Development of peptide-based biosensors for measuring cross-linking and deamidation activities of transglutaminases

doi:10.21203/rs.3.rs-1603071/v1

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Development of peptide-based biosensors for measuring cross-linking and deamidation activities of transglutaminases

https://doi.org/10.21203/rs.3.rs-1603071/v1

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published 10 May, 2023

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Transglutaminases (TGs) are a protein family that catalyzes isopeptide bond formation between glutamine and lysine residues of various proteins. There are eight TG isozymes in humans, and each is involved in diverse biological phenomena due to their characteristic distribution. Abnormal activity of TG1 and TG2, which are major TG isozymes, is believed to cause various diseases, such as ichthyosis, and celiac disease. To elucidate TGs’ mechanisms of action and develop new therapeutic strategies, it is essential to develop bioprobes that can specifically examine the activity of each TG isozyme, which has not been sufficiently studied. We previously have identified several substrate peptide sequences containing Gln residues for each isozyme and developed a method to detect isozyme-specific activities by incorporating a labeled substrate peptide into lysine residues of proteins. We prepared the fluorescein isothiocyanate (FITC)-labeled Gln substrate peptide (FITC-K5 and FITC-T26) and Rhodamine B-labeled Lys substrate peptide (RhoB-Kpep). Each TG reaction specifically cross-linked these probe pairs, and the proximity of FITC and Rhodamine B significantly decreased the fluorescence intensity of FITC depending on the concentration and reaction time of each TG. In this study, we developed a peptide-based biosensor that quickly and easily measures TG isozyme-specific activity. This probe is expected to be helpful in elucidating TG’s physiological and pathological functions and in developing compounds that modulate TG activity.

Transglutaminase

Cross-link

Biosensor

Substrate peptide

Förster resonance energy transfer

Transglutaminases (TGs) are a family of enzymes that catalyze the Ca²⁺-dependent cross-linking formation of an ε(γ-glutamyl)lysine isopeptide bond between specific glutamine (Gln) and a lysine (Lys) residue in the proteins, and of incorporation of a primary amine into Gln residue (Lorand and Graham 2003; Iismaa et al. 2009; Eckert et al. 2014; Hitomi et al. 2015). A deamidation reaction occurs without an appropriate amine donor or a specific substrate, converting Gln to glutamic acid (Glu) in a protein. TGs are widely present in mammals and invertebrates, amphibians, fish, birds, plants, and microorganisms. The TG family consists of eight isozymes designated as coagulation factor XIII and TG1–TG7, which are widely distributed and involved in multiple biological processes. The abnormal activity of TGs causes the development of various diseases. Several mutations in human TG1 are associated with autosomal recessive congenital ichthyosis, characterized by abnormal skin formation caused by a dramatic decrease in TG1 cross-linking activity (Huber et al. 1995; Russell et al. 1995). In addition, TG1 mutations have recently been suggested to be associated with Alzheimer’s disease (Tripathy et al. 2020). TG2 has been implicated in autoimmune diseases, infectious diseases, fibrosis, cancer, and neurodegenerative diseases (Siegel and Khosla 2007; Jeitner et al. 2009; Mehta et al. 2010; Sollid and Jabri 2011; Tatsukawa et al. 2016, 2021). Celiac disease, an autoimmune disease triggered by gluten, affects 1% of the population in western countries. Gliadin, from digested gluten, is modified by TG2, causing an abnormal immune response due to the antigen modification (Lebwohl et al. 2018). Thus, each TG isozyme is essential for various biological processes in mammalian tissues, and in many cases, these enzymes are expressed at varying levels in identical cells. Therefore, it is necessary to evaluate their enzymatic activities accurately in an isozyme-dependent manner. In addition, developing rapid and accurate biosensors that can specifically examine these TG isozyme activities is essential to elucidate the molecular mechanisms by which TGs influence disease pathogenesis and to develop new therapeutic strategies to target each TG.

Many in vitro assay systems have been developed to measure TG activity. A commonly used method quantifies the amount of radio- or biotin-labeled amine incorporated into Gln residues in proteins, such as casein, by utilizing the cross-linking reaction that occurs between Gln and Lys residue in the proteins and incorporation of a primary amine (Folk et al. 1980). TG activity can be measured in these assays as the amount of amine incorporated into casein after protein precipitation or coated casein on a multi-well plate. In a similar assay, Lys polypeptide is coated on a plate, and the amount of cross-linking with a labeled peptide, including Gln residue (TVQQEL), is examined. Although both methods are relatively simple, they require a large amount of measurement time and include many complicated steps, such as coating the plate, washing, and adding several reagents. In addition, these methods to measure TG activity lack isozyme specificity.

To distinguish each TG isozyme, we have previously determine the peptide sequences including Gln residue that react specifically with each isozyme (Sugimura et al. 2006, 2008). These peptide sequences, some as long as 12 amino acids, appeared to act as substrates with high reactivity and isozyme specificity. The isozyme specificity of these substrate peptides has made it possible to detect differences in each enzyme activity at the cellular and tissue level (Perez Alea et al. 2009; Hitomi et al. 2009a; Itoh et al. 2011, 2013; Watanabe et al. 2013; Tatsukawa et al. 2017b, 2018; Takeuchi et al. 2021). In addition, we have developed a Förster resonance energy transfer (FRET)-based probes to measure TG activity in an isozyme-specific manner by producing probes consisting of a fluorescent protein and specific substrate peptides of each TG isozyme (Tatsukawa et al. 2017a). Although these protein-based probes are useful for rapidly and conveniently monitoring TG activity, they require a considerable amount of proteinaceous probe for one measurement of TG activity in multiple samples. Furthermore, in these probes, unexpected Gln and Lys residues in the fluorescent proteins are cross-linked, resulting in the formation of multimers.

