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The underlying pathology of immunoglobulin A (IgA) nephropathy (IgAN), the most common glomerulonephritis worldwide, is driven by the deposition of immune complexes containing galactose-deficient IgA1 [Tn(+)IgA1] in the glomerular mesangium. Here, we report that novel anti-Tn circulating immune complexes (anti-Tn CICs) contain predominantly IgM, representing large macromolecular complexes of ~1.2 megadaltons to several megadalton sizes together with Tn(+)IgA1 and some IgG. These complexes are significantly elevated in sera of patients with IgAN, which contains higher levels of complement C3, compared to healthy individuals. Anti-Tn CICs are bioactive and induce specific proliferation of human renal mesangial cells. We found that these anti-Tn CICs can be dissociated with small glycomimetic compounds, which mimic the Tn antigen of Tn(+)IgA1, releasing IgA1 from anti-Tn CICs. This glycomimetic compound can also significantly inhibit the proliferative activity of anti-Tn CICs of patients with IgAN. These findings could enhance both the diagnosis of IgAN and its treatment, as specific drug treatments are now unavailable.

(1) Background: Barrett’s esophagus is a major risk factor for esophageal adenocarcinoma. In this pilot study, we employed precision mass spectrometry to map global (phospho)protein perturbations in Barrett’s esophagus lesions and adjacent normal tissue to glean insights into disease progression. (2) Methods: Biopsies were collected from two small but independent cohorts. Comparative analyses were performed between Barrett’s esophagus samples and adjacent matched (normal) tissues from patients with known pathology, while specimens from healthy patients served as additional controls. (3) Results: We identified and quantified 6810 proteins and 6395 phosphosites in the discovery cohort, revealing hundreds of statistically significant differences in protein abundances and phosphorylation states. We identified a robust proteomic signature that accurately classified the disease status of samples from the independent patient cohorts. Pathway-level analysis of the phosphoproteomic profiles revealed the dysregulation of specific cellular processes, including DNA repair, in Barrett’s esophagus relative to paired controls. Comparative analysis with previously published transcriptomic profiles provided independent evidence in support of these preliminary findings. (4) Conclusions: This pilot study establishes the feasibility of using unbiased quantitative phosphoproteomics to identify molecular perturbations associated with disease progression in Barrett’s esophagus to define potentially clinically actionable targets warranting further assessment.

Mucins are large gel-forming polymers inside the mucus barrier that inhibit the yeast-to-hyphal transition of Candida albicans, a key virulence trait of this important human fungal pathogen. However, the molecular motifs in mucins that inhibit filamentation remain unclear despite their potential for therapeutic interventions. Here, we determined that mucins display an abundance of virulence-attenuating molecules in the form of mucin O-glycans. We isolated and cataloged >100 mucin O-glycans from three major mucosal surfaces and established that they suppress filamentation and related phenotypes relevant to infection, including surface adhesion, biofilm formation and cross-kingdom competition between C. albicans and the bacterium Pseudomonas aeruginosa. Using synthetic O-glycans, we identified three structures (core 1, core 1 + fucose and core 2 + galactose) that are sufficient to inhibit filamentation with potency comparable to the complex O-glycan pool. Overall, this work identifies mucin O-glycans as host molecules with untapped therapeutic potential to manage fungal pathogens.

Bladder cancer is the ninth most frequently diagnosed cancer worldwide, and there is a need to develop new biomarkers for staging and prognosis of this disease. Here we report that cell lines derived from low-grade and high-grade bladder cancers exhibit major differences in expression of glycans in surface glycoproteins. We analyzed protein glycosylation in three low-grade bladder cancer cell lines RT4 (grade-1-2), 5637 (grade-2), and SW780 (grade-1), and three high-grade bladder cancer cell lines J82COT (grade-3), T24 (grade-3) and TCCSUP (grade-4), with primary bladder epithelial cells, A/T/N, serving as a normal bladder cell control. Using a variety of approaches including flow cytometry, immunofluorescence, glycomics and gene expression analysis, we observed that the low-grade bladder cancer cell lines RT4, 5637 and SW780 express high levels of the fucosylated Lewis-X antigen (Lex, CD15) (Galβ1–4(Fucα1–3)GlcNAcβ1-R), while normal bladder epithelial A/T/N cells lack Lex expression. T24 and TCCSUP cells also lack Lex, whereas J82COT cells express low levels of Lex. Glycomics analyses revealed other major differences in fucosylation and sialylation of N-glycans between these cell types. O-glycans are highly differentiated, as RT4 cells synthesize core 2-based O-glycans that are lacking in the T24 cells. These differences in glycan expression correlated with differences in RNA expression levels of their cognate glycosyltransferases, including α1–3/4-fucosyltransferase genes. These major differences in glycan structures and gene expression profiles between low- and high-grade bladder cancer cells suggest that glycans and glycosyltransferases are candidate biomarkers for grading bladder cancers.

