| Malignant behaviors in transformed cells and human tumors such as migration and invasion are highly associated with the significant increase of asparagine-linked glycans (N-glycans) containing GlcNAcβ1,6 branching. The β1-6 branching of carbohydrates is the result of β1-6 N-acetylglucosaminyltransferase V , a trans Golgi enzyme encoded by the Mgat5 (mannoside acetylglucosaminyltransferase 5) gene, which adds N-acetylglucosamine (GlcNAc) to the mannose of the trimannosyl core in a β1-6 linkage. Results from recent studies showed the expression of β1,6-GlcNAc branched N-linked oligosaccharides in human mammary, colon, hepatic, and glial tumors and confirm the notion that the GlcNAcβ1,6 branched structure acquires the properties of cancer invasion and metastasis. Therefore, GnT-V was taken as one of the most important glycosyltransferases in tumor growth and metastasis . Integrins are composed of a and β subunits, which are all major carriers of N-glycans .Changes in N-glycans of these integrins have the key effects on cell-cell and cell-extracellular matrix (ECM) interactions.Reportedly, the minimal catalytic domain of GnT-V was existed in its carboxyl -terminus, and deletion of as few as 4-8 amino acids from its carboxyl-terminus destroys its catalytic activity .In this study, an enzymatic inactive mutant of GnT-V (AcGnT-V) was constructed. Positive (pcDNA-wtGnT-V or pcDNA3-AcGnT-V) and negative (empty vector) clones were selected, and transfectants with higher expression levels of wt- or Ac- GnT-V were used for the experiments described herein. The transfectants from pcDNA3, pcDNA3-wtGnT-V and pcDNA3-AcGnT-V were designated as Mock, wt-7721 and Ac-7721 cells, respectively. We investigated the integrin β1 expression pattern in the Mock, wt-7721 and Ac-7721 cells. Our results showed that integrin β1 was increased in wt-7721 cells compared with Mock cells, while decreased in Ac-7721 cells .Integrin β1 in AcGnT-V transfectants (Ac-7721) showed attenuation of the number of GlcNAcβ1,6 branching, whereas those inwtGnT-V transfectants (wt-7721) presented a GlcNAcβ1,6-rich pattern. High integrin β1 expression was observed in wt-7721 compared with Mock cells ( 7721 cell transfected with the vector pcDNA3), while transfection of AcGnT-V decreased the integrin β1, despite of no significant changes on integrin β1 mRNA level in these cell lines. Pulse-chase experiment showed that Integrin β1 in Ac-7721 was prone to quick degradation and its half-life was less than 3 h, on the contrary, the alleviating degradation of β1 subunit was observed in wt-7721 where the β1 subunit half-life was about 16 h, meanwhile, the degradation rate of β1 subunit in Mock cells was in between, about 10h.More effective in promoting cell migration toward fibronectin and invasion through Matrigel was observed in wt-7721 while this was almost suppressed in Ac-7721. In wt-7721 cells, the total level of PKB and FAK did not change, but the levels of p-PKB Ser473,p-GSK3-β Ser9 and p-FAK were significantly increased; On the contrary,in Ac-7721 cells, the levels of p-PKB Ser473, p-GSK3-p- Ser9 and p-FAK levels were markedly decreased.Our results suggest that the addition of GlcNAcβ1,6 branching caused more fully glycosylated mature form on integrin piand inhibited β1 protein degradation; on the other hand,changes in N-linked glycosylation caused by A cGnT-V directed degradation of β1 integrin and subsequently inhibited cell migration and invasion. Glycosylation caused by GnT-V directs integrin β1 stability and more delivery to plasma membrane, subsequently promotes FN-based cell migration and invasion. Thus, our findings support the notion that GnT-V up-regulates β1 integrin preferably through enhancing its half-life in the enzymatic activity-dependent manner.
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