N-Glycosylation of Laminin-332 Regulates Its Biological Functions: A NOVEL FUNCTION OF THE BISECTING GlcNAc*

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 β1,6-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 β1,6-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 α3β1 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

The C-terminal Region of Laminin β Chains Modulates the Integrin Binding Affinities of Laminins*S⃞

Laminins are major cell-adhesive proteins in basement membranes that are capable of binding to integrins. Laminins consist of three chains (α, β, and γ), in which three laminin globular modules in the α chain and the Glu residue in the C-terminal tail of the γ chain have been shown to be prerequisites for binding to integrins. However, it remains unknown whether any part of the β chain is involved in laminin-integrin interactions. We compared the binding affinities of pairs of laminin isoforms containing the β1 or β2 chain toward a panel of laminin-binding integrins, and we found that β2 chain-containing laminins (β2-laminins) bound more avidly to α3β1 and α7X2β1 integrins than β1 chain-containing laminins (β1-laminins), whereas α6β1, α6β4, and α7X1β1 integrins did not show any preference toward β2-laminins. Because α3β1 contains the “X2-type” variable region in the α3 subunit and α6β1 and α6β4 contain the “X1-type” region in the α6 subunit, we hypothesized that only integrins containing the X2-type region were capable of discriminating between β1-laminins and β2-laminins. In support of this possibility, a putative X2-type variant of α6β1 was produced and found to bind preferentially to β2-laminins. Production of a series of swap mutants between the β1 and β2 chains revealed that the C-terminal 20 amino acids in the coiled-coil domain were responsible for the enhanced integrin binding by β2-laminins. Taken together, the results provide evidence that the C-terminal region of β chains is involved in laminin recognition by integrins and modulates the binding affinities of laminins toward X2-type integrins