Chain-specificity of laminin 1-5 LG45 modules in the recognition of carbohydrate-linked receptors and intramolecular binding

Laminins are a family of heterotrimers composed of alpha-, beta-, and gamma-chains in the basement membrane. Five alpha chains contain laminin globular (LG) domain consisting of five tandem modules (LG1-5 modules) at their C-terminus. Each LG45 modules is connected to a compact cloverleaf-shaped structure of LG1-3 through a flexible linker. Although the accumulated studies of the LG45 modules have suggested differences in each alpha chain regarding the binding of carbohydrate chain and intramolecular interaction, this remains unclear. In this study, to characterize their functions comparatively, we produced recombinant proteins of LG45 modules of human laminin alpha 1-5 chains. Dystroglycan (DG) modified with matriglycan readily bound to the LG45 modules of alpha 1 and alpha 2 chains but not to the other alpha chains. In contrast, heparin bound to the LG45 modules of the alpha chains, except for alpha 2. The binding of heparan sulfate/heparin-linked syndecans (SDCs) to LG45 modules was influenced by their core proteins. Furthermore, the alpha 1 and alpha 4LG45 modules bound to SDCs in a pH-dependent manner. A cell adhesion assay showed that HEK293 cells could readily adhere to the LG45 modules of alpha 3-5 chains through a combination of SDCs and integrins. Moreover, alpha 5LG45 modules bound to the E8 fragment, which includes the C-terminus of the laminin coiled-coil (LCC) domain and LG1-3 modules, but alpha 2LG45 modules did not. The results suggested that although alpha 5LG45 modules was fixed within the LG domain, alpha 2LG45 modules was freely placed in the vicinity of LG1-3. Our findings provide information for investigation of the structural and functional diversity of basement membranes.