Ubiquitinylation of Igβ Dictates the Endocytic Fate of the B Cell Antigen Receptor

Abstract

In both infection and autoimmunity, the development of high-affinity Abs and memory requires B cells to efficiently capture and process Ags for presentation to cognate T cells. Although a great deal is known about how Ags are processed, the molecular mechanisms by which the BCR captures Ag for processing are still obscure. In this study, we demonstrate that the Igβ component of the BCR is diubiquitinylated and that this is dependent on the E3 ligase Itch. Itch−/− B lymphocytes manifest both a defect in ligand-induced BCR internalization and endocytic trafficking to late endosomal Ag-processing compartments. In contrast, analysis of ubiquitinylation-defective receptors demonstrated that the attachment of ubiquitins to Igβ is required for endosomal sorting and for the presentation of Ag to T cells, yet, ubiquitinylation is dispensable for receptor internalization. Membrane-bound Igμ was not detectably ubiquitinylated nor were the conserved lysines in the mu cytosolic tail required for trafficking to late endosomes. These results demonstrate that ubiquitinylation of a singular substrate, Igβ, is required for a specific receptor trafficking event. However, they also reveal that E3 ligases play a broader role in multiple processes that determine the fate of Ag-engaged BCR complexes.

The Syk-binding Ubiquitin Ligase c-Cbl Mediates Signaling-dependent B Cell Receptor Ubiquitination and B Cell Receptor-mediated Antigen Processing and Presentation

“…In that report, itch-deficient B cells were shown to exhibit alterations in the intracellular trafficking of Ag⅐BCR complexes and impaired BCR-mediated antigen processing/ presentation, which was correlated with a change in ubiquitination of CD79b (17). Although the finding in this work may initially appear to be at odds with that previous work, that is not necessarily the case.…”

“…Previously, a report from the group of Marcus Clark implicated the ubiquitin ligase itch in ubiquitination of the CD79b subunit of the BCR complex and BCR-mediated antigen processing (17). In that report, itch-deficient B cells were shown to exhibit alterations in the intracellular trafficking of Ag⅐BCR complexes and impaired BCR-mediated antigen processing/ presentation, which was correlated with a change in ubiquitination of CD79b (17).…”

“…Binding of antigen to the BCR results in ubiquitination of the BCR cytoplasmic tail, which targets delivery of internalized Ag⅐BCR complexes to MVB-like MIIC, where they are converted to antigenic peptide class II complexes (16,17). Previous reports have established that BCR ubiquitination is signaling-dependent and restricted to lipid raft-resident Ag⅐BCR complexes (18).…”

Endocytic sequestration of the B cell antigen receptor and toll-like receptor 9 in anergic cells

“…When we examined aggregation-induced BCR internalization using a flow cytometric assay, it was similar between B cells from 3H9, V 8, or 3H9/V 8 mice (Fig. S3 A) (23). Also, transit of the ligated BCR through TfR ϩ early endosomes was similar between all 3 populations of splenic B cells (Fig.…”

“…Confocal microscopy was performed as previously described (23). Splenic B cells were labeled with either FITC-conjugated goat anti-mouse IgGϩIgM (heavy and light chain) F(abЈ) 2 antibodies (Jackson Immunoresearch) or CG50-biotin linked to streptavidin-Qdot655, and then warmed to 37°C for the indicated times.…”