Abstract
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.
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Abbreviations
- ACN:
-
Acetonitrile
- DC:
-
Dansylcadaverine
- E/S:
-
Enzyme to substrate ratio
- IFN:
-
Human interferon β-1a
- PEG:
-
Polyethylene glycol
- TFA:
-
Trifluoroacetic acid
- TGase:
-
Transglutaminase
- TCEP:
-
Tris(2-carboxyethyl)phosphine
- ZQG:
-
Carbobenzoxy-l-glutaminyl-glycine
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Acknowledgements
We acknowledge Silvia Gelio for conducting some experiments. This work was supported by the University of Padua (60A04-3887/12 and 60A04-8780/15).
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Spolaore, B., Forzato, G. & Fontana, A. Site-specific derivatization of human interferon β-1a at lysine residues using microbial transglutaminase. Amino Acids 50, 923–932 (2018). https://doi.org/10.1007/s00726-018-2563-1
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DOI: https://doi.org/10.1007/s00726-018-2563-1