Supplementary Information (Nektarios Tavernarakis, Nature T05081A TH/mah)
Specific aspartyl and calpain proteases are required for neurodegeneration in C. elegans
Popi Syntichaki, Keli Xu, Monica Driscoll & Nektarios Tavernarakis
Supplementary Information
Supplementary Methods
GeneBank accession numbers of the aspartyl protease sequences used for the analysis are: Human
hCathepsinD, NP_001900; mouse mCathepsinD, NP_034113; Drosophila dRE02351p, AAL48533; yeast
yPep4p, NP_015171; ASP-1 (ORF: Y39B6A.20), AAF19442; ASP-2 (ORF: T18H9.2), AAA83331;
ASP-3 (ORF: H22K11.1), AAK39240; ASP-4 (ORF: R12H7.2), NP_510191; ASP-5 (ORF: F21F8.3),
AAC47991; ASP-6 (ORF: F21F8.7), AAC47996; Y39B6A.23, CAD31820; Y39B6A.24, CAD31821;
Y39B6A.22, CAD31819; ZK384.3, T26490; Y39B6A.21, CAD31818; F59D6.3, T31770; F59D6.2,
T31771; F21F8.4, T29411; C11D2.2, T32964; F21F8.6, T29409; F28A12.4, T28954; C15C8.3, T19309;
F21F8.2, T29413 and K10C2.3, T25812. Accession numbers of the calpain sequences are: Human hmCalpain, NP_001739; human hCalpain1, NP_008989; mouse mCalpain1, NP_031626; Drosophila
dCalpain, CAA55297; Drosophila dSol, P27398; CLP-1 (ORF: C06G4.2), P34308; CLP-2 (ORF:
T04A8.16), T24431; CLP-3 (ORF: Y47H10A.1), NP_493052; CLP-4 (ORF: Y39A3CL.5), AAF60521;
TRA-3/CLP-5 (ORF: LLC1.1), AAB60256; CLP-6 (ORF: Y77E11A.10), AAF36079; CLP-7 (ORF:
Y77E11A.11), AAF36080; T11A5.6, T24825; W05G11.4, T32871; F44F1.3, T22177; F47F6.5,
NP_493933; F44F1.1, T22175; M04F3.4, NP_491447; Y53H1B.6, NP_492903; H25P06.4, T23122;
W04A4.4, T26132;and T21H3.3, T31737.
BLAST searches1 were performed with the National Center for Biotechnology Information web
based servers (NCBI; http://www.ncbi.nlm.nih.gov/BLAST/). Multiple sequence alignments were
1
Supplementary Information (Nektarios Tavernarakis, Nature T05081A TH/mah)
generated using the ClustalW algorithm2 and displayed with SeqVu (The Garvan Institute of Medical
Research, Sydney, Australia) and BoxShade 3.21 (EMBNet;
http://www.ch.embnet.org/software/BOX_form.html). Dendrograms showing phylogenetic distance
relationships among aspartyl and calpain proteases were constructed with the neighbor-joining method3,
based on pair wise distance estimates of the expected number of amino acid replacements per site, and
visualized with TreeView (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). Prosite searches for
conserved motifs in protein sequences were performed with the Expert Protein Analysis System
(ExPASy) proteomics web based server at the Swiss Institute of Bioinformatics (SIB;
http://www.expasy.ch/tools/scanprosite/).
Supplementary References
1.
Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database
search programs. Nucleic Acids Res 25, 3389-3402. (1997).
2.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. CLUSTAL W: improving the sensitivity of
progressive multiple sequence alignment through sequence weighting, position-specific gap
penalties and weight matrix choice. Nucleic Acids Res 22, 4673-4680. (1994).
3.
Saitou, N. & Nei, M. The neighbor-joining method: a new method for reconstructing phylogenetic
trees. Mol Biol Evol 4, 406-425. (1987).
4.
Tcherepanova, I., Bhattacharyya, L., Rubin, C. S. & Freedman, J. H. Aspartic proteases from the
nematode Caenorhabditis elegans. Structural organization and developmental and cell-specific
expression of asp-1. J Biol Chem 275, 26359-26369 (2000).
5.
Moldoveanu, T. et al. A Ca(2+) switch aligns the active site of calpain. Cell 108, 649-660 (2002).
2
Supplementary Information (Nektarios Tavernarakis, Nature T05081A TH/mah)
Legends
Figure 5. ClustalW-generated, multiple alignment of nematode aspartyl proteases.
Representative members from other species are also included for comparison (hCathepsinD,
human; mCathepsinD, mouse; dRE02351p, Drosophila; yPep4p, yeast). The symbol # in the
consensus line denotes the conserved lysosomal targeting, N-glycosylation site (asparagine 71
in ASP-1), which is absent in ASP-3 and ASP-4, as well as the potential N-glycosylation site in
ASP-3 and ASP-4, common in non-lysosomal cathepsin E proteases. Of the 20 predicted
nematode proteins included in the alignment, at least 6 appear to be expressed (ASP-1 to 6)4.
Figure 6. Dendrogram showing phylogenetic relationships among aspartyl proteases aligned in
Figure 5. ASP-3 and ASP-4, shown in bold, are required for neurodegeneration and cluster
within a distinct branch that also includes mammalian proteases. Red branches indicate
nematode proteins (green for ASP-3 and ASP-4). Non-nematode proteins are in blue. Branch
lengths represent the expected number of amino acid replacements per site (0.1 in the scale
bar).
Figure 7. mec-4(d)-induced neurodegeneration is suppressed in aspartyl protease-deficient
genetic backgrounds and is restored by elevated expression of ASP-3 and ASP-4. a, Images of
vacuolated touch receptor neurons (indicated by arrows) in animals of the indicated genetic
background. mec-4(d) L1 larvae exhibit large prominent vacuoles in mid-body and tail areas
corresponding to dying ALM and PLM touch receptor neurons respectively. In cad-1(j1);mec4(d), daf-4(e1364);mec-4(d) and unc-52(su250);mec-4(d) double mutants, where
neurodegeneration is significantly suppressed, not only is the number of vacuoles lower, but
incident vacuoles are also qualitatively different; they are smaller and less pronounced than in
mec-4(d) single mutants. b, Introduction of ASP-3 and ASP-4 restores neurodegeneration in
3
Supplementary Information (Nektarios Tavernarakis, Nature T05081A TH/mah)
daf-4(e1364);mec-4(d) and unc-52(su250);mec-4(d) double mutants. Bars signify degenerating
touch receptors at L1 stage. Ex[asp-3] and Ex[asp-4] restored cell death in daf-4(e1364);mec4(d) and unc-52(su250);mec-4(d) double mutants (n=100, P<0.001, unpaired t-test). Values for
mec-4(d), daf-4(e1364);mec-4(d) and unc-52(su250);mec-4(d) controls are those in Fig. 1a and
are included here for comparison.
Figure 8. Multiple alignment of nematode calpain proteases. Representative members from
other species are also included for comparison (hm-Calpain, human; hCalpain1, human;
mCalpain1, mouse; dCalpain, Drosophila; dSol, Drosophila Small optic lobes protease). The
symbol # in the consensus line denotes the conserved catalytic residues (glutamine 269,
cysteine 275, histidine 431, asparagine 455 and tryptophan 457 in CLP-1)5. Of the 17 predicted
nematode proteins included in the alignment, 7 show significant identity to mammalian calpains
over their entire length and have been annotated as such in WormBase
(http://www.wormbase.org).
Figure 9. Dendrogram showing distance relationships among calpain proteases aligned in
Figure 8. CLP-1 and TRA-3, shown in bold, are involved in neurodegeneration. Red branches
indicate nematode proteins (green for CLP-1 and TRA-3). Non-nematode proteins are in blue.
Branch lengths represent the expected number of amino acid replacements per site (0.1 in the
scale bar).
Figure 10. Details of protease gene expression in C. elegans. Aspartyl proteases ASP-3 and
ASP-4 appear to be highly expressed in the intestine of adult animals and at relatively lower
levels in many other tissues including body wall muscles, the hypodermis, neurons, the uterus
and others. Expression of both genes is detectable in two-fold stage embryos. Calpain clp-1 is
strongly expressed in the nervous system, muscles and the hypodermis. Expression is also
4
Supplementary Information (Nektarios Tavernarakis, Nature T05081A TH/mah)
observed in the intestine, the canal cell and the pharynx. a, images of animals carrying ASP3::GFP. b, images of animals carrying ASP-4::GFP. c, images of animals carrying pclp-1GFP.
Various cells and organs are indicated by arrows.
5
Supplementary Information (Nektarios Tavernarakis, Nature T05081A TH/mah)
Table 1. The conserved catalytic pentad of amino acid residues in calpains (glutamine 269, cysteine 275,
histidine 431, asparagine 455 and tryptophan 457 in CLP-1)5 is intact in CLP-1, CLP-2, TRA-3, CLP-6
and CLP-7 (shown in bold). The rest of the 17 nematode sequences are missing one or more specific
residues (+/-: presence/absence of catalytic residues).
Q269 C275 H431 N455 W457
+
+
+
+
+
1 CLP-1
+
+
+
+
+
2 CLP-2
+
3 CLP-3
+
+
4 CLP-4
+
+
+
+
+
5 TRA-3
+
+
+
+
+
6 CLP-6
+
+
+
+
+
7 CLP-7
+
+
+
+
8 F44F1.3
+
+
+
+
9 F44F1.1
+
+
+
10 F47F6.5
+
11 H25P06.4
12 M04F3.4
+
+
+
+
13 T11A5.6
14 T21H3.3
+
15 W04A4.4
+
+
+
+
16 W05G11.4
+
17 Y53H1B.6
Table 2. Neurodegeneration inflicted by overexpression of asp-4 cannot be bypassed by calpain protease
deficiency.
Strain
%a
Ex[pmec-4ASP-4]
13.9±1.4
Ex[pmec-4ASP-4] clp-1(RNAi)
13.2±1.8
Ex[pmec-4ASP-4] tra-3(RNAi)
12.8±1.6
Ex[pmec-4ASP-4] clp-1(RNAi);tra-3(RNAi)
13.5±2.1
a
Percentage (± standard deviation) of animals with vacuolated touch receptor neurons at L1 stage.
