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Development of in-cell imaging assay systems for MMP-2 and MMP-9 based on trans-localizing molecular beacon proteins

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Abstract

A sensitive in-cell imaging MMP-2 and MMP-9 detection systems that enables direct fluorescence detection of a target protease and its inhibition inside living cells has been developed. This in-cell imaging system utilizes the concept of fluorescent molecular beacon reporter (MBR) protein comprising a masking protein, a mitochondrial targeting sequence, a protease specific cleavage sequence and a fluorescent marker sequence, green fluorescent protein (GFP). The MBR protein is designed to change its intracellular location upon cleavage by either MMP-2 or MMP-9 from cytosol to mitochondria. Full and partial MMP-2 and MMP-9 were tested for optimal expression and activity in the cell. The activity of MMP-2 and MMP-9 was approximately 65–71 %. Among MMP clones, MMP-2 catalytic domain and MMP-9 clone containing pro, catalytic and hemopexin domain were most active. Both MMP-2 and MMP-9 required divalent ions Ca and Zn for its activity and MMP-9 was more active at higher Ca/Zn ratio. With the in-cell imaging assay the protease activity can be measured in cellular environment and cellular toxicity of candidate molecules can be monitored at the same time. These are great advantage when compared to other currently used in vitro biochemical assays. The in-cell imaging assay developed in this study can be modified for other MMPs and can be used in various life science and drug discovery researches including the high throughput screening and high contents screening applications.

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References

  • Allan, J.A., A.J. Docherty, P.J. Barker, N.S. Huskisson, J.J. Reynolds, et al. 1995. Binding of gelatinases A and B to type-I collagen and other matrix components. Biochemical Journal 309: 299–306.

    CAS  PubMed Central  PubMed  Google Scholar 

  • Berg, G., V. Miksztowicz, and L. Schreier. 2011. Metalloproteinases in metabolic syndrome. Clinica Chim Acta 412: 1731–1739.

    Article  CAS  Google Scholar 

  • Brew, K., D. Dinakarpandian, and H. Nagase. 2000. Tissue inhibitors of metalloproteinases: Evolution, structure and function. Biochimica et Biophysica Acta 1477: 267–283.

    Article  CAS  PubMed  Google Scholar 

  • Briknarová, K., M. Gehrmann, L. Bányai, H. Tordai, L. Patthy, et al. 2001. Gelatin binding region of human matrix metalloproteinase-2: Solution structure, dynamics, and function of the COL-23 two-domain construct. Journal of Biological Chemistry 276: 27618–27621.

    Article  Google Scholar 

  • Cha, H.J., E. Kopetzki, R. Huber, M. Lanzendörfer, and H. Brandstetter. 2002. Structural basis of the adaptive molecular recognition by MMP9. Journal of Molecular Biology 320: 1065–1079.

    Article  CAS  PubMed  Google Scholar 

  • Chen, J., C.H. Tung, J.R. Allport, S. Chen, R. Weissleder, et al. 2005. Near-infrared fluorescent imaging of matrix metalloproteinase activity after myocardial infarction. Circulation 111: 1800–1805.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Chen, P., and W.C. Parks. 2009. Role of matrix metalloproteinases in epithelial migration. Journal of Cellular Biochemistry 108: 1233–1243.

    Article  CAS  PubMed  Google Scholar 

  • Cheng, D., Q. Shen, F. Nan, Z. Qian, and Q.Z. Ye. 2003. Purification and characterization of catalytic domains of gelatinase A with or without fibronectin insert for high-throughput inhibitor screening. Protein Expression and Purification 27: 63–74.

    Article  CAS  PubMed  Google Scholar 

  • Corbitt, C.A., J. Lin, and M.L. Lindsey. 2007. Mechanisms to inhibit matrix metalloproteinase activity: Where are we in the development of clinically relevant inhibitors? Recent Patents on Anti-Cancer Drug Discovery 2: 135–142.

    Article  CAS  PubMed  Google Scholar 

  • Dragutinović, V.V., S.B. Tatić, S. Nikolić-Mandić, S. Savin, D. Cvejić, et al. 2012. Matrix metalloproteinase-9 and the Cu/Zn ratio as ancillary diagnostic tools in distinguishing between the classical and follicular variants of papillary thyroid carcinoma. Biological Trace Element Research 149: 29–33.

    Article  PubMed  Google Scholar 

  • Emery, A., D.A. Sorrell, S. Lawrence, E. Easthope, M. Stockdale, et al. 2011. A novel cell-based, high-content assay for phosphorylation of Lats2 by Aurora A. Journal of Biomolecular Screening 16: 925–931.

    Article  CAS  PubMed  Google Scholar 

  • Fang, Y., A.G. Frutos, and R. Verklereen. 2008. Label-free cell-based assays for GPCR screening. Combinatorial Chemistry & High Throughput Screening 11: 357–369.

    Article  CAS  Google Scholar 

  • Geurts, N., E. Martens, I. Van Aelst, P. Proost, G. Opdenakker, et al. 2008. Beta-hematin interaction with the hemopexin domain of gelatinase B/MMP-9 provokes autocatalytic processing of the propeptide, thereby priming activation by MMP-3. Biochemistry 47: 2689–2699.

    Article  CAS  PubMed  Google Scholar 

  • Gioia, M., G.F. Fasciglione, S. Marini, S. D’Alessio, G. De Sanctis, et al. 2002. Modulation of the catalytic activity of neutrophil collagenase MMP-8 on bovine collagen I. Role of the activation cleavage and of the hemopexin-like domain. Journal of Biological Chemistry 277: 23123–23130.

    Article  CAS  PubMed  Google Scholar 

  • Gross, J., and C.M. Lapiere. 1962. Collagenolytic activity in amphibian tissues: A tissues culture assay. Proceedings of the National Academy of Sciences of the United States of America 48: 1014–1022.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hai, X., E. Adams, and A. Van Schepdael. 2013. Screening of matrix metalloproteinase inhibitors by microanalysis with fluorescence detection. Methods in Molecular Biology 919: 97–109.

    CAS  PubMed  Google Scholar 

  • Hansel, A., L. Kuschel, S. Hehl, C. Lemke, H.J. Agricola, et al. 2002. Mitochondrial targeting of the human peptide methionine sulfoxide reductase (MSRA), an enzyme involved in the repair of oxidized proteins. FASEB 16: 911–913.

    CAS  Google Scholar 

  • Hernandez-Barrantes, S., M. Toth, M.M. Bernardo, M. Yurkova, D.C. Gervasi, et al. 2000. Binding of active (57 kDa) membrane type 1-matrix metalloproteinase (MT1-MMP) to tissue inhibitor of metalloproteinase (TIMP)-2 regulates MT1-MMP processing and pro-MMP-2 activation. Journal of Biological Chemistry 275: 12080–12089.

    Article  CAS  PubMed  Google Scholar 

  • Khokha, R., A. Murthy, and A. Weiss. 2013. Metalloproteinases and their natural inhibitors in inflammation and immunity. Nature Reviews Immunology 13: 649–665.

    Article  CAS  PubMed  Google Scholar 

  • Korn, K., and E. Krausz. 2007. Cell-based high-content screening of small-molecule libraries. Current Opinion in Chemical Biology 11: 503–510.

    Article  CAS  PubMed  Google Scholar 

  • Kuschel, L., A. Hansel, R. Schonherr, H. Weissbach, N. Brot, et al. 1999. Molecular cloning and functional expression of a human peptide methionine sulfoxide reductase (hMsrA). FEBS 456: 17–21.

    Article  CAS  Google Scholar 

  • Makowski, G.S., and M.L. Ramsby. 2005. Autoactivation profiles of calcium-dependent matrix metalloproteinase-2 and -9 in inflammatory synovial fluid: Effect of pyrophosphate and bisphosphonates. Clinica Chimica Acta 358: 182–191.

