Glycan microarrays prepared by immobilization of amino-functionalized glycans on NHS-activated glass slides have been successfully used to study protein-glycan interactions. Fluorescently tagged glycans with an amino functional group can be prepared from natural glycans released from glycoproteins. These tagged glycans can be enzymatically modified with various glycosyltransferases, phosphotransferases, sulfotransferases, etc., to quickly expand the size and diversity of the tagged glycan libraries (TGLs). The TGLs, presented in the format of microarrays, provide a convenient platform for identifying the glycan ligands of glycan-binding proteins (GBPs). The chapter provides the background to prepare a defined glycan microarray and uses as an example glycans generated as phosphodiesters and phosphomonoesters of high-mannose type N-glycans. The method describes the preparation of high-mannose type glycan-AEAB conjugates (GAEABs), the purification of their phosphodiesters, and the subsequent mild acid hydrolysis to obtain corresponding phosphomonoesters. These GAEABs are covalently printed as a phosphorylated glycan microarray and used for analysis of the glycan ligand specificities of P-type lectins, such as the mannose-6-phosphate receptors (Man-6-P receptors or MPRs).
by
Anubama Rajan;
Matthew J. Robertson;
Hannah E. Carter;
Nina M. Poole;
Justin R. Clark;
Sabrina I. Green;
Zachary K. Criss;
Boyang Zhao;
Umesh Karandikar;
Yikun Xing;
Mar Margalef-Catala;
Nihil Jain;
Reid L. Wilson;
Fan Bai;
Joseph M. Hyser;
Joseph Petrosino;
Noah F. Shroyer;
Sarah E. Blutt;
Cristian Coarfa;
Xuezheng Song;
B.V. Venkataram Prasad;
Manuel R. Amieva;
Jane Grande-Allen;
Mary K. Estes;
Pablo C. Okhuysen;
Anthony W. Maresso
Enteroaggregative Escherichia coli (EAEC) is a significant cause of acute and chronic diarrhea, foodborne outbreaks, infections of the immunocompromised, and growth stunting in children in developing nations. There is no vaccine and resistance to antibiotics is rising. Unlike related E. coli pathotypes that are often associated with acute bouts of infection, EAEC is associated with persistent diarrhea and subclinical long-term colonization. Several secreted virulence factors have been associated with EAEC pathogenesis and linked to disease in humans, less certain are the molecular drivers of adherence to the intestinal mucosa. We previously established human intestinal enteroids (HIEs) as a model system to study host-EAEC interactions and aggregative adherence fimbriae A (AafA) as a major driver of EAEC adherence to HIEs. Here, we report a large-scale assessment of the host response to EAEC adherence from all four segments of the intestine across at least three donor lines for five E. coli pathotypes. The data demonstrate that the host response in the duodenum is driven largely by the infecting pathotype, whereas the response in the colon diverges in a patient-specific manner. Major pathways altered in gene expression in each of the four enteroid segments differed dramatically, with responses observed for inflammation, apoptosis and an overwhelming response to different mucin genes. In particular, EAEC both associated with large mucus droplets and specific mucins at the epithelial surface, binding that was ameliorated when mucins were removed, a process dependent on AafA. Pan-screening for glycans for binding to purified AafA identified the human ligand as heparan sulfate proteoglycans (HSPGs). Removal of HSPG abrogated EAEC association with HIEs. These results may mean that the human intestine responds remarkably different to distinct pathobionts that is dependent on the both the individual and intestinal segment in question, and uncover a major role for surface heparan sulfate proteoglycans as tropism-driving factor in adherence and/or colonization.
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 glycopeptides, 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.
