Site-specific de-N-glycosylation of diglycosylated ovalbumin in hen oviduct by endogenous peptide: N-glycanase as a quality control system for newly synthesized proteins.
Site-specific de-N-glycosylation of diglycosylated ovalbumin in hen oviduct by endogenous peptide: N-glycanase as a quality control system for newly synthesized proteins.
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DOI：
10.1073/pnas.94.12.6244
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发表时间：
1997-06
影响因子：
11.1
通讯作者：
Tadashi Suzuki;K. Kitajima;Yasufumi Emori;Y. Inoue;Sadako Inoue
中科院分区：
文献类型：
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作者：
Tadashi Suzuki;K. Kitajima;Yasufumi Emori;Y. Inoue;Sadako Inoue
已知鸡卵清蛋白（OVA）在鸡蛋白色中以单N-糖基化形式存在，聚糖链位于Asn-292上。先前的研究表明，在Asn-292和Asn-311上具有两个N-聚糖链的二-N-糖基化形式的OVA [二-OVA; CHO-Asn-292/CHO-Asn-311（CHO，N-聚糖链）]仅在母鸡输卵管中瞬时表达。在鸡蛋白色中未发现二-OVA，表明该形式不能正常分泌，可能在合成和分泌过程中通过肽：N-聚糖酶（PNGase）的作用转化为单-N-糖基化OVA（CHO-Asn-292/Asp-311）。在这项研究中，我们已经确定了假定的PNGase活性的母鸡输卵管匀浆，纯化1,000倍，并指定为PNGase HO。我们检查了Di-OVA与PNGase HO的反应性，发现该酶在Asn-311位点特异性切割聚糖链，将Di-OVA转化为单-N-糖基化形式（CHO-Asn-292/Asp-311）。相比之下，当将其作为底物进行测试时，发现该酶不作用于在鸡蛋白色中发现的单-N-糖基化的OVA（CHO-Asn-292/Asn-311）。目前的研究结果支持我们的观点，PNGase催化的去N-糖基化可能参与新合成蛋白质的质量控制，通过将其二糖基化形式转化为可以分泌的单糖基化形式。然而，不能排除去N-糖基化可能引发异常糖基化糖蛋白的胞质降解的替代可能性。
Hen ovalbumin (OVA) is known to exist as a singly N-glycosylated form with a glycan chain on Asn-292 in egg white. Previous studies showed that di-N-glycosylated form of OVA [Di-OVA; CHO-Asn-292/CHO-Asn-311 (CHO, N-glycan chain)], which has two N-glycan chains on Asn-292 and Asn-311, was expressed only transiently in hen oviduct. Di-OVA was not found in egg white, suggesting that this form cannot be secreted normally and may possibly be converted to mono-N-glycosylated OVA (CHO-Asn-292/Asp-311) by the action of peptide:N-glycanase (PNGase) during synthesis and secretion. In this study, we have identified the putative PNGase activity in the homogenate of hen oviduct, purified 1,000-fold, and designated as PNGase HO. We examined the reactivity of Di-OVA to PNGase HO and found that this enzyme site-specifically cleaved off the glycan chain at Asn-311 to convert Di-OVA into the mono-N-glycosylated form (CHO-Asn-292/Asp-311). In contrast, this enzyme was found not to act on the mono-N-glycosylated OVA (CHO-Asn-292/Asn-311) found in egg white when it was tested as a substrate. The present findings support our view that de-N-glycosylation catalyzed by PNGase may be involved in quality control of newly synthesized proteins by converting its diglycosylated form into the mono-N-glycosylated form that can be secreted. However, the alternative possibility that de-N-glycosylation may trigger cytosolic degradation of the aberrantly glycosylated glycoprotein cannot be ruled out.