Functional analysis of tryptophans alpha 62 and beta 120 on HLA-DM.
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 alphaW62A,betaW120A (DM(W62A/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 DM(W62A/W120A) removes CLIP as efficiently as its wild-type counterpart. DM(W62A/W120A) was found in the endocytic pathway by immunofluorescence, and DM-DR complexes were immunoprecipitated from these cells at pH 5. Finally, mutations alphaW62A and betaW120A 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 DM(W62A/)W120A were co-immunoprecipitated at pH 7. We conclude that the alpha62 and beta120 tryptophan residues are not required for the activity of DM, nor are they directly implicated in the interaction with DR or DO.
Faubert
Amélie
A
Laboratoire d'Immunologie Moléculaire, Département de Microbiologie et d'Immunologie, Université de Montréal, Montréal, Québec, H3C 3J7, Canada.
Samaan
Angela
A
Thibodeau
Jacques
J
D015153:Blotting, Western; D018076:DNA, Complementary / Q000378:metabolism; D004705:Endocytosis; D005434:Flow Cytometry; D006681:HLA-D Antigens / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014162:Transfection; D014364:Tryptophan / Q000737:chemistry* / Q000502:physiology*
D018076:DNA, Complementary; D006681:HLA-D Antigens; C084245:HLA-DM antigens; D014364:Tryptophan