This higher level of product formation was almost completely recovered in a

This higher level of product formation was almost

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This higher level of product formation was almost completely recovered in a high-speed supernatant fraction after mem- brane solubilization in a final concentration of 1% Triton X-100 (lane 4). In contrast, the supernatant fraction from un- extracted membranes demonstrated little activity (lane 3). It was important to determine whether the a -1,3 mannosyl- transferase activity was present in membranes from the model yeast S. cerevisiae . The availability of genome sequences and abundant genetic tools for this organism could greatly facilitate the study of such an enzyme. However, as S. cerevisiae does not produce a similar polysaccharide capsule, perhaps expression of the same transferase would not be expected. Tests of S. cerevisiae membranes prepared and assayed under conditions identical to those used with C. neoformans demonstrated that they produced no detectable amounts of product I in standard reactions (not shown). Membranes of Candida albicans , a non- encapsulated pathogenic fungus, were also tested in standard assays. These membranes produced no product I but instead modified the assay substrate to form both product II and the branched mannose trimer Man- a -1,6-(Man- a -1,3)-Man, a structure similar to that formed at several branch points of N -glycan synthesis (11). DISCUSSION The C. neoformans polysaccharide capsule is a fascinating structure with multiple roles in the biology of this fungal or- ganism. Mutant cells which are defective in capsule production are avirulent in animal models; correction of the defects by complementation restores both capsule production and the ability to cause fatal infection (4–6). Encapsulated organisms can deplete host complement by fixing it with great efficiency and are resistant to phagocytosis and killing by host effector cells. This leads to a reduction in host immune responses, such as cytokine production and antigen presentation (reviewed in reference 3). GXM is the dominant capsule component and plays a clear role in inhibiting host response. High serum or cerebrospinal fluid levels of this antigen also correlate with poor clinical prognosis (21). GXM is also the best-described capsule component in terms of its structure, due to extensive study by Cherniak and coworkers (8). Because of the unique structure of GXM, understanding its biosynthesis will yield FIG. 5. Effect of pH and cations on formation of products I and II. Assays were performed at pH 7.5 (dashed lines) or 8.5 (solid lines) in the presence of Mg or Mn as indicated, and the TLC plate was scanned. The abscissa is expanded to clarify the separation between the two products, and the region towards the solvent front (containing only free mannose) is not shown. The peak extending beyond the plot frame is at the plate origin. The ordinate is in arbitrary scanner units, and product peaks are identified above the top panel.
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  • Fall '20
  • Enzyme, Saccharomyces cerevisiae, Cryptococcus neoformans, Cryptococcus, C. neoformans

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