Ubiquitination of HLA‐DO by MARCH family E3 ligases

Abstract

HLA-DO (DO) is a nonclassical MHC class II (MHCII) molecule that negatively regulates the ability of HLA-DM to catalyse the removal of invariant chain-derived CLIP peptides from classical MHCII molecules. Here, we show that DO is posttranslationally modified by ubiquitination. The location of the modified lysine residue is shared with all classical MHCII beta chains, suggesting a conserved function. Three membrane-associated RING-CH (MARCH1, 8 and 9) family E3 ligases that polyubiquitinate MHCII induce similar profiles of polyubiquitination on DOβ. All three MARCH proteins also influenced trafficking of DO indirectly by a mechanism that required the DOβ encoded di-leucine and tyrosine-based endocytosis motifs. This may be the result of MARCH-induced ubiquitination of components of the endocytic machinery. MARCH9 was by far the most efficient at inducing intracellular redistribution of DO but did not target molecules for lysosomal degradation. The specificity of MARCH9 for HLA-DQ and HLA-DO suggests a need for common regulation of these two MHC-encoded molecules.

HLA-DM and HLA-DO, key regulators of MHC-II processing and presentation

“…Consistent with this prior finding, Xiu et al used B cell and HeLa cell transfectants to show that DM redistributes to the limiting membrane from the internal vesicles of lysosomal multivesicular bodies though its interaction with DO, in a process mediated by the sorting motif present in the HLA-DO beta subunit cytoplasmic tail [50]. Jahnke et al report evidence supporting ubiquitination as a direct regulator of DO intracellular localization, with additional indirect effects on endocytic machinery [51]. Thus, intracellular sorting and localization might provide the spatial regulation hypothesized by Gu et al to generate a different spectrum of self and foreign peptide antigens bound to MHC-II in the presence or absence of DO.…”

Protein Homeostasis at the Plasma Membrane

“…Endogenous or overexpressed MARCHs play a key role in the ubiquitinationdependent lysosomal downregulation of native cell surface receptors (e.g., MHC-I: K3, K5, MARCH-9 and -4; MHC-II: MARCH-2 and -8, CD4, CD44 and CD98, MARCH-4 and -8) (6,7,23,26,41,46,62,65,66,90). MARCH-8 and -1 can recognize a transmembrane hydrophobic segment in concert with charged residues and results in the Ub-conjugation to a Lys residue at the membrane-cytosol interface in the case of the HLA-DR and transferrin receptor (41, 46).…”

HLA-DO Modulates the Diversity of the MHC-II Self-peptidome

“…Binding Affinity and DM Sensitivity Measurements-For three abundant self-peptides, DM sensitivity has been previously characterized: CLIP, the invariant chain chaperone fragment efficiently removed by DM (18,54), DR␣ [52][53][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68] , the human ortholog of the YAe epitope (55) known to be highly sensitive to DM exchange (56), and the transplantation alloepitope A2 104 -117 (39) previously demonstrated to be highly resistant to DM-mediated exchange (38). For these peptides, we summed the intensities of all peptides sharing the respective core epitopes and compared summed intensities between replicate samples of WT and DO-KO-1 cells.…”

“…Analysis of Epitope Source Protein Intracellular Localization-Because DO has been suggested to control the intracellular location of antigen loading through its effects on DM (26,27,66,67), we evaluated the GO-annotated cellular compartments (68,69) for source proteins from which the eluted peptides were derived. These were not appreciably different between WT and DO-KO, suggesting that the location of antigen loading was not substantially altered by the presence of DO (supplemental Fig.…”