Rod Domain Of Dystrophin Research Articles

THE EARLY PHASE OF CHANGES IN DYSTROPHIN AFTER LENGTHENING CONTRACTIONS IN SKELETAL MUSCLE

Exercise-induced myofiber damage is rather a consequence of the mechanical stress than the number of contractions. The mechanical stress during lengthening contractions (LC) is mainly concentrated on the intermediate filament system, dystrophin, and specialized plasma membrane complexes. Disturbances in these structures might in turn render muscle fibers more susceptible to necrotic changes. PURPOSE: The present study was designed to evaluate the early phase of changes in proteins of the contractile system and sarcolemma after LC in rat skeletal muscle. METHODS: Tibialis anterior (TA) muscles of anesthetized rats were subjected to 240 LC. The contra lateral leg was used as a non-exercised control. Muscle samples were taken 0 h and 4 d post-exercise. Muscle β-glucuronidase activity (β-GU, a reliable indicator of muscle damage) was determined and antibodies against actin, dystrophin (C-terminus and rod domain), and β-dystroglycan were used for double immunohistochemical stainigs and were studied with confocal microscopy. RESULTS: The prominent increase in β-GU as well as a great number of necrotic fibers were observed at 4 d. Immediately after LC swollen fibers and dissappearence of actin staining with discontinuous staining of dystrophin c-terminus and fragmental β-dystroglycan staining was observed while in serial sections no changes in dystrophin rod domain were detected. CONCLUSION: These observations indicate the segmental sensitiveness of dystrophin molecule against physical stress and disturbed structural intergity in all studied proteins at the early phase after the LC. Supported by LIKES-Foundation and The Finnish Ministry of Education.

Utrophin Lacks the Rod Domain Actin Binding Activity of Dystrophin

We previously identified a cluster of basic spectrin-like repeats in the dystrophin rod domain that binds F-actin through electrostatic interactions (Amann, K. J., Renley, B. A., and Ervasti, J. M. (1998) J. Biol. Chem. 273, 28419-28423). Because of the importance of actin binding to the presumed physiological role of dystrophin, we sought to determine whether the autosomal homologue of dystrophin, utrophin, shared this rod domain actin binding activity. We therefore produced recombinant proteins representing the cluster of basic repeats of the dystrophin rod domain (DYSR11-17) or the homologous region of the utrophin rod domain (UTROR11-16). Although UTROR11-16 is 64% similar and 41% identical to DYSR11-17, UTROR11-16 (pI = 4. 86) lacks the basic character of the repeats found in DYSR11-17 (pI = 7.44). By circular dichroism, gel filtration, and sedimentation velocity analysis, we determined that each purified recombinant protein had adopted a stable, predominantly alpha-helical fold and existed as a highly soluble monomer. DYSR11-17 bound F-actin with an apparent K(d) of 7.3 +/- 1.3 microM and a molar stoichiometry of 1:5. Significantly, UTROR11-16 failed to bind F-actin at concentrations as high as 100 microM. We present these findings as further support for the electrostatic nature of the interaction of the dystrophin rod domain with F-actin and suggest that utrophin interacts with the cytoskeleton in a manner distinct from dystrophin.

A Cluster of Basic Repeats in the Dystrophin Rod Domain Binds F-actin through an Electrostatic Interaction

The dystrophin rod domain is composed of 24 spectrin-like repeats and was thought to act mainly as a flexible spacer between the amino-terminal actin binding domain and carboxyl-terminal membrane-associated domains. We previously demonstrated that a fragment of the dystrophin rod domain also binds F-actin. However, the nature and extent of rod domain association with F-actin is presently unclear. To begin addressing these questions, we characterized two recombinant proteins representing adjacent regions of the dystrophin rod. DYS1416 (amino acids 1416-1880) bound F-actin with a Kd of 14.2 +/- 5.2 microM and a stoichiometry of 1 mol:mol of actin. However, DYS1030 (amino acids 1030-1494) failed to bind F-actin, suggesting that not all rod domain repeats are capable of binding F-actin. Interestingly, DYS1416 corresponds to a unique region of the dystrophin rod rich in basic amino acids, whereas DYS1030 is composed mainly of acidic repeats. This observation suggested that DYS1416 may interact with acidic actin filaments through an electrostatic interaction. Supporting this hypothesis, actin binding by DYS1416 was dramatically inhibited by increasing ionic strength. We suggest that electrostatic interactions between basic spectrin-like repeats and actin filaments may contribute to the actin binding activity of other members of the actin cross-linking protein family.

Elevation of serum creatine kinase as the only manifestation of an intragenic deletion of the dystrophin gene in three unrelated families

This study reports three children from three unrelated families, aged from 9 to 12 years, who were investigated because of the incidental finding of elevated serum creatine kinase (CK) levels and were found to have a dystrophinopathy. The molecular defect consisted of a deletion of variable extent within the central rod domain of the dystrophin gene, involving either exons 32-44 or 48-51 or 48-53. In each family we found the same deletion in at least one adult male relative aged from 40 to 77 years, who was either completely asymptomatic or had very mild muscle involvement (thin muscles and/or mild scoliosis), with normal or borderline CK levels. This study suggests once again that deletions of the central rod domain of dystrophin may be associated with elevation of serum CK as the only manifestation and that prediction of the clinical severity based solely on the molecular findings should be interpreted with caution.

A point mutation in the glycerol kinase gene associated with a deletion in the dystrophin gene in a familial X-linked muscular dystrophy: non-contiguous gene syndrome involving Becker muscular dystrophy and glycerol kinase loci

We report a family with an X-linked recessive muscular dystrophy characterised by exercise-induced myalgia, recurrent pigmenturia and mild proximal muscle involvement. Immunocytochemical and immunoblotting analysis in muscle, using the antibody directed against the rod domain of dystrophin, revealed a loss of immunoreactivity, but the immunolabelling using the antibodies directed against the COOH and NH 2 domains of dystrophin were almost normal. The immunoreactions for α-sarcoglycan, γ-sarcoglycan and β-dystroglycan were normal. In the five male patients of this family with increased serum creatine kinase levels (from ×8 to ×50), mass spectrometry screening of the urine revealed a large increase in glycerol elimination which was quantified by enzymatic assay (from ×14 to ×39). An in-frame deletion of the dystrophin gene (exons 13–29) was found in the same five males and in three carrier females. All the deleted chromosomes also carried a missense mutation at nucleotide 947 of the Xp glycerol kinase (GK) gene resulting in a Thr to Met substitution at codon 278. These findings indicate that the two mutations cosegregate on the same chromosome in this family. This is the first reported case of two physically independent mutations, within the DMD and GK genes, which are contiguous but several hundred kilobases apart.

Dp140: Alternatively Spliced Isoforms in Brain and Kidney

Dp140, a protein composed of the distal rod domain and carboxy-terminal domain of dystrophin, is expressed only in brain and kidney; transcription is initiated at a unique first exon located in dystrophin intron 44. Both tissues express specific isoforms with distinct alternative splicing of exons 71–74 and 78. The carboxy-terminal domain of Dp140 is identical to that of full-length 427-kDa dystrophin, and it is this region that is associated, with the “dystrophin-associated protein” complex in skeletal muscle. Alternative splicing encodes domains that have been shown to be protein-binding regions; thus this alternative splicing may regulate the association of Dp140 with integral membrane proteins. The 5′ flanking region of genomic DNA adjacent to the Dp140 first exon contains a variety of transcription factor-binding motifs, some of which could regulate neuroglial-specific gene expression.

Physical properties of dystrophin rod domain.

