Continuous nonradioactive method for screening trypanosomal transsialidase activity and its inhibitors

En:

Glycobiology 2010;20(8):982-990

Fecha:

2010

Formato:

application/pdf

Tipo de documento:

info:eu-repo/semantics/article
info:ar-repo/semantics/artículo
info:eu-repo/semantics/publishedVersion

Descriptores:

Galactose oxidase -  Inhibitors -  Trans-sialidase -  Trypanosoma cruzi -  fetuin -  sialic acid -  sialidase -  sialidase inhibitor -  anion exchange chromatography

Descripción:

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.

Identificador(es):

http://hdl.handle.net/20.500.12110/paper_09596658_v20_n8_p982_Sartor

Derechos:

info:eu-repo/semantics/openAccess
http://creativecommons.org/licenses/by/2.5/ar