Heparin accelerates the inhibition of cathepsin G by mucus proteinase inhibitor: potent effect of O-butyrylated heparin

Abstract

Heparin tightly binds cathepsin G and so protects the enzyme from inhibition by alpha1-antichymotrypsin, alpha1-proteinase inhibitor and eglin c, three proteins which do not bind heparin [Ermolieff J., Boudier C., Laine A., Meyer B. and Bieth J.G. (1994) J. Biol. Chem. 269, 29502-29508]. Here we show that heparin no longer protects cathepsin G from inhibition when the enzyme is reacted with mucus proteinase inhibitor (MPI), a heparin-binding protein. Heparin fragments of Mr=4500 and 8100 and O-butyrylated heparin of Mr=8000 form tight complexes with cathepsin G (Kd=0.5-2.2 nM) and MPI (Kd=0. 4-0.8 muM) and accelerate the MPI-promoted inhibition of cathepsin G by a factor of 17-26. They also accelerate the inhibition of neutrophil elastase and pancreatic chymotrypsin. The rate acceleration is due to the binding of heparin to MPI. Butyrylation of heparin slightly decreases its affinity for cathepsin G and MPI but sharply decreases the ionic interactions between the positively charged proteins and the negatively charged polyanion. The butyrylated heparin derivative is the best rate accelerator: it increases the rate constant for the MPI-induced inhibition of cathepsin G and elastase by factors of 26 and 23, respectively. This, together with the fact that it has a good bioavailability and a very low anticoagulant activity, suggests that it might be an adjuvant of MPI-based therapy of cystic fibrosis.

Heparin‐like dextran derivatives as well as glycosaminoglycans inhibit the enzymatic activity of human cathepsin G

“…Comparison of the logarithms of these numbers indicates that ∼26% of the RG1503–CatG binding free energy is derived from purely ionic interactions at pH 7.4 and 25°C. These results are very similar to those reported by Ermolieff et al [32] which indicate that about 22% of the heparin–CatG binding energy is due to ionic interactions and that an average of two ionic interactions were required for a 1:1 heparin:CatG binding stoichiometry. On the other hand, chemical characterization of RG1503 indicates that one glucosidic unit of this polymer bears an average of 1.7 negatively charged group (sulfate+carboxylate; see Section 2).…”

Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases

“…Heparin binding to SLPI induces a conformational change that accelerates inhibition of HNE (Faller et al, 1992). Fragments of native heparin (4.5 or 8.1 kDa) or O-butyryl (8 kDa) also accelerate the inhibition of CG by SLPI (Ermolieff et al, 1998).…”

Measurement of free and membrane-bound cathepsin G in human neutrophils using new sensitive fluorogenic substrates

“…K m(obs) values of intramolecularly quenched fluorogenic substrates were obtained by measuring the dissociation constant (K i ) towards a chromogenic pNA substrate. Assays were carried out by adding 5 nM cathepsin G to a mixture of 100-500 µM Suc-AAPF-pNA (whose K m is 1.7p0.2 mM [19]) and 0-50 µM fluorogenic Abz-peptidyl-EDDnp derivative. The hydrolysis of Suc-AAPF-pNA was monitored at 410 nm with less than 5 % of substrate hydrolysed.…”

“…Cathepsin G specificity differs from those of HNE and Pr3, as it prefers a Phe or a Lys at P1 [16]. The chromogenic [p-nitroanilide (pNA)] and fluorogenic (7-amino-4-methyl-coumarin hydrochloride) substrates most commonly used to measure cathepsin G activity have a Pro-Phe pair at P2-P1 [17][18][19]. However, cathepsin G cleaves synthetic subAbbreviations used : Abz, o-aminobenzoic acid ; ACT, α 1 -antichymotrypsin ; CMK, chloromethyl ketone ; DTNB, 5,5h-dithio-bis(2-nitrobenzoic acid) ; EDDnp, N- (2,4-dinitrophenyl)ethylenediamine ; HNE, human neutrophil elastase ; PAR, protease-activated receptor ; PMN, polymorphonuclear neutrophil ; pNA, p-nitroanilide ; Pr3, proteinase 3 ; SBzl, thiobenzyl ester ; Z, benzyloxycarbonyl ; Suc, succinyl ; MeOSuc, methoxysuccinyl.…”