Abstract
Introduction of the beta1-4 N-acetylglucosaminyltransferase (GnT-III) gene was reported to suppress metastasis in highly metastatic B16-hm murine melanoma cells (Yoshimura, M., Nishikawa, A. , Ihara, Y., Taniguchi, S., and Taniguchi, N.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8754-8758). In this study, the effect of GnT-III gene transfer on E-cadherin was studied, since E-cadherin acts as a suppressor of metastasis. E-cadherin expression at cell-cell contacts of B16-hm cells expressing high GnT-III activity was greater than controls without affecting transcription. Lectin blotting showed that E-cadherin from GnT-III transfectants was glycosylated by ectopically expressed GnT-III. The glycosylated E-cadherin exhibited the delayed turnover and the decreased release from cell surface, as compared with the native E-cadherin, resulting in the elevated expression at the cell-cell border of GnT-III transfectants. Furthermore, cell-cell aggregation was enhanced in GnT-III transfectants, indicating that the glycosylated E-cadherin is biologically functional. These results suggest that the glycosylated E-cadherin contributes to the suppression of metastasis by the introduction of GnT-III gene into melanoma cells.
Highlights
The malignant phenotype, including metastatic potential, has been reported to be associated with the 1– 6 branches of N-oligosaccharides, the product of 1– 6 N-acetylglucosaminyltransferase (GnT-V, EC 2.4.1.155)1 in ras-transformed rat fibroblasts, rat mammary carcinoma cells, rat lymphoma cell line [1, 2], and human colon cancer cells [3]
The present investigation was undertaken to examine whether the reduced metastatic potential of B16 murine melanoma cells expressing ectopic GnT-III was due to the altered biological function of E-cadherin that mediates homotypic cellcell adhesion, since the E-cadherin expression correlates inversely to metastatic phenotype in many cancer cells [11,12,13]
Increased Glycosylated E-cadherin in GnT-III Transfectants—erythroagglutinating phytohemagglutinin (E-PHA) lectin was used to analyze the alterations of carbohydrate structures of E-cadherin, since E-PHA has a high affinity for bisecting GlcNAc structure [16]
Summary
GnT-V, 1– 6 N-acetylglucosaminyltransferase; GnT-III, 1– 4 N-acetylglucosaminyltransferase; E-PHA, erythroagglutinating phytohemagglutinin; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-buffered saline; TBS, Tris-HCl-buffered saline; FITC, fluorescein isothiocyanate; PAGE, polyacrylamide gel electrophoresis. Both 1– 4 N-acetylglucosaminyltransferase (GnT-III, EC 2.4.1.144) and GnT-V use the biantennary structure of N-oligosaccharides as a substrate, and substrate specificity studies showed that GnT-V is not able to form any further triantennary structure in the presence of a bisecting GlcNAc residue [6, 7]. In our investigation, the decrease of 1– 6 structure by the competition between the endogeneous GnT-V and ectopically expressed GnT-III led to the suppression of lung metastasis by mouse melanoma cells [9]. Cell aggregation was increased in GnT-III transfectants, indicating that the glycosylated E-cadherin participates in the suppression of metastasis by GnT-III gene transfer
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