Major histocompatibility complex class II (MHC II) is a key molecule in the presentation of antigenic peptides to CD4+ T cells by antigen presenting cells, including dendritic cells and B cells. Its cell surface abundance is controlled via ubiquitination at a single lysine (K225) on the cytosolic tail of MHC II β-chain - controlled by Membrane associated RING-CH 1 (MARCH1) - which targets peptide-MHC II complexes for degradation in the lysosome.
Although several recent studies have addressed the physiological function of MARCH1 (1,2), not much is known about the mechanisms of how it ubiquitinates MHC II and what kind of ubiquitin code it produces. Here, we have undertaken several studies to characterise MHC II ubiquitination: first, we analyzed the ubiquitin chain linkage types that are present on MHC II isolated from primary murine immune cells, using an ubiquitin chain restriction (UbiCRest) assay and absolute quantification (AQUA) mass spectrometry. We report that MARCH1 creates mixed ubiquitin chains containing K11- and K63-linked ubiquitin on both dendritic cells as well as B cells. Second, we screened a genome-wide CRISPR knockout library for new factors regulating surface MHC II. Our analysis has identified the ubiquitin-like protein UBL3 as a potential novel regulator of MHC II ubiquitination and trafficking.
In summary, we have conducted an in-depth analysis of the mechanism of MHC II ubiquitination by MARCH1, both by using primary immune cells and engineered cell models. Our findings shed light on the mechanisms that control key molecules of the immune system, and will contribute to current understanding of membrane receptor ubiquitination.