No abstract

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.

No abstract