6
Supplementary Information (Nektarios Tavernarakis, Nature T05081A TH/mah)
Figure 5
Y39B6A.23
Y39B6A.22
Y39B6A.24
ASP-1
ZK384.3
Y39B6A.21
F59D6.3
F59D6.2
hCathepsinD
mCathepsinD
ASP-4
ASP-3
dRE02351p
yPep4p
C11D2.2
F21F8.2
ASP-5
F21F8.4
ASP-6
F21F8.6
ASP-2
F28A12.4
C15C8.3
K10C2.3
consensus
. . . .10 . . . .20 . . . .30 . . . .40 . . . .50 . . . .60 . . . .70 . . . .80 . . . .90 . . . 100 . . . 110 . . . 120 . . . 130 . . . 140 . . . 150 . . . 160 . . . 170 . . . 180
1:..........................................................................................MRLVFLLFLVPFCSAAVLQLP............TKSTGSLRAKLIQAGKYQ.................EFLASQHAARLQQLNTGSQPLI: 61
1:..........................................................................................MHLIFLLFIATFCSAAVLQLP............TKSTGSLRAKLIQSGKYP.................EFLASQHVSR...LNSGALPLA: 58
1:..........................................................................................MHLIFLLFLAPFCSAAVFQLP............TKSTGSLRAKLIRAGKYQ.................EFLITQHAAR...LNTISQPIS: 58
1:..........................................................................................MQTFVLLALVAACSAAVIQVP............THKTESLRAKLIKEGKYT.................AFLASQHAARAQQLNTGFQPFV: 61
1:............................................................................................MNLLLLLVTTCSAAVFQVP............TTTSGSFRAKLIREGKYS.................SFLAS....RLQQLNTGSQPLL: 55
1:.........................................................................................MKCLLLLFAFIGLATCASFSIP............TRSVSSLRAKLIREGRYQ.................EFLAQQNVARTLQLKQGSQPFI: 62
1:MKQGFLIYYSRLLLDFRAFFCRALALAIFFVQFSICITQLVQKLFICVNTTFLCAERGEMITGGTKILRNTFQSIKLEDNRKIFIFHKMFGKLISLLGLVALCSAGQFSIS............VEKSGSLREQLIREGRY......................GQELARIQQLSTGNVSFF:146
1:........................................................................................MTEKLTILLALLVFCSAGQFSIS............LEKSVPLREQLIREGR.........................ELARLQQLSTGNESIY: 55
1:........................................................................................MQPSSLLPLALCLLAAPASALVR..........IPLHKFTSIRRTMSEVG..........GSVEDLIAKGPVSKYSQAVPAVTEGPIPEVLK: 72
1:........................................................................................MKTPGVLLLILGLLASSSFAIIR..........IPLRKFTSIRRTMTEVG..........GSVEDLILKGPITKYSMQSSPKTTEPVSELLK: 72
1:........................................................................................MNRCILLLLGALLLVQGLHVHKRQ....QKLRTVSLKKQPTLRETLLQAGSFETFAKHRHGYKKYLKTNG.NHHFDKYQALNVEGEIDELLR: 87
1:........................................................................................MSGRVFLLLALVALAS...AIQR..........IKLEKRTYTREQY.KFG..........SIQEHLKAKYVPGYIPN......KDAFNEGLS: 62
1:.........................................................................................MRQLLVLLVLVGIGHSAAKLNR..........VQLQVNKNFTKTHGSVK............AEKTVLASKYSFLAETSFSVSSSGATENLH: 69
1:.......................................................................................MFSLKALLPLALLLVSANQVAAKVHKAKIYKHELSDEMKEVTFEQHLAHLG.........QKYLTQFEKANPEVVFSREHPFFTEGGHDVPLT: 84
1:........................................................................................MALPFFLLLPNAHFSSFCMHLVR..................KEHSTCLMG...............PVVSNRLHSSSFKSKHTIHKGQHQHVA: 59
1:........................................................................................................................................MG...............TMFQDR.....................: 8
1:........................................................................................MQT..FVLLALVAACSAAVIQVP............THKTESLRAKLIKEGKYT............AFLASQHAAR..AQQ...LNTGFQPFV: 61
1:........................................................................................MKTTLLAFVAILGMCSATVVQHK............LIYRESPKMQMMRRGEWG............AYVQHKAALR..DANPAVFASAPQNVN: 66
1:........................................................................................MKT.FILLAVLG.LASASVHQHK............IVWRESKKMGMIRTGQYP............AYLEYQRNLR..AVSPNVLANLPQNVN: 64
1:........................................................................................MRS.IILIISLFVFVSGLVHQSK............ILWKPSKRIEMLRSGEWA............TYKKHLERQR..NRNPWQLQSLPQNVN: 65
1:........................................................................................MGN.LFLLLALVAAALAAVVQVP............LVKIEPYRNRLIREGRWV............QYRKDREIRRFMMNKQSNDMSVGQYVN: 67
1:........................................................................................MGI.ISIIFLLIGSSTAAVLEHQ............LIWKESRMIEMIRNGEYP............AYLEYRDRMV..ASRSSNLATVVQSTT: 65
1:........................................................................................MKT.FIALLALLTVVSAEVHQFN............IGYRPNMRQRMNAKGKLA............EYEKERNELL..SKKSLQLASSSSPVI: 65
1:...............................................................................MKVEVCSVENDRGLSRFFGFYVISSKHERINRRAG......CLEIILSSIMIKTILLLAFVAS..............TSAFVIPFKVHAAVTNITKGGQTL: 81
1:----------------------------------------------------------------------------------------*---********-*-****-***------------*-*--***-****-***---------------------------*--**-**-*-*-:180
. . . 190 . . . 200 . . . 210 . . . 220 . . . 230 . . . 240 . . . 250 . . . 260 . . . 270 . . . 280 . . . 290 . . . 300 . . . 310 . . . 320 . . . 330 . . . 340 . . . 350 . . . 360
Y39B6A.23
62:DYYDDMYLANITVGTP.....PQPASVVLDTASANLWVIDAACNS.................................QACNGNPGSGYTKQKFNPNKSSTFVKGTRRFSIQYGSGTSSGYLGTDVLQLGG...........LTVKAQEFG.VATNLGSVLGSEPMDGIFGLGWPAISVD:191
Y39B6A.22
59:DYMDEIYTANITVGTP.....PQSASVVMGTGSANFWVVGAGCTS.................................PACNGFPDSGHTKLKFYSNKSSSFVDLNRKFFINYPTGACSGYLGTDVLQLGG...........LTVKNQEFA.VTTQISDQMGHFPLDGVLGLAWPPVAVD:188
Y39B6A.24
59:DYSDEVYLGNFTVGTP.....PQPVSLVLDTGSANMWVIDASCDN.................................MFCNGWIGSNYTRQKFDTSKSSSFSRENRKFSIQYGKGLCSGYLGTDTVGLGGG..........LTIRKQELG.IANKLDVDFAVQPMDGIFGLAWPALAVD:189
ASP-1
62:DYFDDFYLGNITLGTP.....PQPATVVLDTGSSNLWVIDAACKT.................................QACNGYPDSGYTKQKFDTTKSTTFVKETRKFSIQYGSGSCNGYLGKDVLNFGG...........LTVQSQEFG.VSTHLADVFGYQPVDGILGLGWPALAVD:191
ZK384.3
56:GFSDDPCIGNITIGSP.....PQSAFVFMDTTSANLWVMGSACTS.................................VNCN.DPLLGIIKHKFNTTKSTSFVKSNRKFNIQYGSGECSGYLGTDTVEIGG...........LKIQKQEFG.VANIVDYDFGTRPIDGIFGLAWPALSVD:184
Y39B6A.21
63:DYMDDFYLGNISVGTP.....GQTLTLVLDTGSSNLWVIDAACNS.................................EACNGDPKSGFAKHKFDTKASSTFVSETRKFSISYGSGSCSGHLARDTITMGG...........LKIDNQEFG.VAETLASVFAEQPVDGILGLGWPALADD:192
F59D6.3
147:DHFDEYYTAGVRIGTP.....AQHFQVAFDTTSSNLWVFGVECRS.................................QNCHGGRGR..RDREYNRTASSTFVAGTSSFNLPYDGGHVSGNVGKDTAQFAG...........FTIQSQDFG.IGTAATRLFG.ETFDGVLGLGWPATALN:273
F59D6.2
56:DHFDEYYTVSVRIGTP.....AQHFEVALDTTSSNLWVFGVECKS.................................QSCQVLKYI..RIREYNRTASSTFIAGTSNFVLPFNDGDVSGDLGKDNIQFAG...........SKIQNQDFG.IGTDATRLSG.VTFDGVLGLGWPATALN:182
hCathepsinD 73:NYMDAQYYGEIGIGTP.....PQCFTVVFDTGSSNLWVPSIHCK....................................L..LDIACWIHHKYNSDKSSTYVKNGTSFDIHYGSGSLSGYLSQDTVSVPCQSASSASALGGVKVERQVFGEATKQPGITFIAAKFDGILGMAYPRISVN:209
mCathepsinD 73:NYLDAQYYGDIGIGTP.....PQCFTVVFDTGSSNLWVPSIHCK....................................I..LDIACWVHHKYNSDKSSTYVKNGTSFDIHYGSGSLSGYLSQDTVSVPCK..SDQSKARGIKVEKQIFGEATKQPGIVFVAAKFDGILGMGYPHISVN:207
ASP-4
88:NYMDAQYFGTISIGTP.....AQNFTVIFDTGSSNLWIPSKKCP....................................F..YDIACMLHHRYDSKSSSTYKEDGRKMAIQYGTGSMKGFISKDSV...........CVAGVCAEDQPFAEATSEPGITFVAAKFDGILGMAYPEIAVL:213
ASP-3
63:DYSNAQYYGPVTIGTP.....PQNFQVLFDTGSSNLWVPCANCP....................................F..GDIACRMHNRFDCKKSSSCTATGASFEIQYGTGSMKGTVDNDVVCFG.......HDTTYCTDKNQGLACATSEPGITFVAAKFDGIFGMGWDTISVN:192
dRE02351p
70:NSMNNEYYGVIAIGTP.....EQRFNILFDTGSANLWVPSASCP....................................A..SNTACQRHNKYDSSASSTYVANGEEFAIEYGTGSLSGFLSNDIVT...........IAGISIQNQTFGEALSEPGTTFVDAPFAGILGLAFSAIAVD:195
yPep4p
85:NYLNAQYYTDITLGTP.....PQNFKVILDTGSSNLWVPSNEC........................................GSLACFLHSKYDHEASSSYKANGTEFAIQYGTGSLEGYISQDTLS...........IGDLTIPKQDFAEATSEPGLTFAFGKFDGILGLGYDTISVD:208
C11D2.2
60:DFRDFAYFGNITLGTPIESTAEQTFLVVLDTGSSNVWVPDNSCG....................................IDDKKSACTYKKKYFGTDSSSYEKDGTPFSISYGTGSASGYFGKDTLCFS.........DTTLCIKSQIFGQANNI.APFFANQEIDGILGLGFTDLAVH:193
