Roles of N-linked glycosylation and glycan-binding proteins in placentation: trophoblast infiltration, immunomodulation, angiogenesis, and pathophysiology.

Protein N-linked glycosylation is a structurally diverse post-translational modification that stores biological information in a larger order of magnitude than other post-translational modifications such as phosphorylation, ubiquitination and acetylation. This gives N-glycosylated proteins a diverse range of properties and allows glyco-codes (glycan-related information) to be deciphered by glycan-binding proteins (GBPs). The intervillous space of the placenta is richly populated with membrane-bound and secreted glycoproteins. Evidence exists to suggest that altering the structural nature of their N-glycans can impact several trophoblast functions, which include those related to interactions with decidual cells. This review summarizes trophoblast-related activities influenced by N-glycan-GBP recognition, exploring how different subtypes of trophoblasts actively adapt to characteristics of the decidualized endometrium through cell-specific expression of N-glycosylated proteins, and how these cells receive decidua-derived signals via N-glycan-GBP interactions. We highlight work on how changes in N-glycosylation relates to the success of trophoblast infiltration, interactions of immunomodulators, and uterine angiogenesis. We also discuss studies that suggest aberrant N-glycosylation of trophoblasts may contribute to the pathogenesis of pregnancy complications (e.g. pre-eclampsia, early spontaneous miscarriages and hydatidiform mole). We propose that a more in-depth understanding of how N-glycosylation shapes trophoblast phenotype during early pregnancy has the potential to improve our approach to predicting, diagnosing and alleviating poor maternal/fetal outcomes associated with placental dysfunction.

The pleiotropic role of galectin-3 in melanoma progression: Unraveling the enigma.

Melanoma is a highly aggressive skin cancer with poor outcomes associated with distant metastasis. Intrinsic properties of melanoma cells alongside the crosstalk between melanoma cells and surrounding microenvironment determine the tumor behavior. Galectin-3 (Gal-3), a ß-galactoside-binding lectin, has emerged as a major effector in cancer progression, including melanoma behavior. Data from melanoma models and patient studies reveal that Gal-3 expression is dysregulated, both intracellularly and extracellularly, throughout the stages of melanoma progression. This review summarizes the most recent data and hypotheses on Gal-3 and its tumor-modulating functions, highlighting its role in driving melanoma growth, invasion, and metastatic colonization. It also provides insight into potential Gal-3-targeted strategies for melanoma diagnosis and treatment.

Hypoxia Controls the Glycome Signature and Galectin-8-Ligand Axis to Promote Protumorigenic Properties of Metastatic Melanoma.

The prognosis for patients with metastatic melanoma (MM) involving distant organs is grim, and treatment resistance is potentiated by tumor-initiating cells (TICs) that thrive under hypoxia. MM cells, including TICs, express a unique glycome featuring i-linear poly-N-acetyllactosamines through the loss of I-branching enzyme, ß1,6 N-acetylglucosaminyltransferase 2. Whether hypoxia instructs MM TIC development by modulating the glycome signature remains unknown. In this study, we explored hypoxia-dependent alterations in MM glycome‒associated genes and found that ß1,6 N-acetylglucosaminyltransferase 2 was downregulated and a galectin (Gal)-8-ligand axis, involving both extracellular and cell-intrinsic Gal-8, was induced. Low ß1,6 N-acetylglucosaminyltransferase 2 levels correlated with poor patient outcomes, and patient serum samples were elevated for Gal-8. Depressed ß1,6 N-acetylglucosaminyltransferase 2 in MM cells upregulated TIC marker, NGFR/CD271, whereas loss of MM cell‒intrinsic Gal-8 markedly lowered NGFR and reduced TIC activity in vivo. Extracellular Gal-8 bound preferentially to i-linear poly-N-acetyllactosamines on N-glycans of the TIC marker and prometastatic molecule CD44, among other receptors, and activated prosurvival factor protein kinase B. This study reveals the importance of hypoxia governing the MM glycome by enforcing i-linear poly-N-acetyllactosamine and Gal-8 expression. This mechanistic investigation also uncovers glycome-dependent regulation of pro-MM factor, NGFR, implicating i-linear poly-N-acetyllactosamine and Gal-8 as biomarkers and therapeutic targets of MM.

Cell-specific bioorthogonal tagging of glycoproteins.

Altered glycoprotein expression is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function.

Strategies to control therapeutic antibody glycosylation during bioprocessing: Synthesis and separation.

Glycosylation can be a critical quality attribute in biologic manufacturing. In particular, it has implications on the half-life, immunogenicity, and pharmacokinetics of therapeutic monoclonal antibodies (mAbs), and must be closely monitored throughout drug development and manufacturing. To address this, advances have been made primarily in upstream processing, including mammalian cell line engineering, to yield more predictably glycosylated mAbs and the addition of media supplements during fermentation to manipulate the metabolic pathways involved in glycosylation. A more robust approach would be a conjoined upstream-downstream processing strategy. This could include implementing novel downstream technologies, such as the use of Fc γ-based affinity ligands for the separation of mAb glycovariants. This review highlights the importance of controlling therapeutic antibody glycosylation patterns, the challenges faced in terms of glycosylation during mAb biosimilar development, current efforts both upstream and downstream to control glycosylation and their limitations, and the need for research in the downstream space to establish holistic and consistent manufacturing processes for the production of antibody therapies.

Major differences in glycosylation and fucosyltransferase expression in low-grade versus high-grade bladder cancer cell lines.

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) (Galß1-4(Fucα1-3)GlcNAcß1-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.

Proteome-wide prediction of bacterial carbohydrate-binding proteins as a tool for understanding commensal and pathogen colonisation of the vaginal microbiome.

Bacteria use carbohydrate-binding proteins (CBPs), such as lectins and carbohydrate-binding modules (CBMs), to anchor to specific sugars on host surfaces. CBPs in the gut microbiome are well studied, but their roles in the vagina microbiome and involvement in sexually transmitted infections, cervical cancer and preterm birth are largely unknown. We established a classification system for lectins and designed Hidden Markov Model (HMM) profiles for data mining of bacterial genomes, resulting in identification of >100,000 predicted bacterial lectins available at unilectin.eu/bacteria. Genome screening of 90 isolates from 21 vaginal bacterial species shows that those associated with infection and inflammation produce a larger CBPs repertoire, thus enabling them to potentially bind a wider array of glycans in the vagina. Both the number of predicted bacterial CBPs and their specificities correlated with pathogenicity. This study provides new insights into potential mechanisms of colonisation by commensals and potential pathogens of the reproductive tract that underpin health and disease states.

A mutation in SLC37A4 causes a dominantly inherited congenital disorder of glycosylation characterized by liver dysfunction.

SLC37A4 encodes an endoplasmic reticulum (ER)-localized multitransmembrane protein required for transporting glucose-6-phosphate (Glc-6P) into the ER. Once transported into the ER, Glc-6P is subsequently hydrolyzed by tissue-specific phosphatases to glucose and inorganic phosphate during times of glucose depletion. Pathogenic variants in SLC37A4 cause an established recessive disorder known as glycogen storage disorder 1b characterized by liver and kidney dysfunction with neutropenia. We report seven individuals who presented with liver dysfunction multifactorial coagulation deficiency and cardiac issues and were heterozygous for the same variant, c.1267C>T (p.Arg423∗), in SLC37A4; the affected individuals were from four unrelated families. Serum samples from affected individuals showed profound accumulation of both high mannose and hybrid type N-glycans, while N-glycans in fibroblasts and undifferentiated iPSC were normal. Due to the liver-specific nature of this disorder, we generated a CRISPR base-edited hepatoma cell line harboring the c.1267C>T (p.Arg423∗) variant. These cells replicated the secreted abnormalities seen in serum N-glycosylation, and a portion of the mutant protein appears to relocate to a distinct, non-Golgi compartment, possibly ER exit sites. These cells also show a gene dosage-dependent alteration in the Golgi morphology and reduced intraluminal pH that may account for the altered glycosylation. In summary, we identify a recurrent mutation in SLC37A4 that causes a dominantly inherited congenital disorder of glycosylation characterized by coagulopathy and liver dysfunction with abnormal serum N-glycans.

Modified recombinant human IgG1-Fc is superior to natural intravenous immunoglobulin at inhibiting immune-mediated demyelination.

Intravenous immunoglobulin (IVIG) is an established treatment for numerous autoimmune conditions. Although Fc fragments derived from IVIG have shown efficacy in controlling immune thrombocytopenia in children, the mechanisms of action are unclear and controversial. The aim of this study was to dissect IVIG effector mechanisms using further adapted Fc fragments on demyelination in an ex vivo model of the central nervous system-immune interface. Using organotypic cerebellar slice cultures (OSCs) from transgenic mice, we induced extensive immune-mediated demyelination and oligodendrocyte loss with an antibody specific for myelin oligodendrocyte glycoprotein (MOG) and complement. Protective effects of adapted Fc fragments were assessed by live imaging of green fluorescent protein expression, immunohistochemistry and confocal microscopy. Cysteine- and glycan-adapted Fc fragments protected OSC from demyelination in a dose-dependent manner where equimolar concentrations of either IVIG or control Fc were ineffective. The protective effects of the adapted Fc fragments are partly attributed to interference with complement-mediated oligodendroglia damage. Transcriptome analysis ruled out signatures associated with inflammatory or innate immune responses. Taken together, our findings show that recombinant biomimetics can be made that are at least two hundred-fold more effective than IVIG in controlling demyelination by anti-MOG antibodies.

Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation.

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine-4-epimerase (GALE) like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.

Altered glycosylation of glycodelin in endometrial carcinoma.

Glycodelin is a major glycoprotein expressed in reproductive tissues, like secretory and decidualized endometrium. It has several reproduction related functions that are dependent on specific glycosylation, but it has also been found to drive differentiation of endometrial carcinoma cells toward a less malignant phenotype. Here we aimed to elucidate whether the glycosylation and function of glycodelin is altered in endometrial carcinoma as compared with a normal endometrium. We carried out glycan structure analysis of glycodelin expressed in HEC-1B human endometrial carcinoma cells (HEC-1B Gd) by mass spectrometry glycomics strategies. Glycans of HEC-1B Gd were found to comprise a typical mixture of high-mannose, hybrid, and complex-type N-glycans, often containing undecorated LacNAc (Galß1-4GlcNAc) antennae. However, several differences, as compared with previously reported glycan structures of normal human decidualized endometrium-derived glycodelin isoform, glycodelin-A (GdA), were also found. These included a lower level of sialylation and more abundant poly-LacNAc antennae, some of which are fucosylated. This allowed us to select lectins that showed different binding to these classes of glycodelin. Despite the differences in glycosylation between HEC-1B Gd and GdA, both showed similar inhibitory activity on trophoblast cell invasion and peripheral blood mononuclear cell proliferation. For the detection of cancer associated glycodelin, we established a novel in situ proximity-ligation based histochemical staining method using a specific glycodelin antibody and UEAI lectin. We found that the UEAI reactive glycodelin was abundant in endometrial carcinoma, but virtually absent in normal endometrial tissue even when glycodelin was strongly expressed. In conclusion, we established a histochemical staining method for the detection of endometrial carcinoma-associated glycodelin and showed that this specific glycodelin is exclusively expressed in cancer, not in normal endometrium. Similar methods can be used for studies of other glycoproteins.

Insights into the hyperglycosylation of human chorionic gonadotropin revealed by glycomics analysis.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone that is essential for the maintenance of pregnancy. Glycosylation of hCG is known to be essential for its biological activity. "Hyperglycosylated" variants secreted during early pregnancy have been proposed to be involved in initial implantation of the embryo and as a potential diagnostic marker for gestational diseases. However, what constitutes "hyperglycosylation" is not yet fully understood. In this study, we perform comparative N-glycomic analysis of hCG expressed in the same individuals during early and late pregnancy to help provide new insights into hCG function, reveal new targets for diagnostics and clarify the identity of hyperglycosylated hCG. hCG was isolated in urine collected from women at 7 weeks and 20 weeks' gestation. hCG was also isolated in urine from women diagnosed with gestational trophoblastic disease (GTD). We used glycomics methodologies including matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) and MS/MS methods to characterise the N-glycans associated with hCG purified from the individual samples. The structures identified on the early pregnancy (EP-hCG) and late pregnancy (LP-hCG) samples corresponded to mono-, bi-, tri-, and tetra-antennary N-glycans. A novel finding was the presence of substantial amounts of bisected type N-glycans in pregnancy hCG samples, which were present at much lower levels in GTD samples. A second novel observation was the presence of abundant LewisX antigens on the bisected N-glycans. GTD-hCG had fewer glycoforms which constituted a subset of those found in normal pregnancy. When compared to EP-hCG, GTD-hCG samples had decreased signals for tri- and tetra-antennary N-glycans. In terms of terminal epitopes, GTD-hCG had increased signals for sialylated structures, while LewisX antigens were of very minor abundance. hCG carries the same N-glycans throughout pregnancy but in different proportions. The N-glycan repertoire is more diverse than previously reported. Bisected and LewisX structures are potential targets for diagnostics. hCG isolated from pregnancy urine inhibits NK cell cytotoxicity in vitro at nanomolar levels and bisected type glycans have previously been implicated in the suppression of NK cell cytotoxicity, suggesting that hCG-related bisected type N-glycans may directly suppress NK cell cytotoxicity.

East-Asian Helicobacter pylori strains synthesize heptan-deficient lipopolysaccharide.

The lipopolysaccharide O-antigen structure expressed by the European Helicobacter pylori model strain G27 encompasses a trisaccharide, an intervening glucan-heptan and distal Lewis antigens that promote immune escape. However, several gaps still remain in the corresponding biosynthetic pathway. Here, systematic mutagenesis of glycosyltransferase genes in G27 combined with lipopolysaccharide structural analysis, uncovered HP0102 as the trisaccharide fucosyltransferase, HP1283 as the heptan transferase, and HP1578 as the GlcNAc transferase that initiates the synthesis of Lewis antigens onto the heptan motif. Comparative genomic analysis of G27 lipopolysaccharide biosynthetic genes in strains of different ethnic origin revealed that East-Asian strains lack the HP1283/HP1578 genes but contain an additional copy of HP1105 and JHP0562. Further correlation of different lipopolysaccharide structures with corresponding gene contents led us to propose that the second copy of HP1105 and the JHP0562 may function as the GlcNAc and Gal transferase, respectively, to initiate synthesis of the Lewis antigen onto the Glc-Trio-Core in East-Asian strains lacking the HP1283/HP1578 genes. In view of the high gastric cancer rate in East Asia, the absence of the HP1283/HP1578 genes in East-Asian H. pylori strains warrants future studies addressing the role of the lipopolysaccharide heptan in pathogenesis.

Serum IgA1 shows increased levels of α2,6-linked sialic acid in breast cancer.

The lectin Helix pomatia agglutinin (HPA) recognizes altered glycosylation in solid cancers and the identification of HPA binding partners in tumour tissue and serum is an important aim. Among the many HPA binding proteins, IgA1 has been reported to be the most abundant in liver metastases. In this study, the glycosylation of IgA1 was evaluated using serum samples from patients with breast cancer (BCa) and the utility of IgA1 glycosylation as a biomarker was assessed. Detailed mass spectrometric structural analysis showed an increase in disialo-biantennary N-linked glycans on IgA1 from BCa patients (p < 0.0001: non-core fucosylated; p = 0.0345: core fucosylated) and increased asialo-Thomsen-Friedenreich antigen (TF) and disialo-TF antigens in the O-linked glycan preparations from IgA1 of cancer patients compared with healthy control individuals. An increase in Sambucus nigra binding was observed, suggestive of increased α2,6-linked sialic acid on IgA1 in BCa. Logistic regression analysis showed HPA binding to IgA1 and tumour size to be significant independent predictors of distant metastases (χ 2 13.359; n = 114; p = 0.020) with positive and negative predictive values of 65.7% and 64.6%, respectively. Immunohistochemical analysis of tumour tissue samples showed IgA1 to be detectable in BCa tissue. This report provides a detailed analysis of serum IgA1 glycosylation in BCa and illustrates the potential utility of IgA1 glycosylation as a biomarker for BCa prognostication.

XBP1s activation can globally remodel N-glycan structure distribution patterns.

Classically, the unfolded protein response (UPR) safeguards secretory pathway proteostasis. The most ancient arm of the UPR, the IRE1-activated spliced X-box binding protein 1 (XBP1s)-mediated response, has roles in secretory pathway maturation beyond resolving proteostatic stress. Understanding the consequences of XBP1s activation for cellular processes is critical for elucidating mechanistic connections between XBP1s and development, immunity, and disease. Here, we show that a key functional output of XBP1s activation is a cell type-dependent shift in the distribution of N-glycan structures on endogenous membrane and secreted proteomes. For example, XBP1s activity decreased levels of sialylation and bisecting GlcNAc in the HEK293 membrane proteome and secretome, while substantially increasing the population of oligomannose N-glycans only in the secretome. In HeLa cell membranes, stress-independent XBP1s activation increased the population of high-mannose and tetraantennary N-glycans, and also enhanced core fucosylation. mRNA profiling experiments suggest that XBP1s-mediated remodeling of the N-glycome is, at least in part, a consequence of coordinated transcriptional resculpting of N-glycan maturation pathways by XBP1s. The discovery of XBP1s-induced N-glycan structural remodeling on a glycome-wide scale suggests that XBP1s can act as a master regulator of N-glycan maturation. Moreover, because the sugars on cell-surface proteins or on proteins secreted from an XBP1s-activated cell can be molecularly distinct from those of an unactivated cell, these findings reveal a potential new mechanism for translating intracellular stress signaling into altered interactions with the extracellular environment.

The mucinous domain of pancreatic carboxyl-ester lipase (CEL) contains core 1/core 2 O-glycans that can be modified by ABO blood group determinants.

Carboxyl-ester lipase (CEL) is a pancreatic fat-digesting enzyme associated with human disease. Rare mutations in the CEL gene cause a syndrome of pancreatic exocrine and endocrine dysfunction denoted MODY8, whereas a recombined CEL allele increases the risk for chronic pancreatitis. Moreover, CEL has been linked to pancreatic ductal adenocarcinoma (PDAC) through a postulated oncofetal CEL variant termed feto-acinar pancreatic protein (FAPP). The monoclonal antibody mAb16D10 was previously reported to detect a glycotope in the highly O-glycosylated, mucin-like C terminus of CEL/FAPP. We here assessed the expression of human CEL in malignant pancreatic lesions and cell lines. CEL was not detectably expressed in neoplastic cells, implying that FAPP is unlikely to be a glycoisoform of CEL in pancreatic cancer. Testing of the mAb16D10 antibody in glycan microarrays then demonstrated that it recognized structures containing terminal GalNAc-α1,3(Fuc-α1,2)Gal (blood group A antigen) and also repeated protein sequences containing GalNAc residues linked to Ser/Thr (Tn antigen), findings that were supported by immunostainings of human pancreatic tissue. To examine whether the CEL glycoprotein might be modified by blood group antigens, we used high-sensitivity MALDI-TOF MS to characterize the released O-glycan pool of CEL immunoprecipitated from human pancreatic juice. We found that the O-glycome of CEL consisted mainly of core 1/core 2 structures with a composition depending on the subject's FUT2 and ABO gene polymorphisms. Thus, among digestive enzymes secreted by the pancreas, CEL is a glycoprotein with some unique characteristics, supporting the view that it could serve additional biological functions to its cholesteryl esterase activity in the duodenum.

Loss of GCNT2/I-branched glycans enhances melanoma growth and survival.

Cancer cells often display altered cell-surface glycans compared to their nontransformed counterparts. However, functional contributions of glycans to cancer initiation and progression remain poorly understood. Here, from expression-based analyses across cancer lineages, we found that melanomas exhibit significant transcriptional changes in glycosylation-related genes. This gene signature revealed that, compared to normal melanocytes, melanomas downregulate I-branching glycosyltransferase, GCNT2, leading to a loss of cell-surface I-branched glycans. We found that GCNT2 inversely correlated with clinical progression and that loss of GCNT2 increased melanoma xenograft growth, promoted colony formation, and enhanced cell survival. Conversely, overexpression of GCNT2 decreased melanoma xenograft growth, inhibited colony formation, and increased cell death. More focused analyses revealed reduced signaling responses of two representative glycoprotein families modified by GCNT2, insulin-like growth factor receptor and integrins. Overall, these studies reveal how subtle changes in glycan structure can regulate several malignancy-associated pathways and alter melanoma signaling, growth, and survival.

Human B Cell Differentiation Is Characterized by Progressive Remodeling of O-Linked Glycans.

Germinal centers (GC) are microanatomical niches where B cells proliferate, undergo antibody affinity maturation, and differentiate to long-lived memory B cells and antibody-secreting plasma cells. For decades, GC B cells have been defined by their reactivity to the plant lectin peanut agglutinin (PNA), which binds serine/threonine (O-linked) glycans containing the asialylated disaccharide Gal-ß1,3-GalNAc-Ser/Thr (also called T-antigen). In T cells, acquisition of PNA binding by activated T cells and thymocytes has been linked with altered tissue homing patterns, cell signaling, and survival. Yet, in GC B cells, the glycobiological basis and significance of PNA binding remains surprisingly unresolved. Here, we investigated the basis for PNA reactivity of GC B cells. We found that GC B cell binding to PNA is associated with downregulation of the α2,3 sialyltransferase, ST3GAL1 (ST3Gal1), and overexpression of ST3Gal1 was sufficient to reverse PNA binding in B cell lines. Moreover, we found that the primary scaffold for PNA-reactive O-glycans in B cells is the B cell receptor-associated receptor-type tyrosine phosphatase CD45, suggesting a role for altered O-glycosylation in antigen receptor signaling. Consistent with similar reports in T cells, ST3Gal1 overexpression in B cells in vitro induced drastic shortening in O-glycans, which we confirmed by both antibody staining and mass spectrometric O-glycomic analysis. Unexpectedly, ST3Gal1-induced changes in O-glycan length also correlated with altered binding of two glycosylation-sensitive CD45 antibodies, RA3-6B2 (more commonly called B220) and MEM55, which (in humans) have previously been reported to favor binding to naïve/GC subsets and memory/plasmablast subsets, respectively. Analysis of primary B cell binding to B220, MEM55, and several plant lectins suggested that B cell differentiation is accompanied by significant loss of O-glycan complexity, including loss of extended Core 2 O-glycans. To our surprise, decreased O-glycan length from naïve to post-GC fates best correlated not with ST3Gal1, but rather downregulation of the Core 2 branching enzyme GCNT1. Thus, our data suggest that O-glycan remodeling is a feature of B cell differentiation, dually regulated by ST3Gal1 and GCNT1, that ultimately results in expression of distinct O-glycosylation states/CD45 glycoforms at each stage of B cell differentiation.

Human Immunodeficiency Virus and Simian Immunodeficiency Virus Maintain High Levels of Infectivity in the Complete Absence of Mucin-Type O-Glycosylation.

A highly conserved threonine near the C terminus of gp120 of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) was investigated for its contributions to envelope protein function and virion infectivity. When this highly conserved Thr residue was substituted with anything other than serine (the other amino acid that can accept O-glycosylation), the resulting virus was noninfectious. We found that this Thr was critical for the association of gp120 with the virion and that amino acid substitution increased the amount of dissociated gp120 in the cell culture supernatant. When HIV virions were generated in cells overexpressing polypeptide N-acetylgalactosaminyltransferase 1 (GalNAcT1), viral infectivity was increased 2.5-fold compared to that of virus produced in wild-type HEK293T cells; infectivity was increased 8-fold when the Thr499Ser mutant was used. These infectivity enhancements were not observed when GalNAcT3 was used. Using HEK293T knockout cell lines totally devoid of the ability to perform O-linked glycosylation, we demonstrated production of normal levels of virions and normal levels of infectivity in the complete absence of O-linked carbohydrate. Our data indicate that O-glycosylation is not necessary for the natural replication cycle of HIV and SIV. Nonetheless, it remains theoretically possible that the repertoire of GalNAc transferase isoforms in natural target cells for HIV and SIV in vivo could result in O-glycosylation of the threonine residue in question and that this could boost the infectivity of virions beyond the levels seen in the absence of such O-glycosylation.IMPORTANCE Approximately 50% of the mass of the gp120 envelope glycoprotein of both HIV and SIV is N-linked carbohydrate. One of the contributions of this N-linked carbohydrate is to shield conserved peptide sequences from recognition by humoral immunity. This N-linked glycosylation is one of the reasons that primary isolates of HIV and SIV are so heavily resistant to antibody-mediated neutralization. Much less studied is any potential contribution from O-linked glycosylation. The literature on this topic to date is somewhat confusing and ambiguous. Our studies described in this report demonstrate unambiguously that O-linked glycosylation is not necessary for the natural replication cycle of HIV and SIV. However, the door is not totally closed because of the diversity of numerous GalNAc transferase enzymes that initiate O-linked carbohydrate attachment and the theoretical possibility that natural target cells for HIV and SIV in vivo could potentially complete such O-linked carbohydrate attachment to further increase infectivity.

Insights from the redefinition of Helicobacter pylori lipopolysaccharide O-antigen and core-oligosaccharide domains.

H. pylori is a Gram-negative extracellular bacterium, first discovered by the Australian physicians Barry Marshall and Robin Warren in 1982, that colonises the human stomach mucosa. It is the leading cause of peptic ulcer and commonly infects humans worldwide with prevalence as high as 90% in some countries. H. pylori infection usually results in asymptomatic chronic gastritis, however 10-15% of cases develop duodenal or gastric ulcers and 1-3% develop stomach cancer. Infection is generally acquired during childhood and persists for life in the absence of antibiotic treatment. H. pylori has had a long period of co-evolution with humans, going back to human migration out of Africa. This prolonged relationship is likely to have shaped the overall host-pathogen interactions and repertoire of virulence strategies which H. pylori employs to establish robust colonisation, escape immune responses and persist in the gastric niche. In this regard, H. pylori lipopolysaccharide (LPS) is a key surface determinant in establishing colonisation and persistence via host mimicry and resistance to cationic antimicrobial peptides. Thus, elucidation of the H. pylori LPS structure and corresponding biosynthetic pathway represents an important step towards better understanding of H. pylori pathogenesis and the development of novel therapeutic interventions.