Fluorometric immunocapture assay for the specific measurement of matrix metalloproteinase-9 activity in biological samples: application to brain and plasma from rats with ischemic stroke
Matrix metalloproteinases are important factors in the molecular mechanisms leading to neuronal injury in many neurological disorders. Matrix metalloproteinase (MMP)-9 is up-regulated after cerebral ischemia and neuroinflammation and is actively involved in blood–brain barrier disruption. Current methods of measuring MMP-9 activity, such as gelatin-substrate zymography, are unspecific and arduous. Here we developed an immunocapture assay with high efficiency, specificity, and sensitivity for quantifying endogenously active as well as total MMP-9 activity.
A fluorescence resonance energy transfer (FRET) peptide-based immunocapture assay was developed that enables the accurate assessment of total and active forms of MMP-9 in complex biological samples. The FRET assay demonstrated correct and efficient binding of MMP-9 to a mouse monoclonal MMP-9 antibody and high specificity of the immunocapture antibody for MMP-9. Total and active levels of MMP-9 were measured in rat brain homogenates, plasma, human HT-1080 conditioned media, and RBE4 endothelial cell lysates. The FRET immunocapture assay yielded highly similar results for total MMP-9 activity when compared to gelatin-substrate zymography.
We suggest that the new FRET peptide-based immunocapture assay is a viable replacement of zymography for sensitive and high throughput quantification of MMP-9 activity in biological samples.
- Birkedal-Hansen H, Yamada S, Windsor J, Pollard AH, Lyons G, Stetler-Stevenson W, Birkedal-Hansen B: Matrix metalloproteinases. In Current protocols in cell biology Edited by: Boni JS. 2008. Chapter 10:Unit 10 18
- Zuo J, Ferguson TA, Hernandez YJ, Stetler-Stevenson WG, Muir D: Neuronal matrix metalloproteinase-2 degrades and inactivates a neurite-inhibiting chondroitin sulfate proteoglycan. J Neurosci 1998,18(14):5203–5211.
- Sternlicht MD, Werb Z: How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol 2001, 17:463–516. CrossRef
- Candelario-Jalil E, Yang Y, Rosenberg GA: Diverse roles of matrix metalloproteinases and tissue inhibitors of metalloproteinases in neuroinflammation and cerebral ischemia. Neuroscience 2009,158(3):983–994. CrossRef
- Van Wart HE, Birkedal-Hansen H: The cysteine switch: a principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family. Proc Natl Acad Sci USA 1990,87(14):5578–5582. CrossRef
- Ogata Y, Enghild JJ, Nagase H: Matrix metalloproteinase 3 (stromelysin) activates the precursor for the human matrix metalloproteinase 9. J Biol Chem 1992,267(6):3581–3584.
- O’Connell JP, Willenbrock F, Docherty AJ, Eaton D, Murphy G: Analysis of the role of the COOH-terminal domain in the activation, proteolytic activity, and tissue inhibitor of metalloproteinase interactions of gelatinase B. J Biol Chem 1994,269(21):14967–14973.
