Shan Weiran, Kung Hsiang-Fu, Ge Ruiguang
Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-Sen University, Guangzhou, China.
State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Sun Yat-Sen University Cancer Center, Guangzhou, China.
Helicobacter. 2016 Jun;21(3):192-200. doi: 10.1111/hel.12266. Epub 2015 Sep 8.
The neutrophil-activating protein (NapA) of Helicobacter pylori (H. pylori), with DNA-binding and iron seizing properties, is a fundamental virulence factor involved in H. pylori-related diseases. Compared with Ser70-NapA strain, Thr70-NapA strain is more intimately correlated with iron-deficiency anemia.
To investigate whether two types of proteins differ in iron-binding ability, mutated Thr70-NapA and Ser70-NapA strains were established. Isothermal titration calorimetry (ITC) method was conducted to measure the binding between the NapA protein and Fe(2+) . The structural changes of NapA protein were also tested during iron interaction by fast protein liquid chromatography (FPLC) and circular dichroism (CD) methods. DNA-binding assay was performed for evaluate the affinity of both mutated and wild types of NapA with DNA.
Mutated Thr70-NapA had higher iron-binding ability than wild Ser70-NapA. The structural stability of Thr70-NapA was disrupted and became more active along with the rising concentration of Fe(2+) , whereas no similar association was observed between Ser70-NapA and Fe(2+) level. When the iron/protein molar ratio ranged from 10 to 20, both Ser70-NapA and Thr70-NapA displayed weaker DNA-binding ability.
Thr70-NapA has much stronger ability to sequester ferrous ion compared with Ser70-NapA in H. pylori. In addition, the DNA-binding property of NapA is dependent upon the Fe(2+) concentration.
幽门螺杆菌的中性粒细胞激活蛋白（NapA）具有DNA结合和铁捕获特性，是参与幽门螺杆菌相关疾病的一种基本毒力因子。与Ser70-NapA菌株相比，Thr70-NapA菌株与缺铁性贫血的相关性更强。
为研究这两种蛋白质在铁结合能力上是否存在差异，构建了Thr70-NapA和Ser70-NapA突变菌株。采用等温滴定量热法（ITC）测定NapA蛋白与Fe(2+)之间的结合。还通过快速蛋白质液相色谱法（FPLC）和圆二色性（CD）方法检测了铁相互作用过程中NapA蛋白的结构变化。进行DNA结合试验以评估突变型和野生型NapA与DNA的亲和力。
突变型Thr70-NapA的铁结合能力高于野生型Ser70-NapA。随着Fe(2+)浓度升高，Thr70-NapA的结构稳定性受到破坏并变得更加活跃，而Ser70-NapA与Fe(2+)水平之间未观察到类似关联。当铁/蛋白摩尔比在10至20之间时，Ser70-NapA和Thr70-NapA的DNA结合能力均减弱。
在幽门螺杆菌中，Thr70-NapA螯合亚铁离子的能力比Ser70-NapA强得多。此外，NapA的DNA结合特性取决于Fe(2+)浓度。
