Multiple forms of α₂-macroglobulin from a bony fish, the common carp (Cyprinus carpio): Striking sequence diversity in functional sites

Unlike mammals, bony fish possess multiple genes encoding the complement component C3, a member of the α₂-macroglobulin (α₂M) protein family, presumably expanding the diversity of immune recognition. To examine whether the α₂M gene has also duplicated and diverged in the bony fish lineage, cDNA cloning of α₂M from a pseudotetraploid teleost, the common carp (Cyprinus carpio), was conducted and resulted in the isolation of three distinct α₂M sequences from a single individual, indicating the presence of multiple α₂M genes in this species. The deduced amino acid sequences contained a post-translational cleavage signal, predicting a C3-like two-chain structure, as in lamprey α₂M. Two distinct α₂M proteins were purified from carp serum; both proved to be M(r) 380,000 dimers, the subunits of which are composed of disulfide-linked α chains (M(r) 93,000) and β chains (M(r) 85,000), as reported for the α₂M from plaice, another teleost species. The presence of an internal thioester in the a chain was demonstrated by its autolytic fragmentation and direct incorporation of [¹⁴C]methylamine. Interestingly, the three forms of carp α₂M exhibited outstanding sequence diversity in the bait region which displays target sequences for various proteases, and in the C-terminal region of the α chain assigned as the receptor-binding domain, while an Asn residue at the position corresponding to the catalytic His in C3 was completely conserved in the carp α₂Ms, as in most α₂Ms of other animals. The possible functional significance of the sequence diversity is discussed.