Molecular cloning and expression of channel catfish, Ictalurus punctatus, complement membrane attack complex...

The channel catfish, Ictalurus punctatus, complement membrane attack complex inhibitor CD59 gene was cloned and analyzed. Total RNA from tissues was isolated and cDNA libraries were constructed by the rapid amplification cDNA end (RACE) method. The gene-specific primers in conjunction with the RACE primers were used to PCR amplify 5'- and 3'-ends of the CD59 transcript. The complete channel catfish CD59 cDNA comprised 1109 bp including a 132-bp 5'-untranslated, a 360-bp open reading frame, and a 617-bp 3'-untranslated region. The open reading frame encodes a putative protein of 119-amino acid residues with calculated molecular mass (without potential glycosylation) of 13.2 kDa. However, the CD59 protein has a potential N-glycosylation site at the Asn35 residue. The degree of conservation of the channel catfish amino acid sequence to mammalian counterparts is 24-32%, while to those of other fish species is 44-54%. One remarkable feature is that the number and position of cysteine residues were conserved in the mature protein among species examined, suggesting that although the primary amino acid sequences are divergent, the three-dimensional structure of CD59 via disulfide linkages may be conserved through the evolutionary process. The putative protein could be further divided into three domains: a 21-amino acid signal peptide at the N-terminus, a 72-amino acid mature protein, and a 26-amino acid glycosylphosphatidylinositol (GPI) anchoring signal peptide at the carboxyl terminus. CD59 was expressed in all channel catfish tissues studied, suggesting that like mammals, channel catfish CD59 is constitutively expressed.