An N-glycosylation site on the beta-propeller domain of the integrin alpha5 subunit plays key roles in both its function and site-specific modification by beta1,4-N-acetylglucosaminyltransferase III.
Recently we reported that N-glycans on the beta-propeller domain of the integrin alpha5 subunit (S-3,4,5) are essential for alpha5beta1 heterodimerization, expression, and cell adhesion. Herein to further investigate which N-glycosylation site is the most important for the biological function and regulation, we characterized the S-3,4,5 mutants in detail. We found that site-4 is a key site that can be specifically modified by N-acetylglucosaminyltransferase III (GnT-III). The introduction of bisecting GlcNAc into the S-3,4,5 mutant catalyzed by GnT-III decreased cell adhesion and migration on fibronectin, whereas overexpression of N-acetylglucosaminyltransferase V (GnT-V) promoted cell migration. The phenomenon is similar to previous observations that the functions of the wild-type alpha5 subunit were positively and negatively regulated by GnT-V and GnT-III, respectively, suggesting that the alpha5 subunit could be duplicated by the S-3,4,5 mutant. Interestingly GnT-III specifically modified the S-4,5 mutant but not the S-3,5 mutant. This result was confirmed by erythroagglutinating phytohemagglutinin lectin blot analysis. The reduction in cell adhesion was consistently observed in the S-4,5 mutant but not in the S-3,5 mutant cells. Furthermore mutation of site-4 alone resulted in a substantial decrease in erythroagglutinating phytohemagglutinin lectin staining and suppression of cell spread induced by GnT-III compared with that of either the site-3 single mutant or wild-type alpha5. These results, taken together, strongly suggest that N-glycosylation of site-4 on the alpha5 subunit is the most important site for its biological functions. To our knowledge, this is the first demonstration that site-specific modification of N-glycans by a glycosyltransferase results in functional regulation.
Sato
Yuya
Y
Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japan.
Isaji
Tomoya
T
Tajiri
Michiko
M
Yoshida-Yamamoto
Shumi
S
Yoshinaka
Tsuyoshi
T
Somehara
Toshiaki
T
Fukuda
Tomohiko
T
Wada
Yoshinao
Y
Gu
Jianguo
J
D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion / Q000502:physiology; D002465:Cell Movement / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D019281:Dimerization; D005353:Fibronectins; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D039482:Integrin alpha5 / Q000235:genetics / Q000378:metabolism*; D039081:Integrin alpha5beta1 / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*
D005353:Fibronectins; D039482:Integrin alpha5; D039081:Integrin alpha5beta1; D017351:N-Acetylglucosaminyltransferases; C054599:beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase