Site-Specific Protein Cross-Linking by Peroxidase-Catalyzed Activation of a Tyrosine-Containing Peptide Tag

Abstract

Protein modification methods represent fundamental techniques that are applicable in many fields. In this study, a site-specific protein cross-linking based on the oxidative tyrosine coupling reaction was demonstrated. In the presence of horseradish peroxidase (HRP) and H(2)O(2), tyrosine residues undergo one-electron oxidation reactions and form radicals in their phenolic moieties, and these species subsequently react with each other to form dimers or further react to generate polymers. Here, a peptide-tag containing a tyrosine residue(s) (Y-tag, of which the amino acid sequences were either GGGGY or GGYYY) was genetically introduced at the C-terminus of a model protein, Escherichia coli alkaline phosphatase (BAP). Following the incubation of recombinant BAPs with HRP and H(2)O(2), Y-tagged BAPs were efficiently cross-linked with each other, whereas wild-type BAP did not undergo cross-linking, indicating that the tyrosine residues in the Y-tags were recognized by HRP as the substrates. To determine the site-specificity of the cross-linking reaction, the Y-tag was selectively removed by thrombin digestion. The resultant BAP without the Y-tag showed no reactivity in the presence of HRP and H(2)O(2). Conversely, Y-tagged BAPs cross-linked by HRP treatment were almost completely digested into monomeric BAP units following incubation with the protease. Moreover, cross-linked Y-tagged BAPs retained ∼95% of their native enzymatic activity. These results show that HRP catalyzed the site-specific cross-linking of BAPs through tyrosine residues positioned in the C-terminal Y-tag. The site-selective enzymatic oxidative tyrosine coupling reaction should offer a practical option for site-specific and covalent protein modifications.

Enzyme Initiated Radical Polymerizations

“…For example, proteins presenting tyrosine moieties at their surface can be gelled in the presence of laccases or peroxidases/H 2 O 2 ( Figure 20) [147][148][149][150]. Next to proteins also natural polymers exhibiting phenolic side chains can be gelled enzymatically.…”

Control of a Tyrosyl Radical Mediated Protein Cross-Linking Reaction by Electrostatic Interaction

“…The EGFP(Y237F) was used because in our previous report, it was shown that the wild-type EGFP cross-linked by itself upon HRP treatment probably due to the tyrosine residue at its penultimate C-terminal position. 22 SA mutants, model proteins, and HRP were mixed together in 10 mM Tris-HCl (pH 8.0) at final concentrations of 0.2 mg/ mL, 0.2 mg/mL, and 0.1 mg/mL, respectively. The heteroconjugation reaction was conducted by adding H 2 O 2 in the same manner as described above, and the sample solutions were analyzed by SDS-PAGE.…”

“…The differences in the resulting bands were caused by the following reason ( Figure 4). The BAP-GY cross-links by itself through its GY-tag mostly in a linear form, 22 which appears as mBAP dimers in SDS-PAGE analysis (lane 2 for BAP-GY in Figure 1A and Figure 4A). Meanwhile, the positively charged Y-tags are capable of cross-linking with the intrinsic tyrosine residues of BAP, resulting in mBAP polymers (lane 2 for BAP-RY and BAP-RYR in Figure 1A and as depicted in Figure 4B).…”

“…The GY-tag gives BAP the ability to intermolecularly cross-link through the Y-tag while minimizing the unfavorable intramolecular cross-linking between the GY-tag and the intrinsic tyrosine residues. The RYR-tag shows slow reactivity upon HRP treatment providing more opportunities for BAP-GY to yield (BAP-GY) n homoconjugates, 22 and it also shows favorable cross-linking formation toward negatively charged BAP-GY due to its positive charges. As a result, the combination of BAP-GY and SA-RYR worked well to obtain the (BAP-GY) n -(SA-RYR) m conjugate of which n > m. The results described herein indicated that, by introducing the GY-tag to BAP and the RYRtags to SA, the heteroconjugation reaction between BAP and SA was kinetically controlled.…”

“…This was suggested from our previous study, where the negatively charged BAP possessing GY-tag was capable of co-cross-linking with the positively charged lysozyme, but it showed no reactivity against the negatively charged ovalbumin (OVA) or bovine serum albumin (BSA). 22 Aspartic acid (Asp, D) and arginine (Arg, R) were used to electrostatically charge the Y-tags. The Y-tag sequences of DDDDY, DDYDD, RRRRY, and RRYRR, which are abbreviated as DY-tag, DYD-tag, RY-tag, and RYR-tag, respectively, were appended to the C-terminus of BAP and SA.…”