Polymorphism at Position 97 in MHC Class I Molecules Affects Peptide Specificity, Cell Surface Stability, and Affinity for β2-Microglobulin

Abstract

The two mouse MHC class I alleles, Ld and Lq, share complete amino acid sequence identity except in the α2 domain, where they differ at six positions. Despite their similarity, Lq has a stronger association with β2-microglobulin (β2m), is expressed at higher levels on the cell surface, demonstrates an increased cell surface half-life, and has fewer open forms on the cell surface than Ld. To determine the basis for their phenotypic differences, Ld molecules containing chimeric Ld-Lq α2 domains were characterized, and these analyses implicated residue 97 (LdTrp and LqArg) as the polymorphic site responsible for the disparity in β2m association between the two alleles. Single substitution analysis at this site (LdW97R and LqR97W) confirmed this. Furthermore, the LdW97R mutant molecule has a longer cell surface half-life than either Lq or Ld, and fewer open forms of LdW97R are observed on the cell surface. In addition, both LdW97R and Lq possess decreased binding affinity for the Ld-restricted tum− P91A14–22 peptide compared with Ld. Collectively, these results and the known location of Trp97 in the peptide binding cleft of Ld strongly suggest that the substitution of Arg for Trp97 in Ld alters the peptide binding cleft, increasing its affinity for endogenous peptides, which results in greater cell surface stability and better retention of β2m. Furthermore, these results imply that Trp97 plays an important role in the ability of Ld to efficiently participate in alternative MHC class I Ag presentation pathways.

Engineering and Characterization of a Stabilized α1/α2 Module of the Class I Major Histocompatibility Complex Product Ld

“…Interestingly, one substitution, Trp-97 3 Arg, in the structurally related L q allele has been shown to confer additional stability on L q (20). Remarkably, this mutation was also identified in the present study using a directed evolution approach.…”

“…A library of ϳ10 6 trans- . This mutation has been shown to stabilize peptide binding and ␤2m association (20). Because Trp-97 3 Arg is the only mutation shared between these two mutants, we examined the influence of the mutation when cloned into the L d -␤2m construct.…”

The Same Major Histocompatibility Complex Polymorphism Involved in Control of HIV Influences Peptide Binding in the Mouse H-2Ld System

“…In addition, it appears that peptides that bind to L d may be influenced by more of their individual residues than peptides that bind to K b , which appear to have dominant anchor residues (31). Relevant to recent information about HIV and HLA-B polymorphisms are previous studies showing that two alleles called L q and L d contained the same polymorphism at residue 97 as in the HLA-B studies, and these alleles exhibited differences in stability between the ternary complexes (peptide, H-2L heavy chain, and ␤ 2 -microglobulin) (32). In an independent approach, the W97R substitution was identified in a "needle in a haystack" experiment in which more stable L d mutants were identified because they conferred higher surface levels of the protein in a yeast display system (33 -pMP71 DNA was incubated with 60 g of Lipofectamine 2000 (Invitrogen) in 3 ml of Opti-MEM media (Invitrogen) for 20 min at room temperature.…”

Identification of naturally processed peptides bound to the class I MHC molecule H‐2Kd of normal and TAP‐deficient cells

“…Results were plotted as the concentration of the test peptide needed to achieve a threefold increase in the mean fluorescence intensity (MFI) over the background cell surface Kd levels. We assumed, based on the results of others, that the increase in Kd reflects the binding properties of the peptide for Kd [59][60][61][62].…”