Tyrosine Coupling Creates a Hyperbranched Multivalent Protein

Article pubs.acs.org/bc

Tyrosine Coupling Creates a Hyperbranched Multivalent Protein Polymer Using Horseradish Peroxidase via Bipolar Conjugation Points Kosuke Minamihata,*,† Sou Yamaguchi,‡ Kei Nakajima,‡ and Teruyuki Nagamune*,†,‡ †

Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan Department of Bioengineering, School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan

‡

S Supporting Information *

ABSTRACT: 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 crosslinked site-specifically with a high cross-linking degree is a challenge. Here, we demonstrate the preparation of a sitespecifically 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 Ytagged 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.

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multivalent binding proteins, such as lectins11 and immunoglobulins.12 They show stronger binding affinity against their ligands than monomeric binding proteins or domains. These phenomena are a multivalent effect. The overall affinity that multivalent binding proteins show is referred to as avidity. The preparation of polymeric protein conjugates can be achieved using the self-assembly of molecules5,6,9 or immobilizing proteins onto other nanostructures such as DNA.13−15 However, excluding any additional components in the polymeric protein conjugates other than the target proteins is essential to increase the local concentration of the target protein, maximizing the benefit of protein assembly. In addition, to prevent the disassembly of the polymeric protein conjugates, the component proteins should be connected via covalent bonding. However, assembling proteins into a polymeric structure, i.e., a protein polymer, via covalent bonding without using any other scaffold molecules is challenging. The proteins need to be cross-linked in a sitespecific manner to retain their function; therefore, chemical

INTRODUCTION Proteins possess distinct functions and have precisely controlled molecular shape and size, which are defined by their amino acid sequence; thus, they are intriguing candidates for building blocks in nanoassembly.1−4 Highly assembled protein conjugates possess the function of the component protein, and they are attractive scaffolds or templates for assembling other functional molecules to produce novel materials.5,6 Moreover, polymeric protein structures often show an enhancement effect on the function because each protein unit works cooperatively.5,7−9 For example, cellulolytic microorganisms evolved their cellulose-degrading enzymes in the form of a huge protein complex called cellulosome.10 Within cellulosome, multiple different cellulolytic enzymes are assembled onto a scaffold protein via the dockerin−cohesin interaction. The scaffold protein, scaffoldin, possesses multiple cohesin domains and an orthogonal dockerin domain that specifically binds to the cohesin expressed on the cell surface, enabling the immobilization of cellulosomal structures. The cellulolytic activity of cellulosome is considerably higher than that of free enzymes due to the cooperative catalytic reaction of enzymes within cellulosome. Another example of enhancing protein function by assembly into polymeric form include © XXXX American Chemical Society

Received: March 10, 2016 Revised: April 15, 2016

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DOI: 10.1021/acs.bioconjchem.6b00138 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

used this reaction to conduct protein conjugations by introducing a peptide tag containing Tyr (hereafter abbreviated as Y-tag) at the terminus of target proteins, and HRP efficiently catalyzes the oxidation reaction of Tyr residue in the Y-tag.27,28 Here we define Y-tag as peptide sequences possessing (X)nY(X)m, where X is not acidic amino acids. If Y-tag is attached to N-terminus, then n ≤ 2 and m ≥ 4. If it is attached to C-terminus, then n ≥ 4 and m ≤ 2. The generated tyrosyl radicals react with each other to cross-link Y-tagged proteins. HRP requires no formation of a tripartite complex; therefore, as long as Tyr residues are available for HRP on the surface of a protein polymer, the polymerization reaction proceeds. HRP is an ideal enzyme for the preparation of protein polymer. Although the cross-linking is a radical coupling reaction, the cross-linking site of Y-tagged proteins are highly selective to the Tyr residue in the Y-tag, causing a negligible effect on the protein function.27 Using this Y-tag and HRP, we have demonstrated the polymerization of dimer or tetramer proteins.27,28 However, for a monomeric protein that has a single Y-tag sequence within a molecule, the major product was its dimer, and the maximum cross-linking degree was a pentamer due to steric hindrance.29 For stability, protein polymers should not contain any noncovalent bonding interactions; hence, the polymerization of Y-tagged monomeric protein by HRP reaction is ideal. In this context, we demonstrate the polymerization of monomeric protein using a Y-tag at the N- and C-termini and the HRP-mediated cross-linking reaction. The model protein we test in this research is an antibody binding protein, protein G (pG). We conduct HRP treatment on the dual Y-tagged pG (Y-pG-Y) (Figure 1) to prepare Y-pG-Y polymer. We evaluate

cross-linking is not applicable to obtain protein polymers. Using enzymatic reactions has potential to achieve covalent cross-linking of proteins and form protein polymers. Transglutaminase (TGase)16 and sortase A (SrtA)17 are promising enzymes for protein−protein cross-linking because these enzymes recognize peptidyl substrates, requiring no introduction of exogenous molecules to the target proteins other than natural amino acids. TGase and SrtA form intermediates with one of their substrates, and the other nucleophilic substrates need to contact the intermediates to complete the ligation of two proteins. The formation of these tripartite complexes becomes difficult as the cross-linking degree of substrate molecules becomes large. Moreover, TGase recognizes a wide variety of peptide sequence as substrates, causing nonspecific cross-linking.16 SrtA has strict substrate recognition, LPXTG and N-terminal oligoglycine; however, because the product of the SrtA reaction contains the LPXTG motif, the reaction is in an equilibrium of cleavage and cross-linking, requiring an excess amount of oligoglycine nucleophiles to drive the reaction forward.18 Therefore, although TGase and SrtA are useful for conjugating protein molecules, obtaining highly cross-linked protein polymers using these enzymes is difficult. Alternative approaches to prepare protein polymers using the isopeptideforming domain from Streptococcus pyogenes have been reported.19,20 The isopeptide-forming unit can be split into two domains, a short peptide fragment at the C-terminus and the remaining protein domain, and after mixing them together, an isopeptide bond forms spontaneously between them.21,22 Matsunaga et al. reported self-polymerizing protein shackles using this chemistry and produced a highly cross-linked protein polymer, which they called a hyperthin nanochain.19 The split C-terminal peptide, isopeptag,21 was fused to the N-terminus of isopeptide-forming domain (Spy0128), and the pocket of Spy0128 for isopeptag was blocked with a capping peptide containing a cysteine residue. The formation of isopeptide bond was prevented by a disulfide bond formed between Spy0128 and the capping peptide. The reduction of the disulfide bond initiated the spontaneous polymerization of Spy0128 through its N-terminal isopeptag. The research group of Howarth engineered the CnaB2 domain of fibronectinbinding protein FbaB from S. pyogenes to develop a protein domain, SpyCatcher, and a 13 amino acid peptide, SpyTag, which form an isopeptide bond between Asp of SpyTag and Lys in SpyCatcher spontaneously.22 They further split SpyCatcher to SpyLigase and KTag, which contains a Lys residue to form an isopeptide bond with SpyTag.20 The KTag and SpyTag were introduced to the N- and C-termini of affibody, and SpyLigase-mediated isopeptide bond formation between KTag and SpyTag yielded a highly polymerized affibody polymer. Although these reports successfully demonstrated the preparation of precisely controlled protein polymers and the affinity enhancement of binding proteins, they have drawbacks, such as the protein shackle seems difficult to apply for other proteins that are sensitive to the reductive condition, and the SpyLigase system requires the addition of large amount of SpyLigase to create protein polymer because of its low turnover number of ligation reaction. Other than the widely studied post-translational enzymes for protein conjugations described above, oxidoreductase enzymes are another candidate for achieving protein polymerization.23 Horseradish peroxidase (HRP) recognizes phenolic compounds including an amino acid, tyrosine (Tyr, Y), and generates a polymer via an enzymatic reaction.24−26 We have

