Immunogold localization of adhalin, alpha-dystroglycan and laminin in normal and dystrophic skeletal muscle.
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Antibodies↗
Biomedical subjects
S L Roberds
Publications and source records attributed to S L Roberds.
Clinical and molecular pathological features of severe childhood autosomal recessive muscular dystrophy in Saudi Arabia.
The clinical, biochemical and histochemical features of 14 patients (nine females and five males) with severe childhood autosomal recessive muscular dystrophy (SCARMD) seen at a tertiary hospital in Riyadh from 1982 to 1993 are described. Onset was at 3 to 9 (median 3) years and four of five children aged > 12 years lost ambulation. Five of the eight pairs of parents were closely consanguineous. The mean creatine kinase was 20 times the upper normal limit. Histochemistry of muscle showed dystrophic features in all cases, and dystrophin was positive in all cases examined (N = 6). Three patients (two girls and a boy) were deficient in adhalin, the 50-kDa dystorphin-associated glycoprotein. A boy aged 13 years had rapidly progressing disease. Another boy of the same age (from a family characterized by early onset and slower progression) had normal dystrophin and adhalin. The clinical features conformed with previous observations from Sudan, North Africa and Qatar in the Arabian Peninsula. The disease is common in Saudi Arabia and seems to be more prevalent than Duchenne muscular dystrophy.
Adolescent↗
Ultrastructural localization of adhalin, alpha-dystroglycan and merosin in normal and dystrophic muscle.
Adhalin and alpha-dystroglycan are two components of a complex of proteins that, in conjunction with dystrophin, provide a link between the subsarcolemmal cytoskeleton and the basal lamina of the extracellular matrix of skeletal muscle. In the absence of dystrophin, in Duchenne muscular dystrophy (DMD) and the mdx mouse, levels of adhalin, alpha-dystroglycan and other components of the complex, are severely reduced, and it has been speculated that this might be an important factor in precipitating myofibre necrosis. However, there is, as yet, little information on how these proteins interact structurally or functionally. From biochemical data it might be predicted that adhalin and alpha-dystroglycan are positioned more peripherally in the muscle cell than dystrophin and more proximal than merosin. Using single and double immunogold labelling we here show that adhalin is localized to the plasma membrane with the majority of the gold probe particles situated on the membrane's outer face, while alpha-dystroglycan labelling is seen on material which projects from the outer face and which, in places, forms strands that stretch to the basal lamina. When double labelling of laminin and alpha-dystroglycan is carried out, laminin is localized to the proximal face of the basal lamina, facing the alpha-dystroglycan. In DMD the labelling of adhalin and alpha-dystroglycan is severely reduced quantitatively (although the vestige that remains is positioned normally) but merosin is expressed normally, showing that its incorporation is independent of that of dystrophin and its associated proteins.
Antibodies, Monoclonal↗
Adhalin mRNA and cDNA sequence are normal in the cardiomyopathic hamster.
Adhalin is deficient in two forms of human muscular dystrophy, one due to mutations in the adhalin gene and one linked to an unidentified gene on chromosome 13. Because adhalin is deficient in skeletal and cardiac muscles of BIO 14.6 hamsters, which experience both myopathy and cardiomyopathy, cDNA encoding adhalin from BIO 14.6 hamster skeletal muscle was cloned and sequenced. Adhalin mRNA was expressed at normal levels in BIO 14.6 hamster cardiac muscle, and no mutation in adhalin coding sequence was found, indicating that the inherited myopathy and cardiomyopathy of the BIO 14.6 hamster are most likely not due to mutations in the adhalin gene.
Amino Acid Sequence↗
Rapsyn may function as a link between the acetylcholine receptor and the agrin-binding dystrophin-associated glycoprotein complex.
