No abstract

Trappin‐2 (also known as pre‐elafin) is an endogenous inhibitor of neutrophil serine proteases and is involved in the control of excess proteolysis, especially in inflammatory events, along with the structurally related secretory leucocyte proteinase inhibitor. Secretory leucocyte proteinase inhibitor has been shown to have antibacterial and antifungal properties, whereas recent data indicate that trappin‐2 has antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus. In the present study, we tested the antibacterial properties of trappin‐2 towards other respiratory pathogens. We found that trappin‐2, at concentrations of 5–20 μm, has significant activity against Klebsiella pneumoniae, Haemophilus influenzae, Streptococcus pneumoniae, Branhamella catarrhalis and the pathogenic fungi Aspergillus fumigatus and Candida albicans, in addition to P. aeruginosa and S. aureus. A similar antimicrobial activity was observed with trappin‐2 A62D/M63L, a trappin‐2 variant that has lost its antiprotease properties, indicating that trappin‐2 exerts its antibacterial effects through mechanisms independent from its intrinsic antiprotease capacity. Furthermore, the antibacterial and antifungal activities of trappin‐2 were sensitive to NaCl and heparin, demonstrating that its mechanism of action is most probably dependent on its cationic nature. This enables trappin‐2 to interact with the membranes of target organisms and disrupt them, as shown by our scanning electron microscopy analyses. Thus, trappin‐2 not only provides an antiprotease shield, but also may play an important role in the innate defense of the human lungs and mucosae against pathogenic microorganisms.

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.