Multiple forms of α 2 -macroglobulin from a bony fish, the common carp ( Cyprinus carpio ): striking sequence diversity in functional sites

Abstract

Unlike mammals, bony fish possess multiple genes encoding the complement component C3, a member of the alpha2-macroglobulin (alpha2M) protein family, presumably expanding the diversity of immune recognition. To examine whether the alpha2M gene has also duplicated and diverged in the bony fish lineage, cDNA cloning of alpha2M from a pseudotetraploid teleost, the common carp (Cyprinus carpio), was conducted and resulted in the isolation of three distinct alpha2M sequences from a single individual, indicating the presence of multiple alpha2M 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 alpha2M. Two distinct alpha2M proteins were purified from carp serum; both proved to be Mr 380,000 dimers, the subunits of which are composed of disulfide-linked alpha chains (Mr 93,000) and beta chains (Mr 85,000), as reported for the alpha2M from plaice, another teleost species. The presence of an internal thioester in the alpha chain was demonstrated by its autolytic fragmentation and direct incorporation of [14C]methylamine. Interestingly, the three forms of carp alpha2M exhibited outstanding sequence diversity in the bait region which displays target sequences for various proteases, and in the C-terminal region of the alpha 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 alpha2Ms, as in most alpha2Ms of other animals. The possible functional significance of the sequence diversity is discussed.

Purification and characterization of α2-macroglobulin from grass carp Ctenopharyngodon idellus: Cloning a segment of the corresponding gene

“…There is a possibility that grass carp a 2 M is encoded by multiple genes. According to previous reports, Limulus has two different forms, which are present in a ratio of 2:1 [14], and three different forms have been reported in common carp [7].…”

“…In the present study, we found that grass carp a 2 M may be composed of two different subunits, with molecular weights of 95 kDa and 80 kDa. Reports on a 2 M for the common carp [7], indicate that it consists of two subunits with M r of 93 kDa and 85 kDa. a 2 M differs from other large protease inhibitors in the mechanism by which it inhibits protease activity.…”

Two Divergent Isotypes of the Fourth Complement Component from a Bony Fish, the Common Carp (Cyprinus carpio)

“…Based on the number of chromosomes (e.g., the common carp, 2n ϭ 100 (50 pairs of chromosomes); the tench Tinca tinca, 2n ϭ 48; the barbel steed Hemibarbus labeo, 2n ϭ 50; the grass carp Ctenopharyngodon idellus, 2n ϭ 48 (data from FishBase, ͗www.fishbase.org/͘)), the common carp is believed to have arisen by allotetraploidization 12-50 million years ago in the cyprinid family (42)(43)(44). Hence, it seems reasonable to assume that the tetraploidization has probably contributed, at least in part, to the gene duplications of other members of the thioestercontaining protein family, such as C3, C5, and ␣ 2 M, of this species (21)(22)(23). However, it is unlikely that carp C4-1 and C4-2 were generated by the tetraploidization event as judged from their substantial sequence divergence.…”

“…probe, representing 2925-3136 of carp ␣ 2 M-1 (23). Of the 20 clones that were negative to both probes, one clone (Cp1) showed closer similarity to human and Xenopus C4 than to C3 and ␣ 2 M (Fig.…”

“…Especially, the major members of the thioester-containing protein family have been shown to be present as multiple isoforms: C3 in trout (16 -18), sea bream (19), medaka (8), zebrafish (20), and carp (21), C5 in carp (22), and ␣ 2 -macroglobulin (␣ 2 M) in carp (23). These findings prompted us to examine the diversity of C4 in bony fish to better understand the biological impact of complement diversity and the genetic mechanisms that increased the isotypic genes of the complement components.…”