Association of the Mr 58,000 postsynaptic protein of electric tissue with Torpedo dystrophin and the Mr 87,000 postsynaptic protein.

Dystrophin was purified by immunoaffinity chromatography from detergent-solubilized Torpedo electric organ postsynaptic membranes using monoclonal antibodies. A major doublet of proteins at Mr 58,000 and minor proteins at Mr 87,000, Mr 45,000, and Mr 30,000 reproducibly copurified with dystrophin. The Mr 58,000 and Mr 87,000 proteins were identical to previously described peripheral membrane proteins (Mr 58,000 protein and 87,000 protein) whose muscle homologs are associated with the sarcolemma (Froehner, S. C., Murnane, A. A., Tobler, M., Peng, H. B., and Sealock, R. (1987) J. Cell Biol. 104, 1633-1646; Carr, C., Fischbach, G. D., and Cohen, J. B. (1989) J. Cell Biol. 109, 1753-1764). The copurification of dystrophin and Mr 58,000 protein was shown to be specific, since dystrophin was also captured with a monoclonal antibody against the Mr 58,000 protein but not by several control antibodies. The Mr 87,000 protein was a major component (along with the Mr 58,000 protein) in material purified on anti-58,000 columns, suggesting that the Mr 58,000 protein forms a distinct complex with the Mr 87,000 protein, as well as with dystrophin. Immunofluorescence staining of skeletal and cardiac muscle from the dystrophin-minus mdx mouse with the anti-58,000 antibody was confined to the sarcolemma as in normal muscle but was much reduced in intensity, even though immunoblotting demonstrated that the contents of Mr 58,000 protein in normal and mdx muscle were comparable. Thus, the Mr 58,000 protein appears to associate inefficiently with the sarcolemmal membrane in the absence of dystrophin. This deficiency may contribute to the membrane abnormalities that lead to muscle necrosis in dystrophic muscle.     

Rapid mapping by transposon mutagenesis of epitopes on the muscular dystrophy protein, dystrophin.

Antibody-binding epitopes in the central helical region of the muscular dystrophy protein, dystrophin, have been mapped using a new strategy of transposon mutagenesis. Tn1000 transposons carrying translation termination codons were introduced randomly by bacterial mating into a large fragment of dystrophin cDNA in a pEX2 plasmid to produce a library of transformants expressing truncated dystrophin fusion proteins. Epitopes were progressively lost as the expressed sequences were shortened, enabling the epitopes recognised by 22 monoclonal antibodies to be placed in order along the dystrophin molecule without in vitro manipulation of DNA. The C-terminus of each truncated fusion protein was precisely located within the dystrophin sequence by direct sequencing of pEX2 transformants using transposon-specific primers. Sequences as short as 7 and 17 amino-acids have been identified as essential for antibody binding in this way. Nineteen of the 22 monoclonal antibodies had been selected for their ability to bind both native and SDS-denatured dystrophin and 15 of these bind to one sequence of 74 amino-acids (residues 1431-1505 of the 3684 residue sequence). This may be an area of high immunogenicity or of close structural similarity between native dystrophin and the SDS-treated recombinant fragment used for immunization.     

Detection of dystrophin on two-dimensional gel electrophoresis

A protein with MW approximately 350 k daltons and pI approximately 5.5, which was deleted in the dystrophic mouse (C57BL/10ScSn-mdx), was detected on two-dimensional gel electrophoresis with silver staining. Deletion of this protein was uniformly observed in the dystrophic mouse extensor digitus longus, soleus and cardiac muscle. This protein specifically reacted with the monoclonal antibody against the chemically synthesized N-terminal fragment of human dystrophin. The protein reacting with this monoclonal antibody was also detected in rabbit back-muscle, rat extensor digitus longus and human skeletal muscle at the same position as the mouse muscle protein, on the two-dimensional gel electrophoresis. Our results show that dystrophin is solubilized in 8M guanidine HCl and that the modified two-dimensional gel electrophoresis can be applied to separate dystrophin.     

