Project description:Endogenous retroviruses (ERV), comprising a substantial portion of the vertebrate genome, are remnants of ancient genetic invaders. ERV with near-intact coding potential reactivate in B cell-deficient mice. Here, we employed an antigen-baiting strategy to enrich B cells reactive to ERV surface antigens. We identified ERV-reactive B-1 cells expressing germline-encoded natural IgM antibodies in naïve mice, the level of which further increases upon innate immune sensor stimulation. B cell receptor repertoire profiling of ERV-reactive B-1 cells revealed increased usage of Igh VH gene that gives rise to glycan-specific antibodies targeting terminal N-acetylglucosamine moieties on ERV glycoproteins, which further engage the complement pathway to protect the host from ERV emergence. These same antibodies also recognize glycoproteins of other enveloped viruses, but not self-proteins. These results reveal an innate antiviral mechanism of germline-encoded antibodies with broad reactivity to enveloped viruses, whose absence leads to the emergence of infectious ERV.
Project description:Symbiotic interactions between humans and our communities of resident gut microbes (microbiota) play many roles in health and disease. Some gut bacteria utilize mucus as a nutrient source and can under certain conditions damage the protective barrier it forms, increasing disease susceptibility. We investigated how Ruminococcus torques—a known mucin-degrader that remains poorly studied despite its implication in inflammatory bowel diseases (IBDs)— degrades mucin glycoproteins or their component O-linked glycans to understand its effects on the availability of mucin-derived nutrients for other bacteria. We found that R. torques utilizes both mucin glycoproteins and released oligosaccharides from gastric and colonic mucins, degrading these substrates with a panoply of mostly constitutively expressed, secreted enzymes. Investigation of mucin oligosaccharide degradation by R. torques revealed strong fucosidase, sialidase and b1,4-galactosidase activities. There was a lack of detectable sulfatase and weak β1,3-galactosidase degradation, resulting in accumulation of glycans containing these structures on mucin polypeptides. While the Gram-negative symbiont, Bacteroides thetaiotaomicron grows poorly on mucin glycoproteins, we demonstrate a clear ability of R. torques to liberate products from mucins, making them accessible to B. thetaiotaomicron. This work underscores the diversity of mucin-degrading mechanisms in different bacterial species and the probability that some species are contingent on others for the ability to more fully access mucin-derived nutrients. The ability of R. torques to directly degrade a variety of mucin and mucin glycan structures and unlock released glycans for other species suggests that it is a keystone mucin degrader, which may contribute to its association with IBD.
Project description:Keating CL, Kuhn E, Cocco AR, Yousif AS, Bals J, Matysiak C, angesland M, Setliff I, Ronsard L, Georgiev I, Balazs AB, Carr SA, Lingwood D. In cells asparagine/N-linked glycans are added to glycoproteins co-translationally, in an attachment process that is thought to be supported by the folding of the nascent polypeptide sequence. We find that following pruning of N-glycan by the amidase PNGase F, the influenza viral spike protein hemagglutinin (HA) autocatalyzed the re-addition of N-glycan to deaminated NXS/T sequons when the amidase was removed from solution. This reaction, which we term N-glycanation, was confirmed by site-specific analysis of HA glycoforms by mass spectrometry prior to PNGase F exposure, during exposure to PNGase F, and after amidase removal. Iterative rounds of de-N-glycosylation followed by N-glycanation that could be repeated at least 3 times. Covalent N-glycan reattachment was dependent on forming a non-covalent assembly between HA and glycan in the presence of the amidase. Linearization of HA prevented the retention of N-glycan and subsequent N-glycanation, indicating that protein surface features can exert dramatic self-organized control over the formation of glycan linkages and that NXS/T sequons may reside within active-site-like configurations that lower the activation threshold for N-glycosylation.
Project description:Aberrant O-glycosylation is a hallmark of cancer, but its contribution to immune evasion is unclear. Using CRISPR–Cas9 to delete GalNAc-transferases, particularly Galnt7, in tumor cells, we reduced mucin-type O-glycan truncation. This remodeling activated dendritic cell– and T cell–dependent immunity, eradicating established tumors, preventing metastasis, and generating durable memory. It enhanced clearance of MHC class I–positive tumors via coordinated CD8⁺ and CD4⁺ T cell responses, and uniquely enabled CD4⁺ T cell–mediated elimination of MHC class I–deficient tumors, which evade cytotoxic T lymphocyte surveillance. Tumor cells with reduced O-glycan truncation served as potent whole-cell vaccines, with efficacy further improved by radiotherapy. These findings establish glycosylation as a central regulator of tumor immune evasion and define a broadly applicable vaccine platform that bypasses the limitations of neoantigen-based approaches for low-immunogenicity cancers.