Publications

Inflammatory bowel disease (IBD) affects 6.8 million people globally. A variety of factors have been implicated in IBD pathogenesis, including host genetics, immune dysregulation and gut microbiota alterations. Emerging evidence implicates intestinal epithelial glycosylation as an underappreciated process that interfaces with these three factors. IBD is associated with increased expression of truncated O-glycans as well as altered expression of terminal glycan structures. IBD genes, glycosyltransferase mislocalization, altered glycosyltransferase and glycosidase expression and dysbiosis drive changes in the glycome. These glycan changes disrupt the mucus layer, glycan-lectin interactions, host-microorganism interactions and mucosal immunity, and ultimately contribute to IBD pathogenesis. Epithelial glycans are especially critical in regulating the gut microbiota through providing bacterial ligands and nutrients and ultimately determining the spatial organization of the gut microbiota. In this Review, we discuss the regulation of intestinal epithelial glycosylation, altered epithelial glycosylation in IBD and mechanisms for how these alterations contribute to disease pathobiology. We hope that this Review provides a foundation for future studies on IBD glycosylation and the emergence of glycan-inspired therapies for IBD.

Mild modification of intravenous immunoglobulin (IVIG) has been reported to result in enhanced polyspecificity and leveraged therapeutic effects in animal models of inflammation. Here, we observed that IVIG modification by ferrous ions, heme or low pH exposure, shifted the repertoires of specificities in different directions. Ferrous ions exposed Fe(II)-IVIG, but not heme or low pH exposed IVIG, showed increased pro-apoptotic effects on neutrophil granulocytes that relied on a FAS-dependent mechanism. These effects were also observed in human neutrophils primed by inflammatory mediators or rheumatoid arthritis joint fluid , or patient neutrophils from acute Crohn's disease. These observations indicate that IVIG-mediated effects on cells can be enhanced by IVIG modification, yet specific modification conditions may be required to target specific molecular pathways and eventually to enhance the therapeutic potential.

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.

MOTIVATION: Glycan microarrays are capable of illuminating the interactions of glycan-binding proteins (GBPs) against hundreds of defined glycan structures, and have revolutionized the investigations of protein-carbohydrate interactions underlying numerous critical biological activities. However, it is difficult to interpret microarray data and identify structural determinants promoting glycan binding to glycan-binding proteins due to the ambiguity in microarray fluorescence intensity and complexity in branched glycan structures. To facilitate analysis of glycan microarray data alongside protein structure, we have built the Glycan Microarray Database (GlyMDB), a web-based resource including a searchable database of glycan microarray samples and a toolset for data/structure analysis. RESULTS: The current GlyMDB provides data visualization and glycan-binding motif discovery for 5203 glycan microarray samples collected from the Consortium for Functional Glycomics. The unique feature of GlyMDB is to link microarray data to PDB structures. The GlyMDB provides different options for database query, and allows users to upload their microarray data for analysis. After search or upload is complete, users can choose the criterion for binder versus non-binder classification. They can view the signal intensity graph including the binder/non-binder threshold followed by a list of glycan-binding motifs. One can also compare the fluorescence intensity data from two different microarray samples. A protein sequence-based search is performed using BLAST to match microarray data with all available PDB structures containing glycans. The glycan ligand information is displayed, and links are provided for structural visualization and redirection to other modules in GlycanStructure.ORG for further investigation of glycan-binding sites and glycan structures. AVAILABILITY AND IMPLEMENTATION: http://www.glycanstructure.org/glymdb. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A hallmark of Gram-negative bacteria is an asymmetric outer membrane containing lipopolysaccharides (LPSs) in the extracellular leaflet. LPS molecules consist of lipid A, which is connected to the inner and outer core oligosaccharides. This LPS core structure is extended in the periplasm by the O antigen, a variable and serotype-defining polysaccharide. In the ABC transporter-dependent LPS biosynthesis pathway, the WzmWzt transporter secretes the complete O antigen across the inner membrane for ligation to the LPS core. In some O antigen transporters, the nucleotide-binding domain of Wzt is fused C-terminally to a carbohydrate-binding domain (CBD) that interacts with the O antigen chain. Here, we present the crystal structure of the Aquifex aeolicus CBD that reveals a conserved flat and a variable twisted jelly-roll surface. The CBD dimer is stabilized by mutual β strand exchange. Microbial glycan array binding studies with the isolated CBD provide insights into its interaction with complex carbohydrates.

