Dystrophin Rod Research Articles

Unusually severe muscular dystrophy upon in-frame deletion of the dystrophin rod domain and lack of compensation by membrane-localized utrophin

Utrophin, a dystrophin homolog, is consistently upregulated in muscles of patients with Duchenne muscular dystrophy (DMD) and is believed to partially compensate for the lack of dystrophin in dystrophic muscle. Even though several animal studies support the idea that utrophin can modulate DMD disease severity, human clinical data are scarce. We describe a patient with the largest reported in-frame deletion in the DMD gene, including exons 10-60 and thus encompassing the entire rod domain. The patient presented with an unusually early and severe progressive weakness, initially suggesting congenital muscular dystrophy. Immunostaining of his muscle biopsy showed that the mutant protein was able to localize at the sarcolemma and stabilize the dystrophin-associated complex. Strikingly, utrophin protein was absent from the sarcolemmal membrane despite the upregulation of utrophin mRNA. Our results suggest that the internally deleted and dysfunctional dystrophin lacking the entire rod domain may exert a dominant-negative effect by preventing upregulated utrophin protein from reaching the sarcolemmal membrane and thus blocking its partial rescue of muscle function. This unique case may set a lower size limit for similar constructs in potential gene therapy approaches. This work was supported by a grant from MDA USA (MDA3896) and by grant number R01AR051999 from NIAMS/NIH to C.G.B.

Efficacy of Multi-exon Skipping Treatment in Duchenne Muscular Dystrophy Dog Model Neonates

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, which codes for dystrophin. Because the progressive and irreversible degeneration of muscle occurs from childhood, earlier therapy is required to prevent dystrophic progression. Exon skipping by antisense oligonucleotides called phosphorodiamidate morpholino oligomers (PMOs), which restores the DMD reading frame and dystrophin expression, is a promising candidate for use in neonatal patients, yet the potential remains unclear. Here, we investigate the systemic efficacy and safety of early exon skipping in dystrophic dog neonates. Intravenous treatment of canine X-linked muscular dystrophy in Japan dogs with a 4-PMO cocktail resulted in ∼3%-27% in-frame exon 6-9 skipping and dystrophin restoration across skeletal muscles up to 14% of healthy levels. Histopathology was ameliorated with the reduction of fibrosis and/or necrosis area and centrally nucleated fibers, significantly in the diaphragm. Treatment induced cardiac multi-exon skipping, though dystrophin rescue was not detected. Functionally, treatment led to significant improvement in the standing test. Toxicity was not observed from blood tests. This is the first study to demonstrate successful multi-exon skipping treatment and significant functional improvement in dystrophic dogs. Early treatment was most beneficial for respiratory muscles, with implications for addressing pulmonary malfunction in patients.

Immunohistochemistry of sarcolemmal membrane-associated proteins in formalin-fixed and paraffin-embedded skeletal muscle tissue: a promising tool for the diagnostic evaluation of common muscular dystrophies

