The site-specific TGase-mediated PEGylation of proteins occurs at flexible sites

Abstract

Transglutaminase (TGase) is able to catalyse the acyl transfer reaction between the γ-carboxamide group of a protein-bound glutamine (Gln) residue and an amino-derivative of poly(ethylene glycol) (PEG-NH 2), thus leading to a PEGylated protein. Several proteins of therapeutic interest have been PEGylated by means of TGase, among them interleukin-2, granulocyte colony-stimulating factor, human growth hormone and erythropoietin. Surprisingly, PEGylation occurred at specific Gln residue(s), despite the fact that these proteins contained several Gln residues. An analysis of the TGase-mediated reactions in terms of structure and dynamics of protein substrates revealed a correlation between sites of TGase attack and chain regions of enhanced backbone flexibility, as detected by the crystallographic profile of the B-factor along the protein polypeptide chain. Moreover, the TGase-mediated reactions often occurred at chain regions characterized by missing electron density, indicating that these regions are disordered. In particular, it was noted that in a number of cases the sites of TGase attack occurred at the same chain regions prone to limited proteolysis phenomena. Since chain flexibility or local unfolding was earlier found to dictate the sites of limited proteolysis of proteins, it is concluded that both TGase and a protease require an unfolded polypeptide substrate in an extended conformation for the site-specific enzymatic attack.