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

Abstract

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 alpha-2,3-linked SA in the host sialoglycoconjugates to terminal beta-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'-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 beta-d-Fuc-(1-->6)-alpha-d-GlcNAc (1) as acceptor. Disaccharide 1 was easily synthesized by regioselective glycosylation of benzyl alpha-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-sialylation activities by running the assay in the absence of acceptor 1.

Scheme 1

Transfer of SA by TcTS from a sialyl donor to a D-Fucp unit in the acceptor substrate. Quantification of the terminal galactose residues generated in the donor after the removal of the SA was performed by a coupled colorimetric/fluorimetric galactose oxidase test.

Scheme 2

Synthesis of benzyl β-d-fucopyranosyl-(1→6)-2-acetamido-2-deoxy-α-d-glucopyranoside (1). Reagents and yield: (i) benzoyl chloride, pyridine, yield 75%; (ii) SnCl₄, CH₂Cl₂, yield 42%; (iii) NaOMe, MeOH, yield 94%.

Fig. 1

Analysis of benzyl β-d-Fuc-(1→6)-α-d-GlcNAc (1) as acceptor substrate in the TcTS reaction. (A) Compound 1 and SL, without enzyme; (B) 1 was incubated with SL and TcTS for 15 min at 25°C and the reaction mixture analyzed by HPAEC-PAD; (C) the same as above but TcTS was preincubated with a neutralizing antibody. A CarboPac PA-100 ion exchange analytical column was eluted with 50 mM NaAcO in 100 mM NaOH at 1 mL/min. L, lactose; SA, sialic acid; SL, sialyllactose; S-1, sialylated disaccharide 1.

Fig. 2

Comparison of sensitivities for the nonradioactive and radioactive assays. (A) Colorimetric assay: different amounts of TcTS were incubated with fetuin and 1 at room temperature for 30 min. Then, galactose oxidase, HRP and Amplex Red Reagent were added and incubation continued at 37°C. The absorbance at 540 nm was measured for each TcTS concentration. An expansion of the more diluted area is shown in the inset. (B) Fluorometric assay: different amounts of TcTS were mixed with SL, disaccharide 1, galactose oxidase, HRP and Amplex Red Reagent. Fluorescence emission at 590 nm was read after 30 min of incubation at room temperature. Fluorescence readings are expressed as arbitrary units. Inset: expansion of values obtained at the lower TcTS amounts tested. (C) Radioactive assay: different amounts of TcTS were incubated with SL and [d-glucose-1-¹⁴C]-lactose. After 30 min of incubation at room temperature, a slurry of quaternary aminoethyl-Sephadex was added to bind the ¹⁴C-sialyllactose product, washed with water and the counts per minute retained were quantified. An expansion of the more diluted area is shown in the inset.

Fig. 3

Assay of sialidase and trans-sialidase activities. TcTS was assayed in the presence (dots) or absence (triangles) of disaccharide 1. (A) TcTS (0.1 µg) was incubated at 37°C with SL as sialyl donor, galactose oxidase, HRP and Amplex Red Reagent and OD_540 nm measured at different times. (B) The same reaction as in A was performed at room temperature in the absence of 1 that was incorporated after 20 min (arrow) and incubation continued. Fluorescence was recorded at different incubation times and expressed as arbitrary units.

Fig. 4

Nonradioactive TcTS inhibition assay. Purified neutralizing antibodies from mouse (mAb 13G9) or from human patients (purified AbsNt, 2 µg) or 15 mM DANA (2,3-dehydro-2-deoxy-N-acetylneuraminic acid) were allowed to react with TcTS (0.3 µg) for 15 min and then fetuin and disaccharide 1 substrates were incorporated. Later, galactose oxidase, HRP and Amplex Red Reagent were added and incubated at 37°C. Two positive controls were included adding asialofetuin (AF) to each well containing TcTS inhibitors (DANA AF and AbsNt AF). Clostridium perfringes neuraminidase was also included as another positive control (C). *P < 0.01.