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- Monteiro, Ana C. S., et al.
(författare)
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Identification, characterization and localization of chagasin, a tight-binding cysteine protease inhibitor in Trypanosoma cruzi
- 2001
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Ingår i: Journal of Cell Science. - 0021-9533. ; 114:21, s. 3933-3942
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Tidskriftsartikel (refereegranskat)abstract
- Lysosomal cysteine proteases from mammalian cells and plants are regulated by endogenous tight-binding inhibitors from the cystatin superfamily. The presence of cystatin-like inhibitors in lower eukaryotes such as protozoan parasites has not yet been demonstrated, although these cells express large quantities of cysteine proteases and may also count on endogenous inhibitors to regulate cellular proteolysis. Trypanosoma cruzi, the causative agent of Chagas heart disease, is a relevant model to explore this possibility because these intracellular parasites rely on their major lysosomal cysteine protease (cruzipain) to invade and multiply in mammalian host cells. Here we report the isolation, biochemical characterization, developmental stage distribution and subcellular localization of chagasin, an endogenous cysteine protease inhibitor in T. cruzi. We used high temperature induced denaturation to isolate a heat-stable cruzipain-binding protein (apparent molecular mass, 12 kDa) from epimastigote lysates. This protein was subsequently characterized as a tight-binding and reversible inhibitor of papain-like cysteine proteases. Immunoblotting indicated that the expression of chagasin is developmentally regulated and inversely correlated with that of cruzipain. Gold-labeled antibodies localized chagasin to the flagellar pocket and cytoplasmic vesicles of trypomastigotes and to the cell surface of amastigotes. Binding assays performed by probing living parasites with fluorescein (FITC)-cruzipain or FITC-chagasin revealed the presence of both inhibitor and protease at the cell surface of amastigotes. The intersection of chagasin and cruzipain trafficking pathways may represent a checkpoint for downstream regulation of proteolysis in trypanosomatid protozoa.
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- Marcos, Nuno T., et al.
(författare)
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Role of the Human ST6GalNAc-I and ST6GalNAc-II in the Synthesis of the Cancer-Associated Sialyl-Tn Antigen
- 2004
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Ingår i: CANCER RESEARCH. - 0008-5472. ; 64:19, s. 7050-7
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Tidskriftsartikel (refereegranskat)abstract
- The Sialyl-Tn antigen (Neu5Acalpha2-6GaINAc-O-Ser/Thr) is highly expressed in several human carcinomas and is associated with carcinoma aggressiveness and poor prognosis. We characterized two human sialyltransferases,(CMP)-C-.-Neu5Ac:GaINAc-R alpha2,6-sialyltransferase (ST6GaINAc)-I and ST6GaINAc-II, that are candidate enzymes for Sialyl-Tn synthases. We expressed soluble recombinant hST6GaINAc-I and hST6GaINAc-II and characterized the substrate specificity of both enzymes toward a panel of glycopeptides, glycoproteins, and other synthetic glycoconjugates. The recombinant ST6GaINAc-I and ST6GaINAc-II showed similar substrate specificity toward glycoproteins and GaINAcalpha-O-Ser/Thr glycopeptides, such as glycopeptides derived from the MUC2 mucin and the HIVgp120. We also observed that the amino acid sequence of the acceptor glycopeptide contributes to the in vitro substrate specificity of both enzymes. We additionally established a gastric cell line, MKN45, stably transfected with the full length of either ST6GaINAc-I or ST6GaINAc-II and evaluated the carbohydrate antigens expression profile induced by each enzyme. MKN45 transfected with ST6GaINAc-I showed high expression of Sialyl-Tn, whereas MKN45 transfected with ST6GaINAc-II showed the biosynthesis of the Sialyl-6T structure [GaIbeta1-3 (Neu5Acalpha2-6)GaINAc-O-Ser/Thr].In conclusion, although both enzymes show similar in vitro activities when Tn antigen alone is available, whenever both Tn and T antigens are present, ST6GaINAc-I acts preferentially on Tn antigen, whereas the ST6GaINAc-II acts preferentially on T antigen. Our results show that ST6GaINAc-I is the major Sialyl-Tn synthase and strongly support the hypothesis that the expression of the Sialyl-Tn antigen in cancer cells is due to ST6GaINAc-I activity.
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