Transglutaminase (TGase, E.C. 2.3.2.13) catalyzes acyl transfer reactions between the gamma-carboxamide groups of protein-bound glutamine (Gln) residues, which serve as acyl donors, and primary amines, resulting in the formation of new gamma-amides of glutamic acid and ammonia. By using an amino-derivative of poly(ethylene glycol) (PEG-NH(2)) as substrate for the enzymatic reaction with TGase it is possible to covalently bind the PEG polymer to proteins of pharmaceutical interest. In our laboratory, we have conducted experiments aimed to modify proteins of known structure using TGase and, surprisingly, we were able to obtain site-specific modification or PEGylation of protein-bound Gln residue(s) in the protein substrates. For example, in apomyoglobin (apoMb, myoglobin devoid of heme) only Gln91 was modified and in human growth hormone only Gln40 and Gln141, despite these proteins having many more Gln residues. Moreover, we noticed that these proteins suffered highly selective limited proteolysis phenomena at the same chain regions being attacked by TGase. We have analysed also the results of other published experiments of TGase-mediated modification or PEGylation of several proteins in terms of protein structure and dynamics, among them alpha-lactalbumin and interleukin-2, as well as disordered proteins. A noteworthy correlation was observed between chain regions of high temperature factor (B-factor) determined crystallographically and sites of TGase attack and limited proteolysis, thus emphasizing the role of chain mobility or local unfolding in dictating site-specific enzymatic modification. We propose that enhanced chain flexibility favors limited enzymatic reactions on polypeptide substrates by TGases and proteases, as well as by other enzymes involved in a number of site-specific post-translational modifications of proteins, such as phosphorylation and glycosylation. Therefore, it is possible to predict the site(s) of TGase-mediated modification and PEGylation of a therapeutic protein on the basis of its structure and dynamics and, consequently, the likely effects of modifications on the functional properties of the protein.

Site-specific modification and PEGylation of pharmaceutical proteins mediated by transglutaminase

FONTANA, ANGELO;SPOLAORE, BARBARA;MERO, ANNA;
2008

Abstract

Transglutaminase (TGase, E.C. 2.3.2.13) catalyzes acyl transfer reactions between the gamma-carboxamide groups of protein-bound glutamine (Gln) residues, which serve as acyl donors, and primary amines, resulting in the formation of new gamma-amides of glutamic acid and ammonia. By using an amino-derivative of poly(ethylene glycol) (PEG-NH(2)) as substrate for the enzymatic reaction with TGase it is possible to covalently bind the PEG polymer to proteins of pharmaceutical interest. In our laboratory, we have conducted experiments aimed to modify proteins of known structure using TGase and, surprisingly, we were able to obtain site-specific modification or PEGylation of protein-bound Gln residue(s) in the protein substrates. For example, in apomyoglobin (apoMb, myoglobin devoid of heme) only Gln91 was modified and in human growth hormone only Gln40 and Gln141, despite these proteins having many more Gln residues. Moreover, we noticed that these proteins suffered highly selective limited proteolysis phenomena at the same chain regions being attacked by TGase. We have analysed also the results of other published experiments of TGase-mediated modification or PEGylation of several proteins in terms of protein structure and dynamics, among them alpha-lactalbumin and interleukin-2, as well as disordered proteins. A noteworthy correlation was observed between chain regions of high temperature factor (B-factor) determined crystallographically and sites of TGase attack and limited proteolysis, thus emphasizing the role of chain mobility or local unfolding in dictating site-specific enzymatic modification. We propose that enhanced chain flexibility favors limited enzymatic reactions on polypeptide substrates by TGases and proteases, as well as by other enzymes involved in a number of site-specific post-translational modifications of proteins, such as phosphorylation and glycosylation. Therefore, it is possible to predict the site(s) of TGase-mediated modification and PEGylation of a therapeutic protein on the basis of its structure and dynamics and, consequently, the likely effects of modifications on the functional properties of the protein.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/2449977
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