Biosynthesis of Glycoproteins in the Human Pathogenic Fungus Sporothrix Schenckii: Synthesis of Dolichol Phosphate Mannose and Mannoproteins by Membrane-Bound and Solubilized Mannosyl Transferases

Abstract

A membrane fraction obtained from the filamentous form of Sporothrix schenckii was able to transfer mannose from GDP-Mannose into dolichol phosphate mannose and from this inTermediate into mannoproteins in coupled reactions catalyzed by dolichol phosphate mannose synthase and protein mannosyl transferase(s), respectively. Although the transfer reaction depended on exogenous dolichol monophosphate, membranes failed to use exogenous dolichol phosphate mannose for protein mannosylation to a substantial extent. Over 95% of the sugar was transferred to proteins via dolichol phosphate mannose and the reaction was stimulated several fold by Mg2+ and Mn2+. Incubation of membranes with detergents such as Brij 35 and Lubrol PX released soluble fractions that transferred the sugar from GDP-Mannose mostly into mannoproteins, which were separated by affinity chromatography on Concanavilin A-Sepharose 4B into lectin-reacting and non-reacting fractions. All proteins mannosylated in vitro eluted with the lectin-reacting proteins and analytical electrophoresis of this fraction revealed the presence of at least nine putative mannoproteins with molecular masses in the range of 26-112 kDa. The experimental approach described here can be used to identify and isolate specific glycoproteins mannosylated in vitro in studies of O-glycosylation.

Purification and biochemica characterisation of endoplasmic reticulum α 1,2-mannosidase from Sporothrix schenckiil

“…The mycelial form was propagated as described (Ruiz-Baca et al 2005). The yeast form was induced and propagated by growing conidia in 2 L Erlenmeyer flasks containing 600 mL of YPD medium, pH 7.3 and shaking at 120 rpm.…”

Isolation of Sporothrix schenckii MNT1 and the biochemical and functional characterization of the encoded α1,2-mannosyltransferase activity

“…The mycelial form of Sp. schenckii was propagated in YPD broth, pH 4.5, as described previously (Ruiz-Baca et al, 2005), while transition to yeast morphology was achieved in YPD medium, pH 7.4, as reported previously (Mora-Montes et al, 2010b). The C. albicans strains were grown at 28 uC in YPD medium or in SC medium [0.67 % (w/v) yeast nitrogen base without amino acids, 2 % (w/v) glucose, 0.077 % (w/v) complete supplement mixture minus uracil] supplemented with 50 mg uridine ml 21 when necessary.…”