A point mutation in the groove of HLA-DO allows egress from the endoplasmic reticulum independent of HLA-DM.
B lymphocytes express the nonclassical class II molecule HLA-DO, which modulates the peptide loading activity of HLA-DM in the endocytic pathway. Binding to HLA-DM is required for HLA-DO to egress from the endoplasmic reticulum (ER). To gain insights into the mode of action of DO and on the role of DM in ER release, we sought to identify DM-binding residues on DO. Our results show that DOalpha encompasses the binding site for HLA-DM. More specifically, mutation of residue DOalpha41 on an exposed lateral loop of the alpha1 domain affects the binding to DM, ER egress, and activity of DO. Using a series of chimeric DR/DO molecules, we confirmed the role of the alpha chain and established that a second DM-binding region is located C-terminal to the DOalpha80 residue, most probably in the alpha2 domain. Interestingly, after mutation of a buried proline (alpha11) on the floor of the putative peptide-binding groove, HLA-DO remained functional but became independent of HLA-DM for ER egress and intracellular trafficking. Collectively, these results suggest that the binding of HLA-DM to DOalpha allows the complex to egress from the ER by stabilizing intramolecular contacts between the N-terminal antiparallel beta-strands of the DOalphabeta heterodimer.
Deshaies
Francis
F
Département de Microbiologie et Immunologie, Université de Montréal, CP 6128 Succursale Centre Ville, Montréal, QC, Canada H3T 1J4.
Brunet
Alexandre
A
Diallo
Djibril A
DA
Denzin
Lisa K
LK
Samaan
Angela
A
Thibodeau
Jacques
J
D000911:Antibodies, Monoclonal / Q000378:metabolism; D001402:B-Lymphocytes / Q000378:metabolism*; D015153:Blotting, Western; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005434:Flow Cytometry; D006681:HLA-D Antigens / Q000235:genetics* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008856:Microscopy, Fluorescence; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D000911:Antibodies, Monoclonal; D006681:HLA-D Antigens; C084245:HLA-DM antigens; C046148:HLA-DO antigens; D011993:Recombinant Fusion Proteins