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Elafin and Its Precursor Trappin-2 Still Inhibit Neutrophil Serine Proteinases when They Are Covalently Bound to Extracellular Matrix Proteins by Tissue Transglutaminase

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INSERM U618 Protéases et Vectorisation Pulmonaires and IFR 135 Imagerie Fonctionnelle, Université François Rabelais, 10 Bd Tonnellé, BP 3223, 37032 Tours Cedex, France, and UMR 6175 Physiologie de la Reproduction et des Comportements, INRA, 37380 Nouzilly, France
Cite this: Biochemistry 2005, 44, 47, 15610–15618
Publication Date (Web):November 5, 2005
Copyright © 2005 American Chemical Society

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    Abstract Image

    Elafin and its precursor trappin-2 (also called pre-elafin) are potent protein inhibitors of neutrophil serine proteases such as leukocyte elastase and proteinase 3. Trappin-2 has unique conserved sequence motifs rich in Gln and Lys residues. These motifs are substrates for transglutaminases that may enable trappin-2 to be cross-linked to extracellular matrix proteins, thus anchoring the inhibitor at its site of action. We have used Western blotting and ELISA-based assays to demonstrate that both elafin and trappin-2 can be conjugated to various extracellular matrix proteins in vitro by a type 2 transglutaminase. Cross-linked elafin and trappin-2 still inhibited their target proteases. Surface plasmon resonance studies allowed the determination of the kinetic constants governing the interaction of fibronectin-bound elafin and trappin-2 with neutrophil elastase and proteinase 3. Both inhibitors were potent inhibitors when cross-linked to fibronectin by transglutamination, with equilibrium dissociation constants Ki for their interaction with target proteases of 0.3 nM (elastase−elafin), 20 nM (proteinase 3−elafin), 0.3 nM (elastase−trappin-2), and 12 nM (proteinase 3−trappin-2). The conjugated inhibitors reacted more slowly with their target enzymes than did the soluble inhibitors, perhaps due to their immobilization, with association rate constants of 2−7 × 105 M-1 s-1 for elastase and 1−4 × 104 M-1 s-1 for proteinase 3. We believe this is the first demonstration that transglutaminase-mediated cross-linking of serine protease inhibitors to proteins preserves their inhibitory capacities.

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     N.G. holds a doctoral fellowship from MENRT (Ministère de l'Education Nationale, de la Recherche et de la Technologie). This work was supported by the French cystic fibrosis association “Vaincre la Mucoviscidose”.

     Université François Rabelais.




     To whom correspondence should be addressed:  INSERM U 618, University François Rabelais, 10 Bd Tonnellé, 37032 Tours Cedex, France. Fax:  +33 247 366 046. Telephone: +33 2 4736 6177. E-mail: [email protected].

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