Molecular cloning and protein analysis of divergent forms of the complement component C3 from a bony fish, the common carp (Cyprinus carpio): presence of variants lacking the catalytic histidine

Abstract

Unlike mammals, some bony fish species have been reported to possess multiple forms of the complement component C3. To explore the structural and functional diversity of bony fish C3, we have isolated eight distinct cDNA clones encoding C3 from a single carp (Cypri-nus carpio). The eight sequences were grouped into five C3 types, designated C3-H1, C3-H2, C3-S, C3-Q1 and C3-Q2, each sharing 80-86 % amino acid sequence identity with the others. A striking amino acid substitution was noted at the position corresponding to the catalytic histidine, which is conserved in C3 from all the animals analyzed to date and provides the thioester with the ability to bind covalently to hydroxy groups on the target cells or to be hydrolyzed quickly; C3-S, C3-Q1 and C3-Q2 have serine, glutamine and glutamine residues, respectively, in place of the histidine which is conserved in C3-H1 and C3-H2. On the other hand, five distinct C3 forms, named C3-1 to C3-5, were purified from the serum of a single carp. N-terminal sequencing and covalent binding to [ 3 H]glycine identified C3-1 as the translated product of C3-S, while C3-2 was that of C3-H1, and C3-5 that of C3-H2. C3-1 showed a hemolytic activity threefold higher than that of C3-2, whereas C3-5 was inactive, suggesting that the thioester catalytic mechanism is not a necessary determinant for C3 activity and that C3 lacking the catalytic histidine plays a significant role in the complement system of carp and probably other bony fish.

Carp (Cyprinus carpio L.) innate immune factors are present before hatching

“…3A), purified carp C3 reacted with the anti-C3 antibody in several bands. The bands over 120 kDa in size are consistent with unreduced C3 protein, the two bands at approximately 115 kDa correspond to the a-chain and the band at approximately 60 kDa corresponds to the b-chain of the carp C3 protein [17]. C3 protein (only the b-chain) was already present in unfertilised eggs.…”

Molecular cloning and characterization of European seabass (Dicentrarchus labrax) and Gilthead seabream (Sparus aurata) complement component C3

“…Interestingly, this important domain was not found in the second isoform of European seabass. Previous studies on carp, showed the existence of an isoform without anaphylotoxin domain and more similar to human C3 rather than to carp C3-Q2 isoform [28] indicating a possible different functional role of this isoform.…”

“…For example, the rainbow trout, a quasi-tetraploid species, contains at least 3 different isoforms of C3 [25]. Multiple C3 isoforms have also been characterized in the Gilthead seabream at protein level [26,27] while in the common carp up to eight C3 forms have been described [28]. Unlike mammals, it has been demonstrated that some species of teleost possess multiple forms of functionally active C3 that may be the products of several genes and may vary in their ability to bind to various surfaces.…”

Recent advances on the complement system of teleost fish

“…In this regard, and as shown in mammals, it is likely that the architecture of the microbial surface would be an important factor in determining the binding of Hf or Bf/C2 to the C3b isoform newly bound into that surface [97]. In addition to being present in multiple isoforms, C3 has also been found to be polymorphic in carp [40]. Although it is tempting to speculate about possible functional differences among the polymorphic C3 forms, this intriguing idea remains to be further investigated in carp and other teleosts.…”

“…It has been suggested that differences in these residues account, at least in part, for the variations in the binding efficiencies of the various trout C3 isoforms [96]. The situation in the carp C3 isoforms is similar although the residue differences appear to occur only at the position equivalent to human His 1126 [40]. It will be important in the future to determine whether the variations in binding are solely due to changes in the aforementioned residues, or whether other residues and domains in the C3 protein are also involved.…”