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The First Complete cDNA Sequence of the Hemocyanin from a Bivalve, the Protobranch Nucula nucleus

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Abstract

By cDNA sequencing we have achieved the first, and complete, hemocyanin sequence of a bivalve (Nucula nucleus). This extracellular oxygen-binding protein consists of two immunologically distinguishable isoforms, here termed NnH1 and NnH2. They share a mean sequence identity of 61%, both contain a linear arrangement of eight paralogous, ca.50-kDa functional units (FUs a-h), and in both isoforms the C-terminal FU-h possesses an extension of ca. 100 amino acids. The cDNA of NnH1 comprises 11,090 bp, subdivided into a 5′utr of 75 bp, a 3′utr of 791 bp, and an open reading frame for a signal peptide of 19 amino acids plus a polypeptide of 3389 amino acids (M r = 385 kDa). The cDNA of NnH2 comprises 10,849 bp, subdivided into a 5′utr of 47 bp, a 3′utr of 647 bp, and an open reading frame for a signal peptide of 16 amino acids plus a polypeptide of 3369 amino acids (M r = 387 kDa). In contrast to other molluscan hemocyanins, which are highly glycosylated, the bivalve hemocyanin sequence exhibits only four potential N-glycosylation sites, and within both isoforms a peculiar indel is present, surrounding the highly conserved copper-binding site CuA. Phylogenetic analyses of NnH1 and NnH2, compared to the known hemocyanin sequences of gastropods and cephalopods, reveal a statistically sound closer relationship between gastropod and protobranch hemocyanin than to cephalopod hemocyanin. Assuming a molecular clock, the last common ancestor of protobranch and gastropods lived 494 million ± 50 million years ago, in conformity with fossil records from the late Cambrian.

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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 Dr. Wolfgang Gebauer for providing the electron micrographs. We also thank Dr. Frank Zal (Roscoff, France) for providing animals. This work was financially supported by DFG grants to J.M. (Ma843), the biosyn company (Fellbach, Germany), and the Feldbausch foundation.

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

    Sandra Bergmann, Jürgen Markl & Bernhard Lieb

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  1. Sandra Bergmann
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  2. Jürgen Markl
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Correspondence to Bernhard Lieb.

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[Reviewing Editor: Dr. Rüdiger Cerff]

The sequence reported in this paper has been deposited in the EMBL/GenBank database under accession number AJ786639 for NnH1 and AJ786640 for NnH2.

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Bergmann, S., Markl, J. & Lieb, B. The First Complete cDNA Sequence of the Hemocyanin from a Bivalve, the Protobranch Nucula nucleus . J Mol Evol 64, 500–510 (2007). https://doi.org/10.1007/s00239-006-0036-8

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