Functional evidence for the existence of a bona-fide C2 component in teleost complement system

The complement system of vertebrates comprises classical, lectin, and alternative pathways, yet the evolutionary origin of the classical pathway remains unclear due to limited functional evidence for complement component C2 in early vertebrates. Common carp possesses two Bf/C2-like genes, B/C2-A and B/C2-B, but their functional assignments have not been fully established. In this study, we investigated whether B/C2-B represents a bona fide C2 by characterizing recombinant B/C2-B (rB/C2-B) expressed using a baculovirus/insect cell system. Western blotting confirmed that the recombinant protein corresponds to the endogenous serum molecule. Functional assays demonstrated that rB/C2-B does not participate in the assembly of the alternative pathway C3-convertase and shows minimal interaction with C3b-like C3i. In contrast, quartz crystal microbalance analyses revealed markedly higher affinity of rB/C2-B for C4b-like C4i. Moreover, rB/C2-B was cleaved by the carp MBL-MASP2 complex, generating a C2b-like fragment analogous to mammalian C2 activation in the lectin pathway. These findings collectively demonstrate that B/C2-B acts as functional C2 in carp, contributing to classical and lectin pathway activation but not to the alternative pathway. The results further indicate that Bf and C2 had already diverged by the time of bony fish evolution, supporting an early emergence of the classical pathway in vertebrate history. This study provides the first direct functional evidence for C2 in teleosts and refines our understanding of complement system evolution.