Reusable glycan microarrays using a microwave assisted wet-erase (MAWE) process.

Modern studies on binding of proteins to glycans commonly involve the use of synthetic glycans and their derivatives in which a small amount of the material is covalently printed onto a functionalized slide in a glycan microarray format. While incredibly useful to explore binding interactions with many types of samples, the common techniques involve drying the slides, which leads to irreversible association of the protein to the spots on slides to which they bound, thus limiting a microarray to a single use. We have developed a new technique which we term Microwave Assisted Wet-Erase (MAWE) glycan microarrays. In this approach we image the slides under wet conditions to acquire the data, after which the slides are cleaned of binding proteins by treatment with a denaturing SDS solution along with microwave treatment. Slides cleaned in this way can be reused multiple times, and an example here shows the reuse of a single array 15 times. We also demonstrate that this method can be used for a single-array per slide or multi-array per slide platforms. Importantly, the results obtained using this technique for a variety of lectins sequentially applied to a single array, are concordant to those obtained via the classical dry approaches on multiple slides. We also demonstrate that MAWE can be used for different types of samples, such as serum for antibody binding, and whole cells, such as yeast. This technique will greatly conserve precious glycans and prolong the use of existing and new glycan microarrays.

Germline C1GALT1C1 mutation causes a multisystem chaperonopathy.

Mutations in genes encoding molecular chaperones can lead to chaperonopathies, but none have so far been identified causing congenital disorders of glycosylation. Here we identified two maternal half-brothers with a novel chaperonopathy, causing impaired protein O-glycosylation. The patients have a decreased activity of T-synthase (C1GALT1), an enzyme that exclusively synthesizes the T-antigen, a ubiquitous O-glycan core structure and precursor for all extended O-glycans. The T-synthase function is dependent on its specific molecular chaperone Cosmc, which is encoded by X-chromosomal C1GALT1C1. Both patients carry the hemizygous variant c.59C>A (p.Ala20Asp; A20D-Cosmc) in C1GALT1C1. They exhibit developmental delay, immunodeficiency, short stature, thrombocytopenia, and acute kidney injury (AKI) resembling atypical hemolytic uremic syndrome. Their heterozygous mother and maternal grandmother show an attenuated phenotype with skewed X-inactivation in blood. AKI in the male patients proved fully responsive to treatment with the complement inhibitor Eculizumab. This germline variant occurs within the transmembrane domain of Cosmc, resulting in dramatically reduced expression of the Cosmc protein. Although A20D-Cosmc is functional, its decreased expression, though in a cell or tissue-specific manner, causes a large reduction of T-synthase protein and activity, which accordingly leads to expression of varied amounts of pathological Tn-antigen (GalNAcα1-O-Ser/Thr/Tyr) on multiple glycoproteins. Transient transfection of patient lymphoblastoid cells with wild-type C1GALT1C1 partially rescued the T-synthase and glycosylation defect. Interestingly, all four affected individuals have high levels of galactose-deficient IgA1 in sera. These results demonstrate that the A20D-Cosmc mutation defines a novel O-glycan chaperonopathy and causes the altered O-glycosylation status in these patients.

Siglec-7 represents a glyco-immune checkpoint for non-exhausted effector memory CD8+ T cells with high functional and metabolic capacities.

While inhibitory Siglec receptors are known to regulate myeloid cells, less is known about their expression and function in lymphocytes subsets. Here we identified Siglec-7 as a glyco-immune checkpoint expressed on non-exhausted effector memory CD8+ T cells that exhibit high functional and metabolic capacities. Seahorse analysis revealed higher basal respiration and glycolysis levels of Siglec-7+ CD8+ T cells in steady state, and particularly upon activation. Siglec-7 polarization into the T cell immune synapse was dependent on sialoglycan interactions in trans and prevented actin polarization and effective T cell responses. Siglec-7 ligands were found to be expressed on both leukemic stem cells and acute myeloid leukemia (AML) cells suggesting the occurrence of glyco-immune checkpoints for Siglec-7+ CD8+ T cells, which were found in patients' peripheral blood and bone marrow. Our findings project Siglec-7 as a glyco-immune checkpoint and therapeutic target for T cell-driven disorders and cancer.

