Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia.
FEBS J. 2013 Jan;280(1):233-43. doi: 10.1111/febs.12063. Epub 2012 Dec 14.
Protein disulfide isomerase (PDI) family members are important enzymes for the correct folding and maturation of proteins that transit or reside in the endoplasmic reticulum (ER). The human PDI family comprises at least 19 members that differ in cell type expression, substrate specificity and post-translational modifications. PDI family A member 2 (PDIA2, previously known as PDIp) has a similar domain structure to prototypical PDI (also known as PDIA1), but the function and post-translational modifications of PDIA2 remain poorly understood. Unlike most PDI family members, PDIA2 contains three predicted N-linked glycosylation sites. By site-directed mutagenesis and enzymatic deglycosylation, we show here that all three Asn residues within the potential N-linked glycosylation sites of human PDIA2 (N127, N284 and N516) are glycosylated in human cells. Furthermore, mutation of N284 to glycosylation-null Gln increases formation of a highly stable disulfide-bonded PDIA2 dimer. Nevertheless, in HeLa cells, both wild-type and N127/284/516Q mutant PDIA2 proteins localize to the ER, but not the ER-Golgi intermediate compartment, suggesting that glycosylation is important for PDIA2 protein-protein interactions but not subcellular localization. Finally, we identified human major histocompatibility complex class 1 antigens (HLA-A,B,C) as potential binding partners of PDIA2, suggesting an involvement for PDIA2 in antigen presentation in addition to its previously described roles in autoimmunity and Parkinson's disease. These results further characterize this poorly defined member of the PDI family.
蛋白质二硫键异构酶（PDI）家族成员是内质网（ER）中蛋白质正确折叠和成熟所必需的重要酶。人类 PDI 家族至少包含 19 个成员，这些成员在细胞类型表达、底物特异性和翻译后修饰方面存在差异。PDI 家族 A 成员 2（PDIA2，以前称为 PDIp）的结构域与典型的 PDI（也称为 PDIA1）相似，但 PDIA2 的功能和翻译后修饰仍知之甚少。与大多数 PDI 家族成员不同，PDIA2 包含三个预测的 N 连接糖基化位点。通过定点突变和酶促糖基化，我们在这里显示，人 PDIA2 潜在 N 连接糖基化位点中的三个 Asn 残基（N127、N284 和 N516）都在人细胞中发生糖基化。此外，将 N284 突变为糖基化无效的 Gln 会增加高度稳定的二硫键结合 PDIA2 二聚体的形成。然而，在 HeLa 细胞中，野生型和 N127/284/516Q 突变 PDIA2 蛋白都定位在内质网，但不在 ER-Golgi 中间隔室，这表明糖基化对于 PDIA2 蛋白-蛋白相互作用很重要，但对于亚细胞定位不重要。最后，我们鉴定出人主要组织相容性复合体 I 类抗原（HLA-A、B、C）作为 PDIA2 的潜在结合伴侣，这表明 PDIA2 除了在自身免疫和帕金森病中描述的作用外，还参与抗原呈递。这些结果进一步描述了 PDI 家族中这个定义不明确的成员。
