淋巴特异的酪氨酸磷酸酶（Lyp）不同突变（S201F、R263Q和R266W）的生化特性与细胞功能研究

发布时间：2018-05-06 05:30

本文选题：淋巴特异蛋白酪氨酸磷酸酶 + T细胞信号途径　；参考：《山东大学》2012年硕士论文

【摘要】：Lyp (Lymphoid-specific tyrosine phosphatase)是由PTPN22编码的非受体型酪氨酸磷酸酶,主要在免疫系统中表达。Lyp由807个氨基酸构成,包括N端300个氨基酸的催化结构域和C端500个氨基酸左右的调节区域构成。当前的研究认为,Lyp可以通过去磷酸化Lck、Fyn、ZAP70等蛋白来负调控T细胞的信号转导。Lyp的单核苷酸多态性(single nucleotide polymorphism, SNP)C1858T使PTPN22在620位的酪氨酸替换为精氨酸(即R620W突变),携带R620W突变的人群更容易患自身免疫性疾病,包括Ⅰ型糖尿病(Type Ⅰ Diabetes, T1D)、系统性红斑狼疮(systemic lupus erythematosus, SLE)、风湿性关节炎(rheumatoid arthritis, RA)、幼年特发性关节炎(juvenile idiopathic arthritis, JIA)(?)Grave's disease等。虽然R620W一直被认为是功能增进型的突变,但是最近研究显示R620W导致自身免疫病可能由于它在T细胞信号转导过程中的功能缺失。同时有研究表明Lyp催化区域的基因多态性R263Q可以降低SLE、RA和溃疡性结肠炎(ulcerative colitis, UC)勺患病率,但增加肺结(pulmonary tuberculosis,PT)的易感性。遗传学研究还发现在人类中存在其他Lyp的基因多态性,包括S20lF,R266W等,其中S201F和R266W不增加Ⅰ型糖尿病的患病风险。为进一步分析这些基因多态性对Lyp生化性质和功能的影响,我们检测了Lyp基因多态性S201F、R263Q和R266W对小分子底物,磷酸多肽底物和蛋白底物的磷酸酶活力；以及这些突变导致Lyp在T细胞信号转导途径和免疫应答中功能的变化。本实验结果说明Lyp的S201F突变虽然改变了Lyp对小分子底物的催化活力,但其对磷酸多肽底物和蛋白底物的活力影响甚小,并对Lyp在T细胞中的功能产生较小的影响。Lyp的R263Q和R266W突变显著减低了其对磷酸多肽底物的活力,并减弱了Lyp对T细胞免疫应答的抑制作用。这些结果预示除R263Q可减低一些自身免疫性疾病的风险外,R266W基因多态性也可能通过影响Lyp的功能而对自身免疫性疾病易感性产生影响。 [研究目的] 研究淋巴组织特异酪氨酸磷酸酶(Lyp)的不同突变(S201F、R263Q和R266W的生化特性和细胞功能。 [研究方法] 1.生化特性 (1)用PrimerX软件设计引物,以PET-16b-Lyp-CD-His-wild type为模板用QuikChange的方法构建不同突变的Lyp的质粒：3201F, R263Q, R266W。 (2)在BL21大肠杆菌中过表达Lyp催化区域的蛋白(wild type, S201F, R263Q,R266W),通过Ni2+-NTA柱和CM阳离子交换柱纯化,以获得可以进行生化分析的高纯度蛋白。 (3)用纯化的蛋白进行酶动力学研究： 1)检测Lyp不同突变对小分子底物pNPP磷酸酶活力。 2)检测Lyp不同突变对含有磷酸化酪氨酸的9个氨基酸短肽pLck394的磷酸酶活力。 3)体外纯化Src蛋白,用western blotting检测Lyp不同突变对其416位点的磷酸酶活力。 4)比较Lyp wild type和R266W在pH值4-8.7时对pNPP的磷酸酶活力。 2.细胞功能用电转的方法将质粒(pcDNA4-Lyp-CD-mycHis-wild type, F201, Q263W266)转染Jurkat T细胞,培养细胞48小时后用培养基(对照)和anti-CD3刺激,用特异性的抗体检测野生型和各突变对ERK和Lck394磷酸化水平的影响,比较Lyp不同突变T细胞信号转导途径中磷酸化水平的影响。 [研究结果] 1.生化特性 (1)质粒构建成功。 (2)通过Ni2+-NTA柱和CM阳离子交换柱,Lyp wild type, S201F, R263Q和R266W的蛋白纯度都可以达到95%以上。 (3)酶动力学研究： 1)对小分子底物pNPP的磷酸酶活力：Kcat/Km值可以反映磷酸酶活力的大小Q263的Kcat/Km值与WT相比,差别没有统计学意义(P0.05)。Lyp的S201F,R266W的Kcat/Km值与野生型相比,分别降低了50%和80%。 2)对含有磷酸化酪氨酸的9个氨基酸短肽pLck394磷酸酶活力：F201,Q263,W266的Kcat/Km值与野生型相比,分别降低了20%,40%和60%。 3)对体外纯化Src蛋白的磷酸酶活力：反应5分钟,Lyp野生型可以使Src蛋白的416位酪氨酸磷酸化水平降低50%。Lyp的F201和Q263分别需要10分钟和30分钟使Src蛋白的416位酪氨酸磷酸化水平降低50%。而W266的活力显著降低,反应30分钟才能Src蛋白的416位酪氨酸磷酸化水平降低20%。 4)在不同的pH值下测Lyp野生型和W266对小分子底物pNPP的酶活变化,计算出酶限速反应阶段的解离常数pKb (wild type R266=6.54, W266=6.01)。 2.细胞功能野生型Lyp和三个突变能不同程度的影响T细胞内Lck和ERK的水平。抑制T细胞的能力：wild type) F201Q263 W266。 [结论] F201降低了Lyp磷酸酶活力,但对Lyp在T细胞信号转导中的作用基本无影响,而Q263、W266相比野生型的磷酸酶活力明显减弱,明显减弱了其对T淋巴细胞的免疫响应的抑制作用,因此称之为功能缺陷型突变。Q263由于功能缺失可以降低自身免疫性疾病的患病率,W266也可能降低某些自身免疫性疾病的患病率。抑制Lyp的磷酸酶活力可以作为治疗自身免疫性疾病的有效方法。
[Abstract]:Lyp is composed of 807 amino acids . Lyp consists of 807 amino acids , including the catalytic domain of 300 amino acids and the regulatory region about 500 amino acids in C terminal . It was found that R263Q could reduce the prevalence of SLE , RA and ulcerative colitis ( UC ) , but also increased the susceptibility of SLE , RA and ulcerative colitis ( UC ) .
These results suggest that Lyp ' s S201F mutation alters the catalytic activity of Lyp in T cell signaling pathway and immune response , but it has little effect on the activity of Lyp in T cells . The R263Q and R266W mutations of Lyp significantly reduce the activity of Lyp on T cell immune response . These results suggest that R263Q can reduce the risk of autoimmune diseases , and R266W gene polymorphism may also affect the susceptibility of autoimmune diseases by affecting the function of Lyp .

