Noiva R, Kimura H, Roos J, Lennarz W J
Department of Biochemistry and Molecular Biology, University of Texas, M. D. Anderson Cancer Center, Houston 77030.
J Biol Chem. 1991 Oct 15;266(29):19645-9.
Previously we had demonstrated by photoaffinity labeling that a 57-kDa protein of the endoplasmic reticulum can bind and become covalently linked to glycosylatable photoreactive peptides containing the sequence-Asn-Xaa-Ser/Thr-. Subsequently, it was found that this protein, called glycosylation site-binding protein, was a multifunctional protein, i.e. it was identical to protein disulfide isomerase (PDI), the beta-subunit of prolyl hydroxylase and thyroid hormone-binding protein. In this study, the peptide specificity for binding to this 57-kDa protein, hereafter called PDI, has been investigated in more detail using photoaffinity probes. The results reveal that although N-glycosylation by oligosaccharyl transferase in the endoplasmic reticulum has an absolute requirement for an hydroxyamino acid in the third amino acid residue of the glycosylation site sequence, no such specificity is observed in the binding of such peptides to PDI. In addition to the lack of specificity for an hydroxyamino acid in the third residue position, no specificity was observed for the asparagine residue in the first position. Thus, binding is not restricted to peptides containing N-glycosylation sites. We have investigated the discrepancy between this apparent lack of sequence specificity and earlier results indicating that binding of peptides to PDI was specific for N-glycosylation site sequences. We now demonstrate that PDI in the lumen of microsomes is more efficiently labeled by peptides containing photoreactive-Asn-Xaa-Ser/Thr- sequences than by nonacceptor site sequences because the former become glycosylated. This increased labeling does not occur because the glycosylated form of the probes are preferentially recognized by PDI. Rather, it appears that increased polarity of the affinity probe after attachment of the oligosaccharide chain prevents its exit from the sealed microsomes, in effect concentrating it within the lumen of the microsome. These results, coupled with other studies on the multifunctional nature of PDI, suggest that the observed peptide binding may be a manifestation of the ability of PDI to recognize the backbone of polypeptides in the lumen of the endoplasmic reticulum.
此前我们通过光亲和标记证明，内质网的一种57 kDa蛋白能够结合并共价连接到含有-Asn-Xaa-Ser/Thr-序列的可糖基化光反应性肽段上。随后发现，这种被称为糖基化位点结合蛋白的蛋白是一种多功能蛋白，即它与蛋白二硫键异构酶（PDI）、脯氨酰羟化酶的β亚基以及甲状腺激素结合蛋白相同。在本研究中，使用光亲和探针更详细地研究了与这种57 kDa蛋白（以下称为PDI）结合的肽段特异性。结果表明，尽管内质网中寡糖基转移酶进行的N-糖基化对糖基化位点序列的第三个氨基酸残基中的羟基氨基酸有绝对要求，但在这些肽段与PDI的结合中未观察到这种特异性。除了对第三个残基位置的羟基氨基酸缺乏特异性外，对第一个位置的天冬酰胺残基也未观察到特异性。因此，结合并不局限于含有N-糖基化位点的肽段。我们研究了这种明显缺乏序列特异性与早期结果之间的差异，早期结果表明肽段与PDI的结合对N-糖基化位点序列具有特异性。我们现在证明，微粒体腔中的PDI被含有光反应性-Asn-Xaa-Ser/Thr-序列的肽段标记的效率高于非受体位点序列，因为前者会被糖基化。这种标记增加并不是因为探针的糖基化形式被PDI优先识别。相反，似乎寡糖链连接后亲和探针极性的增加阻止了它从封闭的微粒体中逸出，实际上使其在微粒体腔内浓缩。这些结果，再加上对PDI多功能性质的其他研究，表明观察到的肽段结合可能是PDI识别内质网腔中多肽主链能力的一种表现。
