Distinct roles for laminin globular domains in laminin alpha1 chain mediated rescue of murine laminin alpha2 chain deficiency.
ABSTRACT: BACKGROUND: Laminin alpha2 chain mutations cause congenital muscular dystrophy with dysmyelination neuropathy (MDC1A). Previously, we demonstrated that laminin alpha1 chain ameliorates the disease in mice. Dystroglycan and integrins are major laminin receptors. Unlike laminin alpha2 chain, alpha1 chain binds the receptors by separate domains; laminin globular (LG) domains 4 and LG1-3, respectively. Thus, the laminin alpha1 chain is an excellent tool to distinguish between the roles of dystroglycan and integrins in the neuromuscular system. METHODOLOGY/PRINCIPAL FINDINGS: Here, we provide insights into the functions of laminin alpha1LG domains and the division of their roles in MDC1A pathogenesis and rescue. Overexpression of laminin alpha1 chain that lacks the dystroglycan binding LG4-5 domains in alpha2 chain deficient mice resulted in prolonged lifespan and improved health. Importantly, diaphragm and heart muscles were corrected, whereas limb muscles were dystrophic, indicating that different muscles have different requirements for LG4-5 domains. Furthermore, the regenerative capacity of the skeletal muscle did not depend on laminin alpha1LG4-5. However, this domain was crucial for preventing apoptosis in limb muscles, essential for myelination in peripheral nerve and important for basement membrane assembly. CONCLUSIONS/SIGNIFICANCE: These results show that laminin alpha1LG domains and consequently their receptors have disparate functions in the neuromuscular system. Understanding these interactions could contribute to design and optimization of future medical treatment for MDC1A patients.
Project description:The laminins are large heterotrimeric glycoproteins with fundamental roles in basement membrane architecture and function. The C-terminus of the laminin alpha chain contains a tandem of five laminin G-like (LG) domains. We report the 2.0 A crystal structure of the laminin alpha2 LG4-LG5 domain pair, which harbours binding sites for heparin and the cell surface receptor alpha-dystroglycan, and is 41% identical to the laminin alpha1 E3 fragment. LG4 and LG5 are arranged in a V-shaped fashion related by a 110 degrees rotation about an axis passing near the domain termini. An extended N-terminal segment is disulfide bonded to LG5 and stabilizes the domain pair. Two calcium ions, one each in LG4 and LG5, are located 65 A apart at the tips of the domains opposite the polypeptide termini. An extensive basic surface region between the calcium sites is proposed to bind alpha-dystroglycan and heparin. The LG4-LG5 structure was used to construct a model of the laminin LG1-LG5 tandem and interpret missense mutations underlying protein S deficiency.
Project description:The biological activities of the laminin alpha2 chain LG4-5 module result from interactions with cell surface receptors, such as heparan sulfate proteoglycans and alpha-dystroglycan. In this study, heparin and alpha-dystroglycan binding sequences were identified using 42 overlapping synthetic peptides from the LG4-5 module and using recombinant LG4-5 protein (rec-alpha2LG4-5). Physiological activities of the active peptides were also examined in explants of submandibular glands. Heparin binding screens showed that the A2G78 peptide (GLLFYMARINHA) bound to heparin and prevented its binding to rec-alpha2LG4-5. Furthermore, alanine substitution of the arginine residue in the A2G78 site on rec-alpha2LG4-5 decreased heparin binding activity. When alpha-dystroglycan binding of the peptides was screened, two peptides, A2G78 and A2G80 (VQLRNGFPYFSY), bound alpha-dystroglycan. A2G78 and A2G80 also inhibited alpha-dystroglycan binding of rec-alpha2LG4-5. A2G78 and A2G80 specifically inhibited end bud formation of submandibular glands in culture. These results suggest that the A2G78 and A2G80 sites play functional roles as heparan sulfate- and alpha-dystroglycan-binding sites in the module. These peptides are useful for elucidating molecular mechanisms of heparan sulfate- and/or alpha-dystroglycan-mediated biological functions of the laminin alpha2 chain.
Project description:The laminin G-like (LG) domains of laminin-111, a glycoprotein widely expressed during embryogenesis, provide cell anchoring and receptor binding sites that are involved in basement membrane assembly and cell signaling. We now report the crystal structure of the laminin alpha1LG4-5 domains and provide a mutational analysis of heparin, alpha-dystroglycan, and galactosylsulfatide binding. The two domains of alpha1LG4-5 are arranged in a V-shaped fashion similar to that observed with laminin alpha2 LG4-5 but with a substantially different interdomain angle. Recombinant alpha1LG4-5 binding to heparin, alpha-dystroglycan, and sulfatides was dependent upon both shared and unique contributions from basic residues distributed in several clusters on the surface of LG4. For heparin, the greatest contribution was detected from two clusters, 2719RKR and 2791KRK. Binding to alpha-dystroglycan was particularly dependent on basic residues within 2719RKR, 2831RAR, and 2858KDR. Binding to galactosylsulfatide was most affected by mutations in 2831RAR and 2766KGRTK but not in 2719RKR. The combined analysis of structure and activities reveal differences in LG domain interactions that should enable dissection of biological roles of different laminin ligands.
