HLA-DM and HLA-DO: bi-directional regulation of function, tuned by pH (APP3P.103)

Peptide processing and loading onto MHC-II in endosomes occurs across a wide pH range. We studied pH effects on DM/DO regulation of peptide loading in vitro, using soluble, recombinant molecules, including a stabilized mutant DO dimer (sDOAP11A) that inhibits DM normally. We tested different DO:DM ratios at pH 4.6-7.2 during loading of 4 HLA alleles. DO alone had no effect. DM inhibition was diminished at lower DO:DM ratios, implying stable DM association is required for DO effect. Inhibition occurred across the pH range, but was reduced at pH 4.6. Using a narrower pH gradient (4.56-5.30), across which DM function is equivalent, we identified DO as the primary target of pH effects. DO activity was reduced after pre-incubation at pH 5.4, and longer exposure, lower pH and higher temperature enhanced this effect. FRET analysis of DM/DO at low pH was consistent with DO release rather than conformational change of intact complexes. SPR, bio-layer inferometry and ELISA assays showed reduced DM/DO association at pH≤4.85, suggesting released DO binds back inefficiently. With pre-incubation at pH 4.6 or 5.4, DM-DOwt complexes acquired DM-like activity; analysis of active SEC fractions indicated presence of free DM. Pre-incubation of sDM alone at these pHs caused aggregation. Complexes of full-length DM and DOwt showed similar behavior to soluble molecules. We conclude that low pH generates free DM by releasing DO, which both inhibits and chaperones DM.