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.
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. The regenerated free reducing glycans can be easily analyzed by routine mass spectrometry or re-tagged with different tags for further studies. This new method can be used to efficiently remove a variety of fluorescent tags installed by reductive amination, including 2-aminobenzoic acid and 2-aminopyridine. NBS treatment essentially transforms the commonly used 2-aminobenzoic linkage to a cleavable linkage. It can be used to cleave printed glycans from microarrays and cleave neoglycopeptides containing a 2-aminobenzoic linker.
Glycomics; free reducing glycan; fluorescent tag; reversible cleavage
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 exhibit unique patterns of reactivity, while exposure of animals to distinct microbes induces specific serological recognition. While adaptive immunity exhibited plasticity toward microbial antigens, immunological tolerance limits reactivity toward self. We discovered that several innate immune galectins exhibit specific recognition of microbes that express self-like antigens, leading to direct killing of a broad range of gram negative and positive microbes. Thus, host protection against microbes appears to represent a balance between adaptive and innate immunity to defend against evolving antigenic determinants while protecting against molecular mimicry.
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.
glycans; glycoconjugates; genome; glycosyltransferases; glycan biosynthesis; schistosomiasis
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.
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.
This study provides comprehensive mechanistic insight into helminth-induced immunomodulation, which can be therapeutically exploited to combat various auto-immune disorders.
Electronic supplementary material
The online version of this article (doi:10.1186/s40478-015-0223-1) contains supplementary material, which is available to authorized users.
Auto-immunity; Multiple sclerosis; Innate immunity; Trichuris suis; Monocytes; Mannose receptor; Protein kinase C; PKCδ
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.
cellular engineering; glyco-conjugate; helminth; LDNF; schistosome
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.
glycan microarrays; glycans; schistosomiasis; schistosomula killing; core xylose-core α3 fucose
Carbohydrates and glycoconjugates have been shown to exert pro-inflammatory effects on the dendritic cell (DC), 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 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.
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.
comparison; glycan microarray; lectin; presentation
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.
Assessing interactions of a glycan-binding protein (GBP) or lectin with glycans on a microarray generates large datasets, making it difficult to identify a glycan structural motif or determinant associated with the highest apparent binding strength of the GBP. We have developed a computational method, termed GlycanMotifMiner, that uses the relative binding of a GBP with glycans within a glycan microarray to automatically reveal the glycan structural motifs recognized by a GBP. We implemented the software with a web-based graphical interface for users to explore and visualize the discovered motifs. The utility of GlycanMotifMiner was determined using five plant lectins, SNA, HPA, PNA, Con A, and UEA-I. Data from the analyses of the lectins at different protein concentrations were processed to rank the glycans based on their relative binding strengths. The motifs, defined as glycan substructures that exist in a large number of the bound glycans and few non-bound glycans, were then discovered by our algorithm and displayed in a web-based graphical user interface (http://glycanmotifminer.emory.edu). The information is used in defining the glycan-binding specificity of GBPs. The results were compared to the known glycan specificities of these lectins generated by manual methods. A more complex analysis was also carried out using glycan microarray data obtained for a recombinant form of human galectin-8. Results for all of these lectins show that GlycanMotifMiner identified the major motifs known in the literature along with some unexpected novel binding motifs.
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 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. The promises of a more complete knowledge of glycomes are immense in that glycan modifications of intracellular and extracellular proteins have critical functions in almost all biological pathways.
