Skip to main content
SpringerLink
Log in

Engineering New Protease Inhibitors Using α2-Macroglobulin

  • Protocol
  • First Online:
Proteases and Cancer

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2747))

  • 385 Accesses

Abstract

Protease inhibitors of the alpha-macroglobulin family (αM) have a unique mechanism that allows them to trap proteases that is dependent not on the protease’s class, but rather on its cleavage specificity. Proteases trigger a conformational change in the αM protein by cleaving within a “bait region,” resulting in the sequestering of the protease inside the αM molecule. This nonspecific inhibitory mechanism appears to have arisen early in the αM family, and the broad protease-trapping capacity that it allows may play a role in pathogen defense.

Human α2-macroglobulin (A2M) is a tetrameric αM whose bait region is permissive to cleavage by most proteases, making it a broad-spectrum protease inhibitor. Recent work has demonstrated that the inhibitory capacity of A2M derives directly from its bait region sequence: modifying the bait region sequence to introduce or remove protease cleavage sites will modify A2M’s inhibition of the relevant proteases accordingly. Thus, changing the amino acid sequence of the bait region presents an effective avenue for protein engineering of new protease inhibitors if the substrate specificity of the target protease is known. The design of new A2M-based protease inhibitors with tailored inhibitory capacities has potential applications in basic research and the clinic. In this chapter, we describe the general approach and considerations for the bait region engineering of A2M.

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 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 199.99
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

Similar content being viewed by others

References

  1. Barrett AJ, Brown MA, Sayers CA (1979) The electrophoretically ‘slow’ and ‘fast’ forms of the alpha 2-macroglobulin molecule. Biochem J 181:401–418

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Christensen U, Simonsen M, Harrit N et al (1989) Pregnancy zone protein, a proteinase-binding macroglobulin. Interactions with proteinases and methylamine. Biochemistry 28:9324–9331

    Article  CAS  PubMed  Google Scholar 

  3. Enghild JJ, Salvesen G, Thogersen IB et al (1989) Proteinase binding and inhibition by the monomeric alpha-macroglobulin rat alpha 1-inhibitor-3. J Biol Chem 264:11428–11435

    Article  CAS  PubMed  Google Scholar 

  4. Galliano MF, Toulza E, Gallinaro H et al (2006) A novel protease inhibitor of the alpha2-macroglobulin family expressed in the human epidermis. J Biol Chem 281:5780–5789

    Article  CAS  PubMed  Google Scholar 

  5. Barrett AJ, Starkey PM (1973) The interaction of alpha 2-macroglobulin with proteinases. Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism. Biochem J 133:709–724

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Imber MJ, Pizzo SV (1981) Clearance and binding of two electrophoretic “fast” forms of human alpha 2-macroglobulin. J Biol Chem 256:8134–8139

    Article  CAS  PubMed  Google Scholar 

  7. Sottrup-Jensen L, Petersen TE, Magnusson S (1980) A thiol-ester in alpha 2-macroglobulin cleaved during proteinase complex formation. FEBS Lett 121:275–279

    Article  CAS  PubMed  Google Scholar 

  8. Marrero A, Duquerroy S, Trapani S et al (2012) The crystal structure of human alpha2-macroglobulin reveals a unique molecular cage. Angew Chem Int Ed Engl 51:3340–3344

    Article  CAS  PubMed  Google Scholar 

  9. Nielsen NS, Zarantonello A, Harwood SL et al (2022) Cryo-EM structures of human A2ML1 elucidate the protease-inhibitory mechanism of the A2M family. Nat Commun 13:3033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Salvesen GS, Sayers CA, Barrett AJ (1981) Further characterization of the covalent linking reaction of alpha 2-macroglobulin. Biochem J 195:453–461

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Gettins P, Cunningham LW (1986) Identification of 1H resonances from the bait region of human alpha 2-macroglobulin and effects of proteases and methylamine. Biochemistry 25:5011–5017

    Article  CAS  PubMed  Google Scholar 

  12. Arbelaez LF, Bergmann U, Tuuttila A et al (1997) Interaction of matrix metalloproteinases-2 and -9 with pregnancy zone protein and alpha2-macroglobulin. Arch Biochem Biophys 347:62–68

    Article  CAS  PubMed  Google Scholar 

  13. Enghild JJ, Salvesen G, Brew K et al (1989) Interaction of human rheumatoid synovial collagenase (matrix metalloproteinase 1) and stromelysin (matrix metalloproteinase 3) with human alpha 2-macroglobulin and chicken ovostatin. Binding kinetics and identification of matrix metalloproteinase cleavage sites. J Biol Chem 264:8779–8785

    Article  CAS  PubMed  Google Scholar 

  14. Ellis V, Scully M, MacGregor I et al (1982) Inhibition of human factor Xa by various plasma protease inhibitors. Biochim Biophys Acta 701:24–31

    Article  CAS  PubMed  Google Scholar 

  15. Virca GD, Travis J (1984) Kinetics of association of human proteinases with human alpha 2-macroglobulin. J Biol Chem 259:8870–8874

    Article  CAS  PubMed  Google Scholar 

  16. Raymond WW, Su S, Makarova A et al (2009) Alpha 2-macroglobulin capture allows detection of mast cell chymase in serum and creates a reservoir of angiotensin II-generating activity. J Immunol 182:5770–5777

    Article  CAS  PubMed  Google Scholar 

  17. Gron H, Pike R, Potempa J et al (1997) The potential role of alpha 2-macroglobulin in the control of cysteine proteinases (gingipains) from Porphyromonas gingivalis. J Periodontal Res 32:61–68

    Article  CAS  PubMed  Google Scholar 

  18. Hall PK, Nelles LP, Travis J et al (1981) Proteolytic cleavage sites on alpha 2-macroglobulin resulting in proteinase binding are different for trypsin and Staphylococcus aureus V-8 proteinase. Biochem Biophys Res Commun 100:8–16

    Article  CAS  PubMed  Google Scholar 

  19. Meier UC, Billich A, Mann K et al (1991) alpha 2-Macroglobulin is cleaved by HIV-1 protease in the bait region but not in the C-terminal inter-domain region. Biol Chem Hoppe Seyler 372:1051–1056

    Article  CAS  PubMed  Google Scholar 

  20. Baramova EN, Shannon JD, Bjarnason JB et al (1990) Interaction of hemorrhagic metalloproteinases with human alpha 2-macroglobulin. Biochemistry 29:1069–1074

    Article  CAS  PubMed  Google Scholar 

  21. Wyatt AR, Kumita JR, Mifsud RW et al (2014) Hypochlorite-induced structural modifications enhance the chaperone activity of human alpha2-macroglobulin. Proc Natl Acad Sci U S A 111:E2081–E2090

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. O’Connor-McCourt MD, Wakefield LM (1987) Latent transforming growth factor-beta in serum. A specific complex with alpha 2-macroglobulin. J Biol Chem 262:14090–14099

    Article  PubMed  Google Scholar 

  23. Umans L, Serneels L, Overbergh L et al (1995) Targeted inactivation of the mouse alpha 2-macroglobulin gene. J Biol Chem 270:19778–19785

    Article  CAS  PubMed  Google Scholar 

  24. Umans L, Serneels L, Overbergh L et al (1999) alpha2-macroglobulin- and murinoglobulin-1- deficient mice. A mouse model for acute pancreatitis. Am J Pathol 155:983–993

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Marijanovic EM, Fodor J, Riley BT et al (2019) Reactive Centre loop dynamics and serpin specificity. Sci Rep 9:3870

    Article  PubMed  PubMed Central  Google Scholar 

  26. Van Rompaey L, Proost P, Van den Berghe H et al (1995) Design of a new protease inhibitor by the manipulation of the bait region of alpha 2-macroglobulin: inhibition of the tobacco etch virus protease by mutant alpha 2-macroglobulin. Biochem J 312(Pt 1):191–195

    Article  PubMed  PubMed Central  Google Scholar 

  27. Van Rompaey L, Ayoubi T, Van De Ven W et al (1997) Inhibition of intracellular proteolytic processing of soluble proproteins by an engineered alpha 2-macroglobulin containing a furin recognition sequence in the bait region. Biochem J 326(Pt 2):507–514

    Article  PubMed  PubMed Central  Google Scholar 

  28. Ikai A, Ookata K, Shimizu M et al (1999) A recombinant bait region mutant of human alpha2-macroglobulin exhibiting an altered proteinase-inhibiting spectrum. Cytotechnology 31:53–60

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Harwood SL, Nielsen NS, Diep K et al (2021) Development of selective protease inhibitors via engineering of the bait region of human alpha2-macroglobulin. J Biol Chem 297:100879

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Zhang Y, Wei X, Browning S et al (2017) Targeted designed variants of alpha-2-macroglobulin (A2M) attenuate cartilage degeneration in a rat model of osteoarthritis induced by anterior cruciate ligament transection. Arthritis Res Ther 19:175

    Article  PubMed  PubMed Central  Google Scholar 

  31. Overall CM, Kleifeld O (2006) Tumour microenvironment - opinion: validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy. Nat Rev Cancer 6:227–239

    Article  CAS  PubMed  Google Scholar 

  32. Coussens LM, Fingleton B, Matrisian LM (2002) Matrix metalloproteinase inhibitors and cancer: trials and tribulations. Science 295:2387–2392

    Article  CAS  PubMed  Google Scholar 

  33. Laronha H, Carpinteiro I, Portugal J et al (2020) Challenges in matrix metalloproteinases inhibition. Biomolecules 10:717

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Uhland K (2006) Matriptase and its putative role in cancer. Cell Mol Life Sci 63:2968–2978

    Article  CAS  PubMed  Google Scholar 

  35. Ulisse S, Baldini E, Sorrenti S et al (2009) The urokinase plasminogen activator system: a target for anti-cancer therapy. Curr Cancer Drug Targets 9:32–71

    Article  CAS  PubMed  Google Scholar 

  36. Harwood SL, Diep K, Nielsen NS et al (2022) The conformational change of the protease inhibitor alpha2-macroglobulin is triggered by the retraction of the cleaved bait region from a central channel. J Biol Chem 298:102230

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Rawlings ND, Barrett AJ, Thomas PD et al (2018) The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res 46:D624–D632

    Article  CAS  PubMed  Google Scholar 

  38. Luo SY, Araya LE, Julien O (2019) Protease substrate identification using N-terminomics. ACS Chem Biol 14:2361–2371

    Article  CAS  PubMed  Google Scholar 

  39. Chen EI, Kridel SJ, Howard EW et al (2002) A unique substrate recognition profile for matrix metalloproteinase-2. J Biol Chem 277:4485–4491

    Article  CAS  PubMed  Google Scholar 

  40. Sottrup-Jensen L, Sand O, Kristensen L et al (1989) The alpha-macroglobulin bait region. Sequence diversity and localization of cleavage sites for proteinases in five mammalian alpha-macroglobulins. J Biol Chem 264:15781–15789

    Article  CAS  PubMed  Google Scholar 

  41. Kan CC, Solomon E, Belt KT et al (1985) Nucleotide sequence of cDNA encoding human alpha 2-macroglobulin and assignment of the chromosomal locus. Proc Natl Acad Sci U S A 82:2282–2286

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Harpel PC (1970) Human plasma alpha 2-macroglobulin. An inhibitor of plasma kallikrein. J Exp Med 132:329–352

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Salvesen G, Enghild JJ (1993) Alpha-macroglobulins: detection and characterization. Methods Enzymol 223:121–141

    Article  CAS  PubMed  Google Scholar 

  44. Bury A (1981) Analysis of protein and peptide mixtures: evaluation of three sodium dodecyl sulphate-polyacrylamide gel electrophoresis buffer systems. J Chromatogr A 213:491–500

    Article  CAS  Google Scholar 

  45. Manwell C (1977) A simplified electrophoretic system for determining molecular weights of proteins. Biochem J 165:487–495

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Harwood SL, Nielsen NS, Jensen KT et al (2020) alpha2-Macroglobulin-like protein 1 can conjugate and inhibit proteases through their hydroxyl groups, because of an enhanced reactivity of its thiol ester. J Biol Chem 295:16732–16742

    Article  CAS  PubMed  Google Scholar 

  47. Harwood SL, Nielsen NS, Pedersen H et al (2020) Substituting the thiol Ester of human A2M or C3 with a Disulfide produces native proteins with altered proteolysis-induced conformational changes. Biochemistry 59:4799–4809

    Article  CAS  PubMed  Google Scholar 

  48. Harwood SL, Lyngso J, Zarantonello A et al (2021) Structural investigations of human A2M identify a hollow native conformation that underlies its distinctive protease-trapping mechanism. Mol Cell Proteomics 20:100090

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgement

This study was supported by the VELUX FONDEN (00014557), the Danish Council for Independent Research-Medical Science (DFF-4004-00471), the LEO Foundation, and the Novo Nordisk Foundation (BIO-MS) (NNF18OC0032724).

Author information

Authors and Affiliations

  1. Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    Seandean Lykke Harwood & Jan J. Enghild

Authors
  1. Seandean Lykke Harwood
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jan J. Enghild
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jan J. Enghild .

Editor information

Editors and Affiliations

  1. Department of Biochemical Sciences, School of Biosciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, UK

    Salvatore Santamaria

Rights and permissions

Reprints and permissions

Copyright information

© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Harwood, S.L., Enghild, J.J. (2024). Engineering New Protease Inhibitors Using α2-Macroglobulin. In: Santamaria, S. (eds) Proteases and Cancer. Methods in Molecular Biology, vol 2747. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3589-6_21

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3589-6_21

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3588-9

  • Online ISBN: 978-1-0716-3589-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation