蛋白质酪氨酸磷酸酶SHP-1催化结构域的克隆表达及抑制剂筛选
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摘要
蛋白质酪氨酸磷酸酶(PTP)大家族,是信号转导中的重要调节因子,与人类疾病密切相关。SHP-1是主要表达于造血细胞的酪氨酸磷酸酶,其基因位于第12号染色体的p12-p13区,全长17Kb。本文以人蛋白质酪氨酸磷酸酶SHP-1基因为模板,克隆了SHP-1的催化结构域(△SHP-1)基因,并连接在表达载体pT7上,将重组体pT7-ΔSHP-1质粒转化到E.coil DE3细胞中进行高效表达,通过Q-Sepharose Fast Flow和SP-Sephadex两步离子交换柱层析,分离纯化得到纯度﹥90%的△SHP-1蛋白纯品。对ΔSHP-1的酶学性质研究表明,当底物为p-NPP时,ΔSHP-1的最适pH为5.0,最适反应温度为36℃,最适离子强度为0;对其进行酶促反应进行动力学分析,得出Km=23.75mM,Vmax=8.882μmol/min。
应用蛋白质酪氨酸磷酸酶SHP-1的催化结构域(ΔSHP-1)为靶标,通过体外酶反应动力学实验,从大量的中药中筛选出两种对其有明显抑制作用的中药:中药A和中药B,它们对?SHP-1的IC_(50)分别为2ug/ml和4ug/ml,而且通过动物实验,证明这两种中药对II型糖尿病模型小鼠均具有显著的降血糖作用。实验结果表明,中药A和中药B有可能通过抑制体内SHP-1的活性,从而降低了II型糖尿病模型小鼠的血糖。
采用靶标进行药物筛选,是创新药物的研究方法之一,药效确切、机理清晰、质量标准可靠,具有特异性和准确性,也可以发现原有药物的新用途,从而实现中药产品的二次开发。本研究为寻找有效的中药降糖药物奠定了基础,也为中药A和中药B在治疗II型糖尿病中的应用提供了理论依据。
Protein tyrosine dephosphorylation by protein tyrosine phosphatase (PTPs) and phosphorylation by protein tyrosine kinases (PTKs) both play an important role in regulating cell growth, cell development and cell signal transduction, which are involved in many physiological and pathological phenomena. A lot of studies have shown that human diseases such as cancers, diabetes, leukemia, immuno-deficiency diseases and result from mutations or abnormality of PTPs genes. Therefore, more and more studies are focusing on PTPs.
SHP-1(SH2-containing tyrosine phosphatase 1), also called HCP, SHPTP1 and PTP1C, is an important member of protein tyrosine phosphotase subfamily with highly conserved sequence. SHP-1 is a kind of cytosolic protein containing two SH2 domains at the N-terminal and one catalytic domain at the C-terminal. It was reported that increased SHP-1 activity led to immuno-deficient syndrome andn leukemia. The most recent studies have shown that SHP-1 is related to type II diabetes and high level of blood sugar is due to elevated SHP-1 activity which means that endogenous SHP-1 plays a critical role in regulating blood sugar metabolism and SHP-1 may be a potential target for reducing the blood sugar of diabetes patients by inhibiting its activity.
The catalytic domain△SHP-1 of SHP-1 was amplified from human SHP-1 cDNA and inserted into pT7 vector which is Amp resistant. High expression of the protein products was performed using E. coli DE3 strain and the enzymatic characteristics of△SHP-1 were determined after isolation and purification through ion-exchange chromatography. Also, purified△SHP-1 was used to stimulate rabbit for polyclonal antibody preparation.
Many traditional Chinese herbs have significant effect on diabetes through different mechanisms. The results of our in vitro assays show that among hundreds of traditional Chinese herbs screened, the water-extracts of herb A and herb B inhibit the activity of ?SHP-1 effectively. The animal assays provide further evidence that both of these two herbs significantly reduce the blood sugar of type II diabetes mice.
1. The Cloning and Expression of Catalytic Domain of SHP-1
The catalytic domain encoding sequence was amplified from human SHP-1 cDNA, and two sites for restriction enzyme Nde I and Hind III were introduced into N-terminal and C-terminal, respectively.ΔSHP-1 in the PCR products investigated by agar electrophoresis is ligated into the PKS clone vector which is digested by EcoRV. After that, PKS-ΔSHP-1 is digested by Nde I and Hind III ,check out then constructed into pT7 expression vector. pT7-ΔSHP-1 is delt with T4 DNA ligase, the recombinant plasmid was subsequently transformed into E.coli DE3 and high expression ofΔSHP-1 protein was achieved.
2. Isolation, Purification and enzyme characterization ofΔSHP-1
Two steps of ion-exchange chromatography, Q-Sepharose Fast Flow and SP-Sephadex, were used to isolate and purifyΔSHP-1 protein obtained from E.coli DE3 strain. The purity of target protein was more than 90% determined by SDS-PAGE and HPLC analysis. The results of enzymatic characteristics determination indicated that when p-NPP was added as substrate, the optimal pH forΔSHP-1 activity was 5.0, optimal temperature 36℃and ion concentration 0. The enzyme was most stable at pH 7.5, 15℃and without ions. The kinetics analysis of its catalytic reaction showed that the Km was 23.75mM and Vmax was 8.882μmol/min.
3. The Preparation and Purification of Polyclonal Antibody ofΔSHP-1
The purifiedΔSHP-1 was used to stimulate rabbit, and anti-ΔSHP-1 polyclonal antibody was gained by applying PVDF column. Before and after purification of antibody, by means of ECL , the both efficency were 1:10000, the sensitivity is 0.01μg and 1 ng respectively which is increased to 10 fold. The purity of antibody is over 90% by the investigation of SDS-GAGE and western blot. These results indicated the antibody match to and surpass the standard for further experiments. The purified antibody is dealt with at 56℃for 30 min ,added by azide sodium with final concentration 1/1000(W/V) then fractioned which was stored under -80 oC.
4. Screening Traditional Chinese Herbs for Inhibitors of Protein Tyrosine Phosphatase SHP-1
4.1 Determine the inhibition ratios of Traditional Chinese Herbs
20g herb was added to 200ml distilled water, soaked for 1 hour, heated until boiling and held for 30min. The mixture was filtered before another 150ml distilled water was added to the residue and boiled for 20 more minutes. Then the mixture was filtered again. The liquors from these two steps were combined and centrifuged at 4℃and the supernate was collected as water extract of herbs. p–NPP was used as substrate for measuring the inhibitory rate of herb extract onΔSHP-1. The reaction system contained 20mM p-NPP, Mops-NaOH buffer (25mM,pH7.0,1mM DTT,0.1M NaCl,1mM EDTA,1mg/ml BSA),ΔSHP-1(10μg/ml)and 10ul extract. The reaction condition was designed as incubation at 37℃for 20min, and then the absorbance was measured at 405nm. Each experiment was performed in duplicate and the inhibitory rate of each extract was calculated.
Two herbs which exhibited more than 95% inhibition onΔSHP-1 were screened from about 160 kins of traditional Chinese herbs, herb A 96.77% and herb B 95.10%, respectively.
4.2 Determination of IC_(50):
IC_(50) was analyzed with 20mM p-NPP as a substrate in a buffer (pH=7.0) containing 20mM Mops, 1mM DTT, 0.1M NaCl, 1mM EDTA, and 1 mg/ml BSA for 20mins at 37℃. 10μg/mlΔSHP-1 and varying concentrations of herb A or herb B extract were added to the buffer. Then the absorbance of p-NPP at 405nm was analyzed after the termination of the reaction with NaOH. IC_(50) values are calculated by constructing dose-response curves, and, as a result, the IC_(50) of P herb A and herb B are 2μg/ml and 4μg/ml, respectively.
5. Herb A and Herb B decrease the blood glucose of TypeⅡDiabetes Mice
5.1 The model of TypeⅡDiabetes Mice
40 mice were selected for establishing type II diabete animal model which were injected with 50mgSTZ/kg per day for 5 days. The control group were 10 mice which were injected with pt aequ citrate buffer solution. After the treatment, each mouse was weighed every day and the blood sugar was detected every week using Blood Glucose Kit. The mouse with more than 11.1mmol/L blood sugar was considered as established model.
Our result showed that after 14 days of treatment, 37 mice out of 40 had significantly increased blood sugar level with average 28.3mmol/L, which meant that the type II diabetes model was well established.
5.2 Experiment of administer
The mice were randomly grouped into control group, herb-treated group, and negative control group. Both the control group (healthy mice) and the negative control group (type II diabetic mice) were administrated with 25ml/kg water according to body weights, while the herb-treated group (type II diabetic mice) was administrated with 10g/kg herbs according to body weights for 25days. Body weight, food intake, water intake, and miction variation were observed everyday. Blood glucose levels were analyzed every five days after mice’being abrosia for 16hrs. The blood glucose values were calculated according to the standard preparation.
Our animal experiments provided strong evidence that both of these two herbs, herb A and herb B significantly reduced the blood sugar of type II diabetes animal model with 19.6mmol/L and19.4mmol/L, respectively.
Screening effective drugs based on their direct targets is one of the methods for new drug discovery, which has multiple advantages including precise efficacy, clear mechanism as well as reliable quality. Besides its specificity and accuracy, this method can be utilized to find out the novel potency of existing drugs, which may lead to the secondary development of Chinese traditianal herbs.
