Evolution of teleostean hatching enzyme genes and their paralogous genes

  • Mari Kawaguchi
  • Shigeki Yasumasu
  • Junya Hiroi
  • Kiyoshi Naruse
  • Masayuki Inoue
  • Ichiro Iuchi
Original Article


We isolated genes for hatching enzymes and their paralogs having two cysteine residues at their N-terminal regions in addition to four cysteines conserved in all the astacin family proteases. Genes for such six-cysteine-containing astacin proteases (C6AST) were searched out in the medaka genome database. Five genes for MC6AST1 to 5 were found in addition to embryo-specific hatching enzyme genes. RT-PCR and whole-mount in situ hybridization evidenced that MC6AST1 was expressed in embryos and epidermis of almost all adult tissues examined, while MC6AST2 and 3 were in mesenterium, intestine, and testis. MC6AST4 and 5 were specifically expressed in jaw. In addition, we cloned C6AST cDNA homologs from zebrafish, ayu, and fugu. The MC6AST1 to 5 genes were classified into three groups in the phylogenetic positions, and the expression patterns and hatching enzymes were clearly discriminated from other C6ASTs. Analysis of the exon–intron structures clarified that genes for hatching enzymes MHCE and MAHCE were intron-less, while other MC6AST genes were basically the same as the gene for another hatching enzyme MLCE. In the basal Teleost, the C6AST genes having the ancestral exon–intron structure (nine exon/eight intron structure) first appeared by duplication and chromosomal translocation. Thereafter, maintaining such ancestral exon–intron structure, the LCE gene was newly diversified in Euteleostei, and the MC6AST1 to 5 gene orthologs were duplicated and diversified independently in respective fish lineages. The HCE gene lost all introns in Euteleostei, whereas in the lineage to zebrafish, it was translocated from chromosome to chromosome and lost some of its introns.


Hatching enzyme Astacin protease family Intron-loss evolution Medaka 



medaka high choriolytic enzyme


medaka acidic high choriolytic enzyme


medaka low choriolytic enzyme


medaka six-cysteine-containing astacin family protease


ayu high choriolytic enzyme


ayu low choriolytic enzyme


ayu nephrosin



We express our cordial thanks to Dr. K. Yamagami, former Professor of Developmental Biology, Life Science Institute, Sophia University, Tokyo, for giving us valuable advice and for reading the present manuscript. The present study was supported in part by a Grant-in-aid for Scientific Research (C) from J.S.P.S. to I. I. (No. 17570189) and S. Y. (No. 15570102).

Supplementary material

427_2006_104_MOESM1_ESM.pdf (3.6 mb)
Fig. S1 A multiple alignment of amino acid sequences deduced from C6AST cDNAs of medaka (MHCE, MAHCE, MLCE and MC6AST1 to 5) and ayu (AyHCE, AyLCE1, AyLCE2 and AyNep). White and black triangles indicate the putative signal sequence cleavage sites and the N-terminals of mature enzymes, respectively. Arrows indicate the intron insertion sites for MLCE (intron 1-7), AyLCE1 (intron 1-8), AyLCE2 (intron 2-8), MC6AST1 and MC6AST4 and 5 (intron 1-8), MC6AST2 (intron 1, 3-8) and MC6AST3 (intron 1, 3-7). Identical residues are boxed. Dashes and asterisks represent gaps and stop codons, respectively. Two active site consensus sequences of the astacin family proteases are indicated in dark and light gray boxes, and conserved cysteine residues are in black boxes. Accession numbers: MHCE, M96170; MAHCE, AB256944; MLCE, M96169; MC6AST1, AB256945; MC6AST2, AB256946; MC6AST3, AB256947; MC6AST4, AB256948; MC6AST5, AB256949; AyHCE, AB256940; AyLCE1, AB256941 (3734 kb)


  1. Abascal F, Zardoya R, Posada D (2005) ProtTest: selection of best-fit models of protein evolution. Bioinformatics 21(9):2104–2105PubMedCrossRefGoogle Scholar
  2. Barbazuk WB, Korf I, Kadavi C, Heyen J, Tate S, Wun E, Bedell JA, McPherson JD, Johnson SL (2000) The syntenic relationship of the zebrafish and human genomes. Genome Res 10:1351–1358PubMedCrossRefGoogle Scholar
  3. Barnes K, Ingram J, Kenny AJ (1989) Proteins of the kidney microvillar membrane. Structural and immunochemical properties of rat endopeptidase-2 and its immunohistochemical localization in tissues of rat and mouse. Biochem J 264:335–346PubMedGoogle Scholar
  4. Bode W, Gomis-Ruth FX, Stockler W (1993) Astacins, serralysins, snake venom and matrix metalloproteinases exhibit identical zinc-binding environments (HEXXHXXGXXH and Met-turn) and topologies and should be grouped into a common family, the ‘metzincins’. FEBS Lett 331:134–140PubMedCrossRefGoogle Scholar
  5. Bond JS, Beynon RJ (1995) The astacin family of metalloendopeptidases. Protein Sci 4:1247–1261PubMedCrossRefGoogle Scholar
  6. Butler PE, McKay MJ, Bond JS (1987) Characterization of meprin, a membrane-bound metalloendopeptidase from mouse kidney. Biochem J 241:229–235PubMedGoogle Scholar
  7. Ehrlich J, Sankoff D, Nadeau JH (1997) Synteny conservation and chromosome rearrangements during mammalian evolution. Genetics 147:289–296PubMedGoogle Scholar
  8. Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52(5):696–704PubMedCrossRefGoogle Scholar
  9. Guindon S, Lethiec F, Duroux P, Gascuel O (2005) PHYML Online—a web server for fast maximum likelihood-based phylogenetic inference. Nucleic Acids Res 33(Web Server issue):W557–559PubMedCrossRefGoogle Scholar
  10. Hiroi J, Maruyama K, Kawazu K, Kaneko T, Ohtani-Kaneko R, Yasumasu S (2004) Structure and developmental expression of hatching enzyme genes of the Japanese eel Anguilla Japonica: an aspect of the evolution of fish hatching enzyme gene. Dev Genes Evol 214:176–184PubMedCrossRefGoogle Scholar
  11. Hoegg S, Brinkmann H, Taylor JS, Meyer A (2004) Phylogenetic timing of the fish-specific genome duplication correlates with the diversification of teleost fish. J Mol Evol 59:190–203PubMedCrossRefGoogle Scholar
  12. Hung CH, Huang HR, Huang CJ, Huang FL, Chang GD (1997) Purification and cloning of carp nephrosin, a secreted zinc endopeptidase of the astacin family. J Biol Chem 272:13772–13778PubMedCrossRefGoogle Scholar
  13. Inohaya K, Yasumasu S, Ishimaru M, Ohyama A, Iuchi I, Yamagami K (1995) Temporal and spatial patterns of gene expression for the hatching enzyme in the teleost embryo, Oryzias latipes. Dev Biol 171:374–385PubMedCrossRefGoogle Scholar
  14. Inohaya K, Yasumasu S, Araki K, Naruse K, Yamazaki K, Yasumasu I, Iuchi I, Yamagami K (1997) Species-dependent migration of fish hatching gland cells that express astacin-like proteases in common. Dev Growth Differ 39:191–197PubMedCrossRefGoogle Scholar
  15. Ishiguro NB, Miya M, Nishida M (2003) Basal euteleostean relationships: a mitogenomic perspective on the phylogenetic reality of the “Protacanthopterygii”. Mol Phylogenet Evol 27:476–488PubMedCrossRefGoogle Scholar
  16. Iwamatsu T (1994) Stages of normal development in the medaka Oryzias latipes. Zoolog Sci 11:825–839Google Scholar
  17. Katagiri C, Maeda R, Yamashika C, Mita K, Sargent TD, Yasumasu S (1997) Molecular cloning of Xenopus hatching enzyme and its specific expression in hatching gland cells. Int J Dev Biol 41:19–25PubMedGoogle Scholar
  18. Kawaguchi M, Yasumasu S, Shimizu A, Hiroi J, Yoshizaki N, Nagata K, Tanokura M, Iuchi I (2005a) Purification and gene cloning of Fundulus heteroclitus hatching enzyme. A hatching enzyme system composed of high choriolytic enzyme and low choriolytic enzyme is conserved between two different teleosts, Fundulus heteroclitus and medaka Oryzias latipes. FEBS J 272:4315–4326PubMedCrossRefGoogle Scholar
  19. Kawaguchi M, Yasumasu S, Hiroi J, Iuchi I (2005b) Evolution of hatching enzyme genes in teleost. Zool Sci 22:1394–1395CrossRefGoogle Scholar
  20. Kijimoto T, Watanabe M, Fujimura K, Nakazawa M, Murakami Y, Kuratani S, Kohara Y, Gojobori T, Okada N (2005) cimp1, a novel astacin family metalloproteinase gene from East African cichlids, is differentially expressed between species during growth. Mol Biol Evol 22:1649–1660PubMedCrossRefGoogle Scholar
  21. Kimmel CB, Warga RM, Schilling TF (1990) Origin and organization of the zebrafish fate map. Development 108:581–594PubMedGoogle Scholar
  22. Lee KS, Yasumasu S, Nomura K, Iuchi I (1994) HCE, a constituent of the hatching enzymes of Oryzias latipes embryos, releases unique proline-rich polypeptides from its natural substrate, the hardened chorion. FEBS Lett 339:281–284PubMedCrossRefGoogle Scholar
  23. Lindsay LL, Wallace MA, Hedrick JL (2001) A hatching enzyme substrate in the Xenopus laevis egg envelope is a high molecular weight ZPA homolog. Dev Growth Differ 43:305–313PubMedCrossRefGoogle Scholar
  24. Miya M, Takeshima H, Endo H, Ishiguro NB, Inoue JG, Mukai T, Satoh TP, Yamaguchi M, Kawaguchi A, Mabuchi K, Shirai SM, Nishida M (2003) Major patterns of higher teleostean phylogenies: a new perspective based on 100 complete mitochondrial DNA sequences. Mol Phylogenet Evol 26:121–138PubMedCrossRefGoogle Scholar
  25. Naruse K, Tanaka M, Mita K, Shima A, Postlethwait J, Mitani H (2004) A medaka gene map: the trace of ancestral vertebrate proto-chromosomes revealed by comparative gene mapping. Genome Res 14:820–828PubMedCrossRefGoogle Scholar
  26. Nelson JS (1994) Fishes of the World, 3rd edn. Wiley, New YorkGoogle Scholar
  27. Olivotto I, Yasumasu S, Gioacchini G, Maradonna F, Cionna C, Carnevali O (2004) Cloning and expression of high choriolytic enzyme, a component of the hatching enzyme system, during embryonic development of the marine ornamental fish Chrysiptera prasema. Mar Biol 145:1235–1241CrossRefGoogle Scholar
  28. Padgett RA, Grabowski PJ, Konarska MM, Seiler S, Sharp PA (1986) Splicing of messenger RNA precursors. Ann Rev Biochem 55:1119–1150PubMedCrossRefGoogle Scholar
  29. Piccolo S, Agius E, Lu B, Goodman S, Dale L, De Robertis EM (1997) Cleavage of Chordin by Xolloid metalloprotease suggests a role for proteolytic processing in the regulation of Spemann organizer activity. Cell 91:407–416PubMedCrossRefGoogle Scholar
  30. Postlethwait JH, Woods IG, Ngo-Hazelett P, Yan YL, Kelly PD, Chu F, Huang H, Hill-Force A, Talbot WS (2000) Zebrafish comparative genomics and the origins of vertebrate chromosomes. Genome Res 10:1890–1902PubMedCrossRefGoogle Scholar
  31. Sharp PA (1981) Speculations on RNA splicing. Cell 23:643–646PubMedCrossRefGoogle Scholar
  32. Shibata Y, Iwamatsu T, Oba Y, Kobayashi D, Tanaka M, Nagahama Y, Suzuki N, Yoshikuni M (2000) Identification and cDNA cloning of alveolin, an extracellular metalloproteinase, which induces chorion hardening of medaka (Oryzias latipes) eggs upon fertilization. J Biol Chem 275:8349–8354PubMedCrossRefGoogle Scholar
  33. Shimell MJ, Ferguson EL, Childs SR, O’Connor MB (1991) The Drosophila dorsal–ventral patterning gene tolloid is related to human bone morphogenetic protein 1. Cell 67:469–481PubMedCrossRefGoogle Scholar
  34. Taylor JS, Braasch I, Frickey T, Meyer A, Van de Peer Y (2003) Genome duplication, a trait shared by 22000 species of ray-finned fish. Genome Res 13:382–390PubMedCrossRefGoogle Scholar
  35. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680PubMedGoogle Scholar
  36. Titani K, Torff HJ, Hormel S, Kumar S, Walsh KA, Rodl J, Neurath H, Zwilling R (1987) Amino acid sequence of a unique protease from the crayfish Astacus fluviatilis. Biochemistry 26:222–226PubMedCrossRefGoogle Scholar
  37. Whelan S, Goldman N (2001) A general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach. Mol Biol Evol 18:691–699PubMedGoogle Scholar
  38. Wittbrodt J, Meyer A, Schartl M (1998) More genes in fish? Bioessays 20:511–515CrossRefGoogle Scholar
  39. Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, Hewick RM, Wang EA (1988) Novel regulators of bone formation: molecular clones and activities. Science 242:1528–1534PubMedCrossRefGoogle Scholar
  40. Yamagami K (1972) Isolation of a choriolytic enzyme (hatching enzyme) of the teleost, Oryzias latipes. Dev Biol 29:343–348PubMedCrossRefGoogle Scholar
  41. Yamagami K (1992) Studies on the hatching enzyme and its substrate, egg envelope of Oryzias latipes. Zoological Science 9:1131Google Scholar
  42. Yamagami K (1996) Studies on the hatching enzyme (choriolysin) and its substrate, egg envelope, constructed of the precursors (choriogenins) in Oryzias latipes: a sequel to the information in 1991/1992. Zoological Science 13:331–340PubMedCrossRefGoogle Scholar
  43. Yan L, Pollock GH, Nagase H, Sarras MP Jr (1995) A 25.7×10(3) M(r) hydra metalloproteinase (HMP1), a member of the astacin family, localizes to the extracellular matrix of Hydra vulgaris in a head-specific manner and has a developmental function. Development 121:1591–1602PubMedGoogle Scholar
  44. Yasumasu S, Iuchi I, Yamagami K (1988) Medaka hatching enzyme consists of two kinds of proteases which act cooperatively. Zoological Science 5:191–195Google Scholar
  45. Yasumasu S, Iuchi I, Yamagami K (1989a) Purification and partial characterization of high choriolytic enzyme (HCE), a component of the hatching enzyme of the teleost, Oryzias latipes. J Biochem 105:204–211PubMedGoogle Scholar
  46. Yasumasu S, Iuchi I, Yamagami K (1989b) Isolation and some properties of low choriolytic enzyme (LCE), a component of the hatching enzyme of the teleost, Oryzias latipes. J Biochem 105:212–218PubMedGoogle Scholar
  47. Yasumasu S, Yamada K, Akasaka K, Mitsunaga K, Iuchi I, Shimada H, Yamagami K (1992a) Isolation of cDNAs for LCE and HCE, two constituent proteases of the hatching enzyme of Oryzias latipes, and concurrent expression of their mRNAs during development. Dev Biol 153:250–258PubMedCrossRefGoogle Scholar
  48. Yasumasu S, Katow S, Hamazaki TS, Iuchi I, Yamagami K (1992b) Two constituent proteases of a teleostean hatching enzyme: concurrent syntheses and packaging in the same secretory granules in discrete arrangement. Dev Biol 149:349–356PubMedCrossRefGoogle Scholar
  49. Yasumasu S, Iuchi I, Yamagami K (1994) cDNAs and the genes of HCE and LCE, two constituents of the medaka hatching enzyme. Dev Growth Differ 36:241–250CrossRefGoogle Scholar
  50. Yasumasu S, Shimada H, Inohaya K, Yamazaki K, Iuchi I, Yasumasu I, Yamagami K (1996) Different exon–intron organizations of the genes for two astacin-like proteases, high choriolytic enzyme (choriolysin H) and low choriolytic enzyme (choriolysin L), the constituents of the fish hatching enzyme. Eur J Biochem 237:752–758PubMedCrossRefGoogle Scholar
  51. Yasumasu S, Mao KM, Sultana F, Sakaguchi H, Yoshizaki N (2005) Cloning of a quail homologue of hatching enzyme: its conserved function and additional function in egg envelope digestion. Dev Genes Evol 215:489–498PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Mari Kawaguchi
    • 1
  • Shigeki Yasumasu
    • 1
  • Junya Hiroi
    • 2
  • Kiyoshi Naruse
    • 3
  • Masayuki Inoue
    • 4
  • Ichiro Iuchi
    • 1
  1. 1.Life Science InstituteSophia UniversityTokyoJapan
  2. 2.Department of AnatomySt. Marianna University School of MedicineKawasakiJapan
  3. 3.Department of Biological Sciences, Graduate School of ScienceThe University of TokyoTokyoJapan
  4. 4.Chiba Prefectural Fisheries Research Center Fresh-Water StationChibaJapan

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