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.

A combinatorial DNA assembly approach to biosynthesis of N-linked glycans in E. coli.

Glycoengineering of recombinant glycans and glycoconjugates is a rapidly evolving field. However, the production and exploitation of glycans has lagged behind that of proteins and nucleic acids. Biosynthetic glycoconjugate production requires the coordinated cooperation of three key components within a bacterial cell: a substrate protein, a coupling oligosaccharyltransferase, and a glycan biosynthesis locus. While the acceptor protein and oligosaccharyltransferase are the products of single genes, the glycan is a product of a multigene metabolic pathway. Typically, the glycan biosynthesis locus is cloned and transferred en bloc from the native organism to a suitable Escherichia coli strain. However, gene expression within these pathways has been optimized by natural selection in the native host and is unlikely to be optimal for heterologous production in an unrelated organism. In recent years, synthetic biology has addressed the challenges in heterologous expression of multigene systems by deconstructing these pathways and rebuilding them from the bottom up. The use of DNA assembly methods allows the convenient assembly of such pathways by combining defined parts with the requisite coding sequences in a single step. In this study, we apply combinatorial assembly to the heterologous biosynthesis of the Campylobacter jejuni  N-glycosylation (pgl) pathway in E. coli. We engineered reconstructed biosynthesis clusters that faithfully reproduced the C. jejuni heptasaccharide glycan. Furthermore, following a single round of combinatorial assembly and screening, we identified pathway clones that outperform glycan and glycoconjugate production of the native unmodified pgl cluster. This platform offers a flexible method for optimal engineering of glycan structures in E. coli.

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.

Development of a novel glycoengineering platform for the rapid production of conjugate vaccines.

Conjugate vaccines produced either by chemical or biologically conjugation have been demonstrated to be safe and efficacious in protection against several deadly bacterial diseases. However, conjugate vaccine assembly and production have several shortcomings which hinders their wider availability. Here, we developed a tool, Mobile-element Assisted Glycoconjugation by Insertion on Chromosome, MAGIC, a novel biotechnological platform that overcomes the limitations of the current conjugate vaccine design method(s). As a model, we focused our design on a leading bioconjugation method using N-oligosaccharyltransferase (OTase), PglB. The installation of MAGIC led to at least twofold increase in glycoconjugate yield via MAGIC when compared to conventional N-OTase based bioconjugation method(s). Then, we improved MAGIC to (a) allow rapid installation of glycoengineering component(s), (b) omit the usage of antibiotics, (c) reduce the dependence on protein induction agents. Furthermore, we show the modularity of the MAGIC platform in performing glycoengineering in bacterial species that are less genetically tractable than the commonly used Escherichia coli. The MAGIC system promises a rapid, robust and versatile method to develop vaccines against serious bacterial pathogens. We anticipate the utility of the MAGIC platform could enhance vaccines production due to its compatibility with virtually any bioconjugation method, thus expanding vaccine biopreparedness toolbox.

Measurement of erythrocyte membrane mannoses to assess splenic function.

Red blood cells (RBCs) lose plasma membrane in the spleen as they age, but the cells and molecules involved are yet to be identified. Sickle cell disease and infection by Plasmodium falciparum cause oxidative stress that induces aggregates of cross-linked proteins with N-linked high-mannose glycans (HMGs). These glycans can be recognised by mannose-binding lectins, including the mannose receptor (CD206), expressed on macrophages and specialised phagocytic endothelial cells in the spleen to mediate the extravascular haemolysis characteristic of these diseases. We postulated this system might also mediate removal of molecules and membrane in healthy individuals. Surface expression of HMGs on RBCs from patients who had previously undergone splenectomy was therefore assessed: high levels were indeed observable as large membrane aggregates. Glycomic analysis by mass spectrometry identified a mixture of Man5-9 GlcNAc2 structures. HMG levels correlated well with manual pit counts (r = 0.75-0.85). To assess further whether HMGs might act as a splenic reticuloendothelial function test, we measured levels on RBCs from patients with potential functional hyposplenism, some of whom exhibited high levels that may indicate risk of complications.

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.