- Gu Z, Kaul M, Yan B, Kridel SJ, Cui J, Strongin A, Smith JW, Liddington RC, Lipton SA: S-nitrosylation of matrix metalloproteinases: signaling pathway to neuronal cell death. Science 2002,297(5584):1186–1190. CrossRef
- Okamoto T, Akaike T, Sawa T, Miyamoto Y, van der Vliet A, Maeda H: Activation of matrix metalloproteinases by peroxynitrite-induced protein S-glutathiolation via disulfide S-oxide formation. J Biol Chem 2001,276(31):29596–29602. CrossRef
- Fu X, Kassim SY, Parks WC, Heinecke JW: Hypochlorous acid oxygenates the cysteine switch domain of pro-matrilysin (MMP-7). A mechanism for matrix metalloproteinase activation and atherosclerotic plaque rupture by myeloperoxidase. J Biol Chem 2001,276(44):41279–41287. CrossRef
- Bannikov GA, Karelina TV, Collier IE, Marmer BL, Goldberg GI: Substrate binding of gelatinase B induces its enzymatic activity in the presence of intact propeptide. J Biol Chem 2002,277(18):16022–16027. CrossRef
- Rosenberg GA: Matrix metalloproteinases and their multiple roles in neurodegenerative diseases. Lancet Neurol 2009,8(2):205–216. CrossRef
- Wang X, Rosell A, Lo EH: Targeting extracellular matrix proteolysis for hemorrhagic complications of tPA stroke therapy. CNS Neurol Disord Drug Targets 2008,7(3):235–242. CrossRef
- Rosell A, Lo EH: Multiphasic roles for matrix metalloproteinases after stroke. Curr Opin Pharmacol 2008,8(1):82–89. CrossRef
- Candelario-Jalil E, Thompson J, Taheri S, Grossetete M, Adair JC, Edmonds E, Prestopnik J, Wills J, Rosenberg GA: Matrix metalloproteinases are associated with increased blood–brain barrier opening in vascular cognitive impairment. Stroke 2011,42(5):1345–1350. CrossRef
- Romanic AM, White RF, Arleth AJ, Ohlstein EH, Barone FC: Matrix metalloproteinase expression increases after cerebral focal ischemia in rats: inhibition of matrix metalloproteinase-9 reduces infarct size. Stroke 1998,29(5):1020–1030. CrossRef
- Rosenberg GA, Cunningham LA, Wallace J, Alexander S, Estrada EY, Grossetete M, Razhagi A, Miller K, Gearing A: Immunohistochemistry of matrix metalloproteinases in reperfusion injury to rat brain: activation of MMP-9 linked to stromelysin-1 and microglia in cell cultures. Brain Res 2001,893(1–2):104–112. CrossRef
- Rosenberg GA, Navratil M, Barone F, Feuerstein G: Proteolytic cascade enzymes increase in focal cerebral ischemia in rat. J Cereb Blood Flow Metab 1996,16(3):360–366. CrossRef
- Park KP, Rosell A, Foerch C, Xing C, Kim WJ, Lee S, Opdenakker G, Furie KL, Lo EH: Plasma and brain matrix metalloproteinase-9 after acute focal cerebral ischemia in rats. Stroke 2009,40(8):2836–2842. CrossRef
- Heo JH, Lucero J, Abumiya T, Koziol JA, Copeland BR, del Zoppo GJ: Matrix metalloproteinases increase very early during experimental focal cerebral ischemia. J Cereb Blood Flow Metab 1999,19(6):624–633. CrossRef
- Asahi M, Asahi K, Jung JC, del Zoppo GJ, Fini ME, Lo EH: Role for matrix metalloproteinase 9 after focal cerebral ischemia: effects of gene knockout and enzyme inhibition with BB-94. J Cereb Blood Flow Metab 2000,20(12):1681–1689. CrossRef
- Tejima E, Guo S, Murata Y, Arai K, Lok J, van Leyen K, Rosell A, Wang X, Lo EH: Neuroprotective effects of overexpressing tissue inhibitor of metalloproteinase TIMP-1. J Neurotrauma 2009,26(11):1935–1941. CrossRef
- Shigemori Y, Katayama Y, Mori T, Maeda T, Kawamata T: Matrix metalloproteinase-9 is associated with blood–brain barrier opening and brain edema formation after cortical contusion in rats. Acta Neurochir Suppl 2006, 96:130–133. CrossRef
- Higashida T, Kreipke CW, Rafols JA, Peng C, Schafer S, Schafer P, Ding JY, Dornbos D 3rd, Li X, Guthikonda M: The role of hypoxia-inducible factor-1alpha, aquaporin-4, and matrix metalloproteinase-9 in blood–brain barrier disruption and brain edema after traumatic brain injury. J Neurosurg 2011,114(1):92–101. CrossRef
- Wang X, Jung J, Asahi M, Chwang W, Russo L, Moskowitz MA, Dixon CE, Fini ME, Lo EH: Effects of matrix metalloproteinase-9 gene knock-out on morphological and motor outcomes after traumatic brain injury. J Neurosci 2000,20(18):7037–7042.
