Chain-specificity of laminin α1-5 LG45 modules in the recognition of carbohydrate-linked receptors and intramolecular binding.

Laminins are a family of heterotrimers composed of α-, ß-, and γ-chains in the basement membrane. Five α 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 α 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 α1-5 chains. Dystroglycan (DG) modified with matriglycan readily bound to the LG45 modules of α1 and α2 chains but not to the other α chains. In contrast, heparin bound to the LG45 modules of the α chains, except for α2. The binding of heparan sulfate/heparin-linked syndecans (SDCs) to LG45 modules was influenced by their core proteins. Furthermore, the α1 and α4LG45 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 α3-5 chains through a combination of SDCs and integrins. Moreover, α5LG45 modules bound to the E8 fragment, which includes the C-terminus of the laminin coiled-coil (LCC) domain and LG1-3 modules, but α2LG45 modules did not. The results suggested that although α5LG45 modules was fixed within the LG domain, α2LG45 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.

Laminin α5_CD239_Spectrin is a candidate association that compensates the linkage between the basement membrane and cytoskeleton in skeletal muscle fibers.

The linkage between the basement membrane (BM) and cytoskeleton is crucial for muscle fiber stability and signal transduction. Mutations in the linkage molecules can cause various types of muscular dystrophies. The different severities and times of onset suggest that compensatory linkages occur at the sarcolemma. Cluster of differentiation 239 (CD239) binds to the α5 subunit of laminin-511 extracellularly and is connected to spectrin intracellularly, resulting in a linkage between the BM and cytoskeleton. In this study, we explored the linkage of laminin α5_CD239_spectrin in skeletal muscles. Although laminin α5, CD239, and spectrin were present in embryonic skeletal muscles, they disappeared in adult skeletal muscle tissues, except for the soleus and diaphragm. Laminin α5_CD239_spectrin was localized in the skeletal muscle tissues of Duchenne muscular dystrophy and congenital muscular dystrophy mouse models. The experimental regeneration of skeletal muscle increased the CD239-mediated linkage, indicating that it responds to regeneration, but not to genetic influence. Furthermore, in silico analysis showed that laminin α5_CD239_spectrin was upregulated by steroid therapy for muscular dystrophy. Therefore, CD239-mediated linkage may serve as a therapeutic target to prevent the progression of muscular dystrophy.

RGDX1 X2 motif regulates integrin αvß5 binding for pluripotent stem cell adhesion.

The arginine-glycine-aspartic acid (RGD) motif is a cell adhesion sequence that binds to integrins. Some RGD-containing peptides promote adhesion of both embryonic stem cells and induced pluripotent stem cells (iPSCs); however, not all such RGD-containing peptides are active. In this study, we elucidated the role of RGD-neighboring sequences on iPSC adhesion using diverse synthetic peptides and recombinant proteins. Our results indicate that iPSC adhesion requires RGDX1 X2  sequences, such as RGDVF and RGDNY, and that the X1 X2 residues are essential for the adhesion via integrin αvß5 but not αvß3. iPSCs express integrin αvß5 but not αvß3; therefore, iPSC adhesion requires the RGDX1 X2 -containing sequences. The importance of the X1 X2 residues was confirmed with both HeLa and A549 cells, which express integrin αvß5 but not αvß3. Analysis of RGD-neighboring sequences provides important insights into ligand-binding specificity of integrins. Identification of integrin αvß5-binding motifs is potentially useful in drug development, drug delivery, cell culture, and tissue engineering.

Laminin ß2 variants associated with isolated nephropathy that impact matrix regulation.

Mutations in LAMB2, encoding laminin ß2, cause Pierson syndrome and occasionally milder nephropathy without extrarenal abnormalities. The most deleterious missense mutations that have been identified affect primarily the N-terminus of laminin ß2. On the other hand, those associated with isolated nephropathy are distributed across the entire molecule, and variants in the ß2 LEa-LF-LEb domains are exclusively found in cases with isolated nephropathy. Here we report the clinical features of mild isolated nephropathy associated with 3 LAMB2 variants in the LEa-LF-LEb domains (p.R469Q, p.G699R, and p.R1078C) and their biochemical characterization. Although Pierson syndrome missense mutations often inhibit laminin ß2 secretion, the 3 recombinant variants were secreted as efficiently as WT. However, the ß2 variants lost pH dependency for heparin binding, resulting in aberrant binding under physiologic conditions. This suggests that the binding of laminin ß2 to negatively charged molecules is involved in glomerular basement membrane (GBM) permselectivity. Moreover, the excessive binding of the ß2 variants to other laminins appears to lead to their increased deposition in the GBM. Laminin ß2 also serves as a potentially novel cell-adhesive ligand for integrin α4ß1. Our findings define biochemical functions of laminin ß2 variants influencing glomerular filtration that may underlie the pathogenesis of isolated nephropathy caused by LAMB2 abnormalities.