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Ralph N. D. Luetscher;
Tanya R. McKitrick;
Chao Gao;
Akul Y. Mehta;
Alyssa M. McQuillan;
Robert Kardish;
Kayluz Frias Boligan;
Xuezheng Song;
Lenette Lu;
Jamie Heimburg-Molinaro;
Stephan von Gunten;
Galit Alter;
Richard Cummings
Humoral immunity to pathogens and other environmental challenges is paramount to maintain normal health, and individuals lacking or unable to make antibodies are at risk. Recent studies indicate that many human protective antibodies are against carbohydrate antigens; however, little is known about repertoires and individual variation of anti-carbohydrate antibodies in healthy individuals. Here we analyzed anti-carbohydrate antibody repertoires (ACARs) of 105 healthy individual adult donors, aged 20–60+ from different ethnic backgrounds to explore variations in antibodies, as defined by binding to glycan microarrays and by affinity purification. Using microarrays that contained > 1,000 glycans, including antigens from animal cells and microbes, we profiled the IgG and IgM ACARs from all donors. Each donor expressed many ACAs, but had a relatively unique ACAR, which included unanticipated antibodies to carbohydrate antigens not well studied, such as chitin oligosaccharides, Forssman-related antigens, globo-type antigens, and bacterial glycans. We also saw some expected antibodies to ABO(H) blood group and α-Gal-type antigens, although these also varied among individuals. Analysis suggests differences in ACARs are associated with ethnicity and age. Thus, each individual ACAR is relatively unique, suggesting that individualized information could be useful in precision medicine for predicting and monitoring immune health and resistance to disease.
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 retagged 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.
Sensitive glycomics analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is of great importance but significantly hampered by their low ionization efficiency and labile sialic acid moieties. Chemical derivatization offers a viable way to improve both the ionization efficiency and analytical sensitivity of the glycans in MS analysis by altering their hydrophobicity or charge property. Here we employed Girard's reagent T (GT) for on-target derivatization (GTOD) of reducing glycan under mild acid condition to form stable hydrazones at room temperature, allowing rapid and sensitive identification of neutral and sialylated glycans in positive-ion mode as only permanently positive charged molecular ions without multiple ion adducts by MALDI-TOF-MS. The MS signal intensities of lactose, sialylated N-glycans derived from bovine fetuin and neutral N-glycans derived from RNaseB and ovalbumin were boosted by 7.44, 9.13, 12.96 and 13.47 folds on average (n = 3), respectively. More importantly, after GTOD strategy, unwanted desialylation of sialylated glycans during MS was suppressed. The detection limit of the assay is desirable since the nanogram of N-glycans derived from 0.16 μg ovalbumin could be detected. The assay demonstrated good stability (RSD≤2.95%, within 10 days), reliable reproducibility (RSD = 2.96%, n = 7) and a desirable linear dynamic range from 78 nmol/mL to 10 μmol/mL. The strategy has been successfully applied to MS analysis of reducing glycans from human milks, neutral and sialylated O-, N-glycans from glycoproteins, and reducing glycans derived from glycosphingolipids, presenting neater [M] + signals which allow detection of more low-abundance glycans and assignation of Neu5Ac vs. Neu5Gc or fucose vs. hexose in glycans due to the absence of the ambiguous interpretation from multiple peaks (ion adducts [M+Na] + and [M+K] + ). Moreover, the GTOD assay prevents desialylation during MALDI-TOF-MS profiling and enables distinct linkage-specific characterization of terminal sialic acids of N-glycans derived from human serum protein when combines with an esterification.
The advancement of glycoscience is critically dependent on the access to a large number of glycans for their functional study. Naturally occurring glycans are considered a viable source for diverse and biologically relevant glycan libraries. A mixture of free reducing glycans released from natural sources can be fluorescently tagged and separated by chromatography to produce a natural glycan library. Anthranilic acid (AA) has been widely used to fluorescently tag reducing glycans for HPLC or LC/MS analysis. However, AA conjugated glycans are not efficiently immobilized on microarray slides due to the lack of a primary alkylamine functional group. In this study, we have developed simple and efficient chemistry for bioconjugation and further functionalization of glycan-AA conjugates. This new approach enables quick preparation of glycan microarrays and neoglycoproteins from glycan-AA conjugates, which can be separated by weak anion exchange (WAX) and C18 reversed-phase HPLC.
