| Abstract
| - Elafin and its precursor trappin-2 (also called pre-elafin) are potent protein inhibitors ofneutrophil serine proteases such as leukocyte elastase and proteinase 3. Trappin-2 has unique conservedsequence motifs rich in Gln and Lys residues. These motifs are substrates for transglutaminases that mayenable trappin-2 to be cross-linked to extracellular matrix proteins, thus anchoring the inhibitor at its siteof action. We have used Western blotting and ELISA-based assays to demonstrate that both elafin andtrappin-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 studiesallowed the determination of the kinetic constants governing the interaction of fibronectin-bound elafinand 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 interactionwith 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 targetenzymes than did the soluble inhibitors, perhaps due to their immobilization, with association rate constantsof 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 firstdemonstration that transglutaminase-mediated cross-linking of serine protease inhibitors to proteins preservestheir inhibitory capacities.
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