Two trans-sialidase forms with different sialic acid transfer and sialidase activities from Trypanosoma congolense.
Trypanosomes express an enzyme called trans-sialidase (TS), which enables the parasites to transfer sialic acids from the environment onto trypanosomal surface molecules. Here we describe the purification and characterization of two TS forms from the African trypanosome Trypanosoma congolense. The purification of the two TS forms using a combination of anion exchange chromatography, isoelectric focusing, gel filtration, and subsequently, antibody affinity chromatography resulted, in both cases, in the isolation of a 90-kDa monomer on SDS-PAGE, which was identified as trans-sialidase using micro-sequencing. Monoclonal antibody 7/23, which bound and partially inhibited TS activity, was found in both cases to bind to a 90-kDa protein. Both TS forms possessed sialidase and transfer activity, but markedly differed in their activity ratios. The TS form with a high transfer-to-sialidase activity ratio, referred to as TS-form 1, possessed a pI of pH 4-5 and a molecular mass of 350-600 kDa. In contrast, the form with a low transfer-to-sialidase activity ratio, referred to as TS-form 2, exhibited a pI of pH 5-6.5 and a molecular mass of 130-180 kDa. Both TS forms were not significantly inhibited by known sialidase inhibitors and revealed no significant differences in donor and acceptor substrate specificities; however, TS-form 1 utilized various acceptor substrates with a higher catalytic efficiency. Interestingly, glutamic acid-alanine-rich protein, the surface glycoprotein, was co-purified with TS-form 1 suggesting an association between both proteins.
Tiralongo
Evelin
E
Biochemisches Institut, Universität zu Kiel, Olshausenstrasse 40, Kiel 24098 Germany.
Schrader
Silke
S
Lange
Hans
H
Lemke
Hilmar
H
Tiralongo
Joe
J
Schauer
Roland
R
D000818:Animals; D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006023:Glycoproteins; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D009439:Neuraminidase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D014348:Trypanosoma congolense / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D006023:Glycoproteins; C069715:trans-sialidase; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid