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Journal of Comparative Physiology B

, Volume 174, Issue 2, pp 169–180 | Cite as

Putative phenoloxidases in the tunicate Ciona intestinalis and the origin of the arthropod hemocyanin superfamily

  • A. Immesberger
  • T. Burmester
Original Paper

Abstract

In addition to the respiratory copper-containing proteins for which it is named, the arthropod hemocyanin superfamily also includes phenoloxidases and various copperless storage proteins (pseudo-hemocyanins, hexamerins and hexamerin receptors). It had long been assumed that these proteins are restricted to the arthropod phylum. However, in their analysis of the predicted genes in the Ciona intestinalis (Urochordata:Tunicata) genome, Dehal et al. (Science 298:2157–2167) proposed that the sea squirt lacks hemoglobin but uses hemocyanin for oxygen transport. While there are, nevertheless, four hemoglobin genes present in Ciona, we have identified and cloned two cDNA sequences from Ciona that in fact belong to the arthropod hemocyanin superfamily. They encode for proteins of 794 and 775 amino acids, respectively. The amino acids required for oxygen binding and other structural important residues are conserved in these hemocyanin-like proteins. However, phylogenetic analyses and mRNA expression data suggest that the Ciona hemocyanin-like proteins rather act as phenoloxidases, possibly involved in humoral immune response. Nevertheless, the putative Ciona phenoloxidases demonstrate that the hemocyanin superfamily emerged before the Protostomia and Deuterostomia diverged and allow for the first time the unequivocal rooting of the arthropod hemocyanins and related proteins. Phylogenetic analyses using neighbor-joining and Bayesian methods show that the phenoloxidases form the most ancient branch of the arthropod proteins, supporting the idea that respiratory hemocyanins evolved from ancestors with an enzymatic function. The hemocyanins evolved in agreement with the expected phylogeny of the Arthropoda, with the Onychophora diverged first, followed by the Chelicerata and Pancrustacea. The position of the myriapod hemocyanins is not resolved.

Keywords

Hemocyanin Hemoglobin Phenoloxidase Oxygen transport Tyrosinase 

Abbreviations

EST

expressed sequence tags

Notes

Acknowledgements

We wish to thank T. Hankeln and B. Ebner for the Ciona cDNA library, K. Kusche and S. Hagner-Holler for their help with the cloning experiments, and J. Markl for continuous support. We also thank J. R. Harris and T. Hankeln for critical reading of the manuscript. This work is supported by the Deutsche Forschungsgemeinschaft (Bu956/3 and Bu956/5). The nucleotide sequences reported in this paper have been deposited at the GenBank/EMBL databases with the accession numbers AJ547813 and AJ547814.

