Marie-Louise Zani

Proteolytic susceptibility of the serine protease inhibitor trappin-2 (pre-elafin): evidence for tryptase-mediated generation of elafin.

Abstract A number of serine, cysteine, metallo- and acid proteases were evaluated for their ability to proteolytically cleave the serine protease inhibitor trappin-2, also known as pre-elafin, and to release elafin from its precursor. None of the metalloproteases or acid proteases examined cleaved trappin-2, while serine and cysteine proteases preferentially cleaved trappin-2 within its non-inhibitory N-terminal moiety. Cathepsin L, cathepsin K, plasmin, trypsin and tryptase were able to release elafin by cleaving the Lys38-Ala39 peptide bond in trappin-2. However, purified tryptase appeared to be efficient at releasing elafin. Incubation of trappin-2 with purified mast cells first challenged with anti-immunoglobulin E or calcium ionophore A23187 resulted in the rapid generation of elafin. This proteolytic release of elafin from trappin-2 was inhibited in the presence of a tryptase inhibitor, suggesting that this mast cell enzyme was involved in the process. Finally, ex vivo incubation of trappin-2 with sputum from cystic fibrosis patients indicated the production of a proteolytic immunoreactive fragment with the same mass as that of native elafin. This cleavage did not occur when preincubating the sputum with polyclonal antibodies directed against tryptase. Taken together, these findings indicate that tryptase could likely be involved in the maturation of trappin-2 into elafin under physiological conditions.

SLPI and trappin-2 as therapeutic agents to target airway serine proteases in inflammatory lung diseases: current and future directions

It is now clear that NSPs (neutrophil serine proteases), including elastase, Pr3 (proteinase 3) and CatG (cathepsin G) are major pathogenic determinants in chronic inflammatory disorders of the lungs. Two unglycosylated natural protease inhibitors, SLPI (secretory leucocyte protease inhibitor) and elafin, and its precursor trappin-2 that are found in the lungs, have therapeutic potential for reducing the protease-induced inflammatory response. This review examines the multifaceted roles of SLPI and elafin/trappin-2 in the context of their possible use as inhaled drugs for treating chronic lung diseases such as CF (cystic fibrosis) and COPD (chronic obstructive pulmonary disease).

Purification and characterization of active recombinant rat kallikrein rK9

The rat tissue kallikrein rK9 is most abundant in the submandibular gland and the prostate. It has been successfully expressed in the Pichia pastoris yeast expression system. A full-length cDNA coding for the mature rK9 was fused in frame with yeast alpha-factor cDNA. The fusion protein was secreted into the medium with high yield without being processed by the yeast KEX2 signal peptidase. Mature rK9 was efficiently released from the fusion protein by trypsin and was purified to homogeneity by one-step affinity chromatography using soya bean trypsin inhibitor (SBTI) as affinity ligand. The identity of the recombinant enzyme was checked by N-terminal amino acid sequencing, Western blot analysis and kinetic studies. The dual trypsin- and chymotrypsin-like enzymatic specificity of rK9 was assessed by determining specificity constants (k(cat)/K(m)) for the hydrolysis of fluorogenic substrates, the peptide sequences of which were derived from proparathyroid hormone (pro-PTH) and from semenogelin-I. Our results confirmed the presence of an extended binding site in the rK9 active site. We also identified a far more sensitive substrate of this enzyme than those previously described, Abz-VKKRSARQ-EDDnp, which was hydrolysed with a catalytic efficiency k(cat)/K(m) of 420000 M(-1)s(-1). Finally, we showed that four of the five major proteins contained in secretions of rat seminal vesicles were rapidly degraded by recombinant rK9.

New insights into the substrate specificity of macrophage elastase MMP-12

Abstract Macrophage elastase, or MMP-12, is mainly produced by alveolar macrophages and is believed to play a major role in the development of chronic obstructive pulmonary disease (COPD). The catalytic domain of MMP-12 is unique among MMPs in that it is very highly active on numerous substrates including elastin. However, measuring MMP-12 activity in biological fluids has been hampered by the lack of highly selective substrates. We therefore synthesized four series of fluorogenic peptide substrates based on the sequences of MMP-12 cleavage sites in its known substrates. Human MMP-12 efficiently cleaved peptide substrates containing a Pro at P3 in the sequence Pro-X-X↓Leu but lacked selectivity towards these substrates compared to other MMPs, including MMP-2, MMP-7, MMP-9 and MMP-13. On the contrary, the substrate Abz-RNALAVERTAS-EDDnp derived from the CXCR5 chemokine was the most selective substrate for MMP-12 ever reported. All substrates were cleaved more efficiently by full-length MMP-12 than by its catalytic domain alone, indicating that the C-terminal hemopexin domain influences substrate binding and/or catalysis. Docking experiments revealed unexpected interactions between the peptide substrate Abz-RNALAVERTAS-EDDn and MMP-12 residues. Most of our substrates were poorly cleaved by murine MMP-12 suggesting that human and murine MMP-12 have different substrate specificities despite their structural similarity.

