DNA binds neutrophil elastase and mucus proteinase inhibitor and impairs their functional activity

Abstract

DNA binds neutrophil elastase and mucus proteinase inhibitor as evidenced by affinity chromatography on elastaseSepharose, inhibitor-Sepharose and DNA-cellulose. DNA is a potent hyperbolic inhibitor of elastase. The polynucleotide-en~yme complex is partially active on synthetic substrates and on elastin. DNA strongly increases kd~ and Ki for the inhibition of elastase by mucus proteinase inhibitor kass t E + I ~ EI, K i = kdi~Jka~s).kdiss ['he above effects are all salt-dependent. At physiological ionic strength, DNA is a potent inhibitor of the elastolytic activity of elastase and increases k~ and K~ for the elastase-mucus proteinase inhibitor interaction 160-fold and 100-fold, respectively.,~ey words': Elastase; Mucus proteinase inhibitor; Enzyme i:inetics; DNA; Cystic fibrosis L. IntroductionNeutrophil elastase (NE) is a 30-kDa cationic glycoprotein .vhose crystal structure is known. This serine proteinase cleaves t number of plasma and extracellular matrix proteins including ,.:lastin. It is stored in the azurophil granules of neutrophils from ,vhich it may be released following excessive phagocytosis or :ell death. Its extracellular proteolytic action is normally pre-,lented by protein proteinase inhibitors such as ~l-proteinase nhibitor, ~2-macroglobulin or mucus proteinase inhibitor MPI) (for a review see [1]). The latter is an 11.7-kDa unglyco~ylated cationic protein composed of two domains of similar ;ize and architecture ([2] and refs. therein).MPI is the most abundant NE inhibitor of airways secretions ~here it occurs in concentrations as high as 5/IM [3]. Despite ~:his high load of inhibitor, bronchial secretions from patients ~vith cystic fibrosis contain significant amounts of active NE [4]. On the other hand, these secretions also contain large quantities ~f neutrophil-derived DNA [5]. This raises the question as to uhether DNA interferes with the inhibition of NE by MPI. The ~resent investigation attempts to answer this question. Materials and methods MaterialsHuman NE was purified from purulent sputum as described in [6]. Recombinant MPI was obtained from Synergen, Boulder, CO through the courtesy of Dr. H.P. Schnebli, Ciba-Geigy, Basel, Switzerland. NE and MPI were active site titrated as indicated in [6]. Stock solutions of Suc-Ala3-pNA and MeOSuc-Ala2-Pro-Val-pNA (Bachem, Bubendorf, Switzerland) were made in N-methylpyrolidone and dimethylformamide, respectively. The final concentration of organic solvent in the reaction mixtures was 2% (v/v) throughout. Remazol Brilliant-blue elastin (RBB-elastin) was purchased from Elastin Products Company (Owenville, MO). Salmon sperm DNA and DNA~cellulose came from Sigma. NE or MPI were coupled to epoxy-activated Sepharose (Pharmacia) as described by the manufacturer. The three affinity supports (4 ml) were poured into HR 10/10 Pharmacia columns and equilibrated with 50 mM HEPES buffer, pH 7.4. Determination of k,,, and K,~The kinetic constants kca t and Km for the hydrolysis of MeOSuc-Ala2-Pro-Val-pNA by 30 nM free or DNA-bound NE were determine...

Inhibition of Neutrophil Serine Proteinases by Suramin

“…Similar differences are also observed with cat G. DNA is another polyanion that reacts with NE. At physiological ionic strength, unfractionated DNA yield 25% inhibition of NE activity (29), whereas in vitro selected DNA sequences with high affinity for NE have no significant inhibitory effect on the enzyme (30). Tight enzyme-ligand binding with lack of inhibition might be explained by assuming ␣ ϭ 1 and ␤ ϭ 1, for instance (see Equation 3).…”

DNA Strongly Impairs the Inhibition of Cathepsin G by α1-Antichymotrypsin and α1-Proteinase Inhibitor

“…The inhibition of 30 nM proteinase 3 by concentrations of 30bp DNA up to 1 M was assessed using 2 mM methoxysuccinyl-Lys(2pico)-Ala-Pro-Val-pNA (14). The effect of 30bp DNA on the elastolytic activity of these two proteinases was tested using Remazol Brilliant Blue elastin as described previously (8). The final concentration of the proteinases was 0.5 M. One single concentration of 30bp DNA (15 M) was used in replicate assays.…”

NETs: a new strategy for using old weapons

“…Under such conditions, NETs can protect bystander cells in two ways: 1) by restricting the dispersal of toxic antimicrobials through anchoring them to the decondensed DNA scaffold and 2) by downregulation of their toxic activity. NE has been shown to interact with DNA and chromatin which leads to a dramatic reduction in its proteolytic activity [64]. It is therefore likely that when bound to NETs, NE has reduced proteolytic activity and may function via its proteolysisindependent antimicrobial ability.…”