Kosuke Minamihata

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

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.

Protein heteroconjugation by the peroxidase-catalyzed tyrosine coupling reaction.

Combining different proteins can integrate the functions of each protein to produce novel protein conjugates with wider ranges of applications. We have previously introduced a peptide containing tyrosine residues (Y-tag) at the C-terminus of Escherichia coli alkaline phosphatase (BAP). The tyrosine residues in the Y-tag were efficiently recognized by horseradish peroxidase (HRP) and were site-specifically cross-linked with each other to yield BAP homoconjugates. In this study, the HRP-catalyzed tyrosine coupling reaction was used for protein heteroconjugation. Streptavidin (SA) was selected as the conjugation partner for BAP. The Y-tag (GGGGY) was genetically introduced to the C-terminus of SA. Prior to heteroconjugation, the reactivity of the Y-tagged SA was examined. The Y-tagged SA cross-linked to form an SA homoconjugate upon HRP treatment, whereas wild-type SA remained essentially intact. In the heteroconjugation reaction of BAP and SA, the Y-tagged BAP and SA were efficiently cross-linked with each other upon HRP treatment. The functions of the BAP-SA conjugates were evaluated by measuring the BAP enzymatic activity on a biotin-coated plate. The BAP-SA conjugate tethered to the plate showed BAP enzymatic activity, indicating that both BAP and SA retained their functions following heteroconjugation. The BAP-SA conjugate prepared from both Y-tagged BAP and SA showed the highest enzymatic activity on the biotin-coated plates. This result illustrates the advantage of the protein conjugation reaction in which multiple numbers of proteins can be conjugated at the same time.

Enzymatic preparation of a redox-responsive hydrogel for encapsulating and releasing living cells

Horseradish peroxidase-mediated oxidative cross-linking of a thiolated poly(ethylene glycol) is promoted in the absence of exogenous hydrogen peroxide, by adding a small amount of a phenolic compound under physiological conditions. The prepared hydrogel can encapsulate and release living mammalian cells.

Protein cell‐surface display through in situ enzymatic modification of proteins with a poly(Ethylene glycol)‐lipid

Cell-surface display of functional proteins is a powerful and useful tool for regulating and reinforcing cellular functions. Direct incorporation of site-specifically lipidated proteins from the extracellular medium is more rapid, easily controllable and reliable in displaying active proteins than expression through gene transfer. However, undesirable amphiphilic reagents such as organic co-solvents and detergents were required for suppressing aggregation of ordinary lipidated proteins in solution. We report here sortase A-catalyzed modification of proteins with a poly(ethylene glycol)(PEG)-lipid in situ on the surface of living cells. Proteins fused with a recognition tag were site-specifically ligated with the PEG-lipid which was preliminary incorporated into cell membranes. Accordingly, target proteins were successfully displayed on living cells without aggregation under an amphiphilic reagent-free condition. Furthermore, to demonstrate the availability of the present method, Fc domains of immunoglobulin G were displayed on cancer cells, and the phagocytosis of cancer cells with dendritic cells were enhanced through the Fc-Fc receptor interaction. Thus, the present facile chemoenzymatic method for protein display can be utilized for modulating cell-cell interactions in cell and tissue engineering fields.

Control of a tyrosyl radical mediated protein cross-linking reaction by electrostatic interaction.

Herein, we demonstrate the control of protein heteroconjugation via a tyrosyl coupling reaction by using electrostatic interaction. Aspartic acid and arginine were introduced to a tyrosine containing peptide tag (Y-tag) to provide electrostatic charge. Designed negatively or positively charged Y-tags were tethered to the C-terminus of Escherichia coli alkaline phosphatase (BAP) and streptavidin (SA), and these model proteins were subjected to horseradish peroxidase (HRP) treatment. The negatively charged Y-tags showed low reactivity due to repulsive interactions between the Y-tags with the negatively charged BAP and SA. In contrast, the positively charged Y-tags showed high reactivity, indicating that the electrostatic interaction between Y-tags and proteins significantly affects the tyrosyl radical mediated protein cross-linking. From the heteroconjugation reaction of BAP and SA, the SA with the positively charged Y-tags exhibited favorable cross-linking toward negatively charged BAP, and the BAP-SA conjugates prepared from BAP with GY-tag (GGGGY) and SA with RYR-tag (RRYRR) had the best performance on a biotin-coated microplate. Encompassing the reactive tyrosine residue with arginine residues reduced the reactivity against HRP, enabling the modulation of cross-linking reaction rates with BAP-GY. Thus, by introducing a proper electrostatic interaction to Y-tags, it is possible to kinetically control the heteroconjugation behavior of proteins, thereby maximizing the functions of protein heteroconjugates.

The lifetime evaluation of vapourised phase-change nano-droplets.

Phase-change nano-droplets (PCNDs) are sub-micron particles that are coated with phospholipid and contain liquid-state perfluorocarbons such as perfluoropentane (boiling point=29°C) and perfluorohexane (boiling point=57°C), which can vapourise upon application of ultrasound. The bubbles generated by such reactions can serve as ultrasound contrast agents or HIFU sensitisers. However, the lifetime of bubbles generated from PCNDs on μs-order is not well known. Knowledge of the condition of PCND-derived bubbles on μs-order is essential for producing bubbles customised for specific purposes. In this study, we use an optical measurement system to measure the vapourisation and stability of the bubbles (bubble-lifetime) as well as the stability-controlling method of the nucleated bubbles on μs-order while changing the internal composition of PCNDs and the ambient temperature. PCND-derived bubbles remain in a bubble state when the boiling point of the internal composition is lower than the ambient temperature, but lose their optical contrast after approximately 10μs by re-condensation or dissolution when the boiling point of the internal composition is higher than the ambient temperature. We reveal that the superheating condition significantly affects the fate of vapourised PCNDs and that the bubble-lifetime can be controlled by changing both the ambient temperature conditions and the internal composition of PCNDs.

