【作者】 余晓玲；

【导师】 陈志南； 蒋华良；

【作者基本信息】 第四军医大学 ， 细胞生物学， 2008， 博士

【摘要】 目的CD147 (EMMPRIN、Basign、HAb18G)是一个I型整合跨膜糖蛋白,属于免疫球蛋白超家族(IgSF)成员。在体内大量涉及分子间相互作用的生理和病理过程,CD147均起着至关重要的作用。HAb18G/CD147是用抗人肝癌单克隆抗体(mAb)HAb18筛选人肝癌细胞cDNA文库而获得的一个新的肝癌相关抗原,具有刺激肝癌细胞和间质成纤维细胞分泌高水平基质金属蛋白酶(Matrix Metalloproteinases, MMPs)的功能,从而在肝癌浸润、转移过程中扮演重要角色。我们以前的研究表明,HAb18G/CD147不仅参与肿瘤侵袭、病毒感染以及内风湿关节炎发病机理,而且可做为一个新的通用型肿瘤生物标志物用于多种类型肿瘤的诊断和预后评估,因而成为相关疾病治疗干预措施的潜在靶标。尽管大量的研究揭示了CD147分子的多种生物学功能,但决定这些功能的结构基础仍是未解之谜,这不仅限制了对其功能机制的深入理解和进一步研究,也阻碍了针对这一抗原的新的抗体和小分子化合物的设计。因此,确定HAb18G/CD147分子的三维空间结构具有重要的科学意义和应用价值。本课题的研究目的是:通过高效的真核和原核表达体系,获得高纯度蛋白用于蛋白质结晶研究,利用X射线晶体衍射分析测定HAb18G/CD147分子的三维空间结构;在此基础上,通过计算机模拟分子对接,找到针对HAb18G/CD147分子的特异性单抗HAb18在该分子上的表位,并通过定点突变研究验证关键表位残基对抗原抗体识别和亲和力的影响。方法1 HAb18G/CD147胞外区(HAb18GEP)的高效表达和纯化1.1 HAb18GEP的定点整合真核分泌表达PcDNA5/FRT/18GEP-Fc重组表达质粒转染Flp-InTM CHO细胞并以潮霉素B筛选稳定高表达细胞株。用生物反应器大规模培养高表达CHO细胞株,收获的培养上清首先利用融合标签进行亲和层析纯化,再用3C蛋白酶特异性切除融合标签并再纯化。1.2 HAb18GEP的原核可溶性表达表达质粒pET21a(+)/HAb18GEP转化E.coli OrigamiB(DE3)菌株;表达产物用阴离子交换层析和凝胶过滤层析纯化。2 HAb18GEP的结晶和结构解析2.1 HAb18GEP的结晶和初步X-ray衍射采用悬滴扩散法对真核和原核表达的HAb18G/CD147胞外区蛋白进行晶体生长条件的筛选和优化,然后在X-ray衍射仪上进行天然和硒代蛋白晶体的初步衍射。2.2数据收集和结构解析在同步辐射光源(Photon Factory, Japan)上分别收集天然蛋白晶体和硒代蛋白晶体衍射数据,用单波长反常散射法(SAD)进行结构解析。首先用硒代蛋白晶体的衍射数据获得相位信息和初始结构模型,再用天然蛋白晶体的衍射数据进行结构确定和精修。3 HAb18单抗分子对接和突变验证利用计算机模拟HAb18单抗的三维结构并与HAb18G/CD147的晶体结构进行分子对接,分析特异性单抗HAb18的可能表位并通过定点突变实验进行验证。结果1 HAb18G/CD147胞外区(HAb18GEP)的高效表达和纯化1.1 HAb18GEP的定点整合真核分泌表达PcDNA5/FRT/18GEP-Fc重组表达质粒转染Flp-InTM CHO细胞并以潮霉素B筛选阳性细胞克隆,获得高表达细胞株H8F8E10。经生物反应器大规模培养后,培养上清中的表达产物可达到17mg/L。表达产物经亲和层析纯化并切除融合标签后,获得高纯度的真核HAb18G/CD147胞外区蛋白用于晶体培养。1.2 HAb18GEP的原核可溶性表达HAb18GEP在OrigamiB(DE3)菌株中以可溶形式表达;表达产物经阴离子交换层析和凝胶过滤层析纯化后,得到高纯度的原核可溶性蛋白用于晶体培养。2 HAb18GEP的结晶和结构解析2.1 HAb18GEP的结晶和初步X-ray衍射经过晶体生长条件的大量筛选和优化,原核表达的HAb18GEP在适宜条件下培养出蛋白单晶;在此基础上,制备硒代蛋白并培养出硒代蛋白晶体,通过在X-ray衍射仪上的初步衍射,筛选出分辨率在2.8～3.0?的天然和硒代蛋白晶体用于同步辐射光源上的数据收集。2.2数据收集和结构解析利用天然蛋白晶体的衍射数据(2.8?)和硒代蛋白晶体衍射数据(3.1?)进行结构解析的结果,确定HAb18G/CD147胞外区晶体的空间群为P41212,在一个不对称单元有四个分子,每个分子由两个Ig样结构域组成,N端结构域为Ig C2型,C端结构域为Ig I型;四个分子相互粘联形成寡聚体。3 HAb18单抗分子对接和突变验证抗原抗体分子对接的结果显示:HAb18G/CD147胞外区残基Glu49、Thr51、Asp65为HAb18单抗的关键表位残基。突变前后的Western Blot显示,突变后的抗原结合抗体的能力明显下降。结论1. HAb18G/CD147分子晶体结构的解析,首次从原子水平揭示了一个具有独特的IgC2–I结构域排列方式的IgSF成员,并认为它可能代表了其它CD147家族成员的一般结构。2.在晶体结构中发现的由IgC2结构域介导的二聚化模式为IgSF的同型粘附模式增添了新的范例。3.首次通过晶体结构观察到CD147分子的寡聚现象,从而为该分子寡聚依赖的重要生物学功能提供了结构的阐释。4.基于HAb18G/CD147抗原的晶体结构,首次通过计算机模拟分子对接和定点突变研究证实HAb18G/CD147胞外区残基Glu49、Thr51、Asp65为单抗HAb18的关键表位残基。更多还原