References

  1. Adams MD, Celniker SE, Holt RA, Evans CA, Gocayne JD, Amanatides PG, Scherer SE, Li PW, Hoskins RA, Galle RF, George RA, Lewis SE, Richards S, Ashburner M, Henderson SN, Sutton GG, Wortman JR, Yandell MD, Zhang Q, Chen LX, Brandon RC, Rogers YH, Blazej RG, Champe M, Pfeiffer BD, Wan KH, Doyle C, Baxter EG, Helt G, Nelson CR, Gabor GL, Abril JF, Agbayani A, An HJ, Andrews-Pfannkoch C, Baldwin D, Ballew RM, Basu A, Baxendale J, Bayraktaroglu L, Beasley EM, Beeson KY, Benos PV, Berman BP, Bhandari D, Bolshakov S, Borkova D, Botchan MR, Bouck J, et al. (2000) The genome sequence of Drosophila melanogaster. Science 287:2185–2195CrossRefPubMedGoogle Scholar
  2. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedGoogle Scholar
  3. Bago B, Zipfel W, Williams RM, Jun J, Arreola R, Lammers PJ, Pfeffer PE, Shachar-Hill Y (2002) Translocation and utilization of fungal storage lipid in the arbuscular mycorrhizal symbiosis. Plant Physiol 128:108–124CrossRefPubMedGoogle Scholar
  4. Beintema JJ, Stam WT, Hazes B, Smidt MP (1994) Evolution of arthropod hemocyanins and insect storage proteins (hexamerins). Mol Biol Evol 11:493–503PubMedGoogle Scholar
  5. Boguski MS, Lowe TM, Tolstoshev CM (1993) dbEST—database for “expressed sequence tags”. Nat Genet 4:332–333PubMedGoogle Scholar
  6. Burmester T (1999a) Evolution and function of the insect hexamerins. Eur J Entomol 96:213–225Google Scholar
  7. Burmester T (1999b) Identification, molecular cloning and phylogenetic analysis of a non-respiratory pseudo-hemocyanin of Homarus americanus. J Biol Chem 274:13217–13222CrossRefPubMedGoogle Scholar
  8. Burmester T (2001) Molecular evolution of the arthropod hemocyanin superfamily. Mol Biol Evol 18:184–195PubMedGoogle Scholar
  9. Burmester T (2002) Origin and evolution of arthropod hemocyanins and related proteins. J Comp Physiol B 172:95–117CrossRefPubMedGoogle Scholar
  10. Burmester T, Scheller K (1996) Common origin of arthropod tyrosinase, arthropod hemocyanin, insect hexamerin and dipteran arylphorin receptor. J Mol Evol 42:713–728Google Scholar
  11. Burmester T, Massey HC, Zakharkin SO, Beneš H (1998) The evolution of hexamerins and the phylogeny of insects. J Mol Evol 47:93–108PubMedGoogle Scholar
  12. Dayhoff MO, Schwartz RM, Orcutt BC (1978) A model of evolutionary change in proteins. In Dayhoff MO (ed) Atlas of protein sequence structure, vol 5, suppl 3. National Biomedical Research Foundation, Washington D.C., pp 345–352Google Scholar
  13. Decker H, Tuczek F (2000) Tyrosinase/catecholoxidase activity of hemocyanins: structural basis and molecular mechanism. Trends Biochem Sci 25:392–397CrossRefPubMedGoogle Scholar
  14. Decker H, Ryan M, Jaenicke E, Terwilliger N (2001) SDS induced phenoloxidase activity of hemocyanins from Limulus polyphemus, Eurypelma californicum and Cancer magister. J Biol Chem 276:17796–17799CrossRefPubMedGoogle Scholar
  15. Dehal P, Satou Y, Campbell RK, Chapman J, Degnan B, De Tomaso A, Davidson B, Di Gregorio A, Gelpke M, Goodstein DM, Harafuji N, Hastings KE, Ho I, Hotta K, Huang W, Kawashima T, Lemaire P, Martinez D, Meinertzhagen IA, Necula S, Nonaka M, Putnam N, Rash S, Saiga H, Satake M, Terry A, Yamada L, Wang HG, Awazu S, Azumi K, Boore J, Branno M, Chin-Bow S, DeSantis R, Doyle S, Francino P, Keys DN, Haga S, Hayashi H, Hino K, Imai KS, Inaba K, Kano S, Kobayashi K, Kobayashi M, Lee BI, Makabe KW, Manohar C, Matassi G, Medina M, Mochizuki Y, Mount S, Morishita T, Miura S, Nakayama A, Nishizaka S, Nomoto H, Ohta F, Oishi K, Rigoutsos I, Sano M, Sasaki A, Sasakura Y, Shoguchi E, Shin-i T, Spagnuolo A, Stainier D, Suzuki MM, Tassy O, Takatori N, Tokuoka M, Yagi K, Yoshizaki F, Wada S, Zhang C, Hyatt PD, Larimer F, Detter C, Doggett N, Glavina T, Hawkins T, Richardson P, Lucas S, Kohara Y, Levine M, Satoh N, Rokhsar DS (2002) The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins. Science 298:2157–2167CrossRefPubMedGoogle Scholar