Figure 1. Molecular image of Y-pG-Y. Tyr residues in Y-tags shown as CPK models. The image was produced with the Molecular Operating Environment (MOE, version 2009.10) software developed by the Chemical Computing Group Inc. (Montreal, Canada).

the reactivity of the N- and C-terminal Y-tags of Y-pG-Y and elucidate the reason for yielding a highly cross-linked Y-pG-Y polymer, implying that the difference in the reactivity of the Nand C-terminal Y-tags is the key for creating hyperbranched protein polymers. Finally, we show that the Y-pG-Y polymer retains its function and shows extremely strong avidity against IgG.

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RESULTS AND DISCUSSION HRP Treatment on pG, pG-Y, and Y-pG-Y. Figure 2 shows the results of SDS−PAGE analyses of the HRP treatment on pG variants at their optimum condition (Figures S1 and S2 in Supporting Information). The mobility of bands for each pG variant was not correlated with their molecular weights; therefore we conducted matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF-MS) analyses to confirm these pG variants were expressed and purified with their additional peptide sequences (Figure S3). Because pG is a small protein domain, the length

B


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Figure 2. HRP treatment of pG variants: lane 1, pGs; lane 2, pGs with H2O2; lane 3, pGs with HRP; lane 4, pGs with HRP and H2O2.

and hydrophobicity of the additional peptides greatly affected its electrophoresis mobility. The N-terminal Y-tag sequence tethered to Y-pG-Y contained a human rhinovirus 3C (HRV 3C) protease recognition sequence, which is hydrophobic, and SDS molecules were attached to the hydrophobic sequence, causing an increase in the mobility of Y-pG-Y. In lane 4 of pG-Y, when HRP and H2O2 were added, the band for monomeric pG-Y, which possesses a single Y-tag sequence at the C-terminus, completely disappeared, and new bands for its oligomers formed in the higher molecular weight region. This result is concordant with those described in our previous report, which states that a monomeric protein possessing a single Y-tag sequence forms only oligomers, at most pentamers, after HRP treatment.29 In contrast, in lane 4 of pG, pG without the Y-tag sequence showed no reactivity against HRP treatment, indicating that the intrinsic tyrosine residues of pG were not recognized by HRP. Intriguingly, the cross-linked product of Y-pG-Y showed a drastic increase in its molecular weight compared with that of pG-Y, causing a band at the edge of the stacking and running gel. Although Y-pG-Y possesses two Y-tag sequences within a molecule, it yielded a polymer of an extraordinarily high polymerization degree and did not show intramolecular cross-linking to close its structure. Validating the Cross-Linking Site of Y-pG-Y. We analyzed the cross-linking site of the Y-pG-Y polymer to exclude the possible involvement of intrinsic tyrosine residues in the formation of the polymer. First, the dityrosine (dTyr) fluorescence of pG-Y and Y-pG-Y was measured before and after HRP treatment. Cross-linking via the tyrosine residues in the Y-tag yields dTyr, which emits fluorescence with a peak at around 400 nm after irradiation at 315 nm.30 Both pG-Y and YpG-Y showed excitation and fluorescence spectra that are unique to dTyr (Figure S4). Next, Y-tag sequences of Y-pG-Y were cleaved by protease treatment. N- and C-terminal Y-tag sequences contain HRV 3C protease (LEVLFQ↓GP) and thrombin (LVPR↓GS) recognition sequences, respectively, enabling a specific cut-off for each Y-tag sequence. Figure 3A shows results of HRP treatments on the protease-treated Y-pGY. When the Y-tag sequences of N- or C-termini were cleaved, the pG-Y(ΔY-tagN) and Y-pG(ΔY-tagC) showed a pronounced decrease of cross-linking degree and only oligomers were formed. Furthermore, cleaving both N- and C-terminal Y-tags of Y-pG-Y (pG(ΔY-tagN,C)) completely diminished its reactivity, showing no change in the band of pG(ΔY-tagN,C)

Figure 3. Protease treatments on Y-pG-Y before and after HRP treatment. (A) Thrombin and HRV 3C protease treatment on Y-pG-Y prior to HRP treatment. HRP (0.1 μM) and H2O2 (75 μM) were added to the solution containing protease-treated Y-pG-Y (75 μM) without any purification. (B) Protease treatments on the Y-pG-Y polymer. (C) MALDI-TOF-MS analysis of cross-linked Y-tag peptides cleaved from the Y-pG-Y polymer by protease treatment. N and C represent N- and C-terminal Y-tag peptides, respectively. The expected N- and C-terminal peptide sequences were GYGGGGLEVLFQ (1196.32 Da) and GSGGGGY (553.53 Da), respectively. A mass equivalent to two H atoms would decrease from the sum of conjugated peptide masses for every degree of conjugation.

after HRP treatment. This result is supported by the HRP treatment on pG-Y and pG shown in Figure 2. The reasons for the blurred bands of the oligomer of pG-Y(ΔY-tagN) and YpG(ΔY-tagC) were the cleaved Y-tag peptides in the solution, and no purification was done before HRP treatment. Subsequently, protease treatments on Y-pG-Y polymer were conducted, and the results are shown in Figure 3B. Protease treatments of both HRV 3C protease and thrombin on Y-pG-Y polymer showed the formation of a single band, indicating that the polymer was completely digested to its monomer units (pG(ΔY-tagN,C)). We also confirmed the formation of pG(ΔYtagN,C) monomer from protease treatment on Y-pG-Y polymer by MALDI-TOF-MS (Figure S5). Cleaving one side of the Ytag of the Y-pG-Y polymer yielded oligomers, showing the presence of branched structures within the polymer. Band intensities of the oligomers generated by the HRV 3C protease treatment, i.e., pG oligomers conjugated at C-terminal Y-tag, were denser than those of oligomers generated by thrombin treatment. This result suggests that the C-terminal Y-tag sequence possesses higher reactivity for HRP treatment and C


