The Role of N-glycanase 1 in in vitro activated B cells

Background and Aims: Antibody-secreting cells (ASCs) synthesize large amounts of antibodies to ensure humoral immunity. The production of large amounts of antibodies requires protein quality control mechanisms. The gene Ngly1 encodes N-glycanase 1 (NGLY1), a pivotal component of endoplasmic reticulum-associated degradation (ERAD). NGLY1 removes N-glycans from misfolded glycoproteins and has been implicated in treating multiple myeloma, but its role in B cells and ASCs remains unexplored. We hypothesize that activated B cells require functional NGLY1 for proper differentiation and antibody secretion. To test this hypothesis, we determined in AIM 1 whether NGLY1 abundance is upregulated in in vitro-activated primary mouse B cells. In AIM 2, we assessed whether the chemical inhibition or genetic ablation of Ngly1 in in vitro activated B cells affected viability, proliferation and differentiation and antibody secretion. Methods: To test our hypothesis, we employed in silico analysis of published RNA expression profiles, qPCR and western blot to determine Ngly1 mRNA and protein abundance in naive and in vitro activated murine B cells. In addition, we used chemical inhibition of NGLY1 by the small molecule WRR139 and genetic ablation to determine the effects of NGLY1 disruption on activated B cell survival, proliferation and differentiation by flow cytometry. To analyze the effect on antibody secretion, an enzyme-linked immunosorbent assay (ELISA) was employed. Results: We demonstrated that NGLY1 is upregulated in in vitro activated B cells at the mRNA and protein level. In addition, induced block of NGLY1 function by a small molecule or genetic deletion of Ngly1 reduces viability and proliferation and alters differentiation of in vitro activated B cells. Finally, we found protein aggregation in Ngly1-deficient activated B cells, suggesting disrupted proteostasis. Surprisingly, ELISA of activated B cell supernatants indicates increased antibody secretion upon NGLY1 dysfunction. Conclusions and discussion: Our findings support the hypothesis that activated primary B cells require functional NGLY1 for survival and proliferation. These findings will now be confirmed in a mouse with a B cell-specific conditional deletion of Ngly1. The increased IgM secretion of NGLY1-deficient cells warrants further investigation into the underlying mechanism.