Trypanosoma cruzi, a Stercorarian trypanosome, causes Chagas' disease, a chronic illness affecting over 20 million people in Central and South America. This obligate intracellular parasite has a complex life cycle, with multiple developmental stages in the insect vector and the mammalian host. The immune system of the host mounts a strong response to the organisms, but sterile immunity is rarely if ever achieved, as the parasite lies protected in the cytoplasm of host cells. A 72 kDa glycoprotein, GP72, is an important developmentally regulated surface antigen of T. cruzi, apparently expressed only in the insect stages (epimastigotes and metacyclic trypomastigotes). Because the antisera that have been used to define GP72 recognize carbohydrate epitopes, the question of whether the gene is expressed in other stages, but differentially posttranslationally modified, has not been resolved.
The aim of this proposal is to study the structure and function of developmentally regulated surface glycoproteins, with special emphasis on the structural characterization of GP72, its gene (or genes, since all characterized T. cruzi surface antigens appear to be encoded by gene families), and the nature of its unusual posttranslational modifications. There are several reasons for focusing on GP72: it is still the best defined of all the described T. cruzi surface antigens; a high-affinity monoclonal antibody (WIC 29.26) is available for its purification; it is developmentally regulated; it has highly unusual, if not unique, posttranslational glycan modifications, which are probably involved in some of its biological functions; it has been associated with several biological functions of the parasite; it is a major acceptor for C3, it is very immunogenic, and it has been shown to be protective upon immunization of mice. GP72 has been purified, and partial amino acid sequence data obtained. This information will be used to clone a cDNA for the protein. The organization of gene(s) encoding GP72 will be studied. We anticipate that multiple genes will be present, so we will determine which of these are expressed, at what stage in the life cycle, and whether the expressed copy varies in an isolate, clone-specific, or stage-specific fashion. If the expressed copy varies, we will determine whether there are significant structural and therefore potential functional differences between the gene products. We will explore the nature and location of the attached glycans. Using the cloned gene and antibodies raised against the corresponding protein, we will study the kinetics of biosynthesis and posttranslational modification of GP72. Ultimately, we hope to investigate the immunoprophylactic potential of recombinant proteins, and synthetic glycopeptides.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI026197-02
Application #
3139920
Study Section
Tropical Medicine and Parasitology Study Section (TMP)
Project Start
1990-09-30
Project End
1993-08-31
Budget Start
1991-09-01
Budget End
1992-08-31
Support Year
2
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Rockefeller University
Department
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
Wirtz, E; Leal, S; Ochatt, C et al. (1999) A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol Biochem Parasitol 99:89-101
Ochatt, C M; Butikofer, P; Navarro, M et al. (1999) Conditional expression of glycosylphosphatidylinositol phospholipase C in Trypanosoma brucei. Mol Biochem Parasitol 103:35-48
Engstler, M; Wirtz, E; Cross, G A (1997) Generation of constitutive and inducible trans-sialylation dominant-negative phenotypes in Trypanosoma brucei and Trypanosoma cruzi. Glycobiology 7:955-64
Haynes, P A; Cross, G A (1996) Differential glycosylation of epitope-tagged glycoprotein Gp72 during the Trypanosoma cruzi life cycle. Mol Biochem Parasitol 83:253-6
Haynes, P A; Russell, D G; Cross, G A (1996) Subcellular localization of Trypanosoma cruzi glycoprotein Gp72. J Cell Sci 109 ( Pt 13):2979-88
Haynes, P A; Ferguson, M A; Cross, G A (1996) Structural characterization of novel oligosaccharides of cell-surface glycoproteins of Trypanosoma cruzi. Glycobiology 6:869-78
Nozaki, T; Cross, G A (1995) Effects of 3' untranslated and intergenic regions on gene expression in Trypanosoma cruzi. Mol Biochem Parasitol 75:55-67
Nozaki, T; Cross, G A (1994) Functional complementation of glycoprotein 72 in a Trypanosoma cruzi glycoprotein 72 null mutant. Mol Biochem Parasitol 67:91-102
Inverso, J A; Medina-Acosta, E; O'Connor, J et al. (1993) Crithidia fasciculata contains a transcribed leishmanial surface proteinase (gp63) gene homologue. Mol Biochem Parasitol 57:47-54
de Jesus, A R; Cooper, R; Espinosa, M et al. (1993) Gene deletion suggests a role for Trypanosoma cruzi surface glycoprotein GP72 in the insect and mammalian stages of the life cycle. J Cell Sci 106 ( Pt 4):1023-33

Showing the most recent 10 out of 12 publications