Research on the primitive complement system in agnathan cyclostomes

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A protein from hagfish serum that cross-reacted with the third component of hagfish complement (C3-1) was purified to homogeneity and its structural properties were compared with those of two-subunit-chain (115 kDa and 72 kDa) C3-1. This protein (termed C3-2), when purified from the plasma, consisted of three disulfide-linked polypeptide chains (77 kDa, 72 kDa and 30kDa). On immunoelectrophoresis, purified C3-2 had a more anodal mobility compared to the beta mobility of C3-1. However, immunochmical analysis revealed that C3-2 after the initial step in the purification consisted of two disulfide-linked polypeptide chains (105 kDa and 72 kDa). Treatment of C3-2 with methylamine, prior to the spectrophotometric titration of free SH content in this molecule, did not significantly affect the titration end point, suggesting the disruption of a thiol ester bond in C3-2. Amino acid sequence analysis of the amino-termini of the subunits of C3-2 revealed that 77 amino acid residues at the amino-terminus of native alpha-chain were missing from the 77-kDa as well as 105-kDa polypeptides from C3-2. These results indicate that C3-2 isolated in this study is analogous to C3b. Furthermore, sequencing data indicate that most of the native C3 from hagfish occur in the serum with an irregular two-subunit (alpha + gamma and beta) chain structure, as a result of the one-sided processing of putative hagfish pro-C3 at the beta-alpha processing site exclusively, and, moreover that only the molecular features of degenerated hagfish C3 (C3-2) are altered during its purification to generate a three-chain structure