F21F8.2
8:.....................................................................................................................VSGYMGQDLVSN....................................IDGVLGLAPSMNYDR: 35
ASP-5
62:DYFDDFYLGNITLGTP.....PQPATVVLDTGSSNLWVPGPSC.........................................DGSCKGKREYQSTKSSTFKANGKPWQIQYGSGNAKGYLGEDTVAFGAVTE......KQLPVPSTTFG.IATHISSDFKNDAAGGILGLAFTSLAVD:188
F21F8.4
67:DFGDVEYLGNITIGTP.....PQQFIVVLDTGSANLWVPGSNC.........................................DGTCKGKSEFDSSKSTTFKKNGQSWQIQYESGSAKGIMGVDTVAFGGASE......QQLLVPSTTFG.IASHISSNFKNDAAEGILGLAFTSLAVD:193
ASP-6
65:DFGDFEYLGNITIGTP.....DQGFIVVLDTGSSNLWIPGPTC.........................................KTNCKTKSKFDSTASSTFVKNGKSWTIQYGSGDAAGILGQDTVRFGAKGD......SQLSVPTTTFG.IASKISADFKNDATDGILGLAFTSLAVD:191
F21F8.6
66:DFADFEYLGNITIGTP.....DQSFIVVLDT...............................................................EQIVS....RNTN.................LTRYCEATGE......NQLAIPKTTFG.IANQVSADLKNDATDGIFGLAFTTLAVD:149
ASP-2
68:DYEDEAYVGNITIGTP.....QQQFKVILDTGSSNLWIPDITCGTKPENCS.......TVPACRGIICQFECDDQACCGAGANYTDSCFYQNKFDASKSSSYKTNGRNFIIEYGTGSARGFLGQDTITFGGIGE......PQLAVPNTVFG.QATSLAAFFEGQPLDGILGLAFKSIAVD:228
F28A12.4
66:DYVYYEYMGNITVGTP.....DQNFIVVLDTGSANLLIPGTNC.........................................TTYCEKKRLFNEKASSTYIATNRPWQIKYASGDAYGTLGIDTVKIGGSGE......AQLAIPRSYLG.VADTVGSDFKWSPKEGIFGLAFTALAVD:192
C15C8.3
66:DYEDMAYMVQISLGSP.....AQNFVLFIDSGSSNLWVPDITCAGGKDATCGSYCKSTPYDACLTFCQEECCTKTVEGVKVLSTTDACQSKHRFNSSLSSSYVTNGQKFDMTYNTGEVKGFFGVDTFCFTNTS.........VCATGQVFG.QATTIGEAFAKQPEDGIIGLGWPALAVN:230
K10C2.3
82:EQTSTFFVANLTMGTP.....GQLFTVVIDTSTADIVIPDMSCKT.......................................ANNCYNKRRFNQAKSSSYYAYGNKYTYKNNLGTFQGFDAKDTVVIGDRKT......DLITIPGVKFM.QATDLGLLMDGLGADGILGLGFTASSQI:210
consensus
181:**-**-***#******-----**#***************---*--------------------------------------------*--*****************-*-*************-***-***-----------***--*-**-***-**--**--*-*!**!*********:360
Y39B6A.23
Y39B6A.22
Y39B6A.24
ASP-1
ZK384.3
Y39B6A.21
F59D6.3
F59D6.2
hCathepsinD
mCathepsinD
ASP-4
ASP-3
dRE02351p
yPep4p
C11D2.2
F21F8.2
ASP-5
F21F8.4
ASP-6
F21F8.6
ASP-2
F28A12.4
C15C8.3
K10C2.3
consensus
. . . 370 . . . 380 . . . 390 . . . 400 . . . 410 . . . 420 . . . 430 . . . 440 . . . 450 . . . 460 . . . 470 . . . 480 . . . 490 . . . 500 . . . 510 . . . 520 . . . 530 . . . 540
192:QVTPPMQNLISQKQLDAPLFSIWVDRKLQVSQGGTGGLITYGAVDTKNCDAQVNYVALSSKTYWQFPMDGVAIGNYA.MMKQ...EQAISDTGSAWLG.....LPNPVLNAIVQQTKATYDWNYEIYTLDCSTMQTQPDLVFTIGGMQYPVKSIEYILDLGLG...............NG:347
189:QITPPMQNLMP..QLDAPIFTLWLDRVLQGSQGGSGGLITFGAIDTKNCDAQVNYVALSSKTFWQFPLDGVAIGSYT.SMKK...DQAISDSGDFGLR.....LPLAVLNAVIQQTKATYDWTNEFYTLDCSTMQTQPDLVFTIGGMQYPVKSVEYILDLGLG...............NG:342
190:QITPPMQNLIS..QLDVPVFSVWLDRKIQASHGGSAGMITYGGIDTKNCDAGVTYVPLTAKTYWQFKMDGFAVGTYS.QYGY...NQVISDTGSSWIS.....APYAMINDIATQTHATWDEMNEIYTVKCSTMKTQPDLVFTIGGALFPVKSVEYILDIGLD...............EG:343
192:QVVPPMQNLIAQKQLDAPLFTVWLDRNLQIAQGTPGGLITYGAIDTVNCAKQVTYVPLSAKTYWQFPLDAFAVGTYS.ETKK...DQVISDTGTSWLG.....APNTIVSAIVKQTKAVFDWSTELYTVDCSTMKTQPDLIFTIGGAQFPVKSVEYVLDLQLG...............GG:347
185:QVTPPMQNLVSQNQLDAPIFTIFFDKRDPDYYYPSNGLITYGGLDTKNCNANISYVPVSSKTYWQFKVDGFQVGTYNRTVNR...DQAIMDTGSSWFG.....LPYSVIAGIATQTNATWDLYASVYTVPCSTMNSLPDLVFTIGAEKFPISAPEYVEDVGLD...............DG:341
193:KVTPPMQNLLP..QLDAKVFTVWMDRKLQGSNGGNGGLITYGALDSVNCADDISYVPLTAKTYWEFALDGFAVGSFSETKTA...KVMYMLCGICYMLYVICYMLYVICYMLYVICYMLYVICYMLYVI.CYMLYGICYMLYVICYMLYVICYMVYVICYMLY...............VI:351
274:GTSTTMQNLLP..QLDQKLFTTYFTKS.NMHNGTAGGDIMFGAIDTTHCQSQVNYVPLAYNSFWSYSVDGFSIGTYS.RTQT...ETTIPDTSSGWTG.....VPNVVLAGIVKATGATYDWNHQAYTLPCSSTATLPDMVFTIGGNSYNVRAVEYVVNLNLP...............NG:426
183:GTSTTMQNLLP..QLDQPLFTTYFTKS.SVHNGTVGGEITFGAIDTTHCQSQINYVRLAYDSLWSYSIDGFSFGNYS.RNQT...DTAIPDTTSSYTG.....VPNLVLAEIVIATGAQYDWNHQAYTLPCSSTATLPDLVFTIGGNSYNVRAVEYVVNLNLP...............NG:335
210:NVLPVFDNLMQQKLVDQN.IFSFYLSRDP..DAQPGGELMLGGTDSKYYKGSLSYLNVTRKAYWQVHLDQVEVASGLTLCKEG..CEAIVDTGTSLMV.....GPVDEVRELQKAIG.AVPLIQGEYMIPCEKVSTLPAITLKLGGKGYKLSPEDYTLKVSQA..............GKT:364
208:NVLPVFDNLMQQKLVDKN.IFSFYLNRDP..EGQPGGELMLGGTDSKYYHGELSYLNVTRKAYWQVHMDQLEVGNELTLCKGG..CEAIVDTGTSLLV.....GPVEEVKELQKAIG.AVPLIQGEYMIPCEKVSSLPTVYLKLGGKNYELHPDKYILKVSQG..............GKT:362
214:GVQPVFNTLFEQKKVPSN.LFSFWLNRNP..DSEIGGEITFGGIDSRRYVEPITYVPVTRKGYWQFKMDKV.VGSGVLGCSNG..CQAIADTGTSLIA.....GPKAQIEAIQNFIG.AEPLIKGEYMISCDKVPTLPPVSFVIGGQEFSLKGEDYVLKVSQG..............GKT:367
193:KISQPMDQIFANSAICKNQLFAFWLSRDAN.DITNGGEITLCETDPNHYVGNIAWEPLVSEDYWRIKLASV.VIDGTTYTSGP..IDSIVDTGTSLLT.....GPTDVIKKIQHKIG.GIPLFNGEYEVECSKIPSLPNITFNLGGQNFDLQGKDYILQMSNGN.............GGS:349
196:GVTPPFDNMISQGLLDEP.VISFYLKRQG..TAVRGGELILGGIDSSLYRGSLTYVPVSVPAYWQFKVNTI.KTNGTLLCNG...CQAIADTGTSLIA.....VPLAAYRKINRQLG.ATD.NDGEAFVRCGRVSSLPKVNLNIGGTVFTLAPRDYIVKVTQN..............GQT:347
209:KVVPPFYNAIQQDLLDEK.RFAFYLGDTSK.DTENGGEATFGGIDESKFKGDITWLPVRRKAYWEVKFEGIGLGDEYAELES...HGAAIDTGTSLIT.....LPSGLAEMINAEIG.AKKGWTGQYTLDCNTRDNLPDLIFNFNGYNFTIGPYDYTLEVSGS.................:360
194:SVPPPFVNAVEQGLVDEP.IFTVYLEHHGIKKEASGGYFTYGGEDPDHCGEIITWIPLTKAAYWQFRMQGIGIDNVNEHKNG...WEVISDTGTSFIG.....GPGKVIQDIANKYGATYDEYSDSYVIPCSEIKNLRSLKMKINDLTFEIDPINLVAHP.................DSS:347
36:VFLSPLLNAIQQGLLDDP.LFTVYLEKHGFTSNVPGGRFTYGAVDIDNCG................PIDGVSIGNYTLTRK....MQVMTDTGTSFIA.....APFYVLNEIIVTLGATYDNTTKSYYVSCNSEK..PNLNIVIGNNIYVVEPNNYVIDVSTEKIQRNITTAPITINQGD:187
189:HVVPPLINAINQGLLDQP.LFTVWLEHKGSANDVGGGVFTYGAVDTTNCGPVIAYQPLSSATYYQFVADSFKRGSYSYS..KK..YQVISDTGTSFLG.....GPKAVVAGLASALGATYHSNDDSYYVPCATNIG..TLDITIGGKVYSINPVNYIVDVGMGD................:340
194:GVTPPLINAINKKILDQP.LFTVFLEHKGTAKNVGGGVFTYGAVDTTNCGPVIAYQPLSSATYYQFVADGFKLGSYSNN..KK..SQVISDTGSSLIG.....GPSAVINGFAQALGAKYHSDDDAYYLPCSTSKG..TLDITIGGNVYSIEPVNYILDVGMGD................:345
192:GVVPPLINAINQGILDQP.LFSVWLEHRGAANNVGGGVFTYGAIDTTNCGALVAYQPLSSATYYQFKAAGFKLGSYSNT..KT..VDVISDTGTSFLG.....GPQSVVDGLAKAAGATYDDFNEVYFIDCAAQPG..TLDITIGTNTYSIQPVNYIVDAGNG.................:342
150:GVVPPLINAIKQGILDQP.LFSVFLEHRGTLTNVGGGVFTYGAVDTTNCEPVIGYQPLSSATYFQFKANRFRLGKYLNA..KV..VDVISDTGTSFLG.....GPSVVINEIAKQAGATYYILGEAYVIDCDATPG..PLEVTIGGHDYQIEHYNYIADVGDG.................:300
229:QITPPFINAINQNLVDLP.LFTVFLEHEGDQNGVQGGVYTYGGIDTKNCGPVIAYQPLSSATYYQFKMSAIGSGSYHSS..KG..WQVISDTGTSLIG.....GPKAYVSAIADAVGATWRDDYGVYILPCSAKIN..TLDITIGNQVYNIDSSNTIIPLGDGSN...............:381
193:NITPPIINAINQGLLDQP.LFTTWFGQRGAPGTSASGAFTYGGLDKNHCGPVIGYAELTNARHFQFQATGFSLGSYVST..TT..YEVITDTATSFLC.....GPQAAIDNLAKAAGATWDPTNQVFNIPCDADAG..PIKMKIGQFNYVIRANNYILKIDTN.................:343
231:QQTPPLFNLMNQGKLDQP.YFVVYLANIGPTSQINGGAFTVGGLDTTHCSSNVDWVPLSTQTFWQFKLGGVSSGSYSQAPNSG..WQAAADTAASFIG.....APKSVVTSLAKAVGATYVPLTGAFFMDCDAVVP..DIVFTINGKTYNMPSTSFVVSAGPG.................:383
211:GGNSPFVQGVNAGDISGTFYSIWLEHFNQTDDLGTHGVIYYGGFDPVHCAPNPTYVPLASAYAYQLTMSSFKVVGSSATNSNNKYIQTYLDTTTAQIG.....LPKTYISQVFDSLGISTNVMNAIYPTIVPCNTKITLTFGFVSGTTVSITERDLVISFFGT.................:368
361:******-*****-***-*-********-*---*--*!*******!*-****-*******************-*****---------************-----**--**-***********-----*-******-***-*******--*-*--**********-----------------:540
7
Supplementary Information (Nektarios Tavernarakis, Nature T05081A TH/mah)
. . . 550 . . . 560 . . . 570 . . . 580 . . . 590 . . . 600 . . . 610 . . . 620 . . . 630 . . . 640 . . . 650 . . . 660 . . . 670 . . . 680 . . . 690 . . . 700 . . . 710 . . . 720
Y39B6A.23
348:RCVLAMLSYSNTGFG.PSYVLGHVFIRQFCNVYDIGNARIGFANAHHSF...................................................................................................................................:395
Y39B6A.22
343:RCVLAMFTDSSTGFG.PSFGFGNVFLRQYCNIFDVGNARIGFAIAHHSF...................................................................................................................................:390
Y39B6A.24
344:KCALAISPLMASGFG.PSWILGDVFIRQYCNIYDIGNARIGFANAHHSF...................................................................................................................................:391
ASP-1
348:KCALAVFSMGSGGFG.PSWILGDTFIRQYCNVYDIGNGQIGFATAVHKGL..................................................................................................................................:396
ZK384.3
342:TCALAMYGIDASGFG.PSVTLGNIFIRRYCSVFDVGNARIGFADAIHPNTLTYYQNMLSKLICIKANDGASFQLDMKLLLFLVTTCSAALFQASINCKRFNQQANRYFNFDDSCIGNITIGTPPQSASVFMDTTSANWWVIGSKCTSANCNGYSGIRKHKFNTTKSTSFVEGNRTFSTEY:520
Y39B6A.21
352:CYMLYVICYMLYVICYMLYVICYMLYVICYMLYVISDTGTSWIGAPHSIIS...........AVVKATGAKFEWTTELYVVPCSTMKPNLT.........................................................................................:431
F59D6.3
427:QCALSLFGTAASQSG.PAWILGDNFLRSYCHVFDFGNSRIGLAKAIQNY...................................................................................................................................:474
F59D6.2
336:QCALSLFGTFSSPSG.PLWVFGDNFLRSYCHIFDFGNSRIGLAKAIQNY...................................................................................................................................:383
hCathepsinD 365:LCLSGFMGMDIPPPSGPLWILGDVFIGRYYTVFDRDNNRVGFAEAARL....................................................................................................................................:412
mCathepsinD 363:ICLSGFMGMDIPPPSGPLWILGDVFIGSYYTVFDRDNNRVGFANAVVL....................................................................................................................................:410
ASP-4
368:ICLSGFMGIDLPERVGELWILGDVFIGRYYSVFDFDQNRVGFAQAKTADGRPVDPAPRPFRSVFDNESEESMEQDDE.......................................................................................................:444
ASP-3
350:TCLSGFMGMDIPAPAGPLWILGDVFIGRFYSVFDHGNKRVGFATSRTGK...................................................................................................................................:398
dRE02351p
348:YCMSAFTYMEGLS....FWILGDVFIGKFYTVFDKGNERIGFARVADY....................................................................................................................................:391
yPep4p
360:.CISAITPMDFPEPVGPLAIVGDAFLRKYYSIYDLGNNAVGLAKAI......................................................................................................................................:405
C11D2.2
348:ECDLTLFDMYGGG.FGPSWILGDPFIRQFCNIHDIKNKRIGLAHSKQQ....................................................................................................................................:394
F21F8.2
188:KCLLAISGLSGYG.MGPTWILGTLFMRQFCHVHDIGNQQIGLAKSLQK....................................................................................................................................:234
ASP-5
341:TCVFAAFAFNNFGFG.PAWILGDPFIRQFCNIYDIGGQRMGFANSLQAN...................................................................................................................................:388
F21F8.4
346:TCVFAAFSFDFGGFG.PSWIFGDPFIRQYCNIYDIGQQRIGFAKSLQPSSK.................................................................................................................................:395
ASP-6
343:QCLFAAFPFDFGGFG.PSWILGDPFIRQYCNIYDIGNKRMGFAPSLQK....................................................................................................................................:389
F21F8.6
301:TCVMALFPMDFGGFG.PSWILGDPFIRQYCNIYDIGNFRMGFAPSLQPAPK.................................................................................................................................:350
ASP-2
382:NCIYAIFPFSSGGFG.PSWILGDPFIRQYCNIYDVGTQRVGFAKSLQSK...................................................................................................................................:429
F28A12.4
344:SCLFAAIPQNYAGFG.PSWILGGPFMRQYCNVHDIGQKRVGFCLSLA.....................................................................................................................................:389
C15C8.3
384:PCMFAFYELTAGGFY.PAWMLGPPFMRAYCHVHDMKSGRLGLAKVL......................................................................................................................................:428
K10C2.3
368:.CRLQIIPT......TDRIILGLPLYRGRCTYFDPIMQRVGFTPALLQD...................................................................................................................................:410
consensus
541:-******-**--***-*********************-*****-**--------------------------------------------------------------------------------------------------------------------------------------:720
. . . 730 . . . 740 . . . 750 . . . 760 . . . 770 . . . 780 . . . 790 . . . 800 . . . 810 . . . 820 . . . 830 . . . .
Y39B6A.23
:......................................................................................................................:
Y39B6A.22
:......................................................................................................................:
Y39B6A.24
:......................................................................................................................:
ASP-1
:......................................................................................................................:
ZK384.3
521:GLCTGYLGTDTVQMGGLTITKQELGIATIVGLGFGLKPYVGIFELAWPALSVDQVTPPMQKLISQNQLDAPMFTIWLDQKDQGVYVGYTGLITYGGFDNKNCDANVTYVALSSKTFWQ:638
Y39B6A.21
:......................................................................................................................:
F59D6.3
:......................................................................................................................:
F59D6.2
:......................................................................................................................:
hCathepsinD
:......................................................................................................................:
mCathepsinD
:......................................................................................................................:
ASP-4
:......................................................................................................................:
ASP-3
:......................................................................................................................:
dRE02351p
:......................................................................................................................:
yPep4p
:......................................................................................................................:
C11D2.2
:......................................................................................................................:
F21F8.2
:......................................................................................................................:
ASP-5
:......................................................................................................................:
F21F8.4
:......................................................................................................................:
ASP-6
:......................................................................................................................:
F21F8.6
:......................................................................................................................:
ASP-2
:......................................................................................................................:
F28A12.4
:......................................................................................................................:
C15C8.3
:......................................................................................................................:
K10C2.3
:......................................................................................................................:
consensus
721:----------------------------------------------------------------------------------------------------------------------:838
8
Supplementary Information (Nektarios Tavernarakis, Nature T05081A TH/mah)
Figure 6
9
Supplementary Information (Nektarios Tavernarakis, Nature T05081A TH/mah)
Figure 7
a
mec-4(d)
cad-1(j1);mec-4(d)
unc-52(su250);mec-4(d)
daf-4(e1364);mec-4(d)
10
Supplementary Information (Nektarios Tavernarakis, Nature T05081A TH/mah)
Figure 8
hm-Calpain
mCalpain1
hCalpain1
dCalpain
CLP-3
CLP-6
CLP-7
CLP-4
CLP-1
T11A5.6
W05G11.4
F44F1.3
F44F1.1
F47F6.5
W04A4.4
Y53H1B.6
H25P06.4
dSol
TRA-3
CLP-2
M04F3.4
T21H3.3
consensus
. . . .10 . . . .20 . . . .30 . . . .40 . . . .50 . . . .60 . . . .70 . . . .80 . . . .90 . . . 100 . . . 110 . . . 120 . . . 130 . . . 140 . . . 150 . . . 160 . . . 170 . . . 180
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
