Characterization of class I MHC folding intermediates and their disparate interactions with peptide and beta 2-microglobulin.
Newly synthesized class I heavy chains achieve domain structure using disulfide bonds, assemble with beta-2 microglobulin (beta 2m), and bind peptide ligand to complete the trimeric complex. Although each of these initial events is thought to be critical for class I folding, their sequential order and effect on class I structure are unknown. Using mAb specific for distinct conformations of H-2Ld and Lq, we have defined folding intermediates of class I molecules. We show here that non-peptide-associated forms of Ld or Lq, detected by mAb 64-3-7 and designated L alt, lack numerous conformational epitopes surrounding their ligand binding sites. These results support the notion that L alt molecules have an open conformation. Interestingly, a significant proportion of L alt molecules were detected in association with beta 2m and these L alt/beta 2m heterodimers were preferentially folded by peptide in cell lysates. These findings indicate that class I heavy chain/beta 2m association can precede ligand binding and that peptide is probably the limiting factor for completion of the Ld/beta 2m/peptide trimeric complex in vivo. The characteristics of L alt molecules were investigated further by ascertaining the disulfide bond status of these molecules and their association with beta 2m and peptide. Treatment of cells with dithiothreitol (DTT), a membrane-permeable reducing agent, demonstrated that L alt molecules constitute a heterogeneous population including reduced, partially reduced and native class I molecules. Furthermore, partially reduced Ld alt molecules, in a cell line expressing a mutant Ld molecule lacking the alpha 2 domain disulfide bond, accumulated intracellularly, were not beta 2m-associated and displayed marginal peptide-induced folding in vitro. In accordance with this latter finding, peptide was found to preferentially convert fully disulfide-bonded forms of Ld alt to conformed Ld. Thus, we propose that intrachain disulfide bond formation precedes the association of class I heavy chain with beta 2m and peptide, and that disulfide bond formation is required for efficient assembly, ligand binding and folding of the class I heavy chain.
Smith
J D
JD
Department of Genetics, Washington University School of Medicine, St Louis, MO 63110, USA.
Solheim
J C
JC
Carreno
B M
BM
Hansen
T H
TH
D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D006183:H-2 Antigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D064532:Histocompatibility Antigen H-2D; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D001613:beta 2-Microglobulin / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D004220:Disulfides; D006183:H-2 Antigens; D064532:Histocompatibility Antigen H-2D; D010455:Peptides; D001613:beta 2-Microglobulin