The 43 kDa AChR-associated protein rapsyn is required for the clustering of nicotinic acetylcholine receptors (AChRs) at the developing neuromuscular junction, but the functions of other postsynaptic proteins colocalized with the AChR are less clear. Here we use a fibroblast expression system to investigate the role of the dystrophin-glycoprotein complex (DGC) in AChR clustering. The agrin-binding component of the DGC, dystroglycan, is found evenly distributed across the cell surface when expressed in fibroblasts. However, dystroglycan colocalizes with AChR-rapsyn clusters when these proteins are coexpressed. Furthermore, dystroglycan colocalizes with rapsyn clusters even in the absence of AChR, indicating that rapsyn can cluster dystroglycan and AChR independently. Immunofluorescence staining using a polyclonal antibody to utrophin reveals a lack of staining of clusters, suggesting that the immunoreactive species, like the AChR, does not mediate the observed rapsyndystroglycan interaction. Rapsyn may therefore be a molecular link connecting the AChR to the DGC. At the neuromuscular synapse, rapsyn-mediated linkage of the AChR to the cytoskeleton-anchored DGC may underlie AChR cluster stabilization.
Agrin↗
Primary adhalinopathy: a common cause of autosomal recessive muscular dystrophy of variable severity.
Marked deficiency of muscle adhalin, a 50 kDa sarcolemmal dystrophin-associated glycoprotein, has been reported in severe childhood autosomal recessive muscular dystrophy (SCARMD). This is a Duchenne-like disease affecting both males and females first described in Tunisian families. Adhalin deficiency has been found in SCARMD patients from North Africa Europe, Brazil, Japan and North America (SLR & KPC, unpublished data). The disease was initially linked to an unidentified gene on chromosome 13 in families from North Africa, and to the adhalin gene itself on chromosome 17q in one French family in which missense mutations were identified. Thus there are two kinds of myopathies with adhalin deficiency: one with a primary defect of adhalin (primary adhalinopathies), and one in which absence of adhalin is secondary to a separate gene defect on chromosome 13. We have examined the importance of primary adhalinopathies among myopathies with adhalin deficiency, and describe several additional mutations (null and missense) in the adhalin gene in 10 new families from Europe and North Africa. Disease severity varies in age of onset and rate of progression, and patients with null mutations are the most severely affected.
Adolescent↗
Non-muscle alpha-dystroglycan is involved in epithelial development.
The dystroglycan complex is a transmembrane linkage between the cytoskeleton and the basement membrane in muscle. One of the components of the complex, alpha-dystroglycan binds both laminin of muscle (laminin-2) and agrin of muscle basement membranes. Dystroglycan has been detected in nonmuscle tissues as well, but the physiological role in nonmuscle tissues has remained unknown. Here we show that dystroglycan during mouse development in nonmuscle tissues is expressed in epithelium. In situ hybridization revealed strong expression of dystroglycan mRNA in all studied epithelial sheets, but not in endothelium or mesenchyme. Conversion of mesenchyme to epithelium occurs during kidney development, and the embryonic kidney was used to study the role of alpha-dystroglycan for epithelial differentiation. During in vitro culture of the metanephric mesenchyme, the first morphological signs of epithelial differentiation can be seen on day two. Northern blots revealed a clear increase in dystroglycan mRNA on day two of in vitro development. A similar increase of expression on day two was previously shown for laminin alpha 1 chain. Immunofluorescence showed that dystroglycan is strictly located on the basal side of developing kidney epithelial cells. Monoclonal antibodies known to block binding of alpha-dystroglycan to laminin-1 perturbed development of epithelium in kidney organ culture, whereas control antibodies did not do so. We suggest that the dystroglycan complex acts as a receptor for basement membrane components during epithelial morphogenesis. It is likely that this involves binding of alpha-dystroglycan to E3 fragment of laminin-1.
Animals↗
A common missense mutation in the adhalin gene in three unrelated Brazilian families with a relatively mild form of autosomal recessive limb-girdle muscular dystrophy.
Autosomal recessive limb-girdle muscular dystrophies (AR LGMD) represent a heterogeneous group of diseases with a wide spectrum of clinical variability, classified phenotypically into two main groups, the most severe forms (Duchenne-like muscular dystrophy, DLMD, or severe childhood autosomal recessive muscular dystrophy, SCARMD) and the milder forms. Four genes causing AR LGMD have been mapped: the 15q (LGMD2a), the 2p (LGMD2b), the 13q locus (LGMD2c) and the adhalin gene on chromosome 17q (LGMD2d). In the present report we have performed linkage analysis with 17q markers in three mild AR LGMD and in four DLMD families with adhalin deficiency and unlinked to 2p, 15q or 13q genes. Linkage was observed only among the mild cases. Patients from these three 17q-linked families showed near or total deficiency of adhalin in muscle biopsies. An identical missense mutation was identified in all three 17q-linked unrelated families. These results indicate that AR LGMD with a mild phenotype is caused by mutations in the adhalin gene. In addition, they demonstrate that there is at least one other locus for DLMD associated with adhalin deficiency.
Base Sequence↗
The expression of dystrophin-associated glycoproteins during skeletal muscle degeneration and regeneration. An immunofluorescence study.
The distribution and expression of dystrophin and three of the dystrophin-associated glycoproteins (DAG), alpha-dystroglycan (156 kDa DAG), beta-dystroglycan (43 kDa DAG) and adhalin (50 kDa DAG) in rat skeletal muscle were studied during a controlled cycle of degeneration and regeneration induced by the injection of a snake venom. Cryosections of muscle at various stages of degeneration and regeneration were labeled using monoclonal antibodies to the three glycoproteins and examined at fixed time points after venom injection. Adhalin and alpha-dystroglycan remained present at the sarcolemma throughout the entire cycle of degeneration and regeneration. beta-Dystroglycan, on the other hand, was lost from the sarcolemma by 12 hours and reappeared 2 days after venom injection when new muscle fibers were being formed. Dystrophin was not lost from the sarcolemma until 24 hours after venom injection and did not reappear at the membrane until 4 days. It is suggested that dystrophin and the glycoprotein complex are synthesized separately, both temporally and spatially, and only become associated at the plasma membrane during the later stages of regeneration. The expression of beta-dystroglycan in the regenerating muscle fibers was first seen at sites of newly forming plasma membrane that were closely associated with the old basal lamina tube. The basal lamina may therefore have a regulatory or modulatory role in the expression of the DAG.
Animals↗
Expression of deletion-containing dystrophins in mdx muscle: implications for gene therapy and dystrophin function.
The expression of full-length dystrophin and various dystrophin deletion mutants was monitored in mdx mouse muscle after intramuscular injection of dystrophin-encoding plasmid DNAs. Recombinant dystrophin proteins, including those lacking either the amino terminus, carboxyl terminus, or most of the central rod domain, showed localization to the plasma membrane. This suggests that there are multiple attachment sites for dystrophin to the plasma membrane. Only those constructs containing the carboxyl terminus were able to stabilize dystrophin-associated proteins (DAP) at the membrane, consistent with other studies that suggest that this domain is critical to DAP binding. Colocalization with DAP was not necessary for membrane localization of the various dystrophin molecules. However, stabilization and co-localization of the DAP did seem to be a prerequisite for expression and/or stabilization of mutant dystrophins beyond 1 wk and these same criteria seemed important for mitigating the histopathological consequences of dystrophin deficiency.
Animals↗
Laminin abnormality in severe childhood autosomal recessive muscular dystrophy.
