Cell Wall β-(1,6)-Glucan of Saccharomyces cerevisiae STRUCTURAL CHARACTERIZATION AND IN SITU SYNTHESIS
Cell Wall β-(1,6)-Glucan of Saccharomyces cerevisiae STRUCTURAL CHARACTERIZATION AND IN SITU SYNTHESIS
复制标题
DOI：
10.1074/jbc.m807667200
复制
发表时间：
2009-05-15
影响因子：
4.8
通讯作者：
Latge, Jean-Paul
中科院分区：
文献类型：
--
作者：
Aimanianda, Vishukumar;Clavaud, Cecile;Latge, Jean-Paul
尽管β-(1，6)-葡聚糖在酵母细胞壁中起着重要的作用，但它的确切组成还没有得到很好的表征。用重组β-(1，3)-葡聚糖酶溶解一株野生型酿酒酵母的细胞壁碱不溶性部分，然后用阴离子交换柱层析，我们观察到在溶剂梯度运行结束时洗脱出的可溶性聚合物。进一步的表征表明，该聚合物的相对分子质量约为38 kDa，只能被β-(1，6)-葡聚糖酶降解。气相色谱-质谱分析和核磁共振(H-1和C-13)分析证实它是一种β-(1，6)-葡聚糖聚合物，平均每5个残基就有一个分支，侧链上有一个或两个β(1，3)连接的葡萄糖单元。在已知在β-(1，6)葡聚糖生物合成中起关键作用的Kre基因突变体(kre9和kre5)的相应酶切中，这一聚合峰显著降低。在目前的研究中，我们开发了一种生化方法，其中UDP-[C-14]葡萄糖与渗透的酿酒酵母细胞孵育导致合成一种与从细胞壁分离的支化的β-(1，6)-葡聚糖化学上相同的聚合物。利用该方法，确定了β-(1，6)-葡聚糖合成活性所必需的参数。
Despite its essential role in the yeast cell wall, the exact composition of the beta-(1,6)-glucan component is not well characterized. While solubilizing the cell wall alkali-insoluble fraction from a wild type strain of Saccharomyces cerevisiae using a recombinant beta-(1,3)-glucanase followed by chromatographic characterization of the digest on an anion exchange column, we observed a soluble polymer that eluted at the end of the solvent gradient run. Further characterization indicated this soluble polymer to have a molecular mass of similar to 38 kDa and could be hydrolyzed only by beta-(1,6)-glucanase. Gas chromatography-mass spectrometry and NMR(H-1 and C-13) analyses confirmed it to be a beta-(1,6)-glucan polymer with, on average, branching at every fifth residue with one or two beta-(1,3)-linked glucose units in the side chain. This polymer peak was significantly reduced in the corresponding digests from mutants of the kre genes (kre9 and kre5) that are known to play a crucial role in the beta-(1,6)glucan biosynthesis. In the current study, we have developed a biochemical assay wherein incubation of UDP-[C-14] glucose with permeabilized S. cerevisiae yeasts resulted in the synthesis of a polymer chemically identical to the branched beta-(1,6)-glucan isolated from the cell wall. Using this assay, parameters essential for beta-(1,6)-glucan synthetic activity were defined.