1:MGTISSVLQWSCTKCNTINPTESLKCFNCGTVRKVFPQQQQQQHRSSSITASWTADDALEQEQAEKGQERDKEKGRAAVARSEYKHVYKSLLRGCLKRPQRNSQNLPANCVDCEDTRKYIKSSIELYRHFSNPALNRRWVCHACGTDNSSVTWHCLICDTVSYLAPIYKDAIAADRGQDL: 180
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
1:------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: 180
hm-Calpain
mCalpain1
hCalpain1
dCalpain
CLP-3
CLP-6
CLP-7
CLP-4
CLP-1
T11A5.6
W05G11.4
F44F1.3
F44F1.1
F47F6.5
W04A4.4
Y53H1B.6
H25P06.4
dSol
TRA-3
CLP-2
M04F3.4
T21H3.3
consensus
. . . 190 . . . 200 . . . 210 . . . 220 . . . 230 . . . 240 . . . 250 . . . 260 . . . 270 . . . 280 . . . 290 . . . 300 . . . 310 . . . 320 . . . 330 . . . 340 . . . 350 . . . 360
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
1:................................MFACSKQQHFQIDRLEERSVSTIFAIEENFYSGSCYKQSKKQKMLLAFFIALLIPRAHSTTLTFSQFQELHSKTADMTSMKVCWMLGGDILPVDANIPEKGHKCTATVDRWSSNAKDAKEACASRIPYHIDSAQYGTKTQCTFLINLA: 148
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
181:AGSLGNRGELLAADHSHPHHHHHYLHQELEEQHQHQLHSQHLHKRHLKGRSASGSGSGPGSGSGLRRTQSLSTAIDKSASGRSCHICYANNQSKDIFNLPQIKPAPQLTGIPPVAACSNSRFAIANDTFCRRKQNNNNKNQNHKVVRESGAKRKYNFTITTLSRSAAKDAGHGQMKPLRQ: 360
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
181:------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: 360
hm-Calpain
mCalpain1
hCalpain1
dCalpain
CLP-3
CLP-6
CLP-7
CLP-4
CLP-1
T11A5.6
W05G11.4
F44F1.3
F44F1.1
F47F6.5
W04A4.4
Y53H1B.6
H25P06.4
dSol
TRA-3
CLP-2
M04F3.4
T21H3.3
consensus
. . . 370 . . . 380 . . . 390 . . . 400 . . . 410 . . . 420 . . . 430 . . . 440 . . . 450 . . . 460 . . . 470 . . . 480 . . . 490 . . . 500 . . . 510 . . . 520 . . . 530 . . . 540
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
149:CAEDHWQIHGKCYRLHNGKYTWAQAKAICQTGVGDDVKSQVAIFYSEALSIYLNDMENIGAAWVNVPNLNDYFTNPNNDHGGVYIQGAAYKYDTRQGSIMMFKAESKHQVICEYTPPTTMAEMFHLAKVYAQIYPIGVYSHGAVFPTSSYVRITQTDIIAYNGKFTDKYGNEVEKFDASG: 328
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
361:VVNLNLNLQQEPQQKSPANPQQLHRKTQREPAAVSMNPTQFTIPRNGVFIAVNEWSEPMASSSSVSSSSNHHHHHHSNSNSNSSGNSNIINNNSSSSSGSNKLYENECVALAQQQLRAAAAQAAQAAATAVAIASSPSAKAMAEPAPTATMPIYAQVNKQHKLKKKQQIASESQTNNNTG: 540
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
361:------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: 540
11
Supplementary Information (Nektarios Tavernarakis, Nature T05081A TH/mah)
hm-Calpain
mCalpain1
hCalpain1
dCalpain
CLP-3
CLP-6
CLP-7
CLP-4
CLP-1
T11A5.6
W05G11.4
F44F1.3
F44F1.1
F47F6.5
W04A4.4
Y53H1B.6
H25P06.4
dSol
TRA-3
CLP-2
M04F3.4
T21H3.3
consensus
. . . 550 . . . 560 . . . 570 . . . 580 . . . 590 . . . 600 . . . 610 . . . 620 . . . 630 . . . 640 . . . 650 . . . 660 . . . 670 . . . 680 . . . 690 . . . 700 . . . 710 . . . 720
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
1:.........................................................................................................................MSDEEEYYEEDGGNDQGGEDYNDDYGGGGGGGGGGYDDQNQFQEQDYGNEQNYGNQDYG: 59
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
329:LEKKCASIGNILNVKSYPVSGHADDFNAMKSLLKGHQCYLTSSYKNDGFKKTDYRAKSGNNSLEGGIFEGASSDEYCNAHSLSMSTTERLPTMAATGACALCAMHQIHYEYGPCPDKPDWIDEVYRFERPSRVFCHYINNHEVETRADAVVRCESFGASLSGVDSDPEFQGLAQKLSPKY: 508
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
541:SGEIADAVSESLTAGLGTSTDGSGEASESESQVEEHSIYAKVWKGPRKATESKIMHDPGSSSRLSGAASAAAGTASAGAIAAGVG.....AAAASRHDNKTQLGNGSRSKMWICIKCSYAYNRLWLQTCEMCEAKAEQQQQQLHLQQQQQQQQQHHHHHHHHLQQQQAEAPRDEPWTCKK: 715
:....................................................................................................................................................................................:
1:.................................................................................................................................................................................MDE:
3
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
541:------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: 720
hm-Calpain
mCalpain1
hCalpain1
dCalpain
CLP-3
CLP-6
CLP-7
CLP-4
CLP-1
T11A5.6
W05G11.4
F44F1.3
F44F1.1
F47F6.5
W04A4.4
Y53H1B.6
H25P06.4
dSol
TRA-3
CLP-2
M04F3.4
T21H3.3
consensus
. . . 730 . . . 740 . . . 750 . . . 760 . . . 770 . . . 780 . . . 790 . . . 800 . . . 810 . . . 820 . . . 830 . . . 840 . . . 850 . . . 860 . . . 870 . . . 880 . . . 890 . . . 900
1:............................................................................................................................................................................MAGIAAKL:
1:..................................................................................................................................................................MTEELITPVYCTGVSAQV:
1:...............................................................................................................................................................................MVAHI:
1:....................................................................................................................................................................MGAAKDVVGSVINEIF:
1:.................................................................MHFIGFFLVFENF...............IDFLLN......FSEFVGDDCGDVAGKLFDQLSSMIPRDSVG...RGLTNIFSS...RELESLAGDLISKNSHDFVAVNSETGGING:
1:.................................................................MSDEEVLNNEDEQ...............EENINNNNLPSIVKEFVSDYTGSIWDELLRQLTALIPRDSSG...RGLTNIFSS...GALESLVGNLISNASHEFFGVNSENGEIIG:
60:QGYGNQQDYGNQQDYQQQNYGGGGYDDEYQQQDYQGGYDDSEQYEEQEEAEPEPEEQEDYSNDVEHSEEEEEEDDDDDGYTQAAANTNYGENDGGNNENHGMGSLIKGAMDGFGGGGFDGVVGQIGSLISQGGGGDSSGGLTNVFSS...GGMESIVGNLISSATHQFFGINPATGAIIG:
1:..MANHAKLVDQEIYRSP..........................................VDDFELNYQEYLNDDDDD...KQEAPVAVSKAPKGKGSNHGLGCRMKGLFDGFGGTDLKTIMRHLRRRRPKSGQ.....ALTNFMLS...GNMEKVVDQHVKDFVHHFGRINPENGRIMG:
1:MAKDLIGGIFDNVVNRKG...........................................KKEQDNYGGGGNYGGGGGNQGGGGGGGFNFNDIGGLINSMGGGGGGGQRQGGGGGGFGDILGGIGSLIGGGGGGQYNGGGGNVNPNNLNGGMVNVIGNLIGEAAHRFLGVDPGTGRIIG:
1:....................................................................MEMTAWNCLYCTLINDPAIFICQGCGSQKPLTKKPAKLTGSFPKIITDTVRDVSNAISDLVNNRDRSPAHNFVRATHFRTPLPNGPFVDGQSNDPRDTHQLSMCSKITTTLK:
1:.........................................................................................................MHSSFYLVFANLSPEHDRAIAQFKCRKN...AYKFPKNTIFLKSIYS.........NKKDT....MFLQITPSLK:
1:...................................................................................................................................................MNSPQEEELLELIEKYNEDKYCTNMNWTIFLGD:
1:.......................................................................................................................................MCSLDDVVFSDREDENDNDEEIEKEEKEEKEEEYEELG.MLVIEN:
509:PPGNTVDITPHSRTVYFGDANKGAIRIDDHYWLGGISPCETDCSGGPTGVREASWDAGVAVNTTFLNNYNHDGHPWNLLPVTQYVSFRNDFNAFHIHPLDHYYTKNGSIVHYEKMFFVCGKSAHLTQSTTIKSALQQTSPAFCYFTVLLLYLFEQKISAFSCMIELFYLTKVYQQMFLIG:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
716:CTLVNYSTAMACVVCGGSKLKSISSIEDMTLRKGEFWTCSHCTLKNSLHSPVCSACKSHRQPQLSMAMEAVRERPDGQSYEEQDAAAVGGGGGSAHQSGANEVKAPTALNLPLTSVALPMPMLQLPTSTAAGLRGSRSPSPRMQLLPSLQQQRNSSSSGAIPKRHSTGGSIVPRNISIAG:
:....................................................................................................................................................................................:
4:KATQSAFELSQKAVYYDQGKKYEEAIFCYTEAGNQLLRLVQEKKCLPIFRKNVMECINRAEFLKSNIQNLQQQYPPTTDDIAHNVEYLMVKAAVFQNYENQVDEARQVYENVVEQCLGASRNRTLPQETLKRLRSTAENALKCIEDLVALKKKVTEVELLFPEVPVDDLSNLNFNNQPSP:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
721:----------------------------------------------------------------------------------------------------------------------------*---------------------------------------------*------*--:
hm-Calpain
mCalpain1
hCalpain1
dCalpain
CLP-3
CLP-6
CLP-7
CLP-4
CLP-1
T11A5.6
W05G11.4
F44F1.3
F44F1.1
F47F6.5
W04A4.4
Y53H1B.6
H25P06.4
dSol
TRA-3
CLP-2
M04F3.4
T21H3.3
consensus
. . . 910 . . . 920 . . . 930 . . . 940 . . . 950 . . . 960 . . . 970 . . . 980 . . . 990 . . .1000 . . .1010 . . .1020 . . .1030 . . .1040 . . .1050 . . .1060 . . .1070 . . .1080
9:AKDREAAEGLGSHERAIKYLN.....................................................................................QDYEALRNECLEAGTLFQDPSFPAIPSALGFKELGPYSSKTRGMRWKRPTEICAD.....PQFIIGGATRTDIC: 98