Purpose of research

The biochemical characteristics and cellular functions of the different mutations ( S201F , R263Q and R266W ) of lymphoid tissue specific tyrosine phosphatase ( Lyp ) were studied .

Methodology of research

1 . Biochemical characteristics

( 1 ) The primers were designed using the PrimerX software , and the plasmids of Lyp with different mutations were constructed by using the method of QuikChange in the form of PET - 16b - Lyp - CD - His - wild type : 3201F , R263Q , R266W .

( 2 ) The protein ( wild type , S201F , R263Q , R266W ) in the Lyp catalytic region was overexpressed in BL21 . coli , and purified by a Ni 2 + - nta column and a CM cation exchange column to obtain a high purity protein that can be subjected to biochemical analysis .

( 3 ) carrying out the enzymatic kinetics study with purified protein :

1 ) The pNPP phosphatase activity of the small molecule substrate was detected by detecting the different mutation of Lyp .

2 ) The phosphatase activity of 9 amino acid short peptide pLck394 containing phosphorylated tyrosine was detected by Lyp mutation .

The phosphatase activity of different mutation of Lyp was detected by western blotting .

4 ) The phosphatase activity of Lyp wild type and R266W at pH 4 - 8.7 was compared .

2 . Cell function

The effects of wild type and mutation on the phosphorylation of ERK and Lck394 were detected by using specific antibody to detect the phosphorylation of ERK and Lck394 from wild type and mutation , and the effect of Lyp on phosphorylation level in T cell signal transduction pathway was compared .

Outcome of the study

1 . Biochemical characteristics

( 1 ) Plasmid construction was successful .

( 2 ) The purity of the protein of Lyp wild type , S201F , R263Q and R266W can reach more than 95 % through Ni 2 + - nta column and CM cation exchange column .

( 3 ) Enzyme kinetics study :

1 ) The phosphatase activity of pNPP of small molecule substrate : Kcat / Km value can reflect the Kcat / Km value of phosphatase activity Q263 . Compared with WT , the difference is not significant ( P0.05 ) . The Kcat / Km value of Lyp is reduced by 50 % and 80 % compared with wild type .

2 ) The Kcat / Km values of 9 amino acid short peptides containing phosphorylated tyrosine were reduced by 20 % , 40 % and 60 % , respectively , compared with wild type in the Kcat / Km values of F201 , Q263 , W266 .

3 ) The phosphatase activity of the src protein was purified in vitro : reaction for 5 minutes , Lyp wild - type could decrease the tyrosine phosphorylation level of 416 - position tyrosine phosphorylation by 50 % . Lyp ' s F201 and Q263 required 10 minutes and 30 minutes to lower the tyrosine phosphorylation level of 416 - position tyrosine phosphorylation by 50 % . However , the activity of W266 decreased significantly , and the tyrosine phosphorylation level of 416 - position tyrosine phosphorylation decreased by 20 % in 30 minutes .

4 ) Under different pH values , Lyp wild type and W266 were tested for the enzyme activity of pNPP , and the dissociation constant pKb ( wild type R266 = 6.54 , W266 = 6.01 ) was calculated .

2 . Cell function

The wild type Lyp and the three mutations can affect the levels of Lck and ERK in T cells in varying degrees . The ability to inhibit T cells : wild type F201 Q263 W266 .

Conclusion

F201 reduced the activity of Lyp phosphatase , but had no effect on the role of Lyp in T cell signal transduction , but the activity of Q263 , W266 was obviously weakened compared with wild type phosphatase , thus it was called the function defect type mutation . Since function deletion could reduce the prevalence of autoimmune diseases , W266 could reduce the prevalence of autoimmune diseases .

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R341

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