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Butelase 1-Mediated Ligation of Peptides and Proteins

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Enzyme-Mediated Ligation Methods

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2012))

Abstract

Structurally, butelase 1 is a cysteine protease of the asparaginyl endoprotease (AEP) family, but functionally, it displays intense Asn/Asp-specific (Asx) ligase activity and is virtually devoid of protease activity. Butelase 1 recognizes specifically a C-terminal Asx-containing tripeptide motif, Asx-His-Val, to form an Asx-Xaa peptide bond (Xaa = any amino acid), either intramolecularly or intermolecularly, resulting in cyclic peptides or site-specific modified peptides/proteins, respectively. Our work in the past 4 years has validated that butelase 1 is a potent and versatile tool for peptide and protein modification. Here we describe our protocols using butelase 1 for efficient and site-specific peptide and protein ligation, N-terminal labeling, preparation of thioesters, and bioconjugation of dendrimers. Additionally, we provide an example using butelase 1 for protein cyclization in combination with genetic code expansion in order to incorporate unnatural building blocks.

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References

  1. Usmani SS, Bedi G, Samuel JS, Singh S, Kalra S, Kumar P, Ahuja AA, Sharma M, Gautam A, Raghava GPS (2017) THPdb: database of FDA-approved peptide and protein therapeutics. PLoS One 12(7):e0181748. https://doi.org/10.1371/journal.pone.0181748

    Article  CAS  Google Scholar 

  2. Fosgerau K, Hoffmann T (2015) Peptide therapeutics: current status and future directions. Drug Discov Today 20(1):122–128. https://doi.org/10.1016/j.drudis.2014.10.003

    Article  CAS  Google Scholar 

  3. Haase J, Lanka E (1997) A specific protease encoded by the conjugative DNA transfer systems of IncP and Ti plasmids is essential for pilus synthesis. J Bacteriol 179:5728–5735. https://doi.org/10.1128/jb.179.18.5728-5735.1997

    Article  CAS  Google Scholar 

  4. Koehnke J, Bent A, Houssen WE, Zollman D, Morawitz F, Shirran S, Vendome J, Nneoyiegbe AF, Trembleau L, Botting CH, Smith MC, Jaspars M, Naismith JH (2012) The mechanism of patellamide macrocyclization revealed by the characterization of the PatG macrocyclase domain. Nat Struct Mol Biol 19(8):767–772. https://doi.org/10.1038/nsmb.2340

    Article  CAS  Google Scholar 

  5. Oueis E, Stevenson H, Jaspars M, Westwood NJ, Naismith JH (2017) Bypassing the proline/thiazoline requirement of the macrocyclase PatG. Chem Comm 53:4. https://doi.org/10.1039/C7CC06550G

    Article  Google Scholar 

  6. Barber CJS, Pujara PT, Reed DW, Chiwocha S, Zhang H, Covello PS (2013) The two-step biosynthesis of cyclic peptides from linear precursors in a member of the plant family caryophyllaceae involves cyclization by a serine protease-like enzyme. J Biol Chem 288(18):12500–12510. https://doi.org/10.1074/jbc.M112.437947

    Article  CAS  Google Scholar 

  7. Chekan JR, Estrada P, Covello PS, Nair SK (2017) Characterization of the macrocyclase involved in the biosynthesis of RiPP cyclic peptides in plants. Proc Natl Acad Sci U S A 114(25):6551–6556. https://doi.org/10.1073/pnas.1620499114

    Article  CAS  Google Scholar 

  8. Nguyen GKT, Wang S, Qiu Y, Hemu X, Lian Y, Tam JP (2014) Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis. Nat Chem Biol 10(9):732–738. https://doi.org/10.1038/nchembio.1586

    Article  CAS  Google Scholar 

  9. Luo H, Hong SY, Sgambelluri RM, Angelos E, Li X, Walton JD (2014) Peptide macrocyclization catalyzed by a prolyl oligopeptidase involved in alpha-amanitin biosynthesis. Chem Biol 21:1610. https://doi.org/10.1016/j.chembiol.2014.10.015

    Article  CAS  Google Scholar 

  10. Mazmanian SK, Liu G, Ton-That H, Schneewind O (1999) Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall. Science 285:760–763. https://doi.org/10.1126/science.285.5428.760

    Article  CAS  Google Scholar 

  11. Mao H, Hart SA, Schink A, Pollok BA (2004) Sortase-mediated protein ligation: a new method for protein engineering. J Am Chem Soc Comm 126(9):2670–2671. https://doi.org/10.1021/ja039915e

    Article  CAS  Google Scholar 

  12. Popp MW, Antos JM, Grotenbreg GM, Spooner E, Ploegh HL (2007) Sortagging: a versatile method for protein labeling. Nat Chem Biol 3(11):707–708. https://doi.org/10.1038/Nchembio.2007.31

    Article  CAS  Google Scholar 

  13. Chang TK, Jackson DY, Burnier JP, Wells JA (1994) Subtiligase: a tool for semisynthesis of proteins. Proc Natl Acad Sci U S A 91(26):12544–12548. https://doi.org/10.1073/pnas.91.26.12544

    Article  CAS  Google Scholar 

  14. Jackson DY, Burnier J, Quan C, Stanley M, Tom J, Wells JA (1994) A designed peptide ligase for total synthesis of ribonuclease A with unnatural catalytic residues. Science 266(5183):243–247. https://doi.org/10.1126/science.7939659

    Article  CAS  Google Scholar 

  15. Atwell S, Wells JA (1999) Selection for improved subtiligases by phage display. Proc Natl Acad Sci U S A 96(17):9497–9502. https://doi.org/10.1073/pnas.96.17.9497

    Article  CAS  Google Scholar 

  16. Nuijens T, Toplak A, Quaedflieg PJLM, Drenth J, Wu B, Janssen DB (2016) Engineering a diverse ligase toolbox for peptide segment condensation. Adv Synth Catal 358(24):4041–4048. https://doi.org/10.1002/adsc.201600774

    Article  CAS  Google Scholar 

  17. Toplak A, Nuijens T, Quaedflieg PJLM, Wu B, Janssen DB (2016) Peptiligase, an enzyme for efficient chemoenzymatic peptide synthesis and cyclization in water. Adv Synth Catal 358(13):2140–2147. https://doi.org/10.1002/adsc.201600017

    Article  CAS  Google Scholar 

  18. Schmidt M, Toplak A, Quaedflieg PJLM, Ippel H, Richelle GJJ, Hackeng TM, van Maarseveen JH, Nuijens T (2017) Omniligase-1: a powerful tool for peptide head-to-tail cyclization. Adv Synth Catal 359(12):2050–2055. https://doi.org/10.1002/adsc.201700314

    Article  CAS  Google Scholar 

  19. Chen J-M, Rawlings ND, Stevens RAE, Barrett AJ (1998) Identification of the active site of legumain links it to caspases, clostripain and gingipains in a new clan of cysteine endopeptidases. FEBS Lett 441(3):361–365. https://doi.org/10.1016/S0014-5793(98)01574-9

    Article  CAS  Google Scholar 

  20. Halfon S, Patel S, Vega F, Zurawski S, Zurawski G (1998) Autocatalytic activation of human legumain at aspartic acid residues. FEBS Lett 438(1-2):114–118. https://doi.org/10.1016/S0014-5793(98)01281-2

    Article  CAS  Google Scholar 

  21. Hiraiwa N, Nishimura M, Hara-Nishimura I (1999) Vacuolar processing enzyme is self-catalytically activated by sequential removal of the C-terminal and N-terminal propeptides. FEBS Lett 447(2–3):213–216. https://doi.org/10.1016/S0014-5793(99)00286-0

    Article  CAS  Google Scholar 

  22. Chen JM, Fortunato M, Barrett AJ (2000) Activation of human prolegumain by cleavage at a C-terminal asparagine residue. Biochem J 352(Pt 2):327–334. https://doi.org/10.1042/bj3520327

    Article  CAS  Google Scholar 

  23. Kuroyanagi M, Nishimura M, Hara-Nishimura I (2002) Activation of arabidopsis vacuolar processing enzyme by self-catalytic removal of an auto-inhibitory domain of the C-terminal propeptide. Plant Cell Physiol 43(2):143–151. https://doi.org/10.1093/pcp/pcf035

    Article  CAS  Google Scholar 

  24. Bernath-Levin K, Nelson C, Elliott AG, Jayasena AS, Millar AH, Craik DJ, Mylne JS (2015) Peptide macrocyclization by a bifunctional endoprotease. Chem Biol 22(5):571–582. https://doi.org/10.1016/j.chembiol.2015.04.010

    Article  CAS  Google Scholar 

  25. Harris KS, Durek T, Kaas Q, Poth AG, Gilding EK, Conlan BF, Saska I, Daly NL, van der Weerden NL, Craik DJ, Anderson MA (2015) Efficient backbone cyclization of linear peptides by a recombinant asparaginyl endopeptidase. Nat Commun 6:10199. https://doi.org/10.1038/ncomms10199

    Article  CAS  Google Scholar 

  26. Zhao L, Hua T, Crowley C, Ru H, Ni X, Shaw N, Jiao L, Ding W, Qu L, Hung LW, Huang W, Liu L, Ye K, Ouyang S, Cheng G, Liu ZJ (2014) Structural analysis of asparaginyl endopeptidase reveals the activation mechanism and a reversible intermediate maturation stage. Cell Res 24(3):344–358. https://doi.org/10.1038/cr.2014.4

    Article  CAS  Google Scholar 

  27. Cao Y, Nguyen GK, Tam JP, Liu CF (2015) Butelase-mediated synthesis of protein thioesters and its application for tandem chemoenzymatic ligation. Chem Commun 51:17289–17292. https://doi.org/10.1039/C5CC07227A

    Article  CAS  Google Scholar 

  28. Nguyen GK, Hemu X, Quek JP, Tam JP (2016) Butelase-mediated macrocyclization of d-amino-acid-containing peptides. Angew Chem Int Ed Engl 55(41):12802–12806. https://doi.org/10.1002/anie.201607188

    Article  CAS  Google Scholar 

  29. Nguyen GKT, Kam A, Loo S, Jansson AE, Pan LX, Tam JP (2015) Butelase 1: a versatile ligase for peptide and protein macrocyclization. J Am Chem Soc Comm 137(49):15398–15401. https://doi.org/10.1021/jacs.5b11014

    Article  CAS  Google Scholar 

  30. Bi X, Yin J, Hemu X, Rao C, Tam JP, Liu CF (2018) Immobilization and intracellular delivery of circular proteins by modifying a genetically incorporated unnatural amino acid. Bioconjug Chem 29(7):2170–2175. https://doi.org/10.1021/acs.bioconjchem.8b00244

    Article  CAS  Google Scholar 

  31. Nguyen GK, Cao Y, Wang W, Liu CF, Tam JP (2015) Site-specific N-terminal labeling of peptides and proteins using butelase 1 and thiodepsipeptide. Angew Chem Int Ed Engl 54(52):15694–15698

    Article  CAS  Google Scholar 

  32. Cao Y, Nguyen GK, Chuah S, Tam JP, Liu CF (2016) Butelase-mediated ligation as an efficient bioconjugation method for the synthesis of peptide dendrimers. Bioconjug Chem 27(11):2592–2596. https://doi.org/10.1021/acs.bioconjchem.6b00538

    Article  CAS  Google Scholar 

  33. Harmand TJ, Bousbaine D, Chan A, Zhang X, Liu DR, Tam JP, Ploegh HL (2018) One-pot dual labeling of IgG 1 and preparation of C-to-C fusion proteins through a combination of sortase A and butelase 1. Bioconjug Chem 29:3245. https://doi.org/10.1021/acs.bioconjchem.8b00563

    Article  CAS  Google Scholar 

  34. Bi X, Yin J, Nguyen GKT, Rao C, Halim NBA, Hemu X, Tam JP, Liu CF (2017) Enzymatic engineering of live bacterial cell surfaces using butelase 1. Angew Chem Int Ed Engl 56(27):7822–7825. https://doi.org/10.1002/anie.201703317

    Article  CAS  Google Scholar 

  35. Nguyen GK, Qiu Y, Cao Y, Hemu X, Liu CF, Tam JP (2016) Butelase-mediated cyclization and ligation of peptides and proteins. Nat Protoc 11(10):1977–1988. https://doi.org/10.1038/nprot.2016.118

    Article  CAS  Google Scholar 

  36. Neumann H, Peak-Chew SY, Chin JW (2008) Genetically encoding N(epsilon)-acetyllysine in recombinant proteins. Nat Chem Biol 4(4):232–234. https://doi.org/10.1038/nchembio.73

    Article  CAS  Google Scholar 

  37. Nguyen DP, Lusic H, Neumann H, Kapadnis PB, Deiters A, Chin JW (2009) Genetic encoding and labeling of aliphatic azides and alkynes in recombinant proteins via a pyrrolysyl-tRNA synthetase/tRNACUA pair and click chemistry. J Am Chem Soc Comm 131:8720–8721. https://doi.org/10.1021/ja900553w

    Article  CAS  Google Scholar 

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Acknowledgments

This research was supported by Academic Research Grant Tier 3 (MOE2016-T3-1-003) from Singapore Ministry of Education (MOE).

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Authors and Affiliations

  1. School of Biological Sciences, Nanyang Technological University, Singapore, Singapore

    Xinya Hemu, Xiaohong Zhang, Xiaobao Bi, Chuan-Fa Liu & James P. Tam

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  1. Xinya Hemu
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  3. Xiaobao Bi
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  4. Chuan-Fa Liu
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  5. James P. Tam
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Corresponding author

Correspondence to James P. Tam .

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Editors and Affiliations

  1. EnzyPep B.V., Geleen, The Netherlands

    Timo Nuijens  & Marcel Schmidt  & 

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Hemu, X., Zhang, X., Bi, X., Liu, CF., Tam, J.P. (2019). Butelase 1-Mediated Ligation of Peptides and Proteins. In: Nuijens, T., Schmidt, M. (eds) Enzyme-Mediated Ligation Methods. Methods in Molecular Biology, vol 2012. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9546-2_6

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  • DOI: https://doi.org/10.1007/978-1-4939-9546-2_6

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9545-5

  • Online ISBN: 978-1-4939-9546-2

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