Side group addition by xylosyltransferase and glucuronyltransferase in biosynthesis of capsular polysaccharide inCryptococcus neoformans

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Abstract
The capsular polysaccharide of Cryptococcus neoformans consists of an O-acetylated mannan backbone with xylosyl and glucuronyl substituents. We have studied two enzymes in the biosynthetic pathway of this polysaccharide. A particulate enzyme preparation contained xylosyltransferase and glucuronyltransferase activities with pH optima of 7.5 and < 6.5, and optimum incubation temperatures of 25.degree. and 37.degree.C respectively. O-acetylated mannan served as an acceptor for glucuronyl residues but xylomannan did not. Glucuronomannan served as an acceptor for xylosyl residues but acetylated mannan did not. These observations suggest that glucuronate is added before xylose and after acetate. This hypothesis was supported by studies of lipid-linked sugars in which a microsomal preparation was incubated with various combinations of nucleotide sugars. With GDP-mannose, UDP-glucuronate and UDP-[14C]xylose; GDP-mannose, UDP-[14C]glucuronate and UDP-xylose; or GDP-mannose and UDP-[14C]glucuronate, no evidence for a lipid-linked monosaccharide was obtained. However, lipid-linked xylose was detected only if GDP-mannose and UDP-[14C]xylose were provided, a finding consistent with a pool of lipid-linked xylose which is only transferred following glucuronylation. The order of addition inferred is mannosyl, acetyl, glucuronyl and xylosyl.