A slimy, hydrated mucus gel lines all wet epithelia in the human body, including the eyes, lungs, and gastrointestinal and urogenital tracts. Mucus forms the first line of defence while housing trillions of microorganisms that constitute the microbiota1. Rarely do these microorganisms cause infections in healthy mucus1, suggesting that mechanisms exist in the mucus layer that regulate virulence. Using the bacterium Pseudomonas aeruginosa and a three-dimensional (3D) laboratory model of native mucus, we determined that exposure to mucus triggers downregulation of virulence genes that are involved in quorum sensing, siderophore biosynthesis and toxin secretion, and rapidly disintegrates biofilms—a hallmark of mucosal infections. This phenotypic switch is triggered by mucins, which are polymers that are densely grafted with O-linked glycans that form the 3D scaffold inside mucus. Here, we show that isolated mucins act at various scales, suppressing distinct virulence pathways, promoting a planktonic lifestyle, reducing cytotoxicity to human epithelia in vitro and attenuating infection in a porcine burn model. Other viscous polymer solutions lack the same effect, indicating that the regulatory function of mucin does not result from its polymeric structure alone. We identify that interactions with P. aeruginosa are mediated by mucin-associated glycans (mucin glycans). By isolating glycans from the mucin backbone, we assessed the collective activity of hundreds of complex structures in solution. Similar to their grafted counterparts, free mucin glycans potently regulate bacterial phenotypes even at relatively low concentrations. This regulatory function is likely dependent on glycan complexity, as monosaccharides do not attenuate virulence. Thus, mucin glycans are potent host signals that ‘tame’ microorganisms, rendering them less harmful to the host. Host mucin glycans downregulate virulence processes of Pseudomonas aeruginosa and can be used therapeutically to attenuate infection in vivo in a burn wound model.

Aims: To assess the effects of walnuts on cardiometabolic outcomes in obese people and to explore the underlying mechanisms using novel methods including metabolomic, lipidomic, glycomic and microbiome analysis, integrated with lipid particle fractionation, appetite-regulating hormones and haemodynamic measurements. Materials and Methods: A total of 10 obese individuals were enrolled in this cross-over, randomized, double-blind, placebo-controlled clinical trial. The participants had two 5-day inpatient stays, during which they consumed a smoothie containing 48 g walnuts or a macronutrient-matched placebo smoothie without nuts, with a 1-month washout period between the two visits. Results: Walnut consumption improved aspects of the lipid profile; it reduced fasting small and dense LDL particles (P < 0.02) and increased postprandial large HDL particles (P < 0.01). Lipoprotein insulin resistance score, glucose and the insulin area under the curve (AUC) decreased significantly after walnut consumption (P < 0.01, P < 0.02 and P < 0.04, respectively). Consuming walnuts significantly increased 10 N-glycans, with eight of them carrying a fucose core. Lipidomic analysis showed a robust reduction in harmful ceramides, hexosylceramides and sphingomyelins, which have been shown to mediate effects on cardiometabolic risk. The peptide YY AUC significantly increased after walnut consumption (P < 0.03). No major significant changes in haemodynamic or metabolomic analysis or in microbiome host health-promoting bacteria such as Faecalibacterium were found. Conclusions: These data provide a more comprehensive mechanistic perspective of the effect of dietary walnut consumption on cardiometabolic variables. Lipidomic and lipid nuclear magnetic resonance spectroscopy analysis showed an early but significant reduction in ceramides and other atherogenic lipids with walnut consumption, which may explain the longer-term benefits of walnuts or other nuts on insulin resistance, cardiovascular risk and mortality.

High levels of proinflammatory cytokines have been associated with a loss of tissue function in ocular autoimmune diseases, but the basis for this relationship remains poorly understood. Here we investigate a new role for tumor necrosis factor α in promoting N-glycan-processing deficiency at the surface of the eye through inhibition of N-acetylglucosaminyltransferase expression in the Golgi. Using mass spectrometry, complex-type biantennary oligosaccharides were identified as major N-glycan structures in differentiated human corneal epithelial cells. Remarkably, significant differences were detected between the efficacies of cytokines in regulating the expression of glycogenes involved in the biosynthesis of N-glycans. Tumor necrosis factor α but not IL-1β had a profound effect in suppressing the expression of enzymes involved in the Golgi branching pathway, including N-acetylglucosaminyltransferases 1 and 2, which are required for the formation of biantennary structures. This decrease in gene expression was correlated with a reduction in enzymatic activity and impaired N-glycan branching. Moreover, patients with ocular mucous membrane pemphigoid were characterized by marginal N-acetylglucosaminyltransferase expression and decreased N-glycan branching in the conjunctiva. Together, these data indicate that proinflammatory cytokines differentially influence the expression of N-glycan-processing enzymes in the Golgi and set the stage for future studies to explore the pathophysiology of ocular autoimmune diseases.

Lupus nephritis (LN) is a serious complication occurring in 50% of patients with systemic lupus erythematosus (SLE) for which there is a lack of biomarkers, a lack of specific medications, and a lack of a clear understanding of its pathogenesis. The expression of calcium/calmodulin kinase IV (CaMK4) is increased in podocytes of patients with LN and lupus-prone mice, and its podocyte-targeted inhibition averts the development of nephritis in mice. Nephrin is a key podocyte molecule essential for the maintenance of the glomerular slit diaphragm. Here, we show that the presence of fucose on N-glycans of IgG induces, whereas the presence of galactose ameliorates, podocyte injury through CaMK4 expression. Mechanistically, CaMK4 phosphorylates NF-κB, upregulates the transcriptional repressor SNAIL, and limits the expression of nephrin. In addition, we demonstrate that increased expression of CaMK4 in biopsy specimens and in urine podocytes from people with LN is linked to active kidney disease. Our data shed light on the role of IgG glycosylation in the development of podocyte injury and propose the development of “liquid kidney biopsy” approaches to diagnose LN.

Patient-derived organoid models are proving to be a powerful platform for both basic and translational studies. Here we conduct a methodical analysis of pancreatic ductal adenocarcinoma (PDAC) tumor organoid drug response in paired patient-derived xenograft (PDX) and PDX-derived organoid (PXO) models grown under WNT-free culture conditions. We report a specific relationship between area under the curve value of organoid drug dose response and in vivo tumor growth, irrespective of the drug treatment. In addition, we analyzed the glycome of PDX and PXO models and demonstrate that PXOs recapitulate the in vivo glycan landscape. In addition, we identify a core set of 57 N-glycans detected in all 10 models that represent 50%–94% of the relative abundance of all N-glycans detected in each of the models. Last, we developed a secreted biomarker discovery pipeline using media supernatant of organoid cultures and identified potentially new extracellular vesicle (EV) protein markers. We validated our findings using plasma samples from patients with PDAC, benign gastrointestinal diseases, and chronic pancreatitis and discovered that 4 EV proteins are potential circulating biomarkers for PDAC. Thus, we demonstrate the utility of organoid cultures to not only model in vivo drug responses but also serve as a powerful platform for discovering clinically actionable serologic biomarkers., Pancreatic ductal adenocarcinoma tumor organoids are effective models for predicting in vivo drug response and discovering new biomarkers.

SummaryGlycosylation is essential to brain development and function, though prior studies have often been limited to a single analytical technique. Using several methodologies, we analyzed Asn-linked (N-glycans) and Ser/Thr/Tyr-linked (O-glycans) protein glycosylation between brain regions and sexes in mice. Brain N-glycans were surprisingly less complex in sequence and variety compared to other tissues, consisting predominantly of high-mannose precursors and fucosylated/bisected structures. Most brain O-glycans were unbranched, sialylated O-GalNAc and O-mannose structures. A consistent pattern was observed between regions, and sex differences were minimal compared to those observed in plasma. Brain glycans correlate with RNA expression of their synthetic enzymes, and analysis of all glycosylation genes in humans showed a global downregulation in the brain compared to other tissues. We hypothesize that the restricted repertoire of protein glycans arises from their tight regulation in the brain. These results provide a roadmap for future studies of glycosylation in neurodevelopment and disease.

Alpha-1-acid glycoprotein (AGP-1) is a positive acute phase glycoprotein with uncertain functions. Serum AGP-1 (sAGP-1) is primarily derived from hepatocytes and circulates as 12–20 different glycoforms. We isolated a glycoform secreted from platelet-activating factor (PAF)-stimulated human neutrophils (nAGP-1). Its peptide sequence was identical to hepatocyte-derived sAGP-1, but nAGP-1 differed from sAGP-1 in its chromatographic behavior, electrophoretic mobility, and pattern of glycosylation. The function of these 2 glycoforms also differed. sAGP-1 activated neutrophil adhesion, migration, and neutrophil extracellular traps (NETosis) involving myeloperoxidase, peptidylarginine deiminase 4, and phosphorylation of ERK in a dose-dependent fashion, whereas nAGP-1 was ineffective as an agonist for these events. Furthermore, sAGP-1, but not nAGP-1, inhibited LPS-stimulated NETosis. Interestingly, nAGP-1 inhibited sAGP-1-stimulated neutrophil NETosis. The discordant effect of the differentially glycosylated AGP-1 glycoforms was also observed in platelets where neither of the AGP-1 glycoforms alone stimulated aggregation of washed human platelets, but sAGP-1, and not nAGP-1, inhibited aggregation induced by PAF or ADP, but not by thrombin. These functional effects of sAGP-1 correlated with intracellular cAMP accumulation and phosphorylation of the protein kinase A substrate vasodilator-stimulated phosphoprotein and reduction of Akt, ERK, and p38 phosphorylation. Thus, the sAGP-1 glycoform limits platelet reactivity, whereas nAGP-1 glycoform also limits proinflammatory actions of sAGP-1. These studies identify new functions for this acute phase glycoprotein and demonstrate that the glycosylation of AGP-1 controls its effects on 2 critical cells of acute inflammation.

