Molecular Cloning of Equine (Thoroughbred) Myostatin cDNA and Detection of Myostatin Precursor Proteins in the Serum

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Growth/Differentiation Factor 8, or GDF-8, is a member of the TGF-β family and is expressed specifically in myogenic precursor cells of the myotome during development as well as in adult axial and paraxial muscles of the mouse. It acts as a negative regulator of skeletal muscle mass, and is known as myostatin. As a part of our studies to elucidate mechanisms underlying the growth of skeletal muscle mass due to physical training in mammals including horses and humans, we isolated and cloned myostatin cDNA from equine (Thoroughbred) skeletal muscles by an RT-PCR method. The base sequence of the entire coding region of equine myostatin cDNA (DDBJ accession no. AB033541) exhibited a high degree of homology to those of other species so far reported. In the deduced amino acid sequence, 4 amino acid positions were unique to the equine myostatin, i. e., Val¹⁵ instead of Met, Leu¹⁸ instead of Val or Ala, Ala²⁰¹ instead of Thr, Arg²⁴⁴ instead of Gly or Glu in other species. Western blot analysis of the equine skeletal muscles using anti-myostatin antibody, yielded 3 immunoreactive bands, i. e., 26, 45 and 50 kDa. The band at approximately 26 kDa probably represents mature myostatin and another at approximately 50 kDa corresponds in size to the unprocessed precursor myostatin proteins reported in the literature. Western blot analysis of equine sera of 2 independent breeds, Thoroughbred and Kiso-uma, revealed the possible presence of precursor myostatin proteins at relatively high levels. The molecular identity and the biological significance of serum myostatin immunoreactivities remain to be clarified.