Purpose: Thyroid cancer recurrence following curative thyroidectomy is associated with increased morbidity and mortality, but current surveillance strategies are inadequate for early detection. Prior studies indicate that tissue glycosylation is altered in thyroid cancer, but the utility of serum glycosylation in thyroid cancer surveillance remains unexplored. We therefore assessed the potential utility of altered serum glycomic profile as a tumor-specific target for disease surveillance in recurrent thyroid cancer. Experimental design: We employed banked serum samples from patients with recurrent thyroid cancer post thyroidectomy and healthy controls. N-glycans were enzymatically released from serum glycoproteins, labeled via permethylation, and analyzed by MALDI-TOF mass spectrometry. Global level and specific subtypes of glycan structures were compared between patients and controls. Results: We evaluated 28 independent samples from 13 patients with cancer recurrence and 15 healthy controls. Global features of glycosylation, including N-glycan class and terminal glycan modifications were similar between groups, but three of 35 individual glycans showed significant differences. The three glycans were biosynthetically related biantennary core fucosylated N-glycans that only varied by the degree of galactosylation (G0F, G1F, and G2F; G: galactose, F: fucose). The ratio of G0F:G1F that captures reduced galactosylation was observed in patients samples but not in healthy controls (p = 0.004) and predicted thyroid cancer recurrence (AUC = 0.82, CI 95% = 0.64–0.99). Conclusions: Altered N-glycomic profile was associated with thyroid cancer recurrence. This serum-based biomarker would be useful as an effective surveillance tool to improve the care and prognosis of thyroid cancer after prospective validation.
To examine the range of selective processes that potentially operate when poorly binding influenza viruses adapt to replicate more efficiently in alternative environments, we passaged a virus containing an attenuating mutation in the hemagglutinin (HA) receptor binding site in mice and characterized the resulting mutants with respect to the structural locations of mutations selected, the replication phenotypes of the viruses, and their binding properties on glycan microarrays. The initial attenuated virus had a tyrosine-to-phenylalanine mutation at HA1 position 98 (Y98F), located in the receptor binding pocket, but viruses that were selected contained second-site pseudoreversion mutations in various structural locations that revealed a range of molecular mechanisms for modulating receptor binding that go beyond the scope that is generally mapped using receptor specificity mutants. A comparison of virus titers in the mouse respiratory tract versus MDCK cells in culture showed that the mutants displayed distinctive replication properties depending on the system, but all were less attenuated in mice than the Y98F virus. An analysis of receptor binding properties confirmed that the initial Y98F virus bound poorly to several different species of erythrocytes, while all mutants reacquired various degrees of hemagglutination activity. Interestingly, both the Y98F virus and pseudoreversion mutants were shown to bind very inefficiently to standard glycan microarrays containing an abundance of binding substrates for most influenza viruses that have been characterized to date, provided by the Consortium for Functional Glycomics. The viruses were also examined on a recently developed microarray containing glycans terminating in sialic acid derivatives, and limited binding to a potentially interesting subset of glycans was revealed. The results are discussed with respect to mechanisms for HA-mediated receptor binding, as well as regarding the species of molecules that may act as receptors for influenza virus on host cell surfaces.
Protein O-glycosylation has key roles in many biological processes, but the repertoire of O-glycans synthesized by cells is difficult to determine. Here we describe an approach termed Cellular O-Glycome Reporter/Amplification (CORA), a sensitive method used to amplify and profile mucin-type O-glycans synthesized by living cells. Cells convert added peracetylated benzyl-α-N-acetylgalactosamine to a large variety of modified O-glycan derivatives that are secreted from cells, allowing for easy purification for analysis by HPLC and mass spectrometry (MS). Relative to conventional O-glycan analyses, CORA resulted in an ∼100-1,000-fold increase in sensitivity and identified a more complex repertoire of O-glycans in more than a dozen cell types from Homo sapiens and Mus musculus. Furthermore, when coupled with computational modeling, CORA can be used for predictions about the diversity of the human O-glycome and offers new opportunities to identify novel glycan biomarkers for human diseases.