Phosphorylation Status of 72 kDa MMP-2 Determines Its Structure and Activity in Response to Peroxynitrite

<div><p>Matrix metalloproteinase-2 (MMP-2) is a key intra- and extra-cellular protease which contributes to several oxidative stress related pathologies. A molecular understanding of 72 kDa MMP-2 activity, directly mediated by S-glutathiolation of its cysteine residues in the presence of peroxynitrite (ONOO⁻) and by phosphorylation of its serine and threonine residues, is essential to develop new generation inhibitors of intracellular MMP-2. Within its propeptide and collagen binding domains there is an interesting juxtaposition of predicted phosphorylation sites with nearby cysteine residues which form disulfide bonds. However, the combined effect of these two post-translational modifications on MMP-2 activity has not been studied. The activity of human recombinant 72 kDa MMP-2 (hrMMP-2) following <i>in vitro</i> treatments was measured by troponin I proteolysis assay and a kinetic activity assay using a fluorogenic peptide substrate. ONOO⁻ treatment in the presence of 30 µM glutathione resulted in concentration-dependent changes in MMP-2 activity, with 0.1–1 µM increasing up to twofold and 100 µM attenuating its activity. Dephosphorylation of MMP-2 with alkaline phosphatase markedly increased its activity by sevenfold, either with or without ONOO⁻. Dephosphorylation of MMP-2 also affected the conformational structure of the enzyme as revealed by circular dichroism studies, suggesting an increase in the proportion of α-helices and a decrease in β-strands compared to the phosphorylated form of MMP-2. These results suggest that ONOO⁻ activation (at low µM) and inactivation (at high µM) of 72 kDa MMP-2, in the presence or absence of glutathione, is also influenced by its phosphorylation status. These insights into the role of post-translational modifications in the structure and activity of 72 kDa MMP-2 will aid in the development of inhibitors specifically targeting intracellular MMP-2.</p></div

Effect of MMP-2 phosphorylation status in regard to activity changes following ONOO^- treatment using troponin I (TnI) as a known intracellular substrate.

<p>(A) Representative time-dependent troponin I (TnI) hydrolysis by 0.3 µM ONOO⁻ - treated, native (upper panel) or dephosphorylated (lower panel) 72 kDa MMP-2, in the presence of 30 µM GSH, following different incubation times (30, 60, or 120 min) at 37°C with TnI. Representative Coomassie blue stained SDS-PAGE gels. (B) Quantitative analysis of TnI hydrolysis by native (left) or dephosphorylated (right) 72 kDa MMP-2, treated with different concentrations of ONOO⁻ (0–0.3 µM) or DPN, in the presence of 30 µM GSH. Incubation for 120 min at 37°C. Mean ± SEM, N = 4–7/group. * p<0.05 compared with control (C, TnI alone). DPN, decomposed peroxynitrite.</p

Potential phosphorylation sites of MMP-2.

<p>(A) Domain structure, NetPhos predicted phosphorylation sites, cysteine residues and disulphide bonds of human MMP-2. Vertical white, grey and black lines that extend above the demarcated horizontal threshold indicate putative amino acid phosphorylation sites, whether serine, threonine, or tyrosine, respectively. Red vertical lines indicate all cysteine residues present in the MMP-2 sequence, and blue horizontal lines represent the disulphide bonds of human MMP-2. (B) Crystal structure of MMP-2 (PDB ID: 1CK7) is shown in cartoon representation. The pro-peptide (hot pink), collagenase-like domain 1 (deep teal), collagenase-binding domain (forest green), collagenase-like domain 2 (pale green) and hemopexin-like domain (yellow) are shown in the indicated colours. Zn²⁺ ions are indicated as purple spheres. Potential phosphorylation sites (serine, threonine and tyrosine) are shown in Corey-Pauling-Koltun (CPK)/space filling representation, with carbon and oxygen atoms of the side chains in grey and red, respectively.</p

Phosphorylation status of 72 kDa MMP-2 and changes in its secondary structure following dephosphorylation.

<p>(A) In representative Coomassie blue stained SDS-PAGE gels, both native (phosphorylated, +P) and dephosphorylated (−P) 72 kDa MMP-2 run with the same pattern, showing bands only at 72 kDa (left). However, when run on the Phos-tag acrylamide gel, a more slowly migrating band also appeared, indicating that a portion of MMP-2 is phosphorylated. Phos-tag gel revealed at least two different phosphorylation states of 72 kDa MMP-2 and confirmed its dephosphorylation after treatment with alkaline phosphatase (right, N = 4). (B) The ratio of band intensity of total MMP-2 measured by SDS-PAGE (left), as well as ratio of higher to lower band intensities measured in the Phos-tag gel (right, mean ± SEM, N = 4), comparing phosphorylated to dephosphorylated samples, is demonstrated in the bar graphs below. (C) CD spectra of hrMMP-2. Far UV range CD spectrum of native (phosphorylated, black line) and dephosphorylated (red line).</p

Mass spectrometry data obtained for native and dephosphorylated MMP-2 treated with 0.3 µM ONOO⁻ and 30 µM GSH showing modifications in Cys residues.

<p>+P = native, −P = dephosphorylated, G+O = glutathione and ONOO⁻ treated, ND = not detected.</p

Effect of MMP-2 phosphorylation status in regard to activity changes following ONOO⁻ treatment using Omni MMP as a substrate.

<p>Proteolysis of fluorogenic Omni MMP substrate by ONOO⁻-treated, native (+P, left panel) or dephosphorylated (−P, right panel) 72 kDa MMP-2, in the presence of 30 µM GSH. Mean ± SEM, N = 7–8/group. *p<0.05 versus 0 µM ONOO⁻. DPN, decomposed peroxynitrite.</p

S-glutathiolation of phosphorylated and dephosphorylated 72 kDa MMP-2 after ONOO⁻/GSH treatment.

<p>S-glutathiolation of phosphorylated (A) or dephosphorylated (B) MMP-2, treated with 0.3 µM ONOO⁻ in the presence or absence of 30 µM GSH was determined by immunoprecipitation with glutathione (GSH) antibody, or IgG as a control, followed by Western blot for MMP-2. M, MMP-2; O, 0.3 µM ONOO⁻; G, 30 µM GSH; D, 1 mM dithiothreitol.</p