Phylogeny of the Third Complement Component, C3, and Conservation of C3‐Ligand Interactions a

Of the 30 distinct complement proteins recognized to date, C3 is probably the most versatile and multifunctional molecule known, interacting with at least 20 different proteins.'s2 It plays a critical role in both pathways of complement activation and participates in phagocytic and immunoregulatory processes.'V2 The study of C3 molecules from different species gives insight into the structural elements involved in its different functions, the structural features constrained by selection, and the evolutionary history of C3 and complement in general. C3-like activity has been reported in a variety of species, including invertebrates, yet thus far, C3 has been purified only from chordates and has been found to be present in representatives of each of the seven living classes of vertebrates.'S2 The complete primary structures have been deduced for C3 of human, guinea pig, mouse, rat, hagfish, lamprey, and cobra; and partial primary structures have been determined for C3 of rabbit and Xenopus (for review see Lambris2). In this study we purified C3 from different species, analyzed the conservation of its structural and functional features, and obtained the cDNA sequence of frog (Xenopus gilli), trout (Salmo gairdneri), and chicken C3. C3 was purified from swine (Po), rabbit (Rb), mouse (Mo), cobra (Co),Xenopm (Xe), axolotl (Ax), chicken (Ch), and trout (Tr) serum.3 All C3s tested were composed of two chains (a/p-chain) and contain a thioester bond within the a-chain. The two N-linked high-mannose carbohydrates found in human C3 were only conserved in Rb C3. In contrast, Xe, Ax, and Tr C3 have this moiety only in the f3-chain and Po and Mo C3 only the a-chain. Co C3, in contrast to cobra venom factor (CVF), lacks Con A binding carbohydrates in either chain. The NH,-termini of the Xe and Ax C3 p-chains were found to be blocked. TABLE 1 summarizes the features of C3 from different species characterized in this and other studies (for review see Lambris2). The cDNA sequences of Tr, Xe, and Ch C3s were obtained and the amino acid sequence similarity between the different C3s is shown in TABLE 2. Several C3 regions have been found to be highly conserved including that of the thioester bond, which is crucial to the function of C3. The thioester site, GCGEE, is 100% conserved in all species, and the amino acid sequences of the surrounding regions are highly similar. This conservation of the thioester bond and its surrounding hydrophobic amino acids emphasizes the functional importance of this region in maintaining,