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Preferential transfer of truncated oligosaccharides to the first sequon of yeast exoglucanase in Saccharomyces cerevisiae alg3 cells

机译:优先将截短的寡糖转移到酿酒酵母alg3细胞中的酵母外切葡聚糖酶的第一个序列中

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摘要

In addition to the exoglucanases (Exg) secreted into the culture medium by wild type cells, Exgla and Exgib, which have oligosaccharides attached to both potential /V-glycosylation sites, Saccharomyces cerevisiae alg3 mutant secreted substantial amounts (35-44%) of underglycosylated and unglycosylated forms. Quantification of these forms indicated that no more than 78% of the available N-sites were occupied. About 50% of the transferred oligosaccharides were endo H sensitive, indicating that the lipid-linked precursor had completed its synthesis to Glc3-Man9-GlcNAc2. The other 50% remained endo H-resistant and, accordingly, it should be derived from the precursor oligosaccharide Man5-GlcNAc, synthesized by this mutant. A closer analysis of forms that have received two oligosaccharides (Exgib) showed that the first sequon was enriched in truncated residues, whereas the second one was enriched in regular counterparts. Similarly, analysis of the individual underglycosylated glycoforms indicated that 38% of the oligosaccharides attached to the second site were regular. This percentage dropped to 20% for glycoforms carrying the oligosaccharide in the first sequon. The preferential transfer of truncated oligosaccharides to the first glycosylation site seems to be a consequence of (1) the low percentage of truncated Hpid linked oligosaccharides that receives the glucotriose unit, and (2) the effect of the glucotriose unit on the selection of N-sites to be glycosylated.
机译:除了野生型细胞Exgla和Exgib分泌到培养基中的外切葡聚糖酶(Exg)都将寡糖连接到两个潜在的/ V-糖基化位点之外,酿酒酵母alg3突变体还分泌了大量(35-44%)的糖基化不足和非糖基化形式。这些形式的量化表明,不超过78%的可用N位被占用。大约50%转移的寡糖对内H敏感,表明脂质连接的前体已经完成了对Glc3-Man9-GlcNAc2的合成。其余的50%仍然对内H具有抗性,因此,它应源自该突变体合成的前体寡糖Man5-GlcNAc。对已接受两种低聚糖(Exgib)的形式的进一步分析表明,第一个序列中的截短残基富集,而第二个序列中的常规对应物富集。同样,对单个糖基化不足的糖型的分析表明,附着在第二个位点的寡糖中有38%是规则的。对于在第一后代中携带寡糖的糖型,该百分比下降至20%。截短的寡糖向第一糖基化位点的优先转移似乎是以下原因的结果:(1)接受葡萄糖三糖单元的截短的Hpid连接的寡糖百分比低,并且(2)葡萄糖三糖单元对N-的选择的影响被糖基化的位点。

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