b Amphomycin was tested in the presence of 5 mM CaCl 2 the appropriate control

B amphomycin was tested in the presence of 5 mm cacl

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b Amphomycin was tested in the presence of 5 mM CaCl 2 ; the appropriate control is listed in the previous row. 5486 DOERING J. B ACTERIOL .
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albicans . These results are consistent with the idea that the a -1,3 transferase described in this work is unique to C. neofor- mans and that it is likely to play a role in GXM construction. In these experiments radiolabeled products larger than tri- saccharides were not detected. Because the substrate was in substantial molar excess over the radiolabel under standard assay conditions, it is not surprising that only a single round of addition would occur. On the other hand, if this enzyme func- tions in the construction of an extensive linear mannan, it might be expected to display processivity. One possible expla- nation for the absence of evidence for processivity is that a factor required for this behavior was absent or inactive in the membranes prepared for these assays. Another possibility is based on the structure of GXM, which is composed of repeat- ing mannose trimers with their associated side chains (7, 8). It is conceivable that individual trimannose units are constructed and modified with side chains before the polymer is assembled. Kinetic and substrate studies will address these mechanistic questions in the future, once enzyme purification has pro- gressed. A second mannosyltransferase present in wild-type crypto- coccal membranes was discovered in the course of this work and shown to form a -1,2 linkages. The precise position of mannose attachment was not determined in these experiments, as the results from both enzymatic and chemical degradation experiments are consistent with either a linear mannose trimer (Man- a -1,2-Man- a -1,3-Man) or the branched structure Man- a -1,2-(Man- a -1,3)-Man. It is most likely that the product is a linear species, because it migrates fairly close to the linear standard derived from GXM (Fig. 3), separated by a distance similar to that which separates a -1,3- and a -1,2-linked dimer standards (Fig. 2). Branched species exhibit very different be- havior on this TLC system (not shown). Additionally, neither O-linked, N-linked, nor glycosyl phosphatidylinositol struc- tures of yeasts that have been described contain the branched- structure motif (11). Mannose in a -1,2 linkage is not found in either GXM or GalXM (28). It may be present, however, in the mannoprotein component of the capsule (4% by mass [7]). Protein-linked glycans of C. neoformans have not been analyzed directly, but examination of these structures in S. cerevisiae may be instruc- tive in suggesting a role for the a -1,2 mannosyltransferase activity. While adjacent a -1,2 and a -1,3 linkages between man- nose residues do occur in O-linked glycans of S. cerevisiae , they are present in reverse order to the structure probably formed here (24). However, there are two occasions during core N- glycan synthesis when Man- a -1,2-Man- a -1,3-Man is made.
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  • Fall '20
  • Enzyme, Saccharomyces cerevisiae, Cryptococcus neoformans, Cryptococcus, C. neoformans

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