应用蛋白质酪氨酸磷酸酶SHP-1的催化结构域(ΔSHP-1)为靶标,通过体外酶反应动力学实验,从大量的中药中筛选出两种对其有明显抑制作用的中药:中药A和中药B,它们对?SHP-1的IC_(50)分别为2ug/ml和4ug/ml,而且通过动物实验,证明这两种中药对II型糖尿病模型小鼠均具有显著的降血糖作用。实验结果表明,中药A和中药B有可能通过抑制体内SHP-1的活性,从而降低了II型糖尿病模型小鼠的血糖。
采用靶标进行药物筛选,是创新药物的研究方法之一,药效确切、机理清晰、质量标准可靠,具有特异性和准确性,也可以发现原有药物的新用途,从而实现中药产品的二次开发。本研究为寻找有效的中药降糖药物奠定了基础,也为中药A和中药B在治疗II型糖尿病中的应用提供了理论依据。
Protein tyrosine dephosphorylation by protein tyrosine phosphatase (PTPs) and phosphorylation by protein tyrosine kinases (PTKs) both play an important role in regulating cell growth, cell development and cell signal transduction, which are involved in many physiological and pathological phenomena. A lot of studies have shown that human diseases such as cancers, diabetes, leukemia, immuno-deficiency diseases and result from mutations or abnormality of PTPs genes. Therefore, more and more studies are focusing on PTPs.
SHP-1(SH2-containing tyrosine phosphatase 1), also called HCP, SHPTP1 and PTP1C, is an important member of protein tyrosine phosphotase subfamily with highly conserved sequence. SHP-1 is a kind of cytosolic protein containing two SH2 domains at the N-terminal and one catalytic domain at the C-terminal. It was reported that increased SHP-1 activity led to immuno-deficient syndrome andn leukemia. The most recent studies have shown that SHP-1 is related to type II diabetes and high level of blood sugar is due to elevated SHP-1 activity which means that endogenous SHP-1 plays a critical role in regulating blood sugar metabolism and SHP-1 may be a potential target for reducing the blood sugar of diabetes patients by inhibiting its activity.
The catalytic domain△SHP-1 of SHP-1 was amplified from human SHP-1 cDNA and inserted into pT7 vector which is Amp resistant. High expression of the protein products was performed using E. coli DE3 strain and the enzymatic characteristics of△SHP-1 were determined after isolation and purification through ion-exchange chromatography. Also, purified△SHP-1 was used to stimulate rabbit for polyclonal antibody preparation.
Many traditional Chinese herbs have significant effect on diabetes through different mechanisms. The results of our in vitro assays show that among hundreds of traditional Chinese herbs screened, the water-extracts of herb A and herb B inhibit the activity of ?SHP-1 effectively. The animal assays provide further evidence that both of these two herbs significantly reduce the blood sugar of type II diabetes mice.
1. The Cloning and Expression of Catalytic Domain of SHP-1
The catalytic domain encoding sequence was amplified from human SHP-1 cDNA, and two sites for restriction enzyme Nde I and Hind III were introduced into N-terminal and C-terminal, respectively.ΔSHP-1 in the PCR products investigated by agar electrophoresis is ligated into the PKS clone vector which is digested by EcoRV. After that, PKS-ΔSHP-1 is digested by Nde I and Hind III ,check out then constructed into pT7 expression vector. pT7-ΔSHP-1 is delt with T4 DNA ligase, the recombinant plasmid was subsequently transformed into E.coli DE3 and high expression ofΔSHP-1 protein was achieved.
2. Isolation, Purification and enzyme characterization ofΔSHP-1
Two steps of ion-exchange chromatography, Q-Sepharose Fast Flow and SP-Sephadex, were used to isolate and purifyΔSHP-1 protein obtained from E.coli DE3 strain. The purity of target protein was more than 90% determined by SDS-PAGE and HPLC analysis. The results of enzymatic characteristics determination indicated that when p-NPP was added as substrate, the optimal pH forΔSHP-1 activity was 5.0, optimal temperature 36℃and ion concentration 0. The enzyme was most stable at pH 7.5, 15℃and without ions. The kinetics analysis of its catalytic reaction showed that the Km was 23.75mM and Vmax was 8.882μmol/min.
3. The Preparation and Purification of Polyclonal Antibody ofΔSHP-1
The purifiedΔSHP-1 was used to stimulate rabbit, and anti-ΔSHP-1 polyclonal antibody was gained by applying PVDF column. Before and after purification of antibody, by means of ECL , the both efficency were 1:10000, the sensitivity is 0.01μg and 1 ng respectively which is increased to 10 fold. The purity of antibody is over 90% by the investigation of SDS-GAGE and western blot. These results indicated the antibody match to and surpass the standard for further experiments. The purified antibody is dealt with at 56℃for 30 min ,added by azide sodium with final concentration 1/1000(W/V) then fractioned which was stored under -80 oC.
4. Screening Traditional Chinese Herbs for Inhibitors of Protein Tyrosine Phosphatase SHP-1
4.1 Determine the inhibition ratios of Traditional Chinese Herbs
20g herb was added to 200ml distilled water, soaked for 1 hour, heated until boiling and held for 30min. The mixture was filtered before another 150ml distilled water was added to the residue and boiled for 20 more minutes. Then the mixture was filtered again. The liquors from these two steps were combined and centrifuged at 4℃and the supernate was collected as water extract of herbs. p–NPP was used as substrate for measuring the inhibitory rate of herb extract onΔSHP-1. The reaction system contained 20mM p-NPP, Mops-NaOH buffer (25mM,pH7.0,1mM DTT,0.1M NaCl,1mM EDTA,1mg/ml BSA),ΔSHP-1(10μg/ml)and 10ul extract. The reaction condition was designed as incubation at 37℃for 20min, and then the absorbance was measured at 405nm. Each experiment was performed in duplicate and the inhibitory rate of each extract was calculated.
Two herbs which exhibited more than 95% inhibition onΔSHP-1 were screened from about 160 kins of traditional Chinese herbs, herb A 96.77% and herb B 95.10%, respectively.
4.2 Determination of IC_(50):
IC_(50) was analyzed with 20mM p-NPP as a substrate in a buffer (pH=7.0) containing 20mM Mops, 1mM DTT, 0.1M NaCl, 1mM EDTA, and 1 mg/ml BSA for 20mins at 37℃. 10μg/mlΔSHP-1 and varying concentrations of herb A or herb B extract were added to the buffer. Then the absorbance of p-NPP at 405nm was analyzed after the termination of the reaction with NaOH. IC_(50) values are calculated by constructing dose-response curves, and, as a result, the IC_(50) of P herb A and herb B are 2μg/ml and 4μg/ml, respectively.
5. Herb A and Herb B decrease the blood glucose of TypeⅡDiabetes Mice
5.1 The model of TypeⅡDiabetes Mice
40 mice were selected for establishing type II diabete animal model which were injected with 50mgSTZ/kg per day for 5 days. The control group were 10 mice which were injected with pt aequ citrate buffer solution. After the treatment, each mouse was weighed every day and the blood sugar was detected every week using Blood Glucose Kit. The mouse with more than 11.1mmol/L blood sugar was considered as established model.
Our result showed that after 14 days of treatment, 37 mice out of 40 had significantly increased blood sugar level with average 28.3mmol/L, which meant that the type II diabetes model was well established.
5.2 Experiment of administer
The mice were randomly grouped into control group, herb-treated group, and negative control group. Both the control group (healthy mice) and the negative control group (type II diabetic mice) were administrated with 25ml/kg water according to body weights, while the herb-treated group (type II diabetic mice) was administrated with 10g/kg herbs according to body weights for 25days. Body weight, food intake, water intake, and miction variation were observed everyday. Blood glucose levels were analyzed every five days after mice’being abrosia for 16hrs. The blood glucose values were calculated according to the standard preparation.
Our animal experiments provided strong evidence that both of these two herbs, herb A and herb B significantly reduced the blood sugar of type II diabetes animal model with 19.6mmol/L and19.4mmol/L, respectively.
Screening effective drugs based on their direct targets is one of the methods for new drug discovery, which has multiple advantages including precise efficacy, clear mechanism as well as reliable quality. Besides its specificity and accuracy, this method can be utilized to find out the novel potency of existing drugs, which may lead to the secondary development of Chinese traditianal herbs.
引文
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[17] 戚向阳,陈维军,宋云飞,等.罗汉果提取物对糖尿病小鼠的降血糖作用.中国公共卫生,2003,19(10):1226-1227.
[18] 郝海平,许惠琴,朱荃,等.环烯醚萜总苷对糖尿病血管并发症大鼠NO、NOS 和 ET 的影响.南京中医药大学学报,2003,19(3):157-158.
[19] 高小平,张蔚瑜,邹文俊,等.中药提取物中 α-葡萄糖苷酶抑制剂的筛选.天然产物研究与开发,2003,15(6):536-538.
[20] 刘 霞,冯长根.酶抑制剂在抗糖尿病药物中的应用研究.中国药学杂志, 2003, 38(2): 89.
[21] 侯宁宁,隋国良,刘国庆,等.第59届美国糖尿病协会(ADA)年会会议纪要.中国糖尿病杂志, 1999, 7(5): 313.
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