An N-glycosylation site on the β-propeller domain of the integrin α5 subunit plays key roles in both its function and site-specific modification by β1,4-N-acetylglucosaminyltransferase III

Abstract

Recently we reported that N-glycans on the β-propeller domain of the integrin α5 subunit (S-3, 4, 5) are essential for α5β1 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 Glc-NAc 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 α5 subunit were positively and negatively regulated by GnT-V and GnT-III, respectively, suggesting that the α5 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 α5. These results, taken together, strongly suggest that N-glycosylation of site-4 on the α5 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. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.