FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2000572756> ?p ?o ?g. }

• W2000572756 endingPage "32130" @default.
• W2000572756 startingPage "32122" @default.
• W2000572756 abstract "N-Acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of β1,6-GlcNAc branching of N-glycans, which contributes to metastasis. N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the formation of a bisecting GlcNAc structure in N-glycans, resulting in the suppression of metastasis. It has long been hypothesized that the suppression of GnT-V product formation by the action of GnT-III would also exist in vivo, which will consequently lead to the inhibition of biological functions of GnT-V. To test this, we draw a comparison among MKN45 cells, which were transfected with GnT-III, GnT-V, or both, respectively. We found that α3β1 integrin-mediated cell migration on laminin 5 was greatly enhanced in the case of GnT-V transfectant. This enhanced cell migration was significantly blocked after the introduction of GnT-III. Consistently, an increase in bisected GlcNAc but a decrease in β1,6-GlcNAc-branched N-glycans on integrin α3 subunit was observed in the double transfectants of GnT-III and GnT-V. Conversely, GnT-III knockdown resulted in increased migration on laminin 5, concomitant with an increase in β1,6-GlcNAc-branched N-glycans on the α3 subunit in CHP134 cells, a human neuroblastoma cell line. Therefore, in this study, the priority of GnT-III for the modification of the α3 subunit may be an explanation for why GnT-III inhibits GnT-V-induced cell migration. Taken together, our results demonstrate for the first time that GnT-III and GnT-V can competitively modify the same target glycoprotein and furthermore positively or negatively regulate its biological functions. N-Acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of β1,6-GlcNAc branching of N-glycans, which contributes to metastasis. N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the formation of a bisecting GlcNAc structure in N-glycans, resulting in the suppression of metastasis. It has long been hypothesized that the suppression of GnT-V product formation by the action of GnT-III would also exist in vivo, which will consequently lead to the inhibition of biological functions of GnT-V. To test this, we draw a comparison among MKN45 cells, which were transfected with GnT-III, GnT-V, or both, respectively. We found that α3β1 integrin-mediated cell migration on laminin 5 was greatly enhanced in the case of GnT-V transfectant. This enhanced cell migration was significantly blocked after the introduction of GnT-III. Consistently, an increase in bisected GlcNAc but a decrease in β1,6-GlcNAc-branched N-glycans on integrin α3 subunit was observed in the double transfectants of GnT-III and GnT-V. Conversely, GnT-III knockdown resulted in increased migration on laminin 5, concomitant with an increase in β1,6-GlcNAc-branched N-glycans on the α3 subunit in CHP134 cells, a human neuroblastoma cell line. Therefore, in this study, the priority of GnT-III for the modification of the α3 subunit may be an explanation for why GnT-III inhibits GnT-V-induced cell migration. Taken together, our results demonstrate for the first time that GnT-III and GnT-V can competitively modify the same target glycoprotein and furthermore positively or negatively regulate its biological functions. Malignant transformation is accompanied by increased β1,6-GlcNAc branching of N-glycans attached to Asn-X-Ser/Thr sequences in mature glycoproteins (1Yamashita K. Totani K. Iwaki Y. Kuroki M. Matsuoka Y. Endo T. Kobata A. J. Biol. Chem. 1989; 264: 17873-17881Abstract Full Text PDF PubMed Google Scholar, 2Pierce M. Arango J. J. Biol. Chem. 1986; 261: 10772-10777Abstract Full Text PDF PubMed Google Scholar, 3Granovsky M. Fata J. Pawling J. Muller W.J. Khokha R. Dennis J.W. Nat. Med. 2000; 6: 306-312Crossref PubMed Scopus (468) Google Scholar). N-Acetylglucosaminyltransferase V (GnT-V) 3The abbreviations used are: GnT-V, N-acetylglucosaminyltransferase V; GnT-III, N-acetylglucosaminyltransferase III; ECM, extracellular matrix; LN5, laminin 5; FN, fibronectin; COL, collagen I; PBS, phosphate-buffered saline; PHA, phytohemagglutinin; LC, liquid chromatography; MS, mass spectrometry; FT-ICR, Fourier transform ion cyclotron resonance. 3The abbreviations used are: GnT-V, N-acetylglucosaminyltransferase V; GnT-III, N-acetylglucosaminyltransferase III; ECM, extracellular matrix; LN5, laminin 5; FN, fibronectin; COL, collagen I; PBS, phosphate-buffered saline; PHA, phytohemagglutinin; LC, liquid chromatography; MS, mass spectrometry; FT-ICR, Fourier transform ion cyclotron resonance. catalyzes the addition of β1,6-linked GlcNAc (see Fig. 8) and defines this subset of N-glycans (4Cummings R.D. Trowbridge I.S. Kornfeld S. J. Biol. Chem. 1982; 257: 13421-13427Abstract Full Text PDF PubMed Google Scholar, 5Shoreibah M. Perng G.S. Adler B. Weinstein J. Basu R. Cupples R. Wen D. Browne J.K. Buckhaults P. Fregien N. J. Biol. Chem. 1993; 268: 15381-15385Abstract Full Text PDF PubMed Google Scholar). A relation between GnT-V and cancer metastasis has been reported by Dennis et al. (6Dennis J.W. Laferte S. Waghorne C. Breitman M.L. Kerbel R.S. Science. 1987; 236: 582-585Crossref PubMed Scopus (857) Google Scholar) and Yamashita et al. (1Yamashita K. Totani K. Iwaki Y. Kuroki M. Matsuoka Y. Endo T. Kobata A. J. Biol. Chem. 1989; 264: 17873-17881Abstract Full Text PDF PubMed Google Scholar). Studies on transplantable tumors in mice indicate that the product of GnT-V directly contributes to the growth of cancer and subsequent metastasis (7Demetriou M. Nabi I.R. Coppolino M. Dedhar S. Dennis J.W. J. Cell Biol. 1995; 130: 383-392Crossref PubMed Scopus (252) Google Scholar, 8Seberger P.J. Chaney W.G. Glycobiology. 1999; 9: 235-241Crossref PubMed Scopus (69) Google Scholar). On the other hand, somatic tumor cell mutants that are deficient in GnT-V activity produce fewer spontaneous metastases and grow more slowly than wild-type cells (6Dennis J.W. Laferte S. Waghorne C. Breitman M.L. Kerbel R.S. Science. 1987; 236: 582-585Crossref PubMed Scopus (857) Google Scholar, 9Lu Y. Pelling J.C. Chaney W.G. Clin. Exp. Metastasis. 1994; 12: 47-54Crossref PubMed Scopus (38) Google Scholar). The suppression of tumor growth and metastasis has been reported in GnT-V-deficient mice (3Granovsky M. Fata J. Pawling J. Muller W.J. Khokha R. Dennis J.W. Nat. Med. 2000; 6: 306-312Crossref PubMed Scopus (468) Google Scholar). Moreover, Partridge et al. (10Partridge E.A. Le Roy C. Di Guglielmo G.M. Pawling J. Cheung P. Granovsky M. Nabi I.R. Wrana J.L. Dennis J.W. Science. 2004; 306: 120-124Crossref PubMed Scopus (589) Google Scholar) reported that GnT-V-modified N-glycans with poly-N-acetyllactosamine, the preferred ligand for galectin-3, on surface receptors oppose their constitutive endocytosis and result in promoting intracellular signaling and consequently cell migration and tumor metastasis. These results indicate that inhibition of GnT-V might be useful in the treatment of malignancies by targeting their roles in metastasis. N-Acetylglucosaminyltransferase III (GnT-III) participates in the branching of N-glycans (see Fig. 8), catalyzing the formation of a unique sugar chain structure-bisecting GlcNAc (11Narasimhan S. J. Biol. Chem. 1982; 257: 10235-10242Abstract Full Text PDF PubMed Google Scholar). GnT-III is generally regarded to be a key glycosyltransferase in the N-glycan biosynthetic pathway, since in vitro the introduction of the bisecting GlcNAc results in the suppression of further processing and the elongation of N-glycans as the result of catalysis by other glycosyltransferases, which are unable to use the bisected oligosaccharide as a substrate (12Schachter H. Biochem. Cell Biol. 1986; 64: 163-181Crossref PubMed Scopus (490) Google Scholar, 13Gu J. Nishikawa A. Tsuruoka N. Ohno M. Yamaguchi N. Kangawa K. Taniguchi N. J. Biochem. (Tokyo). 1993; 113: 614-619Crossref PubMed Scopus (137) Google Scholar). It is interesting to note that the metastatic capabilities of B16 mouse melanoma cells are down-regulated by introduction of the GnT-III gene (14Yoshimura M. Nishikawa A. Ihara Y. Taniguchi S. Taniguchi N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8754-8758Crossref PubMed Scopus (255) Google Scholar). E-cadherin, a homophilic type of adhesion molecule (15Takeichi M. Curr. Opin. Cell Biol. 1993; 5: 806-811Crossref PubMed Scopus (829) Google Scholar), is highly associated with the prevention of metastasis (16Hirohashi S. Am. J. Pathol. 