Continuous nonradioactive method for screening trypanosomal trans-sialidase activity and its inhibitors.
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.
Sartor
Paula A
PA
Departamento de Microbiología, Facultad de Medicina, Universidad de Buenos Aires, 1121 Buenos Aires, Argentina.
Agusti
Rosalía
R
Leguizamón
Maria S
MS
Campetella
Oscar
O
de Lederkremer
Rosa M
RM
D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005690:Galactose / Q000138:chemical synthesis / Q000737:chemistry; D006023:Glycoproteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D009439:Neuraminidase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D009844:Oligosaccharides / Q000737:chemistry; D012794:Sialic Acids / Q000737:chemistry / Q000378:metabolism; D013194:Staining and Labeling; D013329:Structure-Activity Relationship; D014349:Trypanosoma cruzi / Q000201:enzymology*; D000509:alpha-Fetoproteins / Q000737:chemistry
C421467:3'-sialyllactose; D004791:Enzyme Inhibitors; D006023:Glycoproteins; D009844:Oligosaccharides; D012794:Sialic Acids; D000509:alpha-Fetoproteins; C069715:trans-sialidase; D009439:Neuraminidase; D005690:Galactose