Design,Expression And Application Of Gene Encoding FRET Fluorescence Proteins Sensing Glycosylation Of CD147

CD147, belonging to the immunoglobulin family (IgSF), is a highly glycosylated transmembrane protein. It is widely expressed on numerous cells and plays an important role in many physiological and pathological processes with interacting with other ligands, such as cell movement, tissue reconstruction,inflammation and so on. CD147, also named EMMPRIN, is highly expressed on many tumor cells, correlating with tumor progression under experimental and clinical conditions. It can induce adjacent fibroblasts and tumor cells themselves to produce extracellular matrix metalase (MMPs), which could degrade the extracellular matrix. A significant biochemical property of CD147 is its high level of glycosylation. Two forms of glycosylated CD147 are reported: high-glycosylation CD147 and low-glycosylation CD147. Many researches demonstrated that only the high-glycosylation of CD147 contributes to its MMPs induction activity. So far,most of the sensors targeting to CD147 are developed based on the antibody and could not sense the glycosylation status of CD147 in real time. In order to better understand the physiological and pathological functions of CD147 and its role in tumor metastasis, it is indispensable to develop sensors that could be used to image the glycosylation of CD147 in living cells.In this work, we designed series of novel gene coding FRET flurescence sensors sensing the glycosylation of CD147. Firstly, we desgined series of sensors using proteins or peptides interacting with CD147 specifically. To target CD147, we synthetised sensors and studied the colocalization with CD147 in living cells. The group of sensor targeting CD147 best in live cell imaging was selected. Then, we measured glycosylation of CD147 under different levels with the sensor by fluorescence imaging, and established a semi-quantitative relationship between the flurescence measurement and CD147 glycosylation. Finally, we verified the targeting specificity of the sensor. We want to further optimize the specificity and sensitivity of the sensors, and investigate the mechanism of CD147 glycosylation and tumor metastasis with the sensors in the future.