Abstract
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.
Highlights
The flagellated protozoa, trypanosomes, the agents of several diseases, express a unique type of glycosyltransferase, called trans-sialidase (TS)1, which is believed to play an important role in maintaining pathogenicity of the parasites [1;2]
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-Polyacrylamide Gel Electrophoresis (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
Summary
The flagellated protozoa, trypanosomes, the agents of several diseases, express a unique type of glycosyltransferase, called trans-sialidase (TS), which is believed to play an important role in maintaining pathogenicity of the parasites [1;2]. Since native and recombinant enzymes can differ in their glycosylation, antibody specificity and biochemical properties, it is important that the native enzyme be purified and characterized, with the subsequent aim of obtaining sequence information. This is especially important, as several genes encoding TS/SA enzymes, or even silent genes may exist in trypanosomes, as has been shown for T.cr. We present characterization studies which reveal significant differences between both TS forms concerning their transfer to SA ratios and catalytic efficiencies using various acceptor substrates
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