Biosynthesis of Oligogalactosyl Side Chains of the Cell Envelope Glycoprotein of Cryptococcus laurentii

Abstract

Abstract A particulate enzyme preparation from the fungus imperfectus Cryptococcus laurentii var. flavescens (NNRL Y-1401) catalyzes the transfer of galactosyl residues from UDP-[14C]-galactose to an endogenous acceptor. The radioactive reaction product is particle-bound, but is solubilized by pronase treatment. The solubilized radioactive material migrates as a single peak on a column of Sepharose 4-B, indicating that the galactosyl residues are transferred to an endogenous polymeric acceptor of high molecular weight. After elution from Sepharose 4B the material was found to contain 70% carbohydrate and 30% protein. The monosaccharides released after acid hydrolysis are mannose, galactose, and xylose, galactose being the only radioactive monosaccharide constituent. The radioactive reaction product is retained on DEAE-cellulose, from which over 80% of it is eluted with 0.5 m NaCl together with endogenous carbohydrate and protein. After treatment with NaOH-NaBH4, the radioactive material no longer binds to DEAE-cellulose. Subsequent gel filtration on Sephadex G-25 results in a symmetrical radioactive peak of approximately 2000 molecular weight, suggesting liberation of a dodecasaccharide chain. Treatment with NaOH-NaB3H4 followed by complete acid hydrolysis allows the isolation of tritium-labeled alanine and α-aminobutyric acid, indicating that carbohydrate side chains are linked through seryl and threonyl residues to protein. The major radioactive disaccharides isolated after partial acid hydrolysis are 6-O-α-[14C]galactosyl-O-[14C]galactose and O-β-[14C]-galactosyl-O-mannose. The data suggest addition of multiple galactosyl residues to an endogenous glycoprotein acceptor: the first galactosyl residue is linked to a mannosyl residue forming a β linkage; subsequently additional galactosyl residues are transferred sequentially forming linear chains containing 6-O-α-galactosyl-O-galactosyl linkages.