Expression, Purification And Preliminary Analysis Of Glycosylation Of Murine CD147 Extracellular Domain

Characteristics of malignant tumor invasion and metastasis make it one of the three diseases threat to human survival. Tumor metastasis and invasion is a complex multi-step process. Tumor invasion and metastasis are major obstacles to treatment of cancer and causes lead cancer patients to death. The main methods of tumor metastasis are lymphatic metastasis, hematogenous metastasis, and implantation metastases. Studies have shown that glycoprotein glycosylation form and glycons structure of tumor cell surface is deeply related with tumor metastasis.CD 147,also known as EMMPRIN,are immunoglobulin superfamily (immune-globulin superfamily, IGSF) of the transmembrane glycoprotein, express in many tumor cell surface which is monitor molecules of cell membrane. CD147 is composed of two extracellular Ig domains, a trans-membrane domain and an intracellular structural domains. Extracel-lular domain has three Asn glycosylation sites. As a result of heterogeneous N-glycosylation, CD147 exists in both a highly glycosylated form (HG-CD147) and lowly glycosylated form (LG-CD147).CD147 is involved in tissue remodeling, cell differentiation, angiogenesis, inflamm-ation and tumor metastasis mainly via induction of matrix metallopro-teinases (matrix metallo-proteinases, MMPs). Caveolae is a cell surface membrane structure by invagination. Cav-1 is membrane marker protein of Caveolae, and play a crucial role in regulation the tumor occurrence and developmentOur laboratory studies have shown that:CD147 glycosylation is related with tumor metastasis. HG-CD147 in the high potential of lymphatic metastasis of mouse liver cells (Hca-F)express high and express lower i(?) low metastatic cell line (Hca-P), while the LG-CD147 in the absence of metastatic potential mouse hepatoma cell line (Hepal-6) comprise a major part. In addition,CD147 glycosylation and the Cav-1 were positively correlated. In Hca-F, in HG-CD147 expression decreased as Cav-1 expres-sion decreased, while LG-CD147 expression increased.Stable expression of Cav-1 in Hepal-6 led to HG-CD147 expression increased,while the LG-CD147 expression was significantly decreased.In this paper, we constructed secreted extracellular domain of mouse CD147 plasmid, and we expressed and purified CD147 extracellular domain in Hepal-6 and Hepa1-6/Cav-l cells. By comparing the glycosylation form and glycons structure in the existence of Cav-1 or not, we expect to investegate the association between CD 147 glycosylation and tumor metastasis, and Speculate the molecular mechanism of Cav-1 regulate CD147 glycosylation.Method:1. Construction of recombinant vector PCDNA3.1/CD147We amplified the extracellular domain of mouse CD147 by PCR. The secretory signal peptide and CD147 extracellular chimeric gene was inserted into the expression vector pCDNA3.1 to construct the eukaryotic secreted expression vector pCDNA3.1/CD147.2. Expression and purification of CD147-His fusion protein in Hepal-6 and Hepal-6/Cav-1The recombinant vector pCDNA3.1/CD147 was transient transfect into Hepa1-6 and Hepa1-6/Cav-1 cells. ELISA was used to detect the expression of fusion protein in cell culture supernatant of 24h,48h,72h. Based on this, a great deal of transfection, Ni-NTA affinity chromatography was used to purify protein,and SDS-PAGE, Western-blot was used to identify target protein.3. Detection of biological activity of CD147-His fusion protein and preliminary analysis of glycosylationMouse fibroblast cells was stimulated with the purified fusion protein, the change in its secretion of MMP9 was detected by gelatin zymo-graphy.Lectin blotting, mass spectrometry and other methods are used to analyze the glycosylation of fusion protein Result:1.The extracellular domain of mouse CD 147 gene is amplified by PCR, the size is about 560bp.By insert the secreted signaling peptide, we constructed secereted eukaryotic expression vector pCDNA3.1/CD147.2.ELISA showed that the protein was secreted mainly at 24h after transfaction. A large number of transfection, the protein was purified by affinity chromatography. SDS-PAGE, and Western-blot showed that the target protein molecular weight is about 48KD.3. Gelatin Zymography showed that the target protein have biological activity, which could induce fibroblast cells produce MMPs;ELISA and Dot-blot showed that the target protein could specialy bind to lectin PHA-E.Conclusion:1.Successfully constructed recombinant expression vector pCDNA3.1/ CD147.2.Achieve the expression and purified of CD 147 in Hepal-6 and Hepal-6/Cav-1 cells.3.The purified glycoprotein in Hepal-6 cells have biological activity, and the protein glycon contains a split-type GlcNAc branches.