Date of Award

2016-01-01

Degree Name

Doctor of Philosophy

Department

Chemistry

Advisor(s)

Katja Michael

Abstract

The protozoan parasite, Trypanosoma cruzi, the etiologic agent of Chagas disease (ChD), has a cell surface covered by immunogenic glycoconjugates. A synthetic glycoarray containing non-reducing α-galactopyranosyl moieties related to mucin O-glycans of the parasite T. cruzi was evaluated by a chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) with sera from patients with chronic ChD, revealing the disaccharide Galα(1,3)Galβ as the immunodominant glycotope (Chapter 2). This disaccharide comprises the two terminal sugars of the known immunodominant glycotope Galα(1,3)Galβ(1,4)GlcNAcα, which 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. The mercaptopropyl glycoside of that glycotope was synthesized and conjugated to bovine serum albumin (BSA). CL-ELISA revealed that Galα(1,3)Galβ(1,4)GlcNAcα-BSA is strongly recognized by both purified anti-α-Gal Abs and pooled sera from chronic ChD patients, but not significantly by anti-α-Gal Abs nor normal sera of healthy individuals. The underlying disaccharide Galβ(1,4)GlcNAcα, and monosaccharides GlcNAcα or GlcNAcβ are poorly or not recognized by purified anti-α-Gal Abs or sera from Chagasic patients or healthy individuals. The Galα(1,3)Galβ(1,4)GlcNAcα-BSA NGP was used to immunize α1,3-galactosyltransferase-knockout mice, which produced antibody titers 40-fold higher as compared to pre-immunization titers. These 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 (Chapter 3).The glycoprotein gp72 is expressed in all life cycle stages of T. cruzi, and contains the unusual saccharide α-L-Rhamnose (Rhaα) both terminally and internally linked to Fucose. Rhaα-containing mono-, di-, and trisaccharides were synthesized and compared to saccharides where the Rhaα is replaced with Galα, both to investigate Chagasic antibody binding to Rhaα-containing NGPs, as well as Rhaα’s known ability to mimic Galα in some circumstances. Strong binding was found to all Rhaα-containing NGPs from both ChHSP and NHS, indicating anti-Rha antibody cross-reactivity. While these results do not represent a useful tool toward diagnosis of ChD, it highlights an abundance of anti-Rha Abs in the serum of Chagasic patients, particularly toward Rhaα(1,3)Galβ(1,4)Glcβ, as well as an abundance of anti-Rha Abs in normal human sera, particularly toward Rhaα(1,4)Galβ (Chapter 4). Lastly, a series of neoglycopeptides (NGPeps) were synthesized, consisting of a major histocompatibility complex class II (MHCII)-binding peptide, either tetanus toxoid peptide (TTp) or ovalbumin peptide (OVAp), covalently linked to the Galα(1,3)Galβ(1,4)GlcNAcα glycotope. This was accomplished by pre-forming a glycosyl amino acid, which was incorporated by solid-phase peptide synthesis (SPPS) at either the N-terminal or C-terminal of the peptide chain, for structural comparisons. These synthetic NGPeps were then used for the immunization of α1,3GalT-KO mice and are awaiting the examination of B and T cell-mediated immune responses (Chapter 5).

Language

en

Provenance

Received from ProQuest

File Size

252 pages

File Format

application/pdf

Rights Holder

Nathaniel Scott Schocker

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