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.

Elafin, an Elastase-specific Inhibitor, Is Cleaved by Its Cognate Enzyme Neutrophil Elastase in Sputum from Individuals with Cystic Fibrosis

“…Transglutaminases are a family of enzymes catalyzing the formation of an isopeptide bond between the side chains of a Gln and a Lys residue belonging to two different proteins. By this mechanism, both elafin and trappin-2 are able to be cross-linked with a variety of proteins, although trappin-2 was shown to be more rapidly immobilized, likely due to a higher amount of transglutaminase substrate motif (5). In vivo, trappin-2/elafin have been found covalently complexed in several epithelia and colocalized with type-1 transglutaminase, suggesting that the immobilization process results from transglutaminase activities.…”

“…In the present study we have demonstrated that elafin is rapidly cleaved in P. aeruginosa-positive CF sputum compared with P. aeruginosa-negative CF samples. Airways from CF individuals is known to be low (pH [5][6], as demonstrated in exhaled breath condensate from patients with exacerbated and stable CF (34).…”

“…In tracheal epithelium, trappin-2/elafin is also found in a complexed form (4). Previous in vitro studies showed that trappin-2 and/or elafin can covalently bind to a number of ECM proteins including elastin, fibronectin, laminin, fibrinogen, collagen, and ␤-crystallin by transglutamination (4,5,48). The transglutaminase-mediated cross-linking of trappin-2 and elafin to fibronectin and elastin was demonstrated to preserve their antiprotease activity and to protect the associated ECM molecule against proteolysis mediated by NE (5,48).…”

“…Previous in vitro studies showed that trappin-2 and/or elafin can covalently bind to a number of ECM proteins including elastin, fibronectin, laminin, fibrinogen, collagen, and ␤-crystallin by transglutamination (4,5,48). The transglutaminase-mediated cross-linking of trappin-2 and elafin to fibronectin and elastin was demonstrated to preserve their antiprotease activity and to protect the associated ECM molecule against proteolysis mediated by NE (5,48). Hence, the immobilization of trappin-2/elafin ECM proteins in elastic tissues such as lung and skin may play a protective role by preserving the structural integrity of the tissue against damages caused by neutrophilic infiltrations during inflammation.…”

“…In addition to its ability to inhibit porcine pancreatic elastase, elafin is a potent inhibitor of two neutrophil serine proteases, neutrophil elastase (NE) and proteinase 3 (1,2), and is therefore thought to protect tissue from degradation by these enzymes. Elafin is released by proteolytic cleavage from a larger molecule called trappin-2 or pre-elafin, which possesses at the N terminus of the whey acidic protein domain a cementoin domain containing several motifs having the consensus sequence GQDPVK that can act as transglutaminase substrate, allowing the cross-linking of the inhibitor to extracellular matrix proteins (3)(4)(5). It has been shown that tryptase, a mast cell-derived protease, may be involved in the proteolytic processing of trappin-2 into elafin (6).…”