Subunit structure and carbohydrate composition of the extracellular acid phosphatase of Rhodotorula glutinis.

The extracellular acid phosphatase (EC 3.1.3.2) of Rhodotomla glutinis is a glycoenzyme composed of 608 protein, 35% mannose, and 5% N-acetylglucosamine by weight. The native enzyme elutes from a column of BioGel A-5M agarose with an M, 5 185,000 but on sodium dodecyl sulfate-acrylamide gel electrophoresis migrates with an M, = 93,000. The acid phosphatase thus appears to consist of two subunits of equal size, a conclusion also supported by cross-linking experiments with dimethyl suberimidate. Digestion of denatured acid phosphatase with endo-8-N-acetylglucosaminidase H from Streptomyces plicatus removes 80% of the mannose and 50% of the N-acetylglucosamine, with the remaining mannose liberated either by a-mannosidase or by alkaline /3 elimination. The carbohydratedepleted protein has an Mr = 59,500 by sodium dodecyl sulfate-acrylamide gel electrophoresis and by ultracentrifugation in 6 M guanidine hydrochloride. The NH2 terminus appears to be blocked, as no amino acid residues are released on 10 cycles of automated Edman degradation. However, COOH-terminal sequencing with carboxypeptidases A, B, and Y releases, in order, 1 mol of -Ser-Lys-Ala-OH/59,500 g of protein, suggesting that the subunits are identical. Chemical analyses of acid phosphatase indicate that the subunits contain an average of 11 asparagine-linked oligosaccharide chains. The mannose resistant to the endoglycosidase is shown to be distributed in 15 0-glycosidic chains/ subunit, 9 linked to threonine and 6 to serine. Chromatography on a calibrated Bio-Gel P-4 column reveals the N-linked oligosaccharide chains to contain from 9 to 18 mannoses, while those associated with serine and threonine contain from 1 to 5 mannose residues. These studies show that mannosyl oligosaccharides coexist n both 0and N-linked configurations on the subunits of R glutinis acid phosphatase, an occurrence which appears to be unique among the fungal glycoproteins characterized thus far.