The epitope-mediated MMP activation assay: detection and quantification of the activation of Mmp2 in vivo in the zebrafish embryo

Jeffrey, Emma J.; Crawford, Bryan D.

doi:10.1007/s00418-018-1634-4

Short Communication
Published: 19 January 2018

The epitope-mediated MMP activation assay: detection and quantification of the activation of Mmp2 in vivo in the zebrafish embryo

Histochemistry and Cell Biology volume 149, pages 277–286 (2018)Cite this article

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Abstract

Matrix remodeling is a consequence of tightly regulated matrix metalloproteinase (MMP) activity. MMPs are synthesized as inactive precursors with auto-inhibitory N-terminal propeptides, the proteolytic removal of which exposes the catalytic zinc ion, rendering the protease active. The regulation of MMP activation has been investigated primarily in tissue culture and biochemical assays that lack important biological context. Here we present the epitope-mediated MMP activation (EMMA) assay and use it to observe the activation of Mmp2 (gelatinase A) by endogenous mechanisms in the intact zebrafish embryo. The hemagglutinin (HA) and GFP-tagged reporter construct becomes activated on the surface of specific cells and this activation is abolished by broad-spectrum inhibition of metalloproteinase activity, consistent with existing models of gelatinase A activation. The mechanism(s) acting on the construct are spatially restricted, metalloproteinase-dependent and replacing the HA tag with mCherry abolishes activation, showing that the mechanism(s) are sensitive to the structure of the N-terminal domain. The construct is activated strongly in maturing myotome boundaries, but also intracellularly within myofibrils, consistent with reports implicating this protease in muscle development and function. In addition to general-purpose tools for the production of “EMMAed” MMPs and other proteins, we have established a transgenic line of zebrafish expressing EMMAedMmp2 under control of an inducible promoter to facilitate further investigation into the regulation of this ubiquitous ECM-remodeling protease in vivo.

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Abbreviations

ECM:: Extracellular matrix
EMMA:: Epitope-mediated MMP activation
HA:: Hemagglutinin
GFP:: Green fluorescent protein
hpf:: Hours post-fertilization
hpHS:: Hours post-heat shock
MMP:: Matrix metalloproteinase
TIMP:: Tissue inhibitor of metalloproteinases

