Publications

Mucin-type O-glycans are a class of glycans initiated with N-acetylgalactosamine (GalNAc) α-linked primarily to Ser/Thr residues within glycoproteins and often extended or branched by sugars or saccharides. Most secretory and membrane-bound proteins receive this modification, which is important in regulating many biological processes. Alterations in mucin-type O-glycans have been described across tumor types and include expression of relatively small-sized, truncated O-glycans and altered terminal structures, both of which are associated with patient prognosis. New discoveries in the identity and expression of tumor-associated O-glycans are providing new avenues for tumor detection and treatment. This chapter describes mucin-type O-glycan biosynthesis, altered mucin-type O-glycans in primary tumors, including mechanisms for structural changes and contributions to the tumor phenotype, and clinical approaches to detect and target altered O-glycans for cancer treatment and management.

The mammalian immune system responds to eukaryotic glycan antigens during infections, cancer, and autoimmune disorders, but the immunological bases for such responses are unclear. Conjugate vaccines containing bacterial polysaccharides linked to carrier proteins (neoglycoconjugates) have proven successful, but these often contain repeating epitopes and the reducing end of the glycan is less important, unlike typical glycan determinants in eukaryotes, which are shorter in length and may include the reducing end. Here, we have compared the effects of two linkage methods, one that opens the ring at the reducing end of the glycan, and one that leaves the reducing end closed, on the glycan specificity of the vaccine response in rabbits and mice. We immunized rabbits and mice with bovine serum albumin (BSA) conjugates of synthetic open- and closed-ring forms (OR versus CR) of a simple tetrasaccharide lacto-N-neotetraose (LNnT, Galβ1-4GlcNAcβ1-3Galβ1-4Glc), and tested reactivity to the immunogens and several related glycans in both OR and CR versions on glycan microarrays. We found that in rabbits the immune response to the CR conjugate was directed toward the glycan, whereas the OR conjugate elicited antibodies to the reducing end of the glycan and linker region but not specifically to the glycan itself. Unexpectedly, mice did not generate a glycan-specific response to the CR conjugate. Our findings indicate that the reducing end of the sugar is crucial for generation of a glycan-specific response to some eukaryotic vaccine epitopes, and that there are species-specific differences in the ability to make a glycan-specific response to some glycoconjugates. These findings warrant further investigation with regard to rational design of glycoconjugate vaccines.

The T-synthase (core 1 β3-galactosyltransferase) and its molecular chaperone Cosmc regulate the biosynthesis of mucin type O-glycans on glycoproteins, and evidence suggests that both T-synthase and Cosmc are transcriptionally suppressed in several human diseases, although the transcriptional regulation of these two genes is not understood. Here, we characterized the promoters essential for human Cosmc and T-synthase transcription. The upstream regions of the genes lack a conventional TATA box but contain CpG islands, cCpG-I and cCpG-II for Cosmc and tCpG for T-synthase. Using luciferase reporter assays, site-directed mutagenesis, ChIP assays, and mithramycin A treatment, we identified the core promoters within cCpG-II and tCpG, which contain two binding sites for Krüppel-like transcription factors, including SP1/SP3, respectively. Methylome analysis of Tn4 B cells, which harbor a silenced Cosmc, confirmed the hypermethylation of the Cosmc core promoter but not for T-synthase. These results demonstrate that Cosmc and T-synthase are transcriptionally regulated at a basal level by the specificity protein/Krüppel-like transcription factor family of members, which explains their ubiquitous and coordinated expression, and also indicate that they are differentially epigenetically regulated beyond X chromosome imprinting. These results are important in understanding the regulation of these genes that have roles in human diseases, such as IgA nephropathy and cancer.

Inflammatory immune disorders such as inflammatory bowel disease and multiple sclerosis are major health problems. Currently, the intestinal whipworm Trichuris suis is being explored in clinical trials to reduce inflammation in these diseases; however, the mechanisms by which the parasite affects the host immune system are not known. Here we determined the effects of T. suis soluble products (SPs) on Toll-like receptor-4 (TLR4)-stimulated human dendritic cells (DCs) using Illumina bead chip gene arrays. Pathway analysis of lipopolysaccharide-stimulated DCs with or without T. suis treatment showed that co-stimulation with T. suis SPs resulted in a downregulation of both the myeloid differentiation primary response gene 88-dependent and the TIR-domain-containing adaptor-inducing interferon-β-dependent signalling pathways triggered by TLR4. These data were verified using quantitative real-time PCR of several key genes within these pathways and/or defining their protein levels. In addition, T. suis SPs induce Rab7b, a negative regulator of TLR4 signalling that interferes with its trafficking, which coincided with a reduced surface expression of TLR4. These data indicate that the mechanism by which T. suis SPs reduce inflammatory responses is through suppression of both TLR4 signalling and surface expression on DCs.