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GL column (GE Healthcare UK Ltd., Little Chalfont, U.K.), which has an exclusion limit of approximately 1300 kDa. However, most of the polymer eluted out in the void volume of the column (Figure S8), indicating the formation of a polymer possessing an extremely high molecular weight. Therefore, we evaluated the molecular weight of Y-pG-Y polymer using fieldflow fractionation and multiangle light scattering (FFF-MALS; Figure 5).

forms a branched structure more frequently than that of the Nterminal Y-tag. The thrombin treatment was conducted on pGY, and the results were concordant with those of Y-pG-Y (Figure S6). We measured the mass of the cross-linked Y-tag peptides, which were cleaved from the Y-pG-Y polymer by protease treatments, using MALDI-TOF-MS (Figure 3C). We successfully detected several different combinations of cross-linked Ytag peptides, including N + C, N + C + C, N + N, N + C + C + C, and N + N + C. However, we detected no peptides containing solely C-terminal Y-tag peptides, for example, C + C, C + C + C, and C + C + C + C, which would occur at approximately 1105 m/z, 1656 m/z, and 2208 m/z, respectively. MALDI-TOF-MS analysis on the cleaved Y-tag peptides from pG-Y oligomers showed conjugated C-terminal Y-tag peptides (Figure S7); therefore, the Y-pG-Y polymer may not contain a cross-linking point of only C-terminal Y-tag peptides. This result strongly suggests that the C-terminal Y-tag is recognized by HRP more preferably than the N-terminal Ytag, which is also implied from the result in Figure 3B. The Y-tag sequences in Y-pG-Y are responsible for crosslinking; however, the possible involvement of intrinsic tyrosine residues in the cross-linking has not been refuted. Therefore, we performed a co-cross-linking reaction of pG with pG-Y or YpG-Y to evaluate the contribution of the intrinsic tyrosine residues of pG in the cross-linking of Y-tagged pGs. Figure 4 shows the SDS−PAGE analyses. The band intensity of pG treated by HRP in the presence of Y-tagged pGs was the

Figure 5. Molecular weight measurement of Y-pG-Y polymer by FFFMALS. Blue dots show the molecular weight estimated by MALS on the left vertical axis. Black continuous line shows the UV absorbance of the eluted solution on the right vertical axis.

The maximum and average molecular weights of Y-pG-Y polymer were 1595 and 762.3 kDa, respectively, which are approximately 150-mer and 70-mer of Y-pG-Y. There was no precipitation in the solution after HRP treatment, despite the extremely high molecular weight. The Y-pG-Y polymer was completely soluble in water. As far as we know, this Y-pG-Y polymer possesses the highest cross-linking degree among artificial protein polymers,19,20 which are water-soluble and cross-linked via covalent bonding. The Y-pG-Y polymer showed a peak in the molecular weight measurement at around 10 mL of elution volume. In the normal elution mode of FFF, small molecules elute out faster than large molecules, and the molecular weight shows an upward-sloping curve. Hence, the peak in the MALS chart means that the fraction of Y-pG-Y polymer eluted at around 9.5−11.5 mL of elution volume possesses a small hydrodynamic volume for its molecular weight. This finding is also shown in synthetic polymers with highly branched structures, such as dendrimers and hyperbranched polymers.31 The results in Figures 3B and Figures 3C show the presence of branched cross-linking points in the YpG-Y polymer. We roughly evaluated the degree of branching of this Y-pG-Y polymer from the peak intensities of MALDITOF-MS of the peptide fragments shown in Figure 3C, and it turned out to be about 58.5%; therefore, the Y-pG-Y polymer has a hyperbranched polymer-like structure. The peak position of the highest molecular weight was similar to that of the peak in the UV absorbance chromatogram. This result suggests that the fractions of Y-pG-Y polymer, which possess the highest cross-linking degrees, compose the majority of the Y-pG-Y polymer, and the distribution of cross-linking degree is unexpectedly small.

Figure 4. HRP treatment of pG in the presence of pG-Y or Y-pG-Y. M: pG without any treatment as a marker. 1: pG-Y. 2: pG and pG-Y. 3: pG treated by HRP in the presence of pG-Y. 4: HRP-treated pG-Y. 5: Y-pG-Y. 6: pG and Y-pG-Y. 7: pG treated by HRP in the presence of Y-pG-Y. 8: HRP-treated Y-pG-Y. HRP concentrations were 1 μM and 0.1 μM for pG-Y and Y-pG-Y, respectively. Concentrations of pG variants were set at 75 μM each, and 1 equiv of H2O2 was added.

same as that of pG without HRP treatment (lanes 2 and 3, and lanes 6 and 7 in Figure 4). In addition, the band patterns of the cross-linked pGs, formed with or without pG, were identical (lanes 3 and 4, and lanes 7 and 8). From these results, we conclude that the Y-tagged pGs site-specifically cross-linked via a tyrosine residue in the Y-tags without involving intrinsic Tyr residues of pG. Evaluating the Molecular Weight of Y-pG-Y Polymer. We examined the molecular size of the Y-pG-Y polymer using size-exclusion chromatography using a Superdex 200 10/300 D


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Bioconjugate Chemistry Evaluation of How Y-pG-Y Forms Highly Cross-Linked Polymers. Y-pG-Y has two Y-tag sequences in a molecule; therefore, the intramolecular cross-linking of Y-tags is preferred to intermolecular cross-linking. However, the results shown so far disagree with this expectation. We considered that the reason for yielding highly cross-linked Y-pG-Y polymers after HRP treatment is the difference in the cross-linking kinetics of N- and C-terminal Y-tags against HRP recognition. We evaluated the cross-linking kinetics of Y-tags from two different perspectives; one is the substrate preference by HRP, and another is the effect of charges close to the tyrosyl radicals. First, we evaluated the kinetics of the dTyr formation of Nand C-terminal Y-tag peptides by measuring its fluorescence change during HRP treatment (Figure 6A). The C-terminal Ytag peptides showed a faster increase in the fluorescence

intensity of dTyr than that of the N-terminal Y-tag peptides. Furthermore, Y-pG and pG-Y, pGs possessing N- and Cterminal Y-tag sequences, respectively, were treated by HRP, and the dTyr fluorescence change was monitored (Figure 6B). The pGs with Y-tags showed the same trend as the Y-tag peptides. The Y-pG-Y decreased the rate of dTyr formation compared with that of pG-Y, indicating that the N-terminal Ytag is competing with the C-terminal Y-tag for recognition by HRP. Therefore, as suggested by the results in Figure 3, the Cterminal Y-tag can be recognized by HRP more efficiently than the N-terminal Y-tag. Second, we examined the effect of neighboring charges on the cross-linking of tyrosyl radicals. The Y-tags at the N- or Ctermini possess a positive or negative charge, respectively, which are derived from the terminal amino or carboxylic group. We introduced arginine (R) or aspartic acid (D) to the N- and C-terminal Y-tags to alter the local charge of the Y-tags. An Nterminal Y-tag sequence of GYDDGG− (N-DY-tag) and a Cterminal Y-tag sequence of −GGRRY (C-RY-tag) were introduced to pGs to prepare YDD-pG-Y (possessing N-DYtag and original Y-tag, GGGGY), Y-pG-RRY (possessing original Y-tag GYGGGG and C-RYtag), and YDD-pG-RRY (possessing N-DY-tag and C-RY-tag). The Y-tags of YDD-pGY and Y-pG-RRY possess a local charge of −1 and +1, respectively, whereas the N- and C-terminal Y-tags of YDD-pGRRY have local charges −1 and +1, respectively. All the Y-pG-Y variants were treated by HRP, and the rate of dTyr formation was compared (Figure 7A). YDD-pG-Y showed a slight decrease in its reactivity compared with that of Y-pG-Y, whereas Y-pG-RRY showed a great enhancement on the reactivity. By adding N-DYtag to Y-pG-RRY, i.e., YDD-pGRRY, a slower dTyr formation than that of Y-pG-RRY was recorded. The result of SDS−PAGE analyses (Figure 7B) on these HRP-treated Y-pG-Y variants were in good agreement with the trend of the dTyr formation rate in Figure 7A. Y-pGRRY and YDD-pG-RRY showed higher cross-linking degrees than that of the original Y-pG-Y because the products appeared as dense bands at the edge of the running gel. YDD-pG-Y significantly decreased the cross-linking degree, yielding mainly oligomers. The structure of the side chain of Arg is larger than that of Asp, suggesting that steric hindrance can be excluded as the major reason for the slow dTyr formation from the N-DYtag. Thus, the negative charge on the N-DY-tag is responsible for affecting the HRP recognition of Y-tag or the coupling reaction of tyrosyl radicals. An in silico research by Shamovsky et al. showed that the protonation of tyrosyl radical hampers their contact with each other, inhibiting dTyr formation, and the placement of positively charged amino acid residues in the vicinity of tyrosyl radicals increases the efficiency of the coupling reaction.32 This finding is concordant with our data, as well as the results in our previous work, where we attempted to control the coupling reaction of Y-tag by introducing electrostatic interactions to the Y-tags.33 In our previous report, the C-terminal Y-tag sequences of RRRRY and RRYRR showed a great enhancement of reactivity after HRP treatment, cross-linking intrinsic Tyr residues of a model protein that are not directly recognized by HRP. However, the reactivity of the negatively charged Y-tags, DDDDY and DDYDD, were almost completely diminished. The tyrosine residues in the negatively charged Y-tags were recognized by HRP because the model protein, bacterial alkaline phosphatase with negatively charged Y-tags, showed a co-cross-linking ability against positively charged proteins,

Figure 6. Time-course measurements of dTyr fluorescence of the Ytag peptides and Y-tagged pGs during HRP treatment. (A) Measurements on N- and C-terminal Y-tag peptides are shown as blue and red lines, respectively. (B) Measurements on Y-pG, pG-Y, and Y-pG-Y are shown as blue, red, and black lines, respectively. The concentration of peptides and pGs were 100 μM and 30 μM, respectively. HRP concentration was 0.1 μM for peptides and 0.5 μM for pGs, and H2O2 concentration was 50 μM for peptides and 30 μM for pGs. The measurement was started immediately after adding H2O2 to the solution in the quartz cell. Excitation light was at 315 nm, and the fluorescence intensity at 410 nm was monitored. E


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Bioconjugate Chemistry Tyr • + O2−• + H+ → Tyr + O2

The superoxide ion can be generated from the decomposition reaction of H2O2 in basic conditions,35 as well as from the side catalytic cycle of the HRP reaction (Figure S9).25,36,37 The presence of a repairing process of tyrosyl radicals during the coupling reaction is suggested by the inhibition of the dTyr formation at a low pH, where tyrosyl radicals should be protonated.38 Therefore, the tyrosyl radical formed in Nterminal Y-tag of Y-pG-Y is probably stabilized by the positive charge of N-terminal amino group. Michon et al. reported that a positive charge close to Tyr causes an inhibitory effect on HRP recognition.25 They compared the dTyr formation rate of L -Tyr and Nacetyltyrosine, and the latter was a better substrate for HRP. They concluded that the electrostatic repulsion between the Nterminal amino group of L-Tyr and Arg 183 near the substrate pocket of HRP lowered the affinity of L-Tyr to HRP. There is a clear discrepancy between their results and those reported here. However, they also evaluated the cross-linking behavior of GlyTyr, which was also a better substrate than L-Tyr, and the best substrate for HRP among the substrates they tested was a pentapeptide of PQQPY. Although both Gly-Tyr and PQQPY possess a positive charge derived from an N-terminal amino group, they considered their positive charges too distant from the Tyr residue to affect HRP recognition. By considering the results shown in our reports, we believe that the presence of a peptide bond at the N-terminus of Tyr residue is the key for HRP recognition rather than the absence of a positive charge. Michon et al. measured the dTyr formation rate of Nacetyltyrosine amide, which was comparable to that of Nacetyltyrosine,25 suggesting that the negative charge derived from the carboxylic group of C-terminus is not essential for HRP recognition. In our previous report, we showed that the dTyr formation rates of streptavidin with RRYRR were considerably lower than that of RRRRY.33 In addition, enhanced green fluorescent protein has an intrinsic Tyr residue at its penultimate C-terminal position, −YK, and the Tyr residue can be recognized by HRP to yield cross-linked enhanced green fluorescent protein oligomers after HRP treatment. However, the cross-linking efficiency of enhanced green fluorescent protein was considerably lower than that of the proteins tagged with the Y-tag sequence GGGGY.27 These results assert that tethering peptides following the Tyr residue in a Y-tag cause a significant decrease of HRP recognition rate, probably because of steric hindrance. Thus, as a good substrate for HRP, the Tyr residue should possess a peptide bond at its N-terminal side and the C-terminal side of Tyr residue should be vacant. This explains the differences in the reactivity of Nand C-terminal Y-tag sequences shown in Figure 6. Another important factor that Michon et al. reported was the crosslinked products, such as dTyr, exhibited smaller KM values than that of the monomers and can act as a competitive inhibitor.25 For example, the KM values for (N-acetyltyrosine)2 and PQQY were approximately 20 μM and 2 mM, respectively. Although the affinity of the conjugated tyrosine variants against HRP is higher, the rate of conversion into their radical species by HRP is slower than that of monomers. We elucidate the reason for why cross-linked Y-tag peptides containing a C-terminal Y-tag were not detected in MALDITOF-MS analysis shown in Figure 3C and the reason for yielding highly cross-linked Y-pG-Y polymers after HRP treatment as follows. A summary is presented in Figure 8.

Figure 7. Evaluating the cross-linking behaviors of Y-pG-Y variants possessing charged Y-tag sequences. (A) Time-course measurement of dTyr fluorescence on Y-pG-Y variants after HRP treatment. Blue, green, black, and red lines represent Y-pG-RRY, YDD-pG-RRY, Y-pGY, and YDD-pG-Y, respectively. Concentrations of Y-pG-Y variants, HRP, and H2O2 were 30, 0.5, and 30 μM, respectively. Excitation light was 315 nm, and the emission at 410 nm was monitored. (B) SDS− PAGE analyses of HRP-treated Y-pG-Y variants.

lysozyme and ribonuclease A. Thus, we considered that the loss of cross-linking ability of the negatively charged Y-tags was because of repulsion forces between negatively charged Y-tags and negatively charged model proteins, which are used in the study. Building on the report by Shamovsky et al.,32 the results in our previous report show that the negatively charged Y-tags were repelled from the negatively charged proteins and the tyrosyl radicals in the negatively charged Y-tags were efficiently repaired by protonation because of the low pH condition. The sequence of DDDD provides a strong negatively charged condition around the Tyr residue of the Y-tag and to cancel out the negative charges, because of the equivalent number of protons nearby, causing a low pH. Within these conditions, the tyrosyl radicals in the Y-tag are probably protonated. The positive charge on the protonated tyrosyl radical hampers the collision of tyrosyl radicals, reducing the coupling reaction rate and increasing the probability of being repaired by a superoxide ion to become Tyr residue again as shown in the following scheme.34 F


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Figure 8. Proposed polymerization process of Y-pG-Y after HRP treatment.

linking reaction, Y-pG-Y monomer cross-links intermolecularly via the C-terminal Y-tag because of the fast reaction rate. The intramolecular coupling reaction of Y-tags within Y-pG-Y monomer would not occur. Additionally, The N- and C-termini of pG locate to opposite sides of the pG molecule, reducing the probability of intramolecular cross-linking. The following crosslinking process to form Y-pG-Y polymer was described above. Also, stabilization of the tyrosyl radical formed in N-terminal Ytag by the positive charge of the N-terminal amino group might have contribution on efficient cross-linking. In summary, the extremely high cross-linking degree of the Y-pG-Y polymer was achieved by the difference in tyrosyl radical forming rates between N- and C-terminal Y-tags, which prevents intramolecular cross-linking of Y-pG-Y monomer. Moreover, the Nterminal Y-tag possesses positive charge of N-terminal amino group, which might promote efficient cross-linking of Nterminal Y-tags. These two factors successfully form highly cross-linked Y-pG-Y polymers. Evaluating the Binding Function of Y-pG-Y Polymer against IgG. We evaluated the binding function of Y-pG-Y polymer against IgG. The pG domain that we used in this study can bind to IgG at a ratio of two pG domains to one IgG. We first analyzed the stoichiometry of pG binding to IgG using isothermal titration calorimetry (ITC). The ITC results showed that pG retained 83.8% binding capacity, whereas pG-Y oligomers and Y-pG-Y polymers showed 41.9% and 52.7%, respectively (Figure S10). Therefore, approximately half of the pG domains within pG-Y oligomers or Y-pG-Y polymers were able to bind to Fc domains of IgG. Because the intrinsic tyrosine residues of pG-Y and Y-pG-Y were not involved in the cross-linking reaction via Y-tags, the loss of binding capacity of pG domains in those cross-linked conjugates was caused by steric hindrance. Although about half the pG domains were not functioning, the cross-linking degree of Y-pG-Y polymer is as high as 150 at the maximum; thus, a large amount of pG domains retain their function cooperatively within the Y-pG-Y

In the early stage of the Y-pG-Y cross-linking reaction catalyzed by HRP, the C-terminal Y-tag reacts faster than the N-terminal Y-tag, creating oligomers of Y-pG-Y cross-linked mainly through the C-terminal Y-tag and via the combination of N- and C-termini, such as C−C, C−C−C, and N−C. As the C-terminal Y-tag is consumed, the N-terminal Y-tag starts reacting predominantly and connects the Y-pG-Y oligomers to form Y-pG-Y prepolymers. In this stage, the dTyr in the crosslinked Y-tags behaves as an inhibitor, reducing the reaction rate of the N-terminal Y-tag. Simultaneously, the dTyr is activated by HRP. However, the reaction rate of dTyr is slower than that of Tyr, and the concentration of radicals on the cross-linked Ytag is not high enough to promote further cross-linking. Consequently, dTyr radicals in the cross-linked Y-tags crosslink with tyrosyl radicals on the N-terminal Y-tag, forming cross-linking points, such as N−C−C, N−C−C−C, and N− N−C, which can be seen in Figure 3C. This process will use up the cross-linked points only containing C-terminal Y-tags, and the peaks are undetected in the MALDI-TOF-MS analysis shown in Figure 3C. As the cross-linking degree of Y-tags increases further, HRP can no longer recognize the cross-linked Y-tags because of steric hindrance. Therefore, N-terminal Y-tags cross-link with themselves, creating cross-linking points containing solely N-terminal Y-tags. This proposed reaction process is in good agreement with the results of Figure 3B, showing that the cleavage of a C-terminal Y-tag of Y-pG-Y polymer yields mostly Y-pG-Y monomers, whereas that of the N-terminal Y-tag generates a substantial amount of oligomers. Intramolecular cross-linking of N- and C-terminal Y-tags of Y-pG-Y decreases the cross-linking degree. However, this intramolecular cross-linking inhibits further conjugation only when it happens on Y-pG-Y monomer. Because intermolecularly cross-linked Y-pG-Y conjugates possess available Y-tags, more than three, even intramolecular cross-linking occurs, and there are still available cross-linking sites, allowing further conjugation. As described above, in the first stage of the crossG


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sensor chip, as shown by the constant SPR signal of Y-pG-Y polymer at the dissociation phase. This result was attributed to the strong multivalent effect caused by numerous pG domains working cooperatively within the Y-pG-Y polymer. The crosslinked pG-Y conjugate showed a slower dissociation rate than the pG monomer, whereas Y-pG-Y polymer showed no dissociation at any concentration (Figure S12). Although the Y-pG-Y polymer shows distributed cross-linking degrees, because no decrease of SPR signal was observed, most of the polymer possesses a high enough cross-linking degree to show strong avidity. Once the cross-linking degree of polymeric ligands or receptors exceeds a certain point, the avidity would be saturated, and there is no benefit to increase the multivalency further. Thus, reducing the cross-linking degree of the Y-pG-Y polymer is important to future development. To control the cross-linking degree, conducting a co-cross-linking reaction of Y-pG-Y with pG-Y or Y-pG is ideal because pGs with a single Y-tag act as capping molecules. Introducing negatively charged amino acid residues in the Y-tag sequence is another way to tune the cross-linking degree. By modulating the cross-linking degree of protein polymers, each unit contained in protein polymers can work efficiently, enabling the production of highly functional protein polymers. The results and discussion we described in this study guide the way to control the cross-linking behavior of Y-tagged proteins after HRP treatment.

polymer, implying that the Y-pG-Y polymer shows a strong multivalent effect. The association and dissociation kinetics of pG and Y-pG-Y polymer were evaluated using an IgGimmobilized surface plasmon resonance (SPR) sensor chip (Figure 9).

