Human muscle protein degradation in vitro by eosinophil cationic protein (ECP).
To clarify the role of tissue eosinophils in and around inflammatory foci, we purified eosinophil cationic protein (ECP) and examined its effect on muscle protein degradation in vitro. Eosinophil cationic protein was purified from the buffy coat of blood from healthy volunteers. Myofibrillar, soluble sarcoplasmic, and membrane-associated cytoskeletal proteins were fractionated from latissimus dorsi muscle obtained by orthopedic procedures done on a patient with no neurologic abnormalities. After incubation of these fractions with purified ECP, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were performed. Eosinophil cationic protein degraded the myofibrillar proteins, especially the myosin heavy chain (MHC) and alpha-actinin. It also degraded membrane-associated cytoskeletal proteins dystrophin and spectrin, whereas soluble sarcoplasmic proteins did not undergo proteolysis. Quantitative analysis of the MHC degradation showed that the ECP reaction was dose-dependent and that the optimal pH was 7.0. Protein degradation was not inhibited by heparin or the protease inhibitors leupeptin, E-64, and pepstatin A. Our results suggest that ECP functions in the degradation of myofibrillar and membrane-associated cytoskeletal proteins, indicating that tissue eosinophils have a specific role in muscle fiber degradation in some myopathies associated with numerous tissue eosinophils, such as eosinophilic myositis, eosinophilic myalgia syndrome, and eosinophilic endocardial disease.
Sugihara
R
R
Third Department of Internal Medicine, Oita Medical University, Hasama 1-1, Oita 879-5593, Japan.
Kumamoto
T
T
Ito
T
T
Ueyama
H
H
Toyoshima
I
I
Tsuda
T
T
D000185:Actinin / Q000378:metabolism; D001798:Blood Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D016189:Dystrophin / Q000378:metabolism; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000201:enzymology* / Q000276:immunology; D005343:Fibrinolytic Agents / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D018836:Inflammation Mediators / Q000302:isolation & purification / Q000494:pharmacology*; D007976:Leupeptins / Q000494:pharmacology; D009124:Muscle Proteins / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000378:metabolism; D009220:Myositis / Q000276:immunology / Q000378:metabolism*; D010436:Pepstatins / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D012260:Ribonucleases; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D013049:Spectrin / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology
D001798:Blood Proteins; D015853:Cysteine Proteinase Inhibitors; D016189:Dystrophin; D047091:Eosinophil Granule Proteins; D005343:Fibrinolytic Agents; D018836:Inflammation Mediators; D007976:Leupeptins; D009124:Muscle Proteins; D010436:Pepstatins; D011480:Protease Inhibitors; D000185:Actinin; D013049:Spectrin; D006493:Heparin; D012260:Ribonucleases; D018995:Myosin Heavy Chains; D015032:Zinc; C032854:leupeptin; C031375:pepstatin