Synthesis of Galα(1,3)Galβ(1,4)GlcNAcα-, Galβ(1,4)GlcNAcα- and GlcNAc-containing neoglycoproteins and their immunological evaluation in the context of Chagas disease

Abstract

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.

Enhancing glycan isomer separations with metal ions and positive and negative polarity ion mobility spectrometry-mass spectrometry analyses

“…Understanding these O -glycan structures is of importance for the identification of potential T. cruzi α-Gal-containing biomarkers for the diagnosis of Chagas disease, follow-up of chemotherapy, and the development of preventative and therapeutic vaccines for Chagas disease. The synthetic glycans analyzed were mercaptopropyl glycosides, where mercaptopropyl groups were installed to allow conjugation of these glycans to maleimide-activated bovine serum albumin needed for the generation of a glycan array [36, 37]. …”

“…The O -glycans α- d -Gal-(1 → 3)-β- d -Gal-(1 → 4)-β- d -Glc-(CH 2 ) 3 SH (1)[36], α- d -Gal-(1 → 6)-[α- d -Gal-(1 → 2)]-β- d -Gal-(CH 2 ) 3 SH (2) [36], α- d -Gal-(1 → 3)-[α- d -Gal-(1 → 2)]-β- d -Gal-(CH 2 ) 3 SH (3) [Schocker, N.S., Almeida, I.C., Michael, K., unpublished data], β- d -Gal f -(1 → 4)- [β- d -Gal-(1 →6)]-α- d -GlcNAc-(CH 2 ) 3 SH (4) [Schocker, N.S., 2016, Ph.D. Dissertation, University of Texas at El Paso], α- d -Gal-(1 → 3)-β- d -Gal-(1 → 4)-α- d -GlcNAc-(CH 2 ) 3 SH (5)[37], α- l -Rha-(1 → 2)-α- l -Fuc-(CH 2 ) 3 SH (6), and α- l -Rha-(1 → 3)-α- l -Fuc-(CH 2 ) 3 SH (7) [Khamsi, J., Michael, K., unpublished data] were synthesized in the Michael lab. The glycans were dissolved in a MeOH/H 2 O/formic acid solution (49.45:49.45:0.1, v / v / v ) to a final concentration of ∼5 μM or lower.…”

“…Here we use an IMS-QTOF-MS platform to characterize standard glycans in both positive and negative polarities and also explore the effects of metal ions on enhancing glycan isomer separations. We next applied the optimized IMS-MS method to study synthetic α-Gal-containing O -glycans, some of which contain an immunodominant T. cruzi glycotope that is a potential biomarker for the diagnosis of Chagas disease [12, 36, 37]. …”

The design and synthesis of an α-Gal trisaccharide epitope that provides a highly specific anti-Gal immune response

“…20,21 We note that synthetic methods for the preparation of α-Gal trisaccharides have been reported, however, these involved a need to control selectivity during the glycosylation step. 22,23 We devised a strategy utilizing a chemoenzymatic reaction mediated by α(1,3)galactosyltransferase (α1,3GalT) for incorporating the terminal galactose to disaccharides, which resulted in the facile preparation of our targeted α-Gal epitopes. 24 …”

The Trypanosoma cruzi Surface, a Nanoscale Patchwork Quilt

“…Notably, and at stark variance with insect-dwelling stages mucins, certain βGal p units at the non-reducing end of tGPI-mucins may be modified with αGal p residues within the secretory pathway, while en route to the parasite surface [30]. These α-galactosylations provide further diversification to tGPI-mucins and lead to the eventual display on the parasite surface of the αGal glycotope [31–33]. Importantly, intracellular α-galactosylation of tGPI-mucins ‘titter out’ terminal βGal p units, and thus putative SA acceptors on the parasite surface.…”