- Mori T, Wang X, Aoki T, Lo EH: Downregulation of matrix metalloproteinase-9 and attenuation of edema via inhibition of ERK mitogen activated protein kinase in traumatic brain injury. J Neurotrauma 2002,19(11):1411–1419. CrossRef
- Candelario-Jalil E, Taheri S, Yang Y, Sood R, Grossetete M, Estrada EY, Fiebich BL, Rosenberg GA: Cyclooxygenase inhibition limits blood–brain barrier disruption following intracerebral injection of tumor necrosis factor-alpha in the rat. J Pharmacol Exp Ther 2007,323(2):488–498. CrossRef
- Meli DN, Christen S, Leib SL: Matrix metalloproteinase-9 in pneumococcal meningitis: activation via an oxidative pathway. J Infect Dis 2003,187(9):1411–1415. CrossRef
- Leib SL, Clements JM, Lindberg RL, Heimgartner C, Loeffler JM, Pfister LA, Tauber MG, Leppert D: Inhibition of matrix metalloproteinases and tumour necrosis factor alpha converting enzyme as adjuvant therapy in pneumococcal meningitis. Brain 2001,124(Pt 9):1734–1742. CrossRef
- Asahi M, Wang X, Mori T, Sumii T, Jung JC, Moskowitz MA, Fini ME, Lo EH: Effects of matrix metalloproteinase-9 gene knock-out on the proteolysis of blood–brain barrier and white matter components after cerebral ischemia. J Neurosci 2001,21(19):7724–7732.
- Gu Z, Cui J, Brown S, Fridman R, Mobashery S, Strongin AY, Lipton SA: A highly specific inhibitor of matrix metalloproteinase-9 rescues laminin from proteolysis and neurons from apoptosis in transient focal cerebral ischemia. J Neurosci 2005,25(27):6401–6408. CrossRef
- Rosenberg GA, Estrada EY, Dencoff JE: Matrix metalloproteinases and TIMPs are associated with blood–brain barrier opening after reperfusion in rat brain. Stroke 1998,29(10):2189–2195. CrossRef
- Pfefferkorn T, Rosenberg GA: Closure of the blood–brain barrier by matrix metalloproteinase inhibition reduces rtPA-mediated mortality in cerebral ischemia with delayed reperfusion. Stroke 2003,34(8):2025–2030. CrossRef
- Tsuji K, Aoki T, Tejima E, Arai K, Lee SR, Atochin DN, Huang PL, Wang X, Montaner J, Lo EH: Tissue plasminogen activator promotes matrix metalloproteinase-9 upregulation after focal cerebral ischemia. Stroke 2005,36(9):1954–1959. CrossRef
- Montaner J, Alvarez-Sabin J, Molina C, Angles A, Abilleira S, Arenillas J, Gonzalez MA, Monasterio J: Matrix metalloproteinase expression after human cardioembolic stroke: temporal profile and relation to neurological impairment. Stroke 2001,32(8):1759–1766. CrossRef
- Montaner J, Alvarez-Sabin J, Molina CA, Angles A, Abilleira S, Arenillas J, Monasterio J: Matrix metalloproteinase expression is related to hemorrhagic transformation after cardioembolic stroke. Stroke 2001,32(12):2762–2767. CrossRef
- Montaner J, Molina CA, Monasterio J, Abilleira S, Arenillas JF, Ribo M, Quintana M, Alvarez-Sabin J: Matrix metalloproteinase-9 pretreatment level predicts intracranial hemorrhagic complications after thrombolysis in human stroke. Circulation 2003,107(4):598–603. CrossRef
