African trypanosomiasis is a parasitic disease affecting large areas of the African continent. It presents a threat to man and drastically impedes effective cattle-rearing. The causative agents of this infectious disease are members of the genus Trypanosoma. The parasite evades the mammalian host's immunological response to its presence in body fluids by antigenic variation: the expression of a sequence of immunologically distinct glycoprotein coats over the entire surface of the cells of the invading organism. These variant surface glycoproteins (VSGs) have one or more N-linked glycans and are anchored in the trypanosome membrane by a glycosyl-phosphatidyl-inositol(GPI) moiety. Of the three classes of VSGs (distinguished on the basis of the C-terminal amino acid), the covalent structures of the GPI anchors and N-glycans of only two VSGs, both of class I, of only one trypanosome species (T. b. brucei) have been fully elucidated. The primary aim of the proposed research is to structurally characterize the GPI anchors and N-glycans of a variety of trypanosome VSGs to determine whether such oligosaccharides are significantly different to those of mammalian glycoproteins. Preliminary data indicate that the GPI anchors and the N-glycans of trypanosomes are different from those of the mammalian host. The GPI anchors in particular are considered prime potential targets for chemotherapeutic treatment of trypanosomiasis. We propose to characterize the carbohydrate structures of the VSGs of six trypanosome variant antigen types, and compare them with those of mammalian glycoproteins. In particular we propose to: 1. Isolate the VSGs and their carbohydrate moieties from T. b. rhodesiense LouTat 1 and 1A (both class I), and from T. b. rhodesiense LouTat 1.5 and T. congolense YNat 1.1 and 1.3 (all class II), grown in rodents and/or in vitro. Obtain a class III VSG and the GPI anchor from a mammalian glycoprotein (human erythrocyte acetylcholinesterase, AChE) from collaborators. 2. Characterize the structures of the VSG GPI anchors and compare them with each other. Look for host influence on VSG GPI glycosylation. Compare the structures to the structure of the GPI anchor of AChE. 3. Structurally characterize the N-glycans from the VSGs and investigate the presence of unusual structural elements. Look for host influence on VSG N-glycosylation. The long-term aim is to find a target for the development of chemotherapeutic agents. The proposed studies should provide the foundation necessary for further research leading to the development of new reagents for the treatment of African trypanosomiasis.
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