Major histocompatibility class II in murine antigen presenting cells is modified with a branched K63 and K11-linked...

Major histocompatibility class II (MHC II) is critical for adaptive immunity. MHC II intracellular trafficking and degradation is regulated by ubiquitination. Poly-ubiquitination (Ub) of MHC II directs it away from the plasma membrane and is a critical determinant of MHC II turnover. The MHC II Ub chain has not been characterized. Here, we describe the poly-Ub chain associated with MHC II in primary murine antigen presenting cells; conventional dendritic cells (cDCs) and B cells. Analysis was conducted for endogenous murine MHC II isoforms H2-A and H2-E immunoprecipitated from primary cells. We show that ubiquitination of both I-A and I-E expressed by cDCs and B cells is dependent on the E3 Ub ligase MARCH1. Using mass spectrometry Ub chain linkage analysis and innovative Ub "clipping" proteomics we comprehensively defined the features of the MHC II poly-Ub chain. This revealed the MARCH1-dependent poly-Ub chain associated with MHC II is composed of a branched chain with K11 and K63 Ub chain linkages. This is the first description of the Ub chain linkages and architecture associated with MARCH1-mediated ubiquitination of MHC II in primary antigen presenting cells. This is important because it creates possibilities for manipulation of MHC II function in adaptive immunity.