Activation and Inhibition of Human Matrix Metalloproteinase-9 (MMP9) by HOCl, Myeloperoxidase and Chloramines

Abstract

Matrix metalloproteinase-9 (MMP9, gelatinase B) plays a key role in the degradation of extracellular-matrix (ECM) proteins in both normal physiology and multiple pathologies, including those linked with inflammation. MMP9 is excreted as an inactive proform (proMMP9) by multiple cells, and particularly neutrophils. The proenzyme undergoes subsequent processing to active forms, either enzymatically (e.g., via plasmin and stromelysin-1/MMP3), or via the oxidation of a cysteine residue in the prodomain (the “cysteine-switch”). Activated leukocytes, including neutrophils, generate O2− and H2O2 and release myeloperoxidase (MPO), which catalyzes hypochlorous acid (HOCl) formation. Here, we examine the reactivity of HOCl and a range of low-molecular-mass and protein chloramines with the pro- and activated forms of MMP9. HOCl and an enzymatic MPO/H2O2/Cl− system were able to generate active MMP9, as determined by fluorescence-activity assays and gel zymography. The inactivation of active MMP9 also occurred at high HOCl concentrations. Low (nM—low μM) concentrations of chloramines formed by the reaction of HOCl with amino acids (taurine, lysine, histidine), serum albumin, ECM proteins (laminin and fibronectin) and basement membrane extracts (but not HEPES chloramines) also activate proMMP9. This activation is diminished by the competitive HOCl-reactive species, methionine. These data indicate that HOCl-mediated oxidation and MMP-mediated ECM degradation are synergistic and interdependent. As previous studies have shown that modified ECM proteins can also stimulate the cellular expression of MMP proteins, these processes may contribute to a vicious cycle of increasing ECM degradation during disease development.

Hypochlorous Acid and Chloramines Induce Specific Fragmentation and Cross-Linking of the G1-IGD-G2 Domains of Recombinant Human Aggrecan, and Inhibit ADAMTS1 Activity

“…Next, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), amino acid and protein chloramines were prepared as described previously [12,58]. Thus, HEPES-chloramines (HEPES-CA), Lys-(Lys-CA) and taurine-chloramines (Tau-CA) were prepared by adding HOCl to a fivefold molar excess of HEPES, Nα-acetyl-Lys and taurine, with the parent compounds present at 1 M, 100 mM, and 100 mM respectively, in 5 mM sodium phosphate buffer (pH 7.4).…”

“…HSA-chloramines (HSA-CA), human plasma fibronectin-chloramines (FN-CA), laminin-111-chloramines (laminin-CA) and murine basement membrane extract chloramines (BME-CA) were generated by adding a 50-fold molar excess of HOCl over the protein concentration (40 µM HSA, 26 µM fibronectin, 14 µM laminin-111, 20 µM basement membrane extract based on the molecular mass of laminin). Chloramine concentrations were determined by quantifying the oxidation of 5-thio-2-nitrobenzoic acid (TNB) to 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB) as described previously [12,58]. TNB was generated from DTNB by treatment of 1.3 mM DTNB with 50 mM NaOH for 15 min at 21 • C; this material was then diluted 1:50 in PBS buffer.…”

“…As buffer A contains HEPES, which might yield chloramines (cf. [64], but also [58], which suggests that this may be limited), HEPES chloramines were generated deliberately [58] and incubated with rhAggrecan at similar molar excesses to those used in the HOCl experiments, to examine differences between the buffers (see Section 3.1) in more detail. These experiments (Figure 5) indicate that high molar excesses of HEPES chloramines are required to generate significant cross-links and fragmention of rhAggrecan.…”

“…These data suggest that HEPES chloramines, if formed in buffer A, do not markedly interfere (c.f. [58]), though HEPES may scavenge some HOCl when this is present at high concentrations.…”

“…As HOCl and other oxidants can both activate and inactivate MMPs [58,[66][67][68][69][70], it was of interest to determine whether this also occurred with ADAMTS1. rhADAMTS1 was treated with HOCl (0-500-fold molar excesses over enzyme) for 15 min at 37 • C, then samples of the treated and native enzyme were added to rhAggrecan and further incubated for 24 h at 37 • C in buffer A, with generation of the neoepitope (ARGSV) from rhADAMTS1-mediated cleavage at the aggrecanase site detected using the BC-3 mAb (cf.…”