GlycoPattern: a web platform for glycan array mining.

Unlabelled: GlycoPattern is Web-based bioinformatics resource to support the analysis of glycan array data for the Consortium for Functional Glycomics. This resource includes algorithms and tools to discover structural motifs, a heatmap visualization to compare multiple experiments, hierarchical clustering of Glycan Binding Proteins with respect to their binding motifs and a structural search feature on the experimental data.

Availability And Implementation: GlycoPattern is freely available on the Web at http://glycopattern.

Identification of distinct glycoforms of IgA1 in plasma from patients with immunoglobulin A (IgA) nephropathy and healthy individuals.

Immunoglobulin A nephropathy (IgAN) is the most common form of glomerulonephritis worldwide and is histologically characterized by the deposition of IgA1 and consequent inflammation in the glomerular mesangium. Prior studies suggested that serum IgA1 from IgAN patients contains aberrant, undergalactosylated O-glycans, for example, Tn antigen and its sialylated version, SialylTn (STn), but the mechanisms underlying aberrant O-glycosylation are not well understood. Here we have used serial lectin separation technologies, Western blot, enzymatic modifications, and mass spectrometry to explore whether there are different glycoforms of IgA1 in plasma from patients with IgAN and healthy individuals.

Structural characterization by multistage mass spectrometry (MSn) of human milk glycans recognized by human rotaviruses.

We have shown that recombinant forms of VP8* domains of the human rotavirus outer capsid spike protein VP4 from human neonatal strains (N155(G10P[11]) and RV3(G3P[6]) and a bovine strain (B223) recognize unique glycans within the repertoire of human milk glycans. The accompanying study by Yu et al.(2), describes a human milk glycan shotgun glycan microarray that led to the identification of 32 specific glycans in the human milk tagged glycan library that were recognized by these human rotaviruses.

Human milk contains novel glycans that are potential decoy receptors for neonatal rotaviruses.

Human milk contains a rich set of soluble, reducing glycans whose functions and bioactivities are not well understood. Because human milk glycans (HMGs) have been implicated as receptors for various pathogens, we explored the functional glycome of human milk using shotgun glycomics. The free glycans from pooled milk samples of donors with mixed Lewis and Secretor phenotypes were labeled with a fluorescent tag and separated via multidimensional HPLC to generate a tagged glycan library containing 247 HMG targets that were printed to generate the HMG shotgun glycan microarray (SGM).

Investigating virus-glycan interactions using glycan microarrays.

While all viruses must transit the plasma membrane of mammalian cells to initiate infection, we know little about the complex processes involved in viral attachment, which commonly involve recognition of glycans by viral proteins. Glycan microarrays derived from both synthetic glycans and natural glycans isolated through shotgun glycomics approaches provide novel platforms for interrogating diverse glycans as potential viral receptors. Recent studies with influenza and rotaviruses using such glycan microarrays provide examples of their utility in exploring the challenging questions raised in efforts to define the complex mechanistic protein-glycan interactions that regulate virus attachment to host cells.

Galatrox is a C-type lectin in Bothrops atrox snake venom that selectively binds LacNAc-terminated glycans and can induce acute inflammation.

Previous studies indicate that snake venom contains glycan-binding proteins (GBPs), although the binding specificity and biological activities of many of these GBPs is unclear. Here we report our studies on the glycan binding specificity and activities of galatrox, a Bothrops atrox snake venom-derived GBP. Glycan microarray analysis indicates that galatrox binds most strongly to glycans expressing N-acetyllactosamine (LacNAc), with a significant preference for Galβ1-4GlcNAcβ over Galβ1-3GlcNAcβ compounds.

Deletion of atbf1/zfhx3 in mouse prostate causes neoplastic lesions, likely by attenuation of membrane and secretory proteins and multiple signaling pathways.

The ATBF1/ZFHX3 gene at 16q22 is the second most frequently mutated gene in human prostate cancer and has reduced expression or mislocalization in several types of human tumors. Nonetheless, the hypothesis that ATBF1 has a tumor suppressor function in prostate cancer has not been tested. In this study, we examined the role of ATBF1 in prostatic carcinogenesis by specifically deleting Atbf1 in mouse prostatic epithelial cells.

Shotgun glycomics of pig lung identifies natural endogenous receptors for influenza viruses.

Influenza viruses bind to host cell surface glycans containing terminal sialic acids, but as studies on influenza binding become more sophisticated, it is becoming evident that although sialic acid may be necessary, it is not sufficient for productive binding. To better define endogenous glycans that serve as viral receptors, we have explored glycan recognition in the pig lung, because influenza is broadly disseminated in swine, and swine have been postulated as an intermediary host for the emergence of pandemic strains. For these studies, we used the technology of "shotgun glycomics" to identify natural receptor glycans.

Microbial glycan microarrays define key features of host-microbial interactions.

Genomic approaches continue to provide unprecedented insight into the microbiome, yet host immune interactions with diverse microbiota can be difficult to study. We therefore generated a microbial microarray containing defined antigens isolated from a broad range of microbial flora to examine adaptive and innate immunity. Serological studies with this microarray show that immunoglobulins from multiple mammalian species have unique patterns of reactivity, whereas exposure of animals to distinct microbes induces specific serological recognition.

Molecular factors in dendritic cell responses to adsorbed glycoconjugates.

Carbohydrates and glycoconjugates have been shown to exert pro-inflammatory effects on the dendritic cells (DCs), supporting pathogen-induced innate immunity and antigen processing, as well as immunosuppressive effects in the tolerance to self-proteins. Additionally, the innate inflammatory response to implanted biomaterials has been hypothesized to be mediated by inflammatory cells interacting with adsorbed proteins, many of which are glycosylated. However, the molecular factors relevant for surface displayed glycoconjugate modulation of dendritic cell (DC) phenotype are unknown.

Differential expression of anti-glycan antibodies in schistosome-infected humans, rhesus monkeys and mice.

Schistosomiasis is a debilitating parasitic disease of humans, endemic in tropical areas, for which no vaccine is available. Evidence points to glycan antigens as being important in immune responses to infection. Here we describe our studies on the comparative humoral immune responses to defined schistosome-type glycan epitopes in Schistosoma mansoni-infected humans, rhesus monkeys and mice.

Immunization with recombinantly expressed glycan antigens from Schistosoma mansoni induces glycan-specific antibodies against the parasite.

Schistosomiasis caused by infection with parasitic helminths of Schistosoma spp. is a major global health problem due to inadequate treatment and lack of a vaccine. The immune response to schistosomes includes glycan antigens, which could be valuable diagnostic markers and vaccine targets.

Cross-platform comparison of glycan microarray formats.

Carbohydrates participate in almost every aspect of biology from protein sorting to modulating cell differentiation and cell-cell interactions. To date, the majority of data gathered on glycan expression has been obtained via analysis with either anti-glycan antibodies or lectins. A detailed understanding of the specificities of these reagents is critical to the analysis of carbohydrates in biological systems.

The Cosmc connection to the Tn antigen in cancer.

The Tn antigen is a tumor-associated carbohydrate antigen that is not normally expressed in peripheral tissues or blood cells. Expression of this antigen, which is found in a majority of human carcinomas of all types, arises from a blockage in the normal O-glycosylation pathway in which glycans are extended from the common precursor GalNAcα1-O-Ser/Thr (Tn antigen). This precursor is generated in the Golgi apparatus on newly synthesized glycoproteins by a family of polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAcTs) and then extended to the common core 1 O-glycan Galβ1-3GalNAcα1-O-Ser/Thr (T antigen) by a single enzyme termed the T-synthase (core 1 β3-galactosyltransferase or C1GalT).

Glycan array analysis of influenza H1N1 binding and release.

Influenza viruses initiate infection by attaching to sialic acid receptors on the surface of host cells. It has been recognized for some time that avian influenza viruses usually bind to terminal sialic acid that is linked in the α2-3 configuration to the next sugar while human viruses show preference for α2-6 linked sialic acid. With developments in synthetic chemistry and chemo-enzymatic methods of synthesizing quite complex glycans, it has become clear that the binding specificity extends beyond the sialic acid, and this has led to considerable interest in developing glycan reagents that could be used either as a diagnostic tool for particular influenza viruses, or to identify cells that are susceptible to infection by certain influenza viruses.

Identification of a novel protein binding motif within the T-synthase for the molecular chaperone Cosmc.