The molecular mechanisms regulating lymphocyte homing into lymph nodes are only partly understood. Here, we report that B cell-specific deletion of the X-linked gene, Cosmc, and the consequent decrease of protein O-glycosylation, induces developmental blocks of mouse B cells. After transfer into wild-type recipient, Cosmc-null B cells fail to home to lymph nodes as well as non-lymphoid organs. Enzymatic desialylation of wild-type B cells blocks their migration into lymph nodes, indicating a requirement of sialylated O-glycans for proper trafficking. Mechanistically, Cosmc-deficient B cells have normal rolling and firm arrest on high endothelium venules (HEV), thereby attributing their inefficient trafficking to alterations in the subsequent transendothelial migration step. Finally, Cosmc-null B cells have defective chemokine signaling responses. Our results thus demonstrate that Cosmc and its effects on O-glycosylation are important for controlling B cell homing., Migration and homing of B cells to lymph nodes are important for B cell functions, but their regulation is poorly understood. Here, the authors show that B cell-specific deletion of Cosmc results in decreased protein O-glycosylation, loss of B cell homing to both lymphoid and nonlymphoid organs, and altered transendothelial migration implicated in this loss.

Alpha-1-acid glycoprotein (AGP-1) is a positive acute phase glycoprotein with uncertain functions. Serum AGP-1 (sAGP-1) is primarily derived from hepatocytes and circulates as 12 to 20 different glycoforms. We isolated a glycoform secreted from stimulated human neutrophils (nAGP-1). Its peptide sequence was identical to hepatocyte-derived sAGP-1, but nAGP-1 differed from sAGP-1 in its chromatographic behaviour, electrophoretic mobility, and glycosylation. The function of these two glycoforms also differed. sAGP-1 activated neutrophil adhesion, migration and NETosis in a dose-dependent fashion, while nAGP-1 was ineffective as an agonist for these events. Furthermore, sAGP-1, but not nAGP-1, inhibited LPS-stimulated NETosis. However, nAGP-1 inhibited sAGP-1-stimulated neutrophil NETosis. The discordant effect of the differentially glycosylated AGP-1 glycoforms was also observed in platelets where neither of the AGP-1 glycoforms alone stimulated aggregation of washed human platelets, but sAGP-1, and not nAGP-1, inhibited aggregation induced by Platelet-activating Factor (PAF) or ADP, but not by thrombin. These functional effects of sAGP-1 correlated with intracellular cAMP accumulation and were accompanied by phosphorylation of the PKA substrate Vasodialator stimulated phosphoprotein (VASP) and reduction of Akt, ERK, and p38 phosphorylation. Thus, the sAGP-1 glycoform limits platelet reactivity while nAGP-1 glycoform also limits pro-inflammatory actions of sAGP-1. These studies identify new functions for this acute phase glycoprotein and demonstrate that the glycosylation of AGP-1 controls its effects on two critical cells of acute inflammation.

Filariases are diseases caused by infection with filarial nematodes and transmitted by insect vectors. The filarial roundworm Dirofilaria immitis causes heartworm disease in dogs and other carnivores. D. immitis is closely related to Onchocerca volvulus, Wuchereria bancrofti and Brugia malayi, which cause onchocerciasis (river blindness) and lymphatic filariasis (elephantiasis) in humans and are neglected tropical diseases. Serum N-glycosylation is very sensitive to both pathological infections and changes in mammalian biology due to normal aging or lifestyle choices. Here, we report significant changes in the serum N-glycosylation profiles of dogs infected with D. immitis. Our data derive from analysis of serum from dogs with established patent infections and from a longitudinal infection study. Overall, galactosylation and core fucosylation increase, while sialylation decreases in infected dog sera. We also identify individual glycan structures that change significantly in their relative abundance during infection. Notably, the abundance of the most dominant N-glycan in canine serum (biantennary, disialylated A2G2S2) decreases by over 10 percentage points during the first 6 months of infection in each dog analyzed. This is the first longitudinal study linking changes in mammalian serum N-glycome to progression of a parasitic infection.

Objective To better understand the role of B cells, the potential mechanisms responsible for their aberrant activation, and the production of autoantibodies in the pathogenesis of Sjogren's syndrome (SS), this study explored patterns of selection pressure and sites of N-glycosylation acquired by somatic mutation (acN-glyc) in the IgG variable (V) regions of antibody-secreting cells (ASCs) isolated from the minor salivary glands of patients with SS and non-SS control patients with sicca symptoms. Methods A novel method to produce and characterize recombinant monoclonal antibodies (mAb) from single cell-sorted ASC infiltrates was applied to concurrently probe expressed genes (all heavy- and light-chain isotypes as well as any other gene of interest not related to immunoglobulin) in the labial salivary glands of patients with SS and non-SS controls. V regions were amplified by reverse transcription-polymerase chain reaction, sequenced, and analyzed for the incidence of N-glycosylation and selection pressure. For specificity testing, the amplified regions were expressed as either the native mAb or mutant mAb lacking the acN-glyc motif. Protein modeling was used to demonstrate how even an acN-glyc site outside of the complementarity-determining region could participate in, or inhibit, antigen binding. Results V-region sequence analyses revealed clonal expansions and evidence of secondary light-chain editing and allelic inclusion, of which neither of the latter two have previously been reported in patients with SS. Increased frequencies of acN-glyc were found in the sequences from patients with SS, and these acN-glyc regions were associated with an increased number of replacement mutations and lowered selection pressure. A clonal set of polyreactive mAb with differential framework region 1 acN-glyc motifs was also identified, and removal of the acN-glyc could nearly abolish binding to autoantigens. Conclusion These findings support the notion of an alternative mechanism for the selection and proliferation of some autoreactive B cells, involving V-region N-glycosylation, in patients with SS.

