Skip to main content
SpringerLink
Log in

Purification and Activity Determination of ADAMTS-4 and ADAMTS-5 and Their Domain Deleted Mutants

  • Protocol
  • First Online:
ADAMTS Proteases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2043))

Abstract

A disintegrin-like and metalloproteinase with thrombospondin type-1 motifs-4 (ADAMTS-4) and ADAMTS-5 are zinc-dependent metalloproteinases that are involved in the maintenance of cartilage extracellular matrix (ECM) and are currently considered the major aggrecanases in the development of osteoarthritis. In this chapter we describe the establishment and cultivation of cell lines expressing ADAMTS-4,-5 and their domain deletion mutants; the collection of medium containing expressed ADAMTS-4,-5; the subsequent purification of this medium through anti-FLAG affinity chromatography; and the characterization of ADAMTS-4,-5 activity using synthetic Förster resonance energy transfer (FRET) peptide substrates.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Kelwick R, Desanlis I, Wheeler GN, Edwards DR (2015) The ADAMTS (A disintegrin and metalloproteinase with thrombospondin motifs) family. Genome Biol 16:113. https://doi.org/10.1186/s13059-015-0676-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Rodríguez-Manzaneque JC, Fernández-Rodriguez R, Rodríguez-Baena FJ, Iruela-Arispe ML (2015) ADAMTS proteases in vascular biology. Matrix Biol 44–46:38–45. https://doi.org/10.1016/j.matbio.2015.02.004

    Article  PubMed  PubMed Central  Google Scholar 

  3. Verma P, Dalal K (2011) ADAMTS-4 and ADAMTS-5: key enzymes in osteoarthritis. J Cell Biochem 112(12):3507–3514. https://doi.org/10.1002/jcb.23298

    Article  CAS  PubMed  Google Scholar 

  4. Bondeson J, Wainwright S, Hughes C, Caterson B (2008) The regulation of the ADAMTS4 and ADAMTS5 aggrecanases in osteoarthritis: a review. Clin Exp Rheumatol 26(1):139–145

    CAS  PubMed  Google Scholar 

  5. Gottschall PE, Howell MD (2015) ADAMTS expression and function in central nervous system injury and disorders. Matrix Biol 44–46:70–76. https://doi.org/10.1016/j.matbio.2015.01.014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Kashiwagi M, Enghild JJ, Gendron C, Hughes C, Caterson B, Itoh Y, Nagase H (2004) Altered proteolytic activities of ADAMTS-4 expressed by C-terminal processing. J Biol Chem 279(11):10109–10119. https://doi.org/10.1074/jbc.M312123200

    Article  CAS  PubMed  Google Scholar 

  7. Gendron C, Kashiwagi M, Lim NH, Enghild JJ, Thøgersen IB, Hughes C, Caterson B, Nagase H (2007) Proteolytic activities of human ADAMTS-5: comparative studies with ADAMTS-4. J Biol Chem 282(25):18294–18306. https://doi.org/10.1074/jbc.M701523200

    Article  CAS  PubMed  Google Scholar 

  8. Flannery CR, Zeng W, Corcoran C, Collins-Racie LA, Chockalingam PS, Hebert T, Mackie SA, McDonagh T, Crawford TK, Tomkinson KN, LaVallie ER, Morris EA (2002) Autocatalytic cleavage of ADAMTS-4 (Aggrecanase-1) reveals multiple glycosaminoglycan-binding sites. J Biol Chem 277(45):42775–42780. https://doi.org/10.1074/jbc.M205309200

    Article  CAS  PubMed  Google Scholar 

  9. Mosyak L, Georgiadis K, Shane T, Svenson K, Hebert T, Mcdonagh T, Mackie S, Olland S, Lin L, Zhong X, Kriz R, Reifenberg EL, Collins-Racie LA, Corcoran C, Freeman B, Zollner R, Marvell T, Vera M, Sum PE, Lavallie ER, Stahl M, Somers W (2008) Crystal structures of the two major aggrecan degrading enzymes, ADAMTS4 and ADAMTS5. Protein Sci 17(1):16–21. https://doi.org/10.1110/ps.073287008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Verhoeven JW (1996) Glossary of terms used in photochemistry (IUPAC Recommendations 1996). Pure Appl Chem 68(12):2223–2286. https://doi.org/10.1351/pac199668122223

    Article  CAS  Google Scholar 

  11. Gompels LL, Lim NH, Vincent T, Paleolog EM (2010) In vivo optical imaging in arthritis—an enlightening future? Rheumatology (Oxford) 49(8):1436–1446. https://doi.org/10.1093/rheumatology/keq012

    Article  Google Scholar 

  12. Wayne GJ, Deng SJ, Amour A, Borman S, Matico R, Carter HL, Murphy G (2007) TIMP-3 inhibition of ADAMTS-4 (Aggrecanase-1) is modulated by interactions between aggrecan and the C-terminal domain of ADAMTS-4. J Biol Chem 282(29):20991–20998. https://doi.org/10.1074/jbc.M610721200

    Article  CAS  PubMed  Google Scholar 

  13. Troeberg L, Fushimi K, Scilabra SD, Nakamura H, Dive V, Thogersen IB, Enghild JJ, Nagase H (2009) The C-terminal domains of ADAMTS-4 and ADAMTS-5 promote association with N-TIMP-3. Matrix Biol 28(8):463–469. https://doi.org/10.1016/j.matbio.2009.07.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Williams JW, Morrison JF (1979) The kinetics of reversible tight-binding inhibition. Methods Enzymol 63:437–467. https://doi.org/10.1016/0076-6879(79)63019-7

    Article  CAS  PubMed  Google Scholar 

  15. Bieth JG (1995) Theoretical and practical aspects of proteinase inhibition kinetics. Methods Enzymol 248:59–84. https://doi.org/10.1016/0076-6879(95)48007-2

    Article  CAS  PubMed  Google Scholar 

  16. Knight CG (1995) Active-site titration of peptidases. Methods Enzymol 248:85–101. https://doi.org/10.1016/0076-6879(95)48008-0

    Article  CAS  PubMed  Google Scholar 

  17. Fushimi K, Troeberg L, Nakamura H, Lim NH, Nagase H (2008) Functional differences of the catalytic and non-catalytic domains in human ADAMTS-4 and ADAMTS-5 in aggrecanolytic activity. J Biol Chem 283(11):6706–6716. https://doi.org/10.1074/jbc.M708647200

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Kennedy Institute of Rheumatology, University of Oxford, Oxford, UK

    Milan M. Fowkes & Ngee H. Lim

Authors
  1. Milan M. Fowkes
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ngee H. Lim
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ngee H. Lim .

Editor information

Editors and Affiliations

  1. Department of Biomedical Engineering-ND20, Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA

    Suneel S. Apte

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Fowkes, M.M., Lim, N.H. (2020). Purification and Activity Determination of ADAMTS-4 and ADAMTS-5 and Their Domain Deleted Mutants. In: Apte, S. (eds) ADAMTS Proteases. Methods in Molecular Biology, vol 2043. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9698-8_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9698-8_7

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9697-1

  • Online ISBN: 978-1-4939-9698-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation