGRIEDGPCLKQCVRDTECEFRATLQRTSIAQYITGS
EETEDTVGSLLHCSTQLPTPDTAHGRIGDGPCLKQCVRDSECEYRATLQRTSIAQYITGS
------------------------------------------------------------------------------------------------------YRACLQRTSLFSLLTGA
-----------------------------------------------------------------------------------------------------MNWDTLKGVLKTRRLTKR
469
469
478
466
466
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
LLEATTSLGARSGLLSTFG-GSTGRMMLKERQPGPSVANSNALPSSSAGISKELIDLQPL
LLEATTSLGARSGLLSTFG-GSTGRMMLKERQPGPSVANSNALPSSSAGISKELIDLQPL
LLEATTSLGARSGLLSTFG-GSTGRMMLKERQPGTSMANSSALPSSSAGISKELIDLQPL
LLEATTSLGARSGLLSSFG-GSTGRIMLKERQLGTSMANSNPVPSSSAGISKELIDLQPL
LLEATTSLGARSSLLSSFG-GSTGRIMLKERQPGTSMANSSPVPSSSAGISKELIDLQPL
------------------------MYLFLERHPCSTMATSSSSPASAAGLSKELVDLQHL
LLEAIAALGARSALPYSFPQGSNSHVVLKERQLANSMTSSSALSPAVSGISKELAEMRHL
------------------------------RQLCSSMANSSILPSAVTGISKELADLRHL
TIPAYIHPTSRS---------------DSTSSTQSATAGFILNEEPITLFRLELERLQYI
: :
. : : ** :: :
528
528
537
525
525
36
77
30
63
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
RasGEF-CDC25_(aa551-725)_______
____
IQFPEEVASILMEQEQTIYRRVLPVDYLCFLTRDLGTP---ECQ-SSLPCLKASISASIL
IQFPEEVASILMEQEQTIYRRVLPVDYLCFLTRDLGTP---ECQ-SSLPCLKASISASIL
IQFPEEVASILMEQEQNIYRRVLPVDYLYFLTRDLGTP---ECQ-TPLPCLKASISASIL
IQFPEEVASILTEQEQNIYRRVLPMDYLCFLTRDLSSP---ECQ-RSLPRLKASISESIL
IQFPEEVASILTEQEQNIYRRVLPMDYLCFLTRDLSSP---ECQ-RSLPRLKACISESIL
IQFPEEIASILTEQEQEIYRKVLPVDYLYFLTKDLSNG---ECD-TNLSDIKTSLSASLR
VQFPEEIACILTEQEQQLYQRVFPLDYLCFLTRDLGSP---ECQSKHHPSLKASLSVPAM
IQFPEEIATILTEQEQQLYRRVFPLDYLSFLTRDLGSP---ECH-KRHPHLKASLSAPIM
LHFPEEVAFQLSSTEYQLFYSIQPMDYVRYVSCDLTSVPVS------------------::****:* * . * :: : *:**: ::: ** .
584
584
593
581
581
92
134
86
104
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
____________________________________________________________
TTQNGEHNALEDLVMRFNEVSSWVTWLILTAGSMEEKREVFSYLVHVAKCCWNMGNYNAV
TTQNGEHNALEDLVMRFNEVSSWVTWLILTAGSMEEKREVFSYLVHVAKCCWNMGNYNAV
SSQNGEHNALEDLVMRFNEVSSWVTWLILTAGSMEEKREVFSYLVHVAKCCWNMGNYNAV
TSQSGEHNALEDLVMRFNEVSSWVTWLILTAGSMEEKREVFSYLVHVAKCCWNMGNYNAV
MSQSGEHNALEDLVMRFNEVSSWVTWLILTAGSMEEKREVFSYLVHVAKCCWNMGNYNAV
KLKNGEHDAVEGLVARFNEVSSWVTWLILTAGSMEEKREVFSHVVHIAKCCWNMGNYNAV
STQSSRHNAVEDLVARFNEVSSWVTWLILTAGSMEEKREVFSYLVNVAKCCWNMGNYNGV
PTQNDNHNTVEDLVTRFNEVSSWVTWLILTAGSMEEKREFFSYLVHVAKCSWNMGNYNAV
----ENPSPVRNLVKRLSEVSSWITHVIVSQPTHDDRKVALTAILRIVETCWNIGNFNAA
. ..:..** *:.*****:* :*:: : :::: :: ::.:.: .**:**:*..
644
644
653
641
641
152
194
146
160
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
____________________________________________________________
MEFLAGLRSRKVLKMWQFMDQSDIETMRSLKDAMAQHESSCE---YRKVVTRALHIPGCK
MEFLAGLRSRKVLKMWQFMDQSDIETMRSLKDAMAQHESSCE---YRKVVTRALHIPGCK
MEFLAGLRSRKVLKMWQFMDQSDLETMRSLKDAMAQHESSCE---YRKVITRALHIPGCK
MEFLAGLRSRKVLKMWQFMDQSDIETMRSLKDAMAQHESSVE---YKKVVTRALHIPGCK
MEFLAGLRSRKVLKMWQFMDQSDIETMRSLKDAMAQHESSVE---YKKVVTRALHIPGCK
MEFLAGLRTRKVLKMWQFMDQSDIETMRSLKDAMAQHESSSE---YRKVVNRALNIPGCK
MEFLAGLRSRKVLKMWQFMDQTDIETMRSLKDAMAQHESSSE---YKKVVTRALNIPGCK
MEFLAGLRSRKVLKMWQFMDQADIETMRGLKDAMAQHESSSE---YKKVVSRALNIPGCK
VEVLMGLKSEKLRPFWLSLRQEEKSQFDSLCETLLPANQALPSQAYINAVQRALRMPQSR
:*.* **::.*: :* : * : . : .* :::
:.:
* :.: ***.:* .:
701
701
710
698
698
209
251
203
220
2
17
18
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
________________________
VVPFCGVFLKELCEVLDGASGLMKLCPRYNSQEETLEFVADYSGQDNFLQRVGQNGLKNS
VVPFCGVFLKELCEVLDGASSLMKLCPRYNSQEETLEFVADYSGQDNFLQRVGQNGLKNS
VVPFCGVFLKELCEVLDGASGLMKLCPRYNSQEETLEFVADYSGQDNFLQRVGHNGLKNS
VVPFCGVFLKELCEVLDGASGLLKLCPRYSSQEEALEFVADYSGQDNFLQRVGQNGLKNS
VVPFCGVFLKELCEVLDGASGLLKLCPRYSSQEEALEFVADYSGQDNFLQRVGQNGLKNP
VVPFCGVFLKELCEVLDGAASIISLCPQYDAQSETLEFVSDYNGQDNFLQRIGKDGLKNT
VVPFCGVFLKELSDALDGTASIISLKSPLENSEDSIEFVSDYSGQHNFLLRSGPDGLHIP
VVPFCGVFLKELSEALDGAASIIGLRPSFDSQEDPVEFVTDYNGQQHFLQRLGSDGLHSS
VIPFFGIFLRDLYAIVNDLPNIVVIGQ--EGETQKLEFMSDPNGEDHFSSRIGVGGLLNA
*:** *:**::*
::. ..:: :
. . : :**::* .*:.:* * * .** .
761
761
770
758
758
269
311
263
278
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
EKESTVNSIFQVIRSCNRSLETDE-EDSPSEGNSSRKSSLKDKSRWQFIIGDLLDSDNDI
EKESTVNSIFQVIRSCNRSLETDE-EDSPSEGNSSRKSSLKDKSRWQFIIGDLLDSDNDI
EKESTVNSIFQIIRSCSRSLEAEE-EDSPSEGNSSRKNSLRDKARWQFIIGDLLDSENDI
EKELTVNSIFQVIRSCSRSLEMEE-EDSASEGSGSRKNSLKDKARWQFIIGDLLDSENDI
EKELTVNSIFQIIRSCSRSLETED-EESASEGSGSRKNSLKDKTRWQFIIGDLLDSDNDI
EKESTVNSIMQTIRSCNRSLESEEGEDNLSDAGGIRKSTVMDRTRFQFIMGDLSDSESDI
EKEATVSNILQIIRSCNRSLEVEDTDDGSTSPSSSLSFLFHALLFRFMVGDLSDSDGDLP
DKEATVSNILQTIRSCNRSLEAEEPEERAREITVCPKNSFKDKSRNQFSIGDLSDSEGDP
DKINLVAIVLDNLELFHR-------------------HSRTMIKLLEEQAVPPIQIPQNE
:*
* ::: :.
*
.
820
820
829
817
817
329
371
323
319
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
PH domain (aa852-933)_
___
FEQSK-EYDSHGSEDSQKAFDHGTELIPWYVLSIQADVHQFLLQGATVIHYDQDTHLSAR
FEQSK-EYDSRGSEDSQKAFDHGTELIPWYVLSIQADVHQFLLQGATVIHYDQDTHLSAR
FEQSK-EWDSPSSEEPQKAFDHGTELIPWYVLSIRADVHQFLLQGATVLRYDQDTHLSAR
FEKSK-ECDPHGSEESQKAFDHGTELIPWYVLSIQADVHQFLLQGATVIHYDQDTHLSAR
FEKSK-ECDPHGSEESQKAFDHGTELIPWYVLSIQADVHQFLLQGATVIHYDQDTHLSAR
FEQSK-EWDLHRREEQQKAFSHGTELIPWYVLSMRADVYQFLQQGVTVIRYDQETHISVR
SEPAVKEGEFQGTEETHKAFNHGTELIPWYVLSLQPDVHQFLLQGATVIHYDQDSHLSAR
LETVKDVVDLQTTEDVRGPFSHGTELIPWYVLSLQPDIHQFLLQGATVIHYDPESHLTAR
REQKEKEAKTYEPVQVVRGSSHGVALIPLDTLTFDLDVIQRLQHGTTVIHYEPDSGRSNL
*
.
:
.**. *** .*:: *: * * :*.**::*: :: :
879
879
888
876
876
388
431
383
379
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
____________________________________________________________
CFLQLQPDNSTLTWVKPTTASPASSKAKLGVLNNTAEPGKFPLLGN-------------A
CFLQLQPDNSTLTWVKPTTASPASSKAKLGVLNNTAEPGKFPLLGN-------------A
CFLQLQPDNSTLTWIKPTAASPASARAKLGVLSNTSEPGKFPSPGS-------------A
CFLQLQPDNSTLTWMKPPTASPAGARPKLGVLSNMAEPGKFPSPGN-------------A
CFLQLQPDNSTLTWMKPPTASPAGARLKLGVLSNVAEPGKFPSLGN-------------A
CFLQLQPDNSTLTWTKPTTTCLANTKNKLGTVS-SSAEIKFQFLAN-------------A
CLLRLQPDNTTLTWGNPQK-------------GGASPTEPPLGLGQ-------------A
CLLRLQPDNCFLTWCKPHSSCSLYGRART--FMGHPASPDHLHIGQ-------------P
CLLRLDPSCGQINWHKISYSVNKDPKEKDVLAKVSVSNLQPLDSGRGAPSPMPSGRTPGT
*:*:*:*.
:.* :
.
.
926
926
935
923
923
434
465
428
439
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
_______
GLSSLTEGVLDLFAVKAVYMGHPG-IDIHTVCVQNKLGSMFLSETGVTLLYGLQTTDNRL
GLSSLTEGVLDLFAVKAVYMGHPG-IDIHTVCVQNKLGSMFLSETGVTLLYGLQTTDNRL
GLSGLAEGVLDLFSAKAVYMGHPS-IDIHTVCVQNKLGSMFLSETGVTLLYGLQTTDNRL
GVSGLAEGILDLFSVKAVYMGHPG-IDIHTVCVQNKLSSMLLSETGVTLLYGLQTTDNRL
GVSGLVEGILDLFSVKAVYMGHPG-IDIHTVCVQNKLSSMLLSETGVTLLYGLQTTDNRL
GMNGLAEGFLDLFSVKAVYMGHPG-IDMHTVCVQNKLCNMNLEENGVTLLYGLHTTDNKL
VVAGLAEGLLDLGVVKAVFLGHQG-VDVHAVCLQNKLSHMTVEENTLSLLYGVSTTDNRL
VHCGLSDGLLDLNVVKAVFMGHPG-VDVNYVCLQHKLCNMNPGENGVTLLYGLHTTDNRL
GGVGVEEGELKLSVVKGVELVDSYDIDIEAIYRRHSMEEMSVPVSCWKVSHGQLLSDNEF
.: :* *.* .*.* : .
:*:. : ::.: *
. .: :*
:**.:
985
985
994
982
982
493
524
487
499
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
LHFVAPKHTAKMLFSGLLELTRAVRKMRKFPDQRQQWLRKQYVSLYQEDGR-YEGPTLAH
LHFVAPKHTAKMLFSGLLELTRAVRKMRKFPDQRQQWLRKQYVSLYQEDGR-YEGPTLAH
LHFVAPKHTAKMLFSGLLELTRAVRKMRKFPDQRQQWLRKQYVSLYQEDGR-YEGPTLAH
LHFVAPKHTAEMLFSGLLELTTAVRKIRRFPDQRQQWLRKQYVSLYQEDGR-YEGPTLAH
LHFVAPKHTAKMLFSGLLELTTAVRKIRKFPDQRQQWLRKQYVSFYQEDGR-YEGPTLAH
LHFVAPKYTARTLYDGLLELTKAVRKIKRFPDQRLQWLRKQYVSLYQEENR-FEGPALAQ
LHFVAPNHTTQMLHKGLSELVTATRKLKKFPDQRLQWLRRQYVSLYQEDGR-YEGPTLAQ
LHFVAPKHTARMLHEGLQELLNSIRKIRKFPDQRLQWLRKQYVSLYQEDGR-FEGPTLAH
IYFLAPQQIAQFWTNGLQSVVKSLQGQQRYPDRRMLWIKNVYLSLYEITGESNCGPRPFE
::*:**: :.
.** .: : : :::**:* *::. *:*:*: ..
**
.
3
1044
1044
1053
1041
1041
552
583
546
559
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
AVELFGGRRWSARNPSPGTSAKNAEKPNMQRNNTLGIS-TTKKKKKILMRGESGEVTDDE
AVDSFGGRRWSARNPSPGTSAKNAEKPNMQRNNTLGIS-TTKKKKKILMRGESGEVTDDE
AVELFGGRRWSTRNPSPGTSAKSAEKPSVQRNNTLGIS-TTKKKKKILIRGESGEAADDE
AVELFGGRRWSTRNPSPGMSAKNAEKPNMQRNNTLGIS-TTKKKKKMLMRGESGEVTDDE
AVELFGGRRWSTRNPSPGMSAKNAEKPNMQRNNTLGIS-TTKKKKKMLMRGESGEVTDDE
AIELFGGRRWSTRNTSTGTLTKSTEKPNVQRNNTLGIN-TAKKKKKVLMRGESGDAADDE
AIELFGGRRWNMSTGG-------TEKSAHQKNSPLSINDKTKKKKKVLVRGDSGDATDDE
AIELFGGRRWNMGTSGPGSASRGAEKNSAQKNSPLGINSNVKKKKKALVRGDSGDGTDDE
ALQAFGLSQTNTNATRPNDSSLSSEPGGAKSRLKNLKNAMQKKLRGASREGSRSQSPQPH
*:: ** : .
:*
: .
.
** :
.*. .: .: .
1103
1103
1112
1100
1100
611
636
606
619
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
MATRKAKMHKECRSRSGSDPQDINEQEE---SEV-NAIA--NPPNPLPSRRAHSLTTAGS
MAARKAKMHKECRSRSGSDPQDINEQEE---SEV-NAIA--NPPNPLPSRRAHSLTTAGS
MATRKAKMHRECRSRSGSDPQDMNEQEE---SEA-NAIM--SPPNTLPSRRAHSLTTAGS
MATRKAKXXRECRSRSGSDPQDVNEQEE---SEA-NVIT--NPPNPLHSRRAYSLTTAGS
MATRKAKMYRECRSRSGSDPQEANEQED---SEA-NVIT--NPPNPLHSRRAYSLTTAGS
MATRKTKSCKESRSRSGSDPPEIDEQEE---QDL-NIIAGYSPSQMLPSRRAHSMSTSGS
MVARKTRSCKEGTYRNGPESDSIDHEDPGFMTGSN------------------------MTARKTRSCKETLGRRESD----------------------------------------SPLVRPPSIKSQISSQSGPPGPNSPGYLLKPRGE------PANSDAGDIDSIYTPRSRTP
:.
:.
1157
1157
1166
1154
1154
667
671
625
673
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
PNLAAGTSSPIRPVSSPVLSSSNKSPSSAWSSSSWHGRIKGGMKGFQSFMVSDSNMSFVE
PNLAAGTSSPIRPVSSPVLSSSNKSPSSAWSSSSWHGRIKGGMKGFQSFMVSDSNMSFVE
PNLSAGTSSPIRPVSSPVLSSSNKSPSSAWSSSSWHGRIKGGMKGFQSFMVSDSNMSFVE
PNLATG----------------MSSPISAWSSSSWHGRIRGGMQGFQSFMVSDSNMSFVE
PNLATG----------------MSS-PIAWSSSSWHGRIKGGMKGFQSFMVSDSNMSFIE
PNLTPGPSIPLRPASSPILSNSNKPQSNTWSSSSWHGRVKGGMKGFQSFMVSDSNMNFTE
---------QSRPQSSPTLSGTVKAQPGAWSSRSWHGRGKGCFRGFQNLMISDSTMSFVE
------------------ALENVEQEEAAWSSRSWHGRGKGCFRGFQDLMISDSIMSFVE
TSSSYGGRSVGGRSCKSWRSRGGETPNGSISSSGQMSIQVSGLSGPSGKEFQEKPLTLVE
.
: ** . .
. : * .. ..:. :.: *
1217
1217
1226
1198
1197
727
722
667
733
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
FVELFKSFSVRSRKDLKDLFDVYAVPCNR----SGSESAPLYTNLTIDENTSDLQPDLDL
FVELFKSFSVRSRKDLKDLFDVYAVPCNR----SGSESAPLYTNLTIDENTSDLQPDLDL
FVELFKSFSVRSRKDLKDLFDIYAVPCNR----AGSESAPLYTNLTIDENTSDLQPDLDL
FVELFKSFSIRSRKDLKDIFDIYSVPCNR----SASESAPLYTNLTIEENTSDLQPDLDL
FVELFKSFSIRSRKDLKDIFDIYSVPCNR----SASESAPLYTNLTIEENTNDLQPDLDL
FVELFKSFSVRSRKDLKDIFDVYAVACNR----SGAESVPLYTNLTIDENVVGVQPDLDL
FVELFKSFSIRSRKDLKELFDTFAVPCIR----SDPESVPLYTNLRIDDKDTGLQPDLDL
FVELFKSFSIRSRKDLKELFDTYAVPCSR----SGPESVPLYTTLRIDDKLTGLQPDLDL
FAELFRLFNTRMRKDLRDVFNDVLSTATTPQHCPKRERDRHSPRMQSRLASVSNSYNADF
*.***: *. * ****:::*:
..
. *
. :
. . : *:
1273
1273
1282
1254
1253
783
778
723
793
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
EF-hand (aa1299-1375)___________________
LTRNVSDLGLFIKSKQQLSDNQRQISDAIAAASIVTNGTGIESTSLGIFGVGILQLNDFL
LTRNVSDLGLFIKSKQQLSDNQRQISDAIAAASIVTNGTGIESTSLGIFGVGILQLNDFL
LTRNVSDLGLFIKSKQQLSDNQRQISDAIAAASIVTNGTGIESTSLGIFGVGILQLNDFL
LTRNVSDLGLFIKSKQQLSDNQRQISDAIAAASIVTNGTGIESTSLGIFGVGILQLNDFL
LTRNVSDLGLFMKSKQQLSDNQRQISDAIAAASIVTNGTGIESTSLGIFGVGILQLNDFL
LTRNVSDLGLFIRSRQQLSENQRQISDAIAAASIVTNGTGVESTSLGIFGMGILQFNDFL
LTRNGSDLGLFIRTRQQMSDNQKQISDAIAAASIVTNGTGVENASLGVLGLGIPQLNDFL
LTRNGSDLGLFIRTRQQMSENQKQISDAIAAASIVTNGTGVENSSLGVLGLAISQLNDFL
LSNDFLTRNTAVTS-HHISEKQNKIYNALALASVNSMGGLMDTSRSSMLTP--QMLRAFV
*:.:
. : : :::*::*.:* :*:* **: : * ::.: .::
:. *:
1333
1333
1342
1314
1313
843
838
783
850
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
__________________________________________
__
VNCQGEHCTYDEILSIIQKFEPSISMCHQGLMSFEGFARFLMDKENFASKNDESQENIKE
VNCQGEHCTYDEILSIIQ--EPSISMCHQGLMSFHGFARFLMDKENFASKNDESQENIKE
VNCQGEHCTYDEILSIIQKFEPSISMCHQGLLSFEGFARFLMDKDNFASKNDESQENIKD
VNCQGEHCTYDEILSIIQKFEPSVSMCHQGLLSFEGFARFLMDKDNFASKNDESRENKKE
VNCQGEHCTYDEILSIIQKFEPNISMCHQGLLSFEGFARFLMDKDNFASKNDESRENKKD
VNCQGEHYTYDEVLSIIQKFEPSISMRQQGLMSFEGFARFLMDKDNFASRNDESQVNTEE
VNCQREHLSYDEILSIIQKFEPSSSMRQMGWMSFEGFSRFLMDKDNFASHIEESQMNPEE
VNCQREHLSYDEILSIIQKFEPSSNMRQMGWMSFEGFARFLMDKDNFASKNEESQVNLDE
NTHQMEQIDEQTAIKLIQDHEPDGICRQKNQMSFEGFTRFLCDPVNFAFVPETIEPDEED
. * *:
: :.:** **.
: . :**.**:*** * ***
: . : .:
1393
1391
1402
1374
1373
903
898
843
910
4
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
PLC-X__(aa1393-1541)_____________________________________ _
LQLPLSYYYIESSHNTYLTGHQLKGESSVELYSQVLLQGCRSVELDCWDGDDGMPIIYHG
LQLPLSYYYIESSHNTYLTGHQLKGESSVELYSQVLLQGCRSVELDCWDGDDGMPIIYHG
LQLPLSYYYIESSHNTYLTGHQLKGESSVELYSQVLLQGCRSVELDCWDGDDGMPIIYHG
LQLPLSYYYIESSHNTYLTGHQLKGESSVELYSQVLLQGCRSIELDCWDGDDGMPIIYHG
LQLPLSYYYIESSHNTYLTGHQLKGESSVELYSQVLLQGCRSIELDCWDGDDGMPIIYHG
LQLPLSYYFIESSHNTYLTGHQLKGESSVELYSQVLLQGCRSVELDCWDGDDGMPIIYHG
LQHPLSYYYIQSSHNTYLTGHQLKGESSVELYSQVLLQGCRSVELDCWDGDDGMPVIYHG
LQHPLSYYYIESSHNTYLTGHQLKGESSVELYSQVLLQGCRSVELDCWDGDDGMPVIYHG
LRYPLSHYYINSSHNTYLTGHQLKGPSSSEMYRQVLLTGCRCVELDCWDGDDGLPLIYHG
*: ***:*:*:************** ** *:* **** ***.:**********:*:****
(S1484L, A601, homozygous)
____________________________________________________________
HTLTTKIPFKEVVEAIDRSAFINSDLPIIISIENHCSLPQQRKMAEIFKTVFGEKLVTKF
HTLTTKIPFKEVVEAIDRSAFINSDLPIIISIENHCSLPQQRKMAEIFKTVFGEKLVTKF
HTLTTKIPFKEVVEAIDRSAFINSDLPIIISIENHCSLPQQRKMAEIFKTVFGEKLVAKF
HTLTTKIPFKEVVEAIDRSAFITSDLPIIISIENHCSLPQQRKMAEIFKSVFGEKLVAKF
HTLTTKIPFKEVVEAIDRSAFITSDLPIIISIENHCSLPQQRKMAEIFKSVFGEKLVAKF
HTLTTKIPFKDVIEAIGRSAFITSEMPIVLSIENHCSLPQQRKMADIFKNVFGEKLVTKF
HTLTTKIPFKDVVEAISRSAFVNSNMPVVLSIENHCSLPQQRKMAEIFKTVFGERLVTRF
HTLTTKIPFKDVVEAINRAAFVNSEMPVILSIENHCSLPQQRKMAEIFKMVFGEKLVTKF
HTLVSKIGFRQVVEIIKKSAFITSDLPVILSIENHCSLQQQAKMAQMFKTVLGDLLVSNF
***.:** *::*:* * ::**:.*::*:::******** ** ***::** *:*: **:.*
1453
1451
1462
1434
1433
963
958
903
970
1513
1511
1522
1494
1493
1023
1018
963
1030
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
_______________________{____
LFETDFSDDPMLPSPDQLRKKVLLKNKKLKAH-QTPVDILKQKAHQLASMQVQAYNGGNA
LFETDFSDDPMLPSPDQLRKKVLLKNKKLKAH-QTPVDILKQKAHQLASMQAQAYNGGNA
LFESDFSDDPMLPSPDQLRRKVLLKNKKLKAH-QTPVDILKQKAHQLASMQAQAYNGGNV
LFETDFSDDPMLPSPDQLRRKVLLKNKKLKAH-QTPVDILKQKAHQLASMQAQAFTGGNA
LFETDFSDDPMLPSPDQLRRKVLLKNKKLKAH-QTPVDILKQKAHQLASMQTQAFTGGNA
LFESDFSDDPMLPSPWQLRNKVLLKNKKLKAH-QTPVDILKQKAHQLASMQAQASNGSQM
LFESDFSDDPHLPSPLQLQGRILLKNKKLKAH-QAPVDILKQKVEQPVPFQMDQFRFYMY
LFESDFADEPLLPSPLQLRGKILLKNKKLKAH-QAPVDILKQKAHQLAHMQAQANNGTVS
LFEADFSDSPRLPCPLQMKNKILIKNKKMIVDPPTPLPMIERGAVQRGETQLNLHRKQSK
***:**:*.* **.* *:: ::*:****: .. :*: :::: . *
* :
1572
1570
1581
1553
1552
1082
1077
1022
1090
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
Ab CS117 (Katan)
NPRPANNEEEEDEEDEYDYDYESLSDDNILEDRPENKSCNDKLQFEYNEEIPKRIKKADN
NPPPANNEEEEDEEDEYDYDYESLSDDNILEDRPENKSCNDKLQFEYNEEIPKRIKKADN
NPPPANNEEEEDEEDEYDYDYESLSDDNILEDRPENKSCNDKLQFEYNEEIPKRIKKADN
NPPPASNEEEEDEEDEYDYDYESLSDDNILEDRPENKSCADKLQFEYNEEVPKRIKKADN
NPPPASNEEEEDEEDEYDYDYESLSDDNILEDRPENKSCADKLQFEYNEEVPKRIKKADN
VSPSTNNEEEEDEEDEYDYDYESLSDDNILDDRSETKTNSDKLQFEYNEEASKRIKKTDG
HYFNGSGSP-----------LCSFKSNNLLDDKPEVKSSADKEEQPVDEIPKRMKKPDNT
TTPLGNNDEEEEEEDEYDYDYESLSDDNILDDKPEGKSSTEKLQYESNDE---MPKRFKK
NSYESSTVDEVEDDDLDEFLDDEENEEDDQEEVQVRSEKEDSPKTSKRAEKSARNIKQQD
.
. ..:: ::
.
:. :
.
1632
1630
1641
1613
1612
1142
1126
1079
1150
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
SAC--------------NKGKVYDMELGEEFYLDQNKK------------ESRQIAPELS
SAC--------------NKGKVYDMELGEEFYLDQNKK------------ESRQIAPELS
SAF--------------NKGKVYDMELGEEFYLPQNKK------------ESRQIAPELS
SSG--------------NKGKVYDMELGEEFYLPQNKK------------ESRQIAPELS
SSG--------------NKGKVYDMELGEEFYLPQNKK------------ESRQIAPELS
SSIN-------------TKGKVYDMELGEEFYLPQNKK------------ESRQIAPELS
TQS---------------KGKVFDMELGEEFYLPQNKK------------ESRQIAQELS
AGS---------------KGKMFDMELGEEFYLPQNEK------------ESRQIAQELS
SLCSDHSVEQAKPSTSKTTSKTNDRKTEDEVLYAQLAQNAIRNQQPRKNNTGVQIAPELS
:
..* * : :*.
* :
. *** ***
1666
1664
1675
1647
1646
1177
1159
1112
1210
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
DLVIYCQAVKFPGLSTLNASGSSRGKERKSRKSIFGNNPGRMSPGETASFNKTSGKSSCE
DLVIYCQAVKFPGLSTLNASGSSRGKERKSRKSIFGNNPGRMSPGETASFNKTSGKSSCE
DLVIYCQAVKFPGLSTLNASGSNRGKERKSRKSIFGNNPGRMSPGETASFNKASGKSSCE
DLVIYCQAVKFPGLSTLNSSGSSRGKERKSRKSIFGNNPGRMSPGETAPFNRTSGKGSCE
DLVIYCQAVKFPGLSTLNSSGSGRGKERKSRKSIFGNNPGRMSPGETASFNRTSGKSSCE
DLIIYCQAVKFPGLTTLNPCGSGRGKERKSRKSIFGNNPGRTSPGEPTALAKTSGKGTNE
DLVIYCQAVKFPGTNVFISSSHFH-----SLDFLKVACSSEMFCCFSTALEFNRSNRGAE
DLVIYCQAIKFPGSLEGIR----------------------------------------DIVIYMQATKFKGFPPVDGIQSPR-----IMEEGPASASLSFSSRARTPSNLLNTPAPPR
*::** ** ** *
1726
1724
1735
1707
1706
1237
1214
1131
1265
5
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
PLC-Y_(aa1744-1846)______________________
GIRQTWEESSSPLNPTTSLSAIIRTPKCYHISSLNENAAKRLCRRYSQKLTQHTACQLLR
GIRQTWEESSSPLNPATSLSAIIRTPKCYHISSLNENAAKRLCRRYSQKLIQHTACQLLR
GMRQAWEESSSPLNPTTSLSAIIRTPKCYHISSLNENAAKRLCRRYSQKLIQHTACQLLR
GMRHTWEESS-PLSPSTSLSAIIRTPKCYHISSLNENAAKRLCRRGSQKLIQHTAYQLLR
GIRQIWEEP--PLSPNTSLSAIIRTPKCYHISSLNENAAKRLCRRYSQKLIQHTACQLLR
TRQSWEEPCSPPFNPSTSLSAIIRTPRCYHISSLNENAAKRLCRRYSQKLIQHTAYQLLR
RLSWEEQQTSPVLSPPTSLSAIIRTPKCYHISSVNENAAKRLCRRYSQKLIQHTVCQLLR
----MNSEDQLCLSPSTSLSSIIRTPKCYHISSVNENAAKRLCRRYSQKLIQHTSCQLLR
RQRSSTQLSQELAAEFLGSVRANATATCYQVTSLNENAAKKLMKRHPAKCVSYTRDHLIR
.
.
*. **:::*:******:* :* . * .:* :*:*
1786
1784
1795
1766
1764
1297
1274
1187
1325
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
____________________________________________________________
TYPAATRIDSSNPNPLMFWLHGIQLVALNYQTDDLPLHLNAAMFEANGGCGYVLKPPVLW
TYPAATRIDSSNPNPLMFWLHGIQLVALNYQTDDLPLHLNAAMFEANGGCGYVLKPPVLW
TYPAATRIDSSNPNPLLFWLHGIQLVALNYQTDDLPLHLNAAMFEANGGCGYVLKPPVLW
TYPAATRIDSSNPNPIMFWLHGIQLVALNYQTDDLPLHLNAAMFEANGGCGYVLKPPVLW
TYPAATRIDSSNPNPLMFWLHGIQLVALNYQTDDLPLHLNAAMFEANGGCGYVLKPPVLW
TYPAATRIDSSNPHPLIFWLHGVQLVALNYQTDDLPLHLNAAMFEANGGCGYVLKPPVQW
TYPAATRIDSTNPNPLLFWLHGIQLVALNYQTDDLPMQLNTALFEANGGCGYVLKPAVLW
TYPAATRIDSANPNPLIFWLHGVQLVALNYQTDDLPMQLNAALFEANGHCGFVLKPPVLW
TYPSAKHYDSSNFNPINCWAHGMQMVALNFQTPDVIMAVNQAMFEQSGNCGYQLKPRCLW
***:*.: **:* :*: * **:*:****:** *: : :* *:** .* **: ***
*
1846
1844
1855
1826
1824
1357
1334
1247
1385
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
C2 motif (aa1871-1953)________ __
DKNCPMYQKFSPLERDLDSMDPAVYSLTIVSGQNVCPSNSMGSPCIEVDVLGMPLDSCHF
DKNCPMYQKFSPLERDLDSMDPAVYSLTIVSGQNVCPSNSMGSPCIEVDVLGMPLDSCHF
DKNCPMYQKFSPLERDLDNMEPAVYSLTIVSGQNVCPSSSTGSPCIEVDVLGMPLDSCHF
DKSCPMYQKFSPLERDLDNLDPAIYSLTIISGQNVCPSNSTGSPCIEVDVLGMPLDSCHF
DKSCPMYQKFSPLERDLDAMDPATYSLTIISGQNVCPSNSTGSPCIEVDVLGMPLDSCHF
DRTCPMYQLFSPLERDLENMEPAIYSLTIVSGQNVCPGNSSGSPCIEIDVLGMPVDSCHF
DRNCPMYQQFCPMERDVEKMSPAVYSLAIVSGQNVCPGNSSGSPCIEVDVLGMPVDSAHF
DRSCPLYQHFYPLDRDLENMTPTLYTLTIVSGQNVCPGNSNGSPCVEVEVLGMPADSCHF
DESHLLYNKFLPLSKDIAGHSALLLNLTIISGQHVYPNTHYASLYVEIEVIGIHNDCVRE
*.. :*: * *:.:*:
.
