Reconstitution by MHC-restricted peptides of HLA-A2 heavy chain with β2-microglobulin, in vitro

Abstract

Cytotoxic T lymphocytes kill virally infected cells when they detect antigenic fragments presented by class I major histocompatibility complex (MHC) antigens (HLA in humans). The crystal structures of HLA-A2 and HLA-Aw68 reveal that peptide-antigen forms an integral part of the HLA structure, being retained in a prominent groove even after purification and crystallization. Here we report that the heavy chain and beta 2-microglobulin of HLA-A2, after separation and fractionation in denaturants, reassemble efficiently under renaturing conditions only in the presence of MHC-restricted peptides. A complex of heavy chain, beta 2-microglobulin, and viral peptide in the ratio 1:1:1 is formed in up to 46% yield. Reconstitution is not stimulated by either of two peptides not restricted to HLA-A2. The reconstituted complex of HLA-A2 and the influenza virus (B/Lee/40) nucleoprotein peptide, Np (85-94), crystallizes under conditions previously used to crystallize HLA-A2. Peptide-linked folding and assembly suggests mechanisms for the unusual capacity of HLA to bind many peptides of diverse sequence.

Dipeptides promote folding and peptide binding of MHC class I molecules

“…The human MHC class I allotype HLA-A*02:01 (A2) has been reported to require specific full-length peptide for folding in vitro (8). To confirm this, we expressed A2 in Escherichia coli, dissolved the inclusion bodies in urea buffer, and folded the denatured protein with its light chain, human β-2 microglobulin (hβ 2 m), and with or without the A2-specific peptide NLVPMVATV (from human cytomegalovirus pp65; single-letter amino acid code).…”

“…This knowledge has been used to predict the binding affinity of any peptide sequence to a given class I allotype (6), and, together with theoretical simulations of the conformational movements of class I-peptide complexes, it has allowed the design of altered peptide ligands with higher binding affinities (7). The high-affinity peptides thus identified can be used to fold many bacterially expressed denatured class I molecules into their native state (8). Consequently, high-affinity peptides have been called the "essential third subunit of MHC class I" (9).…”

Influenza B virus-specific CD8+ T-lymphocytes strongly cross-react with viruses of the opposing influenza B lineage

“…Indeed, out of six in vitro confirmed influenza B epitopes described previously (Robbins et al, 1989(Robbins et al, , 1995(Robbins et al, , 1997 were not predicted at all. As these programs predict epitopes based on binding affinity of the epitope with the selected HLA allele, they do not take into account other possible factors that might play a role, such as the dissociation rate of the epitope (van der Burg et al, 1996), folding of the MHC class I molecules (Silver et al, 1991) or antigen processing (van de Sandt et al, 2012). To further test the robustness of the prediction algorithms, we determined the reactivity of polyclonal influenza B virus-specific polyclonal CD8…”

The Role of CD8+T Cells and Major Histocompatibility Complex Class I Expression in the Central Nervous System of Mice Infected with Neurovirulent Sindbis Virus

“…Functional cell surface expression of MHC class I molecules depends on the production of both MHC class I heavy chain and ␤2 microglobulin, which noncovalently bind to each other (1,41). Antigenic peptide presentation also requires the heterodimer of TAP1 and TAP2 proteins that transports short peptides from the cytosol into the endoplasmic reticulum lumen for loading onto assembled MHC class I molecules (3,29,42).…”