trans-sialidase of Trypanosoma cruzi: location of galactose-binding site(s).
Trypanosoma cruzi expresses a trans-sialidase on its surface, which catalyzes the transfer of sialic acid from mammalian host glycans to its own surface glycoproteins. It has been proposed that the enzyme consists of three domains prior to a long C-terminal repeating sequence that is not required for enzyme activity. The first of these domains shares significant sequence identity with bacterial sialidases which catalyse the hydrolysis of sialic acid. Here we report the sequence of the N-terminal domains of the TS19y trans-sialidase gene, which was expressed in bacteria with the same specific activity as natural enzyme of T. cruzi. Various deletion mutants of TS19y, without the C-terminal tandem repeat, have been cloned and expressed and their trans-sialidase and sialidase activities measured. These experiments show that all three N-terminal domains are required for full trans-sialidase activity, though only the first is necessary for sialidase activity. Some transferase activity is observed, however, even with the shortest construct comprising the first N-terminal domain. Deletion mutants to probe the role of the N-terminal residues of the first domain suggest that the first 33 residues are also required for trans-sialidase activity, but not for sialidase activity. Molecular modelling of the first N-terminal domain of TS19y based on our structures of bacterial sialidases and site-directed mutations suggests the location of a galactose-binding site within this domain.
Chuenkova
M
M
Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, United Kingdom.
Pereira
M
M
Taylor
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology; D001665:Binding Sites; D005690:Galactose / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012799:Sialyltransferases / Q000235:genetics / Q000378:metabolism*; D014349:Trypanosoma cruzi / Q000201:enzymology*; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D008565:Membrane Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid; D005690:Galactose; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase