Davis D P, Khurana R, Meredith S, Stevens F J, Argon Y
Department of Pathology, University of Chicago, IL 60637, USA.
J Immunol. 1999 Oct 1;163(7):3842-50.
Newly synthesized Ig chains are known to interact in vivo with the binding protein (BiP), a major peptide-binding chaperone in the endoplasmic reticulum. The predominant interactions between the light chain and BiP are observed early in the folding pathway, when the light chain is either completely reduced, or has only one disulfide bond. In this study, we describe the in vitro reconstitution of BiP binding to the variable domain of light chains (VL). Binding of deliberately unfolded VL was dramatically more avid than that of folded VL, mimicking the interaction in vivo. Furthermore, VL binding was inhibited by addition of ATP, was competed with excess unlabeled VL, and was demonstrated with several different VL proteins. Using this assay, peptides derived from the VL sequence were tested experimentally for their ability to bind BiP. Four peptides from both beta sheets of VL were shown to bind BiP specifically, two with significantly higher affinity. As few as these two peptide sites, one from each beta sheet of VL, are sufficient to explain the association of BiP with the entire light chain. These results suggest how BiP directs the folding of Ig in vivo and how it may be used in shaping the B cell repertoire.
已知新合成的免疫球蛋白（Ig）链在体内会与结合蛋白（BiP）相互作用，BiP是内质网中一种主要的肽结合伴侣蛋白。轻链与BiP之间的主要相互作用在折叠途径的早期就可观察到，此时轻链要么完全还原，要么只有一个二硫键。在本研究中，我们描述了BiP与轻链可变区（VL）结合的体外重建过程。故意展开的VL的结合比折叠的VL的结合明显更强烈，这与体内的相互作用相似。此外，ATP的添加会抑制VL的结合，过量未标记的VL会与之竞争，并且在几种不同的VL蛋白中都得到了证实。利用这种检测方法，对源自VL序列的肽进行了实验测试，以确定它们结合BiP的能力。来自VL两个β折叠的四种肽被证明能特异性结合BiP，其中两种具有显著更高的亲和力。仅这两个肽位点，一个来自VL的每个β折叠，就足以解释BiP与整个轻链的结合。这些结果表明了BiP在体内如何指导Ig的折叠以及它如何用于塑造B细胞库。
