Pathogenic ACVR1R206H activation by Activin A-induced receptor clustering and autophosphorylation.

Funder: Brain Research UK; Id: http://dx.doi.org/10.13039/100013790Fibrodysplasia ossificans progressiva (FOP) and diffuse intrinsic pontine glioma (DIPG) are debilitating diseases that share causal mutations in ACVR1, a TGF-β family type I receptor. ACVR1R206H is a frequent mutation in both diseases. Pathogenic signaling via the SMAD1/5 pathway is mediated by Activin A, but how the mutation triggers aberrant signaling is not known. We show that ACVR1 is essential for Activin A-mediated SMAD1/5 phosphorylation and is activated by two distinct mechanisms. Wild-type ACVR1 is activated by the Activin type I receptors, ACVR1B/C. In contrast, ACVR1R206H activation does not require upstream kinases, but is predominantly activated via Activin A-dependent receptor clustering, which induces its auto-activation. We use optogenetics and live-imaging approaches to demonstrate Activin A-induced receptor clustering and show it requires the type II receptors ACVR2A/B. Our data provide molecular mechanistic insight into the pathogenesis of FOP and DIPG by linking the causal activating genetic mutation to disrupted signaling

N-Linked Glycosylation Regulates CD22 Organization and Function

The organization and clustering of cell surface proteins plays a critical role in controlling receptor signaling; however, the biophysical mechanisms regulating these parameters are not well understood. Elucidating these mechanisms is highly significant to our understanding of immune function in health and disease, given the importance of B cell receptor (BCR) signaling in directing B cells to produce antibodies for the clearance of pathogens, and the potential deleterious effects of dysregulated BCR signaling, such as in B cell malignancies or autoimmune disease. One of main inhibitory co-receptors on B cells is CD22, a sialic-acid binding protein, which interacts homotypically with other sialylated CD22 molecules, as well as heterotypically with IgM and CD45. Although the importance of CD22 in attenuating BCR signaling is well established, we still do not fully understand what mediates CD22 organization and association to BCRs. CD22 is highly glycosylated, containing 12 N-linked glycosylation sites on its extracellular domain, the function of which remain to be resolved. We were interested in how these glycosylation sites mediate homotypic vs. heterotypic interactions. To this end, we mutated five out of the six N-linked glycosylation residues on CD22 localized closest to the sialic acid binding site. Glycan site N101 was not mutated as this resulted in lack of CD22 expression. We used dual-color super-resolution imaging to investigate the impact of altered glycosylation of CD22 on the nanoscale organization of CD22 and its association with BCR. We show that mutation of these five glycosylation sites increased the clustering tendency of CD22 and resulted in higher density CD22 nanoclusters. Consistent with these findings of altered CD22 organization, we found that mutation of N-glycan sites attenuated CD22 phosphorylation upon BCR stimulation, and consequently, increased BCR signaling. Importantly, we identified that these sites may be ligands for the soluble secreted lectin, galectin-9, and are necessary for galectin-9 mediated inhibition of BCR signaling. Taken together, these findings implicate N-linked glycosylation in the organization and function of CD22, likely through regulating heterotypic interactions between CD22 and its binding partners

Survival pathways harnessed by Follicular Lymphoma and Transformed Follicular Lymphoma

The genetic processes that allow for B-cell humoral immunity can also result in genetic aberrations leading to B-cell lymphoma. Follicular lymphoma (FL) is a common non-Hodgkin’s lymphoma and while FL is indolent, it can transform into an aggressive disease. This thesis investigates the survival pathways harnessed by FL and tFL, namely how features of B-cell biology, such as the role of the BCR itself, downstream signalling, and transcriptional states, are harnessed to drive lymphomagenesis, maintenance and transformation. One understudied functional characteristic of the BCR is isotype usage, with lymphomas generally favouring IgM expression. To investigate the role of the IgG isotype, I conducted a mutational analysis in a primary lymphoma dataset and identified novel mutations, enriched in the tFL molecular subtype, in the IgG cytoplasmic tail that render the IgG receptor similar to IgM. I demonstrated that IgG BCR may be inferior to IgM in supporting tFL growth through the tonic signalling pathway, and these mutations may be a mechanism to enhance competitiveness in class-switched lymphomas. Another distinct feature of the FL-BCR is high-mannose variable region glycosylation. These unprocessed sugars may allow for FL cell maintenance in-vivo through interaction with lectin receptors on immune cells in the tumour microenvironment. Previous research has shown that the lectin DC-SIGN can engage FL-BCRs and trigger downstream signalling. To further understand the signalling differences, I investigated the biophysical differences between DC-SIGN and antigen engagement on immune synapse formation and actin dynamics, demonstrating that DC-SIGN stimulation results in unique immunological synapse formation. Finally, there are multiple pathways of FL transformation, one such high-grade transformation results in TdT+ B-lymphoblastic leukaemia (B-LBL) that interestingly displays features of dedifferentiation into a “pre-B-cell” phenotype. Through conducting a genome-wide CRISPR screen on a TdT+ tFL line, I identified MYB as a novel putative regulator of B-LBL cell growth and maintenance of the de-differentiated cell state

The Role of Glycosylation on the Spatiotemporal Organization and Function of B-cell Co-receptor CD22

An important inhibitory co-receptor on B-cells is CD22, a sialic-acid binding protein. CD22 is highly glycosylated with six glycans located in the first two extracellular domains. The cytosolic domain contains ITIMs that upon phosphorylation lead to recruitment of inhibitory phosphatase SHP-1. Proximity of CD22 to B cell receptor (BCR) is required for dephosphorylation of signalling molecules leading to signal attenuation; however, we do not understand what mediates CD22 organization and association to BCRs. Since glycans on CD22 can be terminally sialylated, we hypothesize these glycans facilitate homotypic CD22 interactions, and therefore affect CD22 heterotypic interaction with BCRs. Here, we utilized a N-terminal glycosylation mutant (5Q-CD22) and, using super-resolution imaging, we demonstrate that 5Q-CD22 displays increased clustering and closer association to IgM BCR compared to WT-CD22. Importantly, abrogation of these glycans leads to loss of CD22 function, as ITIM phosphorylation upon B-cell activation is decreased and consequently BCR signalling is enhanced.M.Sc

Endophilin A2 regulates B‐cell endocytosis and is required for germinal center and humoral responses

Antigen‐specific B‐cell responses require endosomal trafficking to regulate antigen uptake and presentation to helper T cells, and to control expression and signaling of immune receptors. However, the molecular composition of B‐cell endosomal trafficking pathways and their specific roles in B‐cell responses have not been systematically investigated. Here, we report high‐throughput identification of genes regulating B‐cell receptor (BCR)‐mediated antigen internalization using genome‐wide functional screens. We show that antigen internalization depends both on constitutive, clathrin‐mediated endocytosis and on antigen‐induced, clathrin‐independent endocytosis mediated by endophilin A2. Although endophilin A2‐mediated endocytosis is dispensable for antigen presentation, it is selectively required for metabolic support of B‐cell proliferation, in part through regulation of iron uptake. Consequently, endophilin A2‐deficient mice show defects in GC B‐cell responses and production of high‐affinity IgG. The requirement for endophilin A2 highlights a unique importance of clathrin‐independent intracellular trafficking in GC B‐cell clonal expansion and antibody responses