Transmembrane mucins are highly O-glycosylated glycoproteins that coat the apical glycocalyx on mucosal surfaces and represent the first line of cellular defense against infection and injury. Relatively low levels of N-glycans are found on transmembrane mucins, and their structure and function remain poorly characterized. We previously reported that carbohydrate-dependent interactions of transmembrane mucins with galectin-3 contribute to maintenance of the epithelial barrier at the ocular surface. Now, using MALDI-TOF mass spectrometry, we report that transmembrane mucin N-glycans in differentiated human corneal epithelial cells contain primarily complex-type structures with N-acetyllactosamine, a preferred galectin ligand. In N-glycosylation inhibition experiments, we find that treatment with tunicamycin and siRNA-mediated knockdown of the Golgi N-acetylglucosaminyltransferase I gene (MGAT1) induce partial loss of both total and cell-surface levels of the largest mucin, MUC16, and a concomitant reduction in glycocalyx barrier function. Moreover, we identified a distinct role for N-glycans in promoting MUC16's binding affinity toward galectin-3 and in causing retention of the lectin on the epithelial cell surface. Taken together, these studies define a role for N-linked oligosaccharides in supporting the stability and function of transmembrane mucins on mucosal surfaces.

Zika virus (ZIKV) is a global public health issue due to its association with severe developmental disorders in infants and neurological disorders in adults. ZIKV uses glycosylation of its envelope (E) protein to interact with host cell receptors to facilitate entry, these interactions could also be important for designing therapeutics and vaccines. Due to a lack of proper information about Asn-linked (N-glycans) on ZIKV E, we analyzed ZIKV E of various strains derived from different cells. We found ZIKV E proteins being extensively modified with oligomannose, hybrid and complex N-glycans of a highly heterogeneous nature. Host cell surface glycans correlated strongly with the glycomic features of ZIKV E. Mechanistically, we observed that ZIKV N-glycans might play a role in viral pathogenesis, as mannose-specific C-type lectins DC-SIGN and L-SIGN mediate host cell entry of ZIKV. Our findings represent the first detailed mapping of N-glycans on ZIKV E of various strains and their functional significance.

A common missense variant in SLC39A8 is convincingly associated with schizophrenia and several additional phenotypes. Homozygous loss-of-function mutations in SLC39A8 result in undetectable serum manganese (Mn) and a Congenital Disorder of Glycosylation (CDG) due to the exquisite sensitivity of glycosyltransferases to Mn concentration. Here, we identified several Mn-related changes in human carriers of the common SLC39A8 missense allele. Analysis of structural brain MRI scans showed a dose-dependent change in the ratio of T2w to T1w signal in several regions. Comprehensive trace element analysis confirmed a specific reduction of only serum Mn, and plasma protein N-glycome profiling revealed reduced complexity and branching. N-glycome profiling from two individuals with SLC39A8-CDG showed similar but more severe alterations in branching that improved with Mn supplementation, suggesting that the common variant exists on a spectrum of hypofunction with potential for reversibility. Characterizing the functional impact of this variant will enhance our understanding of schizophrenia pathogenesis and identify novel therapeutic targets and biomarkers of disease.SummaryA common variant in the manganese transporter SLC39A8 is associated with numerous phenotypes including schizophrenia. Mealer et. al. presents an in-depth analysis of brain MRI and plasma glycomics in human carriers of the common variant, identifying several manganese-related changes with potential for diagnostic and therapeutic biomarker development.

The Tn antigen is a neoantigen abnormally expressed in many human carcinomas and expression correlates with metastasis and poor survival. To explore its biomarker potential, new antibodies are needed that specifically recognize this antigen in tumors. Here we generated two recombinant antibodies to the Tn antigen, Remab6 as a chimeric human IgG1 antibody and ReBaGs6 as a murine IgM antibody, and characterized their specificities using multiple biochemical and biological approaches. Both Remab6 and ReBaGs6 recognize clustered Tn structures, but most importantly do not recognize glycoforms of human IgA1 that contain potential cross-reactive Tn antigen structures. In flow cytometry and immunofluorescence analyses, Remab6 recognizes human cancer cell lines expressing the Tn antigen, but not their Tn-negative counterparts. In immunohistochemistry (IHC), Remab6 stains many human cancers in tissue array format but rarely stains normal tissues and then mostly intracellularly. We used these antibodies to identify several unique Tn containing glycoproteins in Tn-positive Colo205 cells, indicating their utility for glycoproteomics in future biomarker studies. Thus, recombinant Remab6 and ReBaGs6 are useful for biochemical characterization of cancer cells and IHC of tumors, and represent promising tools for Tn biomarker discovery independently of recognition of IgA1. [191 words].

