Local Unfolding Is Required for the Site-Specific Protein Modification by Transglutaminase

Abstract

The transglutaminase (TGase) from Streptomyces mobaraensis catalyzes transamidation reactions in a protein substrate leading to the modification of the side chains of Gln and Lys residues according to the A-CONH(2) + H(2)N-B → A-CONH-B + NH(3) reaction, where both A and B can be a protein or a ligand. A noteworthy property of TGase is its susbstrate specificity, so that often only a few specific Gln or Lys residues can be modified in a globular protein. The molecular features of a globular protein dictating the site-specific reactions mediated by TGase are yet poorly understood. Here, we have analyzed the reactivity toward TGase of apomyoglobin (apoMb), α-lactalbumin (α-LA), and fragment 205-316 of thermolysin. These proteins are models of protein structure and folding that have been studied previously using the limited proteolysis technique to unravel regions of local unfolding in their amino acid sequences. The three proteins were modified by TGase at the level of Gln or Lys residues with dansylcadaverine or carbobenzoxy-l-glutaminylglycine, respectively. Despite these model proteins containing several Gln and Lys residues, the sites of TGase derivatization occur over restricted chain regions of the protein substrates. In particular, the TGase-mediated modifications occur in the "helix F" region in apoMb, in the β-domain in apo-α-LA in its molten globule state, and in the N-terminal region in fragment 205-316 of thermolysin. Interestingly, the sites of limited proteolysis are located in the same chain regions of these proteins, thus providing a clear-cut demonstration that chain flexibility or local unfolding overwhelmingly dictates the site-specific modification by both TGase and a protease.

The LQSP tetrapeptide is a new highly efficient substrate of microbial transglutaminase for the site‐specific derivatization of peptides and proteins

“…Therefore while in the molecules of Sugimura et al [16] the arginine was found to be more available for productive interactions with the enzyme, in our study broad chain flexibility renders this residue less prone to interaction or even able to disrupt the interaction. This hypothesis fits well with the concept that the catalytic reaction strongly depends on both the chemical and structural environment of the glutamine residue [8,13].…”

“…The substrate specificity of TGases has been investigated by using both synthetic peptides [10][11][12] and model proteins [13]. The literature show we still have much to learn about TGases [14].…”

“…The literature show we still have much to learn about TGases [14]. Fontana et al showed that partial unfolding of polypeptide substrates strongly promote site-specific modifications by M-TGases [13], suggesting that local flexibility could be sufficient to favor enzyme recognition. Meanwhile Sugimura et al, using phage displayed random peptide libraries, identified preferred peptide sequences acting as M-TGase substrates [12,15,16] and used them as specific tags to efficiently immobilize functional proteins via M-TGase reactions [17].…”

DOTA-Functionalized Polylysine: A High Number of DOTA Chelates Positively Influences the Biodistribution of Enzymatic Conjugated Anti-Tumor Antibody chCE7agl

“…Modification of lysine residues with glutamine substrates was difficult because of premature, irreversible, enzymatic hydrolysis of the corresponding glutamine substrates to glutamate. Recently, Spolaore et al [29] showed in an elegant study that site-specific protein modification by transglutaminase requires the glutamine and lysine residues to be either in a flexible or a locally unfolded region of the protein. This could also explain why the site-specific derivatization of antibody chCE7agl by mTGase with a Gln substrate could not be accomplished as there is not a lysine residue embedded in such a flexible or unfolded structure.…”

Biotechnological Applications of Transglutaminases

“…Importantly, the cross-links were introduced by MTG only after the collagen had been at least partially heat-denatured, supporting the correlation between structural disorder of the target and recognition by MTG. To further investigate the importance of secondary structure and MTG’s apparent preference for flexible polypeptide regions, the reactivity of MTG towards apoMb, α-lactalbumin (α-LA) and fragment 205-316 of thermolysin was analyzed [53]. These extensively studied proteins are models of α-helices, β-sheets and unstructured regions, respectively.…”