重组人ADAM15去整合素结构域蛋白的制备及其作用机理
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摘要
ADAM (A Disintegrin And Metalloproteinase)是一类含有七个功能结构域并在细胞间相互识别和相互作用中发挥重要作用的跨膜糖蛋白家族。ADAM15作为该家族唯一在去整合素结构域(disintegrin)含有RGD(Arg-Gly-Asp)基序的成员,在细胞外基质降解、细胞粘附、细胞内信号转导和肿瘤的病理变化等过程中发挥重要作用,但是其抗肿瘤机理需进一步阐释。本文在成功获取高表达水平的重组人ADAM15去整合素结构域蛋白片段(rhddADAM15)的基础上,以小鼠黑色素瘤细胞(B16)为研究模型,研究rhddADAM15对B16细胞体外增殖、粘附和迁移等细胞行为的影响,通过筛选rhddADAM15特异结合的12肽以及rhddADAM15在B16细胞上相互作用的靶点蛋白,探讨rhddADAM15干预肿瘤细胞行为的生理机制。主要研究结果如下:
1)为获得大量具有生物活性的rhddADAM15蛋白,在详尽分析目标蛋白的cDNA序列的基础上,(1)选择能为大肠杆菌稀有密码子提供额外tRNA的大肠杆菌Escherichia coli Rosetta(DE3)作为宿主菌,将目标蛋白以GST(Glutathione-S-transferase)融合蛋白形式表达,在最佳诱导表达条件下融合蛋白GST-ADAM15浓度为298 mg/L,最佳凝血酶酶切条件下,rhddADAM15浓度为42 mg/L; (2)采用PCR体外定点突变技术将凝血酶识别序列附近稀有密码子GGA(Gly)替换为GGC,同时消除Pro-Glu-Phe残基,构建突变表达质粒,提高凝血酶酶切效率。在相同的表达和酶切条件下获得GST-△-ADAM15和rhddADAM15蛋白浓度分别为326 mg/L和68mg/L,比野生型分别提高9.4%和61.9%。这一结果表明,在充分认识目标蛋白特性的基础上定向选择表达宿主并改造表达质粒能实现外源蛋白高水平表达;
2)采用MTT法测定rhddADAM15对B16、MCF-7/W以及HMEC-1细胞体外增殖的作用,采用创伤愈合实验、Matrigel基质胶以及Transwell侵袭小室法观察rhddADAM15对B16细胞体外迁移、粘附和侵袭的影响;采用台盼蓝染色法分析细胞活力、流式细胞仪检测rhddADAM15对B16细胞周期的影响。结果表明rhddADAM15对肿瘤细胞和正常内皮细胞生长均有一定的抑制作用(其IC50分别为9.5、11.8和14.9μg/mL),且明显抑制B16细胞的体外增殖、迁移、粘附和侵袭。台盼蓝染色法表明低剂量的rhddADAM15并不能导致B16细胞出现坏死,且rhddADAM15并不导致B16细胞出现典型的凋亡峰,而是将细胞阻滞于G2/ M期,并且这种作用呈剂量依赖关系;
3)采用亲和甄别磁珠技术筛选rhddADAM15在B16细胞上的结合蛋白。将rhddADAM15采用蛋白生物素化方法标记生物素,借助生物素-亲和素系统,将生物素化的rhddADAM15固定于亲和素标记的免疫磁珠。从B16细胞裂解液中钓出与之相互作用的蛋白,经浓缩富集后,采用双向电泳分离技术分离获得主要的结合蛋白点,然后对这些蛋白点进行电喷雾串联质谱技术(ESI-MS)分析,在蛋白数据库中检索,共鉴定了8个蛋白质,按功能可分为如下三类:(1)临床应用的肿瘤标志物(苹果酸脱氢酶、磷酸甘油醛脱氢酶和谷氨酸脱氢酶);(2)能量代谢相关蛋白(烯醇酶、磷酸丙糖异构酶、原肌球蛋白和血红蛋白);(3)信号转导通路成员(p38MAPK激酶)等。在此基础上通过分子对接软件molsoft ICM模拟计算获得rhddADAM15与p38激酶相互作用的最佳模式和重要的结合位点;
4)采用噬菌体展示技术筛选与rhddADAM15特异性相互作用的多肽序列。采用噬菌体随机十二肽库对固定有rhddADAM15的亲和磁珠系统进行生物淘洗,获得4条与rhddADAM15特异结合的12肽。将获得的12肽与蛋白质库进行序列比对,发现细胞周期相关蛋白如Cdc25和p53,信号通路相关蛋白/受体,如整合素αvβ3的胞外区,丝裂原活化蛋白激酶p38α等与所获得的4条12肽具有高度同源性;
5)基于上述研究所获得的rhddADAM15在B16细胞上潜在的靶点蛋白,进一步研究rhddADAM15与p38MAPK激酶的体外相互作用,结合rhddADAM15对B16细胞行为的抑制作用,采用蛋白点杂交和激酶活性测定方法分析rhddADAM15在体外与重组p38激酶蛋白的结合情况,同时研究不同剂量rhddADAM15对B16细胞中p38MAPK激酶磷酸化水平的影响,并观察rhddADAM15对p38激酶活性受到部分抑制的B16细胞的增殖和细胞周期抑制作用的变化。发现:(1)rhddADAM15在离体低温条件下能够和重组p38激酶蛋白结合,但rhddADAM15(0-10μg/mL)在体外对p38MAPK激酶活性没有影响;(2)同样剂量的rhddADAM15作用于B16细胞24 h后,细胞中p38MAPK激酶的磷酸化水平提高(被激活);(3)p38激酶的特异性抑制剂SB203580(0.5 mmol/L)预处理B16细胞30 min后,rhddADAM15对细胞增殖的抑制作用减弱(IC50由未经处理的9.5μg/mL增加到11.4μg/mL),对细胞周期的阻滞作用也减弱(G2/M期细胞比率从未经处理的的28.77%降到16.59%)。上述结果表明,p38MAPK信号转导通路参与了rhddADAM15对B16细胞增殖和细胞周期的抑制作用,同时也表明生理条件下rhddADAM15与p38MAPK激酶相互作用的复杂性。
A Disintegrin And Metalloproteases (ADAM) proteins are a family of multifunctional proteins containing disintegrin and metalloproteinase domains that perform both adhesive and proteolytic functions in cell-cell and cell-matrix interactions. ADAM15 is unique among these proteins in having an Arg-Gly-Asp (RGD) motif in its disintegrin-like domain. This dissertation chose the B16 murine melanoma cell as a model, to investigate the effect of the recombinant human ADAM15 disintegrin domain (rhddADAM15) on B16 cells gowth, and the target protein of rhddADAM15 on B6 cells, and further study the involvement of the target protein in the anti-cancer effect of rhddADAM15. The main results were described as follows:
1) In order to enhance the production and bioactivity of rhddADAM15 in Escherichia coli, two different strategies were examined based on the genetic analysis of coding sequence of rhddADAM15: to select the suitable host strain, and to optimize the rare codons and delete the amino acids residues. It was found that (1) when choosing E. coli Rosetta (DE3) as the expression host that supplys additional tRNA for rare codons, 298 mg/L GST-ADAM15 and 42 mg/L rhddADAM15 were achieved under the optimal expression and thrombin digestion conditions, respectively; (2) when introducing E. coli preferred codon GGC and deleting“Pro-Glu-Phe”residues by PCR-based site-directed mutagenesis, 326 mg/L GST-△-ADAM15 and 68 mg/L rhddADAM15 were achieved under the same condition, respectively, higher 19.4% and 61.9% than that of the wide type strain. The data presented here demonstrated that high expression of heterologous protein could be achieved by releasing rare codon usage and amino acids residues restriction.
2) The effect of rhddADAM15 on the proliferation of B16, MCF-7/W and HMEC-1 cells were examined by MTT assay. The effect of rhddADAM15 on B16 cell migration, adhesion and invention were tested by the wound migration assay, cell-Matrigel adhesion assay and the Transwell chambers, respectively; The growth and activity of B16 cells were explored by using trypan blue staining. The cell cycle and apoptosis were determined by flow cytometry. The results demonstrated that rhddADAM15 could inhibit the proliferation of normal cells, but exhibit stronger inhibitory effect on the tumor cells. Moreover, rhddADAM15 inhibited B16 cell migration, adhesion and invasion in the dose/time-dependent mode and the necrosis could not be observed in the low concentration of rhddADAM15. The apoptotic peaks were not found in cell cycle analyses and the cells are arrested in G2/M phase.
