Chemoproteomics of matrix metalloproteases in a model of cartilage degeneration suggests functional biomarkers associated with posttraumatic osteoarthritis

Abstract

Active matrix metalloproteases (MMPs) play a significant role in the pathogenesis of many diseases including osteoarthritis (OA), which involves progressive proteolytic degradation of cartilage. Clinical success of OA interventions that target MMPs has been limited by a lack of information about the presence and activity of specific disease-related proteases. We therefore developed a chemoproteomics approach based on MS to characterize the release and activity of MMPs in an in vitro model of the early inflammatory phase of posttraumatic OA (PTOA). We designed and synthesized chemical activity-based probes (ABPs) to identify active MMPs in bovine cartilage explants cultured for 30 days with the proinflammatory cytokine, interleukin-1α. Using these probes in an activity-based protein profiling-multidimensional identification technology (ABPP-MudPIT) approach, we identified active MMP-1, -2, -3, -7, -9, -12, and -13 in the medium after 10 days of culture, the time at which irreversible proteolysis of the collagen network in the explant was detected using proteolytic activation of FRET-quenched MMP substrates. Total MMP levels were quantified by shotgun proteomics, which, taken with ABPP-MudPIT data, indicated the presence of predominantly inactive MMPs in the culture medium. The selectivity of the ABPP-MudPIT approach was further validated by detection of specific endogenous MMPs activated de novo with 4-aminophenylmurcuric acetate. The utility of the new ABPP-MudPIT approach for detecting molecular biomarkers of PTOA disease initiation and potential targets for therapeutics motivates possible application in other diseases involving MMP activity.

Keywords: ADAMTS; matrix metalloproteinase (MMP); osteoarthritis; peptidase; protein engineering.

Figure 1.

ABPs modified with a cleavable detection moiety retain reactivity with recombinant MMP in the context of cell lysate. A, structures of the multifunctional activity-based probes. All the probes contain unique disulfide moieties, which are susceptible to reduction in DTT solution, but cleavable using TCEP. All probes contain biotin as an affinity handle. B, validation of Probe 1 reactivity with recombinant MMP-2 in the context of cell lysate. Visible bands at 75 kDa show expected increase in intensity as the dose of recombinant MMP-2 is increased (0, 15, and 30 ng), indicating specific labeling of active MMP-2 by using the activity-based Probe 1.

Figure 2.

ABPs show expected inhibition of MMP activity assayed by cleavable FRET-based probes and enable detection of global MMP activity by SDS-PAGE in an explant model of osteoarthritis. A, schematic representation of sample preparation. Cartilage disks from the femoropatellar groove were treated with IL-1α or left untreated as controls. All disks were cultured in 24-well plates. B, toluidine blue-stained histology images of bovine cartilage explants untreated or treated with 10 ng/ml of IL-1α for 2, 6, or 12 days to detect GAG. By day 12, GAG is depleted from the ECM of cartilage explants. C, FRET-based assay of MMP activity with soluble MMP substrates (18) in the absence or presence of ABPs. Greater activity of MMPs is seen in days 20 and 30 in IL-1α–treated samples compared with day 10-treated or untreated samples (black). Addition of ABPs to the samples completely suppresses the observed activity of MMPs during kinetics measurement of soluble FRET-peptide cleavage (red). Graph shows average and standard deviation (n = 3). D, activity-based labeling of MMPs in explant culture medium. No MMPs are labeled by ABPs in the untreated control samples, but IL-1α–treated samples show excellent MMP labeling by the multifunctional ABPs that increases over time.

Figure 3.

Identification of individual active MMPs in osteoarthritis model culture supernatants using ABPP-MudPIT. A, summary of identified active MMPs. B, quantitative measurement of active MMPs in the untreated and IL-1α–treated explant culture medium. The graph presents mean values of two biological replicates and standard error are plotted to add visualization of the data and comparison across conditions.

Figure 4.

Total relative concentrations (as spectral counts) of individual metalloproteinases in culture supernatants identified by shotgun proteomics. Amounts shown for each MMP and ADAMTS include zymogens and pro-enzymes as well as active forms. Endogenous inhibitors TIMP-1 and -2 were also identified in IL-1α–treated explant culture medium (data not shown). The graph presents mean values of two biological replicates and standard error are plotted to add visualization of the data and comparison across conditions.

Figure 5.

Comparison of spectral counts of MMP-1, -2, -3, -9, -12, and -13 with ABPP-MudPIT and shotgun methods. Shown are APMA-activated MMPs in day 20 IL-1α–treated explant culture medium. The complex mixtures of proteases were treated with APMA along with ABPs and the activity of the MMPs was profiled using ABPP-MudPIT. The graph presents mean values of two biological replicates and standard error are plotted to add visualization of the data and comparison across conditions.