Polymorphonuclear neutrophils (PMNs) play a critical role in the innate immune response to invading pathogens. However, dysregulated mucosal trafficking of PMNs and associated epithelial tissue damage is a pathological hallmark of numerous inflammatory conditions including inflammatory bowel disease. The glycoprotein CD11b/CD18 plays a well-described role in regulating PMN transepithelial migration and PMN inflammatory functions. Previous studies have demonstrated that targeting of the N-linked glycan Lewis X on CD11b blocks PMN transepithelial migration (TEpM). Given evidence of glycosylation-dependent regulation of CD11b/CD18 function, we performed MALDI TOF Mass Spectrometry (MS) analyses on CD11b/CD18 purified from human PMNs. Unusual glycan epitopes identified on CD11b/CD18 included high Mannose oligosaccharides recognized by the Galanthus Nivalis lectin and biantennary galactosylated N-glycans recognized by the Phaseolus Vulgaris erythroagglutinin lectin. Importantly, we show that selective targeting of glycans on CD11b with such lectins results in altered intracellular signaling events that inhibit TEpM and differentially affect key PMN inflammatory functions including phagocytosis, superoxide release and apoptosis. Taken together, these data demonstrate that discrete glycan motifs expressed on CD11b/CD18 such as biantennary galactose could represent novel targets for selective manipulation of CD11b function and reduction of PMN-associated tissue damage in chronic inflammatory diseases.

Protein glycosylation represents a nearly ubiquitous post-translational modification, and altered glycosylation can result in clinically significant pathological consequences. Here we focus on O-glycosylation in tumor cells of mice and humans. O-glycans are those linked to serine and threonine (Ser/Thr) residues via N-acetylgalactosamine (GalNAc), which are oligosaccharides that occur widely in glycoproteins, such as those expressed on the surfaces and in secretions of all cell types. The structure and expression of O-glycans are dependent on the cell type and disease state of the cells. There is a great interest in O-glycosylation of tumor cells, as they typically express many altered types of O-glycans compared with untransformed cells. Such altered expression of glycans, quantitatively and/or qualitatively on different glycoproteins, is used as circulating tumor biomarkers, such as CA19-9 and CA-125. Other tumor-associated carbohydrate antigens (TACAs), such as the Tn antigen and sialyl-Tn antigen (STn), are truncated O-glycans commonly expressed by carcinomas on multiple glycoproteins; they contribute to tumor development and serve as potential biomarkers for tumor presence and stage, both in immunohistochemistry and in serum diagnostics. Here we discuss O-glycosylation in murine and human cells with a focus on colorectal, breast, and pancreatic cancers, centering on the structure, function and recognition of O-glycans. There are enormous opportunities to exploit our knowledge of O-glycosylation in tumor cells to develop new diagnostics and therapeutics.

MOTIVATION: Glycan structures are commonly represented using symbols or linear nomenclature such as that from the Consortium for Functional Glycomics (also known as modified IUPAC-condensed nomenclature). No current tool allows for writing the name in such format using a graphical user interface (GUI); thus, names are prone to errors or non-standardized representations. RESULTS: Here we present GlycoGlyph, a web application built using JavaScript, which is capable of drawing glycan structures using a GUI and providing the linear nomenclature as an output or using it as an input in a dynamic manner. GlycoGlyph also allows users to save the structures as an SVG vector graphic, and allows users to export the structure as condensed GlycoCT. AVAILABILITY AND IMPLEMENTATION: The application can be used at: https://glycotoolkit.com/Tools/GlycoGlyph/. The application is tested to work in modern web browsers such as Firefox or Chrome. CONTACT: aymehta@bidmc.harvard.edu or rcummin1@bidmc.harvard.edu.

Humoral immunity to pathogens and other environmental challenges is paramount to maintain normal health, and individuals lacking or unable to make antibodies are at risk. Recent studies indicate that many human protective antibodies are against carbohydrate antigens; however, little is known about repertoires and individual variation of anti-carbohydrate antibodies in healthy individuals. Here we analyzed anti-carbohydrate antibody repertoires (ACARs) of 105 healthy individual adult donors, aged 20-60 from different ethnic backgrounds to explore variations in antibodies, as defined by binding to glycan microarrays and by affinity purification. Using microarrays that contained > 1,000 glycans, including antigens from animal cells and microbes, we profiled the IgG and IgM ACARs from all donors. Each donor expressed many ACAs, but had a relatively unique ACAR, which included unanticipated antibodies to carbohydrate antigens not well studied, such as chitin oligosaccharides, Forssman-related antigens, globo-type antigens, and bacterial glycans. We also saw some expected antibodies to ABO(H) blood group and α-Gal-type antigens, although these also varied among individuals. Analysis suggests differences in ACARs are associated with ethnicity and age. Thus, each individual ACAR is relatively unique, suggesting that individualized information could be useful in precision medicine for predicting and monitoring immune health and resistance to disease.