3) The protein pull-down assay using the biotin-avidin system supported by the immol/Lunomagnetic beads was used to capture the binding protein of rhddADAM15 from the cell lysis buffer of B16. The mixture of the binding protein was concentrated and separated by the two-dimensional eletrophoresis (2DE) and eight of the protein spots were identified by the ESI-MS/MS. By using the software BIOWORKS and the protein database ipi MOUSE v3.26, the binding proteins were mainly identified as follows: (1) Tumor markers in the clinical application: malate dehydrogenase, phosphate dehydrogenase and glutamate dehydrogenase; (2) Enzymes in glycolytic pathway: alpha-enolase and triosephosphate isomerase; (3) The important components in cytoskeleton: tropomyosin-3; (4) Proteins in oxygen transportation: hemoglobin subunit beta-1; (5) Members in signal transduction: p38MAPK. In addition, the interaction modes between rhddADAM15 and p38MAPK were preliminarily analyzed using the molecular docking program, molsoft ICM-PRO-3.5.
4) The target peptides of rhddADAM15 were explored by the phage display technique. Firstly, the rhddADAM15 was biotinylated and immol/Lobilized on the immol/Lunomagnetic beads with avidin labeled. Secondly, four specific binding peptides (peptide A, B, C and D) were screened from a phage display-12 peptide library by a screening protocol towards immol/Lobilized rhddADAM15. By using the software BLAST and the relevant protein database, some target proteins were screened, such as the Cdc25 phosphatase, tyrosine kinase p56, integrinαvβ3, and the p38 mitogen activated protein kinase (p38MAPK), related with the cell cycles and the signal transduction.
5) The p38MAPK was selected for further investigation of the involvement in the anti-proliferative effect of rhddADAM15 on melanoma cells. Firstly, the effect of rhddADAM15 on activity of p38 MAPK in vitro was observed. Secondly, phosphorylation of p38 kinase in melanoma cells was detected by Western blot analysis upon treatment with rhddADAM15 (0-10μg/mL). Thirdly, the specific inhibitor of p38 kinase (SB203580) was employed to partly suppress the activity of p38 kinase in B16 cells, and the inhibitory effect of rhddADAM15 was then observed. The results were demonstrated as follows: (1) rhddADAM15 could bind to the recombinant p38 kinase at 4 oC but had no effect on the activity of p38 kinase in vitro; (2) rhddADAM15 had inhibitory effect on the activity of p38 kinase in B16 cells; (3) A decrease in the rhddADAM15 inhibitory effect on melanoma cell proliferation, and the value of IC50 was increased from 9.5μg/mL to11.4μg/mL, when the activity of p38 kinase was partly suppressed; (4) A release in the cell cycle arrest, the cell ratio in G2/M phase was decreased from 28.77% to16.59%, under the condition of suppressing the p38 kinase. These results provided evidences that rhddADAM15 inhibiting the proliferation and the cell cycle of the melanoma cells was partly through p38 kinase activation and the p38MAPK signal transduction pathway was involved in the effect of rhddADAM15 on B16 cells.
1)为获得大量具有生物活性的rhddADAM15蛋白,在详尽分析目标蛋白的cDNA序列的基础上,(1)选择能为大肠杆菌稀有密码子提供额外tRNA的大肠杆菌Escherichia coli Rosetta(DE3)作为宿主菌,将目标蛋白以GST(Glutathione-S-transferase)融合蛋白形式表达,在最佳诱导表达条件下融合蛋白GST-ADAM15浓度为298 mg/L,最佳凝血酶酶切条件下,rhddADAM15浓度为42 mg/L; (2)采用PCR体外定点突变技术将凝血酶识别序列附近稀有密码子GGA(Gly)替换为GGC,同时消除Pro-Glu-Phe残基,构建突变表达质粒,提高凝血酶酶切效率。在相同的表达和酶切条件下获得GST-△-ADAM15和rhddADAM15蛋白浓度分别为326 mg/L和68mg/L,比野生型分别提高9.4%和61.9%。这一结果表明,在充分认识目标蛋白特性的基础上定向选择表达宿主并改造表达质粒能实现外源蛋白高水平表达;
2)采用MTT法测定rhddADAM15对B16、MCF-7/W以及HMEC-1细胞体外增殖的作用,采用创伤愈合实验、Matrigel基质胶以及Transwell侵袭小室法观察rhddADAM15对B16细胞体外迁移、粘附和侵袭的影响;采用台盼蓝染色法分析细胞活力、流式细胞仪检测rhddADAM15对B16细胞周期的影响。结果表明rhddADAM15对肿瘤细胞和正常内皮细胞生长均有一定的抑制作用(其IC50分别为9.5、11.8和14.9μg/mL),且明显抑制B16细胞的体外增殖、迁移、粘附和侵袭。台盼蓝染色法表明低剂量的rhddADAM15并不能导致B16细胞出现坏死,且rhddADAM15并不导致B16细胞出现典型的凋亡峰,而是将细胞阻滞于G2/ M期,并且这种作用呈剂量依赖关系;
3)采用亲和甄别磁珠技术筛选rhddADAM15在B16细胞上的结合蛋白。将rhddADAM15采用蛋白生物素化方法标记生物素,借助生物素-亲和素系统,将生物素化的rhddADAM15固定于亲和素标记的免疫磁珠。从B16细胞裂解液中钓出与之相互作用的蛋白,经浓缩富集后,采用双向电泳分离技术分离获得主要的结合蛋白点,然后对这些蛋白点进行电喷雾串联质谱技术(ESI-MS)分析,在蛋白数据库中检索,共鉴定了8个蛋白质,按功能可分为如下三类:(1)临床应用的肿瘤标志物(苹果酸脱氢酶、磷酸甘油醛脱氢酶和谷氨酸脱氢酶);(2)能量代谢相关蛋白(烯醇酶、磷酸丙糖异构酶、原肌球蛋白和血红蛋白);(3)信号转导通路成员(p38MAPK激酶)等。在此基础上通过分子对接软件molsoft ICM模拟计算获得rhddADAM15与p38激酶相互作用的最佳模式和重要的结合位点;
4)采用噬菌体展示技术筛选与rhddADAM15特异性相互作用的多肽序列。采用噬菌体随机十二肽库对固定有rhddADAM15的亲和磁珠系统进行生物淘洗,获得4条与rhddADAM15特异结合的12肽。将获得的12肽与蛋白质库进行序列比对,发现细胞周期相关蛋白如Cdc25和p53,信号通路相关蛋白/受体,如整合素αvβ3的胞外区,丝裂原活化蛋白激酶p38α等与所获得的4条12肽具有高度同源性;
5)基于上述研究所获得的rhddADAM15在B16细胞上潜在的靶点蛋白,进一步研究rhddADAM15与p38MAPK激酶的体外相互作用,结合rhddADAM15对B16细胞行为的抑制作用,采用蛋白点杂交和激酶活性测定方法分析rhddADAM15在体外与重组p38激酶蛋白的结合情况,同时研究不同剂量rhddADAM15对B16细胞中p38MAPK激酶磷酸化水平的影响,并观察rhddADAM15对p38激酶活性受到部分抑制的B16细胞的增殖和细胞周期抑制作用的变化。发现:(1)rhddADAM15在离体低温条件下能够和重组p38激酶蛋白结合,但rhddADAM15(0-10μg/mL)在体外对p38MAPK激酶活性没有影响;(2)同样剂量的rhddADAM15作用于B16细胞24 h后,细胞中p38MAPK激酶的磷酸化水平提高(被激活);(3)p38激酶的特异性抑制剂SB203580(0.5 mmol/L)预处理B16细胞30 min后,rhddADAM15对细胞增殖的抑制作用减弱(IC50由未经处理的9.5μg/mL增加到11.4μg/mL),对细胞周期的阻滞作用也减弱(G2/M期细胞比率从未经处理的的28.77%降到16.59%)。上述结果表明,p38MAPK信号转导通路参与了rhddADAM15对B16细胞增殖和细胞周期的抑制作用,同时也表明生理条件下rhddADAM15与p38MAPK激酶相互作用的复杂性。
A Disintegrin And Metalloproteases (ADAM) proteins are a family of multifunctional proteins containing disintegrin and metalloproteinase domains that perform both adhesive and proteolytic functions in cell-cell and cell-matrix interactions. ADAM15 is unique among these proteins in having an Arg-Gly-Asp (RGD) motif in its disintegrin-like domain. This dissertation chose the B16 murine melanoma cell as a model, to investigate the effect of the recombinant human ADAM15 disintegrin domain (rhddADAM15) on B16 cells gowth, and the target protein of rhddADAM15 on B6 cells, and further study the involvement of the target protein in the anti-cancer effect of rhddADAM15. The main results were described as follows:
1) In order to enhance the production and bioactivity of rhddADAM15 in Escherichia coli, two different strategies were examined based on the genetic analysis of coding sequence of rhddADAM15: to select the suitable host strain, and to optimize the rare codons and delete the amino acids residues. It was found that (1) when choosing E. coli Rosetta (DE3) as the expression host that supplys additional tRNA for rare codons, 298 mg/L GST-ADAM15 and 42 mg/L rhddADAM15 were achieved under the optimal expression and thrombin digestion conditions, respectively; (2) when introducing E. coli preferred codon GGC and deleting“Pro-Glu-Phe”residues by PCR-based site-directed mutagenesis, 326 mg/L GST-△-ADAM15 and 68 mg/L rhddADAM15 were achieved under the same condition, respectively, higher 19.4% and 61.9% than that of the wide type strain. The data presented here demonstrated that high expression of heterologous protein could be achieved by releasing rare codon usage and amino acids residues restriction.
