Crystal Structure Of HAb18G/CD147-discovery Of A Novel Immunoglobulin Superfamily Dimerization Model

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.