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

Yong-Sam Kim, Soo Young Hwang, Hye-Yeon Kang, Hosung Sohn, Sejeong Oh, Jin-Young Kim, Jong Shin Yoo, Young Hwan Kim, Cheorl-Ho Kim, Jae-Heung Jeon, Jung Mi Lee, Hyun Ah Kang, Eiji Miyoshi, Naoyuki Taniguchi, Hyang-Sook Yoo and Jeong-Heon Ko
Molecular & Cellular Proteomics January 1, 2008, First published on September 18, 2007, 7 (1) 1-14; https://doi.org/10.1074/mcp.M700084-MCP200
Yong-Sam Kim
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Soo Young Hwang
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Hye-Yeon Kang
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Hosung Sohn
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Sejeong Oh
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Jin-Young Kim
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Jong Shin Yoo
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Young Hwan Kim
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Cheorl-Ho Kim
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Jae-Heung Jeon
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Jung Mi Lee
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Hyun Ah Kang
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Eiji Miyoshi
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Naoyuki Taniguchi
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Hyang-Sook Yoo
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Jeong-Heon Ko
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  • Fig. 1.
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    Fig. 1.

    Search for target proteins of GnT-V in colon cancer WiDr cells and the identification of TIMP-1 as a candidate.A, GnT-V-catalyzed addition of β1,6-GlcNAc to the core N-glycan comprised of GlcNAc (□) and mannose (•). N, asparagine; S, serine; T, threonine; x, any amino acid except proline. B, WiDr colon cancer cells were transfected with the MGAT5 gene, and the stable transfectant cells were established. C, protein samples prepared by precipitation of WiDr conditioned serum-free media were displayed on two-dimensional electrophoresis gel. Spots that displayed differentially between WiDr:mock and WiDr:GnT-V cells were used for identification. Proteins indicated by arrows were identified as follows; 1, heparan sulfate proteoglycan perlecan; 2, tissue inhibitor of metalloprotease-1; 3, ribonuclease T2 precursor; 4, cathepsin X precursor; 5, Zn-α-2-glycoprotein; 6, cathepsin D preproprotein; 7, protective protein for β-galactosidase; 8, legumain preproprotein; 9, human Fc binding protein; 10, discoidin receptor tyrosine kinase; 11, angiotensinogen preproprotein; 12, α-1-antitrypsin precursor; 13, β-N-acetylhexosaminidase A; 14, prosaposin; 15, galectin 3 binding protein; 16, N-acetylgalactosamine-6-sulfatase; 17, dipeptidyl peptidase 7 preproprotein; 18, heat shock 70 kDa protein 8 isoform 1; 19, hexosaminidase B preproprotein; 20, granulin isoform 1 precursor; 21, N-acetyl-glucosamine-6-sulfatase; 22, heat shock protein gp96 precursor; 23, DDR1 variant protein; 24, protein-tyrosine kinase-related receptor PTK7; 25, protein tyrosine phosphatase κ. D, peptide derived from spot 2 in panel C was sequence analyzed by ESI/Q-TOF mass spectrometry, blasted against the Mascot database, and identified to be human TIMP-1 from the sequence SEEFLIAGK seen in the mass spectrum together with other sequences.

  • Fig. 2.
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    Fig. 2.

    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.

  • Fig. 3.
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    Fig. 3.

    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.

  • Fig. 4.
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    Fig. 4.

    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.

  • Fig. 5.
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    Fig. 5.

    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.”

  • Fig. 6.
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    Fig. 6.

    Correlation of TIMP-1 aberration with colon cancer progression.A, the normal tissues and cancer tissues from colon cancer cases of each stage were compared with respect to TIMP-1 expression levels, aberrant glycosylation, and transcription levels of the GnT-V gene. Closed circle indicates an acquisition of β1,6-GlcNAc moiety on TIMP-1 molecule; open circle indicates a normal glycosylation of TIMP-1; diameters of circles indicate a relative TIMP-1 level in cancerous tissues compared with that in normal tissues. B, proteins were extracted from normal and cancer tissues from resection materials of colon cancer cases and precleaned with anti-mouse IgM-agarose beads. Immunoprecipitation was performed using the conjugates of an anti-TIMP-1 monoclonal antibody. The immunoprecipitates were subjected to both immunoblot and lectin blot using l-PHA. C, transcription levels of GnT-V were monitored by RT-PCR using two primers: 5′-tgtgtatggcaaagtggata-3′ (forward) and 5′-accatggtttttcacgtaac-3′ (backward). D, actin from normal and cancer tissues were compared by immunoblot analysis.

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    Table I

    Proteins differentially recognized by l-PHA on 2-DE gels and identified by ESI-MS spectrometry and a Mascot blast search against NCBInr

    Accession gi no.IdentitiesPeptides matchedSequence coverage (%)Total scoreaMr/pIbLevelsc (±S.D.)
    177836 α-1-antitrypsin precursor726.343846.7/5.50.26 (0.03)
    15079348 Angiotensinogen preproprotein310.118953.1/5.81.19 (0.07)
    4261632 β-N-acetylhexosaminidase A1022.357560.7/5.01.77 (0.17)
    54697170 Cathepsin D preproprotein511.732044.5/6.13.87 (0.24)
    3650498 Cathepsin X precursor25.910733.9/7.11.19 (0.11)
    68533097 DDR1 variant protein34.414199.0/6.30.66 (0.06)
    16877430 Dipeptidyl peptidase 7 preproprotein311.314654.3/5.92.33 (0.17)
    38327632 Discoidin receptor tyrosine kinase26.19996.9/6.11.40 (0.12)
    4758116 Dystroglycan 1 precursor23.310697.6/8.71.42 (0.15)
    5031863 Galectin 3 binding protein36.814765.3/5.10.53 (0.06)
    4504151 Granulin isoform 1 precursor35.714963.5/6.41.21 (0.09)
    5729877 Heat shock 70 kDa protein 8 isoform 1714.637970.9/5.42.47 (0.15)
    15010550 Heat shock protein gp96 precursor912.048490.2/4.70.34 (0.04)
    11602963 Heparan sulfate proteoglycan perlecan31.1171466.6/6.03.26 (0.33)
    16924217 Hexosaminidase B preproprotein510.426663.1/6.30.87 (0.06)
    5080756 Human Fc binding protein31.6179572.1/5.10.19 (0.02)
    106586 Ig kappa chain V-III325.125223.1/5.81.48 (0.11)
    9845498 Laminin γ1 precursor118.7746177.6/5.01.47 (0.06)
    56682964 Legumain preproprotein312.715249.4/6.11.54 (0.11)
    51095116 Met proto-oncogene75.3471155.4/7.01.76 (0.04)
    4503899 N-acetylgalactosamine-6-sulfatase514.929558.0/6.30.63 (0.06)
    4504061 N-acetylglucosamine-6-sulfatase817.945762.0/8.60.18 (0.02)
    57209715 Prosaposin510.931858.1/5.12.01 (0.08)
    4505989 Protective protein for β-galactosidase25.417854.5/6.20.35 (0.04)
    2136061 Protein-tyrosine kinase-related receptor PTK779.2432118.3/6.70.47 (0.02)
    57160745 Protein tyrosine phosphatase kappa77.8374162.0/5.62.04 (0.07)
    5231228 Ribonuclease T2 precursor525.326229.5/6.70.86 (0.07)
    57210053 Tissue inhibitor of metalloproteinase-1426.121523.2/8.51.97 (0.06)
    120749 Tumor-associated calcium signal transducer 1211.818434.9/7.41.08 (0.09)
    38026 Zn-α-2-glycoprotein419.523434.7/5.70.41 (0.05)
    • ↵ a Total score is a sum of the score values obtained from each of an individual peptide. Score is −10 x Log (P), where P is the probability that the observed match is a random event; it is based on NCBInr database using the MASCOT searching program as MS/MS data.

    • ↵ b Molecular weight (Mr) and isoelectric point (pI) are theoretical values where glycan residues were not considered for the calculations. The theoretical values are prone to be changed by an attachment of glycans to peptides and thus to be different from the experimental values estimated on two-dimensional electrophoresis gels.

    • ↵ c Numbers refer to the relative levels of each protein from WiDr:GnT-V compared to those from WiDr:mock (n = 5).

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    Table II

    Inhibition constants for active gelatinase/TIMP-1 interactions

    TIMP-1Gelatinaseskon (M−1·s−1×105)koff (s−1×103)Ki (nm)
    TIMP-1: mock62 kDa MMP-24.99 ± 0.722.71 ± 0.475.43 ± 1.39
    82 kDa MMP-97.07 ± 0.642.26 ± 0.433.20 ± 1.23
    TIMP-1: GnT-V62 kDa MMP-20.98 ± 0.063.83 ± 0.2439.1 ± 8.38
    82 kDa MMP-90.75 ± 0.072.74 ± 0.3936.5 ± 6.54

Additional Files

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Functional Proteomics Study Reveals That N-Acetylglucosaminyltransferase V Reinforces the Invasive/Metastatic Potential of Colon Cancer through Aberrant Glycosylation on Tissue Inhibitor of Metalloproteinase-,
Yong-Sam Kim, Soo Young Hwang, Hye-Yeon Kang, Hosung Sohn, Sejeong Oh, Jin-Young Kim, Jong Shin Yoo, Young Hwan Kim, Cheorl-Ho Kim, Jae-Heung Jeon, Jung Mi Lee, Hyun Ah Kang, Eiji Miyoshi, Naoyuki Taniguchi, Hyang-Sook Yoo, Jeong-Heon Ko
Molecular & Cellular Proteomics January 1, 2008, First published on September 18, 2007, 7 (1) 1-14; DOI: 10.1074/mcp.M700084-MCP200

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Functional Proteomics Study Reveals That N-Acetylglucosaminyltransferase V Reinforces the Invasive/Metastatic Potential of Colon Cancer through Aberrant Glycosylation on Tissue Inhibitor of Metalloproteinase-,
Yong-Sam Kim, Soo Young Hwang, Hye-Yeon Kang, Hosung Sohn, Sejeong Oh, Jin-Young Kim, Jong Shin Yoo, Young Hwan Kim, Cheorl-Ho Kim, Jae-Heung Jeon, Jung Mi Lee, Hyun Ah Kang, Eiji Miyoshi, Naoyuki Taniguchi, Hyang-Sook Yoo, Jeong-Heon Ko
Molecular & Cellular Proteomics January 1, 2008, First published on September 18, 2007, 7 (1) 1-14; DOI: 10.1074/mcp.M700084-MCP200
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Molecular & Cellular Proteomics: 7 (1)
Molecular & Cellular Proteomics
Vol. 7, Issue 1
January 2008
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