Novel MMP-9 substrates in cancer cells revealed by a label-free quantitative proteomics approach.

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.

• Authors: Danmei Xu, Naoko Suenaga, Mariola J Edelmann, Rafael Fridman, Ruth J Muschel, Benedikt M Kessler
• Journal: Molecular & cellular proteomics : MCP (Mol Cell Proteomics) Vol. 7 Issue 11 Pg. 2215-28 (Nov 2008) ISSN: 1535-9484 [Electronic] United States
• PMID: 18596065 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• APP protein, human
• Amyloid beta-Protein Precursor
• Culture Media, Conditioned
• LIF protein, human
• Leukemia Inhibitory Factor
• Matrix Metalloproteinase Inhibitors
• Protease Nexins
• Receptors, Cell Surface
• SERPINE2 protein, human
• Serpin E2
• Matrix Metalloproteinase 9

Topics

• Amino Acid Sequence
• Amyloid beta-Protein Precursor (metabolism)
• Base Sequence
• Binding Sites
• Cell Line, Tumor
• Culture Media, Conditioned
• Humans
• Leukemia Inhibitory Factor (metabolism)
• Male
• Matrix Metalloproteinase 9 (chemistry, genetics, metabolism)
• Matrix Metalloproteinase Inhibitors
• Molecular Sequence Data
• Peptide Mapping
• Prostatic Neoplasms (enzymology, genetics)
• Protease Nexins
• Proteomics (methods)
• RNA Interference
• Receptors, Cell Surface (metabolism)
• Serpin E2
• Substrate Specificity