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The Hemocyanin from a Living Fossil, the Cephalopod Nautilus pompilius: Protein Structure, Gene Organization, and Evolution

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Abstract

By electron microscopic and immunobiochemical analyses we have confirmed earlier evidence that Nautilus pompilius hemocyanin (NpH) is a ring-like decamer (Mr = ∼3.5 million), assembled from 10 identical copies of an ∼350-kDa polypeptide. This subunit in turn is substructured into seven sequential covalently linked functional units of ∼50 kDa each (FUs a–g). We have cloned and sequenced the cDNA encoding the complete polypeptide; it comprises 9198 bp and is subdivided into a 5′ UTR of 58 bp, a 3′ UTR of 365 bp, and an open reading frame for a signal peptide of 21 amino acids plus a polypeptide of 2903 amino acids (Mr = 335,881). According to sequence alignments, the seven FUs of Nautilus hemocyanin directly correspond to the seven FU types of the previously sequenced hemocyanin “OdH” from the cephalopod Octopus dofleini. Thirteen potential N-glycosylation sites are distributed among the seven Nautilus hemocyanin FUs; the structural consequences of putatively attached glycans are discussed on the basis of the published X-ray structure for an Octopus dofleini and a Rapana thomasiana FU. Moreover, the complete gene structure of Nautilus hemocyanin was analyzed; it resembles that of Octopus hemocyanin with respect to linker introns but shows two internal introns that differ in position from the three internal introns of the Octopus hemocyanin gene. Multiple sequence alignments allowed calculation of a rather robust phylogenetic tree and a statistically firm molecular clock. This reveals that the last common ancestor of Nautilus and Octopus lived 415 ± 24 million years ago, in close agreement with fossil records from the early Devonian.

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References

  • Albrecht U, Keller H, Gebauer W, Markl J (2001) Rhogocytes (pore cells) as the site of hemocyanin biosynthesis in the marine gastropod Haliotis tuberculata. Cell Tissue Res 304:455–462

    Article  CAS  PubMed  Google Scholar 

  • Altenhein B, Markl J, Lieb B (2002) Gene structure and hemocyanin isoform HtH2 from the mollusc Haliotis tuberculata indicate early and late intron hot spots. Gene 301:53–60

    Article  CAS  PubMed  Google Scholar 

  • Bause E (1983) Structural requirements of N-glycosylation of proteins. Studies with proline peptides as conformational probes. Biochem J 209:331–336

    CAS  PubMed  Google Scholar 

  • Benton MJ (1993) In: The fossil record 2. Chapman and Hall, London, pp 125–270

  • Bonaventura C, Bonaventura J, Miller KI, van Holde KE (1981) Hemocyanin of the chambered Nautilus: structure-function relationships. Arch Biochem Biophys 211:589–598

    Article  CAS  PubMed  Google Scholar 

  • Bonnaud L, Boucher-Rodini R, Monnerot M (1997) Phylogeny of cephalopods inferred from mitochondrial DNA Sequences. Mol Phylogenet Evol 7:44–54

    CAS  PubMed  Google Scholar 

  • Bonnaud L, Ozouf-Costaz C, Boucher-Rodini R (2004) A molecular and karyological approach to the taxonomy of Nautilus. CR Biol 327:133–138

    CAS  Google Scholar 

  • Chignell D, van Holde KE, Miller KI (1997) The hemocyanin of the squid Sepioteuthis lessoniana: Structural Comparison with other cephalopod hemocyanins. Comp Biochem Physiol 118:895–902

    Google Scholar 

  • Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159

    Article  CAS  PubMed  Google Scholar 

  • Cuff ME, Miller KI, van Holde KE, Hendrickson WA (1998) Crystal structure of a functional unit from Octopus hemocyanin. J Mol Biol 278:855–870

    Article  CAS  PubMed  Google Scholar 

  • Declerq L, Witters R, Preaux G (1990) Partial sequence determination of Sepia officinalis hemocyanin via cDNA. In: Preaux G, Lontie R (eds). Invertebrate dioxygen carriers. Leuven University Press, Louvain, Belgium, pp 131–134

    Google Scholar 

  • Felsenstein J (2001) PHYLIP (phylogeny inference package), version 3.6α2. Distributed by the author. Department of Genetics, University of Washington, Seattle

  • Gebauer W, Harris JR, Heid H, Süling M, Hillenbrand R, Söhngen S, Wegener-Strake A, Markl J (1994) Quaternary structure, subunits and domain patterns of two discrete forms of keyhole limpet hemocyanin: KLH1 and KLH2. Zoology 98:51–68

    CAS  Google Scholar 

  • Gebauer W, Stoeva S, Voelter W, Danese E, Salvato B, Beltramini M, Markl J (1999) Hemocyanin subunit organization of the gastropod Rapana thomasiana. Arch Biochem Biophys 372:128–134

    Article  CAS  PubMed  Google Scholar 

  • Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis 18:2714–2723

    Article  CAS  PubMed  Google Scholar 

  • Hall RL, Wood EJ, Kamberling JP, Gerwig GJ, Vliegenthart FG (1977) 3-O-Methyl sugars as constituents of glycoproteins. Identification of 3-O-methylgalactose and 3-O-methylmannose in pulmonate gastropod haemocyanins. Biochem J 165:173–176

    CAS  PubMed  Google Scholar 

  • Harris JR, Horne RW (1991) Negative staining. In: Harris JR (eds). Electron microscopy in biology. IRL Press, Oxford, UK, pp 203–228

    Google Scholar 

  • Herskovits TT, Hamilton MG (1991) Higher order assemblies of molluscan hemocyanins. Comp Biochem Physiol B 99:19–34

    CAS  PubMed  Google Scholar 

  • Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17:754–755

    Article  CAS  PubMed  Google Scholar 

  • Idakieva K, Severov S, Svendsen I, Genov N, Stoeva S, Beltramini M, Tognon G, Di Muro P, Salvato B (1993) Structural properties of Rapana thomasiana Grosse hemocyanin: isolation, characterization and N-terminal amino acid sequence of two different dissociation products. Comp Biochem Physiol 106B:53–59

    CAS  Google Scholar 

  • Jeletzky JA (1966) Comparative morphology, phylogeny, and classification of fossil coleoidea. The University of Kansas paleontological contributions. Mollusca 7:1–162

    Google Scholar 

  • Jellie AM, Tate WP, Trotman CN (1996) Evolutionary history of introns in a multidomain globin gene. J Mol Evol 42:641–647

    CAS  PubMed  Google Scholar 

  • Klabunde T, Eicken C, Sacchettini JC, Krebs B (1998) Crystal structure of a plant catechol oxidase containing a discopper center. Nat Struct Biol 5:1084–1090

    CAS  PubMed  Google Scholar 

  • Kroll J (1973) Crossed-line immunoelectrophoresis. Scand J Immunol 2:79–81

    Google Scholar 

  • Kurokawa T, Wuhrer M, Lochnit G, Geyer H, Markl J, Geyer R, (2002) Hemocyanin from the keyhole limpet Megathura crenulata (KLH) carries a novel type of N-glycans with Gal(beta1-6)Man-motifs. Eur J Biochem 269:5459–5473

    Article  CAS  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  • Lang WH, van Holde KE (1991) Cloning and sequencing of Octopus dofleini hemocyanin cDNA: derived sequences of functional units Ode and Odf. Proc Natl Acad Sci USA 88:244–248

    CAS  PubMed  Google Scholar 

  • Lambert O, Boisset N, Penczek P, Lamy J, Taveau JC, Frank J, Lamy JN (1994) Quaternary structure of Octopus vulgaris hemocyanin. Three-dimensional reconstruction from frozen-hydrated specimens and intramolecular location of functional units Ove and Ovb. J Mol Biol 238:75–87

    Article  CAS  PubMed  Google Scholar 

  • Lamy J, Gielens C, Lambert O, Taveau JC, Motta G, Loncke P, De Geest N, Preaux G, Lamy J (1993) Further approaches to the quaternary structure of octopus hemocyanin: A model based on immunoelectron microscopy and image processing. Arch Biochem Biophys 305:17–29

