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

Glycobiology, 2016, vol. 26, no. 1, 39–50 doi: 10.1093/glycob/cwv081 Advance Access Publication Date: 18 September 2015 Original Article Glycan Synthesis 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 Nathaniel S Schocker2, Susana Portillo3, Carlos R N Brito3,4, Alexandre F Marques4, Igor C Almeida3, and Katja Michael1,2 2 Department of Chemistry, 3Department of Biological Sciences, Border Biomedical Research Center, University of Texas at El Paso, 500 W. University Ave., El Paso, TX 79968, USA, and 4Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG 31270-901, Brazil 1 To whom correspondence should be addressed: Tel: +1-915-747-5240; e-mail: Received 29 June 2015; Revised 9 September 2015; Accepted 9 September 2015 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. Key words: biomarkers, carbohydrates, Chagas disease, immunization, neoglycoprotein © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: 39 40 Introduction The surface of the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (ChD), is heavily coated by glycoproteins containing highly immunogenic glycans (Travassos and Almeida 1993; Acosta-Serrano et al. 2007). An immunodominant glycotope, Galα(1,3)Galβ(1,4)GlcNAcα, is abundantly expressed in the mammaldwelling T. cruzi trypomastigote stage (Almeida et al. 1994) and is not expressed on human cells, thus it is highly immunogenic to humans (Travassos and Almeida 1993; Macher and Galili 2008). The Galα(1,3)Galβ(1,4)GlcNAcα epitope contains a terminal, nonreducing αGal residue, which is highly conserved on trypomastigotederived GPI-mucins (tGPI-mucins) of at least four major T. cruzi genotypes causing ChD in humans: TcI, TcII, TcV and TcVI (Almeida et al. 1993; Travassos and Almeida 1993; Soares et al. 2012; Izquierdo et al. 2013). The Galα(1,3)Galβ(1,4)GlcNAcα glycotope contains the disaccharide Galα1,3Galβ, which is strongly recognized by Chagasic (Ch) anti-α-Gal Abs and to a much lesser extent by the natural anti-α-Gal Abs from healthy individuals (NHS anti-α-Gal) (Almeida et al. 1994; Ashmus et al. 2013), which are produced mainly against gram-negative enterobacteria of the human flora (Galili et al. 1999). These enterobacteria (e.g., E. coli, Enterobacter spp., Serratia spp., Salmonella spp., Shigella spp., Klebsiella spp. and Citrobacter spp.) have various types of non-reducing, terminal α-Gal-linked glycans, mostly Galα1,2-R, Galα1,4-R and Galα1,6-R (where R is the remaining side chain or core glycan) on the lipopolysaccharide (LPS) core oligosaccharides or O-antigens (Wilkinson 1996). The glycotope Galα (1,3)Galβ(1,4)GlcNAcα, so far not reported in enterobacteria, and most likely other yet unidentified T. cruzi-specific cell surface saccharides with terminal αGal moieties, induce the major lytic, protective antibodies (Ch anti-α-Gal Abs) produced during both the acute and chronic stages of ChD (Milani and Travassos 1988; Avila et al. 1989; Almeida et al. 1991, 1994; Gazzinelli et al. 1991; Travassos and Almeida 1993). These studies strongly indicate that lytic Ch anti-α-Gal Abs could be one of the main immunological mechanisms for controlling the parasitemia in both stages of the disease in humans. Thus, Galα(1,3)Galβ(1,4)GlcNAcα offers a potential route toward a carbohydrate-based vaccine against ChD. Glycoconjugates are still unexplored as vaccine targets in T. cruzi, although these molecules are the most abundant and immunogenic antigens on the plasma membrane of all T. cruzi developmental stages (Frasch 2000; Buscaglia et al. 2004; Acosta-Serrano et al. 2007). N S Schocker et al. Here we describe the synthesis of glycosides of Galα(1,3)Galβ(1,4) GlcNAcα, and its truncated versions Galβ(1,4)GlcNAcα and GlcNAcα, as well as its diastereomer GlcNAcβ, all equipped with a thiol functionality (glycosides 1–4, Figure 1) for their conjugation to the carrier protein bovine serum albumin (BSA). All neoglycoproteins (NGPs) were immunologically evaluated by chemiluminescentenzyme-linked immunosorbent assay (CL-ELISA) (Almeida et al. 1997), using purified Ch anti-α-Gal Abs vs. NHS anti-α-Gal Abs, and Ch human serum pool (ChHSP) vs. normal human serum pool (NHSP). Lastly, the NGP Galα(1,3)Galβ(1,4)GlcNAcα-BSA was used to immunize α1,3-galactosyltransferase-knockout (α1,3-GalT-KO) mice, which do not express terminal αGal epitopes in their cells (Tearle et al. 1996; Thall et al. 1996). These animals are able to produce lytic anti-α-Gal Abs, mimicking therefore the human humoral immune response against T. cruzi (Almeida et al. unpublished data). The production of the trisaccharide Galα(1,3)Galβ(1,4)GlcNAcα and related analogs has been previously accomplished for a variety of uses, and mostly involves chemoenzymatic syntheses (Vic et al. 1997; Fang et al. 1998; Qian et al. 1999; Brin (...truncated)