BACKGROUND: In skeletal muscle, dystrophin exists in a large oligomeric complex tightly associated with several novel sarcolemmal proteins, including the 50-kDa transmembrane glycoprotein called adhalin. The dystrophin-glycoprotein complex links the subsarcolemmal actin cytoskeleton to the basal lamina component laminin, thus providing stability to the sarcolemma. Disturbance of this linkage due to the absence of dystrophin plays a crucial role in the molecular pathogenesis of muscle fiber necrosis in Duchenne muscular dystrophy. Severe childhood autosomal recessive muscular dystrophy (SCARMD) is similar to Duchenne muscular dystrophy in phenotype but is characterized by the deficiency of adhalin. At present, the status of the link between the dystrophin-glycoprotein complex and laminin is unclear in SCARMD. EXPERIMENTAL DESIGN: We investigated, by immunohistochemistry using confocal laser scanning microscopy, the status of the expression of laminin subunits, A, M, B1, B2, and S chains, in skeletal muscle biopsy specimens of eight SCARMD patients from various human populations. In addition, we correlated the severity of laminin abnormality with the severity of both clinical symptoms and histopathologic changes in these patients. RESULTS: The reduction of laminin B1 chain and the overexpression of the S chain, a homologue of B1, in the extrajunctional basal lamina were observed in the five patients who had advanced clinical symptoms and histopathologic changes. Abnormalities in the expression of laminin were not observed in the three less affected patients. CONCLUSIONS: The expression of laminin is greatly disturbed in severely diseased SCARMD muscle deficient in adhalin. Disturbance of sarcolemma-basal lamina interaction may play an important role in the molecular pathogenesis of muscle fiber necrosis in SCARMD.
Adolescent↗
Missense mutations in the adhalin gene linked to autosomal recessive muscular dystrophy.
Adhalin, the 50 kDa dystrophin-associated glycoprotein, is deficient in skeletal muscle of patients having severe childhood autosomal recessive muscular dystrophy (SCARMD). In several North African families, SCARMD has been linked to chromosome 13q, but SCARMD has been excluded from linkage to this locus in other families. We have now cloned human adhalin cDNA and mapped the adhalin gene to chromosome 17q12-q21.33, excluding it from involvement in 13q-linked SCARMD. However, one allelic variant of a polymorphic microsatellite located within intron 6 of the adhalin gene cosegregated perfectly with the disease phenotype in a large family. Furthermore, missense mutations were identified within the adhalin gene that might cause SCARMD in this family. Thus, the adhalin gene is involved in at least one form of autosomal recessive muscular dystrophy.
Amino Acid Sequence↗
Alpha-dystroglycan deficiency correlates with elevated serum creatine kinase and decreased muscle contraction tension in golden retriever muscular dystrophy.
The dystrophin-glycoprotein complex was examined in dystrophin-deficient dogs with golden retriever muscular dystrophy (GRMD) using immunoblot and immunofluorescence analysis. The dystrophin-associated proteins were substantially reduced in muscle from dogs with GRMD. Interestingly, regression analysis revealed a strong correlation between the amount of alpha-dystroglycan and serum creatine kinase levels and the contraction tension measured for a given peroneus longus muscle.
Animals↗
A role for dystrophin-associated glycoproteins and utrophin in agrin-induced AChR clustering.
Synapse formation is characterized by the accumulation of molecules at the site of contact between pre- and postsynaptic cells. Agrin, a protein implicated in the regulation of this process, causes the clustering of acetylcholine receptors (AChRs). Here we characterize an agrin-binding site on the surface of muscle cells, show that this site corresponds to alpha-dystroglycan, and present evidence that alpha-dystroglycan is functionally related to agrin activity. Furthermore, we demonstrate that alpha-dystroglycan and adhalin, components of the dystrophin-associated glycoprotein complex, as well as utrophin, colocalize with agrin-induced AChR clusters. Thus, agrin may function by initiating or stabilizing a synapse-specific membrane cytoskeleton that in turn serves as a scaffold upon which synaptic molecules are concentrated.
Agrin↗
Adhalin gene polymorphism.
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Base Sequence↗
Abnormal expression of laminin suggests disturbance of sarcolemma-extracellular matrix interaction in Japanese patients with autosomal recessive muscular dystrophy deficient in adhalin.