We have prepared two fragments of the human dystrophin rod domain, each containing eight spectrin-like repeating units, by expression in Escherichia coli. The first corresponds to the central portion of the rod, the other to three repeats from the N-terminal end, fused to five repeats from the C-terminal end. The latter makes up the entire mutant rod, found in a patient with mild (Becker-type) muscular dystrophy. Both fragments were found to possess an ordered, stable structure, and had the form of short rod-like particles in the electron microscope. Molecular weight determinations by sedimentation equilibrium revealed that both polypeptides were monomeric in solution, suggesting that the dystrophin rod domain is incapable of forming an antiparallel homodimer. This supports the inference from sequence analyses [Winder et al., 1995: FEBS Lett. 369:27-33, 1996: Biochem. Soc. Trans. 24:2805] that the dystrophin rod domain lacks the arrangement of sites required for lateral self-association, and that dystrophin, unlike the other known proteins of the spectrin superfamily, may thus exist as a monomer.

Physical properties of dystrophin rod domain

We have prepared two fragments of the human dystrophin rod domain, each containing eight spectrin-like repeating units, by expression in Escherichia coli. The first corresponds to the central portion of the rod, the other to three repeats from the N-terminal end, fused to five repeats from the C-terminal end. The latter makes up the entire mutant rod, found in a patient with mild (Becker-type) muscular dystrophy. Both fragments were found to possess an ordered, stable structure, and had the form of short rod-like particles in the electron microscope. Molecular weight determinations by sedimentation equilibrium revealed that both polypeptides were monomeric in solution, suggesting that the dystrophin rod domain is incapable of forming an antiparallel homodimer. This supports the inference from sequence analyses [Winder et al., 1995: FEBS Lett. 369:27–33, 1996: Biochem. Soc. Trans. 24:2805] that the dystrophin rod domain lacks the arrangement of sites required for lateral self-association, and that dystrophin, unlike the other known proteins of the spectrin superfamily, may thus exist as a monomer. Cell Motil. Cytoskeleton 36:246–252, 1997. © 1997 Wiley-Liss, Inc.

A new model for the interaction of dystrophin with F-actin.

The F-actin binding and cross-linking properties of skeletal muscle dystrophin-glycoprotein complex were examined using high and low speed cosedimentation assays, microcapillary falling ball viscometry, and electron microscopy. Dystrophin-glycoprotein complex binding to F-actin saturated near 0.042 +/- 0.005 mol/ mol, which corresponds to one dystrophin per 24 actin monomers. Dystrophin-glycoprotein complex bound to F-actin with an average apparent Kd for dystrophin of 0.5 microM. These results demonstrate that native, full-length dystrophin in the glycoprotein complex binds F-actin with some properties similar to those measured for several members of the actin cross-linking super-family of proteins. However, we failed to observe dystrophin-glycoprotein complex-induced cross-linking of F-actin by three different methods, each positively controlled with alpha-actinin. Furthermore, high speed cosedimentation analysis of dystrophin-glycoprotein complex digested with calpain revealed a novel F-actin binding site located near the middle of the dystrophin rod domain. Recombinant dystrophin fragments corresponding to the novel actin binding site and the first 246 amino acids of dystrophin both bound F-actin but with significantly lower affinity and higher capacity than was observed with purified dystrophin-glycoprotein complex. Finally, dystrophin-glycoprotein complex was observed to significantly slow the depolymerization of F-actin, Suggesting that dystrophin may lie along side an actin filament through interaction with multiple actin monomers. These data suggest that although dystrophin is most closely related to the actin cross-linking superfamily based on sequence homology, dystrophin binds F-actin in a manner more analogous to actin side-binding proteins.

Stability of the dystrophin rod domain fold: evidence for nested repeating units

An examination of fragments of the human dystrophin rod domain, corresponding to a single structural repeating unit, showed that a critical chain length, defined with a precision of one residue at the C-terminal end, is required for formation of the native tertiary fold. We report here that extending the chain by six residues beyond this minimum results in a large increase in conformational stability. This is not related to a change in association state of the polypeptide. The results support the conjecture that successive repeating units in the rod domain of the spectrinlike proteins form a nested structure, in which the N-terminal part of the three-helix bundle of one repeat packs into the overlapping structure of the preceding repeat. This would be expected to affect functional characteristics related to flexibility of the dystrophin rod domain.

