Oyster Metallothioneins:  Unprecedented Diversity in a Metazoan Species

Matthew J. Jenny¹, Gregory W. Warr^1,2, Amy H. Ringwood³, Kevin Schey⁴, and Robert W. Chapman^1,5

¹ Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC

² Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC

³ Department of Biology, University of North Carolina, Charlotte, NC

⁴ Department of Cell and Molecular Pharmacology & Experimental Therapeutics, Medical University of South Carolina, Charleston, SC

⁵ Marine Resources Research Institute, South Carolina Department of Natural Resources, Charleston, SC

Metallothioneins (MTs) are low molecular weight metal-binding proteins typically comprised of two domains (a & b) with characteristic repeating cysteine motifs (Cys-Xn-Cys).  They play primary roles in metal metabolism, homeostasis and detoxification. The two domains of MTs are capable of binding metals independently of each other and are functionally diverse.  The a-domain is believed to convey structure and stability to the protein while the b-domain participates in metal exchange reactions with zinc and copper-requiring apoproteins.  Utilizing molecular genetic and protein biochemical approaches, we have identified three MT gene families in the American oyster, Crassostrea virginica, with distinct differences in primary structure, domain organization, and patterns of expression, including their inducibility by metal ions and other stressors. The first family of oyster MTs (CvMT-I & II) consists of the traditional ab-domain structure, as well as a sub-family of MTs, which appears to have resulted from a mutation followed by a series of exon duplications and consists only of one to four a-domains. We have also described a new molluscan MT family (CvMT-III) characterized by the presence of two b-domains and the absence of a-domains.  A third MT gene family (CvMT-IV) was isolated from hemocytes by subtractive hybridization techniques and appears to be more closely related to the classic ab-domain MTs.  However, a series of amino acid substitutions has resulted in the occurrence of four additional cysteines which form novel motifs.  Southern blot analysis suggests the presence of at least 6 CvMT-I & II genes, and 2-3 copies of both CvMT-III and CvMT-IV.  Characterization of a 10X BAC library has identified a single locus for the CvMT-I, II & IV gene families and different loci for CvMT-III.  The diversity of oyster MTs appears to have resulted from the evolution of two distinct ancestral MT genes, an unprecedented observation from any metazoan taxon.