19:QKKRDKELGLGRHENAIKYLG.....................................................................................QDYETLRARCLQSGVLFQDEAFPPVSHSLGFKELGPHSSKTYGIKWKRPTELMSN.....PQFIVDGATRTDIC: 108
6:NNSRLKAKGVGQHDNAQNFGN.....................................................................................QSFEELRAACLRKGELFEDPLFPAEPSSLGFKDLGPNSKNVQNISWQRPKDIINN.....PLFIMDGISPTDIC: 95
17:IKKEADTKRVLPSIKNMRVLGEKSSSLGPYSEV.........................................................................QDYETILNSCLASGSLFEDPLFPASNESLQFSRR.PD....RHIEWLRPHEIAEN.....PQFFVEGYSRFDVQ: 113
89:ALAGNEMFNMGGETNSLSSIGKIVLDN......IVSGKF..KRECHVYVPLS........CQP...........................................IYFHEERQKCLDEKRLFEDPQFPANNSSIYVKVRPKD.....HIVWKRPGELFAN.....PRLIGSDKTHFDVK: 199
95:ALAGNAMFNLGGENNSLSSIGKVVLDN......IVSGKF..KRECHSYVPLK........CQP...........................................LYFYEERQKCLDENRLFEDPQFPANNSSIYYKKPPKS.....HIVWLRPAKIFAN.....PRLIVNGKSRFDVR: 205
237:AIAGNIIFQMGGQNNSLSSIGKVVLDN......IISGKF..KRDVQPFTPG.......GGGGPGIGFQQQFQT.................................INFQQERQRCLDQRCLFEDPQFPANDSSLYYKTRPDE.....PIIWKRPGEIYEN.....PQLIVGEKSRFDVK: 358
126:ALRGNDFFNFGGLHNNLGNTGKVYLDN......LMKGKCGKKRKVHKFKPIVVEELDFGAPKPAVPATPKAPVAPVAPVIPAPAVTPQKPKVDEP.TYADSVSDFGLDFETEREKCLRNKTLFEDPEFPATAASLYYRTPPRD.....RIIWKRPGEIIAN.....PQLITQGESRFDVK: 288
138:AVAGNVIMGLGGKDNSLGNIGKVILDN......IISGKF..RRDVDPFVRPGPDPDRGGGGSGPSPISPRPTTEP...............................QDFYELRDQCLESKRLFEDPQFLANDSSLFFSKRPPK.....RVEWLRPGEITRE.....PQLITEGHSRFDVI: 268
113:QEAEKKSDVIYSNIISYCKSSNSPFID...............................................................................DSFLHNKKSIGSLQFTKNGVKR.HEEPDFLNWLRPSQMFTKDGRSSPWSVLNNPR.....PSD........IEQ: 199
60:QAQDN.SDVIYDEIIYFSTLFNSPFID...............................................................................PCFRHNLKSIGTFPSTTNGVQCNNSIIDSLHWLRPSQIRTKDGTSCAWSVFNNPQ.....PSD........IEQ: 146
34:HENEKEANEKFKSILEDCQRSKSAFID...............................................................................KEFPHEPKSYMTFDEQSKVYGKYESWKTWLFPKIWRRITPPSTDVSSSLSVYNPL.....TFT......AYEVF: 123
45:HSNKKESNEKFKQIMEYCEKTKSTFVD...............................................................................KEFPHEPN........................................LSVYDPL.....TFT......AYEVF: 94
689:AYTHEGVLGNWHFVTVLFSGSLSARHT...............................................................................KSPAKSHKLGSVFVDEEFPPNDTSLGDLPIKKDYPIKWAAPQEFVSVPPVRHETD.....SWTVWRDPRPFQVF: 784
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
896:LANYNLQQGQGVGSASVVSASGAGSGAGAVGASTSSKKWQCPACTYDNCAASVVCDICSSPRGLASAVLGEALGRKSVRVALTPADIRQESKLMENLRQLEETEALTKWQNIIQYCRDNSELFVDDSFPPAPKSLYYNPASGAGEGNPVVQWRRPHEINCDGGAYPPWAVFRTPLPSDIC:1075
1:MTRSEKTRHFGN..............................................................................................QNYEKLRKICIKKKQPFVDTLFPPTNQSLFLEQRQSS.....DIVWKRPGELHPD.....PHLFVEGASPNDVT: 76
184:STPSNQPFKSPTRNFPPSPSNSQKFTKEELAVLFTTS.....................................................................NINNKLYVPFHNYDKVNEFKGQQGKFTDPEGKISLTQKQRMKLKCWKRVSELFEN......PTIIFSIDCHTIK: 288
:....................................................................................................................................................................................:
:....................................................................................................................................................................................:
901:---------**-----*-------------------------------------------------------------------------------------------*---*--**----**-**-***---**-**-----------*-*-**-**--*-----*--*----*-***-:1080
12
8
18
5
16
88
94
236
125
137
112
59
33
44
688
895
183
900
Supplementary Information (Nektarios Tavernarakis, Nature T05081A TH/mah)
. . .1090 . . .1100 . . .1110 . . .1120 . . .1130 . . .1140 . . .1150 . . .1160 . . .1170 . . .1180 . . .1190 . . .1200 . . .1210 . . .1220 . . .1230 . . .1240 . . .1250 . . .1260
hm-Calpain
99:QGALGDCWLLAAIASLTLNEEILARVVPLN....QSFQEN....YAGIFHFQFWQYGEWVEVVVDDRLPTKDGELLFVHSAEGS.EFWSALLEKAYAKINGCYEALSGGATTEGF....EDFTGGIAEWYELKKPPP.NLFKIIQKALQ......KGSLLGCSIDITSA.ADSEAITFQK: 257
mCalpain1
109:QGALGDCWLLAAIASLTLNETILHRVVPYG....QSFQDG....YAGIFHFQLWQFGEWVDVVIDDLLPTKDGKLVFVHSAQGN.EFWSALLEKAYAKVNGSYEALSGGCTSEAF....EDFTGGVTEWYDLQKAPS.DLYQIILKALE......RGSLLGCSINISDI.RDLEAITFKN: 267
hCalpain1
96:QGILGDCWLLAAIGSLTTCPKLLYRVVPRG....QSFKKN....YAGIFHFQIWQFGQWVNVVVDDRLPTKNDKLVFVHSTERS.EFWSALLEKAYAKLSGSYEALSGGSTMEGL....EDFTGGVAQSFQLQRPPQ.NLLRLLRKAVE......RSSLMGCSIEVTSD.SELESMTDKM: 254
dCalpain
114:QGELGDCWLLAATANLTQESNLFFRVIPAE....QSFEEN....YAGIFHFRFWQYGKWVDVIIDDRLPTYNGELMYMHSTEKN.EFWSALLEKAYAKLHGSYEALKGGSTCEAM....EDFTGGVSEWYDLKEAPG.NLFTILQKAAE......RNSMMGCSIEPDP..NVTEAETPQG: 271
CLP-3
200:LGRLENRWFLSGAANLSLRDELFYRVVPPD....QSFTEK....YAGIFHFQFWRYGEWVDVVIDDRLPTVNGTLCYMSSPGGS.EFWGPLLEKAYAKLLGTYEHLNDPYTEQALWQASASFTGGLTQWFDIFMADEMAVLAMIMRGVQ......MGSLIVC.LNIDV..NEKGVRQKNG: 361
CLP-6
206:QGSLGNCWFLAAAATLAQRDELFYRVVPPD....QSFTEN....YAGIFHFQFWRYGEWVDVVIDDRLPTVNGKLIYMSSQDGD.ECWGPLLEKAYAKLYGTYEHLDGGTTTEAL....EDFTGGLTEFYDLTSTDKTMILAMIMKGMQ......MGSLFACSIDPDP..REKEAQLANG: 364
CLP-7
359:QGALGDCWLLAAVANLTLRDELFYRVVPPD....QSFTEN....YAGIFHFQFWRYGQWVDVVIDDRLPTVDGRLCYMRSQENN.EFWSALLEKAYAKLYGGYENLDGGSTAEAL....EDFTGGLTEFFDISKGDKSTTLAMLVRGMQ......MGSLFGCSIDADE..NVKEAQLTNG: 517
CLP-4
289:QGALGDCWFLAALANITLYDALFYRIVPPN....QSFTEN....YAGIFHFQFWHYGKWVDVVVDDRLPTVNNQLYYLHSADNT.EFWSALVEKAYAKLHGGYENLDGGTTAEAL....EDFTGGLTEYFDLRKSEKAAVLAALVKGME......MGSLFGCSIDADA..NIKEAQLRNG: 447
CLP-1
269:QGELGDCWLLAAAANLTLKDELFYRVVPPD....QSFTEN....YAGIFHFQFWQYGKWVDVVIDDRLPTSNGELLYMHSASNN.EFWSALLEKAYAKLFGSYEALKGGTTSEAL....EDMTGGLTEFIDLKNPPR.NLMQMMMRGFE......MGSLFGCSIEADP..NVWEAKMSNG: 426
T11A5.6
200:GTLVGDCWLMSAMALIAERPDVLDDIIPK.....KQYSH......YGVYQIKLCVEGKWEVIIVDDFFPCYSKTNSIAMAVGRRNQLWVPLIEKAMAKVLGSYSKLHGASLAQGLSMLTGASCVNYNCPPIPSSVDDVDTFWAQLVSSK.....ESGFLMCCHCGAFENVVAEAEFKAMG: 363
W05G11.4
147:GNLVGDCWLLSAMALIAEKPEVLDAIIPK.....KQYSK......HGVYQVKVCVEGKWEVIIVDDFFPCYRNTNSIAIAVGRRNQLWVPLIEKAMAKVLGSYSKLHGASLARGLSMLTGASCVRYKCPQTLKIDEEVDTFWAQLVSSQ.....ESGFLTCCHCGPFESEEAEAEFKALG: 310
F44F1.3
124:QRKVGDCGLVAALSAISTKPEVIMNIFDS.....LQLSK......YGVYKVKLFVDGQWKTIIIDDYFPYTTDGIR.IGATSGY.QIWAALIEKALVKECGNYKGIHGFQSLSAFSALTGSPVLLI...PVAKMFNNLRKYWKTLMEFR..............NNQYPMACGTLNREVNG: 273
F44F1.1
95:QRKVGDCGLVAALSAISTKPEVIMNIFDS.....LQLSK......YGVYKVKLFVDGQWKTIIIDDYFPYTTDDMR.IGATSGK.QIWAALIEKALAKERGNYKGITGFQNQSAFSALTGSPVLSI...LTTRILKELRKHWKILMDYKYEFLKILIGVVIFQKNQYPMACETTIQRRNG: 258
F47F6.5
785:QLGVGDCWLIAALQTIARRKTLLDQIVPKR....WFTMK......HGIAQIRLLINGEWKVIKIDFHVPSSSASYEIFTPMVRK.QAWAALIQKAFAKLGGSYAKLHGGFADIAFLQLTGSFTSTY...YLNKLSSD.NDIWDFILSMQ....KSKFLVTACSTYCEEGSEEWDIFLKNQ: 945
W04A4.4
1:.....................................MS......HGIVQIRLLINGEWKVLKIDFHLPQKSNSFERYAYMVKK.QIWVAFIEKGFAKIRKSYEKLSGGVAGIALQQLTGAMTFSV...FMEKFNNDENRVWEFIQENR....NSKFILTVSTPTIEEESEKKQLLEEYG: 129
Y53H1B.6
1:.....................................................................MNKQSIEEYAWMIGK.QTWAAFIEKGFAKLFGSYKSLSAKPIDVAFRKLTGAFSKNY...ELRSFKNC.DAVWDMIVEAH....LAGFLLCTSKLSLDEESAEY.IFNSTG: 101
H25P06.4
1:.....................................................................MNKQSIEEYAWMIGK.QTWAAFIEKGFAKLFGSYKSLSAKPIDVAFRKLTGAFSKNY...ELRSFKNC.DAVWDMIVEAH....LAGFLLCTSKLSLDEESAEY.IFNSTG: 101
dSol
1076:QGVLGNCWLLSALAVLAEREDLVKEVLVTK....EICGQ.......GAYQVRLCKDGKWTTVLVDDLLPCDKRGHLVYSQAKRK.QLWVPLIEKAVAKIHGCYEALVSGRAIEGLATLTGAPCESIPLQASSLPMPSEDELDKDLIWAQLLSSRCVRFLMGASCGGGNMKVDEEEYQQKG:1243
TRA-3
77:QGILGNCWFVSACSALTHNFKLLAQVIPDADDQEWSTKHA....YAGIFRFRFWRFGKWVEVVIDDLLPTRDGKLLFARSKTPN.EFWSALLEKAFAKLYGCYENLVGGHLSDALQDVSGGVAETLHVRKFLKDDPNDTELKLFNDLKT...AFDKGALVVAAIAARTK.EEIEESLDCG: 247
CLP-2
289:QTVISDCSFISSLSIAALYEKRFKKQLVTSIIFPQDANGKPIYNPAGKYMFKFHLNGAWRKVLIDDYFPVDENNRMMCSQTENKGELWVSLLEKAYMKVMGGYDFPGSNSNIDLN.....ALTGWIPERIELSDTSKADPDEVFRKLFD......RFHRGDCLITLATGKMTEDMQKRSG: 457
M04F3.4
1:................................................MAFQQPNLQQIFSSVDKDRSGQISSDELQTALSNGT...WNPFNPETCRLMIGMFDSNGDG......................AINFQEFQALWRYINDWT...............................: 76
T21H3.3
1:...........................................MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLG....QNPTEAELQDMINEVDADGNG.....................TIDFPEFLTMMARKMKDTD.....................SEEEIREAFR: 91
consensus 1081:#*-***#*******-**-*****-****------****------**********-**-************--*-****-*--**-***-**********-******-****-***--***-*****---****------********---------********--*----*-*----**:1260
. . .1270 . . .1280 . . .1290 . . .1300 . . .1310 . . .1320 . . .1330 . . .1340 . . .1350 . . .1360 . . .1370 . . .1380 . . .1390 . . .1400 . . .1410 . . .1420 . . .1430 . . .1440
hm-Calpain 258:LVKGHAYSVTGAEEVE..............SNGSLQKLIRIRNPWG.EVEWTGRWND.NCPSWNTIDPEERERLTRRHE.........DGEFWMSFSDFLRHYSRLEICNLTPDTLTSDT..............YKKWKLTKMDGNWRRG.STAGGCRNYPNTFWMNPQYLIKLEEED..: 395
mCalpain1
268:LVRGHAYSVTGAKQVT..............YQGQRVNLIRMRNPWG.EVEWKGPWSD.SSYEWNKVDPYEREQLRVKME.........DGEFWMSFRDFIREFTKLEICNLTPDALKSRT..............LRNWNTTFYEGTWRRG.STAGGCRNYPATFWVNPQFKIRLEEVDDA: 407
hCalpain1
255:LVRGHAYSVTGLQDVH..............YRGKMETLIRVRNPWG.RIEWNGAWSD.SAREWEEVASDIQMQLLHKTE.........DGEFWMSYQDFLNNFTLLEICNLTPDTLSGDY..............KSYWHTTFYEGSWRRG.SSAGGCRNHPGTFWTNPQFKISLPEGDDP: 394
dCalpain
272:LIRGHAYSITKVCLIDIVTP..........NRQGKIPMIRMRNPWGNEAEWNGPWSD.SSPEWRYIPEEQKAEIGLTFDR........DGEFWMSFQDFLNHFDRVEICNLSPDSLTEDQQHS..........GKRKWEMSMYEGEWTPG.VTAGGCRNFLDTFWHNPQYIITLVDPD..: 419
CLP-3
362:LIKGYGYCITGVNLME..............TEWEKAPLIRIRSPWG.KVKWKGDFCY.RSHRWFGLDKEKRE..SFRIKE........DGEFWMSLKDFMVEFTDVYCCNLSADTMHEVEKMTEVKVMEH...QSQQWIQASFEGEWSSRIGTAGGCDDHD.TFCTNLQYEIHFRATD.S: 510
CLP-6
365:LVRGHAYSVTGVHTVE..............TDKEKVALLRIRNPWG.DTEWNGDWSD.KSSLWEQVDQEQREKMEFRIKE........DGEFWMCLDDFMAQFANLDCCNLSADVMHEITEMTEIEVMEK...QSKQWIQKSADGEWCSRKGTAGGCSNNENTFCTNPQYETYFRAT..S: 515
CLP-7
518:LVRGHAYSITAIQTVN..............TYGGQVPLLRIRNPWGNSKEWNGAWSD.GSSEWSQVDPQQREQMGVQFAK........DGEFWMSFDDFMTNFTQMECCNLSADVMDEISEMTGVEVHDK...QKHQWVEKSEDGEWNSRQGTAGGCQNN.DTYCNNPQYGTYFQVPMDS: 670
CLP-4
448:L................................................EWNGAWSD.GSSEWSKIDEATKKQIDVQFAR........DGEFWMSFEDFFSNFTQMEVCNLTAEIFDEIAEMTGVNRATETVEEEHQWHEIMEDGEWSSKKGTAGGCNNNPSTYPKNPQFSTFFTAPQSS: 570
CLP-1
427:LVKGHAYSITGCRIVD..............GPNGQTCILRIRNPWGNEQEWNGPWSD.NSREWRSVPDSVKQDMGLKFDH........DGEFWMSFDDFMRNFEKMEICNLGPDVMDEVYQMTGVKAAGM......VWAANTHDGAWVRN.QTAGGCRNYINTFANNPQFRVQLTDS...: 573
T11A5.6
364:LLTNHAYSILDVIYEQ..................GYRLLRIRNPWG.QFVWNGKWSD.GWPGWPIGMKQKFLNQRKDETG..........AFWMDLEDFVARFASVTVCKLRMD.......................WSELRVTHK..VGGHSDTALQIVITDT.CEVSVTAFQKGAFNK: 487
W05G11.4
311:LVTSHAYSILDVVHEQ..................GHRLLRIRNPWG.QFVWNGKWSD.GWLEWPIDMKRALLDKRSDETG..........AFWMDLQDFVKRFASVTVCKLRMD.......................WSELRATQE..VGRHADSALQIVITEQLCEVSVTAFQKGSFSK: 435
F44F1.3
274:ILPQHAYTIMDIVERD..................GHKLLLLRNPSG.GSVWTRNWSK.EWEWWPENMKDLLEGMIRG.............SFWISWDDFLNVFCSIYVCRHRSN.......................WFAYYAKLVLKYPEDDAFPAIDIKVTEKCMVCISAVDDWISRE: 397
F44F1.1
259:ILSKHVYTIMDIVERD..................GHKLLLLRNPSG.GSVWTGNWSK.EWEWWPENMKDLLEGMIRG.............SFWISWDDFLTMFTFIHVCRYRSN.......................WFAYYAKVFLKYPRGYTFPVIKINVPEKCTICISAVDDWISR.: 381
F47F6.5
946:ISPNHGHGYTEMGSFT..................EIRWQVP....I.GKNILINYDS.EKCLCGNIFDYVLSTTS...............IHWVDLHILSRFFSCFEICHYREG.......................WKEIRFRKT.LVPKNGKTEILKLFLKTRCELVIEVIRRKEALR:1062
W04A4.4
130:IRDCHEYSVLDAQVYM..................GHRLILL....A.GS...GPFEI.REDWCA..VDLGFSEFG...............TFWIDMSELFQYFEYVTVCQYREK.......................WKEIRIRRN.VVANTKNTE........................: 217
Y53H1B.6
102:IKQNHAYAILNSVVFE..................NHRLVQI....G.NTHACDKWKE.LHKEWN.SLHYFYNKFSSKLPRRPYISHLDDMSFWMDIDQYCEHFSSLTVCEYRKD.......................WKEIRFRQTNDLKQSRETEALRMTVDRRCELVVEVTDRVLAKI: 233
H25P06.4
102:IKQNHAYAILNSVVFE..................NHRLVQI....G.NTHACDKWKE.LHKEWN.SLHYFYNKFSSKLPRRPYISHLDDMSFWMDIDQYCEHFSSLTVCEYRKD.......................WKEIRFRQTNDLKQSRETEALRMTVDRRCELVVEVTDRVLAKI: 233
dSol
1244:LRPRHAYSVLDVKDIQ..................GHRLLKLRNPWG.HYSWRGDWSD.DSSLWTDDLRDALMPHGASEG...........VFWISFEDVLNYFDCIDICKVRSG.......................WNEVRLQGTLQPLCSISCVLLTVLEPTEAEFTLFQEGQRNSEK:1369
TRA-3
248:LVKGHAYAVSAVCTIDVTNPNERSFTSFIMGSKRKQNLIRLQNPWG.EKEWNGAWSD.DSPEWQNVSASQLSTMGVQPAN....SDSDDGDFWMPWESFVHYFTDISLCQLFNTSVFSFSRSYDEQIVFS........EWTTNGKKSGAPDDRAGGCHNFKATFCNNPQYIFDIPSPN..: 411
CLP-2
458:LVETHAYAVIDIRCVET..................KRLLKVKNPWT.HSRWKGNFSDKDKVNWTAKMKNALAFDPEVAAE......KDDGIFWIDYESVRHFFDVIYVNWNAD..........................LFPFKSVYHATWTQDTGPIRDVYTVGENPQYTLTVNLNQK.: 585
M04F3.4
76:.NCFRGFDTDGSGNID...............................KSELTNALTQFGYRLSPQFYNILMHKFDRSHSN............RINFDDFIQLCVVLQTLTAAFR........................LEIRAKIDLEI...............................: 157
T21H3.3
92:VFDKDGNGFISAAELR....................................HVMTNLGEKLTDEEVDEMIREADIDGDG............QVNYEEFVTMMTTK..........................................................................: 149
consensus 1261:****#*******--**--------------------******#*#*---****-***--*-**--****-*--*--------------************--**-*******-*-*---------------------*-***-**-*-----*****-*---**--****-*--------:1440
. . .1450 . . .1460 . . .1470 . . .1480 . . .1490 . . .1500 . . .1510 . . .1520 . . .1530 . . .1540 . . .1550 . . .1560 . . .1570 . . .1580 . . .1590 . . .1600 . . .1610 . . .1620
hm-Calpain 396:EDEEDG.................ESGCTFLVGLIQKHRRRQRKMGEDMHTIGFGIYEVP.EELSGQTNIHLSKNFFLTNRARERSDTFINLREVLNRFKLPPGEYILVPSTFEPNKDGDFCIRVFSEKKADYQAVDDEI....EANL.EEFDISEDDID...DGVRRLFAQLAGEDAEIS: 549
mCalpain1
408:DDYDNR.................ESGCSFLLALMQKHRRRERRFGRDMETIGFAVYQVP.RELAGQP.VHLKRDFFLANASRAQSEHFINLREVSNRIRLPPGEYIVVPSTFEPNKEGDFLLRFFSEKKAGTQELDDQI....QANLPDEKVLSEEEID...DNFKTLFSKLAGDDMEIS: 561
hCalpain1
395:EDDAEGN................VVVCTCLVALMQKNWRHARQQGAQLQTIGFVLYAVP.KEFQNIQDVHLKKEFFTKYQDHGFSEIFTNSREVSSQLRLPPGEYIIIPSTFEPHRDADFLLRVFTEKHSESWELDEVN....YAEQLQEEKVSEDDMD...QDFLHLFKIVAGEGKEIG: 550
dCalpain
420:EEDEEG...................QCTVIVALMQKNRRSKRNMGMECLTIGFAIYSLNDRELENRP...QGLNFFRYKSSVGRSPHFINTREVCARFKLPPGHYLIVPSTFDPNEEGEFIIRVFSETQNNMEENDDHVGYGGKADTITPGFPTPKPIDPQKEGLRRLFDSIAGKDMEVD: 577
CLP-3
511:YDSNHK....................CTIIAALFQKNRDHCVYKGLELFLIGLLVYEMP...GPN...EKVTPEMVKSQTPIADSKLFKDSREANIRFTVPLGHYVIVPSTYDPDQDGEFLLRIFTNVDFDKT...............................................: 617
CLP-6
516:PSSNDK....................CTVIAAVFQKYRRNQLHRGLDMLQIGLSVYKMS...GPN...EKVTAEMMKTHAPIASTKLFVDYREAVVRFTVPPGYYVIVPCTFEPNHDAEFLLRTFSNVEFDKTDMHFIWKSAVFLCALLAINFPLTKSSIDLHSHEKVQISINNTSGPRK: 669
CLP-7
671:VEHDGK....................CTVIGAVLQKYRRELRTKGLDNLPIGFSVYKAD...GSGQAIHDVSGQ.....KPIARTKVFINMREVTVRFRVPPGQYVIVPCTFDAHDDASFLLRIFSNAEFQTTRLR............................................: 778
CLP-4
571:IEADGN....................VTVIVAVLQKYRRELRSKGKDVLPIGVSIYSLG...AEGTARSPLTAQFFSQNRPIARTTVFVNTREVTVRFRVPPGQYVIVPCTFDAYDDAEFLLRVYANGTLKSSLL.............................................: 682
CLP-1
573:.DPDDD....................DELCTAGNNRGR................................LSKQFFAANKSAMRSAAFINLREMTGRFRVPPGNYVVVPSTFEPNEEAEFMLRVYTNGFIESEEL.............................................: 655
T11A5.6
487:..........................KDNLNDLMVCVHRISGDRRIGELIEMSSRISDNHFTIDEFFLAPGEYQIVCHSQSSLLTNKKGVVNMVVHTRYPIFGEYIPMSPRTRMESLHHVIIKEGDIVKNTNDGVVIRT.........................................: 600
W05G11.4
435:..........................EDNLTDLMVCVHKISEDGRIGELIKMSYEIADNHFTIDEFFLPRGVYQVVCHSPHSLVTNTTGVVNIIVHTRFPIFGESVPMSPLTRLESLHRVIIEEGVVQNIKLDGVVIRT.........................................: 548
F44F1.3
398:VLLKTVEFQMPKYVQRYSWIAVHKIN.GIFSDNKKVVACEIINDSTQDIDLEPGIYTITVIYLNDWSLDKRDISIHSSRPISVNEGFSRTRKDSVHIKYIVD....KFGQEIVKEQKDKMSIKKYTDNNFTFIAVVAWN.........................................: 531
F44F1.1
381:..............VKSSWITVHMDDKNYESDIKKVVACEIIDDSTGDIDLEPGSYVITVNKLNNSPIGKRDISIHSSRPISVNEGFLVTSEDGEHIKYVVD....KFGQEIVKEPKDKMSIKKYTDNNSTFIAVVAWN.........................................: 502
F47F6.5
1062:...........................LEAEQLRNRCILFCIVYSATADNKCDKILMITHAYDLNLELTPGTLDPGTYLISFV....ILEDYDELELDWVIR....R.........................................................................:1134
W04A4.4
:....................................................................................................................................................................................:
Y53H1B.6
