Continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors
Trypanosoma cruzi, the agent of American trypanosomiasis is unable to synthesize sialic acid (SA). Instead of using the corresponding nucleotide sugar as donor of the monosaccharide, the transfer occurs from α-2,3-linked SA in the host sialoglycoconjugates to terminal β-galactopyranosyl units of the...
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todo:paper_09596658_v20_n8_p982_Sartor2023-10-03T15:53:04Z Continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors Sartor, P.A. Agusti, R. Leguizamón, M.S. Campetella, O. de Lederkremer, R.M. Galactose oxidase Inhibitors Trans-sialidase Trypanosoma cruzi fetuin sialic acid sialidase sialidase inhibitor anion exchange chromatography article carbohydrate analysis enzyme inhibition enzyme substrate glycosylation nonhuman priority journal spectrophotometry Trypanosoma cruzi alpha-Fetoproteins Enzyme Inhibitors Galactose Glycoproteins Neuraminidase Oligosaccharides Sialic Acids Staining and Labeling Structure-Activity Relationship Trypanosoma cruzi Mammalia Tetra Trypanosoma cruzi Trypanosoma cruzi, the agent of American trypanosomiasis is unable to synthesize sialic acid (SA). Instead of using the corresponding nucleotide sugar as donor of the monosaccharide, the transfer occurs from α-2,3-linked SA in the host sialoglycoconjugates to terminal β-galactopyranosyl units of the parasite mucins. For that purpose, T. cruzi expresses a glycosylphosphatidylinositol-anchored transsialidase (TcTS) that is shed into the milieu, being detected in the blood during the acute phase of the infection. The essential role of TcTS in infection and the absence of a similar activity in mammals make this enzyme an attractive target for the development of alternative chemotherapies. However, there is no effective inhibitor toward this enzyme. In vitro, 3′-sialyllactose (SL) as donor and radioactive lactose as acceptor substrate are widely used to measure TcTS activity. The radioactive sialylated product is then isolated by anion exchange chromatography and measured. Here we describe a new nonradioactive assay using SL or fetuin as donor and benzyl β-D-Fuc-(1→6)-α-D-GlcNAc (1) as acceptor. Disaccharide 1 was easily synthesized by regioselective glycosylation of benzyl α-D-GlcNAc with tetra-Obenzoyl-D-fucose followed by debenzoylation. Compound 1 lacks the hydroxyl group at C-6 of the acceptor galactose and therefore is not a substrate for galactose oxidase. Our method relies on the specific quantification of terminal galactose produced by trans-sialylation from the donor to the 6-deoxy-galactose (D-Fuc) unit of 1 by a spectrophotometric galactose oxidase assay. This method may also discriminate sialidase and trans-sialylation activities by running the assay in the absence of acceptor 1. © The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org. Fil:Agusti, R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Leguizamón, M.S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Campetella, O. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:de Lederkremer, R.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_09596658_v20_n8_p982_Sartor |
institution |
Universidad de Buenos Aires |
institution_str |
I-28 |
repository_str |
R-134 |
collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
topic |
Galactose oxidase Inhibitors Trans-sialidase Trypanosoma cruzi fetuin sialic acid sialidase sialidase inhibitor anion exchange chromatography article carbohydrate analysis enzyme inhibition enzyme substrate glycosylation nonhuman priority journal spectrophotometry Trypanosoma cruzi alpha-Fetoproteins Enzyme Inhibitors Galactose Glycoproteins Neuraminidase Oligosaccharides Sialic Acids Staining and Labeling Structure-Activity Relationship Trypanosoma cruzi Mammalia Tetra Trypanosoma cruzi |
spellingShingle |
Galactose oxidase Inhibitors Trans-sialidase Trypanosoma cruzi fetuin sialic acid sialidase sialidase inhibitor anion exchange chromatography article carbohydrate analysis enzyme inhibition enzyme substrate glycosylation nonhuman priority journal spectrophotometry Trypanosoma cruzi alpha-Fetoproteins Enzyme Inhibitors Galactose Glycoproteins Neuraminidase Oligosaccharides Sialic Acids Staining and Labeling Structure-Activity Relationship Trypanosoma cruzi Mammalia Tetra Trypanosoma cruzi Sartor, P.A. Agusti, R. Leguizamón, M.S. Campetella, O. de Lederkremer, R.M. Continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors |
topic_facet |
Galactose oxidase Inhibitors Trans-sialidase Trypanosoma cruzi fetuin sialic acid sialidase sialidase inhibitor anion exchange chromatography article carbohydrate analysis enzyme inhibition enzyme substrate glycosylation nonhuman priority journal spectrophotometry Trypanosoma cruzi alpha-Fetoproteins Enzyme Inhibitors Galactose Glycoproteins Neuraminidase Oligosaccharides Sialic Acids Staining and Labeling Structure-Activity Relationship Trypanosoma cruzi Mammalia Tetra Trypanosoma cruzi |
description |
Trypanosoma cruzi, the agent of American trypanosomiasis is unable to synthesize sialic acid (SA). Instead of using the corresponding nucleotide sugar as donor of the monosaccharide, the transfer occurs from α-2,3-linked SA in the host sialoglycoconjugates to terminal β-galactopyranosyl units of the parasite mucins. For that purpose, T. cruzi expresses a glycosylphosphatidylinositol-anchored transsialidase (TcTS) that is shed into the milieu, being detected in the blood during the acute phase of the infection. The essential role of TcTS in infection and the absence of a similar activity in mammals make this enzyme an attractive target for the development of alternative chemotherapies. However, there is no effective inhibitor toward this enzyme. In vitro, 3′-sialyllactose (SL) as donor and radioactive lactose as acceptor substrate are widely used to measure TcTS activity. The radioactive sialylated product is then isolated by anion exchange chromatography and measured. Here we describe a new nonradioactive assay using SL or fetuin as donor and benzyl β-D-Fuc-(1→6)-α-D-GlcNAc (1) as acceptor. Disaccharide 1 was easily synthesized by regioselective glycosylation of benzyl α-D-GlcNAc with tetra-Obenzoyl-D-fucose followed by debenzoylation. Compound 1 lacks the hydroxyl group at C-6 of the acceptor galactose and therefore is not a substrate for galactose oxidase. Our method relies on the specific quantification of terminal galactose produced by trans-sialylation from the donor to the 6-deoxy-galactose (D-Fuc) unit of 1 by a spectrophotometric galactose oxidase assay. This method may also discriminate sialidase and trans-sialylation activities by running the assay in the absence of acceptor 1. © The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org. |
format |
JOUR |
author |
Sartor, P.A. Agusti, R. Leguizamón, M.S. Campetella, O. de Lederkremer, R.M. |
author_facet |
Sartor, P.A. Agusti, R. Leguizamón, M.S. Campetella, O. de Lederkremer, R.M. |
author_sort |
Sartor, P.A. |
title |
Continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors |
title_short |
Continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors |
title_full |
Continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors |
title_fullStr |
Continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors |
title_full_unstemmed |
Continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors |
title_sort |
continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors |
url |
http://hdl.handle.net/20.500.12110/paper_09596658_v20_n8_p982_Sartor |
work_keys_str_mv |
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