Intermediates in the assembly and degradation of class I major histocompatibility complex (MHC) molecules probed with free heavy chain-specific monoclonal antibodies.
Unassembled (free) heavy chains appear during two stages of the class I MHC molecule's existence: immediately after translation but before assembly with peptide and beta 2-microglobulin, and later, upon disintegration of the heterotrimeric complex. To characterize the structures of folding and degradation intermediates of the class I heavy chain, three monoclonal antibodies have been produced that recognize epitopes along the H-2K(b) heavy chain which are obscured upon proper folding and subsequent assembly with beta 2-microglobulin (KU1: residues 49-54; KU2: residues 23-30; KU4: residues 193-198). The K(b) heavy chain is inserted into the lumen of the endoplasmic reticulum in an unfolded state reactive with KU1, KU2, and KU4. Shortly after completion of the polypeptide chain, reactivity with KU1, KU2, and KU4 is lost synchronously, suggesting that folding of the class I heavy chain is a rapid, cooperative process. Perturbation of the folding environment in intact cells with the reducing agent dithiothreitol or the trimming glucosidase inhibitor N-7-oxadecyl-deoxynojirimycin prolongs the presence of mAb-reactive K(b) heavy chains. At the cell surface, a pool of free K(b) heavy chains appears after 60-120 min of chase, whose subsequent degradation, but not their initial appearance, is impaired in the presence of concanamycin B, an inhibitor of vacuolar acidification. Thus, free heavy chains that arise at the cell surface are destroyed after internalization.
Machold
R P
RP
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Ploegh
H L
HL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001483:Base Sequence; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000276:immunology; D000939:Epitopes / Q000032:analysis; D006183:H-2 Antigens / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D008223:Lymphoma; D046911:Macromolecular Substances; D051379:Mice; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D001613:beta 2-Microglobulin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006183:H-2 Antigens; D046911:Macromolecular Substances; D001613:beta 2-Microglobulin