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Volume 271, Number 28, Issue of July 12, 1996 pp. 16877-16887
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Identification of Complete Precursors for the Glycosylphosphatidylinositol Protein Anchors of Trypanosoma cruzi

(Received for publication, November 3, 1995, and in revised form, April 10, 1996)

Norton Heise , Jayne Raper ^¶ , Laurence U. Buxbaum ^¶ , Tereza M. S. Peranovich and Maria Lucia Cardoso de Almeida

From the  Department of Microbiology, Immunology and Parasitology, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Botucatu 862, 8° andar, São Paulo-SP, CEP 04023-062, Brazil and the ^¶ Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

The survival of Trypanosoma cruzi, the causative agent of Chagas' disease, depends vitally on proteins and glycoconjugates that mediate the parasite/host interaction. Since most of these molecules are attached to the membrane by glycosylphosphatidylinositol (GPI), alternative means of chemotherapeutic intervention might emerge from GPI biosynthesis studies. The structure of the major 1G7 antigen GPI has been fully characterized by us (Güther, M. L. S., Cardoso de Almeida, M. L., Yoshida, N., and Ferguson, M. A. J. (1992) J. Biol. Chem. 267, 6820-6828; Heise, N., Cardoso de Almeida, M. L., and Ferguson, M. A. J. (1995) Mol. Biochem. Parasitol. 70, 71-84), and based on its properties we now report the complete precursor glycolipids predicted to be transferred to the nascent protein. Migrating closely to Trypanosoma brucei glycolipid A on TLC, such species, named glycolipids A-like 1 and A-like 2, were labeled with tritiated palmitic acid, myo-inositol, glucosamine, and mannose, but surprisingly only the less polar glycolipid A-like 1 incorporated ethanolamine. The predicted products following nitrous acid deamination and digestion with phospholipases A₂, C, and D confirmed their GPI nature. Evidence that they may represent the anchor transferred to the 1G7 antigen came from the following analyses: (i) -mannosidase treatments indicated that only one mannose was amenable to removal; (ii) their lipid moiety was identified as sn-1-alkyl-2-acylglycerol due to their sensitivity to phospholipase A₂ (PLA₂), mild base and by direct high performance TLC analysis of the corresponding benzoylated diradylglycerol components; and (iii) both glycolipids incorporated ³H-fatty acid only in the sn-2- and not in the sn-1-alkyl position as previously found in the GPI of the mature 1G7 antigen. Based on the differential [³H]ethanolamine incorporation pattern and the recent report that an aminoethylphosphonic acid (AEP) replaces ethanolamine phosphate (EtNH₂-PO₄) in the GPI in epimastigote sialoglycoproteins (Previato, J. O., Jones, C., Xavier, M. T., Wait, R., Travassos, L. R., Parodi, A. J., and Mendonça-Previato, L. (1995) J. Biol. Chem. 270, 7241-7250) it is proposed that glycolipid A-like 2 contains AEP and A-like 1 EtNH₂-PO₄. In the in vitro cell-free system both glycolipids were synthesized simultaneously and do not seem to bear a precursor/product relationship. Among the various components synthesized in vitro a glycolipid C-like corresponding to a form of glycolipid A-like 1 acylated on the inositol was also characterized. Phenylmethylsulfonyl fluoride, an inhibitor known to block the addition of ethanolamine phosphate in T. brucei but not in mammalian cells, also inhibits the synthesis of glycolipids A-like and C-like in T. cruzi, indicating that the putative trypanosome EtNH₂-PO₄/AEP transferase(s) might represent a potential target for chemotherapy.

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