Duchenne muscular dystrophy (DMD) is a debilitating X-linked muscle disease. We have used sequence information from complementary DNA clones, derived from the gene that is deleted in DMD patients, to generate an antiserum that stains the surface membrane of intact human and mouse skeletal muscle, but not that of DMD patients and mdx mice. Here we identify the protein reacting with this antiserum as a single component of relative molecular mass 210,000 (Mr = 210K) that fractionates with a low-ionic strength extract of intact human and mouse skeletal muscle. It is therefore distinct from the 400 K protein found in the heavy microsomal fraction of normal muscle and identified as a putative product of the DMD gene. We also analyse further the disease specificity of the antiserum. Positive staining is seen in normal controls, and in samples from patients with a wide range of muscular dystrophies other than DMD. Becker muscular dystrophy, which is allelically related to DMD, was the only other exception, and gave a sporadic staining pattern. The demonstration of a specific defect in the surface membrane of DMD muscle fibres substantiates the hypothesis that membrane lesions may initiate muscle degradation in DMD.

No abstract

We previously reported that a protein which has immunological cross-reactivity with and a molecular weight similar to dystrophin, the Duchenne muscular dystrophy (DMD) gene product, is expressed on the muscle cell membrane (Tanaka et al. 1989b). To examine if this is the translation product of the autosomal transcript with homology to dystrophin mRNA identified by Love et al. (1989), we raised an antibody (PDRP) against a synthetic peptide corresponding to the putative protein (DRP) and examined its expression and cellular localization in human and murine skeletal muscle samples. In immunoblotting, PDRP stained a band with a similar molecular weight to dystrophin in samples from DMD and Becker muscular dystrophy (BMD) patients and control (non-DMD/BMD) human. PDRP was expected not to cross-react with dystrophin because the antigenic peptide was not homologous to dystrophin. In fact, PDRP did not cross-react with dystrophin present in a BMD patient. Immunohistochemically, PDRP stained the muscle cell membrane in samples from DMB and BMD patients and from mdx mice. Only a slight staining was observed in muscles from control human and wild type mice. Our results confirm the presence of DRP in human and murine skeletal muscles, and further demonstrate that it is localized on the cell membrane. The abundance of DRP in dystrophin deficient muscles might be related to some compensatory mechanisms.

Recent studies of Kunkel and his associatesl)-5) have clarified the cloned complementary DNA sequences corresponding to the Duchenne muscular dystrophy (DMD) gene. In the end of 1987, Hoffman et al.~>>7) obtained specific antibodies directed to the proteins encoded by fragments of the mouse DMD gene which is very similar to the human DMD gene. These antibodies recognized a specific protein, named "dystrophin" in normal skeletal and cardiac muscle of both humans and mice, which is absent in two DMD patients and mdx mice. By using these antibodies, Hoffman et al.°) have shown that dystrophin is associated with intracellular membrane fraction, especially the triads, on the basis of Western blot analysis using the pellet obtained by the subcellular fractionation of the mouse and rabbit skeletal muscle. Here we report that the antiserum raised against peptide fragment prepared as predicted from the distinctive region of DMD cDNA stained clearly the surface membranes of normal skeletal muscles of both humans and mice, but did not those of both DMD and mdx skeletal muscles, immunohistochemically. Materials and methods. The peptide fragment was predicted from position 1526 to 1675 on the human cDNA map7} and an extra Tyr was added to the N-terminus. The protected peptide was synthesized by the solid phase method on Applied Biosystems model 430A and deprotected with HF.9) The peptide was purified by preparative reversed phase HPLC. It consists of 51 amino acids, having a molecular weight of 6,267. New Zealand white female rabbit was immunized with intradermal injection of 2 mg of the peptide with Freund's complete adjuvant 3 times every 2 weeks to obtain the antibody. The 4 im frozen sections of the biopsied muscles of 5 patients with DMD and 5 other related neuromuscular diseases and 3 normal controls and 3 mdx (C57BL/lOScSn-mdx) and normal control mice (C57BL/1OScSn) hind limb muscles were incubated with antiserum and stained with fluorescence labeled antibody against rabbit IgG and were observed under fluorescence microscope (Zeiss, Axiophot). Results. As shown in Fig. 1B, the antiserum stained the whole surface