Continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors

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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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Autores principales:	Sartor, P.A., Agusti, R., Leguizamón, M.S., Campetella, O., de Lederkremer, R.M.
Formato:	Artículo publishedVersion
Publicado:	2010
Materias:	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 alpha-Fetoproteins Enzyme Inhibitors Galactose Glycoproteins Neuraminidase Oligosaccharides Sialic Acids Staining and Labeling Structure-Activity Relationship Mammalia Tetra
Acceso en línea:	http://hdl.handle.net/20.500.12110/paper_09596658_v20_n8_p982_Sartor http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=artiaex&d=paper_09596658_v20_n8_p982_Sartor_oai
Aporte de:	Repositorio Digital de la Universidad de Buenos Aires (UBA) de Universidad de Buenos Aires

id	I28-R145-paper_09596658_v20_n8_p982_Sartor_oai
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spelling	I28-R145-paper_09596658_v20_n8_p982_Sartor_oai2020-10-19 Sartor, P.A. Agusti, R. Leguizamón, M.S. Campetella, O. de Lederkremer, R.M. 2010 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. application/pdf http://hdl.handle.net/20.500.12110/paper_09596658_v20_n8_p982_Sartor info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar Glycobiology 2010;20(8):982-990 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 Continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors info:eu-repo/semantics/article info:ar-repo/semantics/artículo info:eu-repo/semantics/publishedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=artiaex&d=paper_09596658_v20_n8_p982_Sartor_oai
institution	Universidad de Buenos Aires
institution_str	I-28
repository_str	R-145
collection	Repositorio Digital de la Universidad de Buenos Aires (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	Artículo Artículo publishedVersion
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
publishDate	2010
url	http://hdl.handle.net/20.500.12110/paper_09596658_v20_n8_p982_Sartor http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=artiaex&d=paper_09596658_v20_n8_p982_Sartor_oai
work_keys_str_mv	AT sartorpa continuousnonradioactivemethodforscreeningtrypanosomaltranssialidaseactivityanditsinhibitors AT agustir continuousnonradioactivemethodforscreeningtrypanosomaltranssialidaseactivityanditsinhibitors AT leguizamonms continuousnonradioactivemethodforscreeningtrypanosomaltranssialidaseactivityanditsinhibitors AT campetellao continuousnonradioactivemethodforscreeningtrypanosomaltranssialidaseactivityanditsinhibitors AT delederkremerrm continuousnonradioactivemethodforscreeningtrypanosomaltranssialidaseactivityanditsinhibitors
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Continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors

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