CLONING OF THE CDNA AND NUCLEOTIDE-SEQUENCE OF A SKELETAL-MUSCLE PROTEASE FROM MYOPATHIC HAMSTERS

A neutral protease with an estimated M-r of about 26 kD and responsibl e for cleavage of myosin LC2 was isolated from hamster skeletal muscle . Complementary DNAs were generated by RT-PCR using total hamster musc le RNA and degenerate oligonucleotide primers based on the sequences o f two internal peptides. The nucleotide sequences of the resultant cDN As were subsequently determined and the complete amino acid sequence o f the protease deduced. Although the hamster protein shared 63-85% ide ntity in nucleotide and amino acid sequences with rat and mouse mast c ell proteases, it had a higher degree of specificity for myosin LC2 th an mast cell proteases which also digested myosin LC1 and myosin heavy chains. As a result, the hamster protease was designated mekratin bec ause of its unique enzymatic specificities to distinguish it from othe r mast cell proteases. A polyclonal antibody was raised specific to th e hamster muscle and human cardiac muscle mekratins without apparent c ross-reaction with rat mast cell proteases. We have earlier demonstrat ed the presence in excess of a neutral protease that specifically clea ves LC2 in human hearts obtained at end stage idiopathic dilated cardi omyopathy (IDC). Western analyses revealed that heart tissue from pati ents with IDC contained 5-10 fold more mekratin than control samples. Furthermore, the level of the protease in human IDC tissues was simila r to that seen in myopathic hamster skeletal muscle. No bands were rec ognized by the antibody when IDC myofibrils were probed due to the rem oval of soluble proteins during sample preparation. Thus, these result s strongly suggest that the anti-mekratin antibody will provide positi ve identification of IDC in many cases and diagnosis by exclusion may be replaced.