Site-Specific Derivatization of Avidin Using Microbial Transglutaminase

Avidin conjugates have several important applications in biotechnology and medicine. In this work, we investigated the possibility to produce site-specific derivatives of avidin using microbial transglutaminase (TGase). TGase allows the modification of proteins at the level of Gln or Lys residues using as substrate an alkyl-amine or a Gln-mimicking moiety, respectively. The reaction is site-specific, since Gln and Lys derivatization occurs preferentially at residues embedded in flexible regions of protein substrates. An analysis of the X-ray structure of avidin allowed us to predict Gln126 and Lys127 as potential sites of TGase’s attack, because these residues are located in the flexible/unfolded C-terminal region of the protein. Surprisingly, incubation of avidin with TGase in the presence of alkylamine containing substrates (dansylcadaverine, 5-hydroxytryptamine) revealed a very low level of derivatization of the Gln126 residue. Analysis of the TGase reaction on synthetic peptide analogues of the C-terminal portion of avidin indicated that the lack of reactivity of Gln126 was likely due to the fact that this residue is proximal to negatively charged carboxylate groups, thus hampering the interaction of the substrate at the negatively charged active site of TGase. On the other hand, incubation of avidin with TGase in the presence of carbobenzoxy-l-glutaminyl-glycine in order to derivatize Lys residue(s) resulted in a clean and high yield production of an avidin derivative, retaining the biotin binding properties and the quaternary structure of the native protein. Proteolytic digestion of the modified protein, followed by mass spectrometry, allowed us to identify Lys127 as the major site of reaction, together with a minor modification of Lys58. By using TGase, avidin was also conjugated via a Lys-Gln isopeptide bond to a protein containing a single reactive Gln residue, namely, Gln126 of granulocyte-macrophage colony-stimulating factor. TGase can thus be exploited for the site-specific derivatization of avidin with small molecules or proteins

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

The transglutaminase (TGase) from <i>Streptomyces mobaraensis</i> catalyzes transamidation reactions in a protein substrate leading to the modification of the side chains of Gln and Lys residues according to the A-CONH₂ + H₂N-B → A-CONH-B + NH₃ 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

Site-Specific Transglutaminase-Mediated Conjugation of Interferon α‑2b at Glutamine or Lysine Residues

Interferon α (IFN α) subtypes are important protein drugs that have been used to treat infectious diseases and cancers. Here, we studied the reactivity of IFN α-2b to microbial transglutaminase (TGase) with the aim of obtaining a site-specific conjugation of this protein drug. Interestingly, TGase allowed the production of two monoderivatized isomers of IFN with high yields. Characterization by mass spectrometry of the two conjugates indicated that they are exclusively modified at the level of Gln101 if the protein is reacted in the presence of an amino-containing ligand (i.e., dansylcadaverine) or at the level of Lys164 if a glutamine-containing molecule is used (i.e., carbobenzoxy-l-glutaminyl-glycine, ZQG). We explained the extraordinary specificity of the TGase-mediated reaction on the basis of the conformational features of IFN. Indeed, among the 10 Lys and 12 Gln residues of the protein, only Gln101 and Lys164 are located in highly flexible protein regions. The TGase-mediated derivatization of IFN was then applied to the production of IFN derivatives conjugated to a 20 kDa polyethylene glycol (PEG), using PEG–NH₂ for Gln101 derivatization and PEG modified with ZQG for Lys164 derivatization. The two mono-PEGylated isomers of IFN were obtained in good yields, purified, and characterized in terms of protein conformation, antiviral activity, and pharmacokinetics. Both conjugates maintained a native-like secondary structure, as indicated by far-UV circular dichroism spectra. Importantly, they disclosed good in vitro antiviral activity retention (about only 1.6- to 1.8-fold lower than that of IFN) and half-lives longer (about 5-fold) than that of IFN after intravenous administration to rats. Overall, these results provide evidence that TGase can be used for the development of site-specific derivatives of IFN α-2b possessing interesting antiviral and pharmacokinetic properties

Compounds selected by SBVS/LBVS-docking procedures for OASS-A and OASS-B.

<p>Compounds selected by SBVS/LBVS-docking procedures for OASS-A and OASS-B.</p

List of compounds selected from virtual screening and tested against OASS-B.

*<p>due to the strong emission at 500 nm for excitation at 412 nm, this compound was assayed at concentrations lower than 100 µM and no binding was observed.</p

LigPlot of the wild type tetrapeptide ligand in the active site of <i>Haemophilus influenzae</i> OASS.

<p>The interactions between the Asn-Leu-Asn-Ile tetrapeptide and the active site residues of <i>H. influenzae</i> OASS-A (PDB code: 1Y7L) are reported. The figure was drawn with LigPlot program version 4.5.3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077558#pone.0077558-Laskowski1" target="_blank">[124]</a>.</p

List of compounds selected from virtual screening and tested against OASS-A.

<p>List of compounds selected from virtual screening and tested against OASS-A.</p

Best HINT scored conformations of the compounds selected by the LBVS/docking procedures for OASS-B.

<p>The images were prepared with PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.)</p

Structural comparison of OASS-A and OASS-B.

<p><b>Panel A</b>: Structure-based amino acid sequence alignment of OASS-A and OASS-B from <i>Salmonella typhimurium</i>. The alignment, carried out on the PDB entries 1OAS and 2JC3 using the Flexible structure AlignmenT (FATCAT) method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077558#pone.0077558-Ye1" target="_blank">[122]</a>, gave an overall identity of 40.32% and a similarity of 56.51%. Identical residues have a red background and residues with similar physicochemical properties are shown in red. Similarity scores were calculated by the ESPript program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077558#pone.0077558-Gouet1" target="_blank">[123]</a> using the Blosum62 matrix set at global score of 0.2. Residues of the first active site shell are indicated by dark circles below the alignment. <b>Panel B</b>: Active site of OASS-A. Residues of the first active site shell and PLP are shown in ball and stick style, colored pink and yellow, respectively. <b>Panel C</b>: Active site of OASS-B. Residues of the first active site shell and PLP are shown in ball and stick style, colored cyan and yellow, respectively.</p

Docking pose of best binders to the two isozymes placed into the active sites.

<p><b>Panel A</b>: Docking pose of <b>1</b> in the OASS-A binding pocket. Red and green contours identify the hydrogen bond acceptor and hydrophobic GRID MIFs. Hydrogen bond donor hot spots have not been shown for clarity. <b>Panel B</b>: Docking pose of compound <b>13</b> in the OASS-B binding pocket. Red and green contours identify the hydrogen bond acceptor and hydrophobic GRID MIFs. Hydrogen bond donor hot spots have not been shown for clarity.</p