Glycomics has become an increasingly important field of research since glycans play critical roles in biology processes ranging from molecular recognition and signaling to cellular communication. Glycans often conjugate with other biomolecules, such as proteins and lipids, and alter their properties and functions, so glycan characterization is essential for understanding the effects they have on cellular systems. However, the analysis of glycans is extremely difficult due to their complexity and structural diversity (i.e., the number and identity of monomer units, and configuration of their glycosidic linkages and connectivities). In this work, we coupled ion mobility spectrometry with mass spectrometry (IMS-MS) to characterize glycan standards and biologically important isomers of synthetic αGal-containing O-glycans including glycotopes of the protozoan parasite Trypanosoma cruzi, which is the causative agent of Chagas disease. IMS-MS results showed significant differences for the glycan structural isomers when analyzed in positive and negative polarity and complexed with different metal cations. These results suggest that specific metal ions or ion polarities could be used to target and baseline separate glycan isomers of interest with IMS-MS.

A synthetic glycoarray containing non-reducing α-galactopyranosyl moieties related to mucin O-glycans of the parasite Trypanosoma cruzi was evaluated by a chemiluminescent enzyme-linked immunosorbent assay with sera from patients with chronic Chagas disease. Our data revealed the disaccharide Galα(1,3)Galβ as the immunodominant glycotope, which may eventually be employed as a diagnostic antigen for Chagas disease.

The protozoan parasite, Trypanosoma cruzi, the etiologic agent of Chagas disease (ChD), has a cell surface covered by immunogenic glycoconjugates. One of the immunodominant glycotopes, the trisaccharide Galα(1,3)Galβ(1,4)GlcNAcα, is expressed on glycosylphosphatidylinositol-anchored mucins of the infective trypomastigote stage of T. cruzi and triggers high levels of protective anti-α-Gal antibodies (Abs) in infected individuals. Here, we have efficiently synthesized the mercaptopropyl glycoside of that glycotope and conjugated it to maleimide-derivatized bovine serum albumin (BSA). Chemiluminescent-enzyme-linked immunosorbent assay revealed that Galα(1,3)Galβ(1,4)GlcNAcα-BSA is recognized by purified anti-α-Gal Abs from chronic ChD patients ∼230-fold more strongly than by anti-α-Gal Abs from sera of healthy individuals (NHS anti-α-Gal). Similarly, the pooled sera of chronic Chagas disease patients (ChHSP) recognized Galα(1,3)Galβ(1,4)GlcNAcα ∼20-fold more strongly than pooled NHS. In contrast, the underlying disaccharide Galβ(1,4)GlcNAcα and the monosaccharide GlcNAcα or GlcNAcβ conjugated to BSA are poorly or not recognized by purified anti-α-Gal Abs or sera from Chagasic patients or healthy individuals. Our results highlight the importance of the terminal Galα moiety for recognition by Ch anti-α-Gal Abs and the lack of Abs against nonself Galβ(1,4)GlcNAcα and GlcNAcα glycotopes. The substantial difference in binding of Ch vs. NHS anti-α-Gal Abs to Galα(1,3)Galβ(1,4)GlcNAcα-BSA suggests that this neoglycoprotein (NGP) might be suitable for experimental vaccination. To this end, the Galα(1,3)Galβ(1,4)GlcNAcα-BSA NGP was then used to immunize α1,3-galactosyltransferase-knockout mice, which produced antibody titers 40-fold higher as compared with pre-immunization titers. Taken together, our results indicate that the synthetic Galα(1,3)Galβ(1,4)GlcNAcα glycotope coupled to a carrier protein could be a potential diagnostic and vaccine candidate for ChD.

BackgroundProtozoan parasites from the genus Leishmania cause broad clinical manifestations known as leishmaniases, which affect millions of people worldwide. Cutaneous leishmaniasis (CL), caused by L. major, is one the most common forms of the disease in the Old World. There is no preventive or therapeutic human vaccine available for L. major CL, and existing drug treatments are expensive, have toxic side effects, and resistant parasite strains have been reported. Hence, further therapeutic interventions against the disease are necessary. Terminal, non-reducing, and linear α-galactopyranosyl (α-Gal) epitopes are abundantly found on the plasma membrane glycolipids of L. major known as glycoinositolphospholipids. The absence of these α-Gal epitopes in human cells makes these glycans highly immunogenic and thus potential targets for vaccine development against CL.Methodology/Principal findingsHere, we evaluated three neoglycoproteins (NGPs), containing synthetic α-Gal epitopes covalently attached to bovine serum albumin (BSA), as vaccine candidates against L. major, using α1,3-galactosyltransferase-knockout (α1,3GalT-KO) mice. These transgenic mice, similarly to humans, do not express nonreducing, linear α-Gal epitopes in their cells and are, therefore, capable of producing high levels of anti-α-Gal antibodies. We observed that Galα(1,6)Galβ-BSA (NGP5B), but not Galα(1,4)Galβ-BSA (NGP12B) or Galα(1,3)Galα-BSA (NGP17B), was able to significantly reduce the size of footpad lesions by 96% in comparison to control groups. Furthermore, we observed a robust humoral and cellular immune response with production of high levels of protective lytic anti-α-Gal antibodies and induction of Th1 cytokines.Conclusions/SignificanceWe propose that NGP5B is an attractive candidate for the study of potential synthetic α-Gal-neoglycoprotein-based vaccines against L. major infection.

Bioinformatic tools and databases for glycobiology and glycomics research are playing increasingly important roles in functional studies. However, to verify hypotheses generated by computational glycomics with empirical functional assays is only an emerging field. In this study, we predicted glycan epitopes expressed by a cancer-derived mucin, MUC1, by computational glycomics. MUC1 is expressed by tumor cells with a deficiency in glycosylation. Although numerous diagnostic reagents and cancer vaccines have been designed based on abnormally glycosylated MUC1 sequences, the glycan and peptide sequences responsible for immune responses in vivo are poorly understood. The immunogenicity of synthetic MUC1 glycopeptides bearing Tn or sialyl-Tn antigens have been studied in mouse models, while authentic glyco-epitopes expressed by tumor cells remain unclear. To examine the immunogenicity of authentic cancer derived MUC1 glyco-epitopes, we expressed membrane bound forms of MUC1 tandem repeats in Jurkat, a mutant cancer cell line deficient of mucin-type core-1 β1–3 galactosyltransferase activity, and immunized mice with cancer cells expressing authentic MUC1 glyco-epitopes. Antibody responses to individual glyco-epitopes were determined by chemically synthesized candidate MUC1 glycopeptides predicted through computational glycomics. Monoclonal antibodies can be generated toward chemically synthesized glycopeptide sequences. With RPAPGS(Tn)TAPPAHG as an example, a monoclonal antibody 16A, showed 25-fold higher binding to glycosylated peptide (EC50=9.278±1.059 ng/ml) compared to its non-glycosylated form (EC50=247.3±16.29 ng/ml) as measured by ELISA experiments with plate-bound peptides. A library of monoclonal antibodies toward authentic MUC1 glycopeptide epitopes may be a valuable tool for studying glycan and peptide sequences in cancer, as well as reagents for diagnosis and therapy.

Chagas disease (ChD), caused by the protozoan parasite Trypanosoma cruzi, affects millions of people worldwide. Chemotherapy is restricted to two drugs, which are partially effective and may cause severe side effects, leading to cessation of treatment in a significant number of patients. Currently, there are no biomarkers to assess therapeutic efficacy of these drugs in the chronic stage. Moreover, no preventive or therapeutic vaccines are available. In this chapter, we describe the purification of Trypanosoma cruzi trypomastigote-derived glycosylphosphatidylinositol (GPI)-anchored mucins (tGPI-mucins) for their use as antigens for the reliable primary or confirmatory diagnosis and as prognostic biomarkers for early assessment of cure following ChD chemotherapy. We also describe, as an example, the synthesis of a potential tGPI-mucin-derived α-Gal-terminating glycan and its coupling to a carrier protein for use as diagnostic and prognostic biomarker in ChD.

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Outbreaks of Old World cutaneous leishmaniasis (CL) have significantly increased due to the conflicts in the Middle East, with most of the cases occurring in resource-limited areas such as refugee settlements. The standard methods of diagnosis include microscopy and parasite culture, which have several limitations. To address the growing need for a CL diagnostic that can be field applicable, we have identified five candidate neoglycoproteins (NGPs): Galα (NGP3B), Galα(1,3)Galα (NGP17B), Galα(1,3)Galβ (NGP9B), Galα(1,6)[Galα(1,2)]Galβ (NGP11B), and Galα(1,3)Galβ(1,4)Glcβ (NGP1B) that are differentially recognized in sera from individuals with Leishmania major infection as compared with sera from heterologous controls. These candidates contain terminal, non-reducing α-galactopyranosyl (α-Gal) residues, which are known potent immunogens to humans. Logistic regression models found that NGP3B retained the best diagnostic potential (area under the curve from receiver-operating characteristic curve = 0.8). Our data add to the growing body of work demonstrating the exploitability of the human anti-α-Gal response in CL diagnosis.