References

Ali M, Cho WJ, Hudson B, Kassiri Z, Granzier H, Schulz R (2011) Titin is a target of MMP-2: implications in myocardial ischemia/ reperfusion injury. Circulation 122(20):2039–2047. https://doi.org/10.1161/circulationaha.109.930222
Article Google Scholar
Apte SS, Parks WC (2015) Metalloproteinases: a parade of functions in matrix biology and an outlook for the future. Matrix Biol 44–46:1–6. https://doi.org/10.1016/j.matbio.2015.04.005
Article PubMed Google Scholar
Arpino V, Brock M, Gill SE (2015) The role of TIMPs in regulation of extracellular matrix proteolysis. Matrix Biol 44–46:247–254. https://doi.org/10.1016/j.matbio.2015.03.005
Article PubMed Google Scholar
Bonnans C, Chou J, Werb Z (2014) Remodelling the extracellular matrix in development and disease. Nat Rev Mol Cell Biol 15(12):786–801. https://doi.org/10.1038/nrm3904
CAS Article PubMed PubMed Central Google Scholar
Brew K, Dinakarpandian D, Nagase H (2000) Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim Biophys Acta Protein Struct Mol Enzymol 1477(1–2):267–283
CAS Article Google Scholar
Butler GS, Butler MJ, Atkinson SJ, Will H, Tamura T, Van Westrum SS, Crabbe T, Clements J, Ortho M, Murphy G (1998) The TIMP2 membrane type 1 metalloproteinase “receptor” progelatinase A. J Biol Chem 273(2):871–880
CAS Article PubMed Google Scholar
Chakraborti S, Mandal M, Das S, Mandal A, Chakraborti T (2003) Regulation of matrix metalloproteinases: an overview. Mol Cell Biochem 253(1–2):269–285
CAS Article PubMed Google Scholar
Coker ML, Doscher MA, Thomas CV, Galis ZS, Spinale FG (1999) Matrix metalloproteinase synthesis and expression in isolated LV myocyte preparations. Am J Physiol 277(2):777–787
Google Scholar
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823. https://doi.org/10.1126/science.1231143
CAS Article PubMed PubMed Central Google Scholar
Crabbe T, Zucker S, Cockett MI, Willenbrock F, Tickle S, O’Connell JP, Scothern JM, Murphy G, Docherty J (1994) Mutation of the active site glutamic acid of human gelatinase A: effects on latency, catalysis, and the binding of tissue inhibitor of metalloproteinases-1. Biochemistry 33(21):6684–6690
CAS Article PubMed Google Scholar
Crawford BD, Henry CA, Clason TA, Becker AL, Hille MB (2003) Activity and distribution of paxillin, focal adhesion kinase, and cadherin indicate cooperative roles during zebrafish morphogenesis. Mol Biol Cell 14(8):3065–3081. https://doi.org/10.1091/mbc.E02-08-0537
CAS Article PubMed PubMed Central Google Scholar
Crawford BD, Pilgrim DB (2005) Ontogeny and regulation of matrix metalloproteinase activity in the zebrafish embryo by in vitro and in vivo zymography. Dev Biol 286(2):405–414. https://doi.org/10.1016/j.ydbio.2005.06.035
CAS Article PubMed Google Scholar
DeCoux A, Lindsey ML, Villarreal F, Garcia RA, Schulz R (2014) Myocardial matrix metalloproteinase-2: inside out and upside down. J Mol Cell Cardiol 77:64–72. https://doi.org/10.1016/j.yjmcc.2014.09.016
CAS Article PubMed Google Scholar
Ellis TR, Crawford BD (2016) Experimental dissection of metalloproteinase inhibition-mediated and toxic effects of phenanthroline on zebrafish development. Int J Mol Sci 17(9). https://doi.org/10.3390/ijms17091503
Felber J, Coombs TL, Vallee BL (1962) The mechanisms of inhibition of carboxypeptidase A by 1,10-phenanthroline. Biochemistry 1:231–238
CAS Article PubMed Google Scholar
Gaffney J, Solomonov I, Zehorai E, Sagi I (2014) Multilevel regulation of matrix metalloproteinases in tissue homeostasis indicates their molecular specificity in vivo. Matrix Biol 44–46:191–199. https://doi.org/10.1016/j.matbio.2015.01.012
Google Scholar
Hadler-Olsen E, Fadnes B, Sylte I, Uhlin-Hansen L, Winberg JO (2011) Regulation of matrix metalloproteinase activity in health and disease. FEBS J 278(1):28–45. https://doi.org/10.1111/j.1742-4658.2010.07920.x
CAS Article PubMed Google Scholar
Halloran MC, Sato-Maeda M, Warren JT, Su F, Lele Z, Krone PH, Kuwada JY, Shoji W (2000) Laser-induced gene expression in specific cells of transgenic zebrafish. Development 127(9):1953–1960
CAS PubMed Google Scholar
Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157(6):1262–1278. https://doi.org/10.1016/j.cell.2014.05.010
CAS Article PubMed PubMed Central Google Scholar
Hynes RO, Naba A (2012) Overview of the Matrisome—an inventory of extracellular matrix constituents and functions. Cold Spring Harb Perspect Biol 4(1):a004903. https://doi.org/10.1101/cshperspect.a004903
Article PubMed PubMed Central Google Scholar
Janssens E, Gaublomme D, De Groef L, Darras VM, Arckens L, Delorme N, Claes F, Van Hove I, Moons L (2013) Matrix metalloproteinase 14 in the zebrafish: an eye on retinal and retinotectal development. PLoS One 8(1):e52915. https://doi.org/10.1371/journal.pone.0052915
CAS Article PubMed PubMed Central Google Scholar
Jenkins MH, Alrowaished SS, Goody MF, Crawford BD, Henry CA (2016) Laminin and matrix metalloproteinase 11 regulate fibronectin levels in the zebrafish myotendinous junction. Skelet Muscle 6:18. https://doi.org/10.1186/s13395-016-0089-3
Article PubMed PubMed Central Google Scholar
Kawakami K (2007) Tol2: a versatile gene transfer vector in vertebrates., Genome Biol 8(Suppl 1):S7. https://doi.org/10.1186/gb-2007-8-s1-s7
Article PubMed PubMed Central Google Scholar
Keow JY, Crawford BD (2012) Investigating matrix metalloproteinase regulation in its biological context; detecting MMP activity in vivo. In: Sorg BA, Brown TE (eds) Matrix metalloproteinases. Nova Science Publishers, Hauppauge, N.Y, pp 151–169. (ISBN 9781621007890)
Keow JY, Herrmann KM, Crawford BD (2011) Differential in vivo zymography: a method for observing matrix metalloproteinase activity in the zebrafish embryo. Matrix Biol 30(3):169–177. https://doi.org/10.1016/j.matbio.2011.01.003
CAS Article PubMed Google Scholar
Keow JY, Pond ED, Cisar JS, Cravatt BF, Crawford BD (2012) Activity-based labeling of matrix metalloproteinases in living vertebrate embryos. PLoS One 7(8):e43434. https://doi.org/10.1371/journal.pone.0043434
CAS Article PubMed PubMed Central Google Scholar
Kessenbrock K, Plaks V, Werb Z (2010) Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 141(1):52–67. https://doi.org/10.1016/j.cell.2010.03.015
CAS Article PubMed PubMed Central Google Scholar
Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203(3):253–310. https://doi.org/10.1002/aja.1002030302
CAS Article PubMed Google Scholar
Koo BH, Kim YH, Han JH, Kim DS (2012) Dimerization of matrix metalloproteinase-2 (MMP-2): functional implication in MMP-2 activation. J Biol Chem 287(27):22643–22653. https://doi.org/10.1074/jbc.M111.337949
CAS Article PubMed PubMed Central Google Scholar
Kwan KM, Fujimoto E, Grabher C, Mangum BD, Hardy ME, Campbell DS, Parant JM, Yost HJ, Kanki JP, Chien CB (2007) The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev Dyn 236(11):3088–3099. https://doi.org/10.1002/dvdy.21343
CAS Article PubMed Google Scholar
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GE (2013) RNA-guided human genome engineering via Cas9. Science 339(6121):823–826. https://doi.org/10.1126/science.1232033
CAS Article PubMed PubMed Central Google Scholar
Morrison CJ, Butler GS, Bigg HF, Roberts CR, Soloway PD, Overall CM (2001) Cellular activation of MMP-2 (gelatinase A) by MT2-MMP occurs via a TIMP-2-independent pathway. J Biol Chem 276(50):47402–47410. https://doi.org/10.1074/jbc.M108643200
CAS Article PubMed Google Scholar
Nagase H (2002) Substrate specificity of MMPs. In: Clendeninn NJ, Appelt K (eds) Matrix metalloproteinase inhibitors in cancer therapy. Humana Press, New York, pp 39–66
Google Scholar
Nishida Y, Miyamori H, Thompson EW, Takino T, Endo Y, Sato H (2008) Activation of matrix metalloproteinase-2 (MMP-2) by membrane type 1 matrix metalloproteinase through an artificial receptor for ProMMP-2 generates active MMP-2. Cancer Res 68(21):9096–9104. https://doi.org/10.1158/0008-5472.CAN-08-2522
CAS Article PubMed Google Scholar
Overall CM, King AE, Sam DK, Ong AD, Lau TTY, Wallon UM, Declerck YA, Atherstone J (1999) Identification of the tissue inhibitor of metalloproteinases-2 (TIMP-2) binding site on the hemopexin carboxyl domain of human gelatinase A by site-directed mutagenesis. J Biol Chem 274(7):4421–4429
CAS Article PubMed Google Scholar
Pedersen ME, Vuong TT, Rønning SB, Koslet SO (2015) Matrix metalloproteinases in fish biology and matrix turnover. Matrix Biol 44–46:86–93. https://doi.org/10.1016/j.matbio.2015.01.009
Article PubMed Google Scholar
Preibisch S, Saalfeld S, Tomancak P Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics 25(11):1463–1465. https://doi.org/10.1093/bioinformatics/btp18c4
Ra HJ, Parks WC (2007) Control of matrix metalloproteinase catalytic activity. Matrix Biol 26(8):587–596. https://doi.org/10.1016/j.matbio.2007.07.001
Rozario T, Desimone DW (2010) The extracellular matrix in development and morphogenesis: a dynamic view. Dev Biol 341(1):126–140. https://doi.org/10.1016/j.ydbio.2009.10.026
CAS Article PubMed Google Scholar
Sbardella D, Fasciglione GF, Gioia M, Ciaccio C, Tundo GR, Marini S, Coletta M (2012) Human matrix metalloproteinases: an ubiquitarian class of enzymes involved in several pathological processes. Mol Asp Med 33(2):119–208. https://doi.org/10.1016/j.mam.2011.10.015
CAS Article Google Scholar
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmi B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open source platform for biological-image analysis. Nat Methods 9(7):676–682. https://doi.org/10.1038/nmeth.2019
CAS Article PubMed Google Scholar
Shoji W, Sato-maeda M (2008) Application of heat shock promoter in transgenic zebrafish. Dev Growth Differ 50(6):401–406. https://doi.org/10.1111/j.1440-169X.2008.01038.x
CAS Article PubMed Google Scholar
Small C, Crawford B (2016) Matrix metalloproteinases in neural development: a phylogenetically diverse perspective. Neural Regen Res 11(3):1–6. https://doi.org/10.4103/1673-5374.179030
Google Scholar
Snow CJ, Peterson MT, Khalil A, Henry CA (2008) Muscle development is disrupted in zebrafish embryos deficient for fibronectin. Dev Dyn 237(9):2542–2553. https://doi.org/10.1002/dvdy.21670
Article PubMed PubMed Central Google Scholar
Strongin AY, Collier I, Bannikov G, Marmer BL, Grant GA, Goldberg GI (1995) Mechanism of cell surface activation of 72 kDa type IV collagenase. J Biol Chem 270(10):5331–5338
CAS Article PubMed Google Scholar
Tochowicz A, Goettig P, Evans R, Visse R, Shitomi Y, Palmisano R, Ito N, Richter K, Maskos K, Franke D, Svergun D, Nagase H, Bode W, Itoh Y (2011) The dimer interface of the membrane type 1 matrix metalloproteinase hemopexin domain: crystal structure and biological functions. J Biol Chem 286(8):7587–7600. https://doi.org/10.1074/jbc.M110.178434
CAS Article PubMed Google Scholar
Tsang KY, Cheung MCH, Chan D, Cheah KSE (2010) The developmental roles of the extracellular matrix: beyond structure to regulation. Cell Tissue Res 339(1):93–110. https://doi.org/10.1007/s00441-009-0893-8
CAS Article PubMed Google Scholar
Tulotta C, He S, Van Der Ent W, Chen L, Groenewoud A, Spaink H, Snaar-Jagalska BE (2016) Imaging cancer angiogenesis and metastasis in a zebrafish embryo model. In: Cohen IR, Lajtha A, Lambris JD, Paoletti R (eds) Advances in experimental medicine and biology, Springer, Berlin, pp 239–263
Google Scholar
Van Wart HE, Birkedal-Hansen H (1990) 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 87(14):5578–5582
Article PubMed PubMed Central Google Scholar
Wang W, Schulze CJ, Suarez-Pinzon WL, Dyck JRB, Sawicki G, Schulz R (2002) Intracellular action of matrix metalloproteinase-2 accounts for acute myocardial ischemia and reperfusion injury. Circulation 106(12):1543–1549
CAS Article PubMed Google Scholar
Westerfield M (2000) The Zebrafish book. A Guide for the Laboratory Use of Zebrafish (Danio rerio), fourth ed. University of Oregon Press, Eugene
Google Scholar
Wyatt RA, Keow JY, Harris ND, Haché CA, Li DH, Crawford BD (2009) The zebrafish embryo: a powerful model system for investigating matrix remodeling. Zebrafish 6(4):347–354. https://doi.org/10.1089/zeb.2009.0609
CAS Article PubMed Google Scholar
Wyatt RA, Trieu NPV, Crawford BD (2017) Zebrafish xenograft: an evolutionary experiment in tumour biology. Genes 8(9):220. https://doi.org/10.3390/genes8090220
Article PubMed Central Google Scholar
Yue B (2014) Biology of the extracellular matrix: an overview. J Glaucoma. https://doi.org/10.1097/IJG.0000000000000108
PubMed PubMed Central Google Scholar
Zavzavadjian JR, Couture S, Park WS, Whalen J, Lyon S, Lee G, Fung E, Mi Q, Liu J, Wall E, Santat L, Dhandapani K, Kivork C, Driver A, Zhu X, Verghese M, Maer A, Saunders B, Ning Y, Subramainiam S, Meyer T, Simon MI, O’Rourke N, Chandy G, Fraser IDC (2007) The alliance for cellular signaling plasmid collection: a flexible resource for protein localization studies and signaling pathway analysis. Mol Cell Proteom 6(3):413–424. https://doi.org/10.1074/mcp.M600437-MCP200
CAS Article Google Scholar
Zhang J, Bai S, Zhang X, Nagase H, Sarras M (2003) The expression of gelatinase A (MMP-2) is required for normal development of zebrafish embryos. Dev Genes Evol 213(9):456–463. https://doi.org/10.1007/s00427-003-0346-4
CAS Article PubMed Google Scholar

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Acknowledgements

This work was supported by a Discovery Grant (341540) to BDC from the Natural Sciences and Engineering Research Council (NSERC) of Canada and the New Brunswick Innovation Foundation (NBIF) STEM Scholarship to EJJ. We thank Rachael Wyatt for her assistance in screening the stable transgenic line, image processing, and for providing feedback on this manuscript. We also thank two anonymous reviewers for valuable constructive criticisms of the manuscript. Finally, we gratefully acknowledge Robyn O’Keefe and Robyn Shortt for their dedication to and maintenance of the University of New Brunswick Zebrafish Aquatic Facility.

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Matrix Dynamics Lab, Biology Department, University of New Brunswick, 10 Bailey Drive, Fredericton, NB, E3B 5A3, Canada
Emma J. Jeffrey & Bryan D. Crawford

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Emma J. Jeffrey
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Bryan D. Crawford
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Correspondence to Bryan D. Crawford.

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Supplemental Figure 1: Sequence of the EMMA destination vector. (PDF 49 KB)

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Jeffrey, E.J., Crawford, B.D. The epitope-mediated MMP activation assay: detection and quantification of the activation of Mmp2 in vivo in the zebrafish embryo. Histochem Cell Biol 149, 277–286 (2018). https://doi.org/10.1007/s00418-018-1634-4

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Accepted: 11 January 2018
Published: 19 January 2018
Issue Date: March 2018
DOI: https://doi.org/10.1007/s00418-018-1634-4

Keywords

Matrix metalloproteinase
Gelatinase A
Zymogen
Post-translational regulation
Proteolytic activation
Zebrafish
EMMA assay