Functional Proteomics Study Reveals That N-Acetylglucosaminyltransferase V Reinforces the Invasive/Metastatic Potential of Colon Cancer through Aberrant Glycosylation on Tissue Inhibitor of Metalloproteinase-,

Establishment of stable transfectants of TIMP-1 and the glycosylation mutants.A, wild-type TIMP-1 and three glycosylation mutant genes were cloned. B, the cloned genes were transfected into WiDr:mock and WiDr:GnT-V cells, and the stable transfectants expressing equal amounts of the recombinant proteins were selected. C, the structure of the aberrant glycan on TIMP-1, initiated by the GnT-V-catalyzed attachment of β1,6-GlcNAc, was deduced by two-dimensional electrophoresis combined with lectin and immunoblot (IB) analyses. β1,6-GlcNAc and additional polylactosamine moieties could be deduced from the l-PHA and DSA blot, respectively, in aberrant TIMP-1 molecules. MW, molecular mass.

Profiling of N-glycans of TIMP-1 in WiDr cells.A, profiling of TIMP-1 glycans from WiDr:mock and WiDr:GnT-V was performed. Glycans digested with PNGase F were mass analyzed in a MALDI-TOF mass spectrometer, and the composition of each glycan was deduced from the mass value. The annotated composition was confirmed by treatment with sialidase (B) and β-galactosidase plus sialidase (C). ▪, N-acetylglucosamine; ○, galactose; ♦, N-acetylneuraminic acid.

Effects of the aberrant glycosylation of TIMP-1 on cell migration and invasion. Cells (2 × 10⁴) were allowed to migrate in RPMI 1640 medium plus 0.1% BSA containing, if necessary, gelatinases inhibitor (Igela) for 4 h (A) and a Matrigel-coated 24-well Boyden chamber for 22 h (B), and cells that migrated to the lower surface of the filters, some of which were precoated with fibronectin (FN) (25 μg/ml), were counted. The values are the means of three independent experiments with standard deviations (*, p < 0.05; **, p < 0.01). C, MMP-2 and MMP-9 were retrieved from WiDr conditioned RPMI 1640 media containing 10% FBS. MMP-2 was immunoprecipitated using anti-MMP-2 monoclonal antibody, and MMP-9 was partially purified on a Con A-agarose column. Gelatin zymography was performed on 10% SDS-PAGE gel with 0.5% (w/v) gelatin copolymerized.

Effects of the aberrant glycosylation of TIMP-1 on gelatinase inhibition. Following the incubation of gelatinases with the various inhibitors at 4 °C for 24 h, hydrolytic reactions of gelatinase A/MMP-2 (A) and gelatinase B/MMP-9 (B) were initiated at 37 °C by adding a fluorogenic substrate. Time courses of the activities were traced, and the slope at steady state was used for the relative activity. Immp-2 and Imp-9 refer to specific inhibitor for MMP-2 and MMP-9, respectively. C, effects of aberrant glycans of TIMP-1 on gelatinolytic activity of gelatinases were investigated by gelatin-zymography as described under “Experimental Procedures.”