Laminin N-terminal Domain Research Articles

Systematic Review of Clinical Characteristics and Genotype-Phenotype Correlation in LAMB2-Associated Disease

Laminin subunit beta-2 (LAMB2)-associated disease, termed Pierson syndrome, presents with congenital nephrotic syndrome, ocular symptoms, and neuromuscular symptoms. In recent years, however, the widespread use of next-generation sequencing (NGS) has helped to discover a variety of phenotypes associated with this disease. Therefore, we conducted this systematic review. A literature search of patients with LAMB2 variants was conducted, and 110 patients were investigated, including 12 of our patients. For genotype-phenotype correlation analyses, the extracted data were investigated for pathogenic variant types, the severity of nephropathy, and extrarenal symptoms. Survival analyses were also performed for the onset age of end-stage kidney disease (ESKD). Among all patients, 81 (78%) presented with congenital nephrotic syndrome, and 52 (55%) developed ESKD within 12 months. The median age at ESKD onset was 6.0 months. Kidney survival analysis showed that patients with biallelic truncating variants had a significantly earlier progression to ESKD than those with other variants (median age 1.2 months vs. 60.0 months, P< 0.05). Although the laminin N-terminal domain is functionally important in laminin proteins, and variants in the laminin N-terminal domain are said to result in a severe kidney phenotype such as earlier onset age and worse prognosis, there were no significant differences in onset age of nephropathy and progression to ESKD between patients with nontruncating variants located in the laminin N-terminal domain and those with variants located outside this domain. This study revealed a diversity of LAMB2-associated diseases, characteristics of LAMB2 nephropathy, and genotype-phenotype correlations.

Cryo-EM reveals the molecular basis of laminin polymerization and LN-lamininopathies

Laminin polymerization is the major step in basement membranes assembly. Its failures cause laminin N-terminal domain lamininopathies including Pierson syndrome. We have employed cryo-electron microscopy to determine a 3.7 Å structure of the trimeric laminin polymer node containing α1, β1 and γ1 subunits. The structure reveals the molecular basis of calcium-dependent formation of laminin lattice, and provides insights into polymerization defects manifesting in human disease.

Laminin Polymerization and Inherited Disease: Lessons From Genetics.

The laminins (LM) are a family of basement membranes glycoproteins with essential structural roles in supporting epithelia, endothelia, nerves and muscle adhesion, and signaling roles in regulating cell migration, proliferation, stem cell maintenance and differentiation. Laminins are obligate heterotrimers comprised of α, β and γ chains that assemble intracellularly. However, extracellularly these heterotrimers then assemble into higher-order networks via interaction between their laminin N-terminal (LN) domains. In vitro protein studies have identified assembly kinetics and the structural motifs involved in binding of adjacent LN domains. The physiological importance of these interactions has been identified through the study of pathogenic point mutations in LN domains that lead to syndromic disorders presenting with phenotypes dependent on which laminin gene is mutated. Genotype-phenotype comparison between knockout and LN domain missense mutations of the same laminin allows inferences to be drawn about the roles of laminin network assembly in terms of tissue function. In this review, we will discuss these comparisons in terms of laminin disorders, and the therapeutic options that understanding these processes have allowed. We will also discuss recent findings of non-laminin mediators of laminin network assembly and their implications in terms of basement membrane structure and function.

A deletion in the N-terminal polymerizing domain of laminin β2 is a new mouse model of chronic nephrotic syndrome