Prior studies suggested that the core 1 β3-galactosyltransferase (T-synthase) is a specific client of the endoplasmic reticulum chaperone Cosmc, whose function is required for T-synthase folding, activity, and consequent synthesis of normal O-glycans in all vertebrate cells. To explore whether the T-synthase encodes a specific recognition motif for Cosmc, we used deletion mutagenesis to identify a cryptic linear and relatively hydrophobic peptide in the N-terminal stem region of the T-synthase that is essential for binding to Cosmc (Cosmc binding region within T-synthase, or CBRT). Using this sequence information, we synthesized a peptide containing CBRT and found that it directly interacts with Cosmc and also inhibits Cosmc-assisted in vitro refolding of denatured T-synthase.

Chemistry of natural glycan microarrays.

Glycan microarrays have become indispensable tools for studying protein-glycan interactions. Along with chemo-enzymatic synthesis, glycans isolated from natural sources have played important roles in array development and will continue to be a major source of glycans. N-glycans and O-glycans from glycoproteins, and glycans from glycosphingolipids (GSLs) can be released from corresponding glycoconjugates with relatively mature methods, although isolation of large numbers and quantities of glycans is still very challenging.

Using glycan microarrays to understand immunity.

Host immunity represents a complex array of factors that evolved to provide protection against potential pathogens. While many factors regulate host immunity, glycan binding proteins (GBPs) appear to play a fundamental role in orchestrating this process. In addition, GBPs also reside at the key interface between host and pathogen.

Profiling of glycan receptors for minute virus of mice in permissive cell lines towards understanding the mechanism of cell recognition.

The recognition of sialic acids by two strains of minute virus of mice (MVM), MVMp (prototype) and MVMi (immunosuppressive), is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA) capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM). Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences.

The challenge and promise of glycomics.

Glycomics is a broad and emerging scientific discipline focused on defining the structures and functional roles of glycans in biological systems. The staggering complexity of the glycome, minimally defined as the repertoire of glycans expressed in a cell or organism, has resulted in many challenges that must be overcome; these are being addressed by new advances in mass spectrometry as well as by the expansion of genetic and cell biology studies. Conversely, identifying the specific glycan recognition determinants of glycan-binding proteins by employing the new technology of glycan microarrays is providing insights into how glycans function in recognition and signaling within an organism and with microbes and pathogens.

Hepatic cytochrome P450s, phase II enzymes and nuclear receptors are downregulated in a Th2 environment during Schistosoma mansoni infection.

Inflammation and infection downregulate the activity and expression of cytochrome P450s (P450s) and other drug metabolizing enzymes (DMEs) involved in hepatic drug clearance. Schistosoma mansoni infection was reported to cause a downregulation of hepatic P450-dependent activities in mouse liver, but little is known about the specific enzymes affected or whether phase II DMEs are also affected. Here we describe the effect of murine schistosomiasis on the expression of hepatic P450s, NADPH-cytochrome P450 reductase (Cpr), phase II drug metabolizing enzymes, and nuclear receptors at 30 and 45 days postinfection (dpi).

α3/4 Fucosyltransferase 3-dependent synthesis of Sialyl Lewis A on CD44 variant containing exon 6 mediates polymorphonuclear leukocyte detachment from intestinal epithelium during transepithelial migration.

Polymorphonuclear leukocyte (PMN) migration across the intestinal epithelium closely parallels disease symptoms in patients with inflammatory bowel disease. PMN transepithelial migration (TEM) is a multistep process that terminates with PMN detachment from the apical epithelium into the lumen. Using a unique mAb (GM35), we have previously demonstrated that engagement of the CD44 variant containing exon 6 (CD44v6) blocks both PMN detachment and cleavage of CD44v6.

Glycoconjugates in host-helminth interactions.

Helminths are multicellular parasitic worms that comprise a major class of human pathogens and cause an immense amount of suffering worldwide. Helminths possess an abundance of complex and unique glycoconjugates that interact with both the innate and adaptive arms of immunity in definitive and intermediate hosts. These glycoconjugates represent a major untapped reservoir of immunomodulatory compounds, which have the potential to treat autoimmune and inflammatory disorders, and antigenic glycans, which could be exploited as vaccines and diagnostics.

Novel cleavage of reductively aminated glycan-tags by N-bromosuccinimide to regenerate free, reducing glycans.

Glycans that are fluorescently tagged by reductive amination have been useful for functional glycomic studies. However, the existing tags can introduce unwanted properties to the glycans and complicate structural and functional studies. Here, we describe a facile method using N-bromosuccinimide (NBS) to remove the tags and efficiently regenerate free reducing glycans.