A new method for screening trans-sialidase activity

MUCHNIK DE LEDERKREMER, R; AGUSTI, R; SARTOR, P; LEGUIZAMON, MS; CAMPETELLA, O

Oslo, Norway

Simposio; 24th International Carbohydrate Symposium (ICS 2008); 2008

International Carbohydrate Organization

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 (GPI)-anchored trans-sialidase (TcTS) on its surface that is shed into the milieu, being detected in the blood during the acute phase of the infection [1]. TcTS has two subsites in the active center: the sialic acid (SA) binding site and the galactose binding site. 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. A new nonradioactive assay was developed in our laboratory using SL or fetuin as donor and benzyl D-Fuc (b1¨6)GlcNAc (1) as acceptor. The specific quantification of lactose produced by trans-sialidation from sialyllactose to 6-deoxy galactose (D-Fuc) was performed by a spectrophotometric galactose oxidase assay [2] (Scheme 1).  Disaccharide 1 was easily synthesized in 50% yield by regioselective 6-O glycosidation of benzyl a-D-GlcNAc with tetra-O-benzoyl-D-fucose using SnCl4 as promoter. After debenzoylation and purification, 1 was characterized by 1H and 13C-NMR spectroscopy. The new disaccharide TcTS acceptor properties were evaluated using a recombinant enzyme and SL as donor. The incubation mixtures were analysed by HPAEC-PAD. A KM value = 0.13 mM for 1 was obtained. A competitive radioactive assay was also performed using radioactive lactose and disaccharide 1, displaying similar efficiency as known acceptors. Once established that 1 was a good acceptor of SA, the reaction was coupled to the galactose oxidase assay. Fetuin or SL was used as SA donor and terminal galactose oxidation rate was determined with the Amplex Red reagent (Molecular Probes, USA). The absorbance was dependent on TcTS concentration and time of reaction.  This is the first report describing a real time measure of TcTS activity. Reactivity decreased by preincubation of the recombinant enzyme with DANA or neutralizing antibodies [3]. In a control, ran in the absence of compound 1, the absorbance was significantly lower, thus, the present assay could also discriminate among sialidase and TS activities.