Montagna Georgina N, Donelson John E, Frasch Alberto C C
Instituto de Investigaciones Biotecnológicas, Universidad de General San Martín, 1650 San Martín, Pcia de Buenos Aires, Argentina.
J Biol Chem. 2006 Nov 10;281(45):33949-58. doi: 10.1074/jbc.M604951200. Epub 2006 Sep 6.
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.
布氏锥虫在昆虫媒介中的前循环期表达一种表面结合的转唾液酸酶（TbTS），该酶将环境中糖缀合物上的唾液酸转移至其表面膜上糖基磷脂酰肌醇锚定蛋白。针对TbTS的RNA干扰消除了前循环期细胞中的转唾液酸酶活性，但并未降低唾液酸酶活性，这表明存在一种单独的用于水解唾液酸的唾液酸酶。对布氏锥虫基因组序列的搜索揭示了另外七个推定基因，这些基因编码与TbTS具有不同相似性的蛋白质。针对其中一种蛋白质TbSA C的RNA干扰大大降低了唾液酸酶活性，但对转唾液酸酶活性没有影响。TbSA C推导的氨基酸序列与TbTS仅具有40%的同一性，但保留了催化所需的大部分相关残基。然而，该唾液酸酶在170位有一个色氨酸替代酪氨酸，这对于结合接受转移唾液酸的末端半乳糖至关重要。当将该唾液酸酶中相同的色氨酸替代引入重组转唾液酸酶时，突变酶几乎失去了所有转唾液酸酶活性并增加了其唾液酸酶活性，进一步证实所鉴定的基因和蛋白质对应于寄生虫唾液酸酶。因此，与迄今为止分析的所有其他仅表达转唾液酸酶或唾液酸酶而非两者都表达的锥虫不同，布氏锥虫在两种不同的蛋白质中表达这两种酶活性。这些结果表明，非洲锥虫可以通过调节每种酶的差异表达来调控其表面关键唾液酸残基的数量。
