Cleavage of dolichyl pyrophosphoryl oligosaccharides by endo-beta-N-acetylglucosaminidase H: comparison of enzymatic and acid hydrolysis techniques for saccharide release.
Cleavage of dolichyl pyrophosphoryl oligosaccharides by endo-beta-N-acetylglucosaminidase H: comparison of enzymatic and acid hydrolysis techniques for saccharide release.
复制标题
内切-β-N-乙酰氨基葡萄糖苷酶 H 裂解多甘基焦磷酸寡糖：糖释放的酶解和酸水解技术的比较。
DOI：
10.1016/0003-9861(84)90166-8
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发表时间：
1984
影响因子：
3.9
通讯作者：
Spiro,RG
中科院分区：
文献类型：
--
作者：
Chalifour,RJ;Spiro,RG
发现内切-β-N-乙酰氨基葡萄糖苷酶H（endo H）可使长链焦磷酰低聚糖完全水解。葡糖基化和非葡糖基化的聚甘露糖寡糖都通过酶裂解二-N-乙酰壳二糖序列而释放。通过加入Triton X-100（浓度大于0.03%时的最大刺激）或少量各种其他去污剂促进了endo H对寡糖脂的作用;然而，十二烷基硫酸钠（0.1%）具有强烈的抑制作用。尽管孵育通常在pH 5.2下进行，但注意到该酶在pH 6.5下同样有效，并且在pH 7.4下保留其对寡糖-脂质的75%活性。虽然这些结果扩大了已知的糖苷配基部分的内切H的特异性，但观察到即使在最佳条件下，脂质连接的Glc 3 Man 9 GlcNAc 2的水解速率也比肽序列中连接到天冬酰胺的相同寡糖的水解速率慢得多。使用endo H（一种可以在不含外切糖苷酶的情况下获得的酶）作为切割寡糖-脂质的工具似乎比温和的酸水解具有许多优点。发现后一种方法导致糖链发生少量但可检测的降解，并且在甲醇存在下进行时，导致约10%的寡糖以其β-甲基糖苷的形式释放。此外，由endo H释放的寡糖可以直接与由这种酶从糖蛋白中释放的寡糖进行比较;这可能被证明在处理寡糖脂质组装及其参与蛋白质的N-糖基化的代谢研究中是有用的。
Endo-β-N-acetylglucosaminidase H (endo H) was found to bring about the complete hydrolysis of dolichyl pyrophosphoryl oligosaccharides. Both glucosylated and unglucosylated polymannose oligosaccharides were released by the enzyme through cleavage of the di-N-acetylchitobiose sequence. The action of the endo H on the oligosaccharidelipids was facilitated by the inclusion of Triton X-100 (maximal stimulation at concentrations greater than 0.03%) or small amounts of a variety of other detergents; however, sodium dodecyl sulfate (0.1%) was strongly inhibitory. Although incubations were routinely carried out at pH 5.2, the enzyme was noted to be equally effective at pH 6.5 and to retain 75% of its activity toward oligosaccharide-lipid at pH 7.4. While these results broaden the known specificity of the endo H for the aglycon moiety, it was observed that even under optimal conditions the rate of hydrolysis of lipid-linked Glc3Man9GlcNAc2was substantially slower than that of the same oligosaccharide attached to asparagine in a peptide sequence. The use of endo H, an enzyme which can be obtained free of exoglycosidases, appears to have a number of advantages over mild acid hydrolysis as a tool for cleaving oligosaccharide-lipids. It was found that the latter procedure causes a small but detectable degradation of the sugar chains and, when carried out in the presence of methanol, leads to the release of about 10% of the oligosaccharide as its β-methyl glycoside. Furthermore, the oligosaccharides released by the endo H can be directly compared to those liberated by this enzyme from glycoproteins; this may prove to be useful in metabolic studies dealing with oligosaccharidelipid assembly and their involvement in theN-glycosylation of proteins.