Wearsch Pamela A, Cresswell Peter
Department of Pathology, Case Western Reserve University, Cleveland, OH, USA.
Department of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, USA.
Methods Mol Biol. 2013;960:67-79. doi: 10.1007/978-1-62703-218-6_6.
The stability of the MHC (major histocompatibility complex) class I peptide repertoire is optimized during assembly in the endoplasmic reticulum (ER) and depends on the collective function of components of the peptide-loading complex (PLC). The chaperone-like molecule tapasin is the cornerstone of this complex and acts directly on the MHC class I molecule to promote high-affinity peptide loading. Optimal tapasin activity, however, relies on the ability of ERp57 and calreticulin, two proteins involved in general ER glycoprotein folding, to bridge and thereby stabilize its otherwise weak interaction with the MHC class I heavy chain. Here, we describe methods for the recombinant expression of soluble components of the PLC specifically tailored to generate the post-translational modifications required to support subcomplex assembly in vitro. Using recombinant MHC class I molecules bearing monoglucosylated N-linked glycans, calreticulin, and disulfide-linked tapasin/ERp57 heterodimers, this soluble PLC subcomplex can be employed to study the mechanism of peptide loading or the principles governing peptide selection for particular MHC class I alleles.
主要组织相容性复合体（MHC）I类肽库的稳定性在其在内质网（ER）中组装时得到优化，并且依赖于肽装载复合体（PLC）各组分的协同作用。类分子伴侣蛋白塔帕辛是该复合体的基石，它直接作用于MHC I类分子以促进高亲和力肽的装载。然而，塔帕辛的最佳活性依赖于内质网蛋白57（ERp57）和钙网蛋白这两种参与内质网一般糖蛋白折叠的蛋白质，它们能够形成桥梁，从而稳定塔帕辛与MHC I类重链原本较弱的相互作用。在此，我们描述了PLC可溶性组分的重组表达方法，这些方法经过专门设计，可产生体外支持亚复合体组装所需的翻译后修饰。利用带有单糖基化N - 连接聚糖的重组MHC I类分子、钙网蛋白以及二硫键连接的塔帕辛/ERp57异二聚体，这种可溶性PLC亚复合体可用于研究肽装载机制或特定MHC I类等位基因的肽选择原则。