1998; 153: 333-339Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar), and E-cadherin on GnT-III-transfected cell surfaces was found to be resistant to proteolysis, resulting in an extended half-life of turnover (17Yoshimura M. Ihara Y. Matsuzawa Y. Taniguchi N. J. Biol. Chem. 1996; 271: 13811-13815Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Thus, GnT-III, contrary to GnT-V, has long been thought to inhibit cancer metastasis. Cell-extracellular matrix (ECM) interactions play essential roles during the acquisition of migration and invasive behavior of cells. Cell surface transmembrane glycoprotein-integrin is a major receptor for ECM and connects many biological functions, such as development, control of cell proliferation, protection against apoptosis, and malignant transformation (18Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6851) Google Scholar). Integrin α3β1, the major laminin 5 (LN5) receptor, is widely distributed in almost all tissues, and it has been proposed to be involved in tumor invasion (19Tsuji T. Kawada Y. Kai-Murozono M. Komatsu S. Han S.A. Takeuchi K. Mizushima H. Miyazaki K. Irimura T. Clin. Exp. Metastasis. 2002; 19: 127-134Crossref PubMed Scopus (103) Google Scholar, 20Plopper G.E. Domanico S.Z. Cirulli V. Kiosses W.B. Quaranta V. Breast Cancer Res. Treat. 1998; 51: 57-69Crossref PubMed Scopus (64) Google Scholar, 21Melchiori A. Mortarini R. Carlone S. Marchisio P.C. Anichini A. Noonan D.M. Albini A. Exp. Cell Res. 1995; 219: 233-242Crossref PubMed Scopus (119) Google Scholar). In some malignant tumors, α3β1 integrin was found to be the most predominant integrin expressed (22Tysnes B.B. Larsen L.F. Ness G.O. Mahesparan R. Edvardsen K. Garcia-Cabrera I. Bjerkvig R. Int. J. Cancer. 1996; 67: 777-784Crossref PubMed Scopus (87) Google Scholar) and made an important contribution to pulmonary metastasis (23Wang H. Fu W. Im J.H. Zhou Z. Santoro S.A. Iyer V. DiPersio C.M. Yu Q.C. Quaranta V. Al-Mehdi A. Muschel R.J. J. Cell Biol. 2004; 164: 935-941Crossref PubMed Scopus (167) Google Scholar). On the other hand, the glycosylation of integrins contributes to the tumor metastasis. Guo et al. reported that an increase in β1,6-GlcNAc sugar chains of the integrin β1 subunit resulted in the stimulation of cell migration (24Guo H.B. Lee I. Kamar M. Akiyama S.K. Pierce M. Cancer Res. 2002; 62: 6837-6845PubMed Google Scholar). Interestingly, it has also been reported that the α3β1 integrin expressed by the metastasis human melanoma cell lines, contained a higher level of β1,6-branched structures than that expressed in a nonmetastasis parent cell line (25Pochec E. Litynska A. Amoresano A. Casbarra A. Biochim. Biophys. Acta. 2003; 1643: 113-123Crossref PubMed Scopus (109) Google Scholar). Although it had been assumed that the reaction of GnT-V can be inhibited by the action of GnT-III, as evidenced by substrate specificity studies in vitro, the hypothesis of competition between GnT-III and GnT-V in cell migration and tumor metastasis has not been directly verified so far. In the present study, we examined the functions of α3β1 integrin, which is believed to be highly associated with tumor metastasis, and found that α3β1 integrin can be modified by either GnT-III or GnT-V. Our finding clearly shows that GnT-III inhibits the effects of GnT-V on α3β1 integrin-mediated cell migration by competing with GnT-V for the modification of α3 subunit. Reagents and Antibodies—Antibodies against integrin α3 subunit (P1B5, I-19), monoclonal antibody against β-actin, mouse control IgG, and peroxidase-conjugated rabbit antibody against goat IgG were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Functional blocking antibody against the integrin β1 subunit was purchased from Chemicon International, Inc. (Temecula, CA). A peroxidase-conjugated goat antibody against mouse IgG was from Promega (Madison, WI). Biotinylated leukoagglutinating phytohemagglutinin (L4-PHA), biotinylated erythroagglutinating phytohemagglutinin (E4-PHA), and monoclonal antibodies against GnT-III and GnT-V were from Seikagaku Corp. Cell Culture—Transfected MKN45 Cells were established as previously reported (26Ihara S. Miyoshi E. Ko J.H. Murata K. Nakahara S. Honke K. Dickson R.B. Lin C.Y. Taniguchi N. J. Biol. Chem. 2002; 277: 16960-16967Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Human gastric cancer cell line MKN45 cells were cultured in RPMI 1640 medium (Sigma) containing 10% fetal bovine serum (Invitrogen), penicillin (100 units/ml), and streptomycin (100 μg/ml) under a humidified atmosphere containing 5% CO2. Human GnT-V cDNA (27Saito H. Nishikawa A. Gu J. Ihara Y. Soejima H. Wada Y. Sekiya C. Niikawa N. Taniguchi N. Biochem. Biophys. Res. Commun. 1994; 198: 318-327Crossref PubMed Scopus (100) Google Scholar) or GnT-III cDNA was inserted into a mammalian expression vector pCXNII (28Miwa K. Matsui K. Terabe M. Ito K. Ishida M. Takagi H. Nakamori S. Sano K. Gene (Amst.). 1985; 39: 281-286Crossref PubMed Scopus (79) Google Scholar). Vectors were then transfected into MKN45 cells by means of Lipofectamine (Invitrogen). Selection was performed by the addition of 500 μg/ml G418 (Sigma). CHP134 cells, a human neuroblastoma cell line expressing endogenous GnT-III and GnT-V, were cultured in RPMI 1640 medium (Sigma) containing 10% fetal bovine serum and penicillin (100 units/ml) and streptomycin (100 μg/ml) under a humidified atmosphere containing 5% CO2. Plasmids and Transient Virus Transfection—cDNAs encoding full-length human GnT-III or GnT-III inactive mutant (D317A) were ligated into adenoviral vector, constructed using an adenoviral expression vector kit (Takara Bio). The 3 × 105 MKN45 GnT-V transfectants were then infected with 150 μlof virus solution (2 × 109 plaque-forming units/ml). After a 24-h incubation, the cultured medium was replaced with a fresh medium. 48 h later after infection, cells were subjected to various experiments. Construction of Small Interfering RNA Vector and Retroviral Infection—Small interfering oligonucleotides specific for GnT-III were designed on the Takara Bio site on the World Wide Web, and the oligonucleotide sequences used in the construction of the small interfering RNA vector were as follows: 5′-GATCCGTCAACCACGAGTTCGACCTTCAAGAGAGGTCGAACTCGTGGTTGACTTTTTTAT-3′ and 5′-CGATAAAAAAGTCAACCACGAGTTCGACCTCTCTTGAAGGTCGAACTCGTGGTTGACG-3′. The oligonucleotides were annealed and then ligated into BamHI/ClaI sites of the pSINsi-hU6 vector (Takara Bio). A retroviral supernatant was obtained by transfection of human embryonic kidney 293 cells using the retrovirus packaging kit Ampho (Takara Bio) according to the manufacturer's protocol. CHP134 cells were infected with the viral supernatant, and the cells were then selected with 500 μg/ml G418 for 2–3 weeks. Stable GnT-III knockdown clones were selected and confirmed by GnT-III activity and gene expression. Quantitative real time PCR analyses of GnT-III mRNA expression in these clones were performed with a Smart Cycler II System and the SYBR premix Taq (Takara Bio). Reverse transcription was carried out at 42 °C for 10 min, followed by 95 °C for 2 min using random primers, followed by PCR for 45 cycles at 95 °C for 5 s and 60 °C for 20 s with the following primers: 5′-GCGTCATCAACGCCATCAA-3′ 5′-TGGACTCGCACACCACAAAG-3′. Normalization of the data were performed using the glyceraldehyde-3-phosphate dehydrogenase mRNA levels. GnT-III and GnT-V Activity Assay—The activities of GnT-III and GnT-V were assayed as described previously (29Taniguchi N. Nishikawa A. Fujii S. Gu J.G. Methods Enzymol. 1989; 179: 397-408Crossref PubMed Scopus (70) Google Scholar, 30Nishikawa A. Gu J. Fujii S. Taniguchi N. Biochim. Biophys. Acta. 1990; 1035: 313-318Crossref PubMed Scopus (113) Google Scholar). Briefly, cell lysates were homogenized in phosphate-buffered saline (PBS) containing protease inhibitors. The supernatant, after removal of the nucleus fraction by centrifugation for 15 min at 900 × g, was used in the assays, which involved high performance liquid chromatography methods using a pyridylaminated biantennary sugar chain as an acceptor substrate. Protein concentrations were determined using a bicinchoninic acid kit (BCA kit) (Pierce) with bovine serum albumin as a standard. Western Blot and Lectin Blot Analysis—Cell cultures were harvested in lysis buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Triton, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm phenymethylsulfonyl fluoride). Cell lysates were centrifuged at 15,000 × g for 10 min at 4 °C, the supernatants were collected, and the protein concentrations were determined using a BCA protein assay kit. Proteins were then immunoprecipitated from the lysates using a combination of 2 μg of anti-integrin α3 subunit antibody and 15 μl of protein G-Sepharose 4 Fast Flow (Amersham Biosciences) for 1 h at 4 °C. Immunoprecipitates were suspended in reducing sample buffer, heated to 100 °C for 3 min, resolved on 7.5% SDS-PAGE, and electrophoretically transferred to nitrocellulose membranes (Schleicher & Schuell). The blots were then probed with anti-α3 antibody or biotinylated E4-or L4-PHA. Immunoreactive bands were visualized using the Vectastain ABC kit (Vector Laboratories, CA) and an ECL kit (Amersham Biosciences). For GnT-III, GnT-V, cell lysate, and actin blotting, an equal amount of cell lysates was subjected to SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were incubated with the corresponding primary antibodies and secondary antibodies for 1 h each, and detection was performed by an ECL kit. Cell Surface Biotinylation—Cell surface biotinylation was performed as described previously with minor modifications (31Sato Y. Takahashi M. Shibukawa Y. Jain S.K. Hamaoka R. Miyagawa J. Yaginuma Y. Honke K. Ishikawa M. Taniguchi N. J. Biol. Chem. 2001; 276: 11956-11962Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Briefly, various semiconfluent transfected MKN45 cells were washed twice with ice-cold PBS and then incubated with ice-cold PBS containing 0.2 mg/ml sulfosuccinimidobiotin (Pierce), for 3 h at 4 °C. After incubation, the cells were washed three times with ice-cold PBS, scraped, and lysed with radioimmune precipitation buffer (50 mm Tris-HCl, pH 7.4, 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, 150 mm NaCl, 1 mm sodium orthovanadate, 2 μg/ml aprotinin, 5 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride). The resulting cell lysates were immunoprecipitated with anti-α3 antibody, as described above. The immunocomplex was subjected to 7.5% SDS-PAGE and then transferred to a nitrocellulose membrane. After blocking the membranes with 3% (w/v) skim milk in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBST, pH 7.5), the biotinylated proteins were visualized using a Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA) and an ECL kit. Migration Assay—Transwells (BD Biosciences) were coated with 5 nm recombinant LN5 as described previously (32Kariya Y. Ishida K. Tsubota Y. Nakashima Y. Hirosaki T. Ogawa T. Miyazaki K. J. Biochem. (Tokyo). 2002; 132: 607-612Crossref PubMed Scopus (42) Google Scholar), 10 μg/ml human plasma FN, and collagen I (COL) (Sigma) in PBS by an incubation overnight at 4 °C. Serum-starved cells (2 × 105 cells/well in 500 μl of 5% fetal calf serum medium) were seeded in the upper chamber of the plates. After incubation overnight at 37 °C, cells in the upper chamber of the filter were removed with a wet cotton swab. Cells on the lower side of the filter were fixed and stained with 0.5% crystal violet. Each experiment was performed in triplicate, and counting was done in three randomly selected microscopic fields within each well. Functional Blocking Assay—To identify which integrin is involved in cell migration on LN5, functional blocking antibodies against different types of integrins were individually preincubated with cells for 10 min at 37 °C. The preincubated cells were transferred into transwells coated with LN5 and then incubated overnight at 37 °C. The migrated cells were then quantified as described above. Statistical Analysis—Statistical evaluations were performed using Student's t test; differences among experimental groups were considered significant for p < 0.05. Data were expressed as mean values ± S.D. Purification of α3β1 Integrin—The purification of α3β1 integrin was performed as described previously (33Gehlsen K.R. Sriramarao P. Furcht L.T. Skubitz A.P. J. Cell Biol. 1992; 117: 449-459Crossref PubMed Scopus (109) Google Scholar). Briefly, cells in confluent were detached with TBS(+) (20 mm Tris-HCl, pH 7.5, 130 mm NaCl, 1 mm CaCl2, and 1 mm MgCl2) and washed with TBS(+). The cell pellets were extracted with 50 mm Tris/HCl containing 15 mm NaCl, 1 mm MgCl2, 1 mm MnCl2, pH 7.4, and protease inhibitor mixture (Roche Applied Science), 100 mm octyl-β-d-glucopyranoside at 4 °C. The cell extract was applied to an affinity column prepared by coupling 5 mg of the GD6 peptide of laminin α1 chain (33Gehlsen K.R. Sriramarao P. Furcht L.T. Skubitz A.P. J. Cell Biol. 1992; 117: 449-459Crossref PubMed Scopus (109) Google Scholar) (KQNCLSSRASFRGCVRNLRLSR residues numbered 3011–3032) (Peptide Institute, Inc., Osaka, Japan) to 1 ml of activated CH-Sepharose (Sigma). The bound α3β1 integrin was eluted with 20 mm EDTA in 50 mm Tris/HCl, pH 7.4, containing 100 mm octyl-β-d-glucopyranoside. The elutes containing α3β1 integrin were further purified on a 1-ml wheat germ agglutinin-agarose column (Seikagaku Corp., Tokyo, Japan) and eluted with 0.2 m N-acetyl-d-glucosamine containing 100 mm octyl-β-d-glucopyranoside. Analysis of N-Glycan Structures by Mass Spectrometry (LC/MSn)—Purified α3β1 integrin was applied to SDS-PAGE, and the α3 subunit was excised from the gel and then cut into pieces. The gel pieces were destained and dehydrated with 50% acetonitrile. The protein in the gel was reduced and carboxymethylated by the incubation with dithiothreitol and sodium monoiodoacetate (34Kikuchi M. Hatano N. Yokota S. Shimozawa N. Imanaka T. Taniguchi H. J. Biol. Chem. 2004; 279: 421-428Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). N-Glycans were extracted from the gel pieces as reported by Kustar et al. (35Kuster B. Wheeler S.F. Hunter A.P. Dwek R.A. Harvey D.J. Anal. Biochem. 1997; 250: 82-101Crossref PubMed Scopus (323) Google Scholar) and reduced with NaBH4. Half of the extracted oligosaccharides were incubated with α-neuraminidase from Arthrobacter ureafaciens in 50 mm phosphate buffer, pH 5.0, at 37 °C for 18 h and desalted with Envi-carb (Supelco, Bellefonte, PA). LC/MS and LC/multistage MS (MSn) was carried out on a quadrupole liner ion trap-Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS; Finnigan LTQ FTTM, Thermo Electron Corp., San Jose, CA) connected to a nano-LC system (Paradigm, Michrom BioResource, Inc., Auburn, CA). The eluents were 5 mm ammonium acetate, pH 9.6, 2% CH3CN (pump A) and 5 mm ammonium acetate, pH 9.6, 80% CH3CN (pump B). The borohydride-reduced N-linked oligosaccharides were separated on a Hypercarb (0.1 × 150 mm, Thermo Electron Corp.) with a linear gradient of 5–20% B in 45 min and 20–50% B in 45 min. A full MS1 scan (m/z 450–2000) by FT-ICR MS followed by data-dependent MS2,3 for the most abundant ions was performed in both negative and positive ion modes as previously reported (36Itoh S. Kawasaki N. Hashii N. Harazono A. Matsuishi Y. Hayakawa T. Kawanishi T. J. Chromatogr. A. 2006; 1103: 296-306Crossref PubMed Scopus (33) Google Scholar). Overexpression of GnT-V Stimulated α3β1 Integrin-mediated Cell Motility—It has been reported that overexpression of GnT-V in epithelial cells results in a loss of contact inhibition, increased cell motility in athymic nude mice (7Demetriou M. Nabi I.R. Coppolino M. Dedhar S. Dennis J.W. J. Cell Biol. 1995; 130: 383-392Crossref PubMed Scopus (252) Google Scholar), and an enhanced metastasis (8Seberger P.J. Chaney W.G. Glycobiology. 1999; 9: 235-241Crossref PubMed Scopus (69) Google Scholar). In this study, experiments were first designed to determine whether GnT-V overexpression could affect cell migration on different ECMs. The extent of haptotaxis toward LN5, FN, and COL, specific ligands for α3β1, α5β1, and α1β1 and α2β1 integrin, respectively, was observed in MKN45 cells transfected with mock, GnT-III, or GnT-V. In the case of the GnT-V transfectants on LN5, the number of transwell cells migrating to the lower surface of the membrane was considerably increased (p = 0.001), the overexpression of GnT-III resulted in a decrease in cell migration on LN5 compared with mock (p = 0.0013) (Fig. 1A). However, the migration of these three types of cells on FN was barely detectable. Although GnT-III transfection resulted in a decreased cell migration on COL compared with mock (p = 0.007), GnT-V transfection failed to induce a significant increase in cell migration on COL (Fig. 1B), suggesting that MKN45 cells may favor LN5 as an ECM for cell migration induced by GnT-V. These results further supported the view that α3β1 integrin, one of the most abundant integrins in epithelial cells, is distinct from other integrins, such as α5β1 integrin, and preferentially promotes cell migration (37Gu J. Sumida Y. Sanzen N. Sekiguchi K. J. Biol. Chem. 2001; 276: 27090-27097Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Moreover, the cell migration of GnT-V transfectant on LN5 was strongly inhibited by the presence of function-blocking antibodies against integrin α3 or/and β1 