Mapping the major interaction between binding protein and Ig light chains to sites within the variable domain.
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.
Davis
D P
DP
Department of Pathology, University of Chicago, IL 60637, USA.
Khurana
R
R
Meredith
S
S
Stevens
F J
FJ
Argon
Y
Y
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000276:immunology; D002352:Carrier Proteins / Q000378:metabolism; D006224:Cricetinae; D000091342:Endoplasmic Reticulum Chaperone BiP; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D010449:Peptide Mapping; D010957:Plasmids / Q000138:chemical synthesis; D011485:Protein Binding / Q000276:immunology
D001426:Bacterial Proteins; D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D018832:Molecular Chaperones; D009842:Oligopeptides