  16. Durstewitz G, Terwilliger NB (1997) Developmental changes in hemocyanin expression in the Dungeness crab, Cancer magister. J Biol Chem 272:4347–4350CrossRefPubMedGoogle Scholar
  17. Ebner B, Burmester T, Hankeln T (2003) Globin genes are present in Ciona intestinalis. Mol Biol Evol 20:1526–1536CrossRefPubMedGoogle Scholar
  18. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791Google Scholar
  19. Felsenstein J (2001) PHYLIP (Phylogeny Inference Package) version 3.6alpha2. Distributed by the author. Department of Genetics, University of Washington, SeattleGoogle Scholar
  20. Friedrich M, Tautz D (1995) Ribosomal DNA phylogeny of the major extant arthropod classes and the evolution of myriapods. Nature 376:165–167PubMedGoogle Scholar
  21. Frizzo A, Guidolin L, Ballarin L, Sabbadin A (1999) Purification and partial characterisation of phenoloxidase from the colonial ascidian Botryllus schlosseri. Mar Biol 135:483–488Google Scholar
  22. Gaykema WPJ, Hol WGJ, Vereifken JM, Soeter NM, Bak HJ, Beintema JJ (1984) 3.2 Å structure of the copper-containing, oxygen-carrying protein Panulirus interruptus hemocyanin. Nature 309:23–29Google Scholar
  23. Giribet G, Edgecombe GD, Wheeler WC (2001) Arthropod phylogeny based on eight molecular loci and morphology. Nature 413:157–161CrossRefPubMedGoogle Scholar
  24. Hazes B, Magnus KA, Bonaventura C, Bonaventura J, Dauter Z, Kalk KH, Hol WGJ (1993) Crystal structure of deoxygenated Limulus polyphemus subunit II hemocyanin at 2.18 Å resolution: clues for a mechanism for allosteric regulation. Protein Sci 2:597–619PubMedGoogle Scholar
  25. Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17:754–755CrossRefPubMedGoogle Scholar
  26. Hughes AL (1999) Evolution of the arthropod prophenoloxidase/hexamerin protein family. Immunogenetics 49:106–114CrossRefPubMedGoogle Scholar
  27. Hwang UW, Friedrich M, Tautz D, Park CJ, Kim W (2001) Mitochondrial protein phylogeny joins myriapods with chelicerates. Nature 413:154–157CrossRefPubMedGoogle Scholar
  28. Jackson AD, Smith VJ, Peddie CM (1993) In vitro phenoloxidase activity in the blood of Ciona intestinalis and other ascidians. Develop Comp Immun 17:97–108CrossRefGoogle Scholar
  29. Jaenicke E, Decker H (2003) Tyrosinases from crustaceans form hexamers. Biochem J 371:515–523CrossRefPubMedGoogle Scholar
  30. Jaenicke E, Decker H, Gebauer W, Markl J, Burmester T (1999) Identification, structure and properties of hemocyanins from diplopod Myriapoda. J Biol Chem 274:29071–29074CrossRefPubMedGoogle Scholar
  31. Kawabata T, Yasuhara Y, Ochiai M, Matsuura S, Ashida M (1995) Molecular cloning of insect pro-phenoloxidase: a copper-containing protein homologous to arthropod hemocyanin. Proc Natl Acad Sci USA 92:7774–7778PubMedGoogle Scholar
  32. Koopmanschap AB, Lammers JHM, de Kort CAD (1995) The structure of the gene encoding diapause protein 1 of the Colorado potato beetle (Leptinotarsa decemlineata). J Insect Physiol 41:509–518CrossRefGoogle Scholar
  33. Kurtz DM Jr (1999) Oxygen-carrying proteins: three solutions to a common problem. Essays Biochem 34:85–100PubMedGoogle Scholar
  34. Kusche K, Burmester T (2001) Diplopod hemocyanin sequence and the phylogenetic position of the Myriapoda. Mol Biol Evol 18:1566–1573PubMedGoogle Scholar
  35. Kusche K, Ruhberg H, Burmester T (2002) A hemocyanin from the Onychophora and the emergence of respiratory proteins. Proc Natl Acad Sci USA 99:10545–10548CrossRefPubMedGoogle Scholar
  36. Lieb B, Altenhein B, Markl J, Vincent A, van Olden E, van Holde KE, Miller KI (2001) Structures of two molluscan hemocyanin genes: significance for gene evolution. Proc Natl Acad Sci USA 98:4546–4551PubMedGoogle Scholar
  37. Linzen B, Soeter NM, Riggs AF, Schneider HJ, Schartau W, Moore MD, Behrens PQ, Nakashima H, Takagi T, Nemoto T, Vereijken JM, Bak HJ, Beintema JJ, Volbeda A, Gaykema WPJ, Hol WGJ (1985) The structure of arthropod hemocyanins. Science 229:519–524PubMedGoogle Scholar
  38. Mangum CP, Scott JL, Black REL, Miller KI, van Holde KE (1985) Centipedal hemocyanin: its structure and implication for arthropod phylogeny. Proc Natl Acad Sci USA 82:3721–3725PubMedGoogle Scholar
