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 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.

trans-Sialidase Neutralizing Antibody Detection in Trypanosoma cruzi-Infected Domestic Reservoirs

“…Taken together, the evidence shows that TIA detects T. cruzi infections in domestic animal reservoirs that are missed by CS. The use of radioactive material hampers its use in the field, but major efforts are under way to replace this substrate with a colorimetric or fluorometric one (24,30,32).…”

Separation of Time‐Resolved Phenomena in Surface‐Enhanced Raman Scattering of the Photocatalytic Reduction of p‐Nitrothiophenol

“…Sialidase activity can be measured either by quantifying the amount of sialic acid using thiobarbituric acid reagent,11 or by spectrophotometric assay of p ‐nitrophenol (PNP)9 or 4‐methylumbelliferone (MU)10 released from the substrate. In addition, an enzyme cascade12 or peanut lectin hemagglutination assay13 is described for the detection of sialidase activity. With these methods, there are some limitations, such as low sensitivity, complex manipulation, the need for specialized equipment, the time‐consuming nature of the procedure, or the need for radiolabeling.…”

trans-Sialylation: a strategy used to incorporate sialic acid into oligosaccharides

“…Mice infected with Trypanosoma cruzi produce antibodies against the enzymatic domain of TS that inhibit its activity. 68 Immunological events caused in Chagas disease by trans-sialylation have been described 64,[69][70][71][72] and reviewed. 73 Campetella and coworkers included an interesting discussion about the humoral response to SAPA in the acute phase of the disease and the detection of neutralizing antibodies in chronic patients.…”