Two Novel Techniques for Determination of Polysaccharide Cross-Links Show that Crh1p and Crh2p Attach Chitin to both β(1-6)- and β(1-3)Glucan in the Saccharomyces cerevisiae Cell Wall
Two Novel Techniques for Determination of Polysaccharide Cross-Links Show that Crh1p and Crh2p Attach Chitin to both β(1-6)- and β(1-3)Glucan in the Saccharomyces cerevisiae Cell Wall
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DOI：
10.1128/ec.00228-09
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发表时间：
2009-11-01
期刊：
影响因子：
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通讯作者：
Cabib, Enrico
中科院分区：
文献类型：
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作者：
Cabib, Enrico
先前的工作表明，在用β(1-3)或β(1-6)葡聚糖酶消化之前或之后，利用羧甲基化来增溶酵母细胞壁，然后进行尺寸层析，表明几丁质与β(1-6)葡聚糖的结合是额外需要转糖基酶Crh1p和Crh2p/Utr2p的。通过这种技术，crh1 Delta crh2 Delta突变体似乎仍然含有相当大比例的与Delta(1-3)葡聚糖相连的甲壳素。现在已经开发了两种新的方法来分析细胞壁中的多糖交联物。一种是基于Curdlan，一种β(1-3)葡聚糖，对羧甲基化细胞壁中的β(1-3)葡聚糖链的亲和力。另一种是细胞壁甲壳素的原位脱乙酰化，生成的壳聚糖可以直接用醋酸提取(游离壳聚糖)，也可以用不同的葡聚糖酶消化后提取(结合壳聚糖)。这两种方法都表明，crh1 Delta crh2 Delta菌株中的所有几丁质都是游离的。对以前使用的程序的重新检查发现，所使用的β(1-3)葡聚糖酶制剂(发酵酶)被少量的内切酶污染，这导致与双重突变体的错误结果。从酶中去除几丁质酶后，三种方法都得到了一致的结果。因此，Crh1p和Crh2p催化甲壳素向β(1-3)-葡聚糖和β(1-6)葡聚糖的转移，细胞壁中所有甲壳素交联物的生物合成机制已经建立。
Previous work, using solubilization of yeast cell walls by carboxymethylation, before or after digestion with beta(1-3)- or beta(1-6) glucanase, followed by size chromatography, showed that the transglycosylases Crh1p and Crh2p/Utr2p were redundantly required for the attachment of chitin to beta(1-6) glucan. With this technique, crh1 Delta crh2 Delta mutants still appeared to contain a substantial percentage of chitin linked to Delta(1-3) glucan. Two novel procedures have now been developed for the analysis of polysaccharide cross-links in the cell wall. One is based on the affinity of curdlan, a beta(1-3)glucan, for beta(1-3)glucan chains in carboxymethylated cell walls. The other consists of in situ deacetylation of cell wall chitin, generating chitosan, which can be extracted with acetic acid, either directly (free chitosan) or after digestion with different glucanases (bound chitosan). Both methodologies indicated that all of the chitin in crh1 Delta crh2 Delta strains is free. Reexamination of the previously used procedure revealed that the beta(1-3) glucanase preparation used (zymolyase) is contaminated with a small amount of endochitinase, which caused erroneous results with the double mutant. After removing the chitinase from the zymolyase, all three procedures gave coincident results. Therefore, Crh1p and Crh2p catalyze the transfer of chitin to both beta(1-3)- and beta(1-6) glucan, and the biosynthetic mechanism for all chitin cross-links in the cell wall has been established.