LRRC4 Inhibits Its Own O-glycosylation To Exert Tumor Suppressor Function In Glioma

BackgroundGlioblastoma(GBM)is the most common primary tumor in the central nervous system,and the prognosis of glioblastoma patients is generally poor.As a tumor suppressor gene of glioblastoma,leucine-rich repeat C4(LRRC4)has been widely discussed in terms of its tumor suppressor mechanism.However,studies on the post-translational oglycosylation of LRRC4 protein and the anti-cancer mechanism of Oglycosylation of LRRC4 are still in the blank stage.Methods and ResultsThis study firstly analyzed the expression of LRRC4 in human and mouse brain tissues and nerve cells by bioinformatics methods.Western Blot verified that LRRC4 protein was highly expressed in nerve cells and tissues,and significant glycosylation of LRRC4 protein occurred in nerve tissues,but the glycosylation level was low in cultured nerve cells in vitro.After overexpression of LRRC4 plasmid in HEK293,GBM cell line U251 and primary cultured GBM cell 1124 C,it was found by immunoprecipitation and mass spectrometry that LRRC4 protein was glycosylated in HEK293 cells.In addition,O-glycosylation occurred mainly on the threonine residues at the 390,509,511 and 512 sites.However,the O-glycosylation of LRRC4 protein in GBM cells was significantly reduced,and only the 509 th threonine residue was Oglycosylated.Transwell and CCK-8 experiments confirmed that the 509 glycosylation site of LRRC4 is a key site for LRRC4 protein to exert tumor suppressor effect.Next,we further explored the mechanism by which LRRC4 protein inhibits its own O-glycosylation modification.Through the analysis of RNA-seq results,RT-qPCR and Western Blot experiments,it was found that LRRC4 mainly inhibited the O-glycosylation modification in tumor cells by inhibiting the expression of the O-glycosylase GALNTL6.However,the same experiment in normal cells and knockout mice showed that LRRC4 did not affect o-glycosylation in normal cell and mice.Targeted metabolomics analysis combined with the detection of β-galactosidase activity showed that LRRC4 inhibited the hexosamine biosynthetic pathway(HBP)by inhibiting β-galactosidase activity.This results in a decrease in the carbohydrate synthesis required for O-glycosylation to inhibit O-glycosylation.Conclusions1.LRRC4 mainly functions in the form of glycosylation modification in the central nervous system,while in normal cell lines cultured in vitro,the glycosylation modification of LRRC4 is significantly reduced.2.The glycosylation modification of LRRC4 in HEK293 cells is mainly O-glycosylation.Compared with that,the O-glycosylation modification of LRRC4 in GBM cells is significantly reduced.Among them,the 509 th O-glycosylation modification is a key site for LRRC4 to exert its tumor suppressor function.3.LRRC4 inhibits its own O-glycosylation modification by downregulating the expression of O-glycosyltransferase GALNTL6 and exerts its anti-tumor effect.4.LRRC4 inhibits its own O-glycosylation by promoting the pentose phosphate pathway in GBM cells to inhibit the synthesis of Nacetylgalactose.