Polymerizing laminins in development, health, and disease

Abstract

Polymerizing laminins are multi-domain basement membrane (BM) glycoproteins that self-assemble into cell-anchored planar lattices to establish the initial BM scaffold. Nidogens, collagen-IV and proteoglycans then bind to the scaffold at different domain loci to create a mature BM. The LN domains of adjacent laminins bind to each other to form a polymer node, while the LG domains attach to cytoskeletal-anchoring integrins and dystroglycan, as well as to sulfatides and heparan sulfates. The polymer node, the repeating unit of the polymer scaffold, is organized into a near-symmetrical triskelion. The structure, recently solved by cryo-electron microscopy in combination with AlphaFold2 modeling and biochemical studies, reveals how the LN surface residues interact with each other and how mutations cause failures of self-assembly in an emerging group of diseases, the LN-lamininopathies, that include LAMA2-related dystrophy and Pierson syndrome.

Keywords: Pierson syndrome; basement membrane; cryo-EM; dystroglycan; extracellular matrix; integrin; lamininopathy; muscular dystrophy; myelination; polymerizing laminins; self-assembly.

Figure 1

Basement membranes. These ECMs are found anchored to cell surfaces of epithelial, endothelial, muscle, and fat cells, but generally not fibroblasts, cartilage, or osteocytes. A, epithelial BMs are typically thin (<100 nm) and are adjacent to the stroma. They are invested with α5 and α3 laminins. B, the mature glomerular basement membrane (300–350 nm in humans) is a filtration barrier enriched in Lm521 that results from the fusion of capillary endothelial and podocyte BMs. The endothelium is fenestrated while the podocyte foot processes are separated by slit diaphragms, leaving the GBM as the only continuous barrier between the blood and urinary space. C, mature skeletal myofibers (Lm211) are each coated with a thin BM along their length with a sparse collagenous stroma between each BM. Specialized BMs are found at the neuromuscular junctions (Lm221, 421) and myotendinous junctions. D, capillary BMs of the blood–brain barrier differs from typical capillary BMs in that they are double separated by pericytes. Components are generated by endothelial, pericyte, and glial cell foot processes such that the endothelial BM contains Lm511 and 411 while the glial BM contains Lm111 and 211.

Figure 2

Family of laminins with γ1 or γ2 subunits.A, laminins 111, 211, 121, and 221 share the same domain structure with similar binding domains. Of these, the LN domains, globular in shape, mediate polymerization. The rod-like LE domains, typically containing eight cysteines that are internally paired, act as spacers. The γ1LEb3 domain mediates nidogen-binding. A three-chain α-helical coiled coil that forms the long arm and contains a binding site for agrin. The LG domains are arranged into a cluster of three proximal domains (LG1-3) connected by a flexible linker to two distal domains (LG4-5). LG1-3 and distal coiled-coil mediate the major integrin while LG4 (and LG1-3) mediate α-dystroglycan (αDG) receptor binding. Laminin-receptor complexes are shown for α1/2 laminins. The LG domains of laminins provide the principal ligands for receptor interactions. LG4 binds to sulfated glycolipids such as sulfatides and to heparin/heparan sulfates, and α-dystroglycan (αDG). Secondary αDG binding sites are also present in LG3 (both Lmα1 and Lmα2). Integrins (α6β1, α7β1) bind to a complex of LG1-3 that requires the terminus of the colied-coil domain. The cytoplasmic portion of integrins binds to adaptor proteins that in turn bind to F-actin. B, laminins 511 and 521 differ in their longer α-short arm containing a pair of RGD integrin-binding sequences for αv integrins, a partially different repertoire of integrins, and a binding site for the Lutheran antigen/BCAM receptor. Lm411 and Lm511 have truncated α-short arms (preventing polymerization) and do not bind to αDG. They bind to α6β1 and α7β1 integrins, weakly in the case of Laminin-411. Laminin-421 also binds to CD146/MCAM, affecting cell migration. Laminin-3A32 (Lm3A32) is a non-polymerizing largely hemidesmosome-associated epithelial laminin with strong integrin interactions. Maturation cleavages occur in the β3 and α3 short arms such that it assumes a rod-like appearance. Laminin-3B11 is an α3 splice variant with a polymerizing LN domain that has been reported to be expressed in vasculature and other sites. While it is unclear what biological role it might play, they may be unique in their integrin integrin-binding repertoire in the absence of αDG binding. γ3-laminins (not illustrated), similar in domain organization to γ1, lack a critical C-terminal sequence required for integrin binding.

