Proteolytic processing and oligomerization of bacteriophage-derived endosialidases
Proteolytic processing and oligomerization of bacteriophage-derived endosialidases
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DOI：
10.1074/jbc.m212048200
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发表时间：
2003-04-11
影响因子：
4.8
通讯作者：
Gerardy-Schahn, R
中科院分区：
文献类型：
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作者：
Mühlenhoff, M;Stummeyer, K;Gerardy-Schahn, R
感染神经侵入性病原体大肠杆菌K1的噬菌体需要内切唾液酸酶来穿透宿主的聚唾液酸囊。可获得K1特异性β-phiK 1 E、phiK 1F和63 D的内唾液酸酶endoNE、endoNF和endoN 63 D的序列信息。克隆的序列共享高度保守的催化结构域，但在N-和C-末端部分的长度不同。虽然在endoNE的情况下成功表达了活性重组酶，但对于endoNF则失败了。所有三种内切唾液酸酶序列的蛋白比对产生了这样的假设，即克隆的endoNF的失活是由C-末端截短引起的。通过对phiK 1F基因组中相应基因位点的重新研究，我们鉴定了一个3195 bp的扩展开放阅读框，编码119 kDa蛋白。全长的endoNF包含在所有内唾液酸酶中保守的C-末端结构域，其可以充当分子内伴侣。用endoNE和endoNF进行的比较研究表明，内切唾液酸酶被蛋白水解加工，释放C-末端结构域。使用突变的方法结合蛋白质分析技术，我们证明：（i）C-末端结构域是内唾液酸酶和其他尾纤维蛋白的共同特征;（ii）C-末端结构域的完整性及其在新生蛋白中的存在对于活性酶的形成至关重要;（iii）蛋白水解加工对于酶活性不是必需的;和（iv）功能性折叠是endoNF三聚化的先决条件。
Bacteriophages infecting the neuroinvasive pathogen Escherichia coli K1 require an endosialidase to penetrate the polysialic acid capsule of the host. Sequence information is available for the endosialidases endoNE, endoNF, and endoN63D of the K1-specific phages phiK1E, phiK1F, and 63D, respectively. The cloned sequences share a highly conserved catalytic domain but differ in the length of the N- and C-terminal parts. Although the expression of active recombinant enzyme succeeded in the case of endoNE, it failed for endoNF. Protein alignments of all three endosialidase sequences gave rise to the assumption that inactivity of the cloned endoNF is caused by a C-terminal truncation. By reinvestigation of the respective gene locus in the, phiK1F genome, we identified an extended open reading frame of 3195 bp, encoding a 119-kDa protein. Full-length endoNF contains the C-terminal domain conserved in all endosialidases, which may act as an intramolecular chaperone. Comparative studies carried out with endoNE and endoNF demonstrate that endosialidases are proteolytically processed, releasing the C-terminal domain. Using a mutational approach in combination with protein analytical techniques we demonstrate that (i) the C-terminal domain is a common feature of endosialidases and other tail fiber proteins; (ii) the integrity of the C-terminal domain and its presence in the nascent protein are crucial for the formation of active enzymes; (iii) proteolytic processing is not essential for enzymatic activity; and (iv) functional folding is a prerequisite for trimerization of endoNF.