Choice of Host Cell Line Is Essential for the Functional Glycosylation of the Fc Region of Human IgG1 Inhibitors of Influenza B Viruses.

Abs are glycoproteins that carry a conserved N-linked carbohydrate attached to the Fc whose presence and fine structure profoundly impacts on their in vivo immunogenicity, pharmacokinetics, and functional attributes. The host cell line used to produce IgG plays a major role in this glycosylation, as different systems express different glycosylation enzymes and transporters that contribute to the specificity and heterogeneity of the final IgG-Fc glycosylation profile. In this study, we compare two panels of glycan-adapted IgG1-Fc mutants expressed in either the human endothelial kidney 293-F or Chinese hamster ovary-K1 systems. We show that the types of N-linked glycans between matched pairs of Fc mutants vary greatly and in particular, with respect, to sialylation. These cell line effects on glycosylation profoundly influence the ability of the engineered Fcs to interact with either human or pathogen receptors. For example, we describe Fc mutants that potently disrupted influenza B-mediated agglutination of human erythrocytes when expressed in Chinese hamster ovary-K1, but not in human endothelial kidney 293-F cells.

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.

Site-specific characterization of SARS-CoV-2 spike glycoprotein receptor-binding domain.

The novel coronavirus SARS-CoV-2, the infective agent causing COVID-19, is having a global impact both in terms of human disease as well as socially and economically. Its heavily glycosylated spike glycoprotein is fundamental for the infection process, via its receptor-binding domains interaction with the glycoprotein angiotensin-converting enzyme 2 on human cell surfaces. We therefore utilized an integrated glycomic and glycoproteomic analytical strategy to characterize both N- and O- glycan site-specific glycosylation within the receptor-binding domain. We demonstrate the presence of complex-type N-glycans with unusual fucosylated LacdiNAc at both sites N331 and N343 and a single site of O-glycosylation on T323.

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.

Insertion of N-Terminal Hinge Glycosylation Enhances Interactions of the Fc Region of Human IgG1 Monomers with Glycan-Dependent Receptors and Blocks Hemagglutination by the Influenza Virus.

In therapeutic applications in which the Fc of IgG is critically important, the receptor binding and functional properties of the Fc are lost after deglycosylation or removal of the unique Asn297 N-X-(T/S) sequon. A population of Fcs bearing sialylated glycans has been identified as contributing to this functionality, and high levels of sialylation also lead to longer serum retention times advantageous for therapy. The efficacy of sialylated Fc has generated an incentive to modify the unique N-linked glycosylation site at Asn297, either through chemical and enzymatic methods or by mutagenesis of the Fc, that disrupts the protein-Asn297 carbohydrate interface. In this study, we took an alternative approach by inserting or deleting N-linked attachment sites into the body of the Fc to generate a portfolio of mutants with tailored effector functions. For example, we describe mutants with enhanced binding to low-affinity inhibitory human Fcγ and glycan receptors that may be usefully incorporated into existing Ab engineering approaches to treat or vaccinate against disease. The IgG1 Fc fragments containing complex sialylated glycans attached to the N-terminal Asn221 sequon bound influenza virus hemagglutinin and disrupted influenza A-mediated agglutination of human erythrocytes.

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.

Glycan characterization of pregnancy-specific glycoprotein 1 and its identification as a novel Galectin-1 ligand.

Pregnancy-specific beta 1 glycoprotein (PSG1) is secreted from trophoblast cells of the human placenta in increasing concentrations as pregnancy progresses, becoming one of the most abundant proteins in maternal serum in the third trimester. PSG1 has seven potential N-linked glycosylation sites across its four domains. We carried out glycomic and glycoproteomic studies to characterize the glycan composition of PSG1 purified from serum of pregnant women and identified the presence of complex N-glycans containing poly LacNAc epitopes with α2,3 sialyation at four sites. Using different techniques, we explored whether PSG1 can bind to galectin-1 (Gal-1) as these two proteins were previously shown to participate in processes required for a successful pregnancy. We confirmed that PSG1 binds to Gal-1 in a carbohydrate-dependent manner with an affinity of the interaction of 0.13 µM. In addition, we determined that out of the three N-glycosylation-carrying domains, only the N and A2 domains of recombinant PSG1 interact with Gal-1. Lastly, we observed that the interaction between PSG1 and Gal-1 protects this lectin from oxidative inactivation and that PSG1 competes the ability of Gal-1 to bind to some but not all of its glycoprotein ligands.

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.

The S-layer protein of a Clostridium difficile SLCT-11 strain displays a complex glycan required for normal cell growth and morphology.

Clostridium difficile is a bacterial pathogen that causes major health challenges worldwide. It has a well-characterized surface (S)-layer, a para-crystalline proteinaceous layer surrounding the cell wall. In many bacterial and archaeal species, the S-layer is glycosylated, but no such modifications have been demonstrated in C. difficile. Here, we show that a C. difficile strain of S-layer cassette type 11, Ox247, has a complex glycan attached via an O-linkage to Thr-38 of the S-layer low-molecular-weight subunit. Using MS and NMR, we fully characterized this glycan. We present evidence that it is composed of three domains: (i) a core peptide-linked tetrasaccharide with the sequence -4-α-Rha-3-α-Rha-3-α-Rha-3-ß-Gal-peptide; (ii) a repeating pentasaccharide with the sequence -4-ß-Rha-4-α-Glc-3-ß-Rha-4-(α-Rib-3-)ß-Rha-; and (iii) a nonreducing end-terminal 2,3 cyclophosphoryl-rhamnose attached to a ribose-branched sub-terminal rhamnose residue. The Ox247 genome contains a 24-kb locus containing genes for synthesis and protein attachment of this glycan. Mutations in genes within this locus altered or completely abrogated formation of this glycan, and their phenotypes suggested that this S-layer modification may affect sporulation, cell length, and biofilm formation of C. difficile In summary, our findings indicate that the S-layer protein of SLCT-11 strains displays a complex glycan and suggest that this glycan is required for C. difficile sporulation and control of cell shape, a discovery with implications for the development of antimicrobials targeting the S-layer.

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.

Site-specific glycoproteomic characterization of ES-62: The major secreted product of the parasitic worm Acanthocheilonema viteae.

ES-62 is the major secreted product of the parasitic filarial nematode Acanthocheilonema viteae and has potent anti-inflammatory activities as a consequence of posttranslational decoration by phosphorylcholine (PC). Previously, we showed that ES-62's PC was attached to N-linked glycans, and using fast atom bombardment mass spectrometry, we characterized the structure of the glycans. However, it was unknown at this time which of ES-62's four potential N-glycosylation sites carries the PC-modified glycans. In the present study, we now employ more advanced analytical tools-nano-flow liquid chromatography with high-definition electrospray mass spectrometry-to show that PC-modified glycans are found at all four potential N-glycosylation sites. Also, our earlier studies showed that up to two PC groups were detected per glycan, and we are now able to characterize N-glycans with up to five PC groups. The number per glycan varies in three of the four glycosylation sites, and in addition, for the first time, we have detected PC on the N-glycan chitobiose core in addition to terminal GlcNAc. Nevertheless, the majority of PC is detected on terminal GlcNAc, enabling it to interact with the cells and molecules of the immune system. Such expression may explain the potent immunomodulatory effects of a molecule that is considered to have significant therapeutic potential in the treatment of certain human allergic and autoimmune conditions.

Cellular O-Glycome Reporter/Amplification to explore O-glycans of living cells.

Protein O-glycosylation has key roles in many biological processes, but the repertoire of O-glycans synthesized by cells is difficult to determine. Here we describe an approach termed Cellular O-Glycome Reporter/Amplification (CORA), a sensitive method used to amplify and profile mucin-type O-glycans synthesized by living cells. Cells convert added peracetylated benzyl-α-N-acetylgalactosamine to a large variety of modified O-glycan derivatives that are secreted from cells, allowing for easy purification for analysis by HPLC and mass spectrometry (MS). Relative to conventional O-glycan analyses, CORA resulted in an ∼100-1,000-fold increase in sensitivity and identified a more complex repertoire of O-glycans in more than a dozen cell types from Homo sapiens and Mus musculus. Furthermore, when coupled with computational modeling, CORA can be used for predictions about the diversity of the human O-glycome and offers new opportunities to identify novel glycan biomarkers for human diseases.

The redefinition of Helicobacter pylori lipopolysaccharide O-antigen and core-oligosaccharide domains.

Helicobacter pylori lipopolysaccharide promotes chronic gastric colonisation through O-antigen host mimicry and resistance to mucosal antimicrobial peptides mediated primarily by modifications of the lipid A. The structural organisation of the core and O-antigen domains of H. pylori lipopolysaccharide remains unclear, as the O-antigen attachment site has still to be identified experimentally. Here, structural investigations of lipopolysaccharides purified from two wild-type strains and the O-antigen ligase mutant revealed that the H. pylori core-oligosaccharide domain is a short conserved hexasaccharide (Glc-Gal-DD-Hep-LD-Hep-LD-Hep-KDO) decorated with the O-antigen domain encompassing a conserved trisaccharide (-DD-Hep-Fuc-GlcNAc-) and variable glucan, heptan and Lewis antigens. Furthermore, the putative heptosyltransferase HP1284 was found to be required for the transfer of the third heptose residue to the core-oligosaccharide. Interestingly, mutation of HP1284 did not affect the ligation of the O-antigen and resulted in the attachment of the O-antigen onto an incomplete core-oligosaccharide missing the third heptose and the adjoining Glc-Gal residues. Mutants deficient in either HP1284 or O-antigen ligase displayed a moderate increase in susceptibility to polymyxin B but were unable to colonise the mouse gastric mucosa. Finally, mapping mutagenesis and colonisation data of previous studies onto the redefined organisation of H. pylori lipopolysaccharide revealed that only the conserved motifs were essential for colonisation. In conclusion, H. pylori lipopolysaccharide is missing the canonical inner and outer core organisation. Instead it displays a short core and a longer O-antigen encompassing residues previously assigned as the outer core domain. The redefinition of H. pylori lipopolysaccharide domains warrants future studies to dissect the role of each domain in host-pathogen interactions. Also enzymes involved in the assembly of the conserved core structure, such as HP1284, could be attractive targets for the design of new therapeutic agents for managing persistent H. pylori infection causing peptic ulcers and gastric cancer.

Characterization of H type 1 and type 1 N-acetyllactosamine glycan epitopes on ovarian cancer specifically recognized by the anti-glycan monoclonal antibody mAb-A4.

Cancer-specific glycans of ovarian cancer are promising epitopes for targeting with monoclonal antibodies (mAb). Despite their potential, structural characterization of these glycan epitopes remains a significant challenge in mAb preclinical development. Our group generated the monoclonal antibody mAb-A4 against human embryonic stem cells (hESC), which also bound specifically to N-glycans present on 11 of 19 ovarian cancer (OC) and 8 of 14 breast cancer cell lines tested. Normal cell lines and tissue were unstained by mAb-A4. To characterize the N-linked glycan epitopes on OC cell lines targeted by mAb-A4, we used glycosidases, glycan microarray, siRNA, and advanced high sensitivity matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The mAb-A4 epitopes were found to be Fucα1-2Galß1-3GlcNAcß (H type 1) and Galß1-3GlcNAcß (type 1 LacNAc). These structures were found to be present on multiple proteins from hESC and OC. Importantly, endo-ß-galactosidase coupled with MALDI-MS allowed these two epitopes, for the first time, to be directly identified on the polylactosamines of N-glycans of SKOV3, IGROV1, OV90, and OVCA433. Furthermore, siRNA knockdown of B3GALT5 expression in SKOV3 demonstrated that mAb-A4 binding was dependent on B3GALT5, providing orthogonal evidence of the epitopes' structures. The recognition of oncofetal H type 1 and type 1 LacNAc on OC by mAb-A4 is a novel and promising way to target OC and supports the theory that cancer can acquire stem-like phenotypes. We propose that the orthogonal framework used in this work could be the basis for advancing anti-glycan mAb characterization.