The importance of the glomerular basement membrane (GBM) in glomerular filtration is underscored by the manifestations of Alport and Pierson syndromes, caused by defects in type IV collagen α3α4α5 and the laminin β2 chain, respectively. Lamb2 null mice, which model the most severe form of Pierson syndrome, exhibit proteinuria prior to podocyte foot process effacement and are therefore useful for studying GBM permselectivity. We hypothesize that some LAMB2 missense mutations that cause mild forms of Pierson syndrome induce GBM destabilization with delayed effects on podocytes. While generating a CRISPR/Cas9-mediated analogue of a human LAMB2 missense mutation in mice, we identified a 44-amino acid deletion (LAMB2-Del44) within the laminin N-terminal domain, a domain mediating laminin polymerization. Laminin heterotrimers containing LAMB2-Del44 exhibited a 90% reduction in polymerization invitro that was partially rescued by type IV collagen and nidogen. Del44 mice showed albuminuria at 1.8-6.0 g/g creatinine (ACR) at one to two months, plateauing at an average 200 g/g ACR at 3.7 months, when GBM thickening and hallmarks of nephrotic syndrome were first observed. Despite the massive albuminuria, some Del44 mice survived for up to 15 months. Blood urea nitrogen was modestly elevated at seven-nine months. Eight to nine-month-old Del44 mice exhibited glomerulosclerosis and interstitial fibrosis. Similar to Lamb2-/- mice, proteinuria preceded foot process effacement. Foot processes were widened but not effaced at one-two months despite the high ACRs. At three months some individual foot processes were still observed amid widespread effacement. Thus, our chronic model of nephrotic syndrome may prove useful to study filtration mechanisms, long-term proteinuria with preserved kidney function, and to test therapeutics.

Enrichment of LOVD-USHbases with 152 USH2A genotypes defines an extensive mutational spectrum and highlights missense hotspots.

Alterations of USH2A, encoding usherin, are responsible for more than 70% of cases of Usher syndrome type II (USH2), a recessive disorder that combines moderate to severe hearing loss and retinal degeneration. The longest USH2A transcript encodes usherin isoform b, a 5,202-amino-acid transmembrane protein with an exceptionally large extracellular domain consisting notably of a Laminin N-terminal domain and numerous Laminin EGF-like (LE) and Fibronectin type III (FN3) repeats. Mutations of USH2A are scattered throughout the gene and mostly private. Annotating these variants is therefore of major importance to correctly assign pathogenicity. We have extensively genotyped a novel cohort of 152 Usher patients and identified 158 different mutations, of which 93 are newly described. Pooling this new data with the existing pathogenic variants already incorporated in USHbases reveals several previously unappreciated features of the mutational spectrum. We show that parts of the protein are more likely to tolerate single amino acid variations, whereas others constitute pathogenic missense hotspots. We have found, in repeated LE and FN3 domains, a nonequal distribution of the missense mutations that highlights some crucial positions in usherin with possible consequences for the assessment of the pathogenicity of the numerous missense variants identified in USH2A.

Laminin Network Formation Studied by Reconstitution of Ternary Nodes in Solution

The polymerization of laminins into a cell-associated network is a key process in basement membrane assembly. Network formation is mediated by the homologous short arm tips of the laminin heterotrimer, each consisting of a globular laminin N-terminal (LN) domain followed by a tandem of laminin-type epidermal growth factor-like (LEa) domains. How the short arms interact in the laminin network is unclear. Here, we have addressed this question by reconstituting laminin network nodes in solution and analyzing them by size exclusion chromatography and light scattering. Recombinant LN-LEa1-4 fragments of the laminin α1, α2, α5, β1, and γ1 chains were monomeric in solution. The β1 and γ1 fragments formed the only detectable binary complex and ternary complexes of 1:1:1 stoichiometry with all α chain fragments. Ternary complex formation required calcium and did not occur at 4 °C, like the polymerization of full-length laminins. Experiments with chimeric short arm fragments demonstrated that the LEa2-4 regions of the β1 and γ1 fragments are dispensable for ternary complex formation, and an engineered glycan in the β1 LEa1 domain was also tolerated. In contrast, mutation of Ser-68 in the β1 LN domain (corresponding to a Pierson syndrome mutation in the closely related β2 chain) abolished ternary complex formation. We conclude that authentic ternary nodes of the laminin network can be reconstituted for structure-function studies.

Crystal Structures of the Network-Forming Short-Arm Tips of the Laminin β1 and γ1 Chains

The heterotrimeric laminins are a defining component of basement membranes and essential for tissue formation and function in all animals. The three short arms of the cross-shaped laminin molecule are composed of one chain each and their tips mediate the formation of a polymeric network. The structural basis for laminin polymerisation is unknown. We have determined crystal structures of the short-arm tips of the mouse laminin β1 and γ1 chains, which are grossly similar to the previously determined structure of the corresponding α5 chain region. The short-arm tips consist of a laminin N-terminal (LN) domain that is attached like the head of a flower to a rod-like stem formed by tandem laminin-type epidermal growth factor-like (LE) domains. The LN domain is a β-sandwich with elaborate loop regions that differ between chains. The γ1 LN domain uniquely contains a calcium binding site. The LE domains have little regular structure and are stabilised by cysteines that are disulphide-linked 1–3, 2–4, 5–6 and 7–8 in all chains. The LN surface is not conserved across the α, β and γ chains, but within each chain subfamily there is a striking concentration of conserved residues on one face of the β-sandwich, while the opposite face invariably is shielded by glycans. We propose that the extensive conserved patches on the β and γ LN domains mediate the binding of these two chains to each other, and that the α chain LN domain subsequently binds to the composite β-γ surface. Mutations in the laminin β2 LN domain causing Pierson syndrome are likely to impair the folding of the β2 chain or its ability to form network interactions.

Origin and Evolution of Laminin Gene Family Diversity

Laminins are a family of multidomain glycoproteins that are important contributors to the structure of metazoan extracellular matrices. To investigate the origin and evolution of the laminin family, we characterized the full complement of laminin-related genes in the genome of the sponge, Amphimedon queenslandica. As a representative of the Demospongiae, a group consistently placed within the earliest diverging branch of animals by molecular phylogenies, Amphimedon is uniquely placed to provide insight into early steps in the evolution of metazoan gene families. Five Amphimedon laminin-related genes possess the conserved molecular features, and most of the domains found in bilaterian laminins, but all display domain architectures distinct from those of the canonical laminin chain types known from model bilaterians. This finding prompted us to perform a comparative genomic analysis of laminins and related genes from a choanoflagellate and diverse metazoans and to conduct phylogenetic analyses using the conserved Laminin N-terminal domain in order to explore the relationships between genes with distinct architectures. Laminin-like genes appear to have originated in the holozoan lineage (choanoflagellates + metazoans + several other unicellular opisthokont taxa), with several laminin domains originating later and appearing only in metazoan (animal) or eumetazoan (placozoans + ctenophores + cnidarians + bilaterians) laminins. Typical bilaterian α, β, and γ laminin chain forms arose in the eumetazoan stem and another chain type that is conserved in Amphimedon, the cnidarian, Nematostella vectensis, and the echinoderm, Strongylocentrotus purpuratus, appears to have been lost independently from the placozoan, Trichoplax adhaerens, and from multiple bilaterians. Phylogenetic analysis did not clearly reconstruct relationships between the distinct laminin chain types (with the exception of the α chains) but did reveal how several members of the netrin family were generated independently from within the laminin family by duplication and domain shuffling and by domain loss. Together, our results suggest that gene duplication and loss and domain shuffling and loss all played a role in the evolution of the laminin family and contributed to the generation of lineage-specific diversity in the laminin gene complements of extant metazoans.

Crystal Structure of the Ligand Binding Domain of Netrin G2

Netrin G proteins represent a small family of synaptic cell adhesion molecules related to netrins and to the polymerization domains of laminins. Two netrin G proteins are encoded in vertebrate genomes, netrins G1 and G2, which are known to bind the leucine-rich repeat proteins netrin G ligand (NGL)-1 and NGL-2, respectively. Netrin G proteins share a common multi-domain architecture comprising a laminin N-terminal (LN) domain followed by three laminin epidermal growth factor-like (LE) domains and a C′ region containing a glycosylphosphatidylinositol anchor. Here, we use deletion analysis to show that the LN domain region of netrin Gs contains the binding site for NGLs to which they bind with 1:1 stoichiometry and sub-micromolar affinity. Netrin Gs are alternatively spliced in their LE domain regions, but the binding region, the LN domain, is identical in all splice forms. We determined the crystal structure for a fragment comprising the LN domain and domain LE1 of netrin G2 by sulfur single-wavelength anomalous diffraction phasing and refined it to 1.8 Å resolution. The structure reveals an overall architecture similar to that of laminin α chain LN domains but includes significant differences including a Ca2+ binding site in the LN domain. These results reveal the minimal binding unit for interaction of netrin Gs with NGLs, define structural features specific to netrin Gs, and suggest that netrin G alternative splicing is not involved in NGL recognition.

A Novel Disulfide Pattern in Laminin-Type Epidermal Growth Factor-like (LE) Modules of Laminin β1 and γ1 Chains

In-depth mass spectrometric analysis of disulfide bond patterns in recombinant mouse laminin β1 and γ1 chain N-terminal fragments comprising the laminin N-terminal (LN) domain and the first four laminin epidermal growth factor-like (LE) domains revealed a novel disulfide pattern for LE domains. This showed a (2-3, 4-5, 6-7, 8-1) connectivity with the last cysteine of one LE domain being connected to the first cysteine of the following LE domain. The same pattern was also found in E4, the N-terminal β1 chain fragment derived by elastase digestion of mouse EHS tumor laminin-111, showing that this pattern occurs in native laminin. The strictly linear pattern with an interdomain disulfide has not been described previously for EGF domains. The N-terminal portions of laminin short arms, consisting of the LN domain and LE domains 1-4, are essential for laminin-laminin self-interactions, whereas the internal LE domains 7-9 in the laminin γ1 chain harbor the nidogen binding site and have a conventional disulfide pattern. This suggests that LE domains differing in function also differ in their disulfide patterns.

Expression of Usherin in the Anthozoan Nematostella vectensis

The transmembrane splice variant of usherin is concentrated in the ankle-link complex of stereocilia in the vertebrate cochlea. Mutations in this variant can result in Usher Syndrome, or congenital deafblindness. In order to study the phylogenetically conserved features of this protein, two adjacent putative genes encoding predicted proteins similar to the amino and carboxy halves of vertebrate usherin were identified in the genome of the anthozoan Nematostella vectensis. RT-PCR was used to show that transcripts encoding each predicted protein are expressed during development, and that both predicted proteins are in fact encoded on the same gene. Thus, anthozoan usherin has a domain organization identical to the transmembrane variant of vertebrate usherin, with a laminin N-terminal domain, 10 epidermal growth factor-like domains, four fibronectin-type III domains, two laminin G domains, 28 additional fibronectin-type III domains, a transmembrane domain, and a short intracellular domain. Usherin is expressed widely in planulae and in the ectoderm of the column of juvenile polyps. Searches of genomic databases reveal usherins with a highly conserved domain organization throughout the deuterostome lineage as well as in some (annelids and molluscs), but not all (arthropods and nematodes), protostomes. Thus, usherin sequence and domain organization is remarkably conserved across phyla. However, its pattern of expression in N. vectensis is not consistent with a role for this protein in the mechanosensory ciliary cone of mature cnidoblasts.

Mutations in Lama1 Disrupt Retinal Vascular Development and Inner Limiting Membrane Formation

The Neuromutagenesis Facility at the Jackson Laboratory generated a mouse model of retinal vasculopathy, nmf223, which is characterized clinically by vitreal fibroplasia and vessel tortuosity. nmf223 homozygotes also have reduced electroretinogram responses, which are coupled histologically with a thinning of the inner nuclear layer. The nmf223 locus was mapped to chromosome 17, and a missense mutation was identified in Lama1 that leads to the substitution of cysteine for a tyrosine at amino acid 265 of laminin alpha1, a basement membrane protein. Despite normal localization of laminin alpha1 and other components of the inner limiting membrane, a reduced integrity of this structure was suggested by ectopic cells and blood vessels within the vitreous. Immunohistochemical characterization of nmf223 homozygous retinas demonstrated the abnormal migration of retinal astrocytes into the vitreous along with the persistence of hyaloid vasculature. The Y265C mutation significantly reduced laminin N-terminal domain (LN) interactions in a bacterial two-hybrid system. Therefore, this mutation could affect interactions between laminin alpha1 and other laminin chains. To expand upon these findings, a Lama1 null mutant, Lama1(tm1.1Olf), was generated that exhibits a similar but more severe retinal phenotype than that seen in nmf223 homozygotes. The increased severity of the Lama1 null mutant phenotype is probably due to the complete loss of the inner limiting membrane in these mice. This first report of viable Lama1 mouse mutants emphasizes the importance of this gene in retinal development. The data presented herein suggest that hypomorphic mutations in human LAMA1 could lead to retinal disease.

