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J. Biol. Chem., Vol. 281, Issue 45, 33949-33958, November 10, 2006

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Procyclic Trypanosoma brucei Expresses Separate Sialidase and trans-Sialidase Enzymes on Its Surface Membrane^*

Georgina N. Montagna¹, John E. Donelson, and Alberto C. C. Frasch²

From the Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, Universidad de General San Martín, 1650 San Martín, Pcia de Buenos Aires, Argentina and Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242

The procyclic stage of Trypanosoma brucei in the insect vector expresses a surface-bound trans-sialidase (TbTS) that transfers sialic acid from glycoconjugates in the environment to glycosylphosphatidylinositol-anchored proteins on its surface membrane. RNA interference against TbTS abolished trans-sialidase activity in procyclic cells but did not diminish sialidase activity, suggesting the presence of a separate sialidase enzyme for hydrolyzing sialic acid. A search of the T. brucei genome sequence revealed seven other putative genes encoding proteins with varying similarity to TbTS. RNA interference directed against one of these proteins, TbSA C, greatly decreased the sialidase activity but had no effect on trans-sialidase activity. The deduced amino acid sequence of TbSA C shares only 40% identity with TbTS but conserves most of the relevant residues required for catalysis. However, the sialidase has a tryptophan substitution for a tyrosine at position 170 that is crucial in binding the terminal galactose that accepts the transferred sialic acid. When this same tryptophan substitution in the sialidase was placed into the recombinant trans-sialidase, the mutant enzyme lost almost all of its trans-sialidase activity and increased its sialidase activity, further confirming that the gene and protein identified correspond to the parasite sialidase. Thus, in contrast to all other trypanosomes analyzed to date that express either a trans-sialidase or a sialidase but not both, T. brucei expresses these two enzymatic activities in two separate proteins. These results suggest that African trypanosomes could regulate the amount of critical sialic acid residues on their surface by modulating differential expression of each of these enzymes.

Received for publication, May 23, 2006 , and in revised form, July 12, 2006.

^* This work was supported, in part, by the Agencia Nacional de Promoción Científica y Tecnológica. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the Gen-Bank^TM/EBI Data Bank with accession number(s) DQ 841707.

¹ A research fellow from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET).

² Supported by an International Research Scholar grant from the Howard Hughes Medical Institute. A researcher from the CONICET. To whom correspondence should be addressed: Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomús, Universidad de General San Martín, INTI, Avenida Gral Paz 5445, Edificio 24, Casilla de Correo 30, 1650 San Martín, Pcia de Buenos Aires, Argentina. Tel.: 54-11-4580-7255; Fax: 54-11-4752-9639; E-mail: cfrasch{at}iib.unsam.edu.ar.

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