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. This review will survey current knowledge of the interactions between helminth glycans and host immunity and highlight the gaps in our understanding which are relevant to advancing therapeutics, vaccine development, and diagnostics.
glycans; glycoconjugates; helminths; C-type lectin; innate immunity; adaptive immunity; anti-glycan antibodies; schistosomiasis
Dengue virus (DENV) is an enveloped RNA virus that is mosquito-transmitted and can infect a variety of immune and non-immune cells. Response to infection ranges from asymptomatic disease to a severe disorder known as dengue hemorrhagic fever. Despite efforts to control the disease, there are no effective treatments or vaccines. In our search for new antiviral compounds to combat infection by dengue virus type 1 (DENV-1), we investigated the role of galectin-1, a widely-expressed mammalian lectin with functions in cell-pathogen interactions and immunoregulatory properties. We found that DENV-1 infection of cells in vitro exhibited caused decreased expression of Gal-1 in several different human cell lines, suggesting that loss of Gal-1 is associated with virus production. In test of this hypothesis we found that exogenous addition of human recombinant Gal-1 (hrGal-1) inhibits the virus production in the three different cell types. This inhibitory effect was dependent on hrGal-1 dimerization and required its carbohydrate recognition domain. Importantly, the inhibition was specific for hrGal-1, since no effect was observed using recombinant human galectin-3. Interestingly, we found that hrGal-1 directly binds to dengue virus and acts, at least in part, during the early stages of DENV-1 infection, by inhibiting viral adsorption and its internalization to target cells. To test the in vivo role of Gal-1 in DENV infection, Gal-1-deficient-mice were used to demonstrate that the expression of endogenous Galectin-1 contributes to resistance of macrophages to in vitro-infection with DENV-1 and it is also important to physiological susceptibility of mice to in vivo infection with DENV-1. These results provide novel insights into the functions of Gal-1 in resistance to DENV infection and suggest that Gal-1 should be explored as a potential antiviral compound.
PMN migration across the intestinal epithelium closely parallels disease symptoms in patients with inflammatory bowel disease (IBD). 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 V6 variant of CD44 (CD44v6) blocks both PMN detachment and cleavage of CD44v6. Here, we report that PMN binding to CD44v6 is mediated by protein-specific O-glycosylation with sialyl Lewis A (sLea). Analyses of glycosyltransferase expression identified fucosyltransferase 3 (Fut3) as the key enzyme driving sLea biosynthesis in human intestinal epithelial cells (IECs). Fut3 transfection of sLea-deficient IECs resulted in robust expression of sLea. However, this glycan was not expressed on CD44v6 in these transfected IECs, and therefore engagement of sLea had no effect on PMN TEM across these cells. Analyses of sLea in human colonic mucosa revealed minimal expression in noninflamed areas, with striking upregulation under colitic conditions that correlated with increased expression of CD44v6. Importantly, intraluminal administration of mAb GM35 blocked PMN TEM and attenuated associated increases in intestinal permeability in a murine intestinal model of inflammation. These findings identify a unique role for protein-specific O-glycosylation in regulating PMN-epithelial interactions at the luminal surface of the intestine.
Glycoproteins expressed by Cryptosporidium parvum are
immunogenic in infected individuals but the nature of the epitopes recognised in
C. parvum glycoproteins is poorly understood. Since a known
immunodominant antigen of Cryptosporidium, the 17 kDa
glycoprotein, has previously been shown to bind to lectins that recognise the Tn
antigen (GalNAcα1-Ser/Thr-R), a large number of glycopeptides with
different Tn valency and presentation were prepared. In addition, glycopeptides
were synthesised based on a 40 kDa cryptosporidial antigen, a polymorphic
surface glycoprotein with varying numbers of serine residues, to determine the
reactivity with sera from C. parvum-infected humans. These
glycopeptides and non-glycosylated peptides were used to generate a glycopeptide
microarray to allow screening of sera from C. parvum-infected
individuals for the presence of IgM and IgG antibodies. IgG but not IgM in sera
from C. parvum-infected individuals bound to multivalent Tn
antigen epitopes presented on glycopep-tides, suggesting that glycoproteins from
C. parvum that contain the Tn antigen induce immune
responses upon infection. In addition, molecular differences in glycosylated
peptides (e.g. substituting Ser for Thr) as well as the site of glycosylation
had a pronounced effect on reactivity. Lastly, pooled sera from individuals
infected with either Toxoplasma or Plasmodium
were also tested against the modified Cryptosporidium peptides
and some sera showed specific binding to glycopeptide epitopes. These studies
reveal that specific anti-glycopeptide antibodies that recognise the Tn antigen
may be useful diagnostically and in defining the roles of parasite
glycoconjugates in infections.
