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 glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3'SIA-LN) and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3'SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3'SIA-Le(X) identified in a previous glycan microarray screen.
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. While early studies sought to understand GBP glycan binding specificity, limitations in the availability of test glycans made it difficult to elucidate a detailed understanding of glycan recognition. Recent developments in glycan microarray technology revolutionized analysis of GBP glycan interactions with significant implications in understanding the role of GBPs in host immunity. In this review, we explore different glycan microarray formats with a focus on the impact of these arrays on understanding the binding specificity and function of GBPs involved in immunity.
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). Formation of the active form of the T-synthase requires a unique molecular chaperone termed Cosmc, encoded by Cosmc on the X-chromosome (Xq24 in humans, Xc3 in mice). Cosmc resides in the endoplasmic reticulum (ER) and prevents misfolding, aggregation, and proteasome-dependent degradation of newly synthesized T-synthase. Loss of expression of active T-synthase or Cosmc can lead to expression of the Tn antigen, along with its sialylated version Sialyl Tn antigen as observed in several cancers. Both genetic and epigenetic pathways, in addition to potential metabolic regulation, can result in abnormal expression of the Tn antigen. Engineered expression of the Tn antigen by disruption of either C1GalT (T-syn) or Cosmc in mice is associated with a tremendous range of pathologies and engineered expression of the Tn antigen in mouse embryos leads to embryonic death. Studies indicate that many membrane glycoproteins expressing the Tn antigen and/or truncated O-glycans may be dysfunctional, due to degradation and/or misfolding. Thus, expression of normal O-glycans is associated with health and homeostasis whereas truncation of O-glycans, e.g. the Tn and/or Sialyl Tn antigens is associated with cancer and other pathologies.
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. Glycan microarrays are increasingly used to determine the binding specificity of glycan-binding proteins (GBPs). In this study, six different glycan microarray platforms with different modes of glycan presentation were compared using five well-known lectins; concanavalin A, Helix pomatia agglutinin, Maackia amurensis lectin I, Sambucus nigra agglutinin and wheat germ agglutinin. A new method (universal threshold) was developed to facilitate systematic comparisons across distinct array platforms. The strongest binders of each lectin were identified using the universal threshold across all platforms while identification of weaker binders was influenced by platform-specific factors including presentation of determinants, array composition and self-reported thresholding methods. This work compiles a rich dataset for comparative analysis of glycan array platforms and has important implications for the implementation of microarrays in the characterization of GBPs.
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. We also examined the effect of Atbf1 deletion on gene expression and signaling pathways in mouse prostates. Histopathologic analyses showed that Atbf1 deficiency caused hyperplasia and mouse prostatic intraepithelial neoplasia (mPIN) primarily in the dorsal prostate but also in other lobes. Hemizygous deletion of Atbf1 also increased the development of hyperplasia and mPIN, indicating a haploinsufficiency of Atbf1. The mPIN lesions expressed luminal cell markers and harbored molecular changes similar to those in human PIN and prostate cancer, including weaker expression of basal cell marker cytokeratin 5 (Ck5), cell adhesion protein E-cadherin, and the smooth muscle layer marker Sma; elevated expression of the oncoproteins phospho-Erk1/2, phospho-Akt and Muc1; and aberrant protein glycosylation. Gene expression profiling revealed a large number of genes that were dysregulated by Atbf1 deletion, particularly those that encode for secretory and cell membrane proteins. The four signaling networks that were most affected by Atbf1 deletion included those centered on Erk1/2 and IGF1, Akt and FSH, NF-κB and progesterone and β-estradiol. These findings provide in vivo evidence that ATBF1 is a tumor suppressor in the prostate, suggest that loss of Atbf1 contributes to tumorigenesis by dysregulating membrane and secretory proteins and multiple signaling pathways, and provide a new animal model for prostate cancer.
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. Rhesus anti-glycan responses over the course of infection were screened on a defined glycan microarray comprising semi-synthetic glycopeptides terminating with schistosome-associated or control mammalian-type glycan epitopes, as well as a defined glycan microarray of mammalian-type glycans representing over 400 glycan structures. Infected rhesus monkeys generated a high immunoglobulin G (IgG) antibody response to the core xylose/core α3 fucose epitope of N-glycans, which peaked at 8-11 weeks post infection, coinciding with maximal ability to kill schistosomula in vitro. By contrast, infected humans generated low antibody levels to this epitope. At 18 months following praziquantel therapy to eliminate the parasite, antibody levels were negligible. Mice chronically infected with S. mansoni generated high levels of anti-fucosylated LacdiNAc (GalNAcβ1, 4(Fucα1, 3)GlcNAc) IgM antibodies, but lacked a robust response to the core xylose/core α3 fucose N-glycan antigens compared with other species studied, and their sera demonstrated an intermediate level of schistosomula killing in vitro. These differential responses to parasite glycan antigens may be related to the ability of rhesus monkeys to self-cure in contrast to the chronic infection seen in humans and mice. Our results validate defined glycan microarrays as a useful technology to evaluate diagnostic and vaccine antigens for schistosomiasis and perhaps other infections.
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. Here we describe the use of the Consortium for Functional Glycomics Glycan Array to investigate binding specificity of influenza hemagglutinin and cleavage by neuraminidase, using seasonal and pandemic H1N1 influenza viruses as examples, and compare the results with published data using other array methods.
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. Moreover, engineered T-synthase carrying mutations within CBRT exhibited diminished binding to Cosmc that resulted in the formation of inactive T-synthase. To confirm the general recognition of CBRT by Cosmc, we performed a domain swap experiment in which we inserted the stem region of the T-synthase into the human β4GalT1 and found that the CBRT element can confer Cosmc binding onto the β4GalT1 chimera. Thus, CBRT is a unique recognition motif for Cosmc to promote its regulation and formation of active T-synthase and represents the first sequence-specific chaperone recognition system in the ER/Golgi required for normal protein O-glycosylation.
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. However, no precedent exists for how to design vaccines targeting eukaryotic glycoconjugates. The di- and tri-saccharide motifs LacdiNAc (GalNAcβ1,4GlcNAc; LDN) and fucosylated LacdiNAc (GalNAcβ1,4(Fucα1-3)GlcNAc; LDNF) are the basis for several important schistosome glycan antigens. They occur in monomeric form or as repeating units (poly-LDNF) and as part of a variety of different glycoconjugates. Because chemical synthesis and conjugation of such antigens is exceedingly difficult, we sought to develop a recombinant expression system for parasite glycans. We hypothesized that presentation of parasite glycans on the cell surface would induce glycan-specific antibodies. We generated Chinese hamster ovary (CHO) Lec8 cell lines expressing poly-LDN (L8-GT) and poly-LDNF (L8-GTFT) abundantly on their membrane glycoproteins. Sera from Schistosoma mansoni-infected mice were highly cross-reactive with the cells and with cell-surface N-glycans. Immunizing mice with L8-GT and L8-GTFT cells induced glycan-specific antibodies. The L8-GTFT cells induced a sustained booster response, with antibodies that bound to S. mansoni lysates and recapitulated the exquisite specificity of the anti-parasite response for particular presentations of LDNF antigen. In summary, this recombinant expression system promotes successful generation of antibodies to the glycans of S. mansoni, and it can be adapted to study the role of glycan antigens and anti-glycan immune responses in many other infections and pathologies.
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. Although adaptive immunity exhibited plasticity toward microbial antigens, immunological tolerance limits reactivity toward self. We discovered that several innate immune galectins show specific recognition of microbes that express self-like antigens, leading to direct killing of a broad range of Gram-negative and Gram-positive microbes. Thus, host protection against microbes seems to represent a balance between adaptive and innate immunity to defend against evolving antigenic determinants while protecting against molecular mimicry.
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. Thus, in this study, a model system was developed to establish the role of glycan composition, density, and carrier cationization state on DC response. Thiol modified glycans were covalently bound to a model protein carrier, maleimide functionalized bovine serum albumin (BSA), and the number of glycans per BSA modulated. Additionally, the carrier isoelectric point was scaled from a pI of ∼4.0 to ∼10.0 using ethylenediamine (EDA). The DC response to the neoglycoconjugates adsorbed to wells of a 384-well plate was determined via a high throughput assay. The underlying trends in DC phenotype in relation to conjugate properties were elucidated via multivariate general linear models. It was found that glycoconjugates with more than 20 glycans per carrier had the greatest impact on the pro-inflammatory response from DCs, followed by conjugates having an isoelectric point above 9.5. Surfaces displaying terminal α1-2 linked mannose structures were able to increase the inflammatory DC response to a greater extent than did any other terminal glycan structure. The results herein can be applied to inform the design of the next generation of combination products and biomaterials for use in future vaccines and implanted materials.
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. The total released N- and O-glycans from pig lung glycoproteins and glycolipid-derived glycans were fluorescently tagged and separated by multidimensional HPLC, and individual glycans were covalently printed to generate pig lung shotgun glycan microarrays. All viruses tested interacted with one or more sialylated N-glycans but not O-glycans or glycolipid-derived glycans, and each virus demonstrated novel and unexpected differences in endogenous N-glycan recognition. The results illustrate the repertoire of specific, endogenous N-glycans of pig lung glycoproteins for virus recognition and offer a new direction for studying endogenous glycan functions in viral pathogenesis.
Schistosoma mansoni and other Schistosoma sp. are multicellular parasitic helminths (worms) that infect humans and mammals worldwide. Infection by these parasites, which results in developmental maturation and sexual differentiation of the worms over a period of 5-6 weeks, induces antibodies to glycan antigens expressed in surface and secreted glycoproteins and glycolipids. There is growing interest in defining these unusual parasite-synthesized glycan antigens and using them to understand immune responses, their roles in immunomodulation, and in using glycan antigens as potential vaccine targets. A key problem in this area, however, has been the lack of information about the enzymes involved in elaborating the complex repertoire of glycans represented by the schistosome glycome. Recent availability of the nuclear genome sequences for Schistosoma sp. has created the opportunity to define the glycogenome, which represents the specific genes and cognate enzymes that generate the glycome. Here we describe the current state of information in regard to the schistosome glycogenome and glycome and highlight the important classes of glycans and glycogenes that may be important in their generation.
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. Galatrox also bound immobilized laminin, a LacNAc-dense extracellular matrix component, suggesting that this GBP can bind LacNAc-bearing glycoproteins. As several endogenous mammalian GBPs utilize a similar binding LacNAc binding preference to regulate neutrophil and monocyte activity, we hypothesized that galatrox may mediate B. atrox toxicity through regulation of leukocyte activity. Indeed, galatrox bound neutrophils and promoted leukocyte chemotaxis in a carbohydrate-dependent manner. Similarly, galatrox administration into the mouse peritoneal cavity induced significant neutrophil migration and the release of pro-inflammatory cytokines IL-1α and IL-6. Exposure of bone marrow-derived macrophages to galatrox induced generation of pro-inflammatory mediators IL-6, TNF-α, and keratinocyte-derived chemokine. This signaling by galatrox was mediated via its carbohydrate recognition domain by activation of the TLR4-mediated MyD88-dependent signaling pathway. These results indicate that galatrox has pro-inflammatory activity through its interaction with LacNAc-bearing glycans on neutrophils, macrophages and extracellular matrix proteins and induce the release of pro-inflammatory mediators.
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.emory.edu with all major browsers supported.
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). To investigate the potential role of HMGs as decoy receptors for rotavirus (RV), a leading cause of severe gastroenteritis in children, we interrogated the HMG SGM with recombinant forms of VP8* domains of the RV outer capsid spike protein VP4 from human neonatal strains N155(G10P) and RV3(G3P) and a bovine strain, B223(G10P). Glycans that were bound by RV attachment proteins were selected for detailed structural analyses using metadata-assisted glycan sequencing, which compiles data on each glycan based on its binding by antibodies and lectins before and after exo- and endo-glycosidase digestion of the SGM, coupled with independent MS(n) analyses. These complementary structural approaches resulted in the identification of 32 glycans based on RV VP8* binding, many of which are novel HMGs, whose detailed structural assignments by MS(n) are described in a companion report. Although sialic acid has been thought to be important as a surface receptor for RVs, our studies indicated that sialic acid is not required for binding of glycans to individual VP8* domains. Remarkably, each VP8* recognized specific glycan determinants within a unique subset of related glycan structures where specificity differences arise from subtle differences in glycan structures.
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. Although total plasma IgA in IgAN patients was elevated ∼ 1.6-fold compared with that in healthy donors, IgA1 in all samples was unexpectedly separable into two distinct glycoforms: one with core 1 based O-glycans, and the other exclusively containing Tn/STn structures. Importantly, Tn antigen present on IgA1 from IgAN patients and controls was convertible into the core 1 structure in vitro by recombinant T-synthase. Our results demonstrate that undergalactosylation of O-glycans in IgA1 is not restricted to IgAN and suggest that in vivo inefficiency of T-synthase toward IgA1 in a subpopulation of B or plasma cells, as well as overall elevation of IgA, may contribute to IgAN pathogenesis.
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.
We have shown that recombinant forms of VP8* domains of the human rotavirus outer capsid spike protein VP4 from human neonatal strains (N155(G10P) and RV3(G3P) 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. These microarray analyses also provided a variety of metadata about the recognized glycan structures compiled from anti-glycan antibody and lectin binding before and after specific glycosidase digestions, along with compositional information from mass analysis by matrix-assisted laser desorption ionization-mass spectrometry. To deduce glycan sequence and utilize information predicted by analyses of metadata from each glycan, 28 of the glycan targets were retrieved from the tagged glycan library for detailed sequencing using sequential disassembly of glycans by ion-trap mass spectrometry. Our aim is to obtain a deeper structural understanding of these key glycans using an orthogonal approach for structural confirmation in a single ion trap mass spectrometer. This sequential ion disassembly strategy details the complexities of linkage and branching in multiple compositions, several of which contained isomeric mixtures including several novel structures. The application of this approach exploits both library matching with standard materials and de novo approaches. This combination together with the metadata generated from lectin and antibody-binding data before and after glycosidase digestions provide a heretofore-unavailable level of analytical detail to glycan structure analysis. The results of these studies showed that, among the 28 glycan targets analyzed, 27 unique structures were identified, and 23 of the human milk glycans recognized by human rotaviruses represent novel structures not previously described as glycans in human milk. The functional glycomics analysis of human milk glycans provides significant insight into the repertoire of glycans comprising the human milk metaglycome.