Introduction of bisecting GlcNAc into integrin alpha5beta1 reduces ligand binding and down-regulates cell adhesion and cell migration.
The enzyme beta1,4-N-acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of a bisecting GlcNAc residue to glycoproteins, resulting in a modulation in biological function. Our previous studies showed that the transfection of the GnT-III gene into B16 melanoma cells results in a suppression of invasive ability and lung colonization. The suppression has been postulated to be due to an increased level of E-cadherin expression on the cell surface, which in turn leads to the up-regulation of cell-cell adhesion. In this study, we report on the effects of overexpression of GnT-III on cell-matrix adhesion. The overexpression of GnT-III, but not that of an enzymatic inactive GnT-III (D323A), inhibits cell spreading and migration on fibronectin, a specific ligand for integrin alpha(5)beta(1), and the focal adhesion kinase phosphorylation. E(4)-PHA lectin blot analyses showed that the levels of bisecting GlcNAc structures on the integrin alpha(5) subunit as well as alpha(2) and alpha(3) subunits immunoprecipitated from GnT-III transfectants were substantially increased. In addition, the affinity of the binding of integrin alpha(5)beta(1) to fibronectin was significantly reduced by the introduction of the bisecting GlcNAc, to the alpha(5) subunit. These findings suggest that the modification of N-glycan of integrin by GnT-III inhibits its ligand binding ability, subsequently leading to the down-regulation of integrin-mediated signaling.
Department of Biochemistry, Osaka University Graduate School of Medicine, B1, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Tomoya
T
Isaji
Jianguo
J
Gu
Ryoko
R
Nishiuchi
Yanyang
Y
Zhao
Motoko
M
Takahashi
Eiji
E
Miyoshi
Koichi
K
Honke
Kiyotoshi
K
Sekiguchi
Naoyuki
N
Taniguchi
D000117:Acetylglucosamine / Q000737:chemistry*; D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D015820:Cadherins / Q000378:metabolism; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002465:Cell Movement; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D038982:Integrin alpha2beta1 / Q000378:metabolism; D039201:Integrin alpha3beta1 / Q000378:metabolism; D039081:Integrin alpha5beta1 / Q000737:chemistry* / Q000378:metabolism; D016023:Integrins / Q000378:metabolism; D037102:Lectins / Q000378:metabolism; D008024:Ligands; D008081:Liposomes / Q000378:metabolism; D008546:Melanoma, Experimental; D051379:Mice; D008954:Models, Biological; D017351:N-Acetylglucosaminyltransferases / Q000502:physiology*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation
D015820:Cadherins; D038982:Integrin alpha2beta1; D039201:Integrin alpha3beta1; D039081:Integrin alpha5beta1; D016023:Integrins; D037102:Lectins; D008024:Ligands; D008081:Liposomes; D017351:N-Acetylglucosaminyltransferases; C054599:beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C496051:Ptk2 protein, mouse; D000117:Acetylglucosamine