    Article  CAS  Google Scholar 

  • Makowski, G.S., M.L. Ramsby, and G.R. Ramsby. 2005. An indium:calcium phosphate colloid that specifically targets fibrin. Journal of Biomedical Science 12: 421–429.

    Article  CAS  PubMed  Google Scholar 

  • Malemude, C.J. 2006. Matrix metal proteinase (MMPs) in health and disease; an overview. Front Biosci 11: 1696–1701.

    Article  Google Scholar 

  • Morgunova, E., A. Tuuttila, U. Bergmann, M. Isupov, Y. Lindqvist, et al. 1999. Structure of human pro-matrix metalloproteinase-2: Activation mechanism revealed. Science 284: 1667–1670.

    Article  CAS  PubMed  Google Scholar 

  • Nagase, H., and J.F. Woessner Jr. 1999. Matrix metalloproteinases. Journal of Biological Chemistry 274: 21491–21494.

    Article  CAS  PubMed  Google Scholar 

  • Nicolotti, O., M. Catto, I. Giangreco, M. Barletta, F. Leonetti, et al. 2012. Design, synthesis and biological evaluation of 5-hydroxy, 5-substituted-pyrimidine-2,4,6-triones as potent inhibitors of gelatinases MMP-2 and MMP-9. European Journal of Medicinal Chemistry 58: 368–376.

    Article  CAS  PubMed  Google Scholar 

  • Omanakuttan, A., J. Nambiar, R.M. Harris, C. Bose, N. Pandurangan, et al. 2012. Anacardic acid inhibits the catalytic activity of matrix metalloproteinase-2 and matrix metalloproteinase-9. Molecular Pharmacology 82: 614–622.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ra, H.J., and W.C. Parks. 2007. Control of matrix metalloproteinase catalytic activity. Matrix Biology 26: 587–596.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Raffo, D., O. Pontiggia, and M. Simian. 2011. Role of MMPs in metastatic dissemination: Implications for therapeutic advances. Current Pharmaceutical Biotechnology 12: 1937–1947.

    Article  CAS  PubMed  Google Scholar 

  • Rasch, M.G., I.K. Lund, M. Illemann, G. Høyer-Hansen, and H. Gårdsvoll. 2010. Purification and characterization of recombinant full-length and protease domain of murine MMP-9 expressed in Drosophila S2 cells. Protein Expression and Purification 72: 87–94.

    Article  CAS  PubMed  Google Scholar 

  • Rowsell, S., P. Hawtin, C.A. Minshull, H. Jepson, S.M. Brockbank, et al. 2002. Crystal structure of human MMP9 in complex with a reverse hydroxamate inhibitor. Journal of Molecular Biology 319: 173–181.

    Article  CAS  PubMed  Google Scholar 

  • Sapna, G., S. Gokul, and K. Bagri-Manjrekar. 2013. Matrix metalloproteinases and periodontal diseases. Oral Diseases. doi:10.1111/odi.12159.

    PubMed  Google Scholar 

  • Sasaki, T., W. Göhring, K. Mann, P. Maurer, E. Hohenester, et al. 1997. Limited cleavage of extracellular matrix protein BM-40 by matrix metalloproteinases increase its affinity for collagen. Journal of Biological Chemistry 272: 9237–9243.

    Article  CAS  PubMed  Google Scholar 

  • Shimokawa, K.K., M. Katayama, Y. Mastsuda, M. Takahashi, I. Hara, et al. 2002. Matrix metalloproteinase (MMP)-2 and MMP-9 activities in human seminal plasma. Molecular Human Reproduction 8: 32–36.

    Article  CAS  Google Scholar 

  • Smotrov, N., A. Mathur, I. Kariv, C.M. Moxham, and N. Bays. 2009. Development of a cell-based assay for measurement of c-Met phosphorylation using AlphaScreen technology and high-content imaging analysis. Journal of Biomolecular Screening 14: 404–411.

    CAS  PubMed  Google Scholar 

  • Stamenkovic, I. 2000. Matrix metalloproteinases in tumor invasion and metastasis. Seminars in Cancer Biology 10: 415–433.

    Article  CAS  PubMed  Google Scholar 

  • Sternlicht, M.D., and Z. Werb. 2001. How matrix metalloproteinase regulate cell behavior. Annual Review of Cell and Developmental Biology 17: 463–516.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Swarnakar, S., and S.P. Gupta. 2012. The gelatinases and their inhibitors: The structure–activity relationships. Springer Basel: Matrix Metalloproteinase Inhibitors.

    Google Scholar 

  • Takino, T., N. Koshikawa, H. Miyamori, M. Tanaka, T. Sasaki, et al. 2003. Cleavage of metastasis suppressor gene product KiSS-1 protein/metastin by matrix metalloproteinases. Oncogene 22: 4617–4626.

    Article  CAS  PubMed  Google Scholar 

  • Tocchi, A., and W.C. Parks. 2013. Functional interactions between matrix metalloproteinases and glycosaminoglycans. FEBS Journal 280: 2332–2341.

    Article  CAS  PubMed  Google Scholar 

  • Verslegers, M., K. Lemmens, I. Van Hove, and L. Moons. 2013. Matrix metalloproteinase-2 and -9 as promising benefactors in development, plasticity and repair of the nervous system. Progress in Neurobiology 105: 60–78.

    Article  CAS  PubMed  Google Scholar 

  • Visse, R., and H. Nagase. 2003. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circulation Research 92: 827–839.

    Article  CAS  PubMed  Google Scholar 

  • Wang, Z., R. Juttermann, and P.D. Soloway. 2000. TIMP-2 is required for efficient activation of proMMP-2 in vivo. Journal of Biological Chemistry 275: 26411–26415.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Winberg, J.O., E. Berg, S.O. Kolset, and L. Uhlin-Hansen. 2003. Calcium-induced activation and truncation of promatrix metalloproteinase-9 linked to the core protein of chondroitin sulfate proteoglycans. European Journal of Biochemistry 270: 3996–4007.

    Article  CAS  PubMed  Google Scholar 

  • Xu, X., Z. Chen, Y. Wang, Y. Yamada, and B. Steffensen. 2005. Functional basis for the overlap in ligand interactions and substrate specificities. Biochemical Journal 393: 127–134.

    Google Scholar 

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Acknowledgments

This study was supported by a grant from the Ministry of Science and Technology, Korea (M10620000001-07N2000-00110 to HJH) and by the BIO & Medical Technology Development Program of National Research Foundation of Korea (MEST, 2012M3A9C6049936 to JHK).

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Authors and Affiliations

  1. Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul, 130-701, Korea

    Min Jun Lee & Jeong Hee Kim

  2. Department of Oral Biochemistry and Molecular Biology, School of Dentistry, Kyung Hee University, Seoul, 130-701, Korea

    Jeong Hee Kim

  3. Department of Oral and Maxillofacial Pathology, School of Dentistry, Kyung Hee University, Seoul, 130-701, Korea

    Young-Ah Cho

  4. R&D Center, Ahram Biosystems Inc., Seoul, 133-120, Korea

    Hyun Jin Hwang

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  1. Min Jun Lee
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Correspondence to Jeong Hee Kim.

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Lee, M.J., Cho, YA., Hwang, H.J. et al. Development of in-cell imaging assay systems for MMP-2 and MMP-9 based on trans-localizing molecular beacon proteins. Arch. Pharm. Res. 38, 1099–1107 (2015). https://doi.org/10.1007/s12272-014-0546-7

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  • DOI: https://doi.org/10.1007/s12272-014-0546-7

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