Minimum folding unit of dystrophin rod domain.

Fragments of the rod domain of dystrophin, which consists of spectrin-like repeating sequences, have been prepared by expression in Escherichia coli. The phasing established earlier for the dystrophin rod, as well as for Drosophila spectrin and smooth muscle alpha-actinin, suggested a length of less than 113 residues for the dystrophin repeat that we have chosen. Fragments with a common N-terminus and lengths between 113 and 119 residues were prepared. The formation of the stable native tertiary fold could be recognized by resistance to proteolysis, the circular dichroism spectrum in the regions of both peptide and aromatic absorption bands and the resolution of the long-wavelength component in the tryptophan absorption spectrum. It was found that the critical length for folding was 117 residues: shortening the chain by 1 further residue resulted in loss of the capacity to form a defined tertiary structure. Residue 117 is a glutamine; replacement of this by a methionine residue did not impair the ability of the chain to enter the folded conformation, implying that it is the length of the C-terminal alpha-helix, rather than any specific side-chain interaction, that is critical in determining the stability of the native structure. The fragment of 119 residues forms a significantly more stable structure than that of 117. It appears that the minimum unit capable of forming the native fold extends some residues into the adjoining sequence repeat.

Immunohistochemical analysis of dystrophin-associated proteins in Becker/Duchenne muscular dystrophy with huge in-frame deletions in the NH2-terminal and rod domains of dystrophin.

The absence of dystrophin causes the drastic reduction of the dystrophin-associated proteins (DAPs) in the sarcolemma and the loss of the linkage between the subsarcolemmal cytoskeleton and the extracellular matrix in Duchenne muscular dystrophy (DMD) skeletal muscle. Here, we report a mild reduction of the DAPs in the unique Becker muscular dystrophy patients with huge deletions in the rod domain of dystrophin and a moderate reduction of the DAPs in patients with huge deletions that involve both the NH2-terminal and rod domains of dystrophin. The phenotype of the latter patients was more severe than that of the former. In both cases, however, the reduction in the DAPs was milder than in typical DMD patients or DMD patients lacking the COOH-terminal domains of dystrophin. Our results suggest that (a) the NH2-terminal and rod domains of dystrophin may not be essential for the interaction with the sarcolemmal glycoprotein complex; and (b) defects in the actin binding activity of dystrophin may cause disruption of the anchorage of the dystrophin-glycoprotein complex to the subsarcolemmal cytoskeleton, which may render muscle fibers susceptible to degeneration.

Conformation and Phasing of Dystrophin Structural Repeats

The presumptive rod domain of dystrophin contains a series of degenerate repeating sequences with homology to those of spectrin. To determine the relation of the implied structural repeating units to the sequence repeat (the phasing), recombinant fragments of the domain of dystrophin were prepared by expression in Escherichia coli. The phasing was established by identifying the minimum sequence element that would form a stable fold of high (approx. 75%) α-helicity: by contrast, incorrectly phased fragments had labile structure with an average α-helicity of about 40%. The isolated folded structural repeat showed high stability towards proteolysis and a urea-denaturation profile with a plateau at low denaturant concentration, indicative of a unique folded conformation. The phasing is consistent with a structure inferred from analysis of the amino acid sequence and also found in spectrin, in which each structural repeat comprises a three-stranded coiled-coil, made up of one short helix (approx. 30 residues) and the N and C-terminal halves of two separate long helices, such that each long helix participates in the formation of two contiguous structural units.