- Montaner J, Rovira A, Molina CA, Arenillas JF, Ribo M, Chacon P, Monasterio J, Alvarez-Sabin J: Plasmatic level of neuroinflammatory markers predict the extent of diffusion-weighted image lesions in hyperacute stroke. J Cereb Blood Flow Metab 2003,23(12):1403–1407. CrossRef
- Rosell A, Alvarez-Sabin J, Arenillas JF, Rovira A, Delgado P, Fernandez-Cadenas I, Penalba A, Molina CA, Montaner J: A matrix metalloproteinase protein array reveals a strong relation between MMP-9 and MMP-13 with diffusion-weighted image lesion increase in human stroke. Stroke 2005,36(7):1415–1420. CrossRef
- Rosell A, Cuadrado E, Ortega-Aznar A, Hernandez-Guillamon M, Lo EH, Montaner J: MMP-9-positive neutrophil infiltration is associated to blood–brain barrier breakdown and basal lamina type IV collagen degradation during hemorrhagic transformation after human ischemic stroke. Stroke 2008,39(4):1121–1126. CrossRef
- Vukasovic I, Tesija-Kuna A, Topic E, Supanc V, Demarin V, Petrovcic M: Matrix metalloproteinases and their inhibitors in different acute stroke subtypes. Clin Chem Lab Med CCLM/FESCC 2006,44(4):428–434.
- Ning M, Furie KL, Koroshetz WJ, Lee H, Barron M, Lederer M, Wang X, Zhu M, Sorensen AG, Lo EH: Association between tPA therapy and raised early matrix metalloproteinase-9 in acute stroke. Neurology 2006,66(10):1550–1555. CrossRef
- Castellanos M, Leira R, Serena J, Pumar JM, Lizasoain I, Castillo J, Davalos A: Plasma metalloproteinase-9 concentration predicts hemorrhagic transformation in acute ischemic stroke. Stroke 2003,34(1):40–46. CrossRef
- Castellanos M, Sobrino T, Millan M, Garcia M, Arenillas J, Nombela F, Brea D, Perez de la Ossa N, Serena J, Vivancos J: Serum cellular fibronectin and matrix metalloproteinase-9 as screening biomarkers for the prediction of parenchymal hematoma after thrombolytic therapy in acute ischemic stroke: a multicenter confirmatory study. Stroke 2007,38(6):1855–1859. CrossRef
- Serena J, Blanco M, Castellanos M, Silva Y, Vivancos J, Moro MA, Leira R, Lizasoain I, Castillo J, Davalos A: The prediction of malignant cerebral infarction by molecular brain barrier disruption markers. Stroke 2005,36(9):1921–1926. CrossRef
- England TJ, Bath PM, Sare GM, Geeganage C, Moulin T, O’Neill D, Woimant F, Christensen H, De Deyn P, Leys D: Asymptomatic hemorrhagic transformation of infarction and its relationship with functional outcome and stroke subtype: assessment from the Tinzaparin in Acute Ischaemic Stroke Trial. Stroke 2010,41(12):2834–2839. CrossRef
- Quesada AR, Barbacid MM, Mira E, Fernandez-Resa P, Marquez G, Aracil M: Evaluation of fluorometric and zymographic methods as activity assays for stromelysins and gelatinases. Clin Exp Metastasis 1997,15(1):26–32. CrossRef
- Viappiani S, Nicolescu AC, Holt A, Sawicki G, Crawford BD, Leon H, van Mulligen T, Schulz R: Activation and modulation of 72kDa matrix metalloproteinase-2 by peroxynitrite and glutathione. Biochem Pharmacol 2009,77(5):826–834. CrossRef
- Lauer-Fields JL, Nagase H, Fields GB: Development of a solid-phase assay for analysis of matrix metalloproteinase activity. J Biomolecular Tech JBT 2004,15(4):305–316.
- Nagase H, Fields CG, Fields GB: Design and characterization of a fluorogenic substrate selectively hydrolyzed by stromelysin 1 (matrix metalloproteinase-3). J Biol Chem 1994,269(33):20952–20957.
- Nagase H, Fields GB: Human matrix metalloproteinase specificity studies using collagen sequence-based synthetic peptides. Biopolymers 1996,40(4):399–416. CrossRef
- Lauer-Fields JL, Sritharan T, Stack MS, Nagase H, Fields GB: Selective hydrolysis of triple-helical substrates by matrix metalloproteinase-2 and −9. J Biol Chem 2003,278(20):18140–18145. CrossRef
- Verheijen JH, Nieuwenbroek NM, Beekman B, Hanemaaijer R, Verspaget HW, Ronday HK, Bakker AH: Modified proenzymes as artificial substrates for proteolytic enzymes: colorimetric assay of bacterial collagenase and matrix metalloproteinase activity using modified pro-urokinase. Biochem J 1997,323(Pt 3):603–609.
- Hanemaaijer R, Visser H, Konttinen YT, Koolwijk P, Verheijen JH: A novel and simple immunocapture assay for determination of gelatinase-B (MMP-9) activities in biological fluids: saliva from patients with Sjogren’s syndrome contain increased latent and active gelatinase-B levels. Matrix Biol J Int Soc Matrix Biol 1998,17(8–9):657–665. CrossRef
- Highly sensitive FRET substrates for the assay of MMPs. http://www.anaspec.com/servePdf.asp?f=c_publications24.pdf&t=publication
- Fisher JF, Mobashery S: Recent advances in MMP inhibitor design. Cancer Metastasis Rev 2006,25(1):115–136. CrossRef
- Lee M, Bernardo MM, Meroueh SO, Brown S, Fridman R, Mobashery S: Synthesis of chiral 2-(4-phenoxyphenylsulfonylmethyl)thiiranes as selective gelatinase inhibitors. Org Lett 2005,7(20):4463–4465. CrossRef
- Lok J, Zhao S, Leung W, Seo JH, Navaratna D, Wang X, Whalen MJ, Lo EH: Neuregulin-1 effects on endothelial and blood–brain-barrier permeability after experimental injury. Trans Stroke Res 2012,3(S1):S119-S124. CrossRef
- Candelario-Jalil E, Gonzalez-Falcon A, Garcia-Cabrera M, Leon OS, Fiebich BL: Post-ischaemic treatment with the cyclooxygenase-2 inhibitor nimesulide reduces blood–brain barrier disruption and leukocyte infiltration following transient focal cerebral ischaemia in rats. J Neurochem 2007,100(4):1108–1120. CrossRef
- Zhang JW, Gottschall PE: Zymographic measurement of gelatinase activity in brain tissue after detergent extraction and affinity-support purification. J Neurosci Methods 1997,76(1):15–20. CrossRef
- Zhang JW, Deb S, Gottschall PE: Regional and differential expression of gelatinases in rat brain after systemic kainic acid or bicuculline administration. Eur J Neurosci 1998,10(11):3358–3368. CrossRef
- Fluorometric immunocapture assay for the specific measurement of matrix metalloproteinase-9 activity in biological samples: application to brain and plasma from rats with ischemic stroke
- Open Access
- Available under Open Access This content is freely available online to anyone, anywhere at any time.
- Online Date
- March 2013
- Online ISSN
- BioMed Central
- Additional Links
- Matrix metalloproteinase-9 activity
- Fluorescence resonance energy transfer peptide
- Immunocapture assay
- Focal cerebral ischemia
- Author Affiliations
- 1. Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL, 32610, USA
- 2. Department of Neurology, University of New Mexico Health Sciences Center, Albuquerque, NM, 87131, USA