Publications

Glycan binding proteins (GBPs) possess the unique ability to regulate a wide variety of biological processes through interactions with highly modifiable cell surface glycans. While many studies demonstrate the impact of glycan modification on GBP recognition and activity, the relative contribution of subtle changes in glycan structure on GBP binding can be difficult to define. To overcome limitations in the analysis of GBP-glycan interactions, recent studies utilized glycan microarray platforms containing hundreds of structurally defined glycans. These studies not only provided important information regarding GBP-glycan interactions, but have also resulted in significant insight into the binding specificity and biological activity of the galectin family. We will describe the methods used when employing glycan microarray platforms to examine galectin-glycan binding specificity and function.

Cellular turnover represents a fundamental aspect of immunological homeostasis. While many factors appear to regulate leukocyte removal during inflammatory resolution, recent studies suggest that members of the galectin family play a unique role in orchestrating this process. Unlike cellular removal through apoptotic cell death, several members of the galectin family induce surface expression of phosphatidylserine (PS), a phagocytic marker on cells undergoing apoptosis, in the absence of cell death. However, similar to PS on cells undergoing apoptosis, galectin-induced PS exposure sensitizes cells to phagocytic removal. As galectins appear to prepare cells for phagocytic removal without actually inducing apoptotic cell death, this process has recently been coined preaparesis. Given the unique characteristics of galectin-induced PS exposure in the context of preaparesis, we will examine important considerations when evaluating the potential impact of different galectin family members on PS exposure and cell viability.

Over a century ago, Karl Landsteiner discovered that blood group antigens could predict the immunological outcome of red blood cell transfusion. While the discovery of ABO(H) blood group antigens revolutionized transfusion medicine, many questions remain regarding the development and regulation of naturally occurring anti-blood group antibody formation. Early studies suggested that blood group antibodies develop following stimulation by bacteria that express blood group antigens. While this may explain the development of anti-blood group antibodies in blood group negative individuals, how blood group positive individuals, who cannot generate anti-blood group antibodies, protect themselves against blood group positive microbes remained unknown. Recent studies suggest that several members of the galectin family specifically target blood group positive microbes, thereby providing innate immune protection against blood group antigen positive microbes regardless of the blood group status of an individual. Importantly, subsequent studies suggest that this unique form of immunity may not be limited to blood group expressing microbes, but may reflect a more generalized form of innate immunity against molecular mimicry. As this form of antimicrobial activity represents a unique and unprecedented form of immunity, we will examine important considerations and methodological approaches that can be used when seeking to ascertain the potential antimicrobial activity of various members of the galectin family.

Bacteriophage receptor-binding proteins (RBPs) confer host specificity. We previously identified a putative RBP (Gp047) from the campylobacter lytic phage NCTC 12673 and demonstrated that Gp047 has a broader host range than its parent phage. While NCTC 12673 recognizes the capsular polysaccharide (CPS) of a limited number of Campylobacter jejuni isolates, Gp047 binds to a majority of C. jejuni and related Campylobacter coli strains. In this study, we demonstrate that Gp047 also binds to acapsular mutants, suggesting that unlike the parent phage, CPS is not the receptor for Gp047. Affinity chromatography and far-western analyses of C. jejuni lysates using Gp047 followed by mass spectrometry indicated that Gp047 binds to the major flagellin protein, FlaA. Because C. jejuni flagellin is extensively glycosylated, we investigated this binding specificity further and demonstrate that Gp047 only recognizes flagellin decorated with acetamidino-modified pseudaminic acid. This binding activity is localized to the C-terminal quarter of the protein and both wild-type and coccoid forms of C. jejuni are recognized. In addition, Gp047 treatment agglutinates vegetative cells and reduces their motility. Because Gp047 is highly conserved among all campylobacter phages sequenced to date, it is likely that this protein plays an important role in the phage life cycle.

