Characterization of class I MHC folding intermediates and their disparate interactions with peptide and β2-microglobulin

Joseph D. Smith, Joyce C Solheim, Beatriz M. Carreno, Ted H. Hansen

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

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 Ld2m/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.

Original languageEnglish (US)
Pages (from-to)531-540
Number of pages10
JournalMolecular Immunology
Volume32
Issue number7
DOIs
StatePublished - Jan 1 1995

Fingerprint

Disulfides
Peptides
Ligands
beta 2-Microglobulin
Dithiothreitol
Reducing Agents
Epitopes
Binding Sites
Cell Line
Membranes
Population

Keywords

  • class I MHC
  • folding intermediate

ASJC Scopus subject areas

  • Immunology
  • Molecular Biology

Cite this

Characterization of class I MHC folding intermediates and their disparate interactions with peptide and β2-microglobulin. / Smith, Joseph D.; Solheim, Joyce C; Carreno, Beatriz M.; Hansen, Ted H.

In: Molecular Immunology, Vol. 32, No. 7, 01.01.1995, p. 531-540.

Research output: Contribution to journalArticle

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