N-Glycosylation of Laminin-332 Regulates Its Biological Functions

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 ␤1,6-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 ␤1,6-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 ␣3␤1 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.

Glycans and Cancer

“…At the molecular levels, it has been suggested that GnT-III and the bisecting GlcNAc play roles in cell adhesion and migration by altering the N-glycans in adhesion molecules and the extracellular matrix such as E-cadherin, laminin, and integrin (Gu & Taniguchi, 2008;Isaji et al, 2004;Kariya et al, 2008;Kariya, Kawamura, Tabei, & Gu, 2010;Kitada et al, 2001;Pinho et al, 2012Pinho et al, , 2011Sato et al, 2009;Yoshimura, Ihara, Matsuzawa, & Taniguchi, 1996;Zhao et al, 2006). The expression of GnT-III was found to be upregulated by E-cadherin-mediated cell adhesion.…”

N-Glycosylation in Regulation of the Nervous System

“…Intriguingly, the functions of a number of these glycoproteins are affected by N-glycosylation outside of the nervous system. Thus, β 1,6 -branching GlcNAc modifications were found to modulate cell adhesion and cell motility by affecting the functions of laminin 332 and α3β1 integrins (Zhao et al 2006, Kariya et al 2008). Another example of a carbohydrate structure that can markedly affect molecular interactions is α 2,6 -sialylation.…”

Potential roles of N-glycosylation in cell adhesion

“…Similarly, tetraspanin CD82 with incomplete Nglycosylation exhibits an enhanced association with the α3 and α5 integrin subunits [45]. A modest association between Lm332 and α6β4 integrin mediated by galectin-3 through the N-glycans on both molecules promoted cell adhesion and migration on Lm332 as well as α6β4 integrin clustering on Lm332 [23,43]. Accordingly, an appropriate intermolecular interaction through N-glycan is important for the efficient cellular signaling and the following cellular function.…”

“…Most of the studies have identified the functional domains of Lm332 and revealed the relationship between its activities and the processing of its subunits [42]. In fact, N-glycans on Lm332 also affect its activities [43]. To examine the effects of N-glycans of Lm332 on its activities, we purified Lm332s from the conditioned media of GnT-III and GnT-V overexpressing MKN45 cells.…”