Purification Of Hepatoma Associated Antigen HAb18G/CD147 And Characterization Of Glycosylation

Aims: HAbl8G is a novel hepatoma-associated antigen, recently cloned by hepatoma monoclonal antibody HAb18 screening from human hepatocellular carcinoma cDNA library. The cDNA open reading frame of HAbl8G is identical to that of CD147 molecule, a member of immunoglobulin super family (IgSF), also named as extracellular matrix metalloproteinase inducer (EMMPRIN), Basigin ,0X47 and 5A11/HT7 , and has been characterized in different species. HAbl8G stimulates fibroblasts to produce Matrix Metalloproteinases (MMPs), which may play an importmant role in tumor invasion and metastasis. Since HAb18G is a highly glycosylated protein the glycosylation may have important effect on its molecular structure and founctional activity. To investigate the characteristic of glycosylation of HAb18G may provide some evidences for learning the relationship between HAb18G and tumor high metastasis and high recurrence, and further studying the functions of HAb18G. Therefore, in the present study, our aims are (1) to obtain native HAb18G with high purity , specificity and bioactivity so as to provide experiment material for study on its structure and functions; (2) to investigate the specific glycosylation of native HAbl8G in hepatoma cells, which may be helpful to reveal the relationship between glycosylation and molecular structure and functional activitiy. Methods: 1 .Purification and functional identification of native HAb18G/CD147A stable affinity chromatographic media was generateded by coupling mAb HAb18 to rProtein A Sepharose beads and further stabilized with DSS, abifunctional chemical crosslinker. After screening several candidates by ELISA and BCA, the optimal detergent was chosen to extract HAb18G from the HCC cell membranes. HAbl8G was then captured from the crude detergent extracts.The purity, specificity and founctional activity of purified HAbl8G were detected by SDS-PAGE ,Western blotting and Gelatin zymography respectively. 2. Initial study of glycosylation of native HAbl8G/CD147(1) HHCC cells grown to conflunce were treated with 2-8 μ g/mL of N-glcosylation inhibitor tunicamycin in normal culture media for 16h.The cells were then lysed and lysates were used for Western blotting to detect HAb18G.(2) Endoglycosidase F and H were used to digest the three potential N-glycosylation sites of HAb18G/CD147, then the effect of deglycosylation on molecular mass was detected by Western blotting.Results:1 .purificaton and founctional identification of native HAb18G/CD147The purified HAb18G showed a single band with molecular mass of ~58kD on silver stained SDS-PAGE, which reacted specifically with mAb HAb18 by Western blotting analysis. The Gelatin zymography showed that the purified HAb18G can stimulate human fibroblasts to secrete elevated MMPs.2. Initial study on glycosylation of native HAb18G/CD147 (1) Human hepatoma cells express two major forms of HAbl8G , Treatment of HHCC cells with tunicamycin, an inhibitor of N-linked glycosalation of newly synthesized proteins, affected both the higher molecular mass form and the lower molecular mass form.The 35kD form completely disappeared ,and the level of the 45-66kD(~58kD) form was greatly diminished after tunicamycin treatment. A new band with lower molecular weight (~27kD) appeared.(2) when deglycosylated by endo F or endo H , the 45-66kD and the 35kD forms of HAbl8G disappeared alternatively according to the endoglycosidase used. Furthermore, the two forms of HAbl8G...