Sequencing and two-dimensional structure prediction of a phospholipase A(2) inhibitor from the serum of the common tiger snake (Notechis scutatus)

A phospholipase A, inhibitor has been identified in the serum of the common tiger snake (Notechis scutatus). The inhibitor is composed of two chains, an alpha -chain and a beta -chain, that form a non-covalently associated co mplex capable of inhibiting the enzymatic activity of all phospholipase A. enzymes it was tested against. The alpha and beta -chains have been purifie d to homogeneity, digested and sequenced. From the peptide sequence generat ed, degenerate PCR primers were designed and used to elucidate the complete cDNA sequence of the chains using 5' and 3' RACE PCR. A total of three alp ha -chain isoforms were identified, only one isoform of the beta -chain was detected. The two-dimensional structure of the three alpha -chains and one beta -chain were predicted using five prediction programs (discrimination of secondary structure class; nearest neighbour secondary structure, profil e network from Heidelberg; self-optimised prediction method from multiple a lignment, SSPAL). For each protein chain a consensus prediction was generat ed. Results are discussed in relation to the function of the protein, and h ow they may influence the three-dimensional structure of the inhibitor. Add itionally, the sequences of several snake phospholipase A(2) inhibitors wer e used as the input for a motif prediction algorithm (MEME). The results ar e discussed in relation to the activity of these proteins. (C) 2001 Academi c Press.