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N-terminal protein modification by substrate-activated reverse proteolysis.

Liebscher S, Schöpfel M, Aumüller T, Sharkhuukhen A, Pech A, Höss E, Parthier C, Jahreis G, Stubbs MT, Bordusa F
Angew Chem Int Ed Engl 53 3024-8 (2014)
Related entries: 4niv, 4niw, 4niy

Cited: 28 times
EuropePMC logo PMID: 24520050

Abstract

Although site-specific incorporation of artificial functionalities into proteins is an important tool in both basic and applied research, it can be a major challenge to protein chemists. Enzymatic protein modification is an attractive goal due to the inherent regio- and stereoselectivity of enzymes, yet their specificity remains a problem. As a result of the intrinsic reversibility of enzymatic reactions, proteinases can in principle catalyze ligation reactions. While this makes them attractive tools for site-specific protein bioconjugation, competing hydrolysis reactions limits their general use. Here we describe the design and application of a highly specific trypsin variant for the selective modification of N-terminal residues of diverse proteins with various reagents. The modification proceeds quantitatively under native (aqueous) conditions. We show that the variant has a disordered zymogen-like activation domain, effectively suppressing the hydrolysis reaction, which is converted to an active conformation in the presence of appropriate substrates.

Reviews citing this publication (14)

  1. Natural Occurring and Engineered Enzymes for Peptide Ligation and Cyclization. Nuijens T, Toplak A, Schmidt M, Ricci A, Cabri W. Front Chem 7 829 (2019)
  2. Antibody-drug conjugates: Current status and future perspectives. Gébleux R, Casi G. Pharmacol. Ther. 167 48-59 (2016)
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  6. Amide Bonds Meet Flow Chemistry: A Journey into Methodologies and Sustainable Evolution. Alfano AI, Lange H, Brindisi M. ChemSusChem 15 e202102708 (2022)
  7. Asparaginyl endopeptidases: enzymology, applications and limitations. Tang TMS, Luk LYP. Org Biomol Chem 19 5048-5062 (2021)
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  10. Engineered peptide ligases for cell signaling and bioconjugation. Frazier CL, Weeks AM. Biochem Soc Trans 48 1153-1165 (2020)
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  13. Promiscuous Enzymes for Residue-Specific Peptide and Protein Late-Stage Functionalization. Alexander AK, Elshahawi SI. Chembiochem 24 e202300372 (2023)
  14. Toward Homogenous Antibody Drug Conjugates Using Enzyme-Based Conjugation Approaches. Hussain AF, Grimm A, Sheng W, Zhang C, Al-Rawe M, Bräutigam K, Abu Mraheil M, Zeppernick F, Meinhold-Heerlein I. Pharmaceuticals (Basel) 14 343 (2021)

Articles citing this publication (14)

  1. Versatile and Efficient Site-Specific Protein Functionalization by Tubulin Tyrosine Ligase. Schumacher D, Helma J, Mann FA, Pichler G, Natale F, Krause E, Cardoso MC, Hackenberger CP, Leonhardt H. Angew. Chem. Int. Ed. Engl. 54 13787-13791 (2015)
  2. Site-Specific N-Terminal Labeling of Peptides and Proteins using Butelase 1 and Thiodepsipeptide. Nguyen GK, Cao Y, Wang W, Liu CF, Tam JP. Angew. Chem. Int. Ed. Engl. 54 15694-15698 (2015)
  3. N-lactoyl-amino acids are ubiquitous metabolites that originate from CNDP2-mediated reverse proteolysis of lactate and amino acids. Jansen RS, Addie R, Merkx R, Fish A, Mahakena S, Bleijerveld OB, Altelaar M, IJlst L, Wanders RJ, Borst P, van de Wetering K. Proc. Natl. Acad. Sci. U.S.A. 112 6601-6606 (2015)
  4. Derivatization of antibody Fab fragments: a designer enzyme for native protein modification. Liebscher S, Kornberger P, Fink G, Trost-Gross EM, Höss E, Skerra A, Bordusa F. Chembiochem 15 1096-1100 (2014)
  5. Chemoenzymatic Semisynthesis of Proteins. Thompson RE, Muir TW. Chem Rev 120 3051-3126 (2020)
  6. A two-trick pony: lysosomal protease cathepsin B possesses surprising ligase activity. Lambeth TR, Dai Z, Zhang Y, Julian RR. RSC Chem Biol 2 606-611 (2021)
  7. Site-specific bioorthogonal protein labelling by tetrazine ligation using endogenous β-amino acid dienophiles. Richter D, Lakis E, Piel J. Nat Chem 15 1422-1430 (2023)
  8. Towards Engineering an Orthogonal Protein Translation Initiation System. Lee BS, Choi WJ, Lee SW, Ko BJ, Yoo TH. Front Chem 9 772648 (2021)
  9. Trypsiligase-Catalyzed Labeling of Proteins on Living Cells. Liebscher S, Mathea S, Aumüller T, Pech A, Bordusa F. Chembiochem 22 1201-1204 (2021)
  10. Application of a Dual Internally Quenched Fluorogenic Substrate in Screening for D-Arginine Specific Proteases. Simon AH, Liebscher S, Aumüller TH, Treblow D, Bordusa F. Front Microbiol 10 711 (2019)
  11. Archaeal Connectase is a specific and efficient protein ligase related to proteasome β subunits. Fuchs ACD, Ammelburg M, Martin J, Schmitz RA, Hartmann MD, Lupas AN. Proc Natl Acad Sci U S A 118 e2017871118 (2021)
  12. Self-cyclisation as a general and efficient platform for peptide and protein macrocyclisation. Jia X, Chin YK, Zhang AH, Crawford T, Zhu Y, Fletcher NL, Zhou Z, Hamilton BR, Stroet M, Thurecht KJ, Mobli M. Commun Chem 6 48 (2023)
  13. Synthesis of Multiple Bispecific Antibody Formats with Only One Single Enzyme Based on Enhanced Trypsiligase. Voigt J, Meyer C, Bordusa F. Int J Mol Sci 23 3144 (2022)
  14. Use of an asparaginyl endopeptidase for chemo-enzymatic peptide and protein labeling. Tang TMS, Cardella D, Lander AJ, Li X, Escudero JS, Tsai YH, Luk LYP. Chem Sci 11 5881-5888 (2020)


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