BackgroundThe analysis of fresh frozen muscle specimens is standard following routine muscle biopsy, but this service is not widely available in countries with limited medical facilities, such as Thailand. Nevertheless, immunohistochemistry (IHC) analysis is essential for the diagnosis of patients with a strong clinical suspicion of muscular dystrophy, in the absence of mutations detected by molecular genetics. As the successful labelling of sarcolemmal membrane-associated proteins in formalin-fixed and paraffin-embedded (FFPE) muscle sections using IHC staining has rarely been described, this study aimed to develop a reproducible IHC method for such an analysis.MethodsThirteen cases were studied from the files of the Department of Pathology, Mahidol University. Diagnoses included three Duchenne muscular dystrophy (DMD), one Becker muscular dystrophy (BMD), one dysferlinopathy, and several not-specified muscular dystrophies. IHC was performed on FFPE sections at different thicknesses (3 μm, 5 μm, and 8 μm) using the heat-mediated antigen retrieval method with citrate/EDTA buffer, followed by an overnight incubation with primary antibodies at room temperature. Antibodies against spectrin, dystrophin (rod domain, C-terminus, and N-terminus), dysferlin, sarcoglycans (α, β, and γ), and β-dystroglycan were used. Frozen sections were tested in parallel for comparative analysis.ResultsAntibodies labelling spectrin, dystrophin (rod domain and C-terminus), dysferlin, sarcoglycans (α, β, and γ), and β-dystroglycan clearly exhibited sarcolemmal staining in FFPE sections. However, staining of FFPE sections using the antibody directed against the N-terminus of dystrophin was unsuccessful. The absence of labeling for dystrophins and dysferlin in FFPE sections was documented in all three DMD patients and the dysferlinopathy patient. The BMD diagnosis could not be made using IHC in FFPE sections alone because of a lack of staining for the dystrophin N-terminus, indicating a limitation of this method.ConclusionsWe developed a reliable and reproducible IHC technique using FFPE muscle. This could become a valuable tool for the diagnosis of some muscular dystrophies, dystrophinopathies, sarcoglycanopathies (LGMD2D, LGMD2E, and LGMD2C), and dysferlinopathy, especially in situations where the analysis of fresh frozen muscle samples is not routinely available.

561. CRISPR-Induced Deletion (CinDel) Method Allows Permanent and Efficient Restoration of the DMD Gene Reading Frame in Duchenne Patient Myoblasts and Preserves Truncated Dystrophin Structure

The CRISPR/Cas9 system is a great revolution in biology. This technology allows the modification of genes in vitro and in vivo in a wide variety of living organisms. In most Duchenne Muscular Dystrophy (DMD) patients, expression of dystrophin (DYS) protein is disrupted because exon deletions result in a frame shift. Here we present CRISPR-induced deletion (CinDel), a new gene therapy approach to correct the DMD gene in Duchenne patients with one or more exons deletions. By using adequate pair of gRNAs targeting specifically the exons precede and follow the patient deletion in DMD gene, CinDel induces precise DSB in targeted sequences and allows an additional deletion. The remaining parts of the exons were fusioned by NHEJ to form a hybrid exon and restored the DMD reading frame in 62 % of hybrid exons in vitro in patient myoblasts and in vivo in electroporated muscle hDMD/mdx mice. Moreover, adequate pairs of gRNAs also restored the normal spectrin-like repeat of the dystrophin rod domain; such restoration is not obtained by exon skipping or deletion of complete exons. The expression of an internally deleted dystrophin protein was detected following the formation of myotubes by the unselected treated DMD myoblasts. Given that CinDel induces permanent reparation of the DMD gene this treatment would not have to be repeated as it is the case for exon skipping induced by oligonucleotides.

389. Muscular Dystrophy Genes and the Early Metazoan Transition from Dynein- to Myosin-Powered Locomotion

During early animal evolution, the primary source of locomotive mechanical power shifted from axonemal (flagellar) dynein to densely arrayed class II myosin. Although the emergence of muscle conferred the advantage of three-dimensional scalability, the transition brought with it the evolutionarily unprecedented problem of safely transmitting huge forces across rapidly deforming cell membranes. This task in vertebrates is accomplished in part through the dystrophin-dystroglycan-sarcoglycan complex (DGC), which is implicated in most forms of muscular dystrophy. We present the inferred earliest steps in the molecular evolution of these cell surface and cortical cytoskeletal proteins, using genome sequence data from all early branching metazoan phyla and a broad sampling of unicellular taxa. Results: Surprisingly, the phylogeny suggests that a DGC emerged before the “sarcomeric” clade of myosins, implying conserved function(s) among unicellular lineages closely related to the Metazoa. Furthermore, linkage of the DGC to the cytoskeleton occurred before the massive tandem-repeat expansions (>200) seen in sarcomeric scaffolding proteins of the titin superfamily. The rod-like domains of dystrophin and utrophin were coopted at their full length from a rapidly lengthening cytoskeletal protein with an unrelated function, that of connecting actin filaments to microtubules at large physical distances. Finally, intron positions in the inferred dystrophin gene in a common ancestor to all Bilateria provide clues to the molecular basis of Duchenne muscular dystrophy and to emerging therapies. Conclusions: Our reconstruction suggests that evolution of membrane-spanning “muscular dystrophy protein complexes” was an essential process as one geometric constraint on power transduction was traded for another. 80% of the length of dystrophin is attributable to tandem DNA duplications that pre-dated the evolutionary emergence of sarcomeres. This chronology implies that escalating power output from striated muscle played no role in providing selective pressure for the serial expansion of “spectrin-like” repeats. This finding has therapeutic implications, as it suggests that minimal length dystophins or utrophins may fully complement the physiological role of full length dystrophin in vertebrates as long as the deletional juxtaposition of triple helical repeats does not disrupt force transmission. This interpretation is consistent with the clinical findings of Becker Muscular Dystrophy in selected patients with in-frame exon duplications. In this view, the evolutionary stability of the length of the dystrophin rod is driven by the incompatibility, when placed in close physical proximity by deletion, of divergent triple helical repeats. The evolutionary translocation of the protein to the cytoskeletal cortex as part of the DGC may have greatly reduced the metabolic load associated with synthesis of the strong but scarce protein, also facilitating evolutionary drift to the enormous size of the gene in a common mammalian ancestor.

Efficient Restoration of the Dystrophin Gene Reading Frame and Protein Structure in DMD Myoblasts Using the CinDel Method.

The CRISPR/Cas9 system is a great revolution in biology. This technology allows the modification of genes in vitro and in vivo in a wide variety of living organisms. In most Duchenne muscular dystrophy (DMD) patients, expression of dystrophin (DYS) protein is disrupted because exon deletions result in a frame shift. We present here the CRISPR-induced deletion (CinDel), a new promising genome-editing technology to correct the DMD gene. This strategy is based on the use of two gRNAs targeting specifically exons that precede and follow the patient deletion in the DMD gene. This pair of gRNAs induced a precise large additional deletion leading to fusion of the targeted exons. Using an adequate pair of gRNAs, the deletion of parts of these exons and the intron separating them restored the DMD reading frame in 62% of the hybrid exons in vitro in DMD myoblasts and in vivo in electroporated hDMD/mdx mice. Moreover, adequate pairs of gRNAs also restored the normal spectrin-like repeat of the dystrophin rod domain; such restoration is not obtained by exon skipping or deletion of complete exons. The expression of an internally deleted DYS protein was detected following the formation of myotubes by the unselected, treated DMD myoblasts. Given that CinDel induces permanent reparation of the DMD gene, this treatment would not have to be repeated as it is the case for exon skipping induced by oligonucleotides.

Human Dystrophin Rod 11-15 Sub-Domain: A Membrane Interacting Zone Modulated by Lipid Packing

Dystrophin is essential for skeletal muscle function and confers resistance to the sarcolemma by interacting with cytoskeletal and membrane partners. We investigated here protein-lipid interaction of a five repeat from the central domain of dystrophin (DYS R11-15) also referred as an actin binding domain. In a first step, we demonstrated that DYS R11-15 interacts more strongly with anionic than with zwitterionic small unilamellar vesicles. Using large unilamellar vesicles with different radius and trypsin accessibility assays, we showed that the protein presents different conformation depending on vesicle curvature and lipid nature. Using label-free quantification mass spectroscometry, a protein domain protected from proteolysis in presence of anionic vesicles was observed while a protein domain more accessible to trypsin in the presence of either anionic or zwitterionic vesicles was identified. In a second step, we studied the adsorption behavior of the protein at the air-liquid and lipid-liquid interface in a Langmuir trough. DYS R11-15 displayed a surface activity while maintaining its α-helical secondary structure as shown by PM-IRRAS. At 16mN/m lateral pressure of monolayer lipid film, few protein clusters were observed by AFM, while at 20 and 30mN/m, a striking protein network was formed with both negative and zwitterionic phospholipids. However, image analysis and behaviour of the networks towards trypsination in the trough as revealed by AFM showed that trypsin accessibility to the protein network depends on the surface pressure as well as on the nature of the phospholipid. These results indicate that DYS R11-15 constitutes part of the dystrophin protein for which anchoring and interaction with membrane depend on the packing and the nature of lipids. Such behaviour provides a strong experimental support for a physiological role of dystrophin central domain in contraction-relaxation cycles and dynamics of muscle cells.

Specific anchoring modes of two distinct dystrophin rod sub-domains interacting in phospholipid Langmuir films studied by atomic force microscopy and PM-IRRAS

Dystrophin rod repeats 1–3 sub-domain binds to acidic phosphatidylserine in a small vesicle binding assay, while the repeats 20–24 sub-domain does not. In the present work, we studied the adsorption behaviour of both sub-domains at the air/liquid interface and at the air/lipid interface in a Langmuir trough in order to highlight differences in interfacial properties. The adsorption behaviour of the two proteins at the air/liquid interface shows that they display surface activity while maintaining their alpha-helical secondary structure as shown by PM-IRRAS. Strikingly, R20–24 needs to be highly hydrated even at the interface, while this is not the case for R1–3, indicating that the surface activity is dramatically higher for R1–3 than R20–24. Surface-pressure measurements, atomic force microscopy and PM-IRRAS are used in a Langmuir experiment with DOPC–DOPS monolayers at two different surface pressures, 20 mN/m and 30 mN/m. At the lower surface pressure, the proteins are adsorbed at the lipid film interface while maintaining its alpha-helical structure. After an increase of the surface pressure, R1–3 subsequently produces a stable film, while R20–24 induces a reorganization of the lipid film with a subsequent decrease of the surface pressure close to the initial value. AFM and PM-IRRAS show that R1–3 is present in high amounts at the interface, being arranged in clusters representing 3.3% of the surface at low pressure. By contrast, R20–24 is present at the interface in small amounts bound only by a few electrostatic residues to the lipid film while the major part of the molecule remains floating in the sub-phase. Then for R1–3, the electrostatic interaction between the proteins and the film is enhanced by hydrophobic interactions. At higher surface pressure, the number of protein clusters increases and becomes closer in both cases implying the electrostatic character of the binding. These results indicate that even if the repeats exhibit large structural similarities, their interfacial properties are highly contrasted by their differential anchor mode in the membrane. Our work provides strong support for distinct physiological roles for the spectrin-like repeats and may partly explain the effects of therapeutic replacement of dystrophin deficiency by minidystrophins.

G.P.13.04 In-frame deletion of the entire dystrophin rod domain prevents compensation by utrophin and causes an unusually severe muscular dystrophy

Clinical data to support the ability of the dystrophin-relative utrophin to ameliorate disease in Duchenne muscular dystrophy (DMD) is scarce, though there is ample evidence for animal data suggesting that forced upregulation of utrophin in dystrophic muscle can restore some function. Similarly, double inactivation of both dystrophin and utrophin in the mouse worsens the phenotype considerably. Here we describe a boy presenting with unusually early, severe weakness suggesting congenital muscular dystrophy.

939. Ramifications of Therapeutic Exon Skipping on Dystrophin Rod Protein Stability and Function

939. Ramifications of Therapeutic Exon Skipping on Dystrophin Rod Protein Stability and Function

A Two-amino Acid Mutation Encountered in Duchenne Muscular Dystrophy Decreases Stability of the Rod Domain 23 (R23) Spectrin-like Repeat of Dystrophin

Lack of functional dystrophin causes severe Duchenne muscular dystrophy. The subsarcolemmal location of dystrophin, as well as its association with both cytoskeleton and membrane, suggests a role in the mechanical regulation of muscular membrane stress. In particular, phenotype rescue in a Duchenne muscular dystrophy mice model has shown that some parts of the central rod domain of dystrophin, constituted by 24 spectrin-like repeats, are essential. In this study, we made use of rare missense pathogenic mutations in the dystrophin gene and analyzed the biochemical properties of the isolated repeat 23 bearing single or double mutations E2910V and N2912D found in muscle dystrophy with severity grading. No dramatic effect on secondary and tertiary structure of the repeat was found in mutants compared with wild type as revealed by circular dichroism and NMR. Thermal and chemical unfolding data from circular dichroism and tryptophan fluorescence show significant decrease of stability for the mutants, and stopped-flow spectroscopy shows decreased refolding rates. The most deleterious single mutation is the N2912D replacement, although we observe additive effects of the two mutations on repeat stability. Based on three-dimensional structures built by homology molecular modeling, we discuss the modifications of the mutation-induced repeat stability. We conclude that the main forces involved in repeat stability are electrostatic inter-helix interactions that are disrupted following mutations. This study represents the first analysis at the protein level of the consequences of missense mutations in the human dystrophin rod domain. Our results suggest that it may participate in mechanical weakening of dystrophin-deficient muscle.

Sub-domains of the dystrophin rod domain display contrasting lipid-binding and stability properties

Dystrophin is a muscle scaffolding protein that establishes a structural link between the cytoskeleton and the extracellular matrix. Despite the large body of knowledge about the dystrophin gene and its interactions, the functional importance of the large central rod domain remains highly controversial. It is composed of 24 spectrin-like repeats interrupted by four hinges that delineate three sub-domains. We express repeat 1–3 and repeat 20–24 sub-domains, delineated by hinges 1–2 and 3–4 and the single repeats 2 and 23. We determine their lipid-binding properties, thermal and urea stabilities and refolding velocities. By using intrinsic tryptophan fluorescence spectroscopy and size exclusion chromatography, we show that repeat 2 and the repeat 1–3 sub-domain strongly interact with anionic lipids. By contrast, repeat 23 and the repeat 20–24 sub-domain do not interact with lipids. In addition, the repeat 1–3 sub-domain and repeat 2 are dramatically less stable and refold faster than the repeat 20–24 sub-domain and repeat 23. The contrasting properties of the two sub-domains clearly indicate that they make up two units of the rod domain that are not structurally interchangeable, thus providing molecular evidence supporting the observations on the biological function of dystrophin.

Molecular Extensibility of Mini-dystrophins and a Dystrophin Rod Construct

Muscular dystrophies arise with various mutations in dystrophin, implicating this protein in force transmission in normal muscle. With 24 three-helix, spectrin repeats interspersed with proline-rich hinges, dystrophin's large size is an impediment to gene therapy, prompting the construction of mini-dystrophins. Results thus far in dystrophic mice suggest that at least one hinge between repeats is necessary though not sufficient for palliative effect. One such mini-dystrophin is studied here in forced extension at the single molecule level. Delta2331 consists of repeats (R) and hinges (H) H1-R1-2 approximately H3 approximately R22-24-H4 linked by native (-) and non-native (approximately) sequence. This is compared to its core fragment R2 approximately H3 approximately R22 as well as an eight-repeat rod fragment middle (RFM: R8-15). We show by atomic force microscopy that all repeats extend and unfold at forces comparable to those that a few myosin molecules can generate. The hinge regions most often extend and transmit force while limiting tandem repeat unfolding. From 23-42 degrees C, the dystrophin constructs also appear less temperature-sensitive in unfolding compared to a well-studied betaI-spectrin construct. The results thus reveal new modes of dystrophin flexibility that may prove central to functions of both dystrophin and mini-dystrophins.

Proximal Dystrophin Gene Deletions and Protein Alterations in Becker Muscular Dystrophy

Alterations in production of cytoskeletal protein dystrophin caused by in-frame gene mutations lead to the Becker muscular dystrophy. In this study we analyzed genotype-phenotype correlation in a group of Becker muscular dystrophy patients with deletions affecting the proximal part of dystrophin gene, encompassing exons 3-13. Four patients with deletions affecting N terminal dystrophin domain had early onset and faster progression of the disease, while three patients with deletions in the proximal part of dystrophin's rod domain had a more benign disease course. Our study suggests that proximal gene deletions in Becker muscular dystrophy have various phenotypic effects depending on the affected domain of protein dystrophin.

Interaction of Dystrophin Rod Domain with Membrane Phospholipids: EVIDENCE OF A CLOSE PROXIMITY BETWEEN TRYPTOPHAN RESIDUES AND LIPIDS

Dystrophin is assumed to act via the central rod domain as a flexible linker between the amino-terminal actin binding domain and carboxyl-terminal proteins associated with the membrane. The rod domain is made up of 24 spectrin-like repeats and has been shown to modify the physical properties of lipid membranes. The nature of this association still remains unclear. Tryptophan residues tend to cluster at or near to the water-lipid interface of the membrane. To assess dystrophin rod domain-membrane interactions, tryptophan residues properties of two recombinant proteins of the rod domain were examined by (1)H NMR and fluorescence techniques in the presence of membrane lipids. F114 (residues 439-553) is a partly folded protein as inferred from (1)H NMR, tryptophan fluorescence emission intensity, and the excited state lifetime. By contrast, F125 (residues 439-564) is a folded compact protein. Tryptophan fluorescence quenching shows that both proteins are characterized by structural fluctuations with their tryptophan residues only slightly buried from the surface. In the presence of negatively charged small vesicles, the fluorescence characteristics of F125 change dramatically, indicating that tryptophan residues are in a more hydrophobic environment. Interestingly, these modifications are not observed with F114. Fluorescence quenching experiments confirm that tryptophan residues are shielded from the solvent in the complex F125 lipids by a close contact with lipids. The use of membrane-bound quenchers allowed us to conclude that dystrophin rod domain lies along the membrane surface and may be involved in a structural array comprising membrane and cytoskeletal proteins as well as membrane lipids.

Spectrin-like repeats from dystrophin and alpha-actinin-2 are not functionally interchangeable.

Mutations in the dystrophin gene result in Duchenne muscular dystrophy (DMD). Dystrophin is a multidomain protein that functions to stabilize the sarcolemmal membrane during muscle contraction. The central rod domain has been proposed to act as a shock absorber, as a force transducer or as a spacer separating important N- and C-terminal domains that interact with actin and the dystrophin-glycoprotein complex (DGC). Structure/function studies demonstrated that deletion of large portions of the rod domain can result in the production of smaller, yet highly functional, dystrophin proteins. In a dramatic example, a 'micro-dystrophin' transgene containing only four dystrophin spectrin-like repeats resulted in complete correction of most of the symptoms associated with dystrophy in the mdx mouse model for DMD. Dystrophin shares considerable homology with the multidomain, actin-crosslinking protein alpha-actinin. To explore the hypothesis that the dystrophin rod domain acts as a spacer region, a chimeric micro-dystrophin transgene containing the four-repeat rod domain of alpha-actinin-2 was expressed in mdx mice. This chimeric transgene was incapable of correcting the morphological pathology of the mdx mouse, but still functioned to assemble the DGC at the membrane and provided some protection from contraction-induced injury. These data demonstrated that different spectrin-like repeats are not equivalent, and reinforced the suggestion that the dystrophin rod domain is not merely a spacer but likely contributes an important mechanical role to overall dystrophin function.

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.

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.