title: N-Glycosylation of laminin-332 regulates its biological functions. A novel function of the bisecting GlcNAc.
abstract: Laminin-332 (Lm332) is a large heterotrimeric glycoprotein that has been identified as a scattering factor, a regulator of cancer invasion as well as a prominent basement membrane component of the skin. Past studies have identified the functional domains of Lm332 and revealed the relationships between its activities and the processing of its subunits. However, there is little information available concerning the effects of N-glycosylation on Lm332 activities. In some cancer cells, an increase of beta1,6-GlcNAc catalyzed by N-acetylglucosaminyltransferase V (GnT-V) is related to the promotion of cancer cell motility. By contrast, bisecting GlcNAc catalyzed by N-acetylglucosaminyltransferase III (GnT-III) suppresses the further processing with branching enzymes, such as GnT-V, and the elongation of N-glycans. To examine the effects of those N-glycosylations to Lm332 on its activities, we purified Lm332s from the conditioned media of GnT-III- and GnT-V-overexpressing MKN45 cells. Lectin blotting and mass spectrometry analyses revealed that N-glycans containing the bisecting GlcNAc and beta1,6-GlcNAc structures were strongly expressed on Lm332 purified from GnT-III-overexpressing (GnT-III-Lm332) and GnT-V-overexpressing (GnT-V-Lm332) cells, respectively. Interestingly, the cell adhesion activity of GnT-III-Lm332 was apparently decreased compared with those of control Lm332 and GnT-V-Lm332. In addition, the introduction of bisecting GlcNAc to Lm332 resulted in a decrease in its cell scattering and migration activities. The weakened activities were most likely derived from the impaired alpha3beta1 integrin clustering and resultant focal adhesion formation. Taken together, our results clearly demonstrate for the first time that N-glycosylation may regulate the biological function of Lm332. This finding could introduce a new therapeutic strategy for cancer.
authors.affiliation: Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Sendai, Miyagi 981-8558, Japan.
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Kariya
authors.forename: Rika
authors.initials: R
authors.lastname: Kato
authors.forename: Satsuki
authors.initials: S
authors.lastname: Itoh
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Fukuda
authors.forename: Yukinao
authors.initials: Y
authors.lastname: Shibukawa
authors.forename: Noriko
authors.initials: N
authors.lastname: Sanzen
authors.forename: Kiyotoshi
authors.initials: K
authors.lastname: Sekiguchi
authors.forename: Yoshinao
authors.initials: Y
authors.lastname: Wada
authors.forename: Nana
authors.initials: N
authors.lastname: Kawasaki
authors.forename: Jianguo
authors.initials: J
authors.lastname: Gu
mesh_terms: D001485:Basement Membrane / Q000201:enzymology / Q000473:pathology; D002448:Cell Adhesion / Q000235:genetics; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D039201:Integrin alpha3beta1 / Q000235:genetics / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D009361:Neoplasm Invasiveness / Q000235:genetics / Q000473:pathology; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012867:Skin / Q000201:enzymology / Q000473:pathology; D012878:Skin Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D000097794:Kalinin
chemical_list: D015815:Cell Adhesion Molecules; D039201:Integrin alpha3beta1; D017351:N-Acetylglucosaminyltransferases; D009363:Neoplasm Proteins; D011134:Polysaccharides; D021122:Protein Subunits; D000097794:Kalinin; C054599:beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase