Biosynthesis of glycoproteins in Candida albicans: Solubilization and partial characterization of dolichol phosphate mannose synthase and protein mannosyl transferases |
| |
Authors: | Blanca Lidia Arroyo-Flores Carlos Calvo-Méndez Arturo Flores-Carreón Everardo López-Romero |
| |
Affiliation: | (1) Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Apartado Postal No. 187, Guanajuato, Gto, 36000, México;(2) Departamento de Genética y Biología Molecular, CINVESTAV del IPN, Apartado Postal 14-740, México, D.F, 07000, México |
| |
Abstract: | Incubation of a mixed membrane fraction isolated from C. albicans yeast cells with Nonidet P-40 at a detergent/protein ratio as low of 0.025 (0.016–0.019%, w/v) yielded a soluble fraction that catalyzed the transfer of mannose from GDP-[14C] Man into dolichol phosphate mannose and from this intermediate into mannoproteins. Over 95% of the sugar in mannoproteins was O-linked as judged from its release after -elimination. Mannose was identified as the sole product after this treatment. Transfer activity did not depend on exogenous lipid acceptor indicating that the latter was solubilized along with the mannosyl transferases. Synthesis of mannolipid and mannoproteins occurred at optima temperatures of 20 °C and 37 °C, respectively, and at a pH in the range of 7.5-9.5. Mannosyl transfer into the mannolipid was stimulated by Mg2+and inhibited by Ca2+and Mn2+whereas mannoprotein labeling was stimulated by Mn2+and to a lower extent by Mg2+. When measured as a function of substrate concentration, the synthesis of the mannolipid was a nearly linear function of GDP-Man concentration in the range of 5 to 32 M whereas protein mannosylation exhibited hyperbolic kinetics with saturation reached at about 10 M. The solubilized preparation was able to utilize an exogenous source of mannolipid as sugar donor for protein mannosylation. Dinucleotides and, to a higher extent trinucleotides, inhibited mannosyl transfer into the mannolipid and hence into mannoproteins. |
| |
Keywords: | Candida glycoprotein biosynthesis mannosyl transferases |
本文献已被 SpringerLink 等数据库收录! |
|