Continuous non-radioactive method for screening trypanosomal trans-sialidase activity and its inhibitors

SARTOR, P.; AGUSTI, R.; LEGUIZAMÓN, M. S.; CAMPETELLA, O.; LEDERKREMER, R.M.

GLYCOBIOLOGY

OXFORD UNIV PRESS INC

Lugar: Oxford; Año: 2010 vol. 20 p. 982 - 990

0959-6658

Trypanosoma cruzi, the agent of American trypanosomiasis is unable to synthesize sialic acid. Instead of using the corresponding nucleotide sugar as donor of the monosaccharide the transfer occurs from a-2,3-linked sialic acid in the host sialoglycoconjugates to terminal b-galactopyranosyl units of the parasite mucins. For that purpose, T. cruzi expresses a glycosylphosphatidylinositol-anchored trans-sialidase (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L-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 b-D-Fuc-(1¨6)-a-D-GlcNAc (1) as acceptor. Disaccharide 1 was easily synthesized by regioselective glycosylation of benzyl a-D-GlcNAc with tetra-O-benzoyl-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-sialidase activities by running the assay in the absence of acceptor 1.