Purification, characterization and cDNA cloning of a phospholipase A2 inhibitor from the serum of the non-venomous snake Elaphe quadrivirgata

摘要

pThe serum of a non-venomous striated snake, iElaphe/iiquadriiv/iirgata/i, was found to contain phospholipase Asub2/sub (PLAsub2/sub) inhibitory proteins (PLIs). One of these inhibitors was purified by Sephadex G-200 gel filtration, Q-Sepharose FF ion-exchange chromatography and Butyl Sepharose 4FF hydrophobic chromatography. The purified PLI inhibited the enzymic activities of all PLAsub2/sub groups, including iElapidae/i venom (group-I), iViperidae/i venom (group-II) and honeybee PLAsub2/subs (group-III). The inhibitor was a 130 kDa glycoprotein consisting of two distinct subunits, A and B, of 30 and 29 kDa respectively; each of which was glycosylated with N-linked oligosaccharide chains. The cDNAs encoding the respective inhibitor subunits were isolated from a liver cDNA library by the use of probes, prepared by PCR, based on the partially determined amino-acid sequences of the corresponding subunits. The respective nucleotide sequences encoded 19-amino-acid-residue signal sequences, followed by 183- and 181-residue protein sequences for the A and B subunits respectively. The amino-acid sequences revealed that the iE./iiquadriiv/iirgata/i inhibitor corresponded to PLIγ, one of three kinds of inhibitors purified from venomous snakes. The existence of PLIγ in the serum of this non-venomous snake suggested that, besides having a protective role against the venom PLAsub2/subs of other venomous snakes, PLIγ has other important physiological functions in regulating local PLAsub2/sub activities; and thus it raises the possibility that PLIγ occurs in other animals, including mammals./p