Zika virus (ZIKV) is a global public health issue due to its association with severe developmental disorders in infants and neurological disorders in adults. Because ZIKV uses glycosylation of its envelope (E) protein to interact with host cell receptors to facilitate entry, these interactions could also be important for designing therapeutics and vaccines. Due to a lack of information about Asn-linked (N-glycans) on ZIKV E, we analyzed ZIKV E of various strains derived from different cells. ZIKV E proteins are extensively modified with oligomannose-, hybrid- and complex-N-glycans of a highly heterogeneous nature. Host cell-surface glycans correlated strongly with the glycomic features of ZIKV E. Mechanistically, we discovered that ZIKV N-glycans are important in viral pathogenesis, as mannose-specific C-type lectins DC-SIGN and L-SIGN mediate cell entry of ZIKV. Our findings represent the first detailed mapping of N-glycans on ZIKV E of various strains and their functional significance.

Despite advances in stem cell research, cell transplantation therapy for liver failure is impeded by a shortage of human primary hepatocytes (HPH), along with current differentiation protocol limitations. Several studies have examined the concept of co-culture of human induced pluripotent cells (hiPSCs) with various types of supporting non-parenchymal cells to attain a higher differentiation yield and to improve hepatocyte-like cell functions both in vitro and in vivo. Co-culturing hiPSCs with human endothelial cells (hECs) is a relatively new technique that requires more detailed studies. Using our 3D human embryoid bodies (hEBs) formation technology, we interlaced Human Adipose Microvascular Endothelial Cells (HAMEC) with hiPSCs, leading to a higher differentiation yield and notable improvements across a wide range of hepatic functions. We conducted a comprehensive gene and protein secretion analysis of our HLCs coagulation factors profile, showing promising results in comparison with HPH. Furthermore, a stage-specific glycomic analysis revealed that the differentiated hepatocyte-like clusters (HLCs) resemble the glycan features of a mature tissue rather than cells in culture. We tested our HLCs in animal models, where the presence of HAMEC in the clusters showed a consistently better performance compared to the hiPSCs only group in regard to persistent albumin secretion post-transplantation.

Patient-derived models are transforming translational cancer research. It is not clear if the emergence of patient-derived organoid (PDO) models can extend the utility of the widely used patient-derived xenograft (PDX). In addition, the utility of PDO models for serum biomarker discovery is not known. Here, we demonstrate that PDO models recapitulate the genomics, cell biology, glycomics and drug responses observed in PDX models. Furthermore, we demonstrate the applicability of PDO models for identification of N-glycans that are enriched in the glycome of pancreatic ductal adenocarcinoma (PDAC). Surprisingly, among all the glycans observed in PDX and PDOs, a core set of 57 N-glycans represent 50-94% of the relative abundance of all N-glycans detected, suggesting that only a subset of glycans dominate the cell surface landscape in PDAC. In addition, we outline a tumor organoid-based pipeline to identify surface proteins in extracellular vesicles (EV) from media supernatant of PDO cultures. When combined with the affinity-based validation platform, the EV surface proteins discovered in PDOs are effective in differentiating patients with PDAC from those with benign pancreatitis in the clinic, identifying PDO as powerful discovery platform for serum biomarkers. Thus, PDOs extend the utility of the archival collections of PDX models for translational research and function as a powerful platform for identification of clinically-actionable biomarkers in patients blood.Significance statementTumor organoids extend the utility of PDX models as platforms for investigating drug response, glycosylation changes and function as new platforms for discovering blood-based biomarkers

Glycosylation is the most common post-translational modification of serum proteins, and changes in the type and abundance of glycans in human serum have been correlated with a growing number of human diseases. While the glycosylation pattern of human serum is well studied, little is known about the profiles of other mammalian species. Here, we report detailed glycosylation profiling of canine serum by hydrophilic interaction chromatography-ultraperformance liquid chromatography (HILIC-UPLC) and mass spectrometry. The domestic dog (Canis familiaris) is a widely used model organism and of considerable interest for a large veterinary community. We found significant differences in the serum N-glycosylation profile of dogs compared to that of humans, such as a lower abundance of galactosylated and sialylated glycans. We also compare the N-glycan profile of canine serum to that of canine IgG – the most abundant serum glycoprotein. Our data will serve as a baseline reference for future studies when performing serum analyses of various health and disease states in dogs.

Human immunoglobulin A1 (IgA1), which carries four to six mucin-type O-glycans (O-glycans) on its hinge region (HR), is the most abundant O-glycoprotein in plasma or serum. While normal O-glycans from hematopoietic-originated cells are core 1-based complex structures, many reports showed that the IgA1 from patients with IgA nephropathy (IgAN) carries undergalactosylated or truncated O-glycans such as the Tn antigen and its sialylated version the SialylTn (STn) antigen on the HR. Yet, there is still a debate whether Tn/STn on the HR of IgA1 is specific to the IgA1 from patients with IgAN since these antigens have also been seen in serum IgA1 of healthy individuals. An additional question is whether the O-glycans at all sites on the two HRs of one IgA1 molecule are homogeneous (either all normal or all Tn/STn) or heterogeneous (both normal and Tn/STn O-glycans). To address these questions, we conducted a systematic study on the O-glycans of plasma IgA1 from both IgAN patients and healthy controls using serial HPA and PNA lectin chromatography followed by western blotting and further analysis of O-glycans from HPA-bound and PNA-bound IgA1 fractions by mass spectrometry. Unexpectedly, we found that a variable minor fraction of IgA1 from both IgAN patients and healthy controls had Tn/STn antigens, and that the O-glycoprotein IgA1 molecules from most samples had only two distinct O-glycoforms: one major glycoform with homogeneous normal core 1-based O-glycans and one minor glycoform with homogeneous Tn/STn antigens. These results raised a serious question about the role of Tn/STn antigens on IgA1 in pathogenesis of IgAN, and there is a demand for a practical methodology that any laboratory can utilize to analyze the O-glycans of IgA1. Herein, we describe the methodology we developed in more detail. The method could also be applied to the analysis of any other O-glycosylated proteins.

International audience; The second human beta-galactoside alpha-2,6-sialyltransferase (hST6Gal II) differs from hST6Gal I, the first member of ST6Gal family, in substrate specificity and tissue expression pattern. While ST6GAL1 gene is expressed in almost all human tissues, ST6GAL2 shows a restricted tissue-specific pattern of expression, mostly expressed in embryonic and adult brain. In order to understand the mechanisms involved in the transcriptional regulation of ST6GAL2, we first characterized the transcription start sites (TSS) in SH-SY5Y neuroblastoma cells. 5' RACE experiments revealed multiple TSS located on three first alternative 5' exons, termed EX, EY and EZ, which are unusually close on the genomic sequence and are all located more than 42 kbp upstream of the first common coding exon. Using Taqman duplex Q-PCR, we showed that the ST6GAL2 transcripts initiated by EX or EY are mainly expressed in both brain-related cell lines and human cerebral cortex, testifying for the use of a similar transcriptional regulation in vivo. Furthermore, we also showed for the first time hST6Gal II protein expression in the different lobes of the human cortex. Luciferase reporter assays allowed us to define two sequences upstream EX and EY with a high and moderate promoter activity, respectively. Bioinformatics analysis and site-directed mutagenesis showed that NF-kappaB and NRSF are likely to act as transcriptional repressors, whereas neuronal-related development factors Sox5, Puralpha and Olf1, are likely to act as transcriptional activators of ST6GAL2. This suggests that ST6GAL2 transcription could be potentially activated for specific neuronal functions.

Immunoglobulin A nephropathy (IgAN) is the most common form of glomerulonephritis worldwide and is histologically characterized by the deposition of IgA1 and consequent inflammation in the glomerular mesangium. Prior studies suggested that serum IgA1 from IgAN patients contains aberrant, undergalactosylated O-glycans, for example, Tn antigen and its sialylated version, SialylTn (STn), but the mechanisms underlying aberrant O-glycosylation are not well understood. Here we have used serial lectin separation technologies, Western blot, enzymatic modifications, and mass spectrometry to explore whether there are different glycoforms of IgA1 in plasma from patients with IgAN and healthy individuals. Although total plasma IgA in IgAN patients was elevated ∼ 1.6-fold compared with that in healthy donors, IgA1 in all samples was unexpectedly separable into two distinct glycoforms: one with core 1 based O-glycans, and the other exclusively containing Tn/STn structures. Importantly, Tn antigen present on IgA1 from IgAN patients and controls was convertible into the core 1 structure in vitro by recombinant T-synthase. Our results demonstrate that undergalactosylation of O-glycans in IgA1 is not restricted to IgAN and suggest that in vivo inefficiency of T-synthase toward IgA1 in a subpopulation of B or plasma cells, as well as overall elevation of IgA, may contribute to IgAN pathogenesis.

In many different human disorders, the cellular glycome is altered. An interesting but poorly understood alteration occurs in the mucin-type O-glycome, in which there is aberrant expression of the truncated O-glycans Tn (GalNAcα1-Ser/Thr) and its sialylated version sialyl-Tn (STn) (Neu5Acα2,6GalNAcα1-Ser/Thr). Both Tn and STn are tumor-associated carbohydrate antigens and tumor biomarkers, since they are not expressed normally and appear early in tumorigenesis. Moreover, their expression is strongly associated with poor prognosis and tumor metastasis. The Tn and STn antigens are also expressed in other human diseases and disorders, such as Tn syndrome and IgA nephropathy. The major pathological mechanism for expression of the Tn and STn antigens is compromised T-synthase activity, resulting from alteration of the X-linked gene that encodes for Cosmc, a molecular chaperone specifically required for the correct folding of T-synthase to form active enzyme. This review will summarize our current understanding of the Tn and STn antigens in terms of their biochemistry and role in pathology.

