Shibata N, Senbongi N, Hosoya T, Kawahara K, Akagi R, Suzuki A, Kobayashi H, Suzuki S, Okawa Y
Second Department of Hygienic Chemistry, Tohoku College of Pharmacy, Sendai, Japan.
Eur J Biochem. 1997 Jun 1;246(2):477-85. doi: 10.1111/j.1432-1033.1997.00477.x.
A mild acetolysis of the mannans of Candida stellatoidea was performed after acetylation to yielded an alpha-1,6-branched mannohexaose, the presence of which had been predicted from the appearance of a specific H1-H2-correlated cross-peak in two-dimensional homonuclear Hartmann-Hahn spectroscopy. In this study, we found that the de-O-acetylation of a 4-O-acetyl group at the branching point, the 3,6-di-O-substituted mannose unit, of an acetylated oligosaccharide by sodium methoxide is significantly slower than that of other acetyl groups. We could separate the 4-O-acetylated branching oligosaccharide from linear isomer using high-performance liquid chromatography. Before and after the de-O-acetylation of the purified branching oligosaccharide, their 1H-NMR signals were sequentially assigned by means of the nuclear Overhauser effect. In the sequential NMR assignment study, we showed that the alpha-1,6-linked mannose unit is attached to the 3-O-substituted unit based on the presence of NOE cross-peak between H1 of the branching mannose unit and H6 of the 3-O-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 alpha-1,6-branched oligosaccharide and the beta-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 beta-1,2-linked mannose units which have been reported to participate in pathogenicity via cytokine production and/or adherence. From the assignment results of H1-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.
对星状念珠菌甘露聚糖进行乙酰化后进行温和的乙酰解反应，得到一种α-1,6-分支的甘露六糖，二维同核Hartmann-Hahn光谱中一个特定的H1-H2相关交叉峰的出现预示了其存在。在本研究中，我们发现，用甲醇钠对乙酰化寡糖分支点处的3,6-二-O-取代甘露糖单元上的4-O-乙酰基进行脱O-乙酰化反应，其速度明显慢于其他乙酰基。我们可以使用高效液相色谱法将4-O-乙酰化的分支寡糖与线性异构体分离。在纯化的分支寡糖脱O-乙酰化前后，通过核Overhauser效应依次确定其1H-NMR信号。在连续NMR归属研究中，我们表明基于分支甘露糖单元的H1与3-O-取代甘露糖单元的H6之间存在NOE交叉峰，α-1,6-连接的甘露糖单元连接到3-O-取代单元上。几种寡糖对因子4血清与星状念珠菌甘露聚糖反应性的酶联免疫吸附抑制试验表明，α-1,6-分支寡糖和含β-1,2连接的寡糖具有抑制活性。该结果表明，因子4血清以及因子5和6血清含有针对β-1,2-连接甘露糖单元的抗体，据报道这些单元通过细胞因子产生和/或黏附参与致病性。根据寡糖和甘露聚糖的H1-H2相关交叉峰的归属结果，提出了甘露聚糖侧链的摩尔比。在本研究中，我们证明了星状念珠菌I型菌株的表位结构与白色念珠菌NIH B-792（血清型B）菌株相同。
