Neisseria gonorrhoeae type IV pili undergo multisite, hierarchical modifications with phosphoethanolamine and phosphocholine requiring an enzyme structurally related to lipopolysaccharide phosphoethanolamine transferases
Neisseria gonorrhoeae type IV pili undergo multisite, hierarchical modifications with phosphoethanolamine and phosphocholine requiring an enzyme structurally related to lipopolysaccharide phosphoethanolamine transferases
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DOI：
10.1074/jbc.m604324200
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发表时间：
2006-09-22
影响因子：
4.8
通讯作者：
Koomey, Michael
中科院分区：
文献类型：
--
作者：
Aas, Finn Erik;Egge-Jacobsen, Wolfgang;Koomey, Michael
两性离子磷酸形式磷酸乙醇胺和磷酸胆碱被认为是病原体细胞表面的有影响力的和重要的取代基。PilE是淋球菌IV型菌毛（Tfp）定植因子的主要菌毛蛋白亚基蛋白，其经历这些部分的独特的翻译后修饰。这些磷酸化修饰已被证明是O-连接的一个特定的，保守的丝氨酸残基的PilE交替。然而，参与其添加的酶和前体是未知的，并且PilE翻译后修饰的全谱尚未被定义。在这里，一个完整的蛋白质为基础的质谱方法与生物信息学和反向遗传学相结合，以解决这些问题。具体而言，我们表明，在其分布的致病性奈瑟氏菌物种和结构相关的酶参与磷酸乙醇胺修饰脂多糖的蛋白质是必要的PilE共价修饰磷酸乙醇胺和磷酸胆碱。这些发现强烈表明，蛋白磷酸化修饰的机制类似于原核糖脂聚糖的类似修饰的过程。我们还表明，PilE进行多位点和层次的磷酸化形式的修改和化学计量的网站占有率可以影响PilE的一级结构和丰富的菌毛蛋白样蛋白PilV。总之，这些发现对PilE的结构和抗原性具有重要意义。
The zwitterionic phospho-forms phosphoethanolamine and phosphocholine are recognized as influential and important substituents of pathogen cell surfaces. PilE, the major pilin subunit protein of the type IV pilus (Tfp) colonization factor of Neisseria gonorrhoeae undergoes unique, post-translational modifications with these moieties. These phospho-form modifications have been shown to be O-linked alternately to a specific, conserved serine residue of PilE. However, the enzymes and precursors involved in their addition are unknown, and the full spectrum of PilE post-translational modifications has yet to be defined. Here, an intact protein-based mass spectrometric approach was integrated with bioinformatics and reverse genetics to address these matters. Specifically we show that a protein limited in its distribution to pathogenic Neisseria species and structurally related to enzymes implicated in phosphoethanolamine modification of lipopolysaccharide is necessary for PilE covalent modification with phosphoethanolamine and phosphocholine. These findings strongly suggest that protein phospho-form modification is mechanistically similar to processes underlying analogous modifications of prokaryotic saccharolipid glycans. We also show that PilE undergoes multisite and hierarchical phospho-form modifications and that the stoichiometries of site occupancy can be influenced by PilE primary structure and the abundance of the pilin-like protein PilV. Together, these findings have important implications for the structure and antigenicity of PilE.