subunit, suggesting that the GnT-V-induced cell migration on LN5 was mainly mediated by α3β1 integrin (Fig. 2). These results indicated that overexpression of GnT-V resulted in an increase in α3β1 integrin-mediated cell motility.FIGURE 2GnT-V induced cell migration was mediated by α3β1 integrin. A, GnT-V-transfected MKN45 cells were detached, preincubated with mouse control IgG (a) or function-blocking monoclonal antibodies against α3 (b) or β1 (c) or both (d) for 10 min, and then replated on the upper chamber coated with LN5 (5 nm) and checked by Transwell assay. Representative fields were photographed using a phase-contrast microscope. The arrowheads indicate migrated cells. B, quantification of migration on LN5 (5 nm). The numbers of migrated cells were quantified and expressed as the means ± S.D. from three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Overexpression of GnT-III Inhibited α3β1 Integrin-mediated Cell Migration Induced by GnT-V—The Overexpression of GnT-III has been reported to inhibit cell migration by enhancement of E-cadherin-mediated homotypic adhesion (17Yoshimura M. Ihara Y. Matsuzawa Y. Taniguchi N. J. Biol. Chem. 1996; 271: 13811-13815Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar) and by inhibiting α5β1 integrin-mediated cell migration (38Isaji T. Gu J. Nishiuchi R. Zhao Y. Takahashi M. Miyoshi E. Honke K. Sekiguchi K. Taniguchi N. J. Biol. Chem. 2004; 279: 19747-19754Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). In addition, in vitro GnT-V cannot use the product of GnT-III, a bisected oligosaccharide, as a substrate (12Schachter H. Biochem. Cell Biol. 1986; 64: 163-181Crossref PubMed Scopus (490) Google Scholar), so experiments were then designed to determine whether the introduction of GnT-III prevents α3β1 integrin-mediated cell migration enhanced by GnT-V. The efficiency of transfection was confirmed by immunostaining with anti-GnT-III antibody and determined to be more than 80% (data not shown). As shown in Fig. 3, the transfection of GnT-III into the GnT-V transfectant resulted in a significant decrease in cell migration compared with the GnT-V transfectant (p = 0.002). However, the inhibition was not observed after transfection of the GnT-III-inactive mutant, suggesting that the activity of GnT-III was essential for the negative regulation of GnT-V-induced cell migration. Therefore, we proposed that GnT-III directly counteracted the effect of GnT-V on α3β1 integrin-mediated cell migration. Transfection of GnT-III Had No Effect on the Expression of GnT-V and Integrin α3 Subunit—To explore the possible mechanisms involved in the inhibition of GnT-III- to GnT-V-induced cell migration, we first attempted to determine whether the overexpression of GnT-III affected the expression of GnT-V and α3 subunit expressed on the cell surface by means of blotting a total cell lysate with the GnT-III antibody and the biotinylation of cell surface proteins followed by immunoprecipitation of α3 using the corresponding antibody, since N-glycosylation plays an important role in the quality control of the expression of glycoproteins. As shown in Fig. 4A, the levels of expression of GnT-V were not influenced by the introduction of GnT-III, and equivalent amounts of loaded proteins were verified by blotting an actin antibody. On the other hand, the expression of integrin α3 subunit on the cell surface also remained unchanged among the transfectants of GnT-III plus GnT-V, GnT-III mutant plus GnT-V, and GnT-V (Fig. 4B). These results suggested that the inhibition of GnT-III- to GnT-V-induced cell migration could not be ascribed to a change in the expression levels of GnT-V and/or α3 subu" @default.
• W2000572756 created "2016-06-24" @default.
• W2000572756 creator A5003528751 @default.
• W2000572756 creator A5009882274 @default.
• W2000572756 creator A5018382490 @default.
• W2000572756 creator A5036428916 @default.
• W2000572756 creator A5037222124 @default.
• W2000572756 creator A5044178319 @default.
• W2000572756 creator A5047604331 @default.
• W2000572756 creator A5060576443 @default.
• W2000572756 creator A5061184556 @default.
• W2000572756 creator A5061288484 @default.
• W2000572756 creator A5079606508 @default.
• W2000572756 creator A5080369708 @default.
• W2000572756 date "2006-10-01" @default.
• W2000572756 modified "2023-10-16" @default.
• W2000572756 title "N-Acetylglucosaminyltransferase III Antagonizes the Effect of N-Acetylglucosaminyltransferase V on α3β1 Integrin-mediated Cell Migration" @default.
• W2000572756 cites W11939915 @default.
• W2000572756 cites W1274731803 @default.
• W2000572756 cites W1505476546 @default.
• W2000572756 cites W1531421007 @default.
• W2000572756 cites W1542183560 @default.
• W2000572756 cites W1565489899 @default.
• W2000572756 cites W1576773659 @default.
• W2000572756 cites W1582944414 @default.
• W2000572756 cites W1603050454 @default.
• W2000572756 cites W1806869952 @default.
• W2000572756 cites W1971394991 @default.
• W2000572756 cites W1974405791 @default.
• W2000572756 cites W1978560897 @default.
• W2000572756 cites W2000400400 @default.
• W2000572756 cites W2003879802 @default.
• W2000572756 cites W2007695634 @default.
• W2000572756 cites W2013615451 @default.
• W2000572756 cites W2026180273 @default.
• W2000572756 cites W2026262319 @default.
• W2000572756 cites W2028134580 @default.
• W2000572756 cites W2029352681 @default.
• W2000572756 cites W2036133848 @default.
• W2000572756 cites W2048065251 @default.
• W2000572756 cites W2049405885 @default.
• W2000572756 cites W2056769161 @default.
• W2000572756 cites W2061327866 @default.
• W2000572756 cites W2062136146 @default.
• W2000572756 cites W2063731065 @default.
• W2000572756 cites W2066545420 @default.
• W2000572756 cites W2069532196 @default.
• W2000572756 cites W2076785665 @default.
• W2000572756 cites W2086996769 @default.
• W2000572756 cites W2089970204 @default.
• W2000572756 cites W2091538590 @default.
• W2000572756 cites W2094155175 @default.
• W2000572756 cites W2099560367 @default.
• W2000572756 cites W2102376680 @default.
• W2000572756 cites W2114413533 @default.
• W2000572756 cites W2115504260 @default.
• W2000572756 cites W2132495434 @default.
• W2000572756 cites W2157834399 @default.
• W2000572756 cites W2436001939 @default.
• W2000572756 cites W4361865139 @default.
• W2000572756 doi "https://doi.org/10.1074/jbc.m607274200" @default.
• W2000572756 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16940045" @default.
• W2000572756 hasPublicationYear "2006" @default.
• W2000572756 type Work @default.
• W2000572756 sameAs 2000572756 @default.
• W2000572756 citedByCount "126" @default.
• W2000572756 countsByYear W20005727562012 @default.
• W2000572756 countsByYear W20005727562013 @default.
• W2000572756 countsByYear W20005727562014 @default.
• W2000572756 countsByYear W20005727562015 @default.
• W2000572756 countsByYear W20005727562016 @default.
• W2000572756 countsByYear W20005727562017 @default.
• W2000572756 countsByYear W20005727562018 @default.
• W2000572756 countsByYear W20005727562019 @default.
• W2000572756 countsByYear W20005727562020 @default.
• W2000572756 countsByYear W20005727562021 @default.
• W2000572756 countsByYear W20005727562022 @default.
• W2000572756 countsByYear W20005727562023 @default.
• W2000572756 crossrefType "journal-article" @default.
• W2000572756 hasAuthorship W2000572756A5003528751 @default.
• W2000572756 hasAuthorship W2000572756A5009882274 @default.
• W2000572756 hasAuthorship W2000572756A5018382490 @default.
• W2000572756 hasAuthorship W2000572756A5036428916 @default.
• W2000572756 hasAuthorship W2000572756A5037222124 @default.
• W2000572756 hasAuthorship W2000572756A5044178319 @default.
• W2000572756 hasAuthorship W2000572756A5047604331 @default.
• W2000572756 hasAuthorship W2000572756A5060576443 @default.
• W2000572756 hasAuthorship W2000572756A5061184556 @default.
• W2000572756 hasAuthorship W2000572756A5061288484 @default.
• W2000572756 hasAuthorship W2000572756A5079606508 @default.
• W2000572756 hasAuthorship W2000572756A5080369708 @default.
• W2000572756 hasBestOaLocation W20005727561 @default.
• W2000572756 hasConcept C137738243 @default.
• W2000572756 hasConcept C1491633281 @default.
• W2000572756 hasConcept C185592680 @default.
• W2000572756 hasConcept C195687474 @default.
• W2000572756 hasConcept C55493867 @default.
• W2000572756 hasConcept C86803240 @default.

• next