Identification of a Novel Family of Laminin N-terminal Alternate Splice Isoforms

The laminins are a family of heterotrimeric basement membrane proteins that play roles in cellular adhesion, migration, and tissue morphogenesis. Through in silico analysis of the laminin-encoding genes, we identified a novel family of alternate splice isoforms derived from the 5′-end of the LAMA3 and LAMA5 genes. These isoforms resemble the netrins in that they contain a laminin N-terminal domain followed by a short stretch of laminin-type epidermal growth factor-like repeats. We suggest the terms LaNt (laminin N terminus) α3 and LaNt α5, for the predicted protein products of these mRNAs. RT-PCR confirmed the presence of these transcripts at the mRNA level. Moreover, they exhibit differential, tissue-specific, expression profiles. To confirm the existence of LaNt α3 protein, we generated an antibody to a unique domain within the putative polypeptide. This antibody recognizes a protein at the predicted molecular mass of 64 kDa by immunoblotting. Furthermore, immunofluorescence analyses revealed a basement membrane staining in epithelial tissue for LaNt α3 and LaNt α3 localized along the substratum-associated surface of cultured keratinocytes. We have also tested the functionality LaNt α3 through RNAi-mediated knockdown. Keratinocytes exhibiting specific knockdown of LaNt α3 displayed impaired adhesion, stress resistance, and reduced ability to close scratch wounds in vitro.

A single point mutation in the LN domain of LAMA2 causes muscular dystrophy and peripheral amyelination

Mutations in the gene encoding the basal lamina (BL) component laminin alpha2 (LAMA2) cause merosin-deficient congenital muscular dystrophy 1A (MDC1A), a complex disorder that includes hypomyelination and myodegeneration. In dystrophia muscularis (dy) mice bearing Lama2 mutations, myofibers and Schwann cells fail to assemble stable BLs, which are thought to be crucial for myofiber survival and Schwann cell differentiation. Here, we describe defects in a new allele of Lama2 in mice, nmf417, in which a point mutation substitutes Arg for Cys79 at a universally conserved CxxC motif in the laminin N-terminal (LN) domain; this domain mediates laminin-laminin interactions. nmf417 homozygosity caused progressive myodegeneration and severe peripheral amyelination in nerve roots, similar to previous Lama2 mutations, but without the pervasive BL thinning previously associated with the disorder. In direct contrast to the previously characterized dy and dy2J alleles, nmf417 homozygous myofibers frequently had thickened BLs. Severe amyelination in nmf417-mutant nerve roots suggested complete laminin 2 inactivation for Schwann cells, although myelinated fibers had normal BLs. The results reveal crucial roles for the LN domain CxxC motif in both nerve and muscle, but challenge expected relationships between LN-domain function, Ln2 activity and BL stability. The nmf417 mutation provides a defined animal model in which to investigate mechanisms and treatments for moderate forms of MDC1A.