Functional EGFP-dystrophin fusion proteins for gene therapy vector development

Transfection and transduction studies involving the use of the full-length dystrophin (11 kb) or the truncated mini-gene (6 kb) cDNAs are hampered by the large size of the resulting viral or non-viral expression vectors. This usually results in very low yields of transgene-expressing cells. Moreover, the detection of the few transgene-expressing cells is often tedious and costly. For these reasons, expression vectors containing the enhanced green fluorescent protein (EGFP) fused with the N-termini of mini- and full-length human dystrophin were constructed. These constructs were tested by transfection of Phoenix cells with Effectene, resulting after 48 h in a green fluorescent signal in 20% of cells. Analysis of the cell extracts by immunoblotting with the use of a monoclonal antibody specific to the dystrophin C-terminus confirmed the expression of EGFP-mini- (240 kDa) and EGFP-full-length human dystrophin (450 kDa) fusion proteins. Moreover, following the in vivo electroporation of the plasmids containing the EGFP-mini- and full-length dystrophin in mouse muscles, both fluorescent proteins were observed in cryostat sections in their normal location under the plasma membrane. This indicates that the fusion of EGFP to dystrophin or mini-dystrophin did not interfere with the normal localization of the protein. In conclusion, the fusion of EGFP provides a good tool for the search of the best methods to introduce mini- or full-length dystrophin cDNA in the cells (in vitro) or muscle fibers (in vivo) for the establishment of a treatment by gene therapy of Duchenne muscular dystrophy patients.     

Dystrophin-related protein is localized to neuromuscular junctions of adult skeletal muscle.

Dystrophin-related protein (DRP) is an autosomal gene product with high homology to dystrophin. We have used highly specific antibodies to the unique C-terminal peptide sequences of DRP and dystrophin to examine the subcellular localization and biochemical properties of DRP in adult skeletal muscle. DRP is enriched in isolated sarcolemma from control and mdx mouse muscle, but is much less abundant than dystrophin. Immunofluorescence microscopy localized DRP almost exclusively to the neuromuscular junction region in rabbit and mouse skeletal muscle, as well as mdx mouse muscle and denervated mouse muscle. DRP is also present in normal size and abundance and localizes to the neuromuscular junction region in muscle from the dystrophic mouse model dy/dy. Thus, DRP is a junction-specific membrane cytoskeletal protein that may play an important role in the organization of the postsynaptic membrane of the neuromuscular junction.     

Dystrophin is a component of the subsynaptic membrane

A subsynaptic protein of Mr approximately 300 kD is a major component of Torpedo electric organ postsynaptic membranes and copurifies with the AChR and the 43-kD subsynaptic protein. mAbs against this protein react with neuromuscular synapses in higher vertebrates, but not at synapses in dystrophic muscle. The Torpedo 300-kD protein comigrates in SDS-PAGE with murine dystrophin and reacts with antibodies against murine dystrophin. The sequence of a partial cDNA isolated by screening an expression library with mAbs against the Torpedo 300-kD protein shows striking homology to mammalian dystrophin, and in particular to the b isoform of dystrophin. These results indicate that dystrophin is a component of the postsynaptic membrane at neuromuscular synapses and raise the possibility that loss of dystrophin from synapses in dystrophic muscle may have consequences that contribute to muscular dystrophy.     

Assembly of the dystrophin-associated protein complex does not require the dystrophin COOH-terminal domain

Dystrophin is a multidomain protein that links the actin cytoskeleton to laminin in the extracellular matrix through the dystrophin associated protein (DAP) complex. The COOH-terminal domain of dystrophin binds to two components of the DAP complex, syntrophin and dystrobrevin. To understand the role of syntrophin and dystrobrevin, we previously generated a series of transgenic mouse lines expressing dystrophins with deletions throughout the COOH-terminal domain. Each of these mice had normal muscle function and displayed normal localization of syntrophin and dystrobrevin. Since syntrophin and dystrobrevin bind to each other as well as to dystrophin, we have now generated a transgenic mouse deleted for the entire dystrophin COOH-terminal domain. Unexpectedly, this truncated dystrophin supported normal muscle function and assembly of the DAP complex. These results demonstrate that syntrophin and dystrobrevin functionally associate with the DAP complex in the absence of a direct link to dystrophin. We also observed that the DAP complexes in these different transgenic mouse strains were not identical. Instead, the DAP complexes contained varying ratios of syntrophin and dystrobrevin isoforms. These results suggest that alternative splicing of the dystrophin gene, which naturally generates COOH-terminal deletions in dystrophin, may function to regulate the isoform composition of the DAP complex.     

Construction of dystrophin fusion proteins to raise targeted antibodies to different epitopes.

For the study of the structure and function relationship of dystrophin, defective in DMD, and for diagnostic purposes it is important to dispose of antibodies against different parts of the protein. We have made five different constructs for the expression of fusion proteins containing parts of the four domains of dystrophin. Two different recombinant expression vectors, pATH2 and pEX1, were used. Rabbits were immunized with the fusion products and several polyclonal antibodies were raised. At a later stage, monoclonal antibodies were also raised to some of the fusion proteins. One polyclonal antibody, named P20 AB, is directed against the region covering amino acid sequence 1749-2248 or the nucleotide sequence 5456-6953 of the mRNA, which corresponds to the major deletion-prone region of the DMD gene. We show the particular value, sensitivity and specificity of the P20 AB in dystrophin analysis.     

Purification of dystrophin from skeletal muscle

Dystrophin was purified from rabbit skeletal muscle by alkaline dissociation of dystrophin-glycoprotein complex which was first prepared by derivatized lectin chromatography. Dystrophin-glycoprotein complex was isolated from digitonin-solubilized rabbit skeletal muscle membranes by a novel two-step method involving succinylated wheat germ agglutinin (sWGA) chromatography and DEAE-cellulose ion exchange chromatography. Proteins co-purifying with dystrophin were a protein triplet of Mr 59,000 and four glycoproteins of Mr 156,000, 50,000, 43,000, and 35,000, all previously identified as components of the dystrophin-glycoprotein complex. Alkaline treatment of sWGA/DEAE-purified dystrophin-glycoprotein complex resulted in complete dissociation of the dystrophin-glycoprotein complex. In order to separate dystrophin from its associated proteins, alkaline-dissociated dystrophin-glycoprotein complex was sedimented by sucrose gradient centrifugation. The residual glycoproteins which contaminated peak dystrophin-containing gradient fractions were then removed by WGA-Sepharose adsorption. The resulting protein appeared as a single band with an apparent Mr of 400,000 on overloaded Coomassie Blue-stained gels. The absence of WGA-peroxidase staining on nitrocellulose transfers of the pure protein indicated that the pure protein was devoid of contaminating glycoproteins. Antisera raised against the carboxyl terminus of human skeletal muscle dystrophin (which does not cross-react with the carboxyl terminus of the chromosome 6-encoded dystrophin-related protein) recognized the pure protein as did antisera specific for the amino terminus of human dystrophin. These data indicate that the protein isolated is indeed the intact, predominant skeletal muscle isoform product of the Duchenne muscular dystrophy gene.     

Monoclonal antibodies to rabbit skeletal muscle protein phosphatases C-I and C-II

Rabbit skeletal muscle protein phosphatases C-I and C-II have been previously isolated as two proteins of Mr = approximately 35,000. Both enzymes display broad substrate specificities but have distinct enzymatic properties in regard to their susceptibility to heat-stable protein inhibitor-2 and their response to divalent cations. Monoclonal antibodies against both protein phosphatase C-I and C-II were produced by fusion of spleen cells of immunized BALB/c mice with SP2/0-Ag14 mouse myeloma cells. The products of the hybrid cells were screened by solid phase radioimmunoassay for the production of antibodies to protein phosphatase C-I and C-II. Positive cells were cloned and injected into mice to produce ascitic fluids. Ten monoclonal antibodies against phosphatase C-I and eight monoclonal antibodies against phosphatase C-II were obtained. These antibodies were characterized with regard to their relative binding affinities to the two protein phosphatases and their abilities to inhibit the phosphorylase phosphatase activities of the two enzymes. All ten of the phosphatase C-I monoclonal antibodies inhibited the phosphorylase phosphatase activity of phosphatase C-I, and three of these also inhibited phosphatase C-II. Only one of the eight antibodies to phosphatase C-II was inhibitory and inhibited the activities of both phosphatase C-I and C-II. Examination of the binding of these monoclonal antibodies by a solid phase radioimmunoassay showed that eight of the ten phosphatase C-I antibodies cross-reacted with phosphatase C-II, while all eight of the phosphatase C-II antibodies cross-reacted with phosphatase C-I. These findings show that phosphatases C-I and C-II possess common antigenic determinant(s) and may, therefore, be structurally related proteins.     

gamma-Syntrophin scaffolding is spatially and functionally distinct from that of the alpha/beta syntrophins

The syntrophins are a family of scaffolding proteins with multiple protein interaction domains that link signaling proteins to dystrophin family members. Each of the three most characterized syntrophins (alpha, beta1, beta2) contains a PDZ domain that binds a unique set of signaling proteins including kinases, ion and water channels, and neuronal nitric oxide synthase (nNOS). The PDZ domains of the gamma-syntrophins do not bind nNOS. In vitro pull-down assays show that the gamma-syntrophins can bind dystrophin but have unique preferences for the syntrophin binding sites of dystrophin family members. Despite their ability to bind dystrophin in vitro, neither gamma-syntrophin isoform co-localizes with dystrophin in skeletal muscle. Furthermore, gamma-syntrophins do not co-purify with dystrophin isolated from mouse tissue. These data suggest that the interaction of gamma-syntrophin with dystrophin is transient and potentially subject to regulatory mechanisms. gamma1-Syntrophin is highly expressed in brain and is specifically localized in hippocampal pyramidal neurons, Purkinje neurons in cerebellum, and cortical neurons. gamma2-Syntrophin is expressed in many tissues including skeletal muscle where it is found only in the subsynaptic space beneath the neuromuscular junction. In both neurons and muscle, gamma-syntrophin isoforms localize to the endoplasmic reticulum where they may form a scaffold for signaling and trafficking.     

Patterns of dystrophin expression in developing, adult and regenerating tail skeletal muscle of Amphibian urodeles.

The patterns of expression of dystrophin were investigated by indirect immunofluorescence and by immunoblotting in developing, adult and regenerating tail skeletal muscle of newts Pleurodeles waltl and Notophthalmus viridescens. In this study, a monoclonal antibody H-5A3 directed against the C-terminal region (residues 3357-3660) and a polyclonal antibody raised to the central domain (residues 1173-1738) of the chicken skeletal muscle dystrophin were used. Western blot analysis showed that these antibodies recognized a 400 kDa band of dystrophin (and may be of dystrophin-related protein) in the adult muscle tissues and in newt tail regenerates. During skeletal muscle differentiation or epimorphic regeneration (blastema), anti-dystrophin immunoreactivity gradually accumulated over the periphery of the myofibers. Dystrophin and laminin were first and concomitantly observed at the ends of the newly formed myotubes where they were anchored on connective tissue septa or bone processes by dystrophin-rich myotendinous structures. It is noteworthy that neuromuscular junctions, which most probably also contain dystrophin, are established in urodeles near the ends of the myofibers as shown by histochemical localization of AChE activity or fluorescent bungarotoxin detection of AChRs. In the stump transition zone close to the tail amputation level where tissue regeneration of injured muscle fibers took place, dystrophin staining located on the cytoplasmic surface of myofibers progressively disappeared during the dedifferentiation process which seemed to occur during muscle regeneration as suggested by electron microscopy. Furthermore, double labeling experiments using anti-dystrophin and anti-laminin antibodies showed a good correlation between the remodeling processes of the muscle fiber basal lamina and the loss of dystrophin along the sarcolemma of damaged and presumably dedifferentiating muscle cells.     

Phosphorylation of dystrophin and alpha-syntrophin by Ca(2+)-calmodulin dependent protein kinase II.

A Ca(2+)-calmodulin dependent protein kinase activity (DGC-PK) was previously shown to associate with skeletal muscle dystrophin glycoprotein complex (DGC) preparations, and phosphorylate dystrophin and a protein with the same electrophoretic mobility as alpha-syntrophin (R. Madhavan, H.W. Jarrett, Biochemistry 33 (1994) 5797-5804). Here, we show that DGC-PK and Ca(2+)-calmodulin dependent protein kinase II (CaM kinase II) phosphorylate a common site (RSDS(3616)) within the dystrophin C terminal domain that fits the consensus CaM kinase II phosphorylation motif (R/KXXS/T). Furthermore, both kinase activities phosphorylate exactly the same three fusion proteins (dystrophin fusions DysS7 and DysS9, and the syntrophin fusion) out of a panel of eight fusion proteins (representing nearly 100% of syntrophin and 80% of dystrophin protein sequences), demonstrating that DGC-PK and CaM kinase II have the same substrate specificity. Complementing these results, anti-CaM kinase II antibodies specifically stained purified DGC immobilized on nitrocellulose membranes. Renaturation of electrophoretically resolved DGC proteins revealed a single protein kinase band (M(r) approximately 60,000) that, like CaM kinase II, underwent Ca(2+)-calmodulin dependent autophosphorylation. Based on these observations, we conclude DGC-PK represents a dystrophin-/syntrophin-phosphorylating skeletal muscle isoform of CaM kinase II. We also show that phosphorylation of the dystrophin C terminal domain sequences inhibits their syntrophin binding in vitro, suggesting a regulatory role for phosphorylation.     

Two monoclonal antibodies distinguish between human recombinant interferon-alpha 5s produced by Escherichia coli and by mouse cells

Two monoclonal antibodies against human IFN-alpha--one against natural leukocyte IFN-alpha and the other against recombinant human IFN-alpha 2 produced in E. coli--were prepared, and designated as HT-1, and 104-5-f, respectively. These monoclonal antibodies were used to examine the antigenicities of recombinant human IFN-alpha 5s produced by E. coli and by mouse cells. The HT-1 antibody could bind and neutralize recombinant human IFN-alpha 5 synthesized in mouse cells, but not recombinant human IFN-alpha 5 synthesized in E. coli. On the other hand, the 104-5-f antibody could bind and neutralize recombinant human IFN-alpha 5 synthesized in E. coli but not recombinant human IFN-alpha 5 synthesized in mouse cells. Then these monoclonal antibodies or sheep polyclonal antibody against human IFN-alpha were used to immunoprecipitate the radioactively labeled recombinant human IFN-alpha 5 synthesized either in E. coli or mouse cells, and analysed on polyacrylamide gel electrophoresis in the presence of NaDodSO4. The labeled recombinant human IFN-alpha 5 produced by mouse cells could be immunoprecipitated with the HT-1 monoclonal antibody or sheep anti-(human IFN-alpha) polyclonal antibody but not with the 104-5-f monoclonal antibody and showed a band of Mr. 17,500 on polyacrylamide gel electrophoresis in the presence of NaDodSO4. On the other hand, the labeled recombinant human IFN-alpha 5 produced by E. coli could be immunoprecipitated with the 104-5-f monoclonal antibody but not with the HT-1 monoclonal antibody and showed a band of similar Mr. on polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation and properties of monoclonal and polyclonal antibodies to mouse epidermal growth factor (EGF) receptors: evidence for cryptic EGF receptors in embryonal carcinoma cells

Monoclonal antibodies to mouse epidermal growth factor (EGF) receptor were prepared by the immunization of rats with receptor glycoprotein purified from mouse liver by affinity chromatography on immobilized EGF. Purified mouse EGF receptor retained EGF-inducible autophosphorylating activity and was antigenic in rats and rabbits. The monoclonal antibodies cross react very poorly with human EGF receptor, while polyclonal rabbit antibodies immunoprecipitate human, rat and mouse EGF receptor equally well. The rabbit antibody blocks EGF binding to mouse fibroblast cells and, at 20-fold higher concentrations, stimulates uptake of tritiated thymidine into DNA. This indicates that antibodies bind at or close to the EGF-binding site and can mimic the effects of the growth factor. None of the monoclonals bind at the EGF site of the receptor. Immunoprecipitation, immunoblotting, 125I-EGF cross linking, 125I-surface labelling, immunohistochemistry and autophosphorylation techniques were used to delineate the basis for the induction of EGF receptors when OC15 embryonal carcinoma (EC) cells differentiate into endodermal derivatives (END). EGF-stimulated autophosphorylation of a 170 X 10(3) Mr protein in solubilized OC15 EC cells is readily detectable, although intact EC cells do not bind or respond to EGF by all other tests. The results suggest that cryptic EGF receptors are present in EC stem cells, a finding with implications in development.     

Reduction of High Background Staining by Heating Unfixed Mouse Skeletal Muscle Tissue Sections Allows for Detection of Thermostable Antigens With Murine Monoclonal Antibodies

Antigen detection with indirect immunohistochemical methods is hampered by high background staining if the primary antibody is from the same species as the examined tissue. This high background can be eliminated in unfixed cryostat sections of mouse skeletal muscle by boiling sections in PBS, and several proteins including even the low abundant dystrophin protein can then be easily detected with murine monoclonal antibodies. However, not all antigens withstand the boiling procedure. Immunoreactivity of some of these antigens can be restored by subsequent washing in Triton X-100, whereas immunoreactivity of other proteins is not restored by this detergent treatment. When such thermolabile proteins are labeled with polyclonal primary antibodies followed by dichlorotriazinylaminofluorescein–conjugated secondary antibodies and boiled, the fluorescence signal persists, and sections can then be processed with a monoclonal antibody for double immunostaining of a protein unaffected by boiling. This stability of certain fluorochromes on heating can also be exploited for double immunofluorescence labeling of two different thermostable proteins with murine monoclonal antibodies as well as for combination with Y-chromosome fluorescence in situ hybridization. Our method should extend the range of monoclonal antibodies applicable to tissues derived from the same species as the monoclonal antibodies. (J Histochem Cytochem 56:969–975, 2008)     

Identification of dystrophin in cardiac sarcolemmal vesicles

We have identified dystrophin in highly purified sarcolemmal vesicles isolated from canine and bovine hearts using specific antibodies against the COOH-terminal region of the protein. Bovine cardiac sarcolemma contained a single immunoreactive protein band (Mr. approximately 400,000) whereas the canine cardiac membrane contained a doublet (Mr. approximately 420,000 and approximately 380,000). The higher molecular weight form of canine cardiac dystrophin was more abundant than the lower molecular weight form. These highly purified preparations of the sarcolemmal vesicles should provide a useful tool for structural and functional analysis of the interaction of dystrophin with the plasma membrane.     

Monoclonal antibodies to human protein C: effects on the biological activity of activated protein C and the thrombin-catalyzed activation of protein C1

Thirteen monoclonal antibodies designated as MFC-1 to MFC-13 were obtained from hybridoma cells cloned after the fusion of mouse myeloma cells with spleen cells of mice immunized with purified human protein C. Studies were made to determine where the antibodies bound to the molecule of protein C and whether they affected the biological actions of protein C. By using the immunoblotting technique, six of these antibodies were shown to bind to the light chain of protein C, and five to the heavy chain of protein C and also activated protein C. The remaining two antibodies bound to neither the light chain nor the heavy chain, though both antibodies bound to the intact protein C. Antibodies specific for the light chain did not bind to the gamma-carboxyglutamic acid-domain. Two of the antibodies specific for the heavy chain (MFC-13 and -1) inhibited the amidolytic activity of activated protein C. The MFC-13 also inhibited the activity of bovine activated protein C, but not that of human Factor IXa, Factor Xa, or thrombin. In addition to these two antibodies, another one for the heavy chain (MFC-10) and two antibodies for the light chain (MFC-9 and -11) inhibited the inactivation of Factor Va by human activated protein C. One of the antibodies which inhibited the enzyme activity (MFC-1) blocked the inhibition of activated protein C by protein C inhibitor. Another one for the heavy chain (MFC-5) inhibited the activation of protein C by thrombin regardless of the presence or absence of thrombomodulin. Based on these results, we have established the positions of some monoclonal antibody-binding sites on the protein C molecule.     

A Dystrophin-Immunoreactive Protein in Mammalian Brain

Abstract: Dystrophin is expressed only in muscle and brain, but is absent from all tissues of the adult mdx mouse, a mutant with a single base substitution in the dystrophin gene. The brains of both normal and mdx mice contain a protein of ∼230 kDa that is recognised by anti-dystrophin antibodies raised to the N-terminal region of the rod-like domain. Although the N-terminal and central rod regions of dystrophin share structural homologies with spectrin, the 230-kDa protein represents neither of the presently described forms of brain spectrin by a variety of criteria (molecular weight, cerebellar localisation, and developmental regulation) and is distinct from the product of the dystrophin gene. Studies of mdx and normal mouse brain show different postnatal developmental regulation of the 230-kDa dystrophin-immunoreactive protein.