RESUMEN
Laminin alpha 5 (LAMA5) is a member of a large family of proteins that trimerise and then polymerise to form a central component of all basement membranes. Consequently, the protein plays an instrumental role in shaping the normal development of the kidney, skin, neural tube, lung and limb, and many other organs and tissues. Pathogenic mutations in some laminins have been shown to cause a range of largely syndromic conditions affecting the competency of the basement membranes to which they contribute. We report the identification of a mutation in the polymerisation domain of LAMA5 in a patient with a complex syndromic disease characterised by defects in kidney, craniofacial and limb development, and by a range of other congenital defects. Using CRISPR-generated mouse models and biochemical assays, we demonstrate the pathogenicity of this variant, showing that the change results in a failure of the polymerisation of α/ß/γ laminin trimers. Comparing these in vivo phenotypes with those apparent upon gene deletion in mice provides insights into the specific functional importance of laminin polymerisation during development and tissue homeostasis.
Asunto(s)
Discapacidades del Desarrollo/genética , Desarrollo Fetal , Laminina/genética , Mutación/genética , Polimerizacion , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Preescolar , Discapacidades del Desarrollo/patología , Feto/embriología , Humanos , Hidronefrosis/patología , Recién Nacido , Riñón/anomalías , Riñón/embriología , Riñón/patología , Laminina/química , Pulmón/anomalías , Pulmón/embriología , Pulmón/patología , Masculino , Ratones , Dominios Proteicos , SíndromeRESUMEN
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 ß2 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 in vitro 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.
Asunto(s)
Síndrome Nefrótico , Trastornos de la Pupila , Animales , Laminina/genética , Ratones , Ratones Noqueados , Síndrome Nefrótico/genética , Trastornos de la Pupila/genéticaRESUMEN
Pierson syndrome is a congenital nephrotic syndrome with eye and neurologic defects caused by mutations in laminin ß2 (LAMB2), a major component of the glomerular basement membrane (GBM). Pathogenic missense mutations in human LAMB2 cluster in or near the laminin amino-terminal (LN) domain, a domain required for extracellular polymerization of laminin trimers and basement membrane scaffolding. Here, we investigated an LN domain missense mutation, LAMB2-S80R, which was discovered in a patient with Pierson syndrome and unusually late onset of proteinuria. Biochemical data indicated that this mutation impairs laminin polymerization, which we hypothesized to be the cause of the patient's nephrotic syndrome. Testing this hypothesis in genetically altered mice showed that the corresponding amino acid change (LAMB2-S83R) alone is not pathogenic. However, expression of LAMB2-S83R significantly increased the rate of progression to kidney failure in a Col4a3-/- mouse model of autosomal recessive Alport syndrome and increased proteinuria in Col4a5+/- females that exhibit a mild form of X-linked Alport syndrome due to mosaic deposition of collagen α3α4α5(IV) in the GBM. Collectively, these data show the pathogenicity of LAMB2-S80R and provide the first evidence of genetic modification of Alport phenotypes by variation in another GBM component. This finding could help explain the wide range of Alport syndrome onset and severity observed in patients with Alport syndrome, even for family members who share the same COL4 mutation. Our results also show the complexities of using model organisms to investigate genetic variants suspected of being pathogenic in humans.
Asunto(s)
Anomalías Múltiples/genética , Anomalías del Ojo/genética , Fallo Renal Crónico/genética , Laminina/genética , Nefritis Hereditaria/genética , Nefritis Hereditaria/metabolismo , Síndrome Nefrótico/genética , Proteinuria/genética , Trastornos de la Pupila/genética , Animales , Autoantígenos/genética , Colágeno Tipo IV/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Anomalías del Ojo/complicaciones , Femenino , Membrana Basal Glomerular/metabolismo , Humanos , Laminina/metabolismo , Ratones , Ratones Transgénicos , Mutación Missense , Síndromes Miasténicos Congénitos , Nefritis Hereditaria/patología , Síndrome Nefrótico/complicaciones , Fenotipo , Polimerizacion , Trastornos de la Pupila/complicacionesRESUMEN
During development, Schwann cells extend lamellipodia-like processes to segregate large- and small-caliber axons during the process of radial sorting. Radial sorting is a prerequisite for myelination and is arrested in human neuropathies because of laminin deficiency. Experiments in mice using targeted mutagenesis have confirmed that laminins 211, 411, and receptors containing the ß1 integrin subunit are required for radial sorting; however, which of the 11 α integrins that can pair with ß1 forms the functional receptor is unknown. Here we conditionally deleted all the α subunits that form predominant laminin-binding ß1 integrins in Schwann cells and show that only α6ß1 and α7ß1 integrins are required and that α7ß1 compensates for the absence of α6ß1 during development. The absence of either α7ß1 or α6ß1 integrin impairs the ability of Schwann cells to spread and to bind laminin 211 or 411, potentially explaining the failure to extend cytoplasmic processes around axons to sort them. However, double α6/α7 integrin mutants show only a subset of the abnormalities found in mutants lacking all ß1 integrins, and a milder phenotype. Double-mutant Schwann cells can properly activate all the major signaling pathways associated with radial sorting and show normal Schwann cell proliferation and survival. Thus, α6ß1 and α7ß1 are the laminin-binding integrins required for axonal sorting, but other Schwann cell ß1 integrins, possibly those that do not bind laminins, may also contribute to radial sorting during peripheral nerve development.
Asunto(s)
Axones/fisiología , Integrina alfa6beta1/fisiología , Integrinas/fisiología , Células de Schwann/fisiología , Animales , Animales Recién Nacidos , Axones/ultraestructura , Proliferación Celular , Células Cultivadas , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Células de Schwann/ultraestructuraRESUMEN
Laminins promote early stages of peripheral nerve myelination by assembling basement membranes (BMs) on Schwann cell surfaces, leading to activation of ß1 integrins and other receptors. The BM composition, structural bonds and ligands needed to mediate this process, however, are not well understood. Mice hypomorphic for laminin γ1-subunit expression that assembled endoneurial BMs with reduced component density exhibited an axonal sorting defect with amyelination but normal Schwann cell proliferation, the latter unlike the null. To identify the basis for this, and to dissect participating laminin interactions, LAMC1 gene-inactivated dorsal root ganglia were treated with recombinant laminin-211 and -111 lacking different architecture-forming and receptor-binding activities, to induce myelination. Myelin-wrapping of axons by Schwann cells was found to require higher laminin concentrations than either proliferation or axonal ensheathment. Laminins that were unable to polymerize through deletions that removed critical N-terminal (LN) domains, or that lacked cell-adhesive globular (LG) domains, caused reduced BMs and almost no myelination. Laminins engineered to bind weakly to α6ß1 and/or α7ß1 integrins through their LG domains, even though they could effectively assemble BMs, decreased myelination. Proliferation depended upon both integrin binding to LG domains and polymerization. Collectively these findings reveal that laminins integrate scaffold-forming and cell-adhesion activities to assemble an endoneurial BM, with myelination and proliferation requiring additional α6ß1/α7ß1-laminin LG domain interactions, and that a high BM ligand/structural density is needed for efficient myelination.
Asunto(s)
Laminina/metabolismo , Vaina de Mielina/metabolismo , Células de Schwann/patología , Animales , Animales Recién Nacidos , Membrana Basal/efectos de los fármacos , Membrana Basal/metabolismo , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Ganglios Espinales/ultraestructura , Células HEK293 , Humanos , Integrinas/metabolismo , Laminina/química , Laminina/genética , Ratones , Atrofia Muscular/metabolismo , Atrofia Muscular/patología , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patología , Unión Proteica/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Proteínas Recombinantes/farmacología , Células de Schwann/efectos de los fármacos , Nervio Ciático/efectos de los fármacos , Nervio Ciático/metabolismo , Nervio Ciático/patologíaRESUMEN
In order to understand the functions of laminins in the renal collecting system, the Lamc1 gene was inactivated in the developing mouse ureteric bud (UB). Embryos bearing null alleles exhibited laminin deficiency prior to mesenchymal tubular induction and either failed to develop a UB with involution of the mesenchyme, or developed small kidneys with decreased proliferation and branching, delayed renal vesicle formation and postnatal emergence of a water transport deficit. Embryonic day 12.5 kidneys revealed an almost complete absence of basement membrane proteins and reduced levels of α6 integrin and FGF2. mRNA levels for fibroblast growth factor 2 (FGF2) and mediators of the GDNF/RET and WNT11 signaling pathway were also decreased. Furthermore, collecting duct cells derived from laminin-deficient kidneys and grown in collagen gels were found to proliferate and branch slowly. The laminin-deficient cells exhibited decreased activation of growth factor- and integrin-dependent pathways, whereas heparin lyase-treated and ß1 integrin-null cells exhibited more selective decreases. Collectively, these data support a requirement of γ1 laminins for assembly of the collecting duct system basement membrane, in which immobilized ligands act as solid-phase agonists to promote branching morphogenesis, growth and water transport functions.
Asunto(s)
Túbulos Renales Colectores/embriología , Túbulos Renales Colectores/metabolismo , Laminina/metabolismo , Animales , Membrana Basal/embriología , Membrana Basal/metabolismo , Diabetes Insípida/embriología , Diabetes Insípida/genética , Diabetes Insípida/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Sustancias de Crecimiento/metabolismo , Heparitina Sulfato/metabolismo , Hidronefrosis/embriología , Hidronefrosis/genética , Hidronefrosis/metabolismo , Integrina beta1/metabolismo , Túbulos Renales Colectores/anomalías , Laminina/deficiencia , Laminina/genética , Masculino , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Morfogénesis , Embarazo , Transducción de SeñalRESUMEN
Null mutations of the Lama2-gene cause a severe congenital muscular dystrophy and associated neuropathy. In the absence of laminin-α2 (Lmα2) there is a compensatory replacement by Lmα4, a subunit that lacks the polymerization and α-dystroglycan (αDG)-binding properties of Lmα2. The dystrophic phenotype in the dy3K/dy3K Lama2-/- mouse were evaluated with transgenes driving expression of two synthetic laminin-binding linker proteins. Transgenic muscle-specific expression of αLNNd, a chimeric protein that enables α4-laminin polymerization, and miniagrin (mag), a protein that increases laminin binding to the receptor αDG, separately improved median mouse survival two-fold. The double transgenes (DT) improved mean survival three-fold with increases in overall body weight, muscle size, and grip strength, but, given absence of neuronal expression, did not prevent hindlimb paresis. Muscle improvements included increased myofiber size and number and reduced fibrosis. Myofiber hypertrophy with increased mTOR and Akt phosphorylation were characteristics of mag-dy3K/dy3K and DT-dy3K/dy3K muscle. Elevations of matrix-bound α4-, ß1 and γ1 laminin subunits were detected in muscle extracts and immunostained sections in response to DT expression. Collectively, these findings reveal a complimentary polymerization and αDG-binding benefit to Lama2-/- mouse muscle largely mediated through modified laminin-411.
Asunto(s)
Músculo Esquelético , Distrofias Musculares , Ratones , Animales , Músculo Esquelético/metabolismo , Laminina/genética , Laminina/metabolismo , Ratones Transgénicos , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , TransgenesRESUMEN
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, ß1 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.
Asunto(s)
Síndrome Nefrótico , Trastornos de la Pupila , Humanos , Laminina/química , Microscopía por Crioelectrón , Polimerizacion , Membrana Basal/químicaRESUMEN
LAMA2 deficiency, resulting from a defective or absent laminin α2 subunit, is a common cause of congenital muscular dystrophy. It is characterized by muscle weakness from myofiber degeneration and neuropathy from Schwann cell amyelination. Previously it was shown that transgenic muscle-specific expression of αLNNd, a laminin γ1-binding linker protein that enables polymerization in defective laminins, selectively ameliorates the muscle abnormality in mouse disease models. Here, adeno-associated virus was used to deliver linker mini-genes to dystrophic dy2J/dy2J mice for expression of αLNNd in muscle, or αLNNdΔG2', a shortened linker, in muscle, nerve, and other tissues. Linker and laminin α2 levels were higher in αLNNdΔG2'-treated mice. Both αLNNd- and αLNNdΔG2'-treated mice exhibited increased forelimb grip strength. Further, αLNNdΔG2'-treated mice achieved hind limb and all-limb grip strength levels approaching those of WT mice as well as ablation of hind limb paresis and contractures. This was accompanied by restoration of sciatic nerve axonal envelopment and myelination. Improvement of muscle histology was evident in the muscle-specific αLNNd-expressing mice but more extensive in the αLNNdΔG2'-expressing mice. The results reveal that an αLN linker mini-gene, driven by a ubiquitous promoter, is superior to muscle-specific delivery because of its higher expression that extends to the peripheral nerve. These studies support a potentially novel approach of somatic gene therapy.
Asunto(s)
Distrofias Musculares , Distrofia Muscular Animal , Animales , Laminina/genética , Laminina/metabolismo , Ratones , Músculo Esquelético/metabolismo , Distrofias Musculares/genética , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patología , Nervios Periféricos/metabolismoRESUMEN
BACKGROUND: Unlike other proteins that exhibit a diffusion pattern after intracerebral injection, laminin displays a vascular pattern. It remains unclear if this unique vascular pattern is caused by laminin-receptor interaction or laminin self-assembly. METHODS: We compared the distribution of various wild-type laminin isoforms in the brain after intracerebral injection. To determine what causes the unique vascular pattern of laminin in the brain, laminin mutants with impaired receptor-binding and/or self-assembly activities and function-blocking antibodies to laminin receptors were used. In addition, the dynamics of laminin distribution and elimination were examined at multiple time points after intracerebral injection. RESULTS: We found that ß2-containing laminins had higher affinity for the vessels compared to ß1-containing laminins. In addition, laminin mutants lacking receptor-binding domains but not that lacking self-assembly capability showed substantially reduced vascular pattern. Consistent with this finding, dystroglycan (DAG1) function-blocking antibody significantly reduced the vascular pattern of wild-type laminin-111. Although failed to affect the vascular pattern when used alone, integrin-ß1 function-blocking antibody further decreased the vascular pattern when combined with DAG1 antibody. EDTA, which impaired laminini-DAG1 interaction by chelating Ca2+, also attenuated the vascular pattern. Immunohistochemistry revealed that laminins were predominantly located in the perivascular space in capillaries and venules/veins but not arterioles/arteries. The time-course study showed that laminin mutants with impaired receptor-engaging activity were more efficiently eliminated from the brain compared to their wild-type counterparts. Concordantly, significantly higher levels of mutant laminins were detected in the cerebral-spinal fluid (CSF). CONCLUSIONS: These findings suggest that intracerebrally injected laminins are enriched in the perivascular space in a receptor (DAG1/integrin)-dependent rather than self-assembly-dependent manner and eliminated from the brain mainly via the perivascular clearance system.
Asunto(s)
Distroglicanos , Laminina , Integrinas , Encéfalo , VenasRESUMEN
Laminin polymerization is a key step of basement membrane assembly that depends on the binding of α, ß and γ N-terminal LN domains to form a polymer node. Nodal assembly can be divided into two steps consisting of ß- and γ-LN dimerization followed by calcium-dependent addition of the α-LN domain. The assembly and structural organization of laminin-111 LN-LEa segments was examined by size-exclusion chromatography (SEC) and electron microscopy. Triskelion-like structures were observed in negatively-stained images of purified α1/ß1/γ1 LN-LEa trimers. Image averaging of these revealed a heel-to-toe organization of the LN domains with angled outward projections of the LEa stem-like domains. A series of single-amino acid substitutions was introduced into the polymerization faces of the α1, ß1 and γ1 LN domains followed by SEC analysis to distinguish between loss of ß-γ mediated dimerization and loss of α-dependent trimerization (with intact ß-γ dimers). Dimer-blocking mutations were confined to the γ1-toe and the ß1-heel, whereas the trimer-only-blocking mutations mapped to the γ1-heel, ß1-toe and the α1-toe and heel. Thus, in the polymer node the γ1-toe pairs with the ß1-heel, the ß1-toe pairs with the α1-heel, and the α1-toe pairs with the γ1-heel.
Asunto(s)
Laminina , Polímeros , Laminina/genética , Morfogénesis , MutaciónRESUMEN
Endoneurial laminins (Lms), beta1-integrins, and dystroglycan (DG) are important for Schwann cell (SC) ensheathment and myelination of axons. We now show that SC expression of galactosyl-sulfatide, a Lm-binding glycolipid, precedes that of Lms in developing nerves. This glycolipid anchors Lm-1 and -2 to SC surfaces by binding to their LG domains and enables basement membrane (BM) assembly. Revealingly, non-BM-forming fibroblasts become competent for BM assembly when sulfatides are intercalated into their cell surfaces. Assembly is characterized by coalescence of sulfatide, DG, and c-Src into a Lm-associated complex; by DG-dependent recruitment of utrophin and Src activation; and by integrin-dependent focal adhesion kinase phosphorylation. Collectively, our findings suggest that sulfated glycolipids are key Lm anchors that determine which cell surfaces can assemble Lms to initiate BM assembly and DG- and integrin-mediated signaling.
Asunto(s)
Fibroblastos/metabolismo , Integrinas/metabolismo , Laminina/metabolismo , Células de Schwann/metabolismo , Sulfoglicoesfingolípidos/metabolismo , Animales , Membrana Basal/metabolismo , Membrana Basal/ultraestructura , Caveolina 1 , Caveolinas/metabolismo , Células Cultivadas , Distroglicanos/metabolismo , Activación Enzimática , Fibroblastos/ultraestructura , Quinasa 2 de Adhesión Focal , Genes src/fisiología , Glucolípidos/metabolismo , Microscopía Electrónica de Transmisión , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Células de Schwann/ultraestructura , Nervio Ciático/metabolismo , Nervio Ciático/ultraestructura , Transducción de Señal/fisiología , Utrofina/metabolismoRESUMEN
An understanding of basement membrane (BM) assembly at a molecular level provides a foundation with which to develop repair strategies for diseases with defects of BM structure. As currently understood, laminins become anchored to cell surfaces through receptor-mediated interactions and polymerize. This provisional matrix binds to proteoglycans, nidogens and type IV collagen to form a mature BM. Identification of BM binding domains and their binding targets has enabled investigators to engineer proteins that link BM components to modify and improve their functions. This approach is illustrated by the development of two linker proteins to repair the LAMA2-deficient muscular dystrophy (LAMA2-MD). Dystrophy-causing mutations of the LAMA2 gene product (Lmα2) disrupt the BM molecular architecture, destabilizing it. In a mild ambulatory type of the dystrophy, α2LN mutations in laminin-211 prevents polymerization. In the more common and severe non-ambulatory type (MDC1A), an absent Lmα2 subunit is replaced by the naturally occurring Lmα4 subunit that is normally largely confined to the microvasculature. The compensatory laminin, however, is a poor substitute because it neither polymerizes nor binds adequately to the anchoring receptor α-dystroglycan. A chimeric laminin-binding protein called αLNNd enables laminins with defective or absent αLN domains to polymerize while another engineered protein, miniagrin (mag), promotes efficient α-dystroglycan receptor-binding in otherwise weakly adhesive laminins. Alone, αLNNd enables Lm211 with a self-assembly defect to polymerize and was used to ameliorate a mouse model of the ambulatory dystrophy. Together, these linker proteins alter Lm411 such that it both polymerizes and binds αDG such that it properly assembles. This combination was used to ameliorate a mouse model of the non-ambulatory dystrophy in which Lm411 replaced Lm211 as seen in the human disease. Collectively, these studies pave the way for the development of somatic gene delivery of repair proteins for treatment of LAMA2-MD. The studies further suggest a more general approach of linker-protein mediated repair in which a variety of existing BM protein domains can be combined together to stabilize BMs in other diseases.
RESUMEN
Laminin polymerization is a key step of basement membrane self-assembly that depends on the binding of the three different N-terminal globular LN domains. Several mutations in the LN domains cause LAMA2-deficient muscular dystrophy and LAMB2-deficient Pierson syndrome. These mutations may affect polymerization. A novel approach to identify the amino acid residues required for polymerization has been applied to an analysis of these and other laminin LN mutations. The approach utilizes laminin-nidogen chimeric fusion proteins that bind to recombinant non-polymerizing laminins to provide a missing functional LN domain. Single amino acid substitutions introduced into these chimeras were tested to determine if polymerization activity and the ability to assemble on cell surfaces were lost. Several laminin-deficient muscular dystrophy mutations, renal Pierson syndrome mutations, and Drosophila mutations causing defects of heart development were identified as ones causing loss of laminin polymerization. In addition, two novel residues required for polymerization were identified in the laminin γ1 LN domain.
Asunto(s)
Laminina/química , Laminina/genética , Glicoproteínas de Membrana/metabolismo , Mutación , Proteínas Recombinantes de Fusión/química , Anomalías Múltiples/genética , Secuencias de Aminoácidos , Animales , Membrana Basal , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Anomalías del Ojo/genética , Células HEK293 , Humanos , Laminina/metabolismo , Modelos Moleculares , Distrofias Musculares/genética , Síndromes Miasténicos Congénitos , Síndrome Nefrótico/genética , Unión Proteica , Multimerización de Proteína , Trastornos de la Pupila/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Laminins are large heterotrimers composed of the α, ß and γ subunits with distinct tissue-specific and developmentally regulated expression patterns. The laminin-α2 subunit, encoded by the LAMA2 gene, is expressed in skeletal muscle, Schwann cells of the peripheral nerve and astrocytes and pericytes of the capillaries in the brain. Mutations in LAMA2 cause the most common type of congenital muscular dystrophies, called LAMA2 MD or MDC1A. The disorder manifests mostly as a muscular dystrophy but slowing of nerve conduction contributes to the disease. There are severe, non-ambulatory or milder, ambulatory variants, the latter resulting from reduced laminin-α2 expression and/or deficient laminin-α2 function. Lm-211 (α2ß1γ1) is responsible for initiating basement membrane assembly. This is primarily accomplished by anchorage of Lm-211 to dystroglycan and α7ß1 integrin receptors, polymerization, and binding to nidogen and other structural components. In LAMA2 MD, Lm-411 replaces Lm-211; however, Lm-411 lacks the ability to polymerize and bind to receptors. This results in a weakened basement membrane leading to the disease. The possibility of introducing structural repair proteins that correct the underlying abnormality is an attractive therapeutic goal. Recent studies in mouse models for LAMA2 MD reveal that introduction of laminin-binding linker proteins that restore lost functional activities can substantially ameliorate the disease. This review discusses the underlying mechanism of this repair and compares this approach to other developing therapies employing pharmacological treatments.
Asunto(s)
Laminina/química , Laminina/deficiencia , Distrofias Musculares/genética , Animales , Membrana Basal/química , Membrana Basal/metabolismo , Distroglicanos/metabolismo , Humanos , Integrinas/metabolismo , Laminina/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Distrofias Musculares/metabolismo , Mutación , Unión ProteicaRESUMEN
Mutations in laminin α2-subunit (Lmα2, encoded by LAMA2) are linked to approximately 30% of congenital muscular dystrophy cases. Mice with a homozygous mutation in Lama2 (dy2J mice) express a nonpolymerizing form of laminin-211 (Lm211) and are a model for ambulatory-type Lmα2-deficient muscular dystrophy. Here, we developed transgenic dy2J mice with muscle-specific expression of αLNNd, a laminin/nidogen chimeric protein that provides a missing polymerization domain. Muscle-specific expression of αLNNd in dy2J mice resulted in strong amelioration of the dystrophic phenotype, manifested by the prevention of fibrosis and restoration of forelimb grip strength. αLNNd also restored myofiber shape, size, and numbers to control levels in dy2J mice. Laminin immunostaining and quantitation of tissue extractions revealed increased Lm211 expression in αLNNd-transgenic dy2J mice. In cultured myotubes, we determined that αLNNd expression increased myotube surface accumulation of polymerization-deficient recombinant laminins, with retention of collagen IV, reiterating the basement membrane (BM) changes observed in vivo. Laminin LN domain mutations linked to several of the Lmα2-deficient muscular dystrophies are predicted to compromise polymerization. The data herein support the hypothesis that engineered expression of αLNNd can overcome polymerization deficits to increase laminin, stabilize BM structure, and substantially ameliorate muscular dystrophy.
Asunto(s)
Laminina , Distrofia Muscular Animal , Mutación , Miofibrillas , Proteínas Recombinantes de Fusión , Animales , Células HEK293 , Humanos , Laminina/biosíntesis , Laminina/genética , Ratones Transgénicos , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patología , Miofibrillas/metabolismo , Miofibrillas/patología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genéticaRESUMEN
LAMA2-related muscular dystrophy (LAMA2 MD or MDC1A) is the most frequent form of early-onset, fatal congenital muscular dystrophies. It is caused by mutations in LAMA2, the gene encoding laminin-α2, the long arm of the heterotrimeric (α2, ß1, and γ1) basement membrane protein laminin-211 (Lm-211). We establish that despite compensatory expression of laminin-α4, giving rise to Lm-411 (α4, ß1, and γ1), muscle basement membrane is labile in LAMA2 MD biopsies. Consistent with this deficit, recombinant Lm-411 polymerized and bound to cultured myotubes only weakly. Polymerization and cell binding of Lm-411 were enhanced by addition of two specifically designed linker proteins. One, called αLNNd, consists of the N-terminal part of laminin-α1 and the laminin-binding site of nidogen-1. The second, called mini-agrin (mag), contains binding sites for laminins and α-dystroglycan. Transgenic expression of mag and αLNNd in a mouse model for LAMA2 MD fully restored basement membrane stability, recovered muscle force and size, increased overall body weight, and extended life span more than five times to a maximum survival beyond 2 years. These findings provide a mechanistic understanding of LAMA2 MD and establish a strong basis for a potential treatment.
Asunto(s)
Membrana Basal/metabolismo , Laminina/metabolismo , Distrofia Muscular Animal/metabolismo , Proteínas Recombinantes/metabolismo , Adolescente , Animales , Membrana Basal/patología , Peso Corporal , Niño , Preescolar , Humanos , Ratones Transgénicos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Distrofia Muscular Animal/patología , TransgenesRESUMEN
Laminins are a major constituent of the basement membranes of the kidney collecting system. Integrins, transmembrane receptors formed by non-covalently bound α and ß subunits, serve as laminin receptors, but their role in development and homeostasis of the kidney collecting system is poorly defined. Integrin α3ß1, one of the major laminin receptors, plays a minor role in kidney collecting system development, while the role of α6 containing integrins (α6ß1 and α6ß4), the other major laminin receptors, is unknown. Patients with mutations in α6 containing integrins not only develop epidermolysis bullosa, but also have abnormalities in the kidney collecting system. In this study, we show that selectively deleting the α6 or ß4 integrin subunits at the initiation of ureteric bud development in mice does not affect morphogenesis. However, the collecting system becomes dilated and dysmorphic as the mice age. The collecting system in both null genotypes was also highly susceptible to unilateral ureteric obstruction injury with evidence of excessive tubule dilatation and epithelial cell apoptosis. Mechanistically, integrin α6-null collecting duct cells are unable to withstand high mechanical force when adhered to laminin. Thus, we conclude that α6 integrins are important for maintaining the integrity of the kidney collecting system by enhancing tight adhesion of the epithelial cells to the basement membrane. These data give a mechanistic explanation for the association between kidney collecting system abnormalities in patients and epidermolysis bullosa.
