title: Elafin and its precursor trappin-2 still inhibit neutrophil serine proteinases when they are covalently bound to extracellular matrix proteins by tissue transglutaminase.
abstract: 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 K(i) 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 x 10(5) M(-)(1) s(-)(1) for elastase and 1-4 x 10(4) 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.
authors.affiliation: INSERM U618 Protéases et Vectorisation Pulmonaires and IFR 135 Imagerie Fonctionnelle, Université François Rabelais, 10 Bd Tonnellé, BP 3223, 37032 Tours Cedex, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Guyot
authors.forename: Marie-Louise
authors.initials: ML
authors.lastname: Zani
authors.forename: Marie-Christine
authors.initials: MC
authors.lastname: Maurel
authors.forename: Sandrine
authors.initials: S
authors.lastname: Dallet-Choisy
authors.forename: Thierry
authors.initials: T
authors.lastname: Moreau
mesh_terms: D053492:Elafin; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053146:Myeloblastin; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D025941:Protein Interaction Mapping; D011498:Protein Precursors / Q000378:metabolism*; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000378:metabolism; D020349:Surface Plasmon Resonance; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D053492:Elafin; D016326:Extracellular Matrix Proteins; D005353:Fibronectins; C506806:PI3 protein, human; D011498:Protein Precursors; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D015842:Serine Proteinase Inhibitors; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D012697:Serine Endopeptidases; D019272:Leukocyte Elastase; D053146:Myeloblastin; D019204:GTP-Binding Proteins