Mucus proteinase inhibitor: a fast-acting inhibitor of leucocyte elastase

doi:10.1016/0167-4838(89)90230-6

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology

Volume 995, Issue 1, 16 March 1989, Pages 36-41

Biochimica et Biophy...

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Abstract

Human mucus proteinase inhibitor is a fast-acting inhibitor of human leucocyte elastase (EC 3.4.21.37) and forms a stable complex with this enzyme. At physiological ionic strength and temperature and in the presence of 10 mg/ml albumin, the kinetic constants characterizing the interaction between elastase and the non-degraded inhibitor are: k_ass = 6.4 · 10⁶M⁻¹ · s⁻¹, k_diss = 2.3 · 10⁻³, K₁ = 3 · 10⁻¹⁰M. The partially degraded inhibitor isolated by chymotrypsin-Sepharose chromatography inhibits elastase with similar efficiency, suggesting that if partial proteolysis of the inhibitor occurs in vivo, the latter may still act as a potent antielastase. Mucus proteinase inhibitor therefore plays a physiological antielastase function in upper respiratory tract secretion, since it inhibits elastase with a delay time of 150 ms and behaves like an irreversible inhibitor.

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Highly efficient inhibition of human chymase by α(2)-macroglobulin
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The inhibition of human chymase by the protease inhibitor α(2)-macroglobulin (α2M) was investigated. Titration of chymase hydrolytic activity with purified α2M showed that approximately 1 mol of α2M tetramer inhibits 1 mol of chymase. Inhibition was associated with cleavage of the α2M bait region and formation of a 200-kDa covalent complex. NH₂-terminal sequencing of chymase-treated α2M revealed cleavage at bonds Phe684–Tyr685 and Tyr685–Glu686 of the bait region. α2M pretreated with methylamine, an inactivator of α2M, did not inhibit chymase. The apparent second-order rate constant for inhibition (k_ass) was 5 × 10⁶ M⁻¹ s⁻¹, making α2M the most efficient natural protein protease inhibitor of chymase so far described. The k_ass value for inhibition was decreased approximately 10-fold by addition of heparin, a glycosaminoglycan produced by mast cells that binds to chymase. Heparin did not change significantly the stoichiometry of inhibition or block covalent complex formation. These results indicate that α2M is an important inhibitor to consider in the regulation of human chymase.
Differential inhibition of mast cell chymases by secretory leukocyte protease inhibitor
1998, Biochimica et Biophysica Acta - General Subjects
The major physiological role of human secretory leukocyte protease inhibitor (SLPI), a low molecular weight inhibitor present in mucus, is the rapid formation of a tight-binding inhibitory complex with neutrophil elastase. It is also the most effective known inhibitor of human mast cell chymase. The inhibitory efficacy of recombinant SLPI towards three other mast cell chymases was therefore investigated. Rat mast cell proteinases-1 and -2 (rMCP-1 and -2, respectively) and sheep mast cell proteinase-1 (sMCP-1), a chymase with additional tryptase-like properties, were treated with the inhibitor. SLPI inhibited rMCP-1 very efficiently in the absence of heparin, with a low dissociation constant, K_i=3×10⁻¹⁰ M and high second order association constant, k_ass=8.0×10⁶ M⁻¹ s⁻¹, and inhibition was enhanced when heparin was present. rMCP-2 was not inhibited by SLPI in the presence or absence of heparin, and did not degrade SLPI on prolonged incubation. SLPI inhibited sMCP-1 very poorly in the absence of heparin (K_i=9×10⁻⁶ M). However, in the presence of heparin, the K_i for inhibition of sMCP-1 by SLPI was reduced to the nanomolar range. sMCP-1 was observed to cleave SLPI with chymase-like specificity at Leu⁷²–Met⁷³ on prolonged incubation in the absence of heparin, but increasing concentrations of heparin reduced the extent of cleavage.
Characterization of placental bikunin, a novel human serine protease inhibitor
1997, Journal of Biological Chemistry
We reported previously the cloning of a novel human serine protease inhibitor containing two Kunitz-like domains, designated as placental bikunin, and the subsequent purification of a natural counterpart from human placental tissue (Marlor, C. W., Delaria, K. A., Davis, G., Muller, D. K., Greve, J. M., and Tamburini, P. P. (1997) J. Biol. Chem. 272, 12202–12208). In this report, the 170 residue extracellular domain of placental bikunin (placental bikunin_(1–170)) was expressed in baculovirus-infected Sf9 cells using its putative signal peptide. The resulting 21.3-kDa protein accumulated in the medium with the signal peptide removed and could be highly purified by sequential kallikrein-Sepharose and C₁₈ reverse-phase chromatography. To provide insights as to the potential in vivo functions of this protein, we performed an extensive investigation of the inhibitory properties of recombinant placental bikunin_(1–170) and both of its synthetically prepared Kunitz domains. All three proteins inhibited a number of serine proteases involved in the intrinsic pathway of blood coagulation and fibrinolysis. Placental bikunin_(1–170) formed inhibitor-protease complexes with a 1:2 stoichiometry and strongly inhibited human plasmin (K _i = 0.1 nm), human tissue kallikrein (K _i = 0.1 nm), human plasma kallikrein (K _i = 0.3 nm) and human factor XIa (K _i = 6 nm). Conversely, this protein was a weaker inhibitor of factor VIIa-tissue factor (K _i = 1.6 μm), factor IXa (K _i = 206 nm), factor Xa (K _i = 364 nm), and factor XIIa (K _i = 430 nm). This specificity profile was to a large extent mimicked, albeit with reduced potency, by the individual Kunitz domains. As predicted from this in vitrospecificity profile, recombinant placental bikunin_(1–170)prolonged the clotting time in an activated partial thromboplastin time assay.