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CONCLUSION We evaluated the cross-linking behavior of pG possessing Nand C-terminal Y-tags after HRP treatment. The Y-pG-Y shows a drastic increase in cross-linking degree compared with that of pG-Y, which has a single Y-tag sequence. The molecular weight estimation on Y-pG-Y polymer by FFF-MALS reveals that the average and maximum molecular weights of the polymer are 70-mer and 150-mer, respectively. These molecular weights are the highest for covalently cross-linked and water-soluble protein conjugates reported so far. Despite the extremely high cross-linking degree, the cross-linking point is strictly limited to the tyrosine residues in the Y-tags, and a hyperbranched polymer-like structure of the Y-pG-Y polymer is predicted. The key for forming the highly cross-linked Y-pGY polymer is the faster tyrosyl radical formation rate of the Cterminal Y-tag than that of the N-terminal Y-tag. The Y-pG-Y polymer exhibits an extremely strong multivalent effect, because no dissociation from an IgG-immobilized SPR sensor chip is observed. The highly cross-linked protein polymer is promising as a nanoscaffold to assemble other molecules, for high affinity material for separation and as a carrier for drug delivery, as well as a scaffold for bulk biomaterials such as protein-based hydrogels.

Figure 9. SPR measurements of (A) pG and (B) Y-pG-Y polymer on an IgG (anti-ovalbumin (chicken), polyclonal) immobilized sensor chip. The concentration of each sample was set at 5 μg/mL, and 40 μL was applied at a flow rate of 20 μL/min.

The association rate of Y-pG-Y polymer was slower than that of pG monomer. In this experiment, the molar concentration of Y-pG-Y polymer was difficult to estimate because of the distribution of cross-linking degrees; thus, we measured the concentrations of both pG and Y-pG-Y polymer using a bicinchoninic acid assay with bovine serum albumin as a standard. The concentrations of the samples of pG and Y-pG-Y polymer were fixed in the units of μg/mL. Therefore, although the molar concentration of pG unit in the samples of pG and YpG-Y polymer measured in Figure 9 is the same, the molar concentration of pG conjugate molecules was different. The YpG-Y polymer concentration was much lower than that of pG. Moreover, the diffusion velocity of the Y-pG-Y polymer should be slower than that of pG monomer because of the high molecular weight. These factors reduce the association rate of Y-pG-Y polymer to IgG compared with that of pG. Y-pG-Y polymer showed no dissociation from the IgG immobilized

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EXPERIMENTAL PROCEDURES Constructing the Expression Vectors of pGs. The pET22b+ vector carrying the gene coding pG2pA-GY29 (a chimera protein of two pG units and one protein A unit in tandem with a hexahistidine tag at its N-terminus and a Y-tag sequence of GGGGY at its C-terminus) was used as the template DNA for vector construction. To construct the vector of pG, the gene coding the second pG, protein A, and Y-tag was deleted from the template DNA by inverse PCR and the selfligation reaction of the PCR product. To construct the vector of pG-Y, the gene coding the second pG and protein A was H


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HiTrap binding buffer. The column was washed with five column volumes of the same buffer, and pGs were eluted by a gradient of elution buffer (25 mM Tris-HCl, 1 M NaCl, pH 8.0) up to 100% with 10 column volumes. The fractions containing pGs were concentrated to a volume of approximately 3 mL using the ultrafiltration membrane, and pGs were purified with size exclusion chromatography using HiLoad 16/ 600 Superdex 75 pg (GE Healthcare UK Ltd.) with 10 mM Tris-HCl (pH 8.0) as a running buffer. The purified pGs were concentrated using the ultrafiltration membrane, and the concentration of pGs was measured by the bicinchoninic acid assay using bovine serum albumin as a standard. For the purification of pG variants fused with MBP, the supernatant containing MBP-pG fusion proteins after sonication and centrifugation was filtered with a 0.2 μm membrane filter and loaded to an MBPTrap HP 5 mL column (GE Healthcare UK Ltd.) pre-equilibrated with 10 mM Tris-HCl (150 mM NaCl, pH 8.0). The column was washed with the same buffer, and MBP-pG fusion was eluted by injecting five column volumes of 10 mM Tris-HCl (pH 8.0) containing 150 mM NaCl and 250 mM maltose. The fractions containing the target protein were gathered and mixed with TEV protease. The cleavage reactions of MBP from pGs were conducted during dialysis against 10 mM Tris-HCl containing 1 mM dithiothreitol at 4 °C overnight. The sample solution was applied to a HisTrap FF crude column to remove the cleaved MBP from the solution in the same manner as that described above. Because the TEV protease we used here was expressed in our laboratory and purified with a HisTrap FF crude 5 mL (GE Healthcare UK Ltd.), the TEV protease remained in the solution containing the target protein, Y-pG-Y. The contaminating TEV protease was successfully removed from Y-pG-Y by anion exchange chromatography and purification using a size-exclusion chromatography column, conducted in the same manner as pG and pG-Y. The obtained Y-pG-Y was concentrated, and its concentration was determined by bicinchoninic acid assay using bovine serum albumin as the standard. Optimizing the Cross-Linking Reaction of pGs. The minimum concentration of H2O2 to consume all of the monomeric pG-Y and Y-pG-Y was determined. The pGs and HRP were mixed in 10 mM Tris-HCl (pH 8.0) at final concentrations of 75 and 5 μM, respectively. The cross-linking reaction was conducted by adding 0−10 equiv (1 mol of H2O2 was equivalent to 2 mol of tyrosine residues in Y-tags, i.e., 2 mol of pG-Y or 1 mol of Y-pG-Y) amounts of H2O2 to the solution. After a 1 h incubation at room temperature, the cross-linking efficiency of pG-Y and Y-pG-Y was evaluated by SDS−PAGE analysis. Subsequently, we optimized the HRP concentration by varying from 0 to 5 μM. The H2O2 concentration was set at 1 equiv against Tyr residues in the Y-tags of pGs, and the concentrations of pGs were 75 μM in 10 mM Tris-HCl (pH 8.0). After adding H2O2 to the solutions and incubating for 1 h at room temperature, the reactivity of pGs was analyzed by SDS−PAGE. Cross-Linking Reaction of pGs at the Optimized Reaction Condition. pG or Y-tagged pGs (75 μM) was mixed with HRP (1 μM for pG and pG-Y, and 0.1 μM for YpG-Y) in 10 mM Tris-HCl (pH 8.0), and 1 equiv of H2O2 against the Tyr residues in the Y-tag was added to conduct the cross-linking reaction. The solution was incubated at 25 °C for 1 h, and the samples were analyzed by SDS−PAGE.

deleted from the template DNA by inverse PCR and the selfligation reaction of PCR product, introducing a DNA sequence of thrombin cleavage sequence (LVPRGS) between the genes of pG and Y-tag. The expression plasmid for Y-pG-Y was constructed using the plasmid of pG-Y as a template. DNA sequences coding tobacco etch virus (TEV) protease recognition sequence (ENLYFQ↓G), Y-tag (YGGGG), and HRV 3C protease recognition sequence (LEVLFQ↓GP) were introduced to the 5′ end of pG-Y by an inverse PCR method as described above to construct the gene of Y-pG-Y with an Nterminal TEV protease sequence. The gene coding Y-pG-Y with TEV protease sequence was amplified with PCR, and the product was inserted into pMAL-c5E (New England BioLabs Inc., Ipswich, MA, USA) after DNA sequence coding maltose binding protein (MBP) and its C-terminal TEV protease recognition sequence by using an In-Fusion HD cloning kit with cloning enhancer (Clontech Laboratories, Inc. Mountain View, CA, USA). The plasmid for Y-pG was constructed by deleting the sequences coding thrombin and C-terminal Y-tag from the pMAL-c5E carrying Y-pG-Y by inverse PCR and the self-ligation reaction. The Y-pG-Y variants possessing charged amino acid residues in the Y-tags were constructed using pMAL-c5E Y-pG-Y as a template, replacing the DNA sequence coding two Gly residues in the Y-tag with the DNA sequences coding either DD or RR. Expression and Purification of pG Variants. The expression of pG variants were conducted on an Escherichia coli BL21 Star(DE3) strain. The plasmid vectors of pG variants were transformed into the cells by a heat shock method and cultured on a lysogeny broth plate containing 100 μg/mL of ampicillin sodium. A single colony was inoculated to 5 mL of lysogeny broth containing the same amount of ampicillin sodium and shaken at 220 rpm and 37 °C overnight. The precultured medium was placed into 1 L of Terrific broth containing the same antibiotics and cultured with shaking at 220 rpm at 37 °C until the optical density at 600 nm reached around 0.6. Expression of pG variants was induced by addition of isopropyl β-D-1-thiogalactopyranoside at a final concentration of 1 mM, and the cells were cultured overnight after lowering the temperature to 15 °C. The cells were harvested by centrifugation at 5000g for 20 min and frozen at −20 °C until purification. For the purification of pG and pG-Y, the pellet was thawed in 50 mL of HisTrap binding buffer (25 mM Tris-HCl, 150 mM NaCl, 20 mM imidazole, pH 7.4) and an amount of 5 μL of Cryonase cold-active nuclease (Takara Bio Inc., Shiga, Japan) was added. The cell suspension was sonicated on ice for 1 min, 15 times with a 3 min interval between each sonication. The cell debris was removed by centrifugation at 22 000g for 30 min at 4 °C, and the supernatant was loaded onto two columns of HisTrap FF crude 5 mL (GE Healthcare UK Ltd.) in tandem, which were pre-equilibrated with the HisTrap binding buffer. The columns were washed with the same buffer until all of the unbound proteinaceous substances eluted out, and pG variants were eluted by a gradient of HisTrap elution buffer (25 mM Tris-HCl, 150 mM NaCl, 500 mM imidazole, pH 7.4) up to 100% with 20 column volumes. The fractions containing pGs were concentrated, and the buffer was exchanged to HiTrap binding buffer (25 mM Tris-HCl, pH 8.0) using an ultrafiltration membrane of 3 kDa MWCO (Amicon Ultra-15 Centrifugal Filter Units, Billerica, MA, USA). The desalted solution containing pGs was applied to a HiTrap Q FF 5 mL column (GE Healthcare UK Ltd.) pre-equilibrated with the I


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Molecular Weight Measurement of Y-pG-Y Polymer by FFF-MALS. Y-pG-Y polymer was prepared by HRP treatment at the optimized condition (Y-pG-Y, 75 μM; HRP, 0.1 μM, and 75 μM of H2O2 at 25 °C for 1 h), and the HRP in the solution was removed by anion exchange chromatography using a 1 mL HiTrap Q HP column. The purified Y-pG-Y polymer solution was desalted into 10 mM Tris-HCl (pH 8.0) by an ultrafiltration membrane (3 kDa MWCO). PBS buffer (pH 7.4) filtered through a 0.22 μm membrane filter was used as a running buffer, and 30 μL of sample solution was used for the measurement of FFF-MALS (FFF, ECLIPSE 2; MALS, DAWN 8+, Wyatt Technology, Goleta, CA, USA). ITC of Cross-Linked pGs. Cross-linked pGs were prepared and purified in the same manner as the sample preparation for FFF-MALS measurement. IgG (anti-ovalbumin (chicken), rabbit polyclonal, catalog no. ab1221, Abcam, Cambridge, U.K.) was dissolved in 10 mM Tris-HCl (pH 8.0). The solution containing purified cross-linked pGs and the IgG solution were placed in microdialysis cups (oscillatory cup (MWCO 3500), Cosmo Bio Co., Ltd., Tokyo, Japan) and dialyzed against 10 mM Tris-HCl (pH 8.0) at 4 °C in the same container to make sure the solvent in the two solutions were the same. The dialysate solution was used for washing the syringes and cells of the ITC equipment (MicroCal iTC200, GE Healthcare UK Ltd.). The IgG solution and the cross-linked pG solutions were diluted with the dialysate solution to concentrations of 0.5 and 0.05 μM, respectively. The sample solutions containing crosslinked pGs were added to the IgG solution in the cell of the ITC equipment, and the temperature change was monitored. The binding stoichiometry of pG against IgG was estimated by the heat change profile. The binding ratio of pG to IgG was set as 2 to 1, and the binding capacity of cross-linked pG was calculated using this ratio. Measurement of SPR of the Cross-Linked Y-pG-Y on an IgG-Immobilized Sensor Chip. IgG (anti-ovalbumin (chicken), rabbit polyclonal, catalog no. ab1221, Abcam) was immobilized onto a CM5 sensor chip by an amine coupling reaction. The IgG immobilized sensor chip (6779.2 RU of IgG was immobilized) was used to determine the affinity of pG and the cross-linked Y-pG-Y. After the cross-linking reaction at the optimized condition (Y-pG-Y, 75 μM; HRP, 0.1 μM, and 75 μM H2O2 at 25 °C for 1 h), the Y-pG-Y conjugate was purified with an anion exchange column and concentrated as described above. The sample solutions were diluted with running buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) to concentrations of 0.3−5.0 μg/mL. The samples were applied to the IgG immobilized sensor chip to obtain SPR sensorgrams using Biacore 2000 (GE Healthcare, Uppsala, Sweden). The flow rate was set at 20 μL/min, and the temperature was 25 °C. The chip was regenerated by the addition of 10 mM Gly-HCl buffer at pH 2.25.

