Loss of enzyme activity in a site-directed mutant of influenza neuraminidase compared to expressed wild-type protein.
Loss of enzyme activity in a site-directed mutant of influenza neuraminidase compared to expressed wild-type protein.
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与表达的野生型蛋白相比，流感神经氨酸酶定点突变体的酶活性丧失。
DOI：
10.1016/0042-6822(86)90404-6
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发表时间：
1986
期刊：
影响因子：
3.7
通讯作者：
Air,GM
中科院分区：
文献类型：
--
作者：
Lentz,MR;Air,GM
将流感病毒A/Tokyo/3/67（N2）神经氨酸酶（NA）基因的全长双链DNA拷贝和体外定点寡核苷酸诱变产生的178位色氨酸被亮氨酸取代的突变体克隆到SV 40晚期置换表达载体中。用这些重组载体感染的细胞的间接免疫荧光显示NA蛋白存在于细胞质中和感染细胞的表面上。表达野生型蛋白质的细胞表现出对胎球蛋白（一种唾液酸化糖蛋白，mol wt = 50，000）和N-乙酰神经氨酰乳糖（一种三糖，mol wt = 600）的神经氨酸酶活性。对N-乙酰神经氨酰乳糖的抑制率为44%，对胎球蛋白的抑制率为98%。与此相反，细胞表达的突变体NA没有检测到的酶活性的底物。在所有已知的NA菌株中178位色氨酸的保守性质、其在三维结构中的底物结合口袋中的位置以及突变蛋白的活性的缺乏表明该残基对于酶活性是必需的。
Full-length double-stranded DNA copies of the neuraminidase (NA) gene of influenza virus A/Tokyo/3/67 (N2) and a mutant generatedin vitroby site-specific, oligonucleotide directed mutagenesis with a substitution of leucine for tryptophan at position 178 were cloned into an SV40 late replacement expression vector. Indirect immunofluorescence of cells infected with these recombinant vectors showed the presence of NA protein in the cytoplasm and on the surface of infected cells. Cells expressing the wild-type protein showed neuraminidase enzyme activity for both fetuin, a sialated glycoprotein (mol wt = 50,000) andN-acetylneuraminyl lactose, a trisaccharide (mol wt = 600). This enzyme activity was inhibited by 44% towardN-acetylneuraminyl lactose and by 98% toward fetuin by adding anti-NA antibody before substrate. In contrast, cells expressing the mutant NA had no detectable enzyme activity for either substrate. The conserved nature of the tryptophan at position 178 in all known NA strains, its location in the substrate binding pocket in the three-dimensional structure and the lack of activity of the mutant protein indicate that this residue is essential for enzyme activity.
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