Molecular and functional characterization of a CD59 analogue from large yellow croaker Pseudosciana crocea.

CD59 is a widely distributed membrane-bound inhibitor of the cytolytic membrane attack complex (MAC) of complement. Here, the cDNA of a CD59 analogue was cloned from large yellow croaker (Pseudosciana crocea), a marine fish (LycCD59), by expressed sequence tags (EST) and RACE techniques. The open reading frame (ORF) of 351 nucleotides (nt) of LycCD59 encodes a polypeptide of 117 amino acids (aa), which includes a putative 20-aa NH(2)-signal peptide and a 97-aa coding region with a putative GPI-anchoring site at Asn(71). The deduced LycCD59 protein shared the structural feature of mammalian CD59, including a conserved cysteine skeleton responsible for the formation of disulfide bonds, and a similar pattern of hydrophobic termini. RT-PCR analysis showed that LycCD59 mRNA was broadly expressed in various tissues examined, except for intestine. And Northern blot analysis revealed a single LycCD59 transcript of approximately 1.0kb. LycCD59 expression in blood, spleen, and kidney was significantly up-regulated during 24h of induction with poly(I:C) or inactivated trivalent bacterial vaccine as determined by a relative quantitative real-time PCR analysis, and a coordinated up-regulation of LycCD59 and complement C3 and C7 mRNA was also found in these three tissues post-induction although their up-regulation pattern and extent were somewhat different in various tissues with poly(I:C) or bacterial vaccine. The recombinant protein of LycCD59 produced in E. coli was shown to significantly inhibit the erythrocyte lysis of tilapia (Oreochromis niloticus) in an in vitro hemolytic system, which was mediated by serum from large yellow croaker and tilapia, respectively, but not from mouse and chicken, suggesting that LycCD59 has a species-selective inhibition of complement activation. These results represent the first functional identification of a CD59 analogue in teleost fish, strongly suggesting the presence of regulatory mechanism for terminal complement pathway in teleost fish.