Lack of IRF6 Disrupts Human Epithelial Homeostasis by Altering Colony Morphology, Migration Pattern, and Differentiation Potential of Keratinocytes.

Variants within the gene encoding for the transcription factor Interferon Regulatory Factor 6 (IRF6) are associated with syndromic and non-syndromic Cleft Lip/Palate (CLP) cases. IRF6 plays a vital role in the regulation of the proliferation/differentiation balance in keratinocytes and is involved in wound healing and migration. Since a fraction of CLP patients undergoing corrective cleft surgery experience wound healing complications, IRF6 represents an interesting candidate gene linking the two processes. However, Irf6 function has been mainly studied in mice and knowledge on IRF6 in human cells remains sparse. Here, we aimed to elucidate the role of IRF6 in human postnatal skin- and oral mucosa-derived keratinocytes. To do so, we applied CRISPR/Cas9 to ablate IRF6 in two TERT-immortalized keratinocyte cultures, which we used as model cell lines. We show that IRF6 controls the appearance of single cells and colonies, with the latter being less cohesive in its absence. Consequently, IRF6 knockout keratinocytes often moved as single cells instead of a collective epithelial sheet migration but maintained their epithelial character. Lack of IRF6 triggered severe keratinocyte differentiation defects, which were already apparent in the stratum spinosum and extended to the stratum corneum in 3D organotypic skin cultures, while it did not alter their growth rate. Finally, proteomics revealed that most of the differentially expressed proteins in the absence of IRF6 could be associated with differentiation, cell-cell adhesion as well as immune response. Our data expand the knowledge on IRF6 in human postnatal keratinocytes, which will help to better understand IRF6-related pathologies.

Parallelism of intestinal secretory IgA shapes functional microbial fitness.

Dimeric IgA secreted across mucous membranes in response to nonpathogenic taxa of the microbiota accounts for most antibody production in mammals. Diverse binding specificities can be detected within the polyclonal mucosal IgA antibody response1-10, but limited monoclonal hybridomas have been studied to relate antigen specificity or polyreactive binding to functional effects on microbial physiology in vivo11-17. Here we use recombinant dimeric monoclonal IgAs (mIgAs) to finely map the intestinal plasma cell response to microbial colonization with a single microorganism in mice. We identify a range of antigen-specific mIgA molecules targeting defined surface and nonsurface membrane antigens. Secretion of individual dimeric mIgAs targeting different antigens in vivo showed distinct alterations in the function and metabolism of intestinal bacteria, largely through specific binding. Even in cases in which the same microbial antigen is targeted, microbial metabolic alterations differed depending on IgA epitope specificity. By contrast, bacterial surface coating generally reduced motility and limited bile acid toxicity. The overall intestinal IgA response to a single microbe therefore contains parallel components with distinct effects on microbial carbon-source uptake, bacteriophage susceptibility, motility and membrane integrity.

Glycan-specific IgG anti-IgE autoantibodies are protective against allergic anaphylaxis in a murine model.

BACKGROUND: IgE causes anaphylaxis in type I hypersensitivity diseases by activating degranulation of effector cells such as mast cells and basophils. The mechanisms that control IgE activity and prevent anaphylaxis under normal conditions are still enigmatic. OBJECTIVE: We aimed to unravel how anti-IgE autoantibodies are induced and we aimed to understand their role in regulating serum IgE level and allergic anaphylaxis. METHODS: We immunized mice with different forms of IgE and tested anti-IgE autoantibody responses and their specificities. We then analyzed the effect of those antibodies on serum kinetics and their in vitro and in vivo impact on anaphylaxis. Finally, we investigated anti-IgE autoantibodies in human sera. RESULTS: Immunization of mice with IgE-immune complexes induced glycan-specific anti-IgE autoantibodies. The anti-IgE autoantibodies prevented effector cell sensitization, reduced total IgE serum levels, protected mice from passive and active IgE sensitization, and resulted in cross-protection against different allergens. Furthermore, glycan-specific anti-IgE autoantibodies were present in sera from subjects with allergy and subjects without allergy. CONCLUSION: In conclusion, this study provided the first evidence that in the murine model, the serum level and anaphylactic activity of IgE may be downregulated by glycan-specific IgG anti-IgE autoantibodies.