RESUMEN
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.
Asunto(s)
Actinas/metabolismo , Distrofias Musculares/metabolismo , Miosinas/metabolismo , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Electroforesis en Gel de Agar , Escherichia coli/genética , Fibrina/metabolismo , Humanos , Distrofias Musculares/enzimología , Reacción en Cadena de la Polimerasa , Pliegue de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidasas/genética , Especificidad por Sustrato , alfa 1-Antitripsina/farmacologíaRESUMEN
Human plasma fibronectin (pFN) contains a cryptic metalloprotease present in the collagen-binding domain. The enzyme could be generated and activated in the presence of Ca2+ from the purified 70-kDa pFN fragment produced by cathepsin D digestion. In this work we cloned and expressed the metalloprotease, designated FN type IV collagenase (FnColA), and a truncated variant (FnColB) in E. coli. The recombinant pFN protein fragment was isolated from inclusion bodies, and subjected to folding and autocatalytic degradation in the presence of Ca2+, and yielded an active enzyme capable of digesting gelatin, helical type II and type IV collagen, alpha- and beta-casein, insulin b-chain, and a synthetic Mca-peptide. In contrast, isolated plasma fibronectin, type I collagen, and the DNP-peptide were no substrates. Both catalytically active recombinant pFN fragments were efficiently inhibited by EDTA, and batimastat, and, in contrast to the glycosylated enzyme isolated from plasma fibronectin, were also inhibited by TIMP-2.