Asunto(s)
Membrana Basal/metabolismo , Integrina alfa6beta1/genética , Integrina alfa6beta4/genética , Túbulos Renales Colectores/metabolismo , Laminina/genética , Obstrucción Ureteral/metabolismo , Animales , Apoptosis , Membrana Basal/patología , Adhesión Celular , Movimiento Celular , Proliferación Celular , Células Epiteliales/metabolismo , Células Epiteliales/patología , Fibrosis , Regulación de la Expresión Génica , Humanos , Integrina alfa6beta1/deficiencia , Integrina alfa6beta4/deficiencia , Túbulos Renales Colectores/patología , Laminina/metabolismo , Ratones , Ratones Noqueados , Unión Proteica , Transducción de Señal , Uréter/cirugía , Obstrucción Ureteral/patología , Obstrucción Ureteral/cirugíaRESUMEN
To understand the role of glycosaminoglycans in bacterial cellular invasion, xylosyltransferase-deficient mutants of Chinese hamster ovary (CHO) cells were created using clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-cas9) gene targeting. When these mutants were compared to the pgsA745 cell line, a CHO xylosyltransferase mutant generated previously using chemical mutagenesis, an unexpected result was obtained. Bacterial invasion of pgsA745 cells by group B Streptococcus (GBS), group A Streptococcus, and Staphylococcus aureus was markedly reduced compared to the invasion of wild-type cells, but newly generated CRISPR-cas9 mutants were only resistant to GBS. Invasion of pgsA745 cells was not restored by transfection with xylosyltransferase, suggesting that an additional mutation conferring panresistance to multiple bacteria was present in pgsA745 cells. Whole-genome sequencing and transcriptome sequencing (RNA-Seq) uncovered a deletion in the gene encoding the laminin subunit α2 (Lama2) that eliminated much of domain L4a. Silencing of the long Lama2 isoform in wild-type cells strongly reduced bacterial invasion, whereas transfection with human LAMA2 cDNA significantly enhanced invasion in pgsA745 cells. The addition of exogenous laminin-α2ß1γ1/laminin-α2ß2γ1 strongly increased bacterial invasion in CHO cells, as well as in human alveolar basal epithelial and human brain microvascular endothelial cells. Thus, the L4a domain in laminin α2 is important for cellular invasion by a number of bacterial pathogens. IMPORTANCE: Pathogenic bacteria penetrate host cellular barriers by attachment to extracellular matrix molecules, such as proteoglycans, laminins, and collagens, leading to invasion of epithelial and endothelial cells. Here, we show that cellular invasion by the human pathogens group B Streptococcus, group A Streptococcus, and Staphylococcus aureus depends on a specific domain of the laminin α2 subunit. This finding may provide new leads for the molecular pathogenesis of these bacteria and the development of novel antimicrobial drugs.
Asunto(s)
Endocitosis , Interacciones Huésped-Patógeno , Laminina/metabolismo , Staphylococcus aureus/fisiología , Streptococcus agalactiae/fisiología , Streptococcus pyogenes/fisiología , Animales , Células CHO , Cricetinae , Cricetulus , Eliminación de Gen , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Laminina/genética , Análisis de Secuencia de ADNRESUMEN
Netrins, a family of laminin-related molecules, have been proposed to act as guidance cues either during nervous system development or the establishment of the vascular system. This was clearly demonstrated for netrin-1 via its interaction with the receptors DCC and UNC5s. However, mainly based on shared homologies with netrin-1, netrin-4 was also proposed to play a role in neuronal outgrowth and developmental/pathological angiogenesis via interactions with netrin-1 receptors. Here, we present the high-resolution structure of netrin-4, which shows unique features in comparison with netrin-1, and show that it does not bind directly to any of the known netrin-1 receptors. We show that netrin-4 disrupts laminin networks and basement membranes (BMs) through high-affinity binding to the laminin γ1 chain. We hypothesize that this laminin-related function is essential for the previously described effects on axon growth promotion and angiogenesis. Our study unveils netrin-4 as a non-enzymatic extracellular matrix protein actively disrupting pre-existing BMs.