Despite the paradigm that carbohydrates are T cell-independent antigens, isotype-switched glycan-specific immunoglobulin G (IgG) antibodies and polysaccharide-specific T cells are found in humans. We used a systems-level approach combined with glycan array technology to decipher the repertoire of carbohydrate-specific IgG antibodies in intravenous and subcutaneous immunoglobulin preparations. A strikingly universal architecture of this repertoire with modular organization among different donor populations revealed an association between immunogenicity or tolerance and particular structural features of glycans. Antibodies were identified with specificity not only for microbial antigens but also for a broad spectrum of host glycans that serve as attachment sites for viral and bacterial pathogens and/or exotoxins. Tumor-associated carbohydrate antigens were differentially detected by IgG antibodies, whereas non-IgG2 reactivity was predominantly absent. Our study highlights the power of systems biology approaches to analyze immune responses and reveals potential glycan antigen determinants that are relevant to vaccine design, diagnostic assays, and antibody-based therapies.

The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) plays an essential role in lysosome biogenesis by targeting ∼ 60 different phosphomannosyl-containing acid hydrolases to the lysosome. This type I membrane glycoprotein has a large extracellular region comprised of 15 homologous domains. Two mannose 6-phosphate (M6P) binding sites have been mapped to domains 3 and 9, whereas domain 5 binds preferentially to the phosphodiester, M6P-N-acetylglucosamine (GlcNAc). A structure-based sequence alignment predicts that the C-terminal domain 15 contains three out of the four conserved residues identified as essential for carbohydrate recognition by domains 3, 5 and 9 of the CI-MPR, but lacks two cysteine residues that are predicted to form a disulfide bond. To determine whether domain 15 of the CI-MPR has lectin activity and to probe its carbohydrate-binding specificity, truncated forms of the CI-MPR were tested for binding to acid hydrolases with defined N-glycans in surface plasmon resonance analyses, and used to interrogate a phosphorylated glycan microarray. The results show that a construct encoding domains 14-15 binds both M6P and M6P-GlcNAc with similar affinity (Kd = 13 and 17 μM, respectively). Site-directed mutagenesis studies demonstrate the essential role of the conserved Tyr residue in domain 15 for phosphomannosyl binding. A structural model of domain 15 was generated that predicted an Arg residue to be in the binding pocket and mutagenesis studies confirmed its important role in carbohydrate binding. Together, these results show that the CI-MPR contains a fourth carbohydrate-recognition site capable of binding both phosphomonoesters and phosphodiesters.

Neoplastic transformation results in a wide variety of cellular alterations that impact the growth, survival, and general behavior of affected tissue. Although genetic alterations underpin the development of neoplastic disease, epigenetic changes can exert an equally significant effect on neoplastic transformation. Among neoplasia-associated epigenetic alterations, changes in cellular glycosylation have recently received attention as a key component of neoplastic progression. Alterations in glycosylation appear to not only directly impact cell growth and survival but also facilitate tumor-induced immunomodulation and eventual metastasis. Many of these changes may support neoplastic progression, and unique alterations in tumor-associated glycosylation may also serve as a distinct feature of cancer cells and therefore provide novel diagnostic and even therapeutic targets.

The glycans displayed on mammalian cells can differ markedly from those on microbes. Such differences could, in principle, be 'read' by carbohydrate-binding proteins, or lectins. We used glycan microarrays to show that human intelectin-1 (hIntL-1) does not bind known human glycan epitopes but does interact with multiple glycan epitopes found exclusively on microbes: β-linked D-galactofuranose (β-Galf), D-phosphoglycerol-modified glycans, heptoses, D-glycero-D-talo-oct-2-ulosonic acid (KO) and 3-deoxy-D-manno-oct-2-ulosonic acid (KDO). The 1.6-Å-resolution crystal structure of hIntL-1 complexed with β-Galf revealed that hIntL-1 uses a bound calcium ion to coordinate terminal exocyclic 1,2-diols. N-acetylneuraminic acid (Neu5Ac), a sialic acid widespread in human glycans, has an exocyclic 1,2-diol but does not bind hIntL-1, probably owing to unfavorable steric and electronic effects. hIntL-1 marks only Streptococcus pneumoniae serotypes that display surface glycans with terminal 1,2-diol groups. This ligand selectivity suggests that hIntL-1 functions in microbial surveillance.

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.

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.