STRUCTURAL STUDIES OF A MAJOR HEMORRHAGIN (RHODOSTOXIN) FROM THE VENOM OF CALLOSELASMA-RHODOSTOMA (MALAYAN PIT VIPER)

The complete amino acid sequence, disulfide Linkages, glycosylation si tes, and carbohydrate structure of rhodostoxin, the major hemorrhagin from Calloselasma rhodostoma (Malayan pit viper), have been determined , This sequence confirmed the deduced amino acid sequence of the putat ive hemorrhagic protein encoded by the prorhodostomin cDNA of C. rhodo stoma. Rhodostoxin contained four disulfide bonds that link Cys19-Cys6 0, Cys117-Cys198, Cys157-Cys182, and Cys159-Cys165. It is the first fo ur-disulfide proteinase reported among all known venom metalloproteina ses, which are either of the two-disulfide or three-disulfide type, Pe ptide-mapping and dot-blotting experiments showed the presence of two glycopeptides. Subsequent sequencing of these peptides established tha t the N-glycosylation sites are located at residues 91 and 181 of the amino acid sequence of the matured protein, Mass spectrometric analyse s of these glycopeptides showed that they contain an oligosaccharide s tructure consisting of 4 units of N-acetylglucosamine, 5 units of hexo se, 1 unit of fucose, and 2 units of neuraminic acids, The complete ca rbohydrate structure was then established by 2-D mapping analysis of t he pyridylamino-oligosaccharides after hydrazinolysis and pyridylamina tion of the glycan chains. (C) 1996 Academic Press, Inc.