Trypanosoma cruzi is the agent of Chagas' disease, which causes a progressive chronic fibrotic myocarditis and degeneration of tissues. The disease is endemic in South America, and has no known cure at present. Recently, a developmentally regulated enzyme was discovered attached to the parasite's surface which transfers sialic acid from host cells to parasite surface glycoconjugates. This trans-sialidase is highly specific for both its donor and acceptor substrates, and differs markedly in this respect from other sialidases which only transfer sialic acid to water. There is mounting evidence that this enzyme is necessary for successful invasion of the mammalian host cell, but it may also play a role in protecting the parasite from lysis by the alternative pathway of the complement system. The uniqueness of the enzyme, its substrate specificity and its high activity at crucial stages in infection make it an ideal target for rational drug design. A prerequisite for such an approach is to determine the atomic structure of the enzyme by X-ray crystallography, and this forms the basis of this application. The objectives are: 1. Crystallization of the active trypomastigote stage T. cruzi trans- sialidase and the shorter epimastigote stage form of the enzyme. 2. X-ray structure determination of these proteins. 3. X-ray structure analysis of inhibitor/substrate complexes, to derive the key residues involved in catalysis and/or cell recognition. 4. Detailed comparative structural analysis with other sialidses, to understand; (i) How the enzyme can act as a trans-sialidase, (ii) Why there is a strict donor/acceptor substrate specificity for sialic acid linked alpha(2-3) to a beta-galactose, (iii) Why the enzyme is more efficient in transferring than in hydrolysing sialic acid. 5. Analysis of the fibronectin-like domain, to understand its relation to the sialidase domain and its function. Similarly, if the whole molecule crystallizes, the same questions will be addressed to the role of the 12 amino-acid tandem repeat. 6. Provide a detailed molecular basis for the rational design and synthesis of inhibitors, by Dr. Mark von Itzstein, Melbourne, and X-ray structure analysis of these synthesized inhibitor complexes.
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