【摘要】：神經(jīng)導(dǎo)向因子Semaphorin4D (Sema4D；CD100)是首先在T淋巴細(xì)胞上發(fā)現(xiàn)的具有促進(jìn)B細(xì)胞分化功能的I型跨膜糖蛋白，進(jìn)一步的研究表明Sema4D通過其受體CD72/Plexin-B1/Plexin-B2參與免疫反應(yīng)、神經(jīng)生長、腫瘤血管新生、血栓形成以及骨生長等。Sema4D可以被金屬蛋白酶17（ADAM17）切割，產(chǎn)生具有生物活性的可溶性蛋白。然而，Sema4D切割和可溶性Sema4D生成的調(diào)控機(jī)制所知甚少。目前認(rèn)為可能有兩種機(jī)制：第一，切割酶的表達(dá)和活性的增強(qiáng)可導(dǎo)致Sema4D的切割；第二，，“底物主導(dǎo)”的切割機(jī)制，Sema4D構(gòu)象改變后可以更接近切割酶，導(dǎo)致自身被切割。本論文根據(jù)Sema4D的切割可能是“底物主導(dǎo)（”substrate-oriented）的設(shè)想，系統(tǒng)分析了Sema4D氨基酸序列和分子結(jié)構(gòu)，發(fā)現(xiàn)Sema4D的胞內(nèi)段近膜區(qū)含有一段18個(gè)氨基酸組成的序列（Arg762-Lys779），這段序列具有保守的極性氨基酸殘基，并且可以形成兩親性α螺旋，這是鈣調(diào)蛋白結(jié)合的特征性結(jié)構(gòu)。利用免疫共沉淀技術(shù)，我們證明在靜息血小板中Sema4D分子與鈣調(diào)蛋白結(jié)合，血小板活化后Sema4D-鈣調(diào)蛋白復(fù)合體解離,提示鈣調(diào)蛋白參與Sema4D切割調(diào)控。 為了探究Sema4D是否通過這段18個(gè)氨基酸組成的基序與鈣調(diào)蛋白結(jié)合，我們合成了Sema4D鈣調(diào)蛋白結(jié)合基序的多肽，命名為Sema4D鈣調(diào)蛋白結(jié)合肽（Calmodulin-binding peptide of Sema4D, CBPS），以這段多肽作為一種工具來進(jìn)一步研究Sema4D-鈣調(diào)蛋白結(jié)合的分子機(jī)制，以及阻斷結(jié)合所產(chǎn)生的生物學(xué)效應(yīng)。利用蛋白標(biāo)記、結(jié)合實(shí)驗(yàn)證明CBPS可與天然和重組Sema4D結(jié)合，其解離常數(shù)為165±40nM，這與血小板表面其他受體與鈣調(diào)蛋白的解離常數(shù)類似。將CBPS多肽作為工具來研究Sema4D切割，首先要明確CBPS是否可以穿過細(xì)胞膜，利用流式細(xì)胞儀以及共聚焦顯微鏡技術(shù)，我們證實(shí)CBPS多肽可穿過細(xì)胞膜，存在于血小板內(nèi)部。功能研究證明5μM CBPS處理血小板30秒即可誘導(dǎo)血小板Sema4D-鈣調(diào)蛋白復(fù)合體的解離，同時(shí)，我們觀察了CBPS對(duì)Sema4D分子切割的影響，結(jié)果顯示，CBPS誘導(dǎo)Sema4D切割并且呈現(xiàn)濃度、時(shí)間依賴性。而且，CBPS誘導(dǎo)的這種切割不依賴于血小板活化和ADAM17的活性增加。 鈣調(diào)蛋白在細(xì)胞內(nèi)以游離形式或者結(jié)合形式存在，兩種形式之間保持一動(dòng)態(tài)平衡。CBPS進(jìn)入血小板內(nèi)部，與鈣調(diào)蛋白結(jié)合，打破動(dòng)態(tài)平衡，導(dǎo)致一些結(jié)合形式的鈣調(diào)蛋白轉(zhuǎn)變?yōu)橛坞x形式，誘導(dǎo)Sema4D分子切割，同時(shí)我們也檢測到CBPS可以誘導(dǎo)其他膜蛋白受體（如GPIbα、GPVI）切割，然而，我們的結(jié)果顯示，5μM的CBPS就可以誘導(dǎo)Sema4D分子切割，而誘導(dǎo)GPVI和GPIbα切割則分別需要10μM和20μM，說明CBPS對(duì)三種分子切割的敏感性不同，這也提示我們鈣調(diào)蛋白調(diào)控切割機(jī)制的普遍性與復(fù)雜性共存。利用鈣調(diào)蛋白抑制劑W7進(jìn)一步證明了鈣調(diào)蛋白調(diào)控Sema4D切割這一生物現(xiàn)象。 以上我們是采用血小板為模型進(jìn)行鈣調(diào)蛋白調(diào)控切割的研究，我們認(rèn)為這種現(xiàn)象也存在于其他細(xì)胞中，而且之前有文獻(xiàn)表明在淋巴細(xì)胞中，Sema4D自發(fā)切割產(chǎn)生可溶性片段。因此，我們通過基因突變方法，構(gòu)建了Sema4D突變質(zhì)粒，刪除Sema4D分子鈣調(diào)蛋白結(jié)合基序，從而阻止Sema4D與鈣調(diào)蛋白結(jié)合。將質(zhì)粒轉(zhuǎn)染中國倉鼠卵巢細(xì)胞，發(fā)現(xiàn)當(dāng)刪除鈣調(diào)蛋白結(jié)合基序后Sema4D分子的自發(fā)切割明顯增加。這一結(jié)果支持之前報(bào)道的自發(fā)切割現(xiàn)象。 綜上所述，我們的研究證明胞內(nèi)鈣調(diào)蛋白與Sema4D的結(jié)合是血小板Sema4D分子切割的調(diào)控機(jī)制之一，其結(jié)果支持Sema4D切割的“底物主導(dǎo)”機(jī)制的設(shè)想。由于Sema4D在多種細(xì)胞表達(dá)并參與多種病理生理過程，Sema4D切割的胞內(nèi)調(diào)控機(jī)制的發(fā)現(xiàn)，將為相關(guān)理論和相關(guān)疾病過程提供理論支持和實(shí)驗(yàn)依據(jù)。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R363;Q51

【共引文獻(xiàn)】

相關(guān)期刊論文 前2條

1 王華;Semaphorins分子與免疫[J];國外醫(yī)學(xué)(免疫學(xué)分冊);2003年03期

2 王華,高杰英,彭虹,石辛甫,易紹瓊,王嵐;小鼠Semcap2在HeLa細(xì)胞的表達(dá)及對(duì)派伊爾結(jié)淋巴細(xì)胞的影響[J];上海免疫學(xué)雜志;2002年06期

相關(guān)博士學(xué)位論文 前3條

1 周虎;CD72在免疫性血小板減少癥中的基因表達(dá)[D];北京協(xié)和醫(yī)學(xué)院;2011年

2 楊玉恒;等劑量國產(chǎn)氯吡格雷與進(jìn)口氯吡格雷的等效性及安全性比較[D];天津醫(yī)科大學(xué);2011年

3 毛國紅;白芷細(xì)胞外鈣調(diào)素結(jié)合蛋白（ECBP21）cDNA克隆及鑒定[D];河北師范大學(xué);2003年

本文編號(hào)：2164393