The long term goal of this proposal is to elucidate the molecular structures on mammalian cells that function as receptors for Trypanosoma cruzi. T. cruzi is the causative agent of Chagas' disease, which affects millions of people causing cardiac arrest, frequently accompanied by death. Despite significant advances in this area very little is known, if any, about host cell receptors for T. cruzi. We have found that the purified surface 74 kDa glycoprotein from heart myoblasts and antibodies to this molecule inhibit T. cruzi trypomastigote binding and internalization into heart myoblasts. This glycoprotein binds to invasive trypomastigote but not to non-invasive epimastigote forms of T. cruzi and is only expressed on cells that can be invaded by trypomastigotes. The recombinant forms of T. cruzi gp83 surface trans-sialidase, which binds to heart myoblasts with a Kd of 1x10-4 muM to mediate trypanosome binding to myoblasts, recognizes this 74 kDa glycoprotein. Binding of trypomastigotes to heart myoblasts and other cells is abolished by mAb 4A4 which recognizes an epitope on the gp83 trans-sialidase or its recombinant form and is required for trypanosome binding to host cells. This monoclonal antibody also abolishes the binding of the 4-gp83 to the 74 kDa glycoprotein or the binding of soluble gp83 trans-sialidase to myoblasts. In view of these findings, we have hypothesized that the host 74 kDa glycoprotein may function as a receptor for T. cruzi to mediate trypanosome binding to facilitate entry. In this proposal we will test this hypothesis and we will investigate the molecular structure of this receptor. To this end, we proposed the following specific aims: a) to identify clones expressing the 74 kDa protein from a cDNA library of heart cells, sequence the full length cDNA encoding the 74 kDa protein and predict its amino acid sequence, b) to express and purify the recombinant 74 kDa glycoprotein for ligand binding studies to trypanosomes, and to assess the ability of the recombinant molecule and its antibodies to inhibit T. cruzi infection, c) to test the ability of the 74 kDa glycoprotein to function as a receptor for T. cruzi by transfecting mammalian cell lines which cannot be invaded by T. cruzi with cDNA coding for the 74 kDa protein, d) to determine the region on the T. cruzi gp83 ligand that binds to the 74 kDa glycoprotein This proposal will generate new information and insights about the molecular structure of a membrane protein of cells that may function as a receptor for invasive forms of T. cruzi. These studies will contributes to establish the molecular basis on T. cruzi recognition by host cells to promote trypanosome entry, which may be important for molecular intervention in yet incurable disease.
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