Amino Acids

, Volume 50, Issue 7, pp 923–932 | Cite as

Site-specific derivatization of human interferon β-1a at lysine residues using microbial transglutaminase

  • Barbara Spolaore
  • Giacomo Forzato
  • Angelo Fontana
Original Article


Microbial transglutaminase (TGase) has been successfully used to produce site-specific protein conjugates derivatized at the level of glutamine (Gln) or lysine (Lys) residues with diverse applications. Here, we study the drug human interferon β-1a (IFN) as a substrate of TGase. The derivatization reaction was performed using carbobenzoxy-l-glutaminyl-glycine to modify Lys residues and dansylcadaverine for Gln residues. The 166 amino acids polypeptide chain of IFN β-1a contains 11 Lys and 11 Gln residues potential sites of TGase derivatization. By means of mass spectrometry analyses, we demonstrate the highly selective derivatization of this protein by TGase at the level of Lys115 and as secondary site at the level of Lys33, while no reactive Gln residue was detected. Limited proteolysis experiments were performed on IFN to determine flexible regions of the protein under physiological conditions. Interestingly, primary and secondary sites of limited proteolysis and of TGase derivatization occur at the same regions of the polypeptide chain, indicating that the extraordinary selectivity of the TGase-mediated reaction is dictated by the conformational features of the protein substrate. We envisage that the TGase-mediated derivatization of IFN can be used to produce interesting derivatives of this important therapeutic protein.


Human interferon β-1a Protein conjugation Transglutaminase Limited proteolysis 







Enzyme to substrate ratio


Human interferon β-1a


Polyethylene glycol


Trifluoroacetic acid









We acknowledge Silvia Gelio for conducting some experiments. This work was supported by the University of Padua (60A04-3887/12 and 60A04-8780/15).

Compliance with ethical standards

Conflicts of interest

The authors declare that there is no conflict of interest with regard to publication of this research work.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

726_2018_2563_MOESM1_ESM.docx (140 kb)
Supplementary material 1 (DOCX 140 kb)


  1. Ando H, Adachi M, Umeda K et al (1989) Purification and characteristics of a novel transglutaminase derived from microorganisms. Agric Biol Chem 53:2613–2617. Google Scholar
  2. Baker DP, Lin EY, Lin K et al (2006) N-terminally PEGylated human interferon-β-1a with improved pharmacokinetic properties and in vivo efficacy in a melanoma angiogenesis model. Bioconjug Chem 17:179–188. CrossRefPubMedGoogle Scholar
  3. Basu A, Yang K, Wang M et al (2006) Structure−function engineering of interferon-β-1b for improving stability, solubility, potency, immunogenicity, and pharmacokinetic properties by site-selective mono-PEGylation. Bioconjug Chem 17:618–630. CrossRefPubMedGoogle Scholar
  4. Cocco E, Marrosu MG (2015) Profile of PEGylated interferon beta in the treatment of relapsing-remitting multiple sclerosis. Ther Clin Risk Manag 11:759–766. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Conradt HS, Egge H, Peter-Katalinic J et al (1987) Structure of the carbohydrate moiety of human interferon-beta secreted by a recombinant Chinese hamster ovary cell line. J Biol Chem 262:14600–14605PubMedGoogle Scholar
  6. Dissing-Olesen L, Thaysen-Andersen M, Meldgaard M et al (2008) The function of the human interferon-1a glycan determined in vivo. J Pharmacol Exp Ther 326:338–347. CrossRefPubMedGoogle Scholar
  7. Folk JE (1983) Mechanism and basis for specificity of transglutaminase-catalyzed epsilon-(gamma-glutamyl) lysine bond formation. Adv Enzymol Relat Areas Mol Biol 54:1–56PubMedGoogle Scholar
  8. Fontana A, Spolaore B, Mero A, Veronese FM (2008) Site-specific modification and PEGylation of pharmaceutical proteins mediated by transglutaminase. Adv Drug Deliv Rev 60:13–28. CrossRefPubMedGoogle Scholar
  9. Fontana A, Spolaore B, Mero A, Veronese FM (2009) The site-specific TGase-mediated PEGylation of proteins occurs at flexible sites. In: Veronese FM (ed) PEGylated protein drugs: basic science and clinical applications. Birkhäuser, Basel, pp 89–112CrossRefGoogle Scholar
  10. Fontana A, de Laureto PP, Spolaore B, Frare E (2012) Identifying disordered regions in proteins by limited proteolysis. In: Uversky VN, Dunker AK (eds) Intrinsically disordered protein analysis, vol 2. Methods and Experimental Tools. Springer, New York, pp 297–318CrossRefGoogle Scholar
  11. Gershon PD (2014) Cleaved and missed sites for trypsin, Lys-C, and Lys-N can be predicted with high confidence on the basis of sequence context. J Proteome Res 13:702–709. CrossRefPubMedGoogle Scholar
  12. Griffin M, Casadio R, Bergamini CM (2002) Transglutaminases: nature’s biological glues. Biochem J 368:377–396. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Jeger S, Zimmermann K, Blanc A et al (2010) Site-specific and stoichiometric modification of antibodies by bacterial transglutaminase. Angew Chem Int Ed 49:9995–9997. CrossRefGoogle Scholar
  14. Karpusas M, Nolte M, Benton CB et al (1997) The crystal structure of human interferon β at 2.2 Å resolution. Proc Natl Acad Sci 94:11813–11818CrossRefPubMedPubMedCentralGoogle Scholar
  15. Klaus W, Gsell B, Labhardt AM et al (1997) The three-dimensional high resolution structure of human interferon α-2a determined by heteronuclear NMR spectroscopy in solution. J Mol Biol 274:661–675. CrossRefPubMedGoogle Scholar
  16. Lee JI, Eisenberg SP, Rosendahl MS et al (2013) Site-specific PEGylation enhances the pharmacokinetic properties and antitumor activity of interferon beta-1b. J Interferon Cytokine Res 33:769–777. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Mariniello L, Porta R, Sorrentino A et al (2014) Transglutaminase-mediated macromolecular assembly: production of conjugates for food and pharmaceutical applications. Amino Acids 46:767–776. CrossRefPubMedGoogle Scholar
  18. Mark DF, Lu SD, Creasey AA et al (1984) Site-specific mutagenesis of the human fibroblast interferon gene. Proc Natl Acad Sci 81:5662–5666CrossRefPubMedPubMedCentralGoogle Scholar
  19. Mastrangeli R, Rossi M, Mascia M et al (2015) In vitro biological characterization of IFN-β-1a major glycoforms. Glycobiology 25:21–29. CrossRefPubMedGoogle Scholar
  20. Mero A, Spolaore B, Veronese FM, Fontana A (2009) Transglutaminase-mediated PEGylation of proteins: direct identification of the sites of protein modification by mass spectrometry using a novel monodisperse PEG. Bioconjug Chem 20:384–389. CrossRefPubMedGoogle Scholar
  21. Nairn NW, Shanebeck KD, Wang A et al (2012) Development of copper-catalyzed azide-alkyne cycloaddition for increased in vivo efficacy of interferon β-1b by site-specific PEGylation. Bioconjug Chem 23:2087–2097. CrossRefPubMedGoogle Scholar
  22. Ohtsuka T, Ota M, Nio N, Motoki M (2000a) Comparison of substrate specificities of transglutaminases using synthetic peptides as acyl donors. Biosci Biotechnol Biochem 64:2608–2613. CrossRefPubMedGoogle Scholar
  23. Ohtsuka T, Sawa A, Kawabata R et al (2000b) Substrate specificities of microbial transglutaminase for primary amines. J Agric Food Chem 48:6230–6233. CrossRefPubMedGoogle Scholar
  24. Piehler J, Thomas C, Garcia KC, Schreiber G (2012) Structural and dynamic determinants of type I interferon receptor assembly and their functional interpretation. Immunol Rev 250:317–334. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Radhakrishnan R, Walter LJ, Hruza A et al (1996) Zinc mediated dimer of human interferon-α2b revealed by X-ray crystallography. Structure 4:1453–1463. CrossRefPubMedGoogle Scholar
  26. Runkel L, Meier W, Pepinsky RB et al (1998a) Structural and functional differences between glycosylated and non-glycosylated forms of human interferon-β (IFN-β). Pharm Res 15:641–649. CrossRefPubMedGoogle Scholar
  27. Runkel L, Pfeffer L, Lewerenz M et al (1998b) Differences in activity between alpha and beta type I interferons explored by mutational analysis. J Biol Chem 273:8003–8008. CrossRefPubMedGoogle Scholar
  28. Runkel L, deDios C, Karpusas M et al (2000) Systematic mutational mapping of sites on human interferon-β-1a that are important for receptor binding and functional activity. Biochemistry 39:2538–2551. CrossRefPubMedGoogle Scholar
  29. Sato H (2002) Enzymatic procedure for site-specific pegylation of proteins. Adv Drug Deliv Rev 54:487–504. CrossRefPubMedGoogle Scholar
  30. Shepard HM, Leung D, Stebbing N, Goeddel DV (1981) A single amino acid change in IFN-[beta]1 abolishes its antiviral activity. Nature 294:563–565. CrossRefPubMedGoogle Scholar
  31. Spolaore B, Raboni S, Ramos Molina A et al (2012) Local unfolding is required for the site-specific protein modification by transglutaminase. Biochemistry 51:8679–8689. CrossRefPubMedGoogle Scholar
  32. Spolaore B, Raboni S, Satwekar AA et al (2016) Site-specific transglutaminase-mediated conjugation of interferon α-2b at glutamine or lysine residues. Bioconjug Chem. PubMedGoogle Scholar
  33. Strop P (2014) Versatility of microbial transglutaminase. Bioconjug Chem 25:855–862. CrossRefPubMedGoogle Scholar
  34. Thom J, Anderson D, McGregor J, Cotton G (2011) Recombinant protein hydrazides: application to site-specific protein PEGylation. Bioconjug Chem 22:1017–1020. CrossRefPubMedGoogle Scholar
  35. Thomas C, Moraga I, Levin D et al (2011) Structural linkage between ligand discrimination and receptor activation by type I interferons. Cell 146:621–632. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Zhou Z, Zhang J, Sun L et al (2014) Comparison of site-specific PEGylations of the N-terminus of interferon beta-1b: selectivity, efficiency, and in vivo/vitro activity. Bioconjug Chem 25:138–146. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Pharmaceutical and Pharmacological SciencesUniversity of PaduaPaduaItaly
  2. 2.CRIBI Biotechnology CentreUniversity of PaduaPaduaItaly

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