Cosmc is the specific molecular chaperone in the endoplasmic reticulum for T-synthase, a Golgi β3-galactosyltransferase that generates the core 1 O-glycan, Galβ1–3GalNAcα-Ser/Thr, in glycoproteins. Dysfunctional Cosmc results in the formation of inactive T-synthase and consequent expression of the Tn antigen (GalNAcα1-Ser/Thr), which is associated with several human diseases. However, the molecular regulation of expression of Cosmc, which is encoded by a single gene on Xq24, is poorly understood. Here we show that epigenetic silencing of Cosmc through hypermethylation of its promoter leads to loss of Cosmc transcripts in Tn4 cells, an immortalized B cell line from a male patient with a Tn-syndrome-like phenotype. These cells lack T-synthase activity and express the Tn antigen. Treatment of cells with 5-aza-2′-deoxycytidine causes restoration of Cosmc transcripts, restores T-synthase activity, and reduces Tn antigen expression. Bisulfite sequencing shows that CG dinucleotides in the Cosmc core promoter are hypermethylated. Interestingly, several other X-linked genes associated with glycosylation are not silenced in Tn4 cells, and we observed no correlation of a particular DNA methyltransferase to aberrant methylation of Cosmc in these cells. Thus, hypermethylation of the Cosmc promoter in Tn4 cells is relatively specific. Epigenetic silencing of Cosmc provides another mechanism underlying the abnormal expression of the Tn antigen, which may be important in understanding aberrant Tn antigen expression in human diseases, including IgA nephropathy and cancer.

The disialoganglioside GD3 is an oncofetal marker of a variety of human tumors including melanoma and neuroblastoma, playing a key role in tumor progression. GD3 and 9-O-acetyl-GD3 are overexpressed in approximately 50% of invasive ductal breast carcinoma, but no relationship has been established between disialoganglioside expression and breast cancer progression. In order to determine the effect of GD3 expression on breast cancer development, we analyzed the biosynthesis of gangliosides in several breast epithelial cell lines including MDA-MB-231, MCF-7, BT-20, T47-D, and MCF10A, by immunocytochemistry, flow cytometry, and real-time PCR. Our results show that, in comparison to tumors, cultured breast cancer cells express a limited pattern of gangliosides. Disialogangliosides were not detected in any cell line and GM3 was only observed at the cell surface of MDA-MB-231 cells. To evaluate the influence of GD3 in breast cancer cell behavior, we established and characterized MDA-MB-231 cells overexpressing GD3 synthase. We show that GD3 synthase expressing cells accumulate GD3, GD2, and GT3 at the cell surface. Moreover, GD3 synthase overexpression bypasses the need of serum for cell growth and increases cell migration. This suggests that GD3 synthase overexpression may contribute to increasing the malignant properties of breast cancer cells.

Bronchial mucins from patients suffering from CF (cystic fibrosis) exhibit glycosylation alterations, especially increased amounts of the sialyl-Lewis(x) (NeuAcalpha2-3Galbeta1-4[Fucalpha1-3]GlcNAc-R) and 6-sulfo-sialyl-Lewis(x) (NeuAcalpha2-3Galbeta1-4[Fucalpha1-3][SO(3)H-6]GlcNAc-R) terminal structures. These epitopes are preferential receptors for Pseudomonas aeruginosa, the bacteria responsible for the chronicity of airway infection and involved in the morbidity and early death of CF patients. However, these glycosylation changes cannot be directly linked to defects in CFTR (CF transmembrane conductance regulator) gene expression since cells that secrete airway mucins express no or very low amounts of the protein. Several studies have shown that inflammation may affect glycosylation and sulfation of various glycoproteins, including mucins. In the present study, we show that incubation of macroscopically healthy fragments of human bronchial mucosa with IL-6 (interleukin-6) or IL-8 results in a significant increase in the expression of alpha1,3/4-fucosyltransferases [FUT11 (fucosyltransferase 11 gene) and FUT3], alpha2-6- and alpha2,3-sialyltransferases [ST3GAL6 (alpha2,3-sialyltransferase 6 gene) and ST6GAL2 (alpha2,6-sialyltransferase 2 gene)] and GlcNAc-6-O-sulfotransferases [CHST4 (carbohydrate sulfotransferase 4 gene) and CHST6] mRNA. In parallel, the amounts of sialyl-Lewis(x) and 6-sulfo-sialyl-Lewis(x) epitopes at the periphery of high-molecular-mass proteins, including MUC4, were also increased. In conclusion, our results indicate that IL-6 and -8 may contribute to the increased levels of sialyl-Lewis(x) and 6-sulfo-sialyl-Lewis(x) epitopes on human airway mucins from patients with CF.