Binding of Netrin-4 to Laminin Short Arms Regulates Basement Membrane Assembly

Netrins were first identified as neural guidance molecules, acting through receptors that are members of the DCC and UNC-5 family. All netrins share structural homology to the laminin N-terminal domains and the laminin epidermal growth factor-like domains of laminin short arms. Laminins use these domains to self-assemble into complex networks. Here we demonstrate that netrin-4 is a component of basement membranes and is integrated into the laminin polymer via interactions with the laminin gamma1 andgamma3 short arms. The binding is mediated through the laminin N-terminal domain of netrin-4. In contrast to netrin-4, other members of the netrin family do not bind to these laminin short arms. Moreover, a truncated form of netrin-4 completely inhibits laminin-111 self-assembly in vitro, and full-length netrin-4 can partially disrupt laminin self-interactions. When added to explant cultures, netrin-4 retards salivary gland branching morphogenesis.

Molecular Analysis of Laminin N-terminal Domains Mediating Self-interactions

The ability of laminins to self-polymerize is crucial for the formation of basement membranes. Development of this polymerized network has profound effects upon tissue architecture as well as on the intracellular organization and differentiation of neighboring cells. The laminin N-terminal (LN) domains have been shown to mediate this interaction and studies using proteolytic fragments derived from laminin-1 led to the theory that network assembly depends on the formation of a heterotrimeric complex between LN domains derived from alpha, beta, and gamma chains in different laminin molecules with homologous interactions being insignificant. The laminin family consists of 15 known isoforms formed from five alpha, three beta, and three gamma chains, of which some are truncated and lack the N-terminal LN domain. To address whether the model of heterotrimeric complex formation is applicable to laminin isoforms other than laminin-1, eight LN domains found in the laminin protein family were recombinantly expressed and tested in three different assays for homologous and heterologous interactions. The results showed that the lack of homologous interactions is an exception, with such interactions being seen for LN domains derived from all alpha chains and from the beta2 and beta3 subunits. The gamma chain-derived LN domains showed a far more limited binding repertoire, particularly in the case of the gamma3 chain, which is found present in a range of non-basement membrane locations. Further, whereas the interactions depended upon Ca2+ ions, with EDTA reversibly abrogating binding, EDTA-induced conformational changes were not reversible. Together these results demonstrate that the assembly model proposed on the basis of laminin-1 may be a simplification, with the assembly of naturally occurring laminin networks being far more complex and highly dependent upon which laminin isoforms are present.

Identification of a novel alternative splicing form of human netrin-4 and analyzing the expression patterns in adult rat brain

Netrins are a family of secreted proteins that function as tropic cues directing axon growth and cell migration during neural development. Beta-netrin or netrin-4 (NTN4), the fourth member of this family, was previously reported by Koch (M. Koch, J.R. Murrell, D.D. Hunter, P.F. Olson, W. Jin, D.R. Keene, W.J. Brunken, R.E. Burgeson. A novel member of the netrin family, beta-netrin, shares homology with the beta chain of laminin: identification, expression, and functional characterization. J. Cell Biol. 151 (2000) 221–234) and Yin (Y. Yin, J.R. Sanes, J.H. Miner, Identification and expression of mouse netrin-4. Mech. Dev. 96 (2000) 115–119), respectively. However, whether there is another isoform of netrin-4 and the expression patterns have not been fully determined. Here we report the cloning of a novel isoform of human netrin-4 using rapid amplification of complementary DNA (cDNA) ends (RACE) technique and analysis on the netrin-4 mRNA expression in adult rat brain using in situ hybridization. The new isoform lacks the signal peptide region and might represent a truncated form at the Laminin-N terminal domain of human netrin-4. Examination of the expression pattern of netrin-4 mRNA in adult rat brain revealed that the netrin-4 mRNA was widely expressed in many regions of the brain. High levels of netrin-4 mRNA was found in the pyramidal cell layer of the cerebral cortex, prepiriform cortex, amygdaloid nuclei, pyramidal layer of hippocampus, Purkinje's cells, medial cerebellar nucleus and interposed cerebellar nucleus, medial nucleus of the trapezoid body and mitral cell layer of the olfactory bulb. Moderate signals were present in frontal cortex, several thalamic and hypothalamic nuclei. The widespread expression of netrin-4 in adult rat brain indicates that netrin-4 is not only important for axon guidance during embryonic development, but also critical to neuronal plasticity in adult stage.