Bisecting GlcNAc Residues on Laminin-332 Down-regulate Galectin-3-dependent Keratinocyte Motility

• Published in: The Journal of biological chemistry 2010-01, Vol.285 (5), p.3330-3340
• Main Authors: Kariya, Yoshinobu, Kawamura, Chihiro, Tabei, Toshiki, Gu, Jianguo
• Format: Article
• Language: English
• Subjects: Acetylglucosamine - chemistry; Cell Adhesion; Cell Movement; Cell/Migration; Down-Regulation; Enzyme Activation; ErbB Receptors - metabolism; Extracellular Matrix/Integrin; Extracellular Matrix/Laminin; Extracellular Signal-Regulated MAP Kinases - metabolism; Galectin 3 - metabolism; Glycobiology and Extracellular Matrices; Glycoproteins/Biosynthesis; Glycoproteins/Surface; Glycosylation; GnT-III; Humans; Integrin alpha3beta1 - metabolism; Integrin alpha6beta4 - metabolism; Keratinocytes - cytology; Laminin - chemistry; N-Acetylglucosaminyltransferases - metabolism; Signal Transduction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.038836

Laminin-332 (Lm332; formerly laminin-5) is a basement membrane protein in the skin, which promotes cell motility in wound healing and cancer invasion. In a previous study, we reported that the introduction of bisecting GlcNAc into Lm332 (GnT-III-Lm332), catalyzed by N-acetylglucosaminyltransferase III (GnT-III), reduced cell migration (Kariya, Y., Kato, R., Itoh, S., Fukuda, T., Shibukawa, Y., Sanzen, N., Sekiguchi, K., Wada, Y., Kawasaki, N., and Gu, J. (2008) J. Biol. Chem. 283, 33036–33045). However, the underlying molecular mechanism by which GnT-III-Lm332 suppresses the normal biological functions of Lm332 remains to be elucidated. In this study, we show that galectin-3, which is a β-galactoside-binding protein, strongly bound to unmodified Lm332 but not to GnT-III-Lm332 and that binding of galectin-3 was completely blocked by lactose. Exogenous galectin-3 significantly enhanced keratinocyte cell motility on control Lm332 but not on GnT-III-Lm332. A functional blocking antibody against galectin-3 inhibited Lm332-induced α3β1 and α6β4 integrin clustering and focal contact formation. Co-immunoprecipitation revealed that galectin-3 associated with both β4 integrin and epidermal growth factor receptor, thereby cross-linking the two molecules. The associations were inhibited by either the presence of lactose or expression of GnT-III. Moreover, galectin-3 consistently enhanced ERK activation. Taken together, the results of this study are the first to clearly identify the molecular mechanism responsible for the inhibitory effects of GnT-III on extracellular matrix-integrin-meditated cell adhesion, migration, and signal transduction. The findings presented herein shed light on the importance of N-glycosylation-mediated supramolecular complex formation on the cell surface.