Elafin and its precursor trappin-2 still inhibit neutrophil serine proteinases when they are covalently bound to extracellular matrix proteins by tissue transglutaminase

Biochemistry. 2005 Nov 29;44(47):15610-8. doi: 10.1021/bi051418i.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Elafin
  • Extracellular Matrix Proteins / metabolism*
  • Fibronectins / metabolism
  • GTP-Binding Proteins / metabolism*
  • Humans
  • Kinetics
  • Leukocyte Elastase / antagonists & inhibitors
  • Leukocyte Elastase / metabolism*
  • Myeloblastin
  • Protein Interaction Mapping
  • Protein Precursors / metabolism*
  • Proteinase Inhibitory Proteins, Secretory
  • Proteins / metabolism*
  • Serine Endopeptidases / metabolism
  • Serine Proteinase Inhibitors / metabolism
  • Surface Plasmon Resonance
  • Transglutaminases / metabolism*

Substances

  • Elafin
  • Extracellular Matrix Proteins
  • Fibronectins
  • PI3 protein, human
  • Protein Precursors
  • Proteinase Inhibitory Proteins, Secretory
  • Proteins
  • Serine Proteinase Inhibitors
  • transglutaminase 2
  • Transglutaminases
  • Serine Endopeptidases
  • Leukocyte Elastase
  • Myeloblastin
  • GTP-Binding Proteins