Isolation of beta-linkage-containing side chain oligosaccharides from the mannan of Candida guilliermondii IFO 10279 strain has been conducted by acetolysis under mild conditions. A structural study of these oligosaccharides by one- and two-dimensional NMR and methylation analyses indicated the presence of extended oligosaccharide side chains with two consecutive beta-1,2-linked mannose units at the nonreducing terminal of alpha-linked oligosaccharides. The linkage sequence present in this mannan, Man beta 1-->2Man alpha 1-->3Man alpha-->, has also been found in the mannan of Saccharomyces kluyveri but not in the mannan of Candida species. Furthermore, these oligosaccharides are branched at position 6 of the 3-O-substituted mannose units as follows. (Carbohydrate sequence in text) Structure 1 and (Carbohydrate sequence in text) Structure 2 The H-1 signals of the mannose units substituted by a 3,6-di-O-substituted unit showed a significant upfield shift (delta delta = 0.04-0.08 ppm) due to a steric effect. The inhibition of an enzyme-linked immunosorbent assay between the mannan of C. guilliermondii and factor 9 serum with oligosaccharides obtained from several mannans indicated that only the oligosaccharides with the above structure were active, suggesting that these correspond to the epitope of antigenic factor 9.

A mild acetolysis of the mannans of Candida stellatoidea was performed after acetylation to yielded an a-1,6-branched mannohexaose, the presence of which had been predicted from the appearance of a specific HI -H2-correlated cross-peak in two-dimensional homonuclear Hartmann-Hahn spectroscopy. In this study, we found that the de-0-acetylation of a 4-0-acetyl group at the branching point, the 3,6-di-0-substituted mannose unit, of an acetylated oligosaccharide by sodium methoxide is significantly slower than that of other acetyl groups. We could separate the 4-0-acetylated branching oligosaccharide from linear isomer using high-performance liquid chromatography. Before and after the de-0-acetylation of , the purified branching oligosaccharide, their 'H-NMR signals were sequentially assigned by means of the nuclear Overhauser effect. In the sequential NMR assignment study, we showed that the a-1,6-linked mannose unit is attached to the 3-0-substituted unit based on the presence of NOE cross-peak between HI of the branching mannose unit and H6 of the 3-0-substituted mannose unit. An enzyme-linked immunosorbent inhibition assay of the reactivity of factor 4 serum to C. stellatoidea mannan by several oligosaccharides indicated that the a-1,6-branched oligosaccharide and the p-1,2 linkage-containing oligosaccharides showed inhibitory activity. This result indicates that factor 4 serum, as well as factor 5 and 6 sera, contains antibodies against p-1,2-linked mannose units which have been reported to participate in pathogenicity via cytokine production and/or adherence. From the assignment results of Hl-H2-correlated cross-peaks of oligosaccharides and mannans, the molar ratio of the mannan side chains was proposed.In this study, we demonstrated that the epitope structure of the C. stellatoidea type I strains was the same as that of the C. albicans NIH B-792 (serotype B) strain.Keywords : Candida stellatoidea ; mannan ; mannooligosaccharide ; NMR ; assignment.Candida stellatoidea is presently treated as a synonym for Candida albicans, an opportunistic yeast-like pathogen known to cause serious infection in immunosuppressed patients. In 1961, Hasenclever and Mitchell [l] reported that the antigenicity of C. stellatoidea cells was related to C. albicans serotype B cells. Later, however, Kwon-Chung et al. [2] reported that the C. stellatoidea species include two karyotypes, I and 11, and that the two types are distinguishable on the basis of their virulence toward mice, resistance to ultraviolet irradiation, growth rate in vitro, and proteinase activity. Furthermore, they showed that type I and type I1 C. stellatoidea express C. albicans serotypes B and A antigens, respectively, using the C. albicans serotype A-specific monoclonal antibody {3]. They also reported that type I1 C. stellatoidea is an a-glucosidase mutant of C. albicans [4] and type I C. stellatoidea is a variant of C. albicans [5].Recently, we found the existence of two a-1,6-branched side chains in the mannan of Candida albicans NIH B-792 (serotype B) stra...