    Article  CAS  PubMed  Google Scholar 

  • Lamy J, You V, Taveau JC, Boisset N, Lamy JN (1998) Intramolecular localization of the functional units of Sepia officinalis hemocyanin by immunoelectron microscopy. J Mol Biol 284:1051–1074

    Article  CAS  PubMed  Google Scholar 

  • Lieb B, Altenhein B, Markl J (2000) The sequence of a gastropod hemocyanin (HtH1 from Haliotis tuberculata). J Biol Chem 275:5675–5681

    Article  CAS  PubMed  Google Scholar 

  • 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 98:4546–4551

    Article  CAS  PubMed  Google Scholar 

  • Lieb B, Boisguerin V, Gebauer W, Markl J (2004) cDNA sequence, protein structure, and evolution of the single hemocyanin from Aplysia californica, an ophistobranch gastropod. J Mol Evol 59:1–10

    Article  Google Scholar 

  • Linn JF, Black P, Derksen K, Rubben H, Thuroff JW (2000) Keyhole limpet haemocyanin in experimental bladder cancer: Literature review and own results. Eur Urol 37:34–40

    Article  CAS  PubMed  Google Scholar 

  • Markl J, Winter S (1989) Subunit-specific monoclonal antibodies to tarantula hemocyanin, and the common epitope shared with calliphorin. J Comp Physiol 159B:139–151

    Google Scholar 

  • Markl J, Lieb B, Gebauer W, Altenhein B, Meissner U, Harris JR (2001) Marine tumor vaccine carriers: structure of the molluscan hemocyanins KLH and HtH. J Cancer Res Clin Oncol 127:R3–R9

    Article  CAS  PubMed  Google Scholar 

  • Meissner U, Dube P, Harris JR, Stark H, Markl J (2000) Structure of a molluscan hemocyanin didecamer (HtH1 from Haliotis tuberculata) at 12 Å resolution by cryoelectron microscopy. J Mol Biol 298:21–34

    Article  CAS  PubMed  Google Scholar 

  • Miller KI, van Holde KE (2003) XIIIth Conference on Dioxygen Binding Proteins, Mainz, Germany

    Google Scholar 

  • Miller KI, Schabtach E, van Holde KE (1990) Arrangement of subunits and domains within the Octopus dofleini hemocyanin molecule. Proc Natl Acad Sci USA 87:1496–1500

    CAS  PubMed  Google Scholar 

  • Miller KI, Cuff ME, Lang WF, Varga-Weisz P, Field KG, van Holde KE (1998) Sequence of the Octopus dofleini hemocyanin subunit: structural and evolutionary implications. J Mol Biol 278:827–842

    Article  CAS  PubMed  Google Scholar 

  • Mouche F, Boisset N, Lamy J, Zal F, Lamy JN (1999) Structural comparison of cephalopod hemocyanins: Phylogenetic significance. J Struct Biol 127:199–212

    Article  CAS  PubMed  Google Scholar 

  • Nicholas KB, Nicholas HB Jr (1997) GeneDoc: Analysis and visualization of genetic variation. Distributed by the authors

  • Perbandt M, Guthohrlein EW, Rypniewski W, Idakieva K, Stoeva S, Voelter W, Genov N, Betzel C (2003) The structure of a functional unit from the wall of a gastropod hemocyanin offers a possible mechanism for cooperativity. Biochemistry 42:6341–6346

    Article  CAS  PubMed  Google Scholar 

  • Pojeta Jr J (1980) Molluscan phylogeny. Tul Stud Geol Paleontol 16:55–80

    Google Scholar 

  • Ruth P, Schipp R, Klüssendorf B (1988) Cytomorphology and copper content of the basal cells in the midgut-gland of Nautilus (Cephalopoda, Tetrabranchiata). A contribution to the localization of hemocyanin synthesis. Zoomorphology 108:1–11

    Article  Google Scholar 

  • Ruth P, Schimmelpfennig R, Schipp R (1999) Comparative immunohistochemical and immunocytochemical investigations on the location of haemocyanin synthesis in dibranchiate and tetrabranchiate Cephalopods (Sepia and Nautilus). In: Olóriz F, Rodriguez-Tovar FJ (eds). Advancing research on living and fossil cephalopods. Plenum Press, New York, pp 189–202

    Google Scholar 

  • Salvato B, Santamaria M, Beltramini M, Alzuet G, Casella L (1998) The enzymatic properties of Octopus vulgaris hemocyanin: o-diphenol oxidase activity. Biochemistry 37:14065–14077

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Russel DW (2001) Molecular cloning. a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  • Sminia T (1977) Haemocyanin-producing cells in gastropod molluscs. In: Bannister JV (eds). Structure and function of haemocyanin. Berlin, Springer, pp 279–288

    Google Scholar 

  • Söhngen SM, Stahlmann A, Harris JR, Muller SA, Engel A, Markl J (1997) Mass determination, subunit organization and control of oligomerization states of keyhole limpet hemocyanin (KLH). Eur J Biochem 248: 602–614

    PubMed  Google Scholar 

  • Stoeva S, Idakieva K, Betzel C, Genov N, Voelter W (2002) Amino acid sequence and glycosylation of functional unit RtH2-e from Rapana thomasiana (gastropod) hemocyanin. Arch Biochem Biophys 399:149–158

    Article  CAS  PubMed  Google Scholar 

  • Streit K, Jackson D, Degnan BM, Lieb B (2005) Developmental expression of two Haliotis asinina hemocyanin isoforms. Differentiation 73:341–349

    Article  CAS  PubMed  Google Scholar 

  • Tajima F (1993) Simple methods for testing molecular clock hypothesis. Genetics 135:599–607

    CAS  PubMed  Google Scholar 

  • 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 24:4876–4882

    Google Scholar 

  • Top A, Gielens C, Witters R, van Beumen J, Preaux G (1990) Partial amino acid sequence and location of the carbohydrate chain in functional unit of Sepia officinalis In: Preaux G, Lontie R (eds). Invertebrate dioxygen carriers. Leuven University Press, Louvain, Belgium, pp 119–124

    Google Scholar 

  • Topham R, Tesh S, Westcott A, Cole G, Mercatante D, Kaufman G, Bonaventura C (1999) Disulfide bond reduction: A powerful, chemical probe for the study of structure-function relationships in the hemocyanins. Arch Biochem Biophys 369:261–266

    Article  CAS  PubMed  Google Scholar 

  • van Gelder CWG, Flurkey WH, Wichers HJ (1997) Sequence and structural features of plant and fungal tyrosinases. Phytochemistry 45:1309–1323

    PubMed  Google Scholar 

  • van Holde KE, Miller KI (1995) Hemocyanins. Adv Protein Chem 47:1–81

    PubMed  Google Scholar 

  • von Boletzky S (1992) Evolutionary aspects of development, life style, and reproductive mode in incirrate octopods (Mollusca, Cephalopoda). Rev Suisse Zool 99:755–770

    Google Scholar 

  • Weeke B (1973) Crossed immunoelectrophoresis. Scand J Immunol 2:47–56

    Google Scholar 

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Acknowledgments

We thank Prof. Dr. J. Robin Harris (Institute of Zoology, University of Mainz) for critically reading the manuscript and correcting the language and our colleague Wolfgang Gebauer for providing the electron micrographs. This work was financially supported by DFG grants to J.M. (Ma843) and by the biosyn company (Fellbach, Germany).

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Authors and Affiliations

  1. Institute of Zoology, Johannes Gutenberg University, Mainz, D-55099, Germany

    Sandra Bergmann, Bernhard Lieb & Jürgen Markl

  2. Institute of Zoology, Justus Liebig University, Giessen, D-35390, Germany

    Peter Ruth

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  1. Sandra Bergmann
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  2. Bernhard Lieb
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  3. Peter Ruth
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  4. Jürgen Markl
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Correspondence to Jürgen Markl.

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[Reviewing Editor: Dr. Axel Meyer]

The sequence reported in this paper has been deposited in the EMBL/GenBank database under accession number AJ619741.

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Bergmann, S., Lieb, B., Ruth, P. et al. The Hemocyanin from a Living Fossil, the Cephalopod Nautilus pompilius: Protein Structure, Gene Organization, and Evolution. J Mol Evol 62, 362–374 (2006). https://doi.org/10.1007/s00239-005-0160-x

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