血小板因子-4与Neuropilin-1（NRP1）相互作用位点的鉴定
摘要
背景:Neuropilin-1 (NRP1)是细胞表面相对分子量(Mr)为102,000Da的Ⅰ型跨膜糖蛋白,通过杂交瘤技术在欧洲蟾蜍的神经系统内首次被发现。进一步研究证明NRP1通常表达在内皮细胞及一些肿瘤细胞,是Semaphorin3A(Sema3A)和血管内皮生长因子165(vascular endothelial growth factor 165,VEGF165)的受体,在神经轴突的生长、血管的生成、发育和肿瘤转移中起着重要的作用[1]。最近有研究表明NRP1在原始T细胞与dendritic cell (DCs)稳定接触中起重要作用,是CD4+CD25+调节性T细胞持续的表面标志之一,不依赖于Treg细胞是否活化,在免疫反应的起始上扮演着重要角色[2]。本课题组的前期研究已显示血小板因子-4(Platelet Factor-4/PF4,又名CXCL4)可以刺激CD4+CD25+调节性T(Treg)细胞的增殖[3],并与NRP1结合[4],同时在PF4刺激Treg细胞增殖过程中NRP1表达量增加[5],故推测NRP1在PF4刺激Tr增殖过程中发挥受体或共受体的作用。本研究为了进一步确定NRP1与PF4作用的结合位点或功能域,为Tr细胞的功能及其调控提供分子依据,并具有应用于治疗自身免疫性疾病的前景。
方法及结果:NRP1在脊椎动物中高度保守,由胞内区、跨膜区和胞外区三部分组成,其中胞外段包括860个氨基酸,由3个不同的结构域组成,分别称为a1/a2、b1/b2和c。其中a1/a2又称CUB结构域(complement-binding protein homology),b1/b2结构域与凝血因子Ⅴ和Ⅷ结构相似同源,在c结构域的中部含有MAM结构域(meprin, A5,μ)。NRP1的胞外段各个区能和不同的分子结合介导不同的信号传导,Sema3A通过a1/a2/b1区与NRP1结合,VEGF165能与NRP1的b1/b2区结合,Heparin亦通过b1/b2区与NRP1结合,所以NRP1具有多种功能[6]。根据NRP1的结构特点,本实验采用原核表达的方式分段表达NRP1的a1/a2(CUB)、b1/b2(F5/8C)、MAM三个结构域,分别得到融合蛋白后,免疫Balb/c鼠后,通过细胞融合制备单克隆抗体并对所制备抗体进行初步鉴定,鉴定后的抗体再与PF4做作用位点的筛选。
首先,搜索NRP1的蛋白序列(Human Protein Reference Database, www.hprd.org),分段截取NRP1的3个domain(CUB、F5/8C、MAM),从人单核细胞cDNA中扩增出3个片段基因。原核表达选择pGEX-5X-1载体作为表达载体,根据载体的酶切图谱设计引物,将NRP1的3个domain基因序列分别插入到pGEX-5X-1载体的EcoR I和Not I两个限制性酶切位点之间,PCR反应后,得到目的条带;将扩增的目的片段从胶上回收后,进行T载体的连接;转化、提取质粒后双酶切,再与同时双酶切的表达载体pGEX-5X-1载体进行连接,然后再转化,各挑取两个阳性克隆,摇菌后,进行测序鉴定。将测序鉴定正确的表达载体转化入BL21菌株后诱导表达,获得正确表达的目的蛋白并纯化。
其次,将纯化的GST-CUB、GST-F5/8C、GST-MAM融合蛋白与等体积福氏完全佐剂进行充分乳化,对Balb/c小鼠进行多点皮下注射。首次免疫后4周加强免疫一次,免疫原用量减为首免的一半,并与福氏不完全佐剂充分乳化。两周后ELISA法检测效价,效价在1:8000以上的小鼠即可进行加强免疫,加强免疫四天后进行细胞融合,ELISA法筛选能够稳定分泌抗CUB、F5/8C、MAM mAb的阳性克隆,并亚克隆,分别获得能够稳定分泌CUB单克隆抗体的细胞系6株,分泌F5/8C单克隆抗体的细胞系5株,分泌MAM单克隆抗体的细胞系2株。
第三,用GST标签蛋白通过Western blot对所获单克隆抗体进行鉴定,其中不识别GST标签蛋白而只识别融合蛋白的细胞株为识别目的蛋白的阳性株。结果显示,识别CUB蛋白的阳性株:UAH058、UDC114、UDG054、UBB067、UCB064,识别F5/8C的阳性株:OCA084、OEG082,无识别MAM的阳性株。用ELISA法分别检测PF4与CUB、F5/8C的相互作用[26],结果为:PF4与F5/8C存在特异的分子间相互作用。
结论:成功获得的NRP1三个片段CUB、F5/8C、MAM的鼠抗人单克隆抗体的可应用于蛋白结构、定位、功能等常规的生命科学研究外,在临床诊断和治疗方面也具有重要的意义,初步证明了PF4可与NRP1的F5/8C区发生特异性的结合,将为今后NRP1分子的功能研究奠定基础。
Background: Neuropilin was 102,000Da spanning transmembrane glycoproteinⅠon cell surface, which was found in the nervous system of Europe toad for the first time. More research proved that NRP1 usually expressed in the endothelial cells and some tumor cells, which function as receptors for semaphorin3A (Sema3A) and VEGF165 (vascular endothelial growth factor165). NRP1 was involved in neuronal cell guidance and axonal growth, vascular formation, growth and metastasis of tumors. Recent research showed that NRP1 play an important role in stable contacts between na?ve T cell and dendritic cell (DCs), that it’s one of continuous surface markers on CD4 + CD25 + regulatory T cells, was constitutively expressed on the surface of CD4+CD25+ Treg cells independently of their activation status, and played an important role in the initial immune response. Our team’s preliminary studies proved that PF4 stimulates proliferation of the human CD4+CD25+Tr cells, bind with PF4. The expression of NRP1 increased when PF4 stimulates proliferation of Treg cell. It is supposed that NRP1 function as receptor or co-receptor when PF4 stimulated proliferation of Treg cell. This study was to further define the binding site with NRP1 and PF4, provides basis for the function and regulation of Treg cell and possess the prospects in the treatment of autoimmune diseases.
