血小板Sema4D钙调蛋白结合基序的鉴定及其调控Sema4D切割的机制研究
发布时间：2018-08-04 16:03
【摘要】：神经导向因子Semaphorin4D (Sema4D；CD100)是首先在T淋巴细胞上发现的具有促进B细胞分化功能的I型跨膜糖蛋白，进一步的研究表明Sema4D通过其受体CD72/Plexin-B1/Plexin-B2参与免疫反应、神经生长、肿瘤血管新生、血栓形成以及骨生长等。Sema4D可以被金属蛋白酶17（ADAM17）切割，产生具有生物活性的可溶性蛋白。然而，Sema4D切割和可溶性Sema4D生成的调控机制所知甚少。目前认为可能有两种机制：第一，切割酶的表达和活性的增强可导致Sema4D的切割；第二，，“底物主导”的切割机制，Sema4D构象改变后可以更接近切割酶，导致自身被切割。本论文根据Sema4D的切割可能是“底物主导（”substrate-oriented）的设想，系统分析了Sema4D氨基酸序列和分子结构，发现Sema4D的胞内段近膜区含有一段18个氨基酸组成的序列（Arg762-Lys779），这段序列具有保守的极性氨基酸残基，并且可以形成两亲性α螺旋，这是钙调蛋白结合的特征性结构。利用免疫共沉淀技术，我们证明在静息血小板中Sema4D分子与钙调蛋白结合，血小板活化后Sema4D-钙调蛋白复合体解离,提示钙调蛋白参与Sema4D切割调控。 为了探究Sema4D是否通过这段18个氨基酸组成的基序与钙调蛋白结合，我们合成了Sema4D钙调蛋白结合基序的多肽，命名为Sema4D钙调蛋白结合肽（Calmodulin-binding peptide of Sema4D, CBPS），以这段多肽作为一种工具来进一步研究Sema4D-钙调蛋白结合的分子机制，以及阻断结合所产生的生物学效应。利用蛋白标记、结合实验证明CBPS可与天然和重组Sema4D结合，其解离常数为165±40nM，这与血小板表面其他受体与钙调蛋白的解离常数类似。将CBPS多肽作为工具来研究Sema4D切割，首先要明确CBPS是否可以穿过细胞膜，利用流式细胞仪以及共聚焦显微镜技术，我们证实CBPS多肽可穿过细胞膜，存在于血小板内部。功能研究证明5μM CBPS处理血小板30秒即可诱导血小板Sema4D-钙调蛋白复合体的解离，同时，我们观察了CBPS对Sema4D分子切割的影响，结果显示，CBPS诱导Sema4D切割并且呈现浓度、时间依赖性。而且，CBPS诱导的这种切割不依赖于血小板活化和ADAM17的活性增加。 钙调蛋白在细胞内以游离形式或者结合形式存在，两种形式之间保持一动态平衡。CBPS进入血小板内部，与钙调蛋白结合，打破动态平衡，导致一些结合形式的钙调蛋白转变为游离形式，诱导Sema4D分子切割，同时我们也检测到CBPS可以诱导其他膜蛋白受体（如GPIbα、GPVI）切割，然而，我们的结果显示，5μM的CBPS就可以诱导Sema4D分子切割，而诱导GPVI和GPIbα切割则分别需要10μM和20μM，说明CBPS对三种分子切割的敏感性不同，这也提示我们钙调蛋白调控切割机制的普遍性与复杂性共存。利用钙调蛋白抑制剂W7进一步证明了钙调蛋白调控Sema4D切割这一生物现象。 以上我们是采用血小板为模型进行钙调蛋白调控切割的研究，我们认为这种现象也存在于其他细胞中，而且之前有文献表明在淋巴细胞中，Sema4D自发切割产生可溶性片段。因此，我们通过基因突变方法，构建了Sema4D突变质粒，删除Sema4D分子钙调蛋白结合基序，从而阻止Sema4D与钙调蛋白结合。将质粒转染中国仓鼠卵巢细胞，发现当删除钙调蛋白结合基序后Sema4D分子的自发切割明显增加。这一结果支持之前报道的自发切割现象。 综上所述，我们的研究证明胞内钙调蛋白与Sema4D的结合是血小板Sema4D分子切割的调控机制之一，其结果支持Sema4D切割的“底物主导”机制的设想。由于Sema4D在多种细胞表达并参与多种病理生理过程，Sema4D切割的胞内调控机制的发现，将为相关理论和相关疾病过程提供理论支持和实验依据。
[Abstract]:Semaphorin4D (Sema4D; CD100) is a I type transmembrane glycoprotein that is first found on T lymphocytes to promote the differentiation of B cells. Further studies suggest that Sema4D is involved in the immune response, nerve growth, neovascularization, thrombosis, and bone growth through its receptor CD72/Plexin-B1/Plexin-B2. 17 (ADAM17) is cut to produce soluble proteins with biological activity. However, little is known about the regulation mechanism of Sema4D cutting and soluble Sema4D formation. There are two mechanisms that may exist. First, the expression and activity of the cleavage enzymes can lead to the cutting of Sema4D; second, "substrate dominated" cutting mechanism, After the Sema4D conformation changes, it can be closer to the cutting enzyme and cause itself to be cut. In this paper, according to the assumption that the cutting of Sema4D may be "substrate dominant" ("substrate-oriented"), the amino acid sequence and molecular structure of the Sema4D are systematically analyzed. It is found that the near membrane region of the intracellular segment of Sema4D contains a sequence of 18 amino acids (Arg762-Lys779). This sequence has a conservative polar amino acid residue and can form a two pro alpha helix, which is a characteristic structure of calmodulin binding. Using immunoprecipitation technique, we have demonstrated that Sema4D molecules in resting platelets are combined with calmodulin, and the Sema4D- calmodulin complex is dissociated after platelet activation, suggesting the calmodulin. Control with Sema4D cutting.
In order to explore whether Sema4D is combined with calmodulin in this 18 amino acid sequence, we synthesize the Sema4D calmodulin binding peptide, named Sema4D calmodulin binding peptide (Calmodulin-binding peptide of Sema4D, CBPS). This polypeptide is used as a tool to further study the combination of Sema4D- Calmodulin The molecular mechanism, and the biological effect produced by blocking the binding, using protein labeling, combined with experiments, shows that CBPS can be combined with natural and recombinant Sema4D with a dissociation constant of 165 + 40nM, which is similar to the dissociation constant of other receptors on the surface of the platelets and calmodulin. CBPS polypeptides are used as a tool to study Sema4D cutting, first to clarify CB Whether PS can pass through the cell membrane, using flow cytometry and confocal microscopy, we confirm that CBPS peptides can pass through the cell membrane and exist in the platelets. Functional studies have shown that the dissociation of platelets Sema4D- calmodulin complex can be induced by the treatment of platelets by 5 u M CBPS for 30 seconds. At the same time, we observed the CBPS to Sema4D molecules. The effects of cutting showed that CBPS induced Sema4D cutting and was time dependent and time dependent. Moreover, the CBPS induced cutting was not dependent on platelet activation and the increase of ADAM17 activity.
Calmodulin exists in free form or binding form in the cell. The two forms maintain a dynamic balance between the two forms and enter the platelets, combine with the calmodulin, break the dynamic balance, cause some binding forms of calmodulin to change into free form, induce Sema4D molecular cutting, and we also detect that CBPS can be induced. Other membrane protein receptors (such as GPIb alpha, GPVI) cut, however, our results show that 5 mu M CBPS can induce Sema4D molecular cutting, while inducement of GPVI and GPIb alpha cutting requires 10 mu M and 20 micron respectively, indicating that CBPS is sensitive to three kinds of molecular cutting, which also suggests that our calmodulin regulates the universality and complexity of the cutting mechanism Coexistence of calmodulin inhibitor W7 further demonstrated calmodulin modulates the biological phenomenon of Sema4D cleavage.
We have used platelets to modulate and regulate calmodulin based on the platelet model. We believe that this phenomenon also exists in other cells, and previous literature showed that Sema4D spontaneously cut into soluble fragments in lymphocytes. Therefore, we constructed Sema4D mutant plasmids by gene mutation and delete Sema4D points. The binding of Sema4D to calmodulin was prevented by the binding of the subcalmodulin. The plasmids were transfected into Chinese hamster ovary cells. The spontaneous cutting of Sema4D molecules increased significantly after the deletion of the calmodulin binding sequence. This result supports the spontaneous cutting phenomenon reported before.
In summary, our study shows that the binding of intracellular calmodulin and Sema4D is one of the regulatory mechanisms of Sema4D molecular cutting in platelets. The results support the assumption of the "substrate dominated" mechanism of Sema4D cutting. The discovery of intracellular regulation mechanism of Sema4D cutting due to the expression of Sema4D in a variety of cells and participation in a variety of pathophysiological processes will be found. It provides theoretical support and experimental evidence for relevant theories and related disease processes.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R363;Q51
本文编号：2164393
[Abstract]:Semaphorin4D (Sema4D; CD100) is a I type transmembrane glycoprotein that is first found on T lymphocytes to promote the differentiation of B cells. Further studies suggest that Sema4D is involved in the immune response, nerve growth, neovascularization, thrombosis, and bone growth through its receptor CD72/Plexin-B1/Plexin-B2. 17 (ADAM17) is cut to produce soluble proteins with biological activity. However, little is known about the regulation mechanism of Sema4D cutting and soluble Sema4D formation. There are two mechanisms that may exist. First, the expression and activity of the cleavage enzymes can lead to the cutting of Sema4D; second, "substrate dominated" cutting mechanism, After the Sema4D conformation changes, it can be closer to the cutting enzyme and cause itself to be cut. In this paper, according to the assumption that the cutting of Sema4D may be "substrate dominant" ("substrate-oriented"), the amino acid sequence and molecular structure of the Sema4D are systematically analyzed. It is found that the near membrane region of the intracellular segment of Sema4D contains a sequence of 18 amino acids (Arg762-Lys779). This sequence has a conservative polar amino acid residue and can form a two pro alpha helix, which is a characteristic structure of calmodulin binding. Using immunoprecipitation technique, we have demonstrated that Sema4D molecules in resting platelets are combined with calmodulin, and the Sema4D- calmodulin complex is dissociated after platelet activation, suggesting the calmodulin. Control with Sema4D cutting.
In order to explore whether Sema4D is combined with calmodulin in this 18 amino acid sequence, we synthesize the Sema4D calmodulin binding peptide, named Sema4D calmodulin binding peptide (Calmodulin-binding peptide of Sema4D, CBPS). This polypeptide is used as a tool to further study the combination of Sema4D- Calmodulin The molecular mechanism, and the biological effect produced by blocking the binding, using protein labeling, combined with experiments, shows that CBPS can be combined with natural and recombinant Sema4D with a dissociation constant of 165 + 40nM, which is similar to the dissociation constant of other receptors on the surface of the platelets and calmodulin. CBPS polypeptides are used as a tool to study Sema4D cutting, first to clarify CB Whether PS can pass through the cell membrane, using flow cytometry and confocal microscopy, we confirm that CBPS peptides can pass through the cell membrane and exist in the platelets. Functional studies have shown that the dissociation of platelets Sema4D- calmodulin complex can be induced by the treatment of platelets by 5 u M CBPS for 30 seconds. At the same time, we observed the CBPS to Sema4D molecules. The effects of cutting showed that CBPS induced Sema4D cutting and was time dependent and time dependent. Moreover, the CBPS induced cutting was not dependent on platelet activation and the increase of ADAM17 activity.
Calmodulin exists in free form or binding form in the cell. The two forms maintain a dynamic balance between the two forms and enter the platelets, combine with the calmodulin, break the dynamic balance, cause some binding forms of calmodulin to change into free form, induce Sema4D molecular cutting, and we also detect that CBPS can be induced. Other membrane protein receptors (such as GPIb alpha, GPVI) cut, however, our results show that 5 mu M CBPS can induce Sema4D molecular cutting, while inducement of GPVI and GPIb alpha cutting requires 10 mu M and 20 micron respectively, indicating that CBPS is sensitive to three kinds of molecular cutting, which also suggests that our calmodulin regulates the universality and complexity of the cutting mechanism Coexistence of calmodulin inhibitor W7 further demonstrated calmodulin modulates the biological phenomenon of Sema4D cleavage.
We have used platelets to modulate and regulate calmodulin based on the platelet model. We believe that this phenomenon also exists in other cells, and previous literature showed that Sema4D spontaneously cut into soluble fragments in lymphocytes. Therefore, we constructed Sema4D mutant plasmids by gene mutation and delete Sema4D points. The binding of Sema4D to calmodulin was prevented by the binding of the subcalmodulin. The plasmids were transfected into Chinese hamster ovary cells. The spontaneous cutting of Sema4D molecules increased significantly after the deletion of the calmodulin binding sequence. This result supports the spontaneous cutting phenomenon reported before.
In summary, our study shows that the binding of intracellular calmodulin and Sema4D is one of the regulatory mechanisms of Sema4D molecular cutting in platelets. The results support the assumption of the "substrate dominated" mechanism of Sema4D cutting. The discovery of intracellular regulation mechanism of Sema4D cutting due to the expression of Sema4D in a variety of cells and participation in a variety of pathophysiological processes will be found. It provides theoretical support and experimental evidence for relevant theories and related disease processes.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R363;Q51
【共引文献】
相关期刊论文 前2条
1 王华;Semaphorins分子与免疫[J];国外医学(免疫学分册);2003年03期
2 王华,高杰英,彭虹,石辛甫,易绍琼,王岚;小鼠Semcap2在HeLa细胞的表达及对派伊尔结淋巴细胞的影响[J];上海免疫学杂志;2002年06期
相关博士学位论文 前3条
1 周虎;CD72在免疫性血小板减少症中的基因表达[D];北京协和医学院;2011年
2 杨玉恒;等剂量国产氯吡格雷与进口氯吡格雷的等效性及安全性比较[D];天津医科大学;2011年
3 毛国红;白芷细胞外钙调素结合蛋白（ECBP21）cDNA克隆及鉴定[D];河北师范大学;2003年
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