Cytometry using anti-DQ antibody L2DQ to confirm activity of the transfected E3 ligases. (B) Intracellular staining for DO performed on saponin-permeabilised Raji cells using anti-DO antibody Mags. DO5. Levels of DO were assessed by flow cytometry. Reduced intracellular staining is seen in cells transduced with MARCH1 and MARCH8 in the absence of chloroquine. In the presence of chloroquine levels of intracellular DO remain unaltered. Data shown are representative of at least three independent experiments performed. (C) Schematic diagram summarising ubiquitination sites and E3 ligases involved in regulating components of the class II antigen presentation machinery. MARCH1 and MARCH8 regulate surface expression of DP, DQ and DR [20, 35]. MARCH9 specifically targets DQ [24, 33] and DR can also be ubiquitinated [25]. HLA-DM is regulated directly by ubiquitination of a DM encoded lysine residue and indirectly through a mechanism involving the tyrosine-based sorting motif [26]. DO is regulated by ubiquitination of the chain-encoded lysine residue and indirectly through the di-leucine and tyrosine motifs.different E3 ligases cause redistribution from the cell surface. In essence, MARCH1 and MARCH8 efficiently target MHCII and to a lesser extent DM and DO. MARCH9 in contrast shows greatest specificity for DO but also targets DM and DQ albeit less efficiently. Why would DO require MARCH regulation and how might this occur Several possibilities are worthy of discussion and these are centered on the major roles of ubiquitination; namely protein degradation and trafficking. First, expression of DO is highly specific and under particularly complex control in developing B cells. It is here that MHCII, DM and DO expression is most dynamic and it is also where DO is proposed to exert its main biological activity. Briefly, MHCII and DM are expressed through proB, pre-B and into immature and mature B-cell stages. DO expression in comparison is initiated as cells mature into IgD+ /CD40+ B cells and then during germinal center reactions expression is reduced. DO levels continue to fall as the cells develop into centroblasts and centrocytes, whereas DM expression remains relatively constant [7, 27]. This pattern is unique to GC reactions because in general downregulation of DO is always associated with reduced DM expression, as is the case for Ig-crosslinkingor PMA activation [22].Deucravacitinib Importantly, the downregulation of DO in GCs is by posttranslational mechanisms, as mRNA transcripts remain unchanged [27].Nemvaleukin alfa The ability of MARCH9 to specifically target DO would provide a mechanism to achieve this.PMID:23522542 However in our hands, although the major target of MARCH9 was DO, ubiquitination did not appear to lead to lysosomal degradation (Fig. 4B). Further studies using GC-derived centroblasts and centrocytes cells will be required to see if MARCH9 is involved in DO downregulation in these cells. A second possible role for DO ubiquitination relates to localisation. Ubiquitination has well-recognised roles in endocytosis and sorting (reviewed in [28]) and may therefore play a role in localisation of DO/DM and MHCII in endosomal compartments. In general, receptor ubiquitylation promotes targeting into the luminal vesicles of MVBs that facilitates protein degradation [28]. DO has been shown to enhance the incorporation of DO/DM complexes and MHCII into the perimeter membrane of MVBs fostering interaction of these molecules without their degradation. This capability requires th.