Tài liệu Báo cáo khoa học: Oxidized elafin and trappin poorly inhibit the elastolytic activity of

Oxidized elafin and trappin poorly inhibit the elastolytic

activity of neutrophil elastase and proteinase 3

Shila M. Nobar1

, Marie-Louise Zani2

, Christian Boudier1

, Thierry Moreau2 and Joseph G. Bieth1

1 Laboratoire d’Enzymologie, INSERM U392, Universite´ Louis Pasteur de Strasbourg, Illkirch, France

2 INSERM U618, Universite´ Franc¸ois Rabelais, Tours, France

Many amino acid residues of proteins are susceptible to

oxidation by reactive oxygen species. Methionine, the

most sensitive of amino acids to oxidation, is readily

transformed into a mixture of the S- and R-epimers of

methionine sulfoxide. The latter may be recycled by

methionine sulfoxide reductases in the presence of thio￾redoxin, which itself may be regenerated by thioredoxin

reductase in an NADPH-dependent reaction. Excessive

methionine sulfoxide production and ⁄ or a defect in its

recycling is believed to be involved in age-related

diseases and in shortening of the maximum life span [1].

Oxidative processes also take place in lung infection

and inflammation, where they are used, in conjunction

with proteolytic enzymes, to kill bacteria and destroy

foreign substances in the phagolysosome of polymor￾phonuclear neutrophils. The membrane of these phago￾cytes contains an NADPH oxidase, which transforms

molecular oxygen into the short-lived superoxide

anion. Superoxide dismutase transforms the latter into

H2O2, an oxidant that further yields hypochloride in

the presence of neutrophil myeloperoxidase. Aliphatic

amines transform hypochloride into chloramines,

which are potent and long-lived oxidants [2].

In inflammatory lung diseases, such as chronic bron￾chitis, emphysema or cystic fibrosis, excessive recruit￾ment, activation or lysis of neutrophils results in

the extracellular release of neutrophil elastase (NE;

EC 3.4.21.37), proteinase 3 (Pr3; EC 3.4.21.76) and

Keywords

elafin; elastase; enzyme kinetics; oxidation;

proteinase 3

Correspondence

J. G. Bieth, INSERM U 392, Faculte´ de

Pharmacie, 74 route du Rhin,

67400 Illkirch, France

Fax: +33 3 90 24 43 08

Tel: +33 3 90 24 41 82

E-mail: [email protected]

(Received 20 May 2005, revised 24 August

2005, accepted 22 September 2005)

doi:10.1111/j.1742-4658.2005.04988.x

Neutrophil proteinase-mediated lung tissue destruction is prevented by

inhibitors, including elafin and its precursor, trappin. We wanted to estab￾lish whether neutrophil-derived oxidants might impair the inhibitory func￾tion of these molecules. Myeloperoxidase ⁄ H2O2 and N-chlorosuccinimide

oxidation of the inhibitors was checked by mass spectrometry and enzy￾matic methods. Oxidation significantly lowers the affinities of the two

inhibitors for neutrophil elastase (NE) and proteinase 3 (Pr3). This

decrease in affinity is essentially caused by an increase in the rate of inhibi￾tory complex dissociation. Oxidized elafin and trappin have, however, rea￾sonable affinities for NE (Ki ¼ 4.0–9.2 · 10)9 m) and for Pr3 (Ki ¼ 2.5–

5.0 · 10)8 m). These affinities are theoretically sufficient to allow the oxi￾dized inhibitors to form tight binding complexes with NE and Pr3 in lung

secretions where their physiological concentrations are in the micromolar

range. Yet, they are unable to efficiently inhibit the elastolytic activity of

the two enzymes. At their physiological concentration, fully oxidized elafin

and trappin do not inhibit more than 30% of an equimolar concentration

of NE or Pr3. We conclude that in vivo oxidation of elafin and trappin

strongly impairs their activity. Inhibitor-based therapy of inflammatory

lung diseases must be carried out using oxidation-resistant variants of these

molecules.

Abbreviations

Lys-(pico), lysyl-(2-picolinoyl); MeOSuc, methoxysuccinyl; NE, human neutrophil elastase; pNA, p-nitroanilide; Pr3, human neutrophil

proteinase 3; RBB–elastin, remazol-Brilliant Blue–elastin.

FEBS Journal 272 (2005) 5883–5893 ª 2005 FEBS 5883