【Abstract】 Aims:CD147 (also known as EMMPRIN, Basign, and HAb18G) is a type I integral transmenbrane glycoprotein that belongs to the immunoglobulin superfamily (IgSF). It plays fundamental roles in intercellular interactions in numerous pathological and physiological processes. HAb18G/CD147, a novel hepatocellular carcinoma (HCC)-associated antigen, was cloned in our lab by screening the HCC cDNA expression library using anti-HCC mAb HAb18. Our studies have found that HAb18G/CD147 stimulates adjacent fibroblasts and HCC cells to produce elevated levels of several MMPs, facilitating invasion and metastasis of the HCC cells. All our findings have indicated that HAb18G/CD147 is an important molecule in tumor progression, viral infection and RA pathogenesis and thereby a potential drug target for therapeutic interventions. Importantly, HAb18G/CD147 has also been shown to be a novel universal cancer biomarker for diagnosis and prognostic assessment of a wide range of cancers. Numerous researches have led to better understanding the biological functions of CD147, but the structural basis underlying its multifunctional character remains to be elucidated. The lack of three- dimensional structure of HAb18G/CD147 hampers the design of new antibodies and small molecule regulators against this antigen; therefore, defining the structure of HAb18G/CD147 is of great interest and pharmaceutical importance. The aim of this research project is to obtain high purity proteins by high efficient prokaryotic and eukaryotic expression, and then crystallize the proteins and solve the crystal structure by X-ray diffraction method. Furthermore, the epitope of specific mAb HAb18 was mapped by molecular docking and mutagenesis validation.Methods1 High efficiency expression and purification of HAb18G/CD147 extracellular portion(HAb18GEP)1.1 Eukaryotic site-directed integration expression of HAb18GEP Expression vector pcDNA5/FRT/HAb18GEP-Fc was transfected in Flp-InTM CHO cells and stable expression cell lines were screened by Hygromycin B. The stable expression CHO cells were cultured with bioreactor and expression products were purified by affinity chromatography; then, fusion-tag was cleaved with HRV 3C protease and the enzymic mixture was further purified.1.3 Prokaryotic expression of HAb18GEP Expression vector pET21a(+)/HAb18GEP was transfected into E.coli OrigamiB (DE3), and expression products were purified by two steps of anion-exchange chromatography and one step of gel-filtration chromatography.2 Crystallization and structure determination of HAb18GEP2.1 Crystallization and preliminary X-ray diffusion Crystals were grown using hanging-drop vapor diffusion method and were preliminary characterized on X-ray diffractometer.2.2 Data collection and structure determination X-ray diffraction data of native and Selenomethionine (SeMet)-substituted crystals were collected respectively on Synchrotron Radiation (Photon Factory, Japan). Structure was solved by single-wavelength anomalous diffusion (SAD) method.3 Molecular docking of mAb HAb18 and mutagenesis validation Based on the crystal structure of HAb18G/CD147, the epitopes of specific mAb HAb18 were mapped by molecular docking with modeled antibody structure. Furthermore, the effect of the key residues on antigen-antibody recognition was evaluated by site-directed mutagenesis.Results1 High efficiency expression and purification of HAb18G/CD147 extracellular portion(HAb18GEP)1.1 Eukaryotic site-directed integration expression of HAb18GEP A stable CHO cell line expressing HAb18GEP-Fc was generated and designated as H8F8E10. After large-scale cell culture, target proteins were harvested by affinity chromatography. Pure proteins were obtained by fusion-tag removal and subsequent purification.1.2 Prokaryotic expression of HAb18GEP Soluble HAb18GEP expressed in E.coli OrigamiB (DE3) was purified by two steps of anion-exchange chromatography and one polishing step of gel-filtration chromatography and was subsequently used for crystallization. 2 Crystallization and structure determination of HAb18GEP2.1 Crystallization and preliminary X-ray diffusion Tetragonal crystals were grown from prokaryotic expressed HAb18GEP after extensive screening of crystalization conditions. Crystals diffracted to 2.8～3.0? were selected by preliminary X-ray diffraction and prepared for data collection on Synchrotron Radiation.2.2 Data collection and structure determination Diffraction data of SeMet-substituted crystals (3.1?) and native crystals (2.8?) were used for structure determination and refinement. HAb18G/CD147 crystallizes in space group P41212 with four monomers in the asymmetric unit. This structure comprises an N-terminal IgC2 domain and a C-terminal IgI domain, which are connected with a 5-residue flexible linker. The four monomers adhere together and form homo-oligomer in the crystal.3 Epitope mapping of mAb HAb18 and mutagenesis validationThe molecular docking mapped the epitope of specific mAb HAb18 to three discrete areas on HAb18G/CD147 N-terminal domain, and three residues Glu49、Thr51、Asp65 are regarded as the key residues of the epitope. Western-blot assay revealed sharply decreased binding affinity after mutation of these residues.Conclusion1. The crystal structure of HAb18G/CD147 reveals for the first time an atomic view of an IgSF member with a C2–I domain organization and may represent a general architecture of other CD147 family members.2. The dimerization mediated by IgC2 domain in the crystal presents a novel paradigm for the existing IgSF homophilic adhesion model. 3. The crystal structure of HAb18G/CD147 also provides a good structural explanation for the oligomerization-dependent multifunction of CD147.4. Based on the results of molecular docking and mutagenesis validation, three residues Glu49, Thr51 and Asp65 of HAb18G/CD147 are believed to be the key epitope residues of mAb HAb18.更多还原