.*:*:***:* *.. .* :*::*:*: *. :
1906
1904
1915
1886
1884
1417
1394
1307
1445
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
________________________________________________
RTKPIHRNTLNPMWNEQFLFHVHFEDLVFLRFAVVENNSSA-VTAQRIIPLKALKRGYRH
RTKPIHRNTLNPMWNEQFLFRVHFEDLVFLRFAVVENNSSA-VTAQRIIPLKALKRGYRH
RTKPIHRNTLNPMWNEQFLFRVHFEDLIFLRFAVVENNSSA-ITAQRIIPLKALKRGYRH
RTKPIHRNTLNPMWNEQFLFRVHFEDLVFLRFAVVENNSSA-ITAQRIIPLRALKRGYRH
RTKPIHRNTLNPMWNEQFLFRVHFEDLVFLRFAVVENNSSA-ITAQRIIPLKALKRGYRH
RTKPIHRNTLNPMWNEQFLFRIYFEDLVFLRFAVVENNSSA-VTAQRIINLKALKRGNRH
RTKPIHRNTLNPMWNEHFQFTVHFEEMCFLRVAVVENNSSQ-TTAQRTLPLKALKSGYRH
RTKPIHRNTLNPMWNEHFQFHVHFEDLAFLRIAVVENNSSQ-VTAQRILPLKTLRAGYRH
KSKVVQRNSVNPIWNHTTQLRIACVDLAFLRIAVCDSGQNGRVVAHRVVPVKCIRPGFRH
::* ::**::**:**.
: :
:: ***.** :....
.*:* : :: :: * **
1965
1963
1974
1945
1943
1476
1453
1366
1505
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
RA1
_
LQLRNLHNEVLEISSLFINSRRMEENSSGNTMSASSMFNTEERKCLQTHRVTVHGVPGPE
LQLRNLHNEVLEISSLFINSRRMEENSSGNTMSASSMFNTEERKCLQTHRVTVHGVPGPE
LQLRNLHNEVLEISSLFINSRRMEENSSSSATPASLMFNTEERKCLQTHRVTVHGVPGPE
LQLRNLHNEILEISSLFINSRRMEENPSGSSMPASLMFNTEERKCSQTHKVTVHGVPGPE
LQLRNLHNEILEISSLFINSRRMEDNPSGSTRPASLMFNTEERKCSQTHKVTVHGVPGPE
LQLRNLHNEPLEVSTLFINSRRMEEIPNGNTLPASLFFSSEERKTPATFKATVHGIPGPE
IQLRTQHNESLEVSSLFIYSRRTEECPTGGDIPSSLLFSSEEKPASQQHRVTVYGAPGPE
LQLRNLHNEPLEVSSLFMFSRRTEESPTG-GPPSASLFSTEERRSVQQHKVTVHGVPGPE
LPLRTPTNLPIDNAMIFLRTRFEQEEHIYLHDDDSNTYCNLEHTLAYRTDLTPNLSPTPI
: **. * :: : :*: :* ::
: : . *:
*
* *
2025
2023
2034
2005
2003
1536
1513
1425
1565
6
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
_
PFTVFTINGGTKAKQLLQQIL-TNEQDIKPVTTDYFLMEEKYFISKEKNECRKQPFQRAI
PFTVFTINGGTKAKQLLQQIL-TNEQDIKPVTTD-FLMEEKYFISKEKNECRKQPFQRAI
PFTVFSISGGTKAKQLLQQIL-TTEQDTKPIVTDYFLMEEKYFISKEKNECRKQPFQRVI
PFAVFTINEGTKAKQLLQQVL-AVDQDTKCTATDYFLMEEKHFISKEKNECRKQPFQRAV
PFAVFTINEGTKAKQLLQQASPLIDQDTKLTAADYFLMEEKHFISKEKNECRKQPFQRAV
PFTVFEISLGTTAKQLLDHILATIQG-NEADITDYFLMEEKCFISKDKNECKKLPFQRVI
PFTVFSVTEQITAKQLLDMVGSVGKYSASAGGNPYFLCEEKVPLTKERSETKRCAQYRPL
PFTVICVDEFTTAKQLLDSLFPT-------ASFKYMLMEERVSLSKE----KKAPQQRPL
LKKQIFVLRITGAFADETAITVHSESGSTVKTVMQQALLNAGKNADQVEEYVLIEESLPA
: :
*
:
:.:
2084
2081
2093
2064
2063
1595
1573
1474
1625
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
_
GPEEEIMQILSSWFPE---------EGYMGRIVLKTQQENLEEKNIVQD----------GPEEEIMQILSSWFPE---------EGYVGRIVLKTQQENLEEKNIVQD----------GPEEEIMQILSSWFPE---------EGYVGRIVLKTQQENLEERSIVQD----------GPEEDIVQILNSWFPE---------EGYVGRIVLKPQQETLEEKSIVFD----------GPEEDIVQILNSWFPE---------EGYVGRIVLKPQQETLEEKNIVHD----------SPEEDILQILNSWFPE---------EGYVGRIVLKTREENMNDKNVQEDKE--------APEEEVVRLVSSWSAE---------EGYVGRICFKLREEKLNEKNAAPEGEEEWSVGG-ANDERLLKLIHSWQPE---------DGYVGRIYLKTREQNCSEKTSVPLESLEELG---PSGEDPIEQRVLPLNEPIMDAVACWNGSMRRFVLRKKGSDPSSRAWITSIIKSGTSGSST
* :.
*
:* : *: :: : . ..:
2124
2121
2133
2104
2103
1637
1622
1521
1685
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
RA2_____
-----------------------------------------DKEVILSSEEESFFVQVHD
-----------------------------------------DKEVILSSEEESFFVQVHD
-----------------------------------------EKEVILSSEEESFFVQVHD
-----------------------------------------DKEVILSSEEESFFVQVHD
-----------------------------------------DREVILSSEEESFFVQVHD
-----------------------------------------EKEVTVSSDEETFFVQVHD
------------------------------------AREGAGGEGAAAAEDDVFLVQIHE
-----------------------------------------------SLEDDTFFVQVHD
SVSPSPLTKDGHVKSASSNQLHGRSLDTDAFGEHLEVTEGKWLNPRARSMGDTFLVCVHN
: *:* :*:
2143
2140
2152
2123
2122
1656
1646
1534
1745
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
H._sapiens
P._troglodytes
C._familiaris
M._musculus
R._norvegicus
X._tropicalis
F._rubripes
D._rerio
C._elegans
.:
Ab CV221 (Katan)
(aa2135-2238)______________________________________________
VSPEQPRTVIKAPRVSTAQDVIQQTLCKAKYSYSILSNPNPSDYVLLEEVVKDTTNKKTT 2203
VSPEQPRTVIKAPRVSTAQDVIQQTLCKAKYSYSILSNPNPSDYVLLEEVVKDTTNKKTT 2200
VSPEQPRTVIKAPRVSTAQDVIQQTLCKAKYSYSILSNPNPSDYVLLEEVVKDAT-KKSS 2211
VSPEQPRTVIKAPRVSTAQDVIQQTLCKAKYSYSILNNPNPCDYVLLEEVLKDAANKKSS 2183
VSPEQPRTVIKAPRVSTAQDVIQQTLCKAKYSYSILNNPNPCDYVLLEEVMKDAPNKKSS 2182
VSPEQPRTVIKALRFSTAQEVIQQALCKAKYSYSILSNPNPSDYVLLEEVIKEASNKKSS 1716
VSPEQPHTVIKAPRYSTAQDIIQQTLFKAKYSYSILSNPNPCDYVLMEEVTKDVGSKKSS 1706
VSPEQPHTVIKAPRYSTAQDIIQQTLSKAKYSLSILSNPNPCDYVLMEELCKDAGGKKSS 1594
VSEDQPYAILRAGIHSTAADIIRQVFVKARR-----SNVDDSEFVLVEETCDDPKLNQGQ 1800
** :** ::::*
*** ::*:*.: **:
.* : .::**:** .:
::
_________________________________________
TP------KSSQRVLLDQECVFQAQSKWKGAGKFILKLKEQVQASREDKKKGISFASELK
TP------KSSQRVLLDQECVFQAQSKWKGAGKFILKLKEQVQASREDKKKGISFASELK
TP------KSSQRVLLDQECVFQAQSKWKGAGKFILRLKEQVQASREDKRKGISFASEFK
TP------KSSQRILLDQECVFQAQSKWKGAGKFILKLKEQVQASREDKRRGISFASELK
TP------KSSQRILLDQECVFQAQSKWKGAGKFILKLKEQVQASREDKRRGISFASELK
TS------KTIQRVLLDQECVFQAQSKWKGAGKFILKLKEQVQAARDDKRKGLSIASELK
TT------KPLQRMLLDHECVYQAQSRWRGAGKFILKLKEQLVR--EDKKKVISFASELK
SA------KPCQRVLQDHECVFQAQSRWKGAGKFILKLKEQLAR--EDKRKGVSFASELR
MTPKYPNNRTTSRVLGQNENVWKAQSRWKSMGRFVLENRKDTVHATLEKGRRVESTTSSS
.
:. .*:* ::* *::***:*:. *:*:*. :::
:* : :. ::.
KLTKSTKQPRGLTSPSQLLTSESIQTKEEKPVGGLSSSDTMDYRQ
KLTKSTKQPRGLTSPSQLLTSESIQTKEEKPVGGLPVTQWITDSD
KFTKSTKQPRGLTS-SQVLASESVQNKEEKPTGSLSSSDTTDSRQ
KLTKSTKQSRGLPSPPQLVASESVQSKEEKPVGALSSSDTVGYQQ
KLTKSTKQTRGLTSPPQLVASESVQSKEEKPMGALASGDTAGYQS
KLAKSSRQSRNFTLSPQIFLSEGTQNRDEKPACSLSFSDINE--KLT-----------------------------------------KLTGRSRSVTVNTNSNDTHSKEEKSACPSMSCVPETSQ------TTTRKISLSSVRSIGLPRKFSKFGKSLTMDAGPK-----------
7
2302
2299
2309
2282
2281
1812
1761
1684
1894
2257
2254
2265
2237
2236
1770
1758
1646
1860
Supplementary Figure 4.
CLUSTAL_W amino acid multiple sequence alignment of PLCε1 throughout evolution.
Deduced amino acid sequences of PLCe1 orthologs had the following GenBank accession
numbers and percentage amino acid sequence identity compared to human sequence: H.
sapiens (CAI16674, 100%); P. troglodytes (ENSPTRP00000004841, 99%); C. familiaris
(ENSCAFP00000011839 89%); M. musculus (NP_062534.1, 84%); R. norvegicus
(NP_446210.1, 84%); X. tropicalis (JGI Xenopus tropicalis v3.0 29459, 77%); F. rubripes
(NEWSINFRUP00000140491, 62%); D. rerio (ENSDARG00000017481, 65%); and C. elegans
(wormbase F31B12.1 WP:CE31495, 30%). Amino acid residues are given as single letter code
with the following coloring for residues: Red if small hydrophobic or aromatic (AVFPMILWY),
blue if acidic (DE), magenta if basic (RHK), green for hydroxyl, amine, basic and glutamine
(STYHCNGQ). The lowermost row indicates conservation among the group of sequences. A
period indicates semi-conserved substitutions. A colon indicates conserved substitutions.
Representations of putative protein domains are drawn above the sequence group and are
marked and color coded as follows: white on red, RasGEF_CDC25 domain (guanine nucleotide
exchange factor for Ras-like small GTPases) (predicted by PFAM PF00617, aa 551-725);
pleckstrin homology domain (PH); PLC_X domain (Phospholipase C, catalytic domain, part X)
(predicted by PFAM PF00388, aa 1393-1541); PLC_Y domain (phospholipase C, catalytic
domain, part Y) (predicted by PFAM PF00387, aa 1744-1846); C2 motif (protein kinase C
conserved region 2, subgroup 2) (predicted by PFAM PF00168, aa 1871-1953); RA1 domain
(RasGTP binding domain from guanine nucleotide exchange factors); RA2 domain (RasGTP
binding domain from guanine nucleotide exchange factors) (predicted by PFAM PF00788, aa
2135-2238). The position of homozygous mutation S1484L in kindred A601 is indicated together
with its evolutionary conservation that includes C. elegans. Amino acid Positions of two peptides
used for antibody production (Katan et al.) are indicated in boxes for the human sequence.
8
Supplementary Table 1. Exon flanking and morpholino oligonucleotide primers used in human and zebrafish PLCE1 studies.
A. Exon-flanking oligonucleotide primers used for PCR in the human PLCE1 gene.
Primer name
SRN3-Ex-2.1_F
SRN3-Ex-2.1_R
SRN3-Ex-2.2_F
SRN3-Ex-2.2_R
SRN3-Ex-2A_F
SRN3-Ex2-A_R
SRN3-Ex-3_F
SRN3-Ex-3_R
SRN3-Ex-4_F
SRN3-Ex-4_R
SRN3-Ex-5_F
SRN3-Ex-5_R
SRN3-Ex-6_F
SRN3-Ex-6_R
SRN3-Ex-7_F
SRN3-Ex-7_R
SRN3-Ex-8.1_F
SRN3-Ex-8.1_R
SRN3-Ex-8.2_F
SRN3-Ex-8.2_R
SRN3-Ex-9_F
SRN3-Ex-9_R
SRN3-Ex-10_F
SRN3-Ex-10_R
SRN3-Ex-11_F
SRN3-Ex-11_R
SRN3-Ex-12-13_F
SRN3-Ex-12-13_R
SRN3-Ex-14_F
SRN3-Ex-14_R
SRN3-Ex-15-16_F
SRN3-Ex-15-16_R
SRN3-Ex-17_F
SRN3-Ex-17_R
SRN3-Ex-18_F
SRN3-Ex-18_R
SRN3-Ex-19_F
SRN3-Ex-19_R
SRN3-Ex-20_F
SRN3-Ex-20_R
SRN3-Ex-21_F
SRN3-Ex-21_R
Sequence (5’ to 3’)
CAGAGTGCAATCCCGAGTAA
GCCCATGGAAGGTCTTTCTA
TGCCAGATTCTGCGAAAAAC
TGTTGTTGCAGTATCAACTCAATC
TTTCACAGAGGTTTTTGCAAT
AAGGAAAGAAACTTCTCTGATCTTC
TTGCACTTGGAGCATCTGAG
TGAACTTAATTTTCCATCAGGAG
AAGATTTGGAATGGCTCTCAAG
AAACCTAGAAGGGGAGGTATGC
CCCAGCCAGGACCTACAG
TGGGTAAAGGTGAGTCCCTG
GAATTTGTATGGAATTTAGGCTCC
TTGGTGAGCACAGACAGGG
TGATTTCATTTCCCTAGCCAAG
TGTCCATCTGAAACTAAGCTGAAG
TTGAATTCATAGTGCGATGAAAAA
TGGGAATTTTCCAGGCTCAGC
CTGGGTAAAGCCCACAACTG
ACTGTCAGAGCTGGGAGCATG
TGTGCTCTTCCACCTCTTGG
CCTGACTTTGTCCTCAACCC
GCAGTATTGAAGGTGGGTAGG
AAATGTTTGCAATGCTTAAATCAC
TGCTCAGACATCCAGAAGCC
TGTAAAGATATGCCCTTCCACAC
AATTGGAGCAAGTCTGGTGG
GTGTGACTGGACCTCTGACC
TTCTCCATTTTAATAACTCCACACC
CCCAGATTTAAAGGCTTTGG
GATGTGGTGGTTTCTTTCCC
TCAAAGGAGTCTGGGGTCAG
CGAGGCTTTATCTCCAGGTG
AACAGAGCGAGAACCCG
CTACCCTGCCTTCTGACCAC
TGACAATTGCAAAACAAGGG
AGCAGGGGACAGCTTCTTTC
GGAATGAAAAGCCCAAAATC
CGATTGTGTTAAACATCAGGG
ATTACTGGTCTTTGGCGCTC
AAGTACAAGTATCTGGATGTCCTCAC
GACAGGGAGCAAGTGGAATC
1
Product
size
414 bp
~1120 bp
601bp
501 bp
480 bp
376 bp
420 bp
376 bp
450 bp
520 bp
392 bp
325 bp
325 bp
502 bp
447 bp
477 bp
267 bp
238 bp
465 bp
321 bp
303 bp
SRN3-Ex-22_F
SRN3-Ex-22_R
SRN3-Ex-23_F
SRN3-Ex-23_R
SRN3-Ex-24_F
SRN3-Ex-24_R
SRN3-Ex-25_F
SRN3-Ex-25_R
SRN3-Ex-26_F
SRN3-Ex-26_R
SRN3-Ex-27_F
SRN3-Ex-27_R
SRN3-Ex-28_F
SRN3-Ex-28_R
SRN3-Ex-29_F
SRN3-Ex-29_R
SRN3-Ex-30_F
SRN3-Ex-30_R
SRN3-Ex-31_F
SRN3-Ex-31_R
SRN3-Ex-32_F
SRN3-Ex-32_R
GAGCTTTGGGAATCCAGAGG
TGAGAGTGTTCACAATGCCC
AATGGGGATGGAAAATGTTG
GGGAAGTGCTTAGACAGTAAAATATC
TGCTATGACTGTTTACTGGGATG
ACATATGGTGTGCCCCAGTC
TTTCATGCTGGAGCTTAGGG
CATGACAGCTTTCCAATGCC
TGTTCTTGGGATTCCTTTGC
TGCTTCTTAATTCAACTTCTTTATAGG
TCCCTGCCCATTTTAAGGTC
ACTCACACCGACCACTTTGC
TGCATTTACATGTTCCTATCCG
CGCATTCATGTGCATCTGTG
AAACCTATCTGAACACCATGAAAG
AGGTTGTGAGTGGTAAATGGC
CATCACCAAGATACAAGCTCAG
GGTCCCTGTTGTTGAGGAAC
AGAATGAATGCAAATGTTGGAG
TCCTAAATTTCACCAGCTTCC
TCCAAAGCTCTAGAGAGAAGAGG
AAAAGACATTGTACTAATTATGCCTTC
278 bp
284 bp
499 bp
379 bp
486 bp
319 bp
347 bp
361 bp
368 bp
373 bp
403 bp
B. Morpholino (MO) antisense oligonucleotides used in zebrafish plce1
knockdown experiments.
Exon 14 donor site invert sense MO
Negative control invert sense MO
GATGTGCTGCAGATGTACCTGGCTG
GTCGGTCCATGTAGACGTCGTGTAG
C. RT-PCR nested primers flanking plce1 exons 12-17 to yield a final
“inside” 630 bp amplicon from wild-type and a 782 bp product from
morphants.
outside forward
outside reverse
inside forward
inside reverse
ATCTCCTCACTCGCAACGGCTCT
AATCTCAGCCATTTTGCGCTGCT`
CGCCAGCAAATGTCTGAAAACCA
CATTTTGCGCTGCTGTGGTAAGG
2