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J. Biol. Chem., Vol. 279, Issue 25, 26134-26142, June 18, 2004

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Hemocyanin of the Molluscan Concholepas concholepas Exhibits an Unusual Heterodecameric Array of Subunits^*

Pablo De Ioannes, Bruno Moltedo, Harold Oliva, Rodrigo Pacheco, Fernando Faunes, Alfredo E. De Ioannes, and María Inés Becker

From the Department of Research and Development, BIOSONDA Corp., Avenida Alcalde Eduardo Castillo Velasco 2902, Santiago 7750269, Chile

We describe here the structure of the hemocyanin from the Chilean gastropod Concholepas concholepas (CCH), emphasizing some attributes that make it interesting among molluscan hemocyanins. CCH exhibits a predominant didecameric structure as revealed by electron microscopy and a size of 8 MDa by gel filtration, and, in contrast with other mollusc hemocyanins, its stabilization does not require additional Ca²⁺ and/or Mg²⁺ in the medium. Polyacrylamide gel electrophoresis studies, analyses by a MonoQ FPLC column, and Western blots with specific monoclonal antibodies showed that CCH is made by two subunits noncovalently linked, named CCH-A and CCH-B, with molecular masses of 405 and 350 kDa, respectively. Interestingly, one of the subunits undergoes changes within the macromolecule; we demonstrated that CCH-A has an autocleavage site that under reducing conditions is cleaved to yield two polypeptides, CCH-A1 (300 kDa) and CCH-A2 (108 kDa), whereas CCH-B remains unchanged. The CCH-A nick occurs at 4 °C, increases at 37 °C, and is not inhibited by the addition of protease inhibitors and/or divalent cations. Since the CCH structure is a heterodimer, we investigated whether subunits would be either intermingled, forming heterodecamers, or assembled as two homogeneous decamers. Light scattering and electron microscope studies of the in vitro reassociation of purified CCH subunits demonstrated that the sole addition of Mg²⁺ is needed for its reassembly into the native decameric molecule; no homodecamer reorganization was found with either CCH-A or CCH-B subunits alone. Our evidence showed that C. concholepas hemocyanin is an unusual example of heterodecameric organization.

Received for publication, January 28, 2004 , and in revised form, April 7, 2004.

^* This work was supported by BIOSONDA Corp., FONTEC-CORFO Grants 1998-1392, 2001-2930, and 2003-3816 (to BIOSONDA Corp.), and FONDECYT Grant 199-0258 (to M. I. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

To whom correspondence should be addressed. Tel.: 56-2-209-6770; Fax: 56-2-274-5462; E-mail: mib{at}biosonda.cl.

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