Peptide Tag-Induced Horseradish Peroxidase-Mediated Preparation of a Streptavidin-Immobilized Redox-Sensitive Hydrogel

Several methods have recently been reported for the preparation of redox-sensitive hydrogels using enzymatic reactions, which are useful for encapsulating sensitive materials such as proteins and cells. However, most of the reported hydrogels is difficult to add further function efficiently, limiting the application of the redox-sensitive hydrogels. In this study, peptide sequences of HHHHHHC and GGGGY (Y-tag) were genetically fused to the N- and C-termini of streptavidin (C-SA-Y), respectively, and C-SA-Y was mixed with horseradish peroxidase and thiol-functionalized 4-arm polyethylene glycol to yield a redox-sensitive C-SA-Y immobilized hydrogel (C-SA-Y gel). The C-SA-Y immobilized in the hydrogel retained its affinity for biotin, allowing for the incorporation of proteins and small molecules to hydrogel via biotin. C-SA-Y gel was further prepared within a water-in-oil (w/o) emulsion system to yield a nanosized hydrogel, to which any intracellular and cytotoxic agent can be modified, making it a potential drug delivery carrier.

Tyrosine Coupling Creates a Hyperbranched Multivalent Protein Polymer Using Horseradish Peroxidase via Bipolar Conjugation Points.

Protein polymers of covalently cross-linked protein monomers are highly attractive biomaterials because each monomer unit possesses distinct protein functions. Protein polymers often show enhancement effects on the function by integrating a large number of molecules into one macromolecule. The cross-linking site of component proteins should be precisely controlled to avoid diminishing the protein function. However, preparing protein polymers that are cross-linked site-specifically with a high cross-linking degree is a challenge. Here, we demonstrate the preparation of a site-specifically cross-linked protein polymer that has a hyperbranched polymer-like structure with a high cross-linking degree. A horseradish peroxidase (HRP) reaction was used to achieve the protein polymerization through a peptide tag containing a tyrosine residue (Y-tag). Y-tag sequences were introduced to both N- and C-termini of a model protein, protein G. The dual Y-tagged protein G (Y-pG-Y) was treated with HRP to form a Y-pG-Y polymer possessing average and maximum cross-linking degree of approximately 70-mer and 150-mer, respectively. The Y-pG-Y polymer shows the highest cross-linking degree among the protein polymers reported, which are completely soluble in water and cross-linked via covalent bonding. The Y-pG-Y was cross-linked site-specifically at the Tyr residue in the Y-tag, retaining its function, and the Y-pG-Y polymer showed extremely strong avidity against immunoglobulin G. The reactivities of N- and C-terminal Y-tags were evaluated, and we revealed that the difference in the radical formation rate by HRP was the key for yielding highly cross-linked protein polymers.

Photosensitizer and polycationic peptide-labeled streptavidin as a nano-carrier for light-controlled protein transduction

Transductions of exogenous proteins into cells enable the precise study of the effect of the transduced proteins on cellular functions. Accordingly, the protein transduction technique, which can control the release of proteins into the cytosol with certainty and high-throughput, is highly desired in various research fields. In this study, streptavidin (SA) labeled with a photosensitizer and cell-permeable peptides (CPP) was proposed as a nano-carrier for light-controlled protein transduction. SA was modified with biotinylated oligo-arginine peptides (Rpep), which were functionalized with Alexa Fluor 546 (AF546), to achieve cell penetrating and endosomal escape functionalities. The SA-Rpep complex was efficiently internalized into living HeLa cells corresponding to the length and the modification number of Rpep. SA conjugated with more than three equimolar AF546-modified Rpep consisting of fifteen arginine residues was achieved to diffuse throughout the cytosol without cytotoxicity by irradiation of the excitation light for AF546. The optimized nano-carrier was confirmed to transduce a biotinylated model cargo protein, enhanced green fluorescent protein fused with thioredoxin (tEGFP) into the cytosol at the light-irradiated area. The results provided proof-of-principle that SA possessing multiple AF546-modified Rpep has the potential to be a versatile and facile carrier for light-controlled protein transduction into the cytosol of mammalian cells.

Site-specific conjugation of an antibody-binding protein catalyzed by horseradish peroxidase creates a multivalent protein conjugate with high affinity to IgG

Cross‐linking proteins offers an approach to enhance the distinct function of proteins due to the multivalent effect. In this study, we demonstrated the preparation of a multivalent antibody‐binding protein possessing high affinity to IgG by conjugating a number of antibody‐binding proteins using the horseradish peroxidase (HRP)‐mediated protein conjugation method. By introducing a peptide tag containing a tyrosine (Y‐tag) to the C‐terminus of the model protein, a chimera protein of protein G and protein A (pG2pA), the Tyr residue in the Y‐tag was efficiently recognized by HRP and cross‐linked with each other to yield a pG2pA conjugate, composed of mainly two to three units of pG2pA. The cross‐linking occurred site specifically at the Tyr residue in the Y‐tag and introduction of the Y‐tag showed no effect on the function of pG2pA. The affinity of the Y‐tagged pG2pA conjugate against IgG clearly increased because of the multivalent effect, demonstrating the benefit of this protein cross‐linking reaction, which yields functional protein oligomers. Such multivalent protein conjugates created by this reaction should have potential to be used in ELISA and Western blotting applications in which highly sensitive detection of target molecules is desired.