牙龈卟啉单胞菌FtsZ的C末端和N末端氨基酸残基缺失对其GTPase活性的影响

doi:10.7694/jldxyxb20130509

吉林大学学报(医学版)

牙龈卟啉单胞菌FtsZ的C末端和N末端氨基酸残基缺失对其GTPase活性的影响

于维先¹，刘歆婵^*，王闻天²，张玉凤³，高爱超¹

(1.吉林大学口腔医院牙发育及颌骨重塑与再生吉林省重点实验室，吉林长春 130021；2.吉林大学口腔医院麻醉科，吉林长春130021；3.吉林大学口腔医院修复科，吉林长春 130021）

收稿日期:2012-12-20 出版日期:2013-09-28 发布日期:2013-09-28
通讯作者: 张玉凤 E-mail:（Tel：0431-88796018，E-mail：568949871@qq.com）
作者简介:张毅（1969-），男，辽宁省锦州市人，主治医师，医学博士，主要从事肝癌的细胞信号转导与凋亡研究。
基金资助:
辽宁省科技厅科学技术计划项目资助课题(201300384)

Effects of C-terminal and N-terminal deletions of FtsZ in Porphyromonas Gingivalis on GTPase activity

YU Wei-xian¹,LIU Xin-chan^*,WANG Wen-tian²,ZHANG Yu-feng³,GAO Ai-chao¹

(1. Key Laboratory of Tooth Development and Bone Remodeling and Regeration,Jilin Province,Stomatology Hospital,Jilin University,Changchun 130021,China;2. Department of Anesthesiology,Stomatology Hospital,Jilin University,Changchun 130021,China;3. Department of Prosthodontics,Stomatology Hospital,Jilin University,Changchun 130021,China)

Received:2012-12-20 Online:2013-09-28 Published:2013-09-28

摘要/Abstract

摘要：

目的：探讨牙龈卟啉单胞菌（Pg）丝状温度敏感蛋白质（FtsZ）的C末端和N末端氨基酸残基缺失对其GTPase活性的影响，阐明PgFtsZ GTPase的功能区域，为牙周病的防治提供实验依据。方法：将质粒pEZ1(Wt PgFtsZ，含野生型PgFtsZ的基因)、pYW1（ZΔC01，从PgFtsZ的C末端去除73个氨基酸残基的变异型）、pYW2（ZΔC02，从PgFtsZ的C末端去除128个氨基酸残基的变异型）、pYWN1（ZΔN01，从PgFtsZ的N末端去除43个氨基酸残基的变异型）和pYWN2（ZΔN02，从PgFtsZ的N末端去除205个氨基酸残基的变异型）分别导入E.coli BL21(DE3)pLysS中表达目的蛋白，然后分离和纯化WtPgFtsZ、ZΔC01、ZΔC02、ZΔN01和ZΔN02，将pET3a 载体导入E.coli BL21(DE3)pLysS中作为空白对照。采用Malachite green assay法检测Wt PgFtsZ、ZΔC01、ZΔC02、ZΔN01和ZΔN02的GTPase活性，沉淀法检测体外聚合功能，光学显微镜下观察Wt PgFtsZ和各种变异型PgFtsZs过表达时E.coli细胞形态变化。结果：除ZΔN01外，ZΔC01、ZΔC02和ZΔN02的GTPase的活性值均较Wt PgFtsZ降低（P<0.05），10 mmol•L^-1 CaCl2可使Wt PgFtsZ和各种变异型PgFtsZs的活性下降（P<0.05）；除ZΔN02以外，10 mmol?L-1 CaCl2均可诱导Wt PgFtsZ、ZΔC01、ZΔC02和ZΔN01的体外聚合反应。Wt PgFtsZ、ZΔC01和ZΔC02在E.coli细胞中过表达时，细胞呈细长丝状，与仅含有pET3a载体作为空白对照的E.coli相比细胞明显变长；ZΔN01和ZΔN02在E.coli中过表达时，E.coli细胞形态与仅含有pET3a载体作为空白对照的E.coli细胞形态相似。结论：PgFtsZ N末端的ZΔN01和ZΔN02之间的162个氨基酸残基及C末端的128个氨基酸残基与PgFtsZ 的GTPase活性有关，二价阳离子Ca2+能抑制PgFtsZ的GTPase活性，促进其体外聚合的功能。