Figure 3

Laminin polymerization. The laminin first assembles into a β-γ dimer through weak LN domain interactions. Adding a third laminin through another β-γ bond is possible. Importantly, the α-LN domain of free laminins binds to the available β-γ intermediate complexes through a higher affinity calcium-dependent interaction, completing the formation of polymer nodes. An image-averaged negative stain of a polymer node is shown in the inset.

Figure 4

Basement membrane assembly through polymerizing laminin. In neuromuscular tissues, Lm211 attaches to the cognate cell surface as it polymerizes. Laminin can form an initial scaffolding by binding to SGLs (sulfatides in Schwann cells), and to laminin-class integrin and α/β-DG receptors. The receptors, in turn, are anchored to the cytoskeleton. Nidogens-1 and -2 and heparan sulfate (HS) chains (when sufficiently sulfated) bind to laminin (Lm) and to collagen-IV, acting as bridges, with the collagen polymerizing through 7S, NC1, and lateral associations to form a second network. Agrin binds through its N-terminal globular domain (NtA) to the laminin coiled-coil and to DG through its C-terminal moiety and perlecan (Perl) domain IV binds to the nidogen G2 domain. Thus, like nidogen, proteoglycan HS chains are thought to form a bridge between the laminin and collagen polymers.

Figure 5

A cryo-EM structure of the laminin polymer node.A, the cryo-EM map (EMD-27542) is color-coded. Laminin subunits α1, β1 and γ1 are shown in green, red and blue, respectively. Flexible parts of the Coulomb map with no model built are shown in gray. The N-glycans are colored in yellow, and a calcium ion is shown as a black sphere. B, a cartoon representation of the laminin polymer node structure (PDB ID: 8DMK). The LN and LE1 domains are labeled in γ1 subunit, whereas the toe and heel regions are highlighted in β1 subunit. C, atomic models of α1 (shown in green), β1 (displayed in red), and γ1 (colored in gray). Different elements constituting the structures are labeled in the figure. Disulfide bridges are displayed in yellow. D, pathogenic laminin mutations implicated in LN-lamininopathies. Twenty-three reported to date pathogenic mutations are mapped onto the cryo-EM structure of the laminin polymer node. The amino acid alternations cluster into four distinct classes. The class 1 involve residues from inter-subunit interfaces. The class 2 consists of mutations located in close proximity to invariant N-glycosylation sites. The mutations from class 3 and class 4 affect formation of disulfide bridges and hydrophobic cores of altered laminin variants, respectively.

Figure 6

Structures of other laminin fragments complexed with various BM components.A, complex formed between the six-bladed tyr-trp-thr-asp nidogen-1 G3 propeller domain and the laminin γ1-LEb EGF-like domains. The dissociation constant is <1 nM and requires Lmα1N802. We have found that the flanking Ty and EGF6 domains contribute to high-affinity binding as well. B, complex formed between the Xyl-GlcA-Xyl-GlcA portion of matriglycan and basic residues of the laminin α2 LG4 domain. A second matriglycan binding site exists in the LG1-3 domains and there are data to suggest that high affinities are seen with LG domain duplication. C, a combination of X-ray crystallography and cryo-EM was used to solve the structure of a laminin-integrin complex, critical for BM structure and functions. The interaction requires the α-subunit LG1-3, terminus of the coiled-coil (α-β-γ subunits) binds to the integrin α6 and β1 subunits, involving five different protein domains The C-terminal segment provides two carboxylate anchor points to bridge the metal-ion dependent adhesion locus of the β1 integrin subunit and N189 of α6.