Demonstration of a peptide:N-glycosidase in the endoplasmic reticulum of rat liver.
Demonstration of a peptide:N-glycosidase in the endoplasmic reticulum of rat liver.
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大鼠肝脏内质网中肽：N-糖苷酶的演示。
DOI：
10.1042/bj3220655
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Spiro,RG
中科院分区：
文献类型：
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作者：
Weng,S;Spiro,RG
根据以前的观察，即聚甘露糖寡糖从新合成的糖蛋白中释放[Anumula和Spiro（1983）J.Biol.Chem.258，15274-15282]，我们检查了大鼠肝内质网（ER）中是否存在内切糖苷酶，该内切糖苷酶可能参与推测为蛋白质质量控制机制的功能的事件。我们的研究表明，一种肽：N-聚糖酶（PNGase）存在于ER膜中，能够从放射性标记的糖肽中释放葡萄糖基化以及非葡萄糖基化的聚甘露糖寡糖，其还原末端终止于二-N-乙酰壳二糖序列（OS-GlcNAc 2）。这种酶，这被发现是管腔的方向，是最活跃的pH范围5.5-7.0，虽然它没有外源的二价阳离子的要求，它被抑制EDTA。对Man 9 GlcNAc 2-肽的详细研究表明，除了游离寡糖（Man 9 GlcNAc 2）外，还释放了一种额外的中性产物，其特征为与尚未鉴定的糖苷配基连接的Man 9 GlcNAc 2，其释放方式表明其作为中间体的作用。我们观察到，与胞质溶胶相反，ER没有内切-β-N-乙酰氨基葡萄糖苷酶活性，这表明终止于单个GlcNAc残基的寡糖（OS-GlcNAc 1），已注意到其在N-糖基化后不久出现在囊外室中[摩尔和斯皮罗（1994）生物化学杂志269，12715-12721]作为OS-GlcNAc 2从蛋白质中释放，并在经历其壳二糖核心的裂解之前以该形式经历ER-至-胞质溶胶的易位。
Prompted by previous observations that polymannose oligosaccharides are released from newly synthesized glycoproteins [Anumula and Spiro (1983) J. Biol. Chem.258, 15274–15282], we examined rat liver endoplasmic reticulum (ER) for the presence of endoglycosidases that could be involved in an event presumed to be a function of the protein quality control machinery. Our investigations indicated that a peptide:N-glycanase (PNGase) is present in ER membranes that has the capacity to release from radiolabelled glycopeptides glucosylated as well as non-glucosylated polymannose oligosaccharides terminating at their reducing end in a di-N-acetylchitobiose sequence (OS-GlcNAc2). This enzyme, which was found to be luminal in orientation, was most active in the pH range 5.5–7.0 and although it had no exogenous bivalent-cation requirements it was inhibited by EDTA. Detailed studies with Man9GlcNAc2-peptides demonstrated that in addition to the free oligosaccharide (Man9GlcNAc2) an additional neutral product characterized as Man9GlcNAc2linked to an as yet unidentified aglycone was released in a manner that suggests its role as an intermediate. Our observation that ER, in contrast with cytosol, had no endo-β-N-acetylglucosaminidase activity would indicate that oligosaccharides terminating in a single GlcNAc residue (OS-GlcNAc1), which have been noted to appear in the extravesicular compartment shortly after N-glycosylation [Moore and Spiro (1994) J. Biol. Chem.269, 12715–12721] are released from the protein as OS-GlcNAc2and undergo an ER-to-cytosol translocation in that form before undergoing cleavage of their chitobiose core.