克雷白氏肺炎桿菌磷酸酪胺酸激酶KpWzc對其下游酵素ManC, Gnd活性影響之分析與Ugd磷酸酪胺酸殘基鑑定與下游受質之搜尋

ManC, Gnd and KpUgd are phosphorylated by tyrosine kinase, KpWzc of Klebsiella pneumoniae-Kinetic analysis of ManC and Gnd, identification of phosphotyrosine residues of KpUgd and search for other phosphorylation target

錢漢聲(Han-Sheng Chien)

指導教授 ： 張晃猷

國立清華大學/生命科學院/分子醫學研究所/碩士(2008年)

https://doi.org/10.6843/NTHU.2008.00275

摘要

Klebsiella pneumoniae, an opportunistic gram-negative bacterium causing community-acquired diseases, is mostly encapsulated by a considerable thick polysaccharidic capsule acting to protect the bacterium from phagocytosis and prevent from damage by serum factors. Our previous study has demonstrated that deletion of yco6, coding for a protein-tyrosine kinase, in K. pneumoniae CG43 reduced significantly the bacterial mucoidy and content of capsular polysaccharide. KpUgd, Gnd and ManC, required for regulation and synthesis of CPS sugar nucleotide precursors, were able to phosphorylated by KpWzcE23. In this study, we want to demonstrate that phosphorylation affects enzyme activity of ManC and Gnd. ManC is a bifunctional protein, with N-terminal GDP-mannose pyrophosphorylase and C-terminal phosphomannose isomerase activity and Gnd is a 6-phosphogluconate dehydrogenase involved in pentose phosphate pathway. Recombinant ManC and Gnd were purified and treated with calf intestine alkaline phosphatase, and we tested their specific enzyme activities after incubation with or without KpWzcE23. The results showed that the VMAX of Gnd and both PMI domain and GMP domain of ManC were elevated after phosphorylated. We also constructed four recombinant truncated KpUgd fragments, each containing 2 to 7 tyrosine residues, to investigate the phosphotyrosine sites. The results showed all four fragments showed phosphorylation signals by isotope autoradiography. We also tried to search other phophorylation proteins in a phosphoproteomic way, but did not find potential targets. In conclusion, this study have suggested KpUgd, ManC and Gnd are involved in KpWzc-dependent CPS synthesis.

關鍵字

克雷白氏肺炎桿菌 ； 磷酸酪胺酸激酶

並列摘要

克雷白氏肺炎桿菌為一株伺機性引起院內感染疾病的格蘭氏陰性菌，其外部包覆著由多醣體所組成的厚莢膜。此一莢膜可以讓細菌逃避細胞的吞噬作用以及避免被血清因子所毒殺。在實驗室之前的研究中，證明了剔除酪胺酸激酶的基因KpWzc會明顯地減少其原本所具有的黏性和莢膜多醣體的量。證實了克雷白氏肺炎桿菌蛋白質酪胺酸激酶KpWzc夠將克雷白氏肺炎桿菌中的KpUgd、Gnd和ManC這三個酵素磷酸化。而磷酸化對KpUgd酵素活性有顯著提升。本實驗目的在驗證蛋白質酪胺酸激酶KpWzc對其下游磷酸化蛋白質Gnd, ManC之酵素活性影響以及尋找KpUgd被磷酸化之酪胺酸殘基。ManC 是一個雙功能酵素，前半段具有磷酸甘露糖異位轉化酶活性，後半段則具有磷酸甘露糖異構酶活性，而Gnd 具有磷酸葡萄糖酸去氫酶活性，過量表現與被純化的兩個酵素先經由牛小腸鹼性去磷酸酶處理後，與蛋白質酪胺酸激酶KpWzcE23進行磷酸化反應，並測試期專有酵素活性，結果顯示兩個酵素的三個酵素最大反應速率皆有上升。在此研究之中，我們亦構築了四段截短的KpUgd片段，用以探討磷酸酪胺酸殘基位置，同位素放射能照像結果顯示，四段皆有被偵測到磷酸化訊號，證明蛋白質酪胺酸激酶KpWzcE23可同時對KpUgd上多個酪胺酸殘基進行磷酸化。另外，我們也用磷酸化蛋白質體學技術來尋找細菌內其他可能被磷酸化的蛋白質，但是並沒有找到可能的磷酸化蛋白質。以上實驗結果可推測KpUgd, ManC與Gnd三個酵素皆參與由蛋白質酪胺酸激酶KpWzc調控之莢膜多醣體合成。

並列關鍵字

Klebsiella pneumoniae ； tyrosine kinase

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