Dityrosine Fluorescence Measurement. The dTyr fluorescence intensity of cross-linked pG-Y and Y-pG-Y was measured to confirm the occurrence of the tyrosine coupling reaction after HRP treatment. pG-Y or Y-pG-Y was cross-linked by the HRP reaction at the optimized condition described above (pGs, 75 μM; HRP, 1 μM for pG-Y, 0.1 μM for Y-pG-Y, and 1 equiv of H2O2). The dTyr fluorescence of the solutions after cross-linking reaction was measured on a spectrofluorometer (FP-6300, Jasco Corporation, Tokyo, Japan). The fluorescence spectra were obtained in the range of 350−550 nm with an excitation at 315 nm. The excitation spectra were measured from 280 to 350 nm while monitoring the fluorescent intensity at 410 nm. Time-course measurements of fluorescence from dTyr after the cross-linking of N- and C-terminal Y-tag peptides of sequences GYGGGGLEVLFQGP and LVPRGSGGGGY, respectively, were conducted. The peptides were dissolved in 10 mM Tris-HCl at a concentration of 100 μM and mixed with 0.1 μM HRP and 50 μM H2O2. As soon as H2O2 was added and mixed, the solution was placed into a quartz cell to measure the dTyr fluorescence, monitoring 410 nm fluorescence from 315 nm excitation at 25 °C. Furthermore, the time-course measurement of dTyr fluorescence of pG variants was performed using the same protocol as that of the peptides. The concentrations of pG variants, HRP, and H2O2 were set at 30 μM, 0.5 μM, and 30 μM, respectively. Protease Treatment on Y-Tagged pGs before and after Cross-Linking Reaction. The pG-Y and Y-pG-Y were treated with thrombin and HRV 3C protease to cleave the Ytags and evaluate the cross-linking ability after HRP treatment. pG-Y (final concentration was 75 μM) was incubated with thrombin (37.5 unit/mL; Takara Bio Inc.) in 10 mM Tris-HCl (pH 8.0) at 37 °C for 16 h. Y-pG-Y (final concentration was 75 μM) was treated with thrombin (37.5 unit/L) and HRV 3C protease (120 unit/mL; Takara Bio Inc.) in 50 mM Tris-HCl, 150 mM NaCl, pH 7.5, at 37 °C for 16 h. HRP was added to the solutions at a final concentration of 0.1 μM, and 75 μM H2O2 was injected to conduct the cross-linking reaction at 25 °C for 1 h. The sample solutions were analyzed by SDS− PAGE. Cross-linked pG-Y and Y-pG-Y at the optimum condition (pGs, 75 μM; HRP, 1 μM for pG-Y, 0.1 μM for YpG-Y, and 1 equiv of H2O2 at 25 °C for 1 h) were treated with these proteases in the same cleavage condition as that shown above and analyzed with SDS−PAGE. Furthermore, the solution containing cross-linked pG-Y or YpG-Y after protease treatment was analyzed with MALDI-TOFMS to detect the cleaved cross-linked Y-tag peptides. The solution was mixed with the same amount of saturated sinapic acid solution in Milli-Q/acetonitrile (50:50) containing 0.1% trifluoroacetic acid. Two microliters of the mixture of sample solution and the matrix solution were cast on the target plate of MALDI-TOF-MS (autoflex speed MALDI TOF/TOF, Bruker, Billerica, MA, USA), and the droplets were dried using a hair dryer. The measurements were performed in linear-positive mode. Co-Cross-Linking Reaction of pG with pG-Y or Y-pG-Y. pG and pG-Y or Y-pG-Y were mixed in 10 mM Tris-HCl (pH 8.0) at a final concentration of 75 μM with HRP. The final concentrations of HRP were 1 and 0.1 μM for the solution containing pG-Y and Y-pG-Y, respectively. One equivalent of H2O2 against the tyrosine residue in the Y-tag was added to the solution to conduct a co-cross-linking reaction of pG and pG-Y or Y-pG-Y. The reaction was performed at room temperature for 1 h, and the solutions were analyzed by SDS−PAGE.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00138. MALDI-TOF-MS analyses on pG variants; dityrosine fluorescence measurement of HRP treated Y-tagged pGs; thrombin treatment on pG-Y; size-exclusion chromatography analyses of Y-pG-Y monomer and polymer; HRP catalytic cycle for generating superoxides ITC measureJ


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ments on pG variants; SPR measurements on pG variants; amino acid sequences of pGs prepared in this study; calculation of degree of branching of Y-pG-Y polymer (PDF)

AUTHOR INFORMATION

Corresponding Authors

*K.M.: phone, +81-3-5841-7219; fax, +81-3-5841-8657; e-mail, [email protected]. *T.N.: phone, +81-3-5841-7328; fax, +81-3-5452-5209; e-mail, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Prof. Kohei Tsumoto, Dr. Hiroki Akiba, and Dr. Ryo Matsunaga for the measurements of the FFF-MALS and ITC.

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ABBREVIATIONS dTyr, dityrosine; FFF-MALS, field flow fractionation and multiangle light scattering; HRP, horseradish peroxidase; HRV 3C, human rhinovirus 3C; IgG, immunoglobulin G; ITC, isothermal titration calorimetry; MALDI-TOF-MS, matrixassisted laser desorption/ionization time-of-flight mass spectrometry; MBP, maltose binding protein; PCR, polymerase chain reaction; pG, protein G; SDS−PAGE, sodium dodecyl sulfate−polyacrylamide gel electrophoresis; SPR, surface plasmon resonance; SrtA, sortase A; TEV, tobacco etch virus; TGase, transglutaminase; Tris, tris(hydroxymethyl)aminomethane

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REFERENCES

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Tyrosine Coupling Creates a Hyperbranched Multivalent Protein

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