The Effect Of RPTP Rho With Altered N-Glycans On Its Phosphatase Activity And Functions

N-Acetylglucosaminyltransferase V (GnT-V), an enzyme that catalyzes the β1,6 branches of N-acetylglucosamine on N-glycans, plays important roles in carcinogenesis and tumor metastasis. Aberrant N-glycosylation on cell-surface proteins can change their functions and subsequent signaling pathway. GnT-V modified β1,6 GlcNAc branched N-glycan has high affinity with galectin-3, which enables its interaction with glycoproteins. Protein tyrosine phosphatases (PTPs), as cellular counterpart of protein tyrosine phosphatases (PTKs), regulate various cellular activities essential for the initiation and maintenance of malignant phenotypes. The type IIB receptor-like protein tyrosine phosphatases (RPTPs) include PTPRT, PTPRU, PTPRM and PTPRK, of which PTPRK has been identified as a substrate of GnT-V by Kim. Due to the important role of RPTPs in maintaining the phosphorylation level of protein tyrosine, it’s of great importance to study their aberrant glycosylation for a better understanding the progression of tumor.We first established the GnT-V overexpressing stable cell line to identify whether there is other substrate of GnT-V. In the first section of our research, we detected the expression of GnT-V in several cell lines and chose SMMC-7721 for further study. SMMC-7721 were infected with GnT-V overexpressing lentivirus or the control lentivirus and designed as Mock-7721 or GnT-V-7721. The overexpression of GnT-V was examined by immnoblotting. Lectin blotting, fluorescent staining and flow cytometry were employed and results implied that β1,6 GlcNAc branched N-glycans on whole cell-surface proteins were increased as a result of GnT-V overexpression. We used confocal microscopy to examine the distribution of PTPRT, PTPRU, PTPRM and PTPRK in Mock-7721 and GnT-V-7721, and chose PTPRT for further study. We predicted its Asn-X-Ser/Thr (NXS/T) sites with NetNglyo 1.0 software and found 16 potential N-glycosyslation sites. Then, the lectin L-PHA was employed to precipitate glycoproteins which may have β1,6 GlcNAc branched N-glycans added by GnT-V. The level of precipitated PTPRT exhibited dramatically higher in the GnT-V cells than that of the Mock cells. By performing immunoprecipitation with PTPRT antibody and then staining with L-PHA or DSL, we confirmed that PTPRT expressed the tri- and tetra-branched N-glycans.Whether increase of β1,6 GlcNAc branched N-glycans on PTPRT have an effect on its protein level? And what’s the molecular mechanism? In the second section of our study, we detected the in situ expression of PTPRT in stable cells. PTPRT glycoprotein obviously accumulated more on the cell membrane in the GnT-V-7721 than that in Mock-7721. Is this phenomenon a result of delayed membrane retention of PTPRT in GnT-V-7721? The cell-surface proteins were biotinylated and cells were re-cultured for different time. We found that PTRT was accumulated in the time course and its level is obviously higher in GnT-V-7721 than Mock-7721, indicating that overexpression of GnT-V extended the protein retention time of PTPRT at cell surface. Interestingly, the protein level of PTPRT increased after the biotinylation, implying the PTPRT may form dimmers. BS3 mediated chemical cross-linking was performed. GnT-V-7721 revealed a higher dimerization level of PTPRT in contrast with Mock-7721.As galectin-3 has a higher affinity with GnT-V modified N-glycans, we proposed galectin-3 form molecular lattice with PTPRT. We biotinylated cell-surface protein and found galectin-3 showed a similar tendency with PTPRT. Confocal microscopy revealed that overexpression of GnT-V led to increased colocalization of galectin-3 and PTPRT at the cell surface. We performed co-immunoprecipitation assay after BS3 cross-linking. The GnT-V overexpressing cells showed an increased binding of galectin-3 with PTPRT. Collectively, these data indicate that the molecular lattice formed by galectin-3 contribute to increased dimerization of PTPRT in GnT-V overexpressing cells.The activity of RPTPs is inhibited by dimerization. It has been reported that PTPRT specifically dephosphorylates STAT3 at tyrosine amino acid 705. So we want to clarify the effect of increased dimerization of PTPRT on its substrate and subsequent biological effect. Here, we found that the phosphorylated STAT3 at Y705 was increased in GnT-V overexprssing cells. Furthermore, the amount of STAT3 located in nucleus was elevated in GnT-V overexpressing cells using nuclear and cytoplasmic protein extraction experiment or with confocal microscopy analysis. We also observed that increased pY705 STAT3 can promote cell migration, which implied that pY705 STAT3 may contribute to the GnT-V medated migration.In summary, we have, for the first evidence, demonstrated that PTPRT is a substrate of GnT-V. Addition of β1,6 GlcNAc branched N-glycans on PTPRT increased its binding with galectin-3, resulting in enhanced dimerization and decreased phosphatase activity on phosphorylated STAT3 at Y705, which may be a possible mechanism for GnT-V mediated migration.