title: Kinetics of the inhibition of neutrophil proteinases by recombinant elafin and pre-elafin (trappin-2) expressed in Pichia pastoris.
abstract: Elafin and its precursor, trappin-2 or pre-elafin, are specific endogenous inhibitors of human neutrophil elastase and proteinase 3 but not of cathepsin G. Both inhibitors belong, together with secretory leukocyte protease inhibitor, to the chelonianin family of canonical protease inhibitors of serine proteases. A cDNA coding either elafin or its precursor, trappin-2, was fused in frame with yeast alpha-factor cDNA and expressed in the Pichia pastoris yeast expression system. Full-length elafin or full-length trappin-2 were secreted into the culture medium with high yield, indicating correct processing of the fusion proteins by the yeast KEX2 signal peptidase. Both recombinant inhibitors were purified to homogeneity from concentrated culture medium by one-step cationic exchange chromatography and characterized by N-terminal amino acid sequencing, Western blot and kinetic studies. Both recombinant elafin and trappin-2 were found to be fast-acting inhibitors of pancreatic elastase, neutrophil elastase and proteinase 3 with k(ass) values of 2-4 x 10(6) m(-1).s(-1), while dissociation rate constants k(diss) were found to be in the 10(-4) s(-1) range, indicating low reversibility of the complexes. The equilibrium dissociation constant K(i) for the interaction of both recombinant inhibitors with their target enzymes was either directly measured for pancreatic elastase or calculated from k(ass) and k(diss) values for neutrophil elastase and proteinase 3. K(i) values were found to be in the 10(-10) molar range and virtually identical for both inhibitors. Based on the kinetic parameters determined here, it may be concluded that both recombinant elafin and trappin-2 may act as potent anti-inflammatory molecules and may be of therapeutic potential in the treatment of various inflammatory lung diseases.
authors.affiliation: INSERM U618, University François Rabelais, Tours, France.
authors.forename: Marie-Louise
authors.initials: ML
authors.lastname: Zani
authors.forename: Shila M
authors.initials: SM
authors.lastname: Nobar
authors.forename: Sandrine A
authors.initials: SA
authors.lastname: Lacour
authors.forename: Soazig
authors.initials: S
authors.lastname: Lemoine
authors.forename: Christian
authors.initials: C
authors.lastname: Boudier
authors.forename: Joseph G
authors.initials: JG
authors.lastname: Bieth
authors.forename: Thierry
authors.initials: T
authors.lastname: Moreau
mesh_terms: D000818:Animals; D006801:Humans; D019272:Leukocyte Elastase / Q000378:metabolism*; D053146:Myeloblastin; D010196:Pancreatic Elastase / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000378:metabolism*; D013552:Swine
chemical_list: D011498:Protein Precursors; D053491:Proteinase Inhibitory Proteins, Secretory; D011506:Proteins; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase; D053146:Myeloblastin