Recombinant cryptic human fibronectinase cleaves actin and myosin: substrate specificity and possible role in muscular dystrophy.
The N-terminal heparin/fibrin binding domain of human plasma fibronectin (pFN) contains a cryptic proteinase. The enzyme could be generated and activated in the presence of Ca2+ from the purified 70 kDa pFN fragment produced by cathepsin D digestion of pFN. In this work we cloned and expressed the serine proteinase, designated fibronectinase (Fnase), in E. coli. The recombinant pFN protein fragment was isolated from inclusion bodies, subjected to folding and autocatalytic degradation in the presence of Ca2+, and yielded an active enzyme capable of digesting fibronectin. Cleavage of pFN and the synthetic peptides Ac-I-E-G-K-pNA and Bz-I-E-G-R-pNA demonstrated identical specificity of the recombinant and the isolated fibronectinase. Further investigations of the substrate specificity revealed for the first time the muscle proteins actin and myosin as being substrates of fibronectinase. The enzyme can be inhibited by alpha1-proteinase inhibitor. In the context of induced cathepsin D release, e. g. from granulocytes under inflammatory conditions, these results indicate an increase in specific proteolytic potential against muscular proteins in dystrophic diseases by the release of cryptic fibronectinase.
Schnepel
J
J
Department of Biochemistry I, Faculty of Chemistry, University of Bielefeld, Germany.
Unger
J
J
Tschesche
H
H
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics; D005337:Fibrin / Q000378:metabolism; D006801:Humans; D009136:Muscular Dystrophies / Q000201:enzymology / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D000515:alpha 1-Antitrypsin / Q000494:pharmacology
D000199:Actins; D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; D005337:Fibrin; D012697:Serine Endopeptidases; C071222:fibronectinase; D009218:Myosins