2) The effect of rhddADAM15 on the proliferation of B16, MCF-7/W and HMEC-1 cells were examined by MTT assay. The effect of rhddADAM15 on B16 cell migration, adhesion and invention were tested by the wound migration assay, cell-Matrigel adhesion assay and the Transwell chambers, respectively; The growth and activity of B16 cells were explored by using trypan blue staining. The cell cycle and apoptosis were determined by flow cytometry. The results demonstrated that rhddADAM15 could inhibit the proliferation of normal cells, but exhibit stronger inhibitory effect on the tumor cells. Moreover, rhddADAM15 inhibited B16 cell migration, adhesion and invasion in the dose/time-dependent mode and the necrosis could not be observed in the low concentration of rhddADAM15. The apoptotic peaks were not found in cell cycle analyses and the cells are arrested in G2/M phase.
3) The protein pull-down assay using the biotin-avidin system supported by the immol/Lunomagnetic beads was used to capture the binding protein of rhddADAM15 from the cell lysis buffer of B16. The mixture of the binding protein was concentrated and separated by the two-dimensional eletrophoresis (2DE) and eight of the protein spots were identified by the ESI-MS/MS. By using the software BIOWORKS and the protein database ipi MOUSE v3.26, the binding proteins were mainly identified as follows: (1) Tumor markers in the clinical application: malate dehydrogenase, phosphate dehydrogenase and glutamate dehydrogenase; (2) Enzymes in glycolytic pathway: alpha-enolase and triosephosphate isomerase; (3) The important components in cytoskeleton: tropomyosin-3; (4) Proteins in oxygen transportation: hemoglobin subunit beta-1; (5) Members in signal transduction: p38MAPK. In addition, the interaction modes between rhddADAM15 and p38MAPK were preliminarily analyzed using the molecular docking program, molsoft ICM-PRO-3.5.
4) The target peptides of rhddADAM15 were explored by the phage display technique. Firstly, the rhddADAM15 was biotinylated and immol/Lobilized on the immol/Lunomagnetic beads with avidin labeled. Secondly, four specific binding peptides (peptide A, B, C and D) were screened from a phage display-12 peptide library by a screening protocol towards immol/Lobilized rhddADAM15. By using the software BLAST and the relevant protein database, some target proteins were screened, such as the Cdc25 phosphatase, tyrosine kinase p56, integrinαvβ3, and the p38 mitogen activated protein kinase (p38MAPK), related with the cell cycles and the signal transduction.
5) The p38MAPK was selected for further investigation of the involvement in the anti-proliferative effect of rhddADAM15 on melanoma cells. Firstly, the effect of rhddADAM15 on activity of p38 MAPK in vitro was observed. Secondly, phosphorylation of p38 kinase in melanoma cells was detected by Western blot analysis upon treatment with rhddADAM15 (0-10μg/mL). Thirdly, the specific inhibitor of p38 kinase (SB203580) was employed to partly suppress the activity of p38 kinase in B16 cells, and the inhibitory effect of rhddADAM15 was then observed. The results were demonstrated as follows: (1) rhddADAM15 could bind to the recombinant p38 kinase at 4 oC but had no effect on the activity of p38 kinase in vitro; (2) rhddADAM15 had inhibitory effect on the activity of p38 kinase in B16 cells; (3) A decrease in the rhddADAM15 inhibitory effect on melanoma cell proliferation, and the value of IC50 was increased from 9.5μg/mL to11.4μg/mL, when the activity of p38 kinase was partly suppressed; (4) A release in the cell cycle arrest, the cell ratio in G2/M phase was decreased from 28.77% to16.59%, under the condition of suppressing the p38 kinase. These results provided evidences that rhddADAM15 inhibiting the proliferation and the cell cycle of the melanoma cells was partly through p38 kinase activation and the p38MAPK signal transduction pathway was involved in the effect of rhddADAM15 on B16 cells.
引文
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