TY - JOUR
T1 - Characterization of class I MHC folding intermediates and their disparate interactions with peptide and β2-microglobulin
AU - Smith, Joseph D.
AU - Solheim, Joyce C.
AU - Carreno, Beatriz M.
AU - Hansen, Ted H.
N1 - Funding Information:
Acknowledgements-The authors thank Dr John Gorka for synthesiso f peptides,D r Wen-Rong Lie for generationo f the rabbit anti-Ld cytoplasmicd omain serum, and MS Rebecca Czerwinski for preparationo f the manuscript.T his work was supportedb y National Instituteso f Health grantA I-19687a nd AI-07163.
PY - 1995/5
Y1 - 1995/5
N2 - Newly synthesized class I heavy chains achieve domain structure using disulfide bonds, assemble with beta-2 microglobulin (β2m), 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 β2m and these L alt β2m heterodimers were preferentially folded by peptide in cell lysates. These findings indicate that class I heavy chain β2m association can precede ligand binding and that peptide is probably the limiting factor for completion of the Ld/β2m/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 β2m 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, partially reduced and native class I molecules. Furthermore, partially reduced Ld alt molecules, in a cell line expressing a mutant Ld molecule lacking the α2 domain disulfide bond, accumulated intracellularly, were not β2m-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 β2m and peptide, and that disulfide bond formation is required for efficient assembly, ligand binding and folding of the class I heavy chain.
AB - Newly synthesized class I heavy chains achieve domain structure using disulfide bonds, assemble with beta-2 microglobulin (β2m), 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 β2m and these L alt β2m heterodimers were preferentially folded by peptide in cell lysates. These findings indicate that class I heavy chain β2m association can precede ligand binding and that peptide is probably the limiting factor for completion of the Ld/β2m/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 β2m 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, partially reduced and native class I molecules. Furthermore, partially reduced Ld alt molecules, in a cell line expressing a mutant Ld molecule lacking the α2 domain disulfide bond, accumulated intracellularly, were not β2m-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 β2m and peptide, and that disulfide bond formation is required for efficient assembly, ligand binding and folding of the class I heavy chain.
KW - class I MHC
KW - folding intermediate
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U2 - 10.1016/0161-5890(95)00013-5
DO - 10.1016/0161-5890(95)00013-5
M3 - Article
C2 - 7783756
AN - SCOPUS:0029046720
SN - 0161-5890
VL - 32
SP - 531
EP - 540
JO - Molecular Immunology
JF - Molecular Immunology
IS - 7
ER -