Analysis Of The Structures And Functions Of N-Glycans Of Met

EGFR-TKI is a most important drug in the treatment of lung cancer and it is important that development of resistance to the drug be overcome. MET amplification has been associated with the drug resistance, but although many agents targeting MET have been studied, they have not yet been clinically applied. To develop new agents targeting MET, it is necessary to clarify its physical property. Considerable evidence has shown that the MET glycans are essential toward their ability to function. MET contains 11 potential sites for N-glycans, but their structures and functions are unclear. The purpose of this study is to investigate the structures and functions of N-glycans of MET. We established a soluble MET stable expression cells, and purified the protein through a series of column chromatography. The purified soluble MET was digested with trypsin, and the glycosylated peptides were then isolated and analyzed by liquid chromatography-tandem mass spectrometry. The results revealed that the dominant population of 11 N-glycans of MET was entirely a complex-type with sialic acids and core fucose. Furthermore, no unglycosylated peptide containing N149, N 399, N 405, N 635, N 785, or N 879 was found in the tryptic peptide mixture, indicating that 6 sites were nearly 100% glycosylated. This indicates that the 6 N-glycans may play a more important role than the others. We are presently determining the functions of each N-glycan by examining HGF signaling of N-glycan deletion mutants. Determining the structures of N-glycans of MET could be crucial for developing a novel platform of MET research. We are also examining the signal suppressing effects of soluble MET and its glycan deletion mutants.