FcμR (TOSO/FAIM3) Is An O-Glycosylated Endocytic Receptor That Shuttles IgM From the Cell Surface to the Lysosome and Whose Expression Is Regulated by TLR Signaling
Authors: Vire Berengere , David Alexandre , Wiestner Adrian 
Published: October 2019

Abstract
Abstract 2828
TOSO/FAIM3 has recently been identified as the long sought after Fc receptor for IgM (FcμR). Previous studies have shown that FcμR is selectively overexpressed in chronic lymphocytic leukemia (CLL) cells as compared to normal B-cells or other B cell malignancies. In this study, we extend the characterization of FcμR and identify the mechanisms regulating its trafficking and expression. In CLL cells, analysis of FcμR protein expression by immunoblotting revealed one major band around 60 kDa, a series of fainter migrating species, and a band at 41 kDa corresponding to its predicted molecular weight. By contrast, normal B cells express predominantly the 60 kDa form. Inspection of FcμR sequence revealed numerous potential O-glycosylation sites in the extracellular domain and treatment with a general inhibitor of O-glycosylation, benzyl-N-acetyl- α-galactosaminide, confirmed that FcμR is indeed heavily O-glycosylated. By immunofluorescence confocal microscopy, we determined that O-glycosylation is critical for trafficking of FcμR to the cell surface as mutations in the predicted O-glycosylation sites led to intracellular retention of FcμR in HeLa cells transfected with the appropriate expression vectors. In addition, pharmacologic inhibition of O-glycosylation decreased FcμR expression on CLL cells. Next, we used biotinylated IgM and streptavidin DyLight 488 to determine the fate of IgM bound to FcμR. Addition of IgM to CLL cells prompted rapid internalization of both IgM and FcμR, reaching half maximal internalization of cell bound IgM within one minute and virtually complete internalization within 5 minutes. By contrast, a monoclonal antibody specific for FcμR was not internalized. Using immunofluorescence confocal microscopy and co-staining with transferrin and anti-LAMP-1 antiserum, we followed IgM trafficking. FcμR transported IgM through the endocytic pathway to the lysosome, where it was degraded. This last step was chloroquine but not bortezomib sensitive, confirming the role of the lysosome in FcμR degradation. Using a series of FcμR deletion mutants, we identified a proline-rich domain that is essential for cell surface expression of FcμR and a second domain, containing a YXX Φ motif, that controls internalization.
Of particular interest, but so far elusive, is a possible functional role of FcμR in CLL pathogenesis. Our data suggest that a major physiologic role of FcμR may be internalization of IgM bound cargo into cells. Given the broad reactivity of IgM such cargo likely includes a variety of infectious agents as well as cellular debris that will be transported into the lysosome and thereby brought into contact with intracellular Toll-like Receptors (TLRs). To address a possible interaction between FcμR and TLRs, we investigated the effect of TLR activation on FcμR expression. There was a striking downregulation of FcμR on CLL cells in response to activation of TLR7 (using imiquimod) or TLR9 (using CpG-ODN) that involved both inhibition of transcription as well as degradation of FcμR through the lysosome. Following CpG stimulation, FcμR mRNA decreased with a half life of approximately 3 hours; that is at the same rate as after actinomycin D treatment. At 24 hours, FcμR protein was greatly reduced in response to either CpG or imiquimod, but interestingly there was less response to CpG in IGHV unmutated than in IGHV mutated CLL samples (p<0.001). In summary, FcμR is an O-glycosylated endocytic receptor that shuttles IgM from the cell surface to the lysosome. In vivo, FcμR could play a role in presenting IgM opsonized immune complexes to intracellular TLRs resulting in B-cell activation. Further studies are needed to clarify a potential contribution of FcμR to the activation of CLL cells in vivo. In addition, owing to the rapid internalization of IgM, we are pursuing FcμR as a potential pathway for the delivery of therapeutic antibody-drug conjugates into CLL cells (to be presented in a separate abstract).
This work was supported by the Intramural Research Program of the National, Heart, Lung and Blood Institute.

Disclosures:
No relevant conflicts of interest to declare.