关键词: 牙龈卟啉单胞菌, 丝状温度敏感蛋白质Z, N末端, C末端, GTPase活性, Ca2+阳离子

Abstract:

Abstract:Objective
To investigate the effects of the C-terminal and N-terminal deletions of FtsZ in Porphyromonas gingivalis (Pg) on the GTPase activity and to clarify the domain of PgFtsZ GTPase,and to provide experimental basis for prevention and treatment of periodontal disease.Methods Plasmids pEZ1 (Wt PgFtsZ,including Wt PgFtsZ gene),pYW1(ZΔC01,missing 73 residues from C-terminus of PgFtsZ),pYW2(ZΔC02,missing 128 residues from C-terminus of PgFtsZ),pYWN1(ZΔN01,missing 43 residues from N-terminus of PgFtsZ),and pYWN2(ZΔN02,missing 205 residues from N-terminus of PgFtsZ) were respectively introduced into E.coli BL21(DE3)pLysS to express，seperate and purify WtPgFtsZ,ZΔC01,ZΔC02,ZΔN01 and ZΔN02.pET3a vector alone was introduced into E.coli BL21(DE3)pLysS as negative control.Malachite green assay was used to measure the GTPase activities of Wt PgFtsZ,ZΔC01,ZΔC02,ZΔN01 and ZΔN02.The assemblies of the proteins were detected by sedimentation in vitro.The morphologies of E.coli that overexpressed Wt PgFtsZ and mutants proteins were observed under light microscope.Results The GTPase activity of ZΔN01 was similar to the GTPase activity of Wt PgFtsZ.However,the GTPase activities of ZΔC01,ZΔC02,and ZΔN02 were lower than that of the Wt PgFtsZ (P<0.05).10 mmol•L^-1 CaCl2 reduced the GTPase activities of Wt PgFtsZ and PgFtsZ mutants (P<0.05).In addition to ZΔN02,10 mmol?L-1 CaCl2 induced the polymerizations of Wt PgFtsZ,PgFtsZ mutants.When wild-type PgFtsZ,ZΔC01 and ZΔC02 overexpressed in E.coli,respectively,and the E.coli cells became long filamentous compared with the E.coli cells containing pET3a vector.However,ZΔN01 and ZΔN02 overexpressed in E.coli,respectively,and the E.coli morphology had similar to that of E.coli cells containing pET3a vector.Conclusion Missing 162 residues from the N-terminus of PgFtsZ between ZΔN01 and ZΔN02 and missing 128 residues from the C-terminus of PgFtsZ has relationship with the GTPase activity of PgFtsZ.Ca2+ cation can inhibit GTPase activity of PgFtsZ and promote its polymerization in vitro.


Key words: prophyromonas qingivalis, filamentous temperature-sensitive protein Z, N-termina, C-terminal, GTPase activity, calcium cationic

中图分类号:

R781.4

于维先，刘歆婵，王闻天，张玉凤，高爱超. 牙龈卟啉单胞菌FtsZ的C末端和N末端氨基酸残基缺失对其GTPase活性的影响[J]. 吉林大学学报(医学版), 2013, 39(5): 898-902.

YU Wei-xian,LIU Xin-chan,WANG Wen-tian,ZHANG Yu-feng,GAO Ai-chao. Effects of C-terminal and N-terminal deletions of FtsZ in Porphyromonas Gingivalis on GTPase activity[J]. Journal of Jilin University Medicine Edition, 2013, 39(5): 898-902.

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牙龈卟啉单胞菌FtsZ的C末端和N末端氨基酸残基缺失对其GTPase活性的影响

Effects of C-terminal and N-terminal deletions of FtsZ in Porphyromonas Gingivalis on GTPase activity

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