Figure Legends for Supplementary Materials: Fig. 1. Nucleotide sequence of the zebrafish lumican (zlum) gene. Exons are indicated by uppercase letters, and introns are indicated by lowercase letters. The amino acid sequence encoded by the exon is given as single uppercase letters beneath the nucleotide sequence. The translation initiation codon ATG and termination codon TAA are indicated in bold. The boxes indicate leucine-rich regions. The polyadenylation signal AATAAA is indicated by an asterisk. Numbers shown beyond the uppercase letters are related to the transcription initiation site T (+1). Figure. 1 tccattaattcgacagaccagagttaaataatccttgtatgccattgtgatcacatctacagttcagattttgtatttca ataaatttgtcaggcttttgtcctcagactgtgaatgtatgcaggaccaggttcttacaaatctcatcaagccttttgtt gacatttgattatatttttgaatgttgtattttgtgaaatcatttggcatagttatgtctgtaatcaaagtccctttgag gcaagtcactttactcaacggccatttttaaaacacctctcgggcagagaatttaatgtttccctctgaatgggaaaaca ttaaattctccaaaactgcttgccaagcttaccactgaattccatattaggtatcaccaataaagttaaacaacaactgt gtctttagtttcatttctaaacatccaaatcacacaaaatcggcagaaactctcgtctggtctgagcacctcccccggag tattgtctgtctatagcaatcgaagattggcttatgtacaagtaggcggagctttattcaccatattgaccattacactt cactattttgcctggaactactttagctgtgcatttatctgaaaaatgtctgcacactttataatgttcacccaccattc agaatcaagcattcaaaaccctgcaatatttttttatcctactccaaagtttatcagtttaattatctttaatggccaag taattgtcaatgaccagacaggattgattagtttagattataatccttattttatatcttcctaattctttacagctttt acagtagtggcccaagaaatgcacaacctaggctctgatacacataaggcttacagaaccaatctatcatctggctctct ggctttgctgcattcctaagactccgcatgtgctattaaagataaactgacctgcaatcagccttgtctttcctggatcc ctcatgagttgtggcccacctcaatgtgctgtgagcttcccatcacagaggaccatgaccacagccagacagaagccgtc tggagtttatggggcttaagattaaaagatggcacactgcttgatgatatacaatctaccagctgttaacgattggatac actctccgttcagatgccgaaggtagacaacacggttatgtaaggctggtttggcccaatacccttttatttggtcagtt tttccaagtcccggttgtctaatagcaggcagatgccattgcaaccagccaatttattttattttccaaatagtttgaaa atagtttcattatcctttgttttgctaggattattacttttacggttgaggtgtttgtaggcttatttgtgtctcacata ataggtgaactgttttgtgggattgtttggaatggatgacatggaaatggaaaagtttgtgtgtgtgtgtgtgtgtgtgg cagcaggtatatatattttgcaaaacctccccagtgagtctttgctcccttctgcatcccacgaaggactacatgtccca agctgcattgctactactcaccattgttaagatccatatgtgccacaactgctgacatctacatactagaaccgcctgac ttctgttttcaatcttgggcgactaaggtctggcttgagtgtctctctctctctctttccctctctctctctatctcttt +1 ctctccactctccctcccttactctttctctccagagtcctcgggccgcacacaactgtacctgaagagagagaagcagg AAGgtaaggatatttagcatctgctgttatgcattgaatgttcatgctagcttttgctttctctggatgagattatcaga aatctgcttggcagattttgggggaggactaaagaaacattatgatcatattgcggaaattcagcgtgatgtttgggaag cttggagttctatctaagctgtttgtggtcaaatataaaggctgtcatttagaattaaatcaaatgatttataggttttt ttaaatgcatgaaattgtacaatggaacaatcaatgcaattttttgcaggaagaaagaaagataaagcagtgcatgcatt tgactagtgattttgcatcaatggattgctataaagaaaacctgggttgcgagattccatgcttgacacctgctgcaaat
tattctatgccgtcataaattattaatggattacccaacttcataaatgtctttcttttccaattccatcttaataatta atcagcatgtcatgcacaaaaaagggaactctaaagtaaagtcaatgtaacttgagatgaacttggatccgaacaccagc aatgtgggtctaatttgtgaagggaggaatgtctgtctgggcctattgcattcttgagaatgaagtcatcaatttccatt agaagagtgtctctggtttttgtttatgtctgaggttcaccgtagaaagttctgttattcttacacattttagttttttt ttaatgtaatattgctccttatttaaccacttctccctttctttcctgttctcttctcctctctactgatctcttgtcta +46 ggtttaatttaagttgtgcagccatgtttgctctgggatccattctgttggctggacttctaagtctgagtttagctcag M F A L G S I L L A G L L S L S L A Q TATGATTACTATGATGAGTACTACATACCCTCCGCACCACTGGAGGGCGTTTCATCACCTTCTTGTGCCCAGGAGTGCGA Y D Y Y D E Y Y I P S A P L E G V S S P S C A Q E C E ATGTCCCATCAACTTTCCCACTGCCATGTACTGCAATGAGCGCAACTTGAAGTTCATCCCCATCGTGCCGACCGGAATCA C P I N F P T A M Y C N E R N L K F I P I V P T G I K AATACCTCTACTTACAGAACAACTTCATTGAGGAGATCAAGGCTGGAGTGTTTGACAATGCCACCGACCTTCGCTGGCTC Y L Y L Q N N F I E E I K A G V F D N A T D L R W L GTCCTCGACAACAACAACATCACCAGTGACAAGATCCAGGCTGGTACTATCGATAAACTAGGAAGCTTGGAGAAGCTCCT V L D N N N I T S D K I Q A G T I D K L G S L E K L L CTTCAGTCATAACAAGCTGACTAAACCCCCTGGTTCTCTTTCGAAGTCTCTGGATGAGTTGAAGCTGATCGGTAACAAGT F S H N K L T K P P G S L S K S L D E L K L I G N K L TAACCTCATTCCCTGCTAACACTTTGGCTGGGATGGAAAACCTGACAACCGTCCACTTGTCCAAGAACAAACTTACCACC T S F P A N T L A G M E N L T T V H L S K N K L T T GAGAGCCTCACAGGAGCCTTCAAGGGCCTCAAGTCCCTCATTCTGCTGGACGTGAGCGAGAACAAGCTCAAGAAACTTCC E S L T G A F K G L K S L I L L D V S E N K L K K L P CTCGGGAGTTCCAGCTTCGCTTTTAATGCTCTACGCTGACAATAATGACATCGACAGCATCCCTAACGGCTACCTGGCAA S G V P A S L L M L Y A D N N D I D S I P N G Y L A K AGCTGCCACTCCTACAGTACCTGCGCATCTCCCACAACAAACTTGTGGATTCAGGAGTCCCTGCAGGAGTGTTCAACGTG L P L L Q Y L R I S H N K L V D S G V P A G V F N V TCCTCTCTTCTTGAGCTTGACCTGTCTTTCAACAAGCTGAAGACCATCCCGGAGATCAACGAATCACTAGAACACCTCTA S S L L E L D L S F N K L K T I P E I N E S L E H L Y CCTGCAAGTCAATGAGATAAACAgtaagtttttttagtctccatctggttaagcgaatcagattttgcattgattttgac L Q V N E I N K tccattttgctttctgtttctttagagtttgagctgacgaatatctgcagatttagctcaccggtcaactactctagact F E L T N I C R F S S P V N Y S R L GAGGACCCTGCGCTTGGATGGAAACAACATCACACACAGCAGTATGCCAGATGACACAGCTAACTGCCTACGCCAGGCCT R T L R L D G N N I T H S S M P D D T A N C L R Q A S CAGAGATCATCTTTGAATAGAGCTAAATCTCGCCAGTAGCTCATCAAACAAAATAAGGCTTTAATTCTTATTAAACCAGG E I I F E 2
GCTGCCCAAAACTTTTCCTGGAGGTCCAGTGTCCTGTAGATTTTAGCTGCTGAACGAGCTAATGAAGACCTTCAGACTCA CTAACAACTTCTCAGCATATGTGATGGAGCTAAAATCTGCAAGACCTTGGCCCTTCTGCAGCTAAATTGAACATCTAAAC CTGTTTCCAGAGGCCGTTATCCTGCAGAGTTTAGCTCCTGAACATGCTGATGAATGTCTTCAGACTCACTTGAACCTTCT TAGAAATTGTGTTGTAGCTAAAATTTGCAGGACTTTGGCTCTTCAGGAGCAGGATTGGATGACTAAACCTGTTCCCAGAT GGCCACTATCTTGTAGAGTTTAGCTCGTGGAAACTAATGAAGGTCTTCAGACTCTTTATAACTTCTTAGTGCGTCGGAGC TAAAACCTGCAGGACTTTGCTCTCTATTAGACACTCTGGATGTCTTTATTCGGGATTCTGGCTACCACATTCGGTCACTT GCACATTGTTGACAATATTTGCTGTTTAAAAAGTTTGTATGAAAAACGGGCAGCAATAGTCTGAAAATCTTGAAAATCTG GCACATAAAAGATTAGTCTTGTATTATAATGTATGAAAAGTGGAAATATTTCAGTCTAAAGAGAGACACAAAATGCTGTG CACTCTATAATGAGTTGCACTACATGTCATCTATTTTTTTTTTTGCATGTTAAAATCTTTAATCACTTGAAACTGTAATC ATATTATTAATAAGCCTGCCAGTAAGGATATTCGACTGTTTTCTACAACCTATGGATATGAACTAAATGCACAGCAGTGC ATTTTAATTTACAAATTAAAAAATAAACATTGTACATTGAGTGAATTCTATCTAATCTCAAAGGGGGTTTATTTAAATGA ****** GAAACTGCAGTGGCAAAATTTATTAATTGCAAACTAGTAGCGGCTTGCATTTCTGAACTCTGCCAACGATTATTCCATAT TCTACTGTATCTGCATGCAGCTCATCTTAATTTTGTAACAGTAATTCCTTTCAGTTCTATGGTACACAGCAATGACTTTT TGA Fig. 2. Amino acid sequence comparison of lumican protein from 18 different species. The deduced amino acid sequence is given as single uppercase letters. Comparison of sequence identities between lumican proteins of different species. The complete amino acid sequences of (dog); (horse); (pig); Bos tourus (cow); (mouse); (rat); (chimpanzee); (macaque); (rhesus mulatta); (human); (Sumatran orangutan); Ornithorhynchus anatinus (platypus); Salmo salar (Atlantic salmon); (zebrafish); Silurana tropicalis (xenopus); (Zebra Finch); (Japanese Quail); (chicken) are aligned. Figure. 2. 3
10 20 30 40 50 60 70 80 ---MNLGVFP LLLALIGGAS STYP------ -DYYEY---- ---YDFPQAL Y-------GR SSPNCAPECN CPESYPSAMY ---MNLGVFT LLLALIGGA- ---------S GQY--YD--- ---YDFSLPI Y-------GL SSPNCAPECN CPESYPSAMY ---MTLNSLP IFLVLISGI- ---------- --FCQYDYGP ADDYGY---- -----DPFGP STAVCAPECN CPLSYPTAMY -------MFA LGSILLAGLL SLSLAQY--- -DY--YDEYY ---------- -IPSAPLEGV SSPSCAQECE CPINFPTAMY ---MNPGAFT FLLALISGTS GYY------- --YDDYD--- ---YNIPLSI Y-------GR SSPNCAPECN CPESYPTAMY ---MTLNSLP IFLVLISGI- ---------- --FCQYDYGP ADDYGYD--- ------PFGP STAVCAPECN CPLSYPTAMY ---MNVCAFS LALALVGSV- ---------S GQY--YD--- ---YDIPLFM Y-------GQ ISPNCAPECN CPHSYPTAMY Ornithorhynchus anatinus ---MALENYL QLWAIL---- -------LLY GKY--Y---- ------LTYI LLVPPLPPGP SSPNCAPECN CPYNYPTAMY ---MSLSAFT LFLALIGGT- ---------S GQY--YD--- ---YDFPLSI Y-------GQ SSPNCAPECN CPESYPSAMY ---MILSAFT LFLALIGGT- ---------S GQYYDYD--- -----FPLSI Y-------GQ SSPNCAPECN CPESYPSAMY ---MNVCTFT LVLALVGSV- ---------S GQYYDYDA-- ------PLFM Y-------GE LSPNCAPECN CPHSYPTAMY MFPLRVPIFA VLVSL----- --------TV GQYDDYDYQP ASQY------ --------GP SSANCAQECE CPINFPTAMY ---MHLGVFT LLLATIGSA- ---------S GHYYDDD--- --DYYFPLSI Y-------GR SSPNCAPECN CPESYPTAMY ---MGLNSLA VFLLLIGGSF C--------- --W--SDYGP GDDYGY---- -----DPFGP SAAVCAPECN CPLSYPTAMY ---MHLHFIP FFAVLISGVL CTF------- -----YDYGY DGGYAGPEGD YYVSSLSLGP STANCAPECT CPFNYPSAMY Consensus G CA EC CP P AMY 90 100 110 120 130 140 150 160 CDELKLKSVP MVPPGIKYLY LRNNQIDHID DKAFENVT-D LQWLILDHNL LENSKIKGKV FSKLKQLKKL HINYNNLTES CDELKLKSVP MVPPGIKYLY LRNNQIDHID EKAFENVT-D LQWLILDHNL LENSKIKGRI FSKLKQLKKL HINHNNLTES CDNLKLKTIP IVPSGIKYLY LRNNMIESIE ENTFDNVT-D LQWLILDHNH LENSKIKGRV FSKLKNLKKL HINYNNLTEA CNERNLKFIP IVPTGIKYLY LQNNFIEEIK AGVFDNAT-D LRWLVLDNNN ITSDKIQAGT IDKLGSLEKL LFSHNKLTKP CDELKLKSVP MVPPGIKYLY LRNNQIDHID EKAFENVT-D LQWLILDHNL LENSKIKGRV FSKLEQLEKL HINHNNQTES CDNLKLKTIP IVPSGIKYLY LRNNMIEAIE ENTFDNVT-D LQWLILDHNH LENSKIKGRV FSKLKNLKKL HINYNNLTEA CDELKLKSVP MVPPGIKYLY LRNNQIDHID EKAFENVT-D LQWLILDHNL LENSKIKGRV FSKLKQLKKL HINHNNLTES CDELKLKSVP MVPPGIKYLY LRNNQIDHID EKAFENVT-D LQWLILDHNL LENSKIKGRV FSKLKQLKKL HINYNNLTES CDELKLKSVP MVPPGIKYLY LRNNQIDHID EKAFENVT-D LQWLILDHNL LENSKIKGRV FSKLKQLKKL HINYNNLTES CDDLKLKSVP MVPPGIKYLY LRNNQIDHID EKAFENVT-D LQWLILDHNL LENSKIKGKV FSKLKQLKKL HINYNNLTES Ornithorhynchus anatinus CDELKLKSIP MVPPGIKYLY LRNNMIESIE EKAFENVT-D LQWLILDHNH LENSKIKGKV FSKLKQLKKL HINYNNLTEV CDELKLKSVP MVPPGIKYLY LRNNQIDHID EKAFENVT-D LQWLILDHNL LENSKIKGRV FSKLKQLKKL HINHNNLTES CDELKLKSVP MVPPGIKYLY LRNNQIDHID EKAFENVT-D LQWFILDHNL LENSKIKGRV FSKLKQLKKL HINHNNLTES CDDLKLKSVP MVPPGIKYLY LRNNQIDHID EKAFENVT-D LQWLILDHNL LENSKIKGKV FSKLKQLKKL HINYNNLTES CDSRNLKFVP IVPSGIKYLY LQNNLIEEIK AGVFDNVTAE LRWLVLDHNQ ITNEKVAKGT IDKLTGLHKI LFSFNKLTEA CDELKLKSVP MVPPGIKYLY LRNNQIDHID DKAFENVT-D LEWLILDHNL LENSKIKGRV FSKLKQLKKL HINYNNLTES CDNLRLKTIP IVPSGIKYLY LRNNMIEGIE ENTFDNVT-D LQWLILDHNH LENSKIKSKV FSKLKNLKKL HINYNNLTEA CDNRKLKVIP MVPAGIQYLY LQSNMIEAIE EDAFKNVT-E LKWLILDHNQ LTNGKIKKNA FSSLKHLVKL YISFNNLTEL Consensus C LK P VP GI YLY L N I I F N T L W LD N K L L K N T 170 180 190 200 210 220 230 240 VGP-LPKSLV DLQLTNNKIS KLGS--FDGL VNLTFIHLQH NQLKEDAVSA ALKGLKSLEY LDLSFNQMTK LPSGLPVSLL VGP-LPKSLV DLQLTHNKIQ KLGS--FDGL VNLTFVHLQH NQLKEDTVSA AFKGLKSLEY LDLSYNQMAK LPSGLPASLL VGP-LPKTLD DLQLSHNKIT KVNPGALEGL VNLTVIHLQN NQLKADSISG AFKGLNSLLY LDLSFNQLTK LPTGLPHSLL PG-SLSKSLD ELKLIGNKLT SFPANTLAGM ENLTTVHLSK NKLTTESLTG AFKGLKSLIL LDVSENKLKK LPSGVPASLL VGP-LPKSLV DLQLTHNKIT KLGS--FDGL VNLTMVHLQH NQLKEDAISA AFRGLKSLEY LDLSFNQMSK LPTGLPVSLL VGP-LPKTLD DLQLSHNKIT KVNPGALEGL VNLTVIHLQN NQLKTDSISG AFKGLNSLLY LDLSFNQLTK LPTGLPHSLL VGP-LPKSLE DLQLTHNKIT KLGS--FEGL VNLTFIHLQH NRLKEDAVSA AFKGLKSLEY LDLSFNQIAR LPSGLPVSLL VGP-LPKSLE DLQLTHNKIT KLGS--FEGL VNLTFIHLQH NRLKEDAVSA ALRGLKSLEY LDLSFNQIAK LPSGLPVSLL VGP-LPKSLE DLQLTHNKIT KLGS--FEGL VNLTFIHLQH NRLKEDAVSA ALRGLKSLEY LDLSFNQIAK LPSGLPVSLL VGP-LPKSLQ DLQLTNNKIS KLGS--FDGL VNLTFIYLQH NQLKEDAVSA SLKGLKSLEY LDLSFNQMSK LPAGLPTSLL Ornithorhynchus anatinus VGP-LPKTLQ DLQLAHNKIS KISSNYLDGL VNLTFVNLQR NQLKAEAISG AFKGLKSLEY LDLSFNQLTK LPTGLPASLM VGP-LPKSLE DLQLTHNKIT KLGS--FEGL VNLTFIHLQH NRLKEDAVSA AFKGLKSLEY LDLSFNQIAK LPSGLPVSLL VGP-LPKSLE DLQLTHNKIT KLGS--FEGL VNLTFIHLQH NRLKEDAVSA AFKGLKSLEY LDLSFNQIAK LPSGLPVSLL VGP-LPKSLQ DLQLANNKIS KLGS--FDGL VNLTFIYLQH NQLKEEAVSA SLKGLKSLEY LDLSFNQMSK LPAGLPTSLL VIPP-SKSLD ELKMMNNQLS KFPAGSLAGM QNLTSVHLQN NELTSKALNG VFKGLNKLVA IDLTNNKLKK LPSGMPSSLE VGP-LPKSLV DLQLTNNKIS KLGS--FDGL VNLTFIHLQH NQLKEDAVSA AFRGLKGLEY LDLSFNQMTK LPSGLPVSLL VGP-LPKTLD DLQLSHNKIT KVNPGAFEGL MNLTVIHLQN NQLKADSISG AFKGLNSLLY LDLSFNQLTK LPTGLPHSLL VGP-LPKTMD DLRVTNNKIS KITPNILEGL ENLTHIHLQY NALKEDSISG AFKGLKQLEY LDLSFNELTK LPKGLPPSIT Consensus K L N G NLT L N L GL L D N LP G P S 250 260 270 280 290 300 310 320 TLYLDNNKIS NIPDEYFKRF SALQYLRLSH NELADSGVPG NSFNVSSLLE LDLSYNKLKS IPTVNENLEN YYLEVNELEK TLYLDNNKIS NIPDEYFKRF NGLQYLRLSH NELADSGVPG NSFNVSSLLE LDLSYNKLKN IPTVNENLEN YYLEVNELEK MLYFDNNQIS NIPDEYFQGF KTLQYLRLSH NKLTDSGIPG NVFNITSLVE LDLSFNQLKS IPTVSENLEN FYLQVNKINK MLYADNNDID SIPNGYLAKL PLLQYLRISH NKLVDSGVPA GVFNVSSLLE LDLSFNKLKT IPEINESLEH LYLQVNEINK ALYLDNNKIS NIPDEYFKRF NGLQYLRLSH NELADGGIPG NSFNVSSLLE LDLSYNKLKN IPTVNENLEN YYLEVNELEK MLYFDNNQIS NIPDEYFQGF KTLQYLRLSH NKLTDSGIPG NVFNITSLVE LDLSFNQLKS IPTVSENLEN FYLQVNKINK TLYLDNNKIS NIPDEYFKRF TGLQYLRLSH NELADSGVPG NSFNISSLLE LDLSYNKLKS IPTVNENLEN YYLEVNELEK Ornithorhynchus anatinus MLYFDNNKIS NIPDEYFKNF NVLQHLRFSH NELSDAGIPG NTFNVSSLIE LDLSYNKLKS IPTVNENLEN YYLEVNEIEK TLYLDNNKIS NIPDEYFKRF NALQYLRLSH NELADSGIPG NSFNVSSLVE LDLSYNKLKN IPTVNENLEN YYLEVNQLES TLYLDNNKIT NIPDEYFNRF TGLQYLRLSH NELADSGVPG NSFNISSLLE LDLSYNKLKS IPTVNENLEN YYLEVNKLEK TFYGDHNDIS SIAAGDLKEL PKLAYLRLAY NQMTDAGIPA GVFNVTSLIE LDLSYNKLQS IPEINEQLEH LYLQVNEINK TLYLDNNKIS NIPDEYFKHF SGLQYLRLSH NELADSGIPG NSFNLSSLLE LDLSYNKLKN IPTVNENLEN YYLEVNELEK MLYFDNNQIS NVPDEYFQAF KALQYLRLSH NKLTDSGIPG NVFNITSLVE LDLSFNQLKS IPIVSENLEN FYLQVNKINK TLYFDNNKIA NIPDEFFQGF KALQYLRLSN NKLKDGGVPG NAFNISSLVE LDLSFNELGS IPAVNEGLEN LYLQVNKIQK Consensus Y D N I L LR N D G P FN SL E LDLS N L IP E LE YL VN.............................. 330 340 350 360 370 FDVKSFCKIL GPLSYSKIKH LRLDGNHITQ TSLPPDMYEC LRVANEITVN FEVKSFCKIL GPLSYSKIKH LRLDGNRLTH TSLPPDMYEC LRVANEITVN FPLSSFCKVV GPLTYSKITH LRLDGNNLTR ADLPQEMYNC LRVAAEISLE FELTNICRFS SPVNYSRLRT LRLDGNNITH SSMPDDTANC LRQASEIIFE FEVKSFCKIL GPLSYSKIKH LRLDGNRLTH TSLPPDMYEC LRVANEITVT FPLSSFCKVV GPLTYSKITH LRLDGNNLTR ADLPQEMYNC LRVAADISLE FDIKSFCKIL GPLSYSKIKH LRLDGNRISE TSLPPDMYEC LRVANEVTLN FDVKSFCKIL GPLSYSKIKH LRLDGNPLTQ SSLPPDMYEC LRVANEITVN Ornithorhynchus anatinus FAVSSFCKVL GPLSYSKVKH LRLDGNKIKR SNLPPEMYEC LRVANDIALE K--------- ---------- ---------- ---------- ---------- FDVKSFCKIL GPLSYSKIKH LRLDGNPLTQ SSLPPDMYEC LRVANEITVN FNLENICKFS GPLNYSRLKH LRLDGNNLTH ADLPHDSSNC LRQASDIIFD FEVKSFCKIL GPLSYSKIKH LRLDGNRITQ TSLPPDMYEC LRVANEITVN FPLSSFCKVV GPTSYSRITH LRLDGNNLTR ADLPQEMYNC LRVAAEISLE FNLNSFCKII GPLEYSKIRH LRLDGNNISR VDLPQDMYSC LRVASEIDLG Consensus 4
5