Abstract
Oligosaccharide moieties of glycoproteins are structurally altered during development, carcinogenesis, and malignant transformations. It is well known that beta1-6 GlcNAc branching, a product of UDP-GlcNAc alpha-mannoside beta1-6-N-acetylglucosaminyltransferase (GnT-V), is associated with malignant transformation as the results of such alterations. However, the mechanism by which beta1-6 GlcNAc branching is linked to metastasis remains unclear, because the identification of specific glycoprotein(s) that are glycosylated by GnT-V and its biological function have not been examined. We herein report that matriptase, which activates both urokinase-type plasminogen activator and hepatocyte growth factor, is a target protein for GnT-V. The overexpression of GnT-V in gastric cancer cells leads to severe peritoneal dissemination in athymic mice, which can be attributed to the increased expression of matriptase. This increase was due to the acquired resistance of matriptase to degradation, since it is glycosylated by GnT-V and a corresponding increase in the active form. These results indicate that this process is a key element in malignant transformation, as the direct result of oligosaccharide modification.
Highlights
N-Glycans are widely distributed on cell surfaces and secreted glycoproteins, where structural change is observed in development, carcinogenesis, and malignant transformation [1,2,3]
Since we reported on the purification and cDNA cloning of human GnT-V [7, 8], numerous studies have reported that 1– 6 GlcNAc branching is associated with malignant transformation, including tumor invasion and metastasis (9 –12)
Functional changes in specific glycoproteins that contain 1– 6 GlcNAc branching have not been described in terms of tumor metastasis, and the biological significance of GnT-V appears to be different for each type of cancer
Summary
Establishment of GnT-V-transfected MKN45 Cells—Human gastric cancer cell line MKN45 was obtained from the Japanese Cancer Research Resource (Tokyo, Japan). Western Blot Analysis—Twenty l of a 10-fold concentrated condition medium from mock and GnT-V transfectants was electrophoresed on an 8% polyacrylamide gel, and transferred onto a nitrocellulose membrane. For immunodepletion of matriptase from GnT-V-transfected MKN45 cells, the conditioned medium was incubated overnight at 4 °C with protein G-Sepharose CL-4B bound with mAb 21-9. The cell lysates were incubated for the indicated times at 37 °C, subjected to SDS-PAGE on an 8% gel, transferred to nitrocellulose membrane, and analyzed by Western blot using mAb 21-9. Pulse-Chase Experiments—Mock and GnT-V transfectants of MKN45 cells in six-well tissue culture plates were preincubated for 2 h at 37 °C with methionine- and cysteine-free medium containing 10% fetal calf serum dialyzed overnight. Data represent mean Ϯ S.D. of three experiments; ND, not detected
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