microbiology, Vol 141, 2289-2294, Copyright © 1995 by Society for General Microbiology
Biosynthesis of glycoproteins in Candida albicans: activity of dolichol phosphate mannose synthase and protein mannosylation in a mixed membrane fraction
BL Arroyo-Flores, C Calvo-Mendez, A Flores-Carreon and E Lopez-Romero
Instituto de Investigacion en Biologia Experimental, Facultad de Quimica, Universidad Autonoma de Guanajuato, Mexico.
A mixed membrane fraction (MMF) was isolated from yeast cells of Candida
albicans with the ability to synthesize dolichol phosphate mannose
(Dol-P-Man) from GDP-Man and dolichol phosphate (Dol-P) and transfer the
sugar to proteins. Temperature of incubation (20-37 degrees C) did not
affect the synthesis of Dol-P-Man but protein mannosylation occurred better
at physiological temperatures (28 degrees C and 37 degrees C). Most of the
sugar (87-93%) in the mannoproteins was O-linked as judged by its release
by beta-elimination. Mannose was identified as the sole product after this
treatment. Following incubation of MMF with the sugar donor, parallel
levels of Dol-P-Man and mannosylated proteins were detected up to 30 min.
Thereafter, Dol-P- Man levels reached a steady value whereas mannoproteins
rapidly accumulated. Lipid-linked oligosaccharides were also detected in
incubation mixtures, though in much lower amounts than those of Dol-P- Man
or mannoproteins. Dol-P-Man synthase activity increased proportionally in
response to increasing concentrations of either of the two enzyme
substrates. A Km value of 0.36 microM for GDP-Man was calculated. MMF
failed to use exogenous Dol-P-Man for protein glycosylation. Specific
inhibition of Dol-P-Man synthesis with amphomycin was concomitant with a
parallel decrease in protein mannosylation, indicating that most of the
sugar is transferred to protein via the carrier lipid. Results are
discussed in terms of the role of Dol-P-Man in protein glycosylation in C.
albicans.