This work describes the synthesis of the 1,2,3-triazole amino acid-derived-3-O-galactosides 1-6 and the 1,2,3-triazole di-lactose-derived glycoconjugate 7 as potential galectin-3 inhibitors. The target compounds were synthesized by Cu(I)-catalyzed azide-alkyne cycloaddition reaction ('click chemistry') between the azido-derived amino acids N3-ThrOBn, N3-PheOBn, N3-N-Boc-TrpOBn, N3-N-Boc-LysOBn, N3-O-tBu-AspOBn and N3-l-TyrOH, and the corresponding alkyne-based sugar 3-O-propynyl-GalOMe, as well as by click chemistry reaction between the azido-lactose and 2-propynyl lactose. Surface plasmon resonance (SPR) assays showed that all synthetic glycoconjugates 1-7 bound to galectin-3 with high affinity, but the highest binders were the amino acids-derived glycoconjugates 2 (KD 7.96μM) and 4 (KD 4.56μM), and the divalent lactoside 7 (KD1 0.15μM/KD2 19μM). Molecular modeling results were in agreement with SPR assays, since more stable interactions with galectin-3 were identified for glycoconjugates 2, 4 and 7. Regarding compounds 2 and 4, they established specific cation-π (Arg144) and ionic (Asp148) interactions, whereas glycoconjugate 7 was capable to bridge two independent galectin-3 CRDs, creating a non-covalent cross-link between two monomers and, thus, reaching a submicromolar affinity towards galectin-3.

Schistosomiasis is a common debilitating human parasitic disease in (sub)tropical areas, however, schistosome infections can also protect against a variety of inflammatory diseases. This has raised broad interest in the mechanisms by which Schistosoma modulate the immune system into an anti-inflammatory and regulatory state. Human dendritic cells (DCs) show many phenotypic changes upon contact with Schistosoma mansoni soluble egg antigens (SEA). We here show that oxidation of SEA glycans, but not heat-denaturation, abrogates the capacity of SEA to suppress both LPS-induced cytokine secretion and DC proliferation, indicating an important role of SEA glycans in these processes. Remarkably, interaction of SEA glycans with DCs results in a strongly increased expression of Suppressor Of Cytokine Signalling1 (SOCS1) and SH2-containing protein tyrosine Phosphatase-1 (SHP1), important negative regulators of TLR4 signalling. In addition, SEA induces the secretion of transforming growth factor β (TGF-β), and the surface expression of the costimulatory molecules Programmed Death Ligand-1 (PD-L1) and OX40 ligand (OX40L), which are known phenotypic markers for the capacity of DCs to polarize naïve T cells into Th2/Treg cell subsets. Inhibition of mannose receptor (MR)-mediated internalization of SEA into DCs by blocking with allyl α-D-mannoside or anti-MR antibodies, significantly reduced SOCS1 and SHP1 expression. In conclusion, we demonstrate that SEA glycans are essential for induction of enhanced SOCS1 and SHP1 levels in DCs via the MR. Our data provide novel mechanistic evidence for the potential of S. mansoni SEA glycans to modulate human DCs, which may contribute to the capacity of SEA to down-regulate inflammatory responses.

Blockade of P-selectin (P-sel)/PSGL-1 interactions holds significant potential for treatment of disorders of innate immunity, thrombosis and cancer. Current inhibitors remain limited due to low binding affinity or by the recognized disadvantages inherent to chronic administration of antibody therapeutics. Here we report an efficient approach for generating glycosulfopeptide mimics of N-terminal PSGL-1 through development of a stereoselective route for multi-gram scale synthesis of the C2 O-glycan building block and replacement of hydrolytically labile tyrosine sulfates with isosteric sulfonate analogues. Library screening afforded a compound of exceptional stability, GSnP-6, that binds to human P-sel with nanomolar affinity (Kd~22 nM). Molecular dynamics simulation defines the origin of this affinity in terms of a number of critical structural contributions. GSnP-6 potently blocks P-sel/PSGL-1 interactions in vitro and in vivo and represents a promising candidate for the treatment of diseases driven by acute and chronic inflammation.

This review discusses the challenges facing research in 'functional glycomics' and the novel technologies that are being developed to advance the field. The structural complexity of glycans and glycoconjugates makes studies of both their structures and recognition difficult. However, these intricate structures can be captured from their natural sources, isolated and fluorescently-tagged for detailed structural analysis and for presentation on glycan microarrays for functional recognition by glycan-binding proteins. These advances in glycan preparation and manipulation enable the streamlining of functional glycomics studies and will help to propel the field forward in studying natural, biologically relevant glycans.

INTRODUCTION: The inverse correlation between prevalence of auto-immune disorders like the chronic neuro-inflammatory disease multiple sclerosis (MS) and the occurrence of helminth (worm) infections, suggests that the helminth-trained immune system is protective against auto-immunity. As monocytes are regarded as crucial players in the pathogenesis of auto-immune diseases, we explored the hypothesis that these innate effector cells are prime targets for helminths to exert their immunomodulatory effects. RESULTS: Here we show that soluble products of the porcine nematode Trichuris suis (TsSP) are potent in changing the phenotype and function of human monocytes by skewing classical monocytes into anti-inflammatory patrolling cells, which exhibit reduced trans-endothelial migration capacity in an in vitro model of the blood-brain barrier. Mechanistically, we identified the mannose receptor as the TsSP-interacting monocyte receptor and we revealed that specific downstream signalling occurs via protein kinase C (PKC), and in particular PKCδ. CONCLUSION: This study provides comprehensive mechanistic insight into helminth-induced immunomodulation, which can be therapeutically exploited to combat various auto-immune disorders.