Abstract
Matrix metalloproteinase-9 (MMP-9) is implicated in tumor metastasis as well as a variety of inflammatory and pathological processes. Although many substrates for MMP-9, including components of the extracellular matrix, soluble mediators such as chemokines, and cell surface molecules have been identified, we undertook a more comprehensive proteomics-based approach to identify new substrates to further understand how MMP-9 might contribute to tumor metastasis. Previous proteomics approaches to identify protease substrates have depended upon differential labeling of each sample. Instead we used a label-free quantitative proteomics approach based on ultraperformance LC-ESI-high/low collision energy MS. Conditioned medium from a human metastatic prostate cancer cell line, PC-3ML, in which MMP-9 had been down-regulated by RNA interference was compared with that from the parental cells. From more than 200 proteins identified, 69 showed significant alteration in levels after depletion of the protease (>+/-2-fold), suggesting that they might be candidate substrates. Levels of six of these (amyloid-beta precursor protein, collagen VI, leukemia inhibitory factor, neuropilin-1, prostate cancer cell-derived growth factor (PCDGF), and protease nexin-1 (PN-1)) were tested in the conditioned media by immunoblotting. There was a strong correlation between results by ultraperformance LC-ESI-high/low collision energy MS and by immunoblotting giving credence to the label-free approach. Further information about MMP-9 cleavage was obtained by comparison of the peptide coverage of collagen VI in the presence and absence of MMP-9 showing increased sensitivity of the C- and N-terminal globular regions over the helical regions. Susceptibility of PN-1 and leukemia inhibitory factor to MMP-9 degradation was confirmed by in vitro incubation of the recombinant proteins with recombinant MMP-9. The MMP-9 cleavage sites in PN-1 were sequenced. This study provides a new label-free method for degradomics cell-based screening leading to the identification of a series of proteins whose levels are affected by MMP-9, some of which are clearly direct substrates for MMP-9 and become candidates for involvement in metastasis.
Highlights
Matrix metalloproteinase-9 (MMP-9) is implicated in tumor metastasis as well as a variety of inflammatory and pathological processes
MMP-9 Levels in Tumor Cells—To select a tumor cell system to use for identification of potential substrates for MMP-9, several invasive tumor cell lines were screened for the expression and activity of MMP-9 through gelatin zymography (Fig. 1A)
Even after treatment with phorbol 12-myristate 13-acetate (PMA), a potent transcriptional activator for several MMPs including MMP-9 [42], the level of MMP-9 released by the knockdown (KD) line remained undetectable, whereas it was significantly increased in PC-3ML
Summary
Cell Culture and Conditioned Medium Collection—PC-3ML cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C with 5% CO2. Quantitative Analysis—The analysis of quantitative changes in protein abundance, which is based on measuring peptide ion peak intensities observed in low collision energy mode in a triplicate set, was carried out using Waters Expression Analysis Software (WEPSTM), which is part of PLGS 2.2.5 (Expression version 2). Identical peptides from each triplicate set per sample were clustered based on mass precision (Ͻ15 ppm, typically ϳ5 ppm) and a retention time tolerance of Ͻ0.25 min using the clustering software included in PLGS 2.2.5. Protease Nexin-1 (PN-1) Peptide Mapping—The determination of MMP-9 cleavage sites in PN-1 as described for Fig. 7 was performed by comparing in-solution tryptic digests of PN-1 control with PN-1 incubated with recombinant MMP-9. Conditioned medium was concentrated and normalized according to cell numbers and intracellular proteins. Data analysis was performed using Mascot using the parameters as described above with the exception of using no restriction for enzyme cleavage
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