Cryptosporidium parvum; Tn antigen; Glycopeptide microarrays; Toxoplasma; Plasmodium
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.
Influenza virus; hemagglutinin; neuraminidase; Glycan Array; Consortium for Functional Glycomics
glycan microarrays; carbohydrate; glycomics
The parasitic blood fluke Schistosoma mansoni synthesizes immunogenic glycans containing the human Lewis x antigen (Lex; Galactose-β1-4(Fucα1-3)N-acetylglucosamine-β-R, also called CD15), but the biological role(s) of this antigen in the parasites and in humans is poorly understood. To develop IgG-based monoclonal antibodies (mAbs) specific for Lex, we harvested splenocytes from S. mansoni-infected Swiss Webster mice at Week 10 postinfection, when peak IgG responses to glycan antigens occur, and generated a panel of hybridomas secreting anti-glycan IgG that recognize periodate-sensitive epitopes in soluble egg antigens of the parasites, and also recognizes a neoglycoprotein containing a pentasaccharide with the Lex sequence. One murine mAb, an IgG3 designated F8A1.1, bound to glycoproteins and glycolipids from schistosome adults and human promyelocytic leukemic HL-60 cells that express Lex antigens, as assessed by a wide variety of approaches including immunofluorescence staining, confocal microscopy, flow cytometry and western blotting, as well as overlay assays of glycolipids after thin-layer chromatography. In contrast, F8A1.1 bound weakly to cercariae, 3-h schistosomula and human Jurkat cells. We also directly compared the glycan specificity of F8A1.1 with commercially available anti-CD15 IgG1 (clone W6D3) using a defined glycan microarray. The results demonstrated that F8A1.1 recognized glycans expressing Lex epitopes in a terminal nonreducing position, whereas anti-CD15 bound to glycans with multiple repeats of Lex epitopes, but not to glycans with a single, terminal Lex epitope. Our results show that F8A1.1 recognizes terminal Lex epitopes and can be used for identification, immunolocalization, immunoprecipitation and purification of Lex-containing glycoconjugates from schistosomes and mammalian cells.
glycans; helminth; Lewis x antigen; monoclonal antibody; Schistosoma mansoni
Major challenges of glycomics are to characterize a glycome and identify functional glycans as ligands for glycan-binding proteins (GBPs). To address these issues we have developed a general strategy termed shotgun glycomics. We focus on glycosphingolipids (GSLs), a challenging class of glycoconjugates recognized by toxins, antibodies, and GBPs. We derivatized GSLs extracted from cells with a heterobifunctional fluorescent tag suitable for covalent immobilization. Fluorescent GSLs were separated by multidimensional chromatography, quantified, and coupled to glass slides to create GSL shotgun microarrays. The microarrays were interrogated with cholera toxin, antibodies, and sera from patients with Lyme disease to identify biologically relevant GSLs that were subsequently characterized by mass spectrometry. Shotgun glycomics incorporating GSLs and potentially glycoprotein-derived glycans provides an approach to accessing the complex glycomes of animal cells and offers a strategy for focusing structural analyses on functionally significant glycans.
glycosphingolipids; glycan array; fluorescent labeling; immobilization; functional glycomics
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.
Modifications of proteins by O-glycosylation determine many of the properties and functions of proteins. We wish to understand the mechanisms of O-glycosylation and develop inhibitors that could affect glycoprotein functions and alter cellular behavior.
We expressed recombinant soluble human Gal- and GlcNAc-transferases that synthesize the O-glycan cores 1 to 4 and are critical for the overall structures of O-glycans. We determined the properties and substrate specificities of these enzymes using synthetic acceptor substrate analogs. Compounds that were inactive as substrates were tested as inhibitors.
Enzymes significantly differed in their recognition of the sugar moieties and aglycone groups of substrates. Core 1 synthase was active with glycopeptide substrates but GlcNAc-transferases preferred substrates with hydrophobic aglycone groups. Chemical modifications of the acceptors shed light on enzyme–substrate interactions. Core 1 synthase was weakly inhibited by its substrate analog benzyl 2-butanamido-2-deoxy-α-D-galactoside while two of the three GlcNAc-transferases were selectively and potently inhibited by bis-imidazolium salts which are not substrate analogs.
This work delineates the distinct specificities and properties of the enzymes that synthesize the common O-glycan core structures 1 to 4. New inhibitors were found that could selectively inhibit the synthesis of cores 1, 2 and 3 but not core 4.
These studies help our understanding of the mechanisms of action of enzymes critical for O-glycosylation. The results may be useful for the re-engineering of O-glycosylation to determine the roles of O-glycans and the enzymes critical for O-glycosylation, and for biotechnology with potential therapeutic applications.
O-Glycans; Specificity; Inhibitors; C1GalT; C3GnT; C2GnT
Glycan microarray technology has become a successful tool for studying protein-carbohydrate interactions, but a limitation has been the laborious synthesis of glycan structures by enzymatic and chemical methods. Here we describe a new method to generate quantifiable glycan libraries from natural sources by combining widely used protease digestion of glycoproteins and Fmoc chemistry. Glycoproteins including chicken ovalbumin, bovine fetuin, and horseradish peroxidase (HRP) were digested by pronase, protected by FmocCl, and efficiently separated by 2D-HPLC. We show that glycans from HRP glycopeptides separated by HPLC and fluorescence monitoring retained their natural reducing end structures, mostly core α1,3-fucose and core α1,2-xylose. After simple Fmoc-deprotection, the glycans were printed on NHS-activated glass slides. The glycans were interrogated using plant lectins and antibodies in sera from mice infected with Schistosoma mansoni, which revealed the presence of both IgM and IgG antibody responses to HRP-glycopeptides. This simple approach to glycopeptide purification and conjugation allows for the development of natural glycopeptide microarrays without the need to remove and derivatize glycans and potentially compromise their reducing end determinants.
Glycan array; fluorescent labeling; immobilization; functional glycomics
A novel strategy for creating naturally-derived glycan microarrays has been developed. Glycosylamines are prepared from free reducing glycans and stabilized by reaction with acryloyl chloride to generate a glycosylamide in which the reducing monosaccharide has a closed ring structure. Ozonolysis of the protected glycan yields an active aldehyde, to which a bifunctional fluorescent linker is coupled by reductive amination. The fluorescent derivatives are easily coupled through a residual primary alkylamine to generate glycan microarrays. This strategy preserves structural features of glycans required for antibody recognition, and allows development of natural arrays of fluorescent glycans in which the cyclic pyranose structure of the reducing-end sugar residue is retained.
Fluorescent labeling; Functional glycomics; Glycan array; Glycosylamine; Immobilization
The expression of ABO(H) blood group antigens causes deletion of cells that generate self anti-blood group antibodies, but this deletion limits adaptive immunity toward pathogens bearing cognate blood group antigens. To explore potential defense mechanisms against these pathogens, given such limitations in adaptive immunity, we screened for innate proteins that could recognize human blood group antigens. Here we report that two innate immune lectins, galectins-4 and -8, which are expressed in the intestinal tract, recognize and kill human blood group antigen-expressing E. coli, while failing to alter viability of other E. coli strains or other gram-negative or gram-positive organisms both in vitro and in vivo. Killing by both galectins-4 and -8 resides within their C-terminal domains, occurs rapidly and independently of complement, and is accompanied by disruption of membrane integrity. These results demonstrate that innate defense lectins can provide immunity against pathogens that display blood group self-antigens on their surface.