Protection of lung epithelial cells from protease-mediated injury by trappin-2 A62L, an engineered inhibitor of neutrophil serine proteases.

Neutrophil serine proteases (NSPs), including elastase, proteinase 3 and cathepsin G, play critical roles in the pathogenesis of chronic inflammatory lung diseases. The release of excess NSPs leads to the destruction of lung tissue and an overexuberant, sustained inflammatory response. Antiproteases could be valuable tools for controlling these NSP-mediated inflammatory events. We have examined the capacity of trappin-2 A62L, a potent engineered inhibitor of all three NSPs, to protect human lung A549 epithelial cells from the deleterious effects of NSPs. Trappin-2 A62L, significantly inhibited the detachment of A549 cells and the degradation of the tight-junction proteins, E-cadherin, β-catenin and ZO-1, induced by each individual NSP and by activated neutrophils. Trappin-2 A62L also decreased the release of the pro-inflammatory cytokines IL-6 and IL-8 from A549 cells that had been stimulated with elastase or LPS. Trappin-2 A62D/M63L, a trappin-2 variant that has no antiprotease activity, has similar properties, suggesting that the anti-inflammatory action of trappin-2 is independent of its antiprotease activity. Interestingly, we present evidence that trappin-2 A62L, as well as wild-type trappin-2, enter A549 cells and move rapidly to the cytoplasm and nucleus, where they are likely to exert their anti-inflammatory effects. We have also demonstrated that trappin-2 A62L inhibits the early apoptosis of A549 cells mediated by NSPs. Thus, our data indicate that trappin-2 A62L is a powerful anti-protease and anti-inflammatory agent that could be used to develop a treatment for patients with inflammatory lung diseases.

Complete 1H, 15N and 13C assignment of trappin-2 and 1H assignment of its two domains, elafin and cementoin

AbstractnTrappin-2 is a serine protease inhibitor with a very narrow inhibitory spectrum and has significant anti-microbial activities. It is a 10xa0kDa cationic protein composed of two distinct domains. The N-terminal domain (38 residues) named cementoin is known to be intrinsically disordered when it is not linked to the elafin. The C-terminal domain (57 residues), corresponding to elafin, is a cysteine-rich domain stabilized by four disulfide bridges and is characterized by a flat core and a flexible N-terminal part. To our knowledge, there is no structural data available on trappin-2. We report here the complete 1H, 15N and 13C resonance assignment of the recombinant trappin-2 and the 1H assignments of cementoin and elafin, under the same experimental conditions. This is the first step towards the 3D structure determination of the trappin-2.

Étude des propriétés de transglutamination de deux inhibiteurs des protéases à sérine de neutrophile à potentiel thérapeutique

Introductionxa0: L’elafine et le SLPI sont deux inhibiteurs de proteases particulierement attractifs dans le cadre d’une utilisation therapeutique anti-inflammatoire visant a cibler les proteases a serine de neutrophile (elastase, protease 3 et cathepsine G) dans les pathologies pulmonaires. L’elafine est liberee a partir d’un precurseur actif, la trappine-2 (ou pre-elafine), qui comporte en plus du domaine elafine inhibiteur, un domaine cementoine a son extremite N-terminale, riche en sequences de type GQDPVK substrat de transglutaminase. Dans nos travaux precedents, nous avons demontre que la trappine-2 et l’elafine, pouvaient se lier de maniere covalente a differentes proteines de matrice extracellulaire dont la fibronectine, grâce a l’action d’une transglutaminase tissulaire. Dans ces conditions, nous avons egalement montre que l’inhibiteur ainsi lie conservait ses proprietes inhibitrices vis-a-vis de ses proteases cibles. Par ailleurs, il a ete demontre que le SLPI pouvait in vivo etre associe aux fibres d’elastine (Histochem., 1986, 86, 165-168). Les objectifs de ce travail sont 1/ d’evaluer la sensibilite du SLPI a l’action d’une transglutaminase et 2/ d’identifier les sites de transglutamination mis en jeu dans la trappine-2 et le SLPI. Methodes et resultatsxa0: Les complexes inhibiteurs/proteines de structure ont ete formes in vitro en presence de transglutaminase 2 et detectes par western blot et ELISA. L’activite inhibitrice des complexes vis-a-vis des proteases a serine de neutrophile a ete mesuree a l’aide de substrats fluorogeniques specifiques de chaque protease, developpes precedemment au laboratoire. Les resultats montrent que le SLPI peut etre lie a la fibronectine et a l’elastine par l’action de la transglutaminase 2 tout en restant inhibiteur de l’elastase et de la cathepsine G.xa0Les sites de transglutamination dans la trappine-2 et le SLPI impliquent a la fois des glutamines et des lysines du motif consensus comme le montre l’analyse en spectrometrie de masse de differents types de conjugues formes avec la transglutaminase 2. Conclusionxa0: L’ancrage des inhibiteurs a des proteines de la matrice extracellulaire par les transglutaminases tissulaires, dont l’expression est fortement augmentee au cours de l’inflammation, pourrait permettre d’augmenter leur biodisponibilite lors d’une administration therapeutique.