This higher level of product formation was almost completelyrecovered in a high-speed supernatant fraction after mem-brane solubilization in a final concentration of 1% TritonX-100 (lane 4). In contrast, the supernatant fraction from un-extracted membranes demonstrated little activity (lane 3).It was important to determine whether thea-1,3 mannosyl-transferase activity was present in membranes from the modelyeastS. cerevisiae. The availability of genome sequences andabundant genetic tools for this organism could greatly facilitatethe study of such an enzyme. However, asS. cerevisiaedoes notproduce a similar polysaccharide capsule, perhaps expressionof the same transferase would not be expected. Tests ofS.cerevisiaemembranes prepared and assayed under conditionsidentical to those used withC. neoformansdemonstrated thatthey produced no detectable amounts of product I in standardreactions (not shown). Membranes ofCandida albicans, a non-encapsulated pathogenic fungus, were also tested in standardassays. These membranes produced no product I but insteadmodified the assay substrate to form both product II and thebranched mannose trimer Man-a-1,6-(Man-a-1,3)-Man, astructure similar to that formed at several branch points ofN-glycan synthesis (11).DISCUSSIONTheC. neoformanspolysaccharide capsule is a fascinatingstructure with multiple roles in the biology of this fungal or-ganism. Mutant cells which are defective in capsule productionare avirulent in animal models; correction of the defects bycomplementation restores both capsule production and theability to cause fatal infection (4–6). Encapsulated organismscan deplete host complement by fixing it with great efficiencyand are resistant to phagocytosis and killing by host effectorcells. This leads to a reduction in host immune responses, suchas cytokine production and antigen presentation (reviewed inreference 3). GXM is the dominant capsule component andplays a clear role in inhibiting host response. High serum orcerebrospinal fluid levels of this antigen also correlate withpoor clinical prognosis (21). GXM is also the best-describedcapsule component in terms of its structure, due to extensivestudy by Cherniak and coworkers (8). Because of the uniquestructure of GXM, understanding its biosynthesis will yieldFIG. 5. Effect of pH and cations on formation of products I and II. Assayswere performed at pH 7.5 (dashed lines) or 8.5 (solid lines) in the presence of Mgor Mn as indicated, and the TLC plate was scanned. The abscissa is expanded toclarify the separation between the two products, and the region towards thesolvent front (containing only free mannose) is not shown. The peak extendingbeyond the plot frame is at the plate origin. The ordinate is in arbitrary scannerunits, and product peaks are identified above the top panel.
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Enzyme, Saccharomyces cerevisiae, Cryptococcus neoformans, Cryptococcus, C. neoformans
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