Experimental regeneration in canine muscular dystrophy—1. Immunocytochemical evaluation of dystrophin and β-spectrin expression

The expression of dystrophin and β-spectrin was examined from 1 to 56 days in regenerating muscle fibres in normal and dystrophic dogs, following necrosis induced by the venom of Notechis scutatis. Normal and dystrophic dog muscle regenerated at an equal rate and new myotubes were present in both at the periphery of necrotic fibres by 3 days. In normal dogs dystrophin was detected in the sarcoplasm of the regenerating fibres by 3 days and was localized to the plasma membrane by 4 days. The localization of dystrophin is independent of β-spectrin and was detected before β-spectrin, which was not observed until 5–6 days. Normal peripheral labelling of both was restored by 14 days in normal dogs. Normal β-spectrin labelling of regenerating dystrophic fibres was also restored by 14 days and is not dependent on the presence of dystrophin in dystrophic dogs. A proportion of regenerating fibres in normal and dystrophic dogs showed weak immunolabelling of β-spectrin prior to 14 days. This is a feature of immature muscle fibres. Antibodies to different domains of dystrophin bound to the periphery and sarcoplasm of regenerating fibres in dystrophic dogs, particularly during the first 7 days of regeneration, but the fluoresence was less intense than in normal dogs. Weak labelling with antibodies corresponding to the C-terminus of the rod domain of dystrophin persisted on dystrophic regenerating fibres up to 21 days. This may relate to developmental isoforms of dystrophin.

Ultrastructural localization of dystropin in human muscle by using gold immunolabelling

Immunolabelling with a 5 nm gold probe was used to localize dystrophin at the ultrastructural level in human muscle. The primary antibody was monoclonal, raised against a segment (amino acids 1181-1388) from the rod domain of dystrophin. The antibody (Dy4/6D3) is specific for dystrophin and shows no immunoreactivity with any protein from mdx mouse muscle or from patients with a gene deletion spanning part of the molecule recognized by the antibody (Nicholson et al. 1989 a; England et al. 1990). Using this antibody, labelling was almost entirely confined to a narrow 75 nm rim at the periphery of the muscle fibres. Histograms of the distance from the gold probe to the cytoplasmic face of the plasma membrane and of the distance between gold probes (nearest neighbour in a plane parallel with the plasma membrane) displayed modes at approximately 15 nm and 120 nm, respectively. The distribution of the probe was the same in longitudinal and transverse sections of the muscle. These observations suggest that the rod portion of the dystrophin molecule is normally arranged close to the cytoplasmic face of the plasma membrane and that the molecules form an interconnecting network. Labelling was not associated with the transverse tubular system.

Structural predictions for the central domain of dystrophin

The animo acid sequence of dystrophin indicates that the molecule has globular N- and C-terminal domains separated by a long central rod domain. The central rod contains multiple repeats, about 100 amino acids long and of variable length. These diverge sufficiently in sequence that, in previous studies, only 14 of the most similar repeats have been aligned and analysed in any detail. We show here that a heptad pattern of hydrophobic residues is preserved across all repeats. Using the heptad pattern together with a consensus sequence template, we identified and aligned 25 repeats in the dystrophin rod sequence. Each repeat consists of a constant-length core helix of 54 residues, coupled via a short linker to a weakly conserved variable-length helix, and then via a second linker to the next core. The variable-length helix appears truncated in repeats 10 and 13 and extended in repeats 4 and 20. The extension of repeat 20 is particularly interesting since it corresponds to a hotspot of dystrophy-inducing mutations. Detailed modelling suggests that the classical Speicher-Marchesi [(1984) Nature 311, 177-180] model for spectrin may not be appropriate to dystrophin without some modification. We propose that whilst the repeating structural motif in dystrophin is probably a bead of triple coiled coil, this bead is twice as massive as, and out of phase with, those proposed for spectrin. Our model raises the possibility that the rod domain of dystrophin may confer elasticity on the molecule. Deletions which truncate this region would then reduce the extensibility of the molecule without affecting actin crosslinking, consistent with their typically producing the relatively benign Becker phenotype of muscular dystrophy.