Project description:The severely debilitating disease Congenital Muscular Dystrophy Type 1A (MDC1A) is caused by mutations in the gene encoding laminin-alpha2. Bax-mediated muscle cell death is a significant contributor to the severe neuromuscular pathology seen in the Lama2-null mouse model of MDC1A. To extend our understanding of pathogenesis due to laminin-alpha2-deficiency, we have now analyzed molecular mechanisms of Bax regulation in normal and laminin-alpha2-deficient muscles and cells, including myogenic cells obtained from patients with a clinical diagnosis of MDC1A. In mouse myogenic cells, we found that, as in non-muscle cells, Bax co-immunoprecipitated with the multifunctional protein Ku70. In addition, cell permeable pentapeptides designed from Ku70, termed Bax-inhibiting peptides (BIPs), inhibited staurosporine-induced Bax translocation and cell death in mouse myogenic cells. We also found that acetylation of Ku70, which can inhibit binding to Bax and can be an indicator of increased susceptibility to cell death, was more abundant in Lama2-null than in normal mouse muscles. Furthermore, myotubes formed in culture from human laminin-alpha2-deficient patient myoblasts produced high levels of activated caspase-3 when grown on poly-L-lysine, but not when grown on a laminin-alpha2-containing substrate or when treated with BIPs. Finally, cytoplasmic Ku70 in human laminin-alpha2-deficient myotubes was both reduced in amount and more highly acetylated than in normal myotubes. Increased susceptibility to cell death thus appears to be an intrinsic property of human laminin-alpha2-deficient myotubes. These results identify Ku70 as a regulator of Bax-mediated pathogenesis and a therapeutic target in laminin-alpha2-deficiency.
Project description:Laminin-211 is a cell-adhesion molecule that is strongly expressed in the basement membrane of skeletal muscle. By binding to the cell surface receptors dystroglycan and integrin ?7?1, laminin-211 is believed to protect the muscle fiber from damage under the constant stress of contractions, and to influence signal transmission events. The importance of laminin-211 in skeletal muscle is evident from merosin-deficient congenital muscular dystrophy type 1A (MDC1A), in which absence of the ?2 chain of laminin-211 leads to skeletal muscle dysfunction. MDC1A is the commonest form of congenital muscular dystrophy in the European population. Severe hypotonia, progressive muscle weakness and wasting, joint contractures and consequent impeded motion characterize this incurable disorder, which causes great difficulty in daily life and often leads to premature death. Mice with laminin ?2 chain deficiency have analogous phenotypes, and are reliable models for studies of disease mechanisms and potential therapeutic approaches. In this review, we introduce laminin-211 and describe its structure, expression pattern in developing and adult muscle and its receptor interactions. We will also discuss the molecular pathogenesis of MDC1A and advances toward the development of treatment.
Project description:The congenital muscular dystrophies (CMD) are a heterogeneous group of autosomal recessive disorders presenting in infancy with muscle weakness, contractures, and dystrophic changes on skeletal-muscle biopsy. Structural brain defects, with or without mental retardation, are additional features of several CMD syndromes. Approximately 40% of patients with CMD have a primary deficiency (MDC1A) of the laminin alpha2 chain of merosin (laminin-2) due to mutations in the LAMA2 gene. In addition, a secondary deficiency of laminin alpha2 is apparent in some CMD syndromes, including MDC1B, which is mapped to chromosome 1q42, and both muscle-eye-brain disease (MEB) and Fukuyama CMD (FCMD), two forms with severe brain involvement. The FCMD gene encodes a protein of unknown function, fukutin, though sequence analysis predicts it to be a phosphoryl-ligand transferase. Here we identify the gene for a new member of the fukutin protein family (fukutin related protein [FKRP]), mapping to human chromosome 19q13.3. We report the genomic organization of the FKRP gene and its pattern of tissue expression. Mutations in the FKRP gene have been identified in seven families with CMD characterized by disease onset in the first weeks of life and a severe phenotype with inability to walk, muscle hypertrophy, marked elevation of serum creatine kinase, and normal brain structure and function. Affected individuals had a secondary deficiency of laminin alpha2 expression. In addition, they had both a marked decrease in immunostaining of muscle alpha-dystroglycan and a reduction in its molecular weight on western blot analysis. We suggest these abnormalities of alpha-dystroglycan are caused by its defective glycosylation and are integral to the pathology seen in MDC1C.
