Procyclic Trypanosoma brucei expresses separate sialidase and trans-sialidase enzymes on its surface membrane.
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.
Montagna
Georgina N
GN
Instituto de Investigaciones Biotecnológicas, Universidad de General San Martín, 1650 San Martín, Pcia de Buenos Aires, Argentina.
Donelson
John E
JE
Frasch
Alberto C C
AC
D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics / Q000378:metabolism; D015152:Blotting, Northern; D006023:Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008018:Life Cycle Stages / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D034622:RNA Interference; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism; D012794:Sialic Acids / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D000953:Antigens, Protozoan; D006023:Glycoproteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; D012794:Sialic Acids; C069715:trans-sialidase; D009439:Neuraminidase