| Resumo: |
Transglutaminase (TGase) catalyzes an acyl transfer reaction between the [gamma]-carboxamide group of glutamine (Gln) and the [epsilon]-amino group of lysine (Lys) residues to form a stable amide bond. The TGase reaction can be used for bioconjugation of an amino-derivative of poly-ethylene glycol (PEG) to protein drugs, leading to PEGylated proteins that display increased bioactivity and stability. The procedure was shown to lead to site-specific bioconjugation of few proteins, thus offering a valid alternative to the chemical methods of PEGylation in current use (1). Moreover, TGase can be used for site-specifically labeling proteins with fluorescent groups at the level of Gln or Lys for diagnostic applications. The TGase attack is not entirely dependent on a specific consensus sequence around the Gln or Lys residues. However, a correlation between the TGase-mediated sites of PEGylation and the chain flexibility has been observed (2). Our study was focused to elucidate the molecular features favoring the TGase mediated site-specific reactions on two extensively
studied model proteins, i.e. apomyoglobin (apoMb) and lysozyme. Besides amino-PEG, we used dansyl-cadaverine as an acyl acceptor
and N-carbobenzoxy-Gln-Gly-OH as acyl donor for the TGase reactions. The sites of protein modification were determined by fingerprinting and ESI mass spectrometry. Myoglobin in its holo form is not susceptible to TGase reactions due to its rigid conformation,but the apo form was conjugated with PEG and fluorescent labels at the level of helix F (chain segment 82–99). The NMR study on apoMb had earlier demonstrated increased flexibility of the helix F (3) and, moreover, several proteases cleave the 153-residue chain of apoMb at the level of helix F only (4). Therefore, the chain region attacked by both TGase and proteases is a flexible or unfolded site(s). Lysozyme in its disulfide crosslinked native state is highly resistant to proteases and TGase attack. However, the more dynamic three-disulphide derivative of lysozyme, lacking the Cys6–Cys127 disulfide bridge, is susceptible to TGase-mediated reactions, as well as limited proteolysis. These results indicate that the sites of TGase reactions and the sites of limited proteolysis have a clear analogy for their presence in flexible/disordered regions of protein substrates. Overall, our studies clearly demonstrate that TGase-mediated reactions occur only at disordered chain regions, as evidenced by the correlation between sites of the TGase reaction and sites of enhanced chain flexibility, the latter deduced from the crystallographic Bfactor.
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