The Study On The Expression And Function Of CD147 Glycosylation Site Mutants In Murine Normal Hepatocyte IAR20 Cells

Objective:Glycosylation is one of the most important post transla- tional modifications of the protein, which is related to many activities of life. With the development of the proteomics, the studies of the glycosylation are attached more and more importance.CD147 is a highly glycosylated transmembrane protein belonging to the immunoglobulin superfamily. CD147, as a cell surface multifunctional glycoprotein, is widely expressed in various cells, such as lymphoblasts, inflammatory cells and especially tumor cells. It plays roles in many physiological and pathological processes involving spermatogenesis and fertilization, neural network formation and development, HIV infection, and tumor metastasis.CD147 is composed of two extracellular Ig domains, a single transmembrane domain, and a cytoplasmic domain. The first Ig domain of CD147 is required for counter receptor binding activity, which is involved in MMP induction and oligomerization. The second Ig domain of CD147 is known to associate with Caveolin-1(Cav-1). As a result of heterogeneous N-glycosylation, CD147 exists in both a highly glycosylated form, HG-CD147 (~40-66kDa) and lowly glycosylated form, LG-CD147 (~33kDa). It has been reported that N-glycosylation of CD147 promotes CD147 exhibits MMP induction activity. But the function of N-glycan on CD147 is not clear.This study is to investigate the effect of N-glycosylation of CD147 on the cellular behavior of murine normal hepatocyte IAR20 cells. This study will supply valuable data and clue for a better understanding of the relationship between CD147 glycosylation and biological function.Methods1. Using pcDNA3.1/CD147(wt) as templete, by PCR site-directed mutagenesis method,the 3 glycosylation sites of CD147 cDNA is mutated, then cloned into pcDNA3.1, assigned as pcDNA3.1/CD147(N154Q) and pcDNA3.1/CD147(N44,190Q), respectively. The constructed plasmids were confirmed by DNA sequencing analysis.2. The plasmid of pcDNA3.1/CD147(wt), along with pcDNA3.1/CD147 (N154Q) and pcDNA3.1/CD147(N44,190Q), was transfected into IAR20 cells by lipofectamine, respectively. The positive cell clone was selected by G418. PCR and Western blot analysis were used to detect the expression of wild type CD147 and mutants (CD147N190Q and CD147N44, 190Q.)3. The cell growth and proliferation of IAR-20 cells were analyzed by MTT method, and the cell migration and invasion ability was observed by Transwell assay.Results1. pcDNA3.1/CD147(N154Q) and pcDNA3.1/CD147(N44,190Q) was obtained by PCR site-directed mutagenesis method.2. Western blot and RT-PCR analysis showed that wild type CD147 and mutants CD147(N154Q)in IAR20 cells as molecular weight of 44 kDa and 45kDa, respectively. But CD147(N44,190Q) expressed as two bands with molecular weight of 37 kDa and 42kDa, respectively..3. IAR-20/CD147(wt) cells and IAR-20/CD147 ( N154Q ) cells exhibited significantly higher proliferation rate and higher cell migration ability than untransfected and Mock IAR-20 cells (p<0.05). But the ability of cell invasion was found no difference among IAR-20/CD147(wt) cells, IAR-20/CD147(N154Q) cells and IAR-20/ CD147(N44,190Q).Conclusions:Glycosylation of CD147 may contribute to the proliferation and migration of IAR-20 cells.