Dystrophin is associated with several novel sarcolemmal proteins, including a laminin-binding extracellular glycoprotein of 156 kD (alpha-dystroglycan) and a transmembrane glycoprotein of 50 kD (adhalin). Deficiency of adhalin characterizes a severe autosomal recessive muscular dystrophy prevalent in Arabs. Here we report for the first time two mongoloid (Japanese) patients with autosomal recessive muscular dystrophy deficient in adhalin. Interestingly, adhalin was not completely absent and was faintly detectable in a patchy distribution along the sarcolemma in our patients. Although the M and B2 subunits of laminin were preserved, the B1 subunit was greatly reduced in the basal lamina surrounding muscle fibers. Our results raise a possibility that the deficiency of adhalin may be associated with the disturbance of sarcolemma-extracellular matrix interaction leading to sarcolemmal instability.
Adult↗
Genetic heterogeneity of severe childhood autosomal recessive muscular dystrophy with adhalin (50 kDa dystrophin-associated glycoprotein) deficiency.
Severe autosomal recessive muscular dystrophy (SCARMD), McKusick n. 253700, has been originally described in North-African populations, in which significant linkage has been established with DNA markers mapping to the proximal region of the long arm of chromosome 13, without evidence for heterogeneity of the SCARMD locus in these populations. A striking feature of this disease is the isolated deficiency of adhalin, a sarcolemmal 50 kDa dystrophin-associated glycoprotein. We report a non-inbred French family with a milder progressive form of muscular dystrophy affecting subjects of both sexes. The parents are not affected suggesting an autosomal recessive transmission. In 4 siblings displaying mild to overt clinical signs of muscular dystrophy, serum creatine kinase was high, and muscle specimens showed variable degree of necrosis-regeneration with little fibrosis. In the 4 cases adhalin was completely absent in muscle sections, whereas dystrophin and the other members of the dystrophin-associated protein complex were normal, except for the 35 kDa dystrophin-associated glycoprotein which was decreased as usually observed in SCARMD. Linkage and homogeneity analysis using 4 microsatellite markers of chromosome 13q that are linked to the North-African SCARMD locus were performed in this family. Results show that the morbid locus involved in this family does not map to the same region as the SCARMD locus. This second locus may be involved in sporadic cases of muscular dystrophy with adhalin deficiency that have been reported in Europe.
Adolescent↗
Primary structure and muscle-specific expression of the 50-kDa dystrophin-associated glycoprotein (adhalin).
The 50-kDa dystrophin-associated glycoprotein (50-DAG) is a component of the dystrophin-glycoprotein complex, which links the muscle cytoskeleton to the extracellular matrix. 50-DAG is specifically deficient in skeletal muscle of patients with severe childhood autosomal recessive muscular dystrophy and in skeletal and cardiac muscles of BIO 14.6 cardiomyopathic hamsters. The lack of 50-DAG leads to a disruption and dysfunction of the dystrophin-glycoprotein complex in these diseases. The cDNA encoding 50-DAG has now been cloned from rabbit skeletal muscle. The 50-DAG deduced amino acid sequence predicts a novel protein having 387 amino acids, a 17-amino acid signal sequence, one transmembrane domain, and two potential sites of N-linked glycosylation. Affinity-purified antibodies against rabbit 50-DAG fusion proteins or synthetic peptides specifically recognized a 50-kDa protein in skeletal muscle sarcolemma and the 50-kDa component of the dystrophin-glycoprotein complex. In contrast to dystroglycan, which is expressed in a wide variety of muscle and non-muscle tissues, 50-DAG is expressed only in skeletal and cardiac muscles and in selected smooth muscles. Finally, 50-DAG mRNA is present in mdx and Duchenne muscular dystrophy (DMD) muscle, indicating that the down-regulation of this protein in DMD and the mdx mouse is likely a post-translational event.
Amino Acid Sequence↗