Method & Result: NRP1 was high conservative in vertebrate, composed of intracellular region, transmenmbrane domain and extracellular region. The extracellular region contains 860 amino acids, was made up of three different structural domain including two complement binding (CUB) domain (a1/a2), two coagulation factor V/VIII homology domain(b1/b2), a MAM (meprin, A5, receptor tyrosine phospatase 1) domain (c), a transmenbrane segment. NRP1 mediated various signal transmission by interaction with three domain and different molecules, such as: Sema3A binds NRP1 through a1/a2/b1, VEGF165 binded NRP1 through b1/b2, and Heparin binds NRP1 through b1/b2 too, so NRP1 possessed multifunction. According to the structural characteristics of NRP1, we expressed fusion protein of three domains (CUB, F5/8C and MAM) by prokaryotic expression system to generate monoclonal antibodies (mAb) against NRP1. Western blot characterized the specificity of mAb. Then we study the interaction site between NRP1 and PF4.
Firstly, we searched for NRP1 sequence in HRPD (Human Protein Reference Database, www.hprd.org), select three domains (CUB, F5/8C, MAM) and amplified this three genes from monocytes cDNA. We designed primers according to cleavage map of pGEX-5X-1expression vectors, inserted three domains into the vector between EcoRⅠand NotⅠrestricted enzyme sites, After PCR, target gene was obtained successfully, double enzyme digestion of target gene and expression vectors, and conjuncted them, transformed into BL21 bacteria to express target proteins, sequencing. GST-CUB, GST-F5/8C and GST-MAM recombination proteins were expressed successfully.
Secondly, purified the recombination protein and emulsify with complete adjuvant were used to immunize Balb/c mice, after four weeks, cut down the dose of the recombination protein, and emulsified with incompelet adjuvant to immunize Balb/c mice, after two weeks, tested the titer of immunized mice. The immunized mice with high titer could be used to generate mAbs by hubridoma technique. Cell clones were screening by ELISA. After cell fusion, 6 hybridomas secreting specific Abs against CUB were established, 5 against F5/8C and 2 against MAM.
Thirdly, The antibody specificity was characterized by Western blot. The results showed that, 5 hybridomas such as UAH058, UDC114, UDG054, UBB067, UCB064 could recognize CUB recombination protein, 2 hybridomas such as OCA084, OEG082 could recognize F5/8C recombination protein, no hybridoma recognize MAM recombination protein. Then we identify interaction between PF4 and CUB, F5/8C, the result showed that F5/8C interact with PF4.
Conclusion: These mAbs could be used in immunoprecipitation, immunohistochemistry and confocal microscopy in the future to study the function of NRP1, such as molecules interact with NRP1and so on. F5/8C identifies initially maybe interact with PF4. And it will be useful for clinical diagnosis and therapy.
方法及结果:NRP1在脊椎动物中高度保守,由胞内区、跨膜区和胞外区三部分组成,其中胞外段包括860个氨基酸,由3个不同的结构域组成,分别称为a1/a2、b1/b2和c。其中a1/a2又称CUB结构域(complement-binding protein homology),b1/b2结构域与凝血因子Ⅴ和Ⅷ结构相似同源,在c结构域的中部含有MAM结构域(meprin, A5,μ)。NRP1的胞外段各个区能和不同的分子结合介导不同的信号传导,Sema3A通过a1/a2/b1区与NRP1结合,VEGF165能与NRP1的b1/b2区结合,Heparin亦通过b1/b2区与NRP1结合,所以NRP1具有多种功能[6]。根据NRP1的结构特点,本实验采用原核表达的方式分段表达NRP1的a1/a2(CUB)、b1/b2(F5/8C)、MAM三个结构域,分别得到融合蛋白后,免疫Balb/c鼠后,通过细胞融合制备单克隆抗体并对所制备抗体进行初步鉴定,鉴定后的抗体再与PF4做作用位点的筛选。
首先,搜索NRP1的蛋白序列(Human Protein Reference Database, www.hprd.org),分段截取NRP1的3个domain(CUB、F5/8C、MAM),从人单核细胞cDNA中扩增出3个片段基因。原核表达选择pGEX-5X-1载体作为表达载体,根据载体的酶切图谱设计引物,将NRP1的3个domain基因序列分别插入到pGEX-5X-1载体的EcoR I和Not I两个限制性酶切位点之间,PCR反应后,得到目的条带;将扩增的目的片段从胶上回收后,进行T载体的连接;转化、提取质粒后双酶切,再与同时双酶切的表达载体pGEX-5X-1载体进行连接,然后再转化,各挑取两个阳性克隆,摇菌后,进行测序鉴定。将测序鉴定正确的表达载体转化入BL21菌株后诱导表达,获得正确表达的目的蛋白并纯化。
其次,将纯化的GST-CUB、GST-F5/8C、GST-MAM融合蛋白与等体积福氏完全佐剂进行充分乳化,对Balb/c小鼠进行多点皮下注射。首次免疫后4周加强免疫一次,免疫原用量减为首免的一半,并与福氏不完全佐剂充分乳化。两周后ELISA法检测效价,效价在1:8000以上的小鼠即可进行加强免疫,加强免疫四天后进行细胞融合,ELISA法筛选能够稳定分泌抗CUB、F5/8C、MAM mAb的阳性克隆,并亚克隆,分别获得能够稳定分泌CUB单克隆抗体的细胞系6株,分泌F5/8C单克隆抗体的细胞系5株,分泌MAM单克隆抗体的细胞系2株。
第三,用GST标签蛋白通过Western blot对所获单克隆抗体进行鉴定,其中不识别GST标签蛋白而只识别融合蛋白的细胞株为识别目的蛋白的阳性株。结果显示,识别CUB蛋白的阳性株:UAH058、UDC114、UDG054、UBB067、UCB064,识别F5/8C的阳性株:OCA084、OEG082,无识别MAM的阳性株。用ELISA法分别检测PF4与CUB、F5/8C的相互作用[26],结果为:PF4与F5/8C存在特异的分子间相互作用。
结论:成功获得的NRP1三个片段CUB、F5/8C、MAM的鼠抗人单克隆抗体的可应用于蛋白结构、定位、功能等常规的生命科学研究外,在临床诊断和治疗方面也具有重要的意义,初步证明了PF4可与NRP1的F5/8C区发生特异性的结合,将为今后NRP1分子的功能研究奠定基础。
Background: Neuropilin was 102,000Da spanning transmembrane glycoproteinⅠon cell surface, which was found in the nervous system of Europe toad for the first time. More research proved that NRP1 usually expressed in the endothelial cells and some tumor cells, which function as receptors for semaphorin3A (Sema3A) and VEGF165 (vascular endothelial growth factor165). NRP1 was involved in neuronal cell guidance and axonal growth, vascular formation, growth and metastasis of tumors. Recent research showed that NRP1 play an important role in stable contacts between na?ve T cell and dendritic cell (DCs), that it’s one of continuous surface markers on CD4 + CD25 + regulatory T cells, was constitutively expressed on the surface of CD4+CD25+ Treg cells independently of their activation status, and played an important role in the initial immune response. Our team’s preliminary studies proved that PF4 stimulates proliferation of the human CD4+CD25+Tr cells, bind with PF4. The expression of NRP1 increased when PF4 stimulates proliferation of Treg cell. It is supposed that NRP1 function as receptor or co-receptor when PF4 stimulated proliferation of Treg cell. This study was to further define the binding site with NRP1 and PF4, provides basis for the function and regulation of Treg cell and possess the prospects in the treatment of autoimmune diseases.
Method & Result: NRP1 was high conservative in vertebrate, composed of intracellular region, transmenmbrane domain and extracellular region. The extracellular region contains 860 amino acids, was made up of three different structural domain including two complement binding (CUB) domain (a1/a2), two coagulation factor V/VIII homology domain(b1/b2), a MAM (meprin, A5, receptor tyrosine phospatase 1) domain (c), a transmenbrane segment. NRP1 mediated various signal transmission by interaction with three domain and different molecules, such as: Sema3A binds NRP1 through a1/a2/b1, VEGF165 binded NRP1 through b1/b2, and Heparin binds NRP1 through b1/b2 too, so NRP1 possessed multifunction. According to the structural characteristics of NRP1, we expressed fusion protein of three domains (CUB, F5/8C and MAM) by prokaryotic expression system to generate monoclonal antibodies (mAb) against NRP1. Western blot characterized the specificity of mAb. Then we study the interaction site between NRP1 and PF4.
Firstly, we searched for NRP1 sequence in HRPD (Human Protein Reference Database, www.hprd.org), select three domains (CUB, F5/8C, MAM) and amplified this three genes from monocytes cDNA. We designed primers according to cleavage map of pGEX-5X-1expression vectors, inserted three domains into the vector between EcoRⅠand NotⅠrestricted enzyme sites, After PCR, target gene was obtained successfully, double enzyme digestion of target gene and expression vectors, and conjuncted them, transformed into BL21 bacteria to express target proteins, sequencing. GST-CUB, GST-F5/8C and GST-MAM recombination proteins were expressed successfully.
Secondly, purified the recombination protein and emulsify with complete adjuvant were used to immunize Balb/c mice, after four weeks, cut down the dose of the recombination protein, and emulsified with incompelet adjuvant to immunize Balb/c mice, after two weeks, tested the titer of immunized mice. The immunized mice with high titer could be used to generate mAbs by hubridoma technique. Cell clones were screening by ELISA. After cell fusion, 6 hybridomas secreting specific Abs against CUB were established, 5 against F5/8C and 2 against MAM.
Thirdly, The antibody specificity was characterized by Western blot. The results showed that, 5 hybridomas such as UAH058, UDC114, UDG054, UBB067, UCB064 could recognize CUB recombination protein, 2 hybridomas such as OCA084, OEG082 could recognize F5/8C recombination protein, no hybridoma recognize MAM recombination protein. Then we identify interaction between PF4 and CUB, F5/8C, the result showed that F5/8C interact with PF4.
Conclusion: These mAbs could be used in immunoprecipitation, immunohistochemistry and confocal microscopy in the future to study the function of NRP1, such as molecules interact with NRP1and so on. F5/8C identifies initially maybe interact with PF4. And it will be useful for clinical diagnosis and therapy.
引文
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10. Neufeld G, Cohen T, Shraga N, et al. The neuropilins: multifunctional semaphorin and VEGF receptors that modulate axon guidance and angiogenesis [J]. Trends Cardiovasc Med, 2002, 12(1): 13-19.
11. Miao HQ, Lee P, Lin H, et al. Neuropilin-1 expression by tumor cells promotes tumor angiogenesis and progression [J]. FASEB J, 2000, 14(15): 2532-2539.
12. Latil A, Bieche I, Pesche S, et a1. VEGF over expression in clinically localized prostate tumors and neuropilin-1 over expression in metastatic forms[J]. Int J Cancer, 2000, 89(2): 167-171.
13. Bachelder RE, Crago A, Chung J, et a1. Vascular endothelial growth factor is an autocrine survival factor for neuropilin-expressing breast carcinoma cells [J]. Cancer research, 2001, 61(15): 5736-40.
14. Basile JR, Barac A, Zhu T, et al. Class IV semaphorins promote angiogenesis by stimulating Rho initiated pathways through plexin-B [J]. Cancer Res, 2004, 64(15): 5212-5224.
15. Takahashi T, Fournier A, Nakamura F, et al. Plexin–neuropilin-1 complexes form functional semaphorin-3A receptors [J]. Cell, 1999, 99(1): 59-69.
16. Antipenko A, Himanen JP, van Leyen K, et al. Structure of the semaphorin3A receptor binding module [J]. Neuron, 2003, 39(4): 589-598.
17. Tordjman R, Lepelletier Y, Lemarchandel V, et al. A neuronal receptor, neuropilin-1, is essential for the initiation of the primary immune response [J]. Nature Immunol, 2002, 3(5): 477-482.
18. Nagler-Andeson C, Bhan AK, Podolsky DK, et al. Control freaks: immune regulatory cells [J]. Nat lmmunol, 2004, 5(2): 119-122.
19. Bruder D, Probst-Kepper M, Westendorf AM, et al. Neuropilin-1: a surface marker of regulatory T cells [J]. Eur J Immunol, 2004, 34(3): 623-630.