ISOLATION, PRIMARY STRUCTURE, AND EVOLUTION OF THE 3RD COMPONENT OF CHICKEN COMPLEMENT AND EVIDENCE FOR A NEW MEMBER OF THE ALPHA(2)-MACROGLOBULIN FAMILY

Although the third component of complement, C3, has been isolated and its primary structure determined from most living classes of vertebrat e, limited information is available on its structure and function for aves, which represent a significant stage in complement evolution. In this study, we present the complete cDNA sequence of chicken C3, the c DNA sequences of the thioester region for two chicken alpha(2)-macrogl obulin (alpha(2)M)-related proteins, a simplified method for purifying chicken C3, and an analysis of the C3 convertase and factor I-mediate d cleavages in chicken C3. Using the reverse-transcriprase PCR, with d egenerate oligonucleotide primers derived from two conserved C3 sequen ces (GCCEQ(N)/(T)M, TWLTA(Y)/V-F) and liver mRNA as template, we isola ted three distinct 220-bp PCR products, one with a high degree of sequ ence similarity to C3 and two to alpha(2)M and pregnancy zone protein from other species. The complete cDNA sequence of chicken C3 was obtai ned by screening a chicken liver lambda gt10 library with the C3 PCR p roduct and probes from the 5' end of the partial-length C3 clones. The obtained sequence is in complete agreement with the protein sequence of several tryptic peptides of purified chicken C3. Chicken pro-C3 con sists of an 18-residue putative signal peptide, a 640-residue beta-cha in (70 kDa), a 989-residue alpha-chain (111 kDa), and an RKRR linker r egion. It contains an internal thioester and three potential N-glycosy lation sites, all in the alpha-chain. The convertase cleavage site, pr edicted to be Arg-Ser, was confirmed by sequencing the zymosan-bound C 3 fragments generated upon complement activation. NH2-terminal sequenc ing of the purified C3 chains showed that 1) pro-C3 is indeed cleaved at the RKRR linker sequence to generate the mature two-chain molecule, and 2) the beta-chain of chicken C3 is blocked. The deduced amino aci d sequence shows 54, 54, 54, 53, 52, 57, and 55% amino acid identities to human, mouse, rat, guinea pig, rabbit, cobra, and Xenopus C3, resp ectively, and an identity of 44, 31, and 33% to trout, hagfish, and la mprey C3, respectively. The identities to human C4, C5, and alpha(2)M are 31, 29 and 23%, respectively. A phylogenetic tree for C3, C4, C5, and alpha(2)M-related proteins was constructed based on the sequence d ata and is discussed.