The trans-sialidase from the african trypanosome Trypanosoma brucei.
Trypanosoma brucei is the cause of the diseases known as sleeping sickness in humans (T. brucei ssp. gambiense and ssp. rhodesiense) and ngana in domestic animals (T. brucei brucei) in Africa. Procyclic trypomastigotes, the tsetse vector stage, express a surface-bound trans-sialidase that transfers sialic acid to the glycosylphosphatidylinositol anchor of procyclin, a surface glycoprotein covering the parasite surface. Trans-sialidase is a unique enzyme expressed by a few trypanosomatids that allows them to scavenge sialic acid from sialylated compounds present in the infected host. The only enzyme extensively characterized is that of the American trypanosome T. cruzi (TcTS). In this work we identified and characterized the gene encoding the trans-sialidase from T. brucei brucei (TbTS). TbTS genes are present at a small copy number, at variance with American trypanosomes where a large gene family is present. The recombinant TbTS protein has both sialidase and trans-sialidase activity, but it is about 10 times more efficient in transferring than in hydrolysing sialic acid. Its N-terminus contains a region of 372 amino acids that is 45% identical to the catalytic domain of TcTS and contains the relevant residues required for catalysis. The enzymatic activity of mutants at key positions involved in the transfer reaction revealed that the catalytic sites of TcTS and TbTS are likely to be similar, but are not identical. As in the case of TcTS and TrSA, the substitution of a conserved tryptophanyl residue changed the substrate specificity rendering a mutant protein capable of hydrolysing both alpha-(2,3) and alpha-(2,6)-linked sialoconjugates.
Montagna
Georgina
G
Instituto de Investigaciones Biotecnológicas, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de General San Martín, Provincia de Buenos Aires, Argentina.
Cremona
M Laura
ML
Paris
Gastón
G
Amaya
M Fernanda
MF
Buschiazzo
Alejandro
A
Alzari
Pedro M
PM
Frasch
Alberto C C
AC
D000349:Africa; D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology; D001665:Binding Sites; D020134:Catalytic Domain; D017124:Conserved Sequence; D018503:Genome, Protozoan; D006023:Glycoproteins; D009439:Neuraminidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics; D014349:Trypanosoma cruzi / Q000201:enzymology*
D006023:Glycoproteins; D011994:Recombinant Proteins; C069715:trans-sialidase; D009439:Neuraminidase