Functional analysis of tryptophans α62 and β120 on HLA-DM

Abstract

In the endocytic pathway of antigen-presenting cells, HLA-DM catalyzes the exchange between class II-associated invariant chain peptide (CLIP) and antigenic peptides onto major histocompatibility complex class II molecules. At low pH of lysosomal compartments, both HLA-DM and HLA-DR undergo conformational changes, and it was recently postulated that two partially exposed tryptophans on HLA-DM might be involved in the interaction between the two molecules. To define contact regions on HLA-DM, we have conducted site-directed mutagenesis on those two hydrophobic residues. The HLA-DM αW62A,βW120A (DMW62A/W120A) double mutant was expressed in HLA-DR+ HeLa cells expressing invariant chain, and the activity of this DM molecule was assessed. Flow cytometry analysis of cell surface DR-CLIP complexes revealed that DMW62A/W120A removes CLIP as efficiently as its wild-type counterpart. DMW62A/W120A was found in the endocytic pathway by immunofluorescence, and DM-DR complexes were immunoprecipitated from these cells at pH 5. Finally, mutations αW62A and βW120A on HLA-DM did not affect the association with HLA-DO. The complex egresses the endoplasmic reticulum and accumulates in endocytic vesicles. Moreover, DO and DMW62A/W120A were co-immunoprecipitated at pH 7. We conclude that the α62 and β120 tryptophan residues are not required for the activity of DM, nor are they directly implicated in the interaction with DR or DO.