Inhibition of human mast cell chymase by secretory leukocyte proteinase inhibitor: Enhancement of the interaction by heparin
1996, Archives of Biochemistry and Biophysics
The inhibition of human chymase, a chymotrypsin-like proteinase stored in mast cell granules, by secretory leukocyte proteinase inhibitor (SLPI) is investigated in this study. SLPI is a serine proteinase inhibitor present in human mucus secretions and tissues. It binds heparin, a highly sulfated glycosaminoglycan also found in mast cell secretory granules, and this interaction increases its effectiveness as an inhibitor of neutrophil elastase. Analysis of the chymase–SLPI interaction by equilibrium and kinetic methods indicates that the inhibition of chymase results from the reversible formation of a stable 1:1 enzyme–inhibitor complex. The dissociation equilibrium constant (K_i) determined in reactions containing 0.18Mor 1.0MNaCl (pH 8.0, 25°C) was 5 × 10⁻⁸and 2 × 10⁻⁸M, respectively. Addition of heparin to the low-salt reaction decreased theK_iapproximately 10-fold to a value of 3 × 10⁻⁹M, making SLPI a more effective inhibitor of human chymase. The decrease was due primarily to an approximately 10-fold increase in the association rate constant (k_ass) from 2 × 10⁴to 3 × 10⁵M⁻¹s⁻¹. The magnitudes of the rate and dissociation equilibrium constants indicate that SLPI has the potential to be a good chymase inhibitorin vivo,especially if chymase and heparin are released from mast cell granules simultaneously. The enhanced interaction in the presence of heparin supports the importance of this glycosaminoglycan to the inhibitory function of SLPI.
Influence of low molecular mass heparin on the kinetics of neutrophil elastase inhibition by mucus proteinase inhibitor
1995, Journal of Biological Chemistry
Commercial low molecular mass heparin accelerates the inhibition of neutrophil elastase by mucus proteinase inhibitor, the predominant antielastase of lung secretions (Faller, B., Mély, Y., Gérard, D., and Bieth, J. G.(1992) Biochemistry 31, 8285-8290). To study the kinetic mechanism of this rate enhancement, we have isolated a 4.5-kDa heparin fragment from commercial heparin. This compound is fairly monodisperse as shown by analytical ultracentrifugation. It binds elastase and inhibitor with a 1:1 stoichiometry and an equilibrium dissociation constant of 3 and 210 nM, respectively. It also forms a tight complex with EI. Flow calorimetry shows that the inhibitor-heparin interaction is characterized by a large negative enthalpy change (ΔH⁰ = −45.2 kJ mol⁻¹) and a small entropy change (ΔS = −23.7 J K⁻¹ mol⁻¹). Stopped-flow kinetics run under pseudo-first-order conditions ([I^o] ⪢ [E^o]) show that in the absence of heparin the inhibition conforms to a simple bimolecular reaction, where, k_a = 3.1 × 10⁶ M⁻¹s⁻¹, k_d= 10⁻⁴ s⁻¹, and K_i= 33 pM, whereas in the presence of heparin, E and I react via a two-step mechanism, where K_i^∗ = 86 nM, k² = 2.2 s⁻¹, k₋₂ = 10⁻⁴ s⁻¹, and K_i= 37 pM. Thus, heparin increases both the rate of inhibition by promoting the formation of a high affinity EI∗ intermediate and the rate of EI dissociation. Since the dissociation is negligible in bronchial secretions where the inhibitor concentration is much higher than K_i, it may be concluded that heparin significantly potentiates the inhibitor's antielastase potential in vivo.

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Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology

Regular paperMucus proteinase inhibitor: a fast-acting inhibitor of leucocyte elastase

Abstract

FEBS Lett.

Biochim. Biophys. Acta

J. Biol. Chem.

Biochem. Med.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

EMBO J.

Scand. J. Lab. Clin. Invest.

Physics-driven identification of clinically approved and investigation drugs against human neutrophil serine protease 4 (NSP4): A virtual drug repurposing study

Highly efficient inhibition of human chymase by α(2)-macroglobulin

Differential inhibition of mast cell chymases by secretory leukocyte protease inhibitor

Characterization of placental bikunin, a novel human serine protease inhibitor

Inhibition of human mast cell chymase by secretory leukocyte proteinase inhibitor: Enhancement of the interaction by heparin

Influence of low molecular mass heparin on the kinetics of neutrophil elastase inhibition by mucus proteinase inhibitor

Regular paper
Mucus proteinase inhibitor: a fast-acting inhibitor of leucocyte elastase