Inhibition of Myeloperoxidase Activity in Cystic Fibrosis Sputum by Peptide Inhibitor of Complement C1 (PIC1)

Abstract

Myeloperoxidase is the major peroxidase enzyme in neutrophil granules and implicated in contributing to inflammatory lung damage in cystic fibrosis. Free myeloperoxidase is present in cystic fibrosis lung fluid and generates hypochlorous acid. Here we report a new inhibitor of myeloperoxidase activity, Peptide Inhibitor of Complement C1 (PIC1). Using TMB as the oxidizing substrate, PIC1 inhibited myeloperoxidase activity in cystic fibrosis sputum soluble fractions by an average of a 3.4-fold decrease (P = 0.02). PIC1 also dose-dependently inhibited myeloperoxidase activity in a neutrophil lysate or purified myeloperoxidase by up to 28-fold (P < 0.001). PIC1 inhibited myeloperoxidase activity similarly, on a molar basis, as the specific myeloperoxidase inhibitor 4-Aminobenzoic acid hydrazide (ABAH) for various oxidizing substrates. PIC1 was able to protect the heme ring of myeloperoxidase from destruction by NaOCl, assayed by spectral analysis. PIC1 incubated with oxidized TMB reversed the oxidation state of TMB, as measured by absorbance at 450 nm, with a 20-fold reduction in oxidized TMB (P = 0.02). This result was consistent with an antioxidant mechanism for PIC1. In summary, PIC1 inhibits the peroxidase activity of myeloperoxidase in CF sputum likely via an antioxidant mechanism.

Fig 1. PIC1 inhibition of MPO peroxidase activity in CF sputum sol samples assayed by TMB.

(A) MPO activity, ± PIC1, in a CF sputum sol at baseline and after addition neutrophils (PMN), killed P. aeruginosa (P. aerug) or heat-aggregated IgG (Agg IgG). (B) PIC1 (7.5 mM) inhibition of MPO activity in 14 CF sputum samples. (C) A dose-response titration of PIC1 inhibition of MPO activity for a CF sputum sol with moderate baseline MPO activity (n = 3). Data are means of independent experiments ±SEM. (D) MPO activity of CF sol in the presence of PIC1 (7.5 mM) or acidified with HCl.

Fig 2. PIC1 inhibition of MPO peroxidase activity in neutrophil lysate or purified MPO.

(A) Dose-response titration of PIC1 into a lysate of 10⁶ neutrophils assaying MPO activity with TMB (n = 4). Data are means of independent experiments ±SEM. (B) PIC1 dose-response inhibition of pure MPO (80 nM) oxidation of TMB (n = 3). Data are means of independent experiments ±SEM. (C) PIC1 inhibition of pure MPO oxidation of ABTS and O-dianisidine. (D) Dose-response inhibition of pure MPO at various concentrations by PIC1 (n = 3). Data are means of independent experiments ±SEM.

Fig 3. Comparison of PIC1 versus ABAH inhibition of purified MPO (80 nM) peroxidase activity.

(A) Dose-response inhibition of MPO oxidation of TMB at various concentrations by PIC1, ABAH and DMSO (n = 3). Data are means of independent experiments ±SEM. (B) Comparison of PIC1 and ABAH inhibition of MPO oxidation of ABTS. (C) Comparison of PIC1 and ABAH inhibition of MPO oxidation of O-dianisidine.

Fig 4. Spectral analysis of purified MPO heme ring.

(A) MPO undergoes oxidative changes when incubated with H₂O₂. PIC1 protects MPO from oxidization in the presence of H₂O₂. Spectral absorption readings were taken from 300 to 550 nm (n = 3). Data are means of independent experiments ±SEM. (B) Spectral analysis of purified MPO when incubated with PIC1 or ABAH. Spectral absorption readings were taken from 300 to 550 nm (n = 3). Data are means of independent experiments ±SEM. (C) MPO undergoes oxidative changes in the presence of bleach. PIC1 protects MPO from oxidization in the presence of bleach. Spectral absorption readings were taken from 300 to 550 nm (n = 2). Data are means of independent experiments ±SEM. (D) Fe release from MPO in the presence of bleach is inhibited by PIC1 as measured by ferrozine assay.

Fig 5. Analysis of reversal of TMB oxidation by PIC1.

(A) Oxidation of TMB, as measured by absorbance, in the presence of MPO is inhibited when PIC1 is added before TMB or after TMB has been incubated with MPO. (B) MPO was bound to a well bottom and TMB was oxidized. The oxidized TMB solution was removed to an uncoated well and PIC1 was added or the solution was acidified with sulfuric acid and absorbance was measured (n = 4). Data are means of independent experiments ±SEM.