Project description:Nodes of Ranvier are specialized axonal domains, at which voltage-gated sodium channels cluster. How axons cluster molecules in discrete domains is mostly unknown. Both axons and glia probably provide constraining mechanisms that contribute to domain formation. Proper sodium channel clustering in peripheral nerves depends on contact from Schwann cell microvilli, where at least one molecule, gliomedin, binds the sodium channel complex and induces its clustering. Furthermore, mice lacking Schwann cell dystroglycan have aberrant microvilli and poorly clustered sodium channels. Dystroglycan could interact at the basal lamina or at the axonglial surface. Because dystroglycan is a laminin receptor, and laminin 2 mutations [merosin-deficient congenital muscular dystrophy (MDC1A)] cause reduced nerve conduction velocity, we asked whether laminins are involved. Here, we show that the composition of both laminins and the dystroglycan complex at nodes differs from that of internodes. Mice defective in laminin 2 have poorly formed microvilli and abnormal sodium clusters. These abnormalities are similar, albeit less severe, than those of mice lacking dystroglycan. However, mice lacking all Schwann cell laminins show severe nodal abnormalities, suggesting that other laminins compensate for the lack of laminin 2. Thus, although laminins are located at a distance from the axoglial junction, they are required for proper clustering of sodium channels. Laminins, through their specific nodal receptors and cytoskeletal linkages, may participate in the formation of mechanisms that constrain clusters at nodes. Finally, abnormal sodium channel clusters are present in a patient with MDC1A, providing a molecular basis for the reduced nerve conduction velocity in this disorder.
Project description:Dystroglycan is a highly glycosylated extracellular matrix receptor with essential functions in skeletal muscle and the nervous system. Reduced matrix binding by ?-dystroglycan (?-DG) due to perturbed glycosylation is a pathological feature of several forms of muscular dystrophy. Like-acetylglucosaminyltransferase (LARGE) synthesizes the matrix-binding heteropolysaccharide [-glucuronic acid-?1,3-xylose-?1,3-]n. Using a dual exoglycosidase digestion, we confirm that this polysaccharide is present on native ?-DG from skeletal muscle. The atomic details of matrix binding were revealed by a high-resolution crystal structure of laminin-G-like (LG) domains 4 and 5 (LG4 and LG5) of laminin-?2 bound to a LARGE-synthesized oligosaccharide. A single glucuronic acid-?1,3-xylose disaccharide repeat straddles a Ca(2+) ion in the LG4 domain, with oxygen atoms from both sugars replacing Ca(2+)-bound water molecules. The chelating binding mode accounts for the high affinity of this protein-carbohydrate interaction. These results reveal a previously uncharacterized mechanism of carbohydrate recognition and provide a structural framework for elucidating the mechanisms underlying muscular dystrophy.
Project description:Congenital muscular dystrophy with laminin ?2 chain deficiency (MDC1A) is one of the most severe forms of muscular disease and is characterized by severe muscle weakness and delayed motor milestones. The genetic basis of MDC1A is well known, yet the secondary mechanisms ultimately leading to muscle degeneration and subsequent connective tissue infiltration are not fully understood. In order to obtain new insights into the molecular mechanisms underlying MDC1A, we performed a comparative proteomic analysis of affected muscles (diaphragm and gastrocnemius) from laminin ?2 chain-deficient dy(3K)/dy(3K) mice, using multidimensional protein identification technology combined with tandem mass tags. Out of the approximately 700 identified proteins, 113 and 101 proteins, respectively, were differentially expressed in the diseased gastrocnemius and diaphragm muscles compared with normal muscles. A large portion of these proteins are involved in different metabolic processes, bind calcium, or are expressed in the extracellular matrix. Our findings suggest that metabolic alterations and calcium dysregulation could be novel mechanisms that underlie MDC1A and might be targets that should be explored for therapy. Also, detailed knowledge of the composition of fibrotic tissue, rich in extracellular matrix proteins, in laminin ?2 chain-deficient muscle might help in the design of future anti-fibrotic treatments. All MS data have been deposited in the ProteomeXchange with identifier PXD000978 (http://proteomecentral.proteomexchange.org/dataset/PXD000978).
Project description:We report the molecular and functional characterization of a new alpha chain of laminin in Drosophila. The new laminin chain appears to be the Drosophila counterpart of both vertebrate alpha2 (also called merosin) and alpha1 chains, with a slightly higher degree of homology to alpha2, suggesting that this chain is an ancestral version of both alpha1 and alpha2 chains. During embryogenesis, the protein is associated with basement membranes of the digestive system and muscle attachment sites, and during larval stage it is found in a specific pattern in wing and eye discs. The gene is assigned to a locus called wing blister (wb), which is essential for embryonic viability. Embryonic phenotypes include twisted germbands and fewer pericardial cells, resulting in gaps in the presumptive heart and tracheal trunks, and myotubes detached from their target muscle attachment sites. Most phenotypes are in common with those observed in Drosophila laminin alpha3, 5 mutant embryos and many are in common with those observed in integrin mutations. Adult phenotypes show blisters in the wings in viable allelic combinations, similar to phenotypes observed in integrin genes. Mutation analysis in the eye demonstrates a function in rhabdomere organization. In summary, this new laminin alpha chain is essential for embryonic viability and is involved in processes requiring cell migration and cell adhesion.