233:...........................WVTRNRDTEVSFVKQDVK.......................................................................................................................................: 251
H25P06.4
233:...........................WVTRNRDTETTCSTNQDKIGLISAVEKNWGSNPDKPNQKTLKRSTISDNSCEPVAK....KPEFDFKTFMQELER....YLACDDEVTPPYEFWTDPLNKSACPRLAKFAAQ.........................................: 337
dSol
1370:SQRSQLD...................LCVVIFRTRSPAAPEIGRLVEHSKRQVRGFVGCHKMLERDIYLLVCLAFNHWHTGIEDPHQYPQCILAIHSSKRLLVEQISPSPHLLADAIISLTLTKGQRHEGREGMTAYYLT........................................:1490
TRA-3
411:..........................CSVMFALIQNDPSEGLKKREPFVTIGMHVMKVE.........NNRQYRVHTAMHPIAISDYASGRSVYLHLQSLPRGRYLLIPTTFAPKEQTLFMLRVYSDEHIHFSPLTKHAP........................................: 516
CLP-2
585:..........................AAAVWILLTRHITAIDDFAVNKEFITLIVYETG.........QKIYIPSNPQPISDGVRINSPLYLCQLSNKKPGITKYTLVVAQYEKTNTINYSLRVFSTTDVKLEPVQLPYS........................................: 690
M04F3.4
:....................................................................................................................................................................................:
T21H3.3
:....................................................................................................................................................................................:
consensus 1441:---------------------------***-*****-**-----**--*-**--**-*-----------**-----------*-*--**--****---***-*-*****-***---**********--------*---------------------------------------------:1620
13
Supplementary Information (Nektarios Tavernarakis, Nature T05081A TH/mah)
. . .1630 . . .1640 . . .1650 . . .1660 . . .1670 . . .1680 . . .1690 . . .1700 . . .1710 . . .1720 . . .1730 . . .1740 . . .1750 . . .1760 . . .1770 . . .1780 . . .1790 . . .1800
hm-Calpain 550:AFELQTILRR..........VLAKRQD.............................IK.....................................SDGFSIETCKIMVDMLDSDGSGKLGLKEFYILWTKIQKYQKIYREIDVDRSGTMNSYEMRKALEEAGFKMPCQLHQVIVARFADD: 653
mCalpain1
562:VKELQTILNR..........IISKHKD.............................LR.....................................TNGFSLESCRSMVNLMDRDGNGKLGLVEFNILWNRIRNYLTIFRKFDLDKSGSMSAYEMRMAIEAAGFKLNKKLHELIITRYSEP: 665
hCalpain1
551:VYELQRLLNR..........MAIKFKS.............................FK.....................................TKGFGLDACRCMINLMDKDGSGKLGLLEFKILWKKLKKWMDIFRECDQDHSGTLNSYEMRLVIEKAGIKLNNKVMQVLVARYADD: 654
dCalpain
578:WMELKRILDHSMRDDLPKPVVFNRFSNNMAFETQAAGPGDDGAGACGLLSLICGPFLKGTPFEEQLGMNDQSNKRLIGDNPADGGPVTANAIVDETHGFSKDVCRSMVAMLDADKSGKLGFEEFETLLSEIAKWKAIFKVYDVENTGRVSGFQLREALNSAGYHLNNRVLNVLGHRYGSR: 757
CLP-3
617:................................................................................................................HALSNG...........................IGLICKSDG....................HLLTVP: 638
CLP-6
670:QVRIRFPEFI...................................................................................................ISGHIFCNGAPKRWLHPQLSTPKNPDEYFNIVTTDAAGMYFLTTGNHFDLDRSVIITVLHQCEMKHLHPEP: 750
CLP-7
:....................................................................................................................................................................................:
CLP-4
:....................................................................................................................................................................................:
CLP-1
:....................................................................................................................................................................................:
T11A5.6
600:..............................................................................................................LTRKFRGMIIMADNCLEKKYLHVGVDCSQSMNIQSSRGFLQVVDVVPPLCRQVLLVLSTIDDSAQYRVSN: 670
W05G11.4
548:..............................................................................................................LNQKFRGSITMVDNYMEQKYLHVKVDNSKSMNVQSSRGSLLIADVVPPRSRQVIAVLSTIDDCAEYKTAN: 618
F44F1.3
531:..............................................................................................................FTYDQFLHAHLRYSITDEQFVSRSLEDKQTVDVL........VVINLEAMPEIVDFPIDIDYKLSKDVDA: 593
F44F1.1
502:..............................................................................................................FAYDQFLHVHLRYSITDEQLVSRSLEDNQTVDVIPPRGVQILVVIEQYAMPEIVDFPIDIDYKLSKDVDS: 572
F47F6.5
:....................................................................................................................................................................................:
W04A4.4
:....................................................................................................................................................................................:
Y53H1B.6
:....................................................................................................................................................................................:
H25P06.4
337:..............................................................................................................FFICPPGSAEVERFFSGAGQILSKYRKSLSPERFN...MLCFLSKNIPLMNKRYRKRVNTEKKEHDAKKK: 404
dSol
1490:................................................................................................................KGWAGLVVMVENRHENKWIHVKCDCQESYNVVSTRGELKTVDSVPPLQRQVIIVLTQLEGSGGFSIAH:1558
TRA-3
516:.................................................................................................KLGLLKCKSAQSVTRLTIHGVDFNSASTGTHNVYAILKDSRKSFRTKTLSGVKSIQWDEQFLFHKSKNRQQYKIEVWEDRKMA: 599
CLP-2
690:.............................................................................................................ISKTTRGNWDGSDKYPIMKLTLHSKSDEIALFMELKAPKQFCVALEMKQNSSDRTVFLETKSSGAYRPGYT: 761
M04F3.4
:....................................................................................................................................................................................:
T21H3.3
:....................................................................................................................................................................................:
consensus 1621:--------------------------------------------------------------------------------------------------------------------*---*---------*----*--------------------------------------------:1800
. . .1810 . . .1820 . . .1830 . . .1840 . . . .
hm-Calpain 654:QLIIDFDNFVRCLVRLETLFKIFKQLDPENTGTIELDLISWLCFSVL..: 700
mCalpain1
666:DLAVDFDNFVCCLVRLETMFRFFKLLDTDLDGVVTFDLFKWLQLTMFA.: 713
hCalpain1
655:DLIIDFDSFISCFLRLKTMFTFFLTMDPKNTGHICLSLEQWLQMTMWG.: 702
dCalpain
758:DGKIAFDDFIMCAVKIKTYIDIFKERDTEKNETATFTLEEWIERTIYS.: 805
CLP-3
:.................................................:
CLP-6
751:CALPYYKTVIPFNSTSSSTTYIRRDLELSKMEAFSSTHCL.........: 790
CLP-7
:.................................................:
CLP-4
:.................................................:
CLP-1
:.................................................:
T11A5.6
671:SLKTLVHRSKCLLPEMWYEAAISAPNAQHYPLLNTSSFDPIHSTVSVF.: 718
W05G11.4
619:SYWLRTNKSTSLLPDW.................................: 634
F44F1.3
594:TINGIKRGCHLPEISSSRFLEYVHREEIISLDDFIPEIEVLSKLN....: 638
F44F1.1
573:TINGIKRGCHLPEISSSRFLEQVHREERISVADCFLDLPSNYCQN....: 617
F47F6.5
:.................................................:
W04A4.4
:.................................................:
Y53H1B.6
:.................................................:
H25P06.4
405:GEDGYEDAEGNSDSDDDFLFG............................: 425
dSol
1559:RLTHRLANSRGLHDWGPPGATHCPPIENVHGLHAPRLIT..........:1597
TRA-3
600:RDHLLAQSVIIALIDNENRDTTLQLTDPRGTVIGTVSVTVSAFDDPMYL: 648
CLP-2
762:VLTLEKVPAGKYYVKISTYTAGDKGPFILRIDSTCKFDLEPIKL.....: 805
M04F3.4
:.................................................:
T21H3.3
:.................................................:
consensus 1801:--------------------------*----------------------:1849
14
Supplementary Information (Nektarios Tavernarakis, Nature T05081A TH/mah)
Figure 9
15
Supplementary Information (Nektarios Tavernarakis, Nature T05081A TH/mah)
Figure 10
a b
intestine
intestine
head sensory neurons
eggs
vulva
pharynx
anal sphincter muscle
ventral nerve cord motorneurons
intestine
c
anus
tail ganglion
neurons
canal cell processes
2-fold embryo
intestine
neuronal processes
pharynx
nerve ring
ventral nerve cord
commissures
16