  39. Markl J, Decker H (1992) Molecular structure of the arthropod hemocyanins. Adv Comp Environ Physiol 13:325–376Google Scholar
  40. Memmel NA, Trewitt PM, Grzelak K, Rajaratnam VS, Kumaran AK (1994) Nucleotide sequence, structure and developmental regulation of LHP82, a juvenile hormone-suppressible hexamerin gene from the waxmoth, Galleria mellonella. Insect Biochem Mol Biol 24:133–144CrossRefPubMedGoogle Scholar
  41. Müller G, Ruppert S, Schmid E, Schütz G (1988) Functional analysis of alternatively spliced tyrosinase gene transcripts. EMBO J 7:2723–2730PubMedGoogle Scholar
  42. Nakai K, Horton P (1999) PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci 24:34–36PubMedGoogle Scholar
  43. Nagai T, Osaki T, Kawabata Si S (2001) Functional conversion of hemocyanin to phenoloxidase by horseshoe crab antimicrobial peptides. J Biol Chem 276:27166–27170PubMedGoogle Scholar
  44. Nicholas KB, Nicholas HB Jr (1997) GeneDoc: Analysis and Visualization of Genetic Variation, http://www.psc.edu/biomed/genedoc/
  45. Pearson WR (2000) Flexible sequence similarity searching with the FASTA3 program package. Meth Mol Biol 132:185–219Google Scholar
  46. Regier JC, Shultz JW (2001) A phylogenetic analysis of Myriapoda (Arthropoda) using two nuclear protein-encoding genes. Zool J Linn Soc 132:469–486CrossRefGoogle Scholar
  47. Rogers JH (1986) Introns between protein domains: selective insertion or frameshifting? Trends Genet 2:223CrossRefGoogle Scholar
  48. Sanchez D, Ganfornina MD, Gutierrez G, Bastiani MJ (1998) Molecular characterization and phylogenetic relationship of a protein with oxygen-binding capabilities in the grasshopper embryo. A hemocyanin in insects? Mol Biol Evol 15:415–426PubMedGoogle Scholar
  49. Smith VJ, Söderhäll K (1991) A comparison of phenoloxidase activity in the blood of marine invertebrates. Develop Comp Immun 15:251–261CrossRefGoogle Scholar
  50. Söderhäll K, Cerenius L (1998) Role of the prophenoloxidase-activating system in invertebrate immunity. Curr Opin Immunol 10:23–28CrossRefPubMedGoogle Scholar
  51. Strimmer K, von Haeseler A (1996) Quartet puzzling: a quartet maximum likelihood method for reconstructing tree topologies. Mol Biol Evol 13:964–969Google Scholar
  52. Telfer WH, Kunkel JG (1991) The function and evolution of insect storage hexamers. Ann Rev Entomol 36:205–228CrossRefGoogle Scholar
  53. Terwilliger NB (1998) Functional adaptations of oxygen-transport proteins. J Exp Biol 201:1085–1098PubMedGoogle Scholar
  54. Terwilliger NB, Dangott LJ, Ryan MC (1999) Cryptocyanin, a crustacean molting protein: evolutionary links to arthropod hemocyanin and insect hexamerins. Proc Natl Acad Sci USA 96:2013–2018CrossRefPubMedGoogle Scholar
  55. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882PubMedGoogle Scholar
  56. Van Gelder CWG, Flurkey WH, Wichers HJ (1997) Sequence and structural features of plant and fungal tyrosinases. Phytochemistry 47:1309–1323Google Scholar
  57. Van Holde KE, Miller KI (1995) Hemocyanins. Adv Protein Chem 47:1-81PubMedGoogle Scholar
  58. Van Holde KE, Miller KI, Decker H (2001) Hemocyanins and invertebrate evolution. J Biol Chem 276:15563–15566PubMedGoogle Scholar
  59. Vinogradov SN (1985) The structure of invertebrate extracellular hemoglobins (erythrocruorins and chlorocruorins). Comp Biochem Physiol B 82:1–15CrossRefPubMedGoogle Scholar
  60. Voll W, Voit R (1990) Characterization of the gene encoding the hemocyanin subunit e from Eurypelma californicum. Proc Natl Acad Sci USA 87:5312–5316PubMedGoogle Scholar
  61. Wada H, Satoh N (1994) Details of the evolutionary history from invertebrates to vertebrates, as deduced from the sequences of 18S rDNA. Proc Natl Acad Sci USA 91:1801–1804PubMedGoogle Scholar
  62. 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

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  1. 1.Institute of ZoologyUniversity of MainzMainzGermany

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