Makiko Nakahara

Expansion of genes encoding complement components in bony fish: biological implications of the complement diversity.

The complement system is a major humoral component of vertebrate defenses for tagging and killing target microorganisms. Recent molecular analyses have uncovered a striking feature of bony fish complement, namely that several complement components are encoded by multiple genes. In this review, the structural diversity of C3, C4, C5, factor B, C2, C1r/s and MASP are discussed with special reference to their functional differentiation, mainly focusing on the common carp (Cyprinus carpio), a tetraploidized teleost. In carp, all the members (C3, C4, C5 and a non-complement protein alpha2-macroglobulin) of the thioester-containing protein family are present in multiple isotypes, differing in the primary structures of various functional sites. Three factor B/C2-like isotypes identified in carp showed distinct expression pattern (sites and inducibility), with one behaving as an acute-phase reactant. Two C1r/C1s/MASP2-like isotypes also contain an amino acid substitution that likely affects their substrate specificity. Overall, the present data suggest that the expanded genes of the carp complement system produce more diversified functional components than are known for mammals. The biological significance of this diversity is discussed.

Molecular cloning of the complement regulatory factor I isotypes from the common carp (Cyprinus carpio)

Abstract.Factor I is a novel serine protease that regulates complement activation. Here we report the complete primary structure of two isotypic factor Is isolated from the common carp (Cyprinus carpio), a pseudotetraploid teleost. A carp hepatopancreas cDNA library was screened using two RT-PCR-amplified cDNA fragments encoding part of the carp factor I-like serine protease domain. Two distinct cDNA clones, designated FI-A and FI-B, were isolated. Their deduced amino acid sequences share 75.2% identity with each other. FI-A has a typical factor I-like domain organization composed of two disulfide-linked polypeptides (H-chain and L-chain). On the other hand, FI-B contains a novel sequence of 115 amino acids inserted at the N-terminus of the H-chain. Genomic Southern hybridization suggests that FI-A and FI-B are encoded by distinct genes in the carp genome. Expression analysis by RT-PCR revealed that the major site of FI-A expression is the ovary, whereas FI-B expression is detected mainly in the hepatopancreas at a level higher than that of FI-A. The present data, taken together, suggest that carp have duplicated genes coding for factor I, and FI-B with the novel insertion plays a dominant role in the complement system. In addition, homology search of the fugu genome database using the carp FI-A and FI-B sequences identified a putative fugu factor I gene, which has an exon/intron organization different from that of the human orthologue.

A complement C3 fragment equivalent to mammalian C3d from the common carp (Cyprinus carpio): generation in serum after activation of the alternative pathway and detection of its receptor on the lymphocyte surface

A terminal degradation product (C3d) of mammalian complement component C3 plays an important role in modulation of the adaptive immune response through the interaction with complement receptor type 2 (CR2) on B cells. The present study is aimed at determining whether this is a functional bridge between the innate and adaptive immune systems in bony fish. The fragmentation of the complement component C3 in carp (Cyprinus carpio) serum, activated with zymosan, was analysed to ascertain if carp C3 also generates a mammalian C3d-like fragment under physiological conditions. A 35 kDa peptide reactive to the anti-carp C3 alpha-chain was detected on the zymosan particles and in the activated serum. Its N-terminal amino acid sequence identified it as carp C3d derived from the C3-H1 isoform. Another C3 isoform, C3-S, of carp was found to yield a C3d fragment at lower efficiency than C3-H1. Recombinant C3d fragments derived from C3-H1 and C3-S were produced in Escherichia coli as fusion proteins with glutathione-S-transferase (GST), and used for ligands to examine the presence of a possible CR2-like C3d receptor on carp lymphocytes. An enzyme-linked immunoadsorbent assay system, using the recombinant C3d proteins and anti-GST on a microplate to which was attached carp peripheral lymphocytes, detected a significant binding of carp C3d to the lymphocyte. The degree of binding of C3-H1-derived C3d was higher than that of C3-S-derived C3d. In addition, the binding of both ligands was inhibited by anti-C3 alpha-chain, but not by EDTA or EGTA, indicating that the putative C3d receptor does not require divalent cation. These properties agree well with those reported for mammalian CR2.