To improve upon these protein-based probes, we have developed new probes based on small fluorescent compounds linked with substrate peptide. Measurements using these peptide-based probes are rapid and convenient. Compared to previous protein-based probes, they are easier to synthesize, are more stable, and provide more reproducible results. These probes should contribute to determining TG activity from multiple cells and tissue samples, revealing detailed functions of each TG family member in various physiological and pathological processes.

Materials

Chemical reagents were purchased from WAKO pure chemicals (Osaka, Japan) and Nacalai Tesk (Kyoto, Japan). Recombinant proteins for human TG1 and TG2 were obtained from Zedira GmbH (Darmstadt, Germany).

Synthesis of fluorescent peptide-based probes

The probes listed below were synthesized by GL Biochem Ltd. (Shanghai, Chaina). FITC-K5: FITC-YEQHKLPSSWPF, FITC-K5QN: FITC-YENHKLPSSWPF, FITC-T26: FITC- HQSYVDPWMLDH, FITC-T26QN: FITC-HNSYVDPWMLDH, Rhodamine B (RhoB)-Kpep: RhoB-GGGSMRHKGS, TAMRA-K5: TAMRA-YEQHKLPSSWPF, TAMRA-T26: TAMRA-HQSYVDPWMLDH, FITC-Kpep: FITC-MRHKGS, TAMRA-Kpep: TAMRA-MRHKGS, Kpep: MRHKGS

Characterization of fluorescent peptide-based probe pairs

The various fluorescent peptide-based probes (50 µM) were evaluated in the reaction buffer containing 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM DTT, with or without 10 mM CaCl₂ at 25°C for 60 min after addition of 2.5 µg/mL TG1 and TG2. The probes were excited at 480 nm (for FITC) and 555 nm (for RhoB and TAMRA), and emission spectrum of the fluorescence, 500–650 nm and 575–650 nm, respectively, was continually measured by fluorospectrometer (EnSpire; Perkin Elmer Inc., MA, USA). Data are indicated as mean ± SEM for triplicate measurements of fluorescent intensities in each probe.

Validation of the specificity for detecting cross-linking reaction in the selected biosensors

The specificity in the selected probe pairs for reflecting cross-linking activity was validated. In the donor probes, point mutants of each reactive Gln-residue of the substrate peptide (change of Gln to Asn), designated as FITC-K5QN and FITC-T26QN, were used instead of FITC-K5 or FITC-T26. In the acceptor probe, no probe, 50 µM Kpep without fluorescent labeling, and 50 µM biotinylated pentylamine (BPA) were used instead of RhoB-Kpep. The probes were excited at 480 nm and emission intensity at 520 nm was continually measured by fluorospectrometer.

Optimization of cross-linking reaction by TG1 and TG2 using the biosensor

Equal dilutions were repeated up to 5 times, starting with 125 ng of TG1 or TG2, and added in a 25 µM of cross-linking reaction buffer including the selected probe pairs. The probes were excited at 480 nm and emission intensity at 520 nm was continually measured by fluorospectrometer.

Optimization of reaction ratios between donor and acceptor of the biosensor

The reaction ratios in the selected probe pairs (FITC-K5/RhoB-Kpep and FITC-T26/RhoB-Kpep) were evaluated in the reaction buffer containing 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM DTT, 10 mM CaCl₂ at 25°C for 60 min after addition of 2.5 µg/mL TG1 and TG2. In each condition, the 10, 20, 30, 40, and 50 µM FITC-K5 or FITC-T26 were reacted with 50 µM RhoB-Kpep. After that, in reverse order, 10 µM FITC-K5 or FITC-T26 were reacted with 10, 20, 30, 40, and 50 µM RhoB-Kpep to determine the optimal conditions.

Comfirmation of calcium-dependency of TG2 in the biosensors

The probe pair FITC-T26/RhoB-Kpep was evaluated in the reaction buffer containing 10 mM Tris-HCl, pH8.0, 150 mM NaCl, 1 mM DTT, in the absence or presence of 0.1–1 mM CaCl₂ at 25°C for 60 min after addition of 2.5 µg/mL TG2. The calcium chelator, EDTA (final 50 mM) was added in the reaction buffer including 10 mM CaCl₂.

Evaluation of pH-dependent reaction of TG1 and TG2 in the biosensors

The probe pairs (FITC-K5/RhoB-Kpep and FITC-T26/RhoB-Kpep) were evaluated in citrate- and Tris-based reaction buffer which adjusted to pH 6 and pH 7–10, respectively, at 25°C for 120 min after addition of 2.5 µg/mL TG1 and TG2. The probes were excited at 480 nm and emission intensity at 520 nm was continually measured by fluorospectrometer. In addition, the reaction solutions before and after the cross-linking reaction were collected and mixed with Laemmli sample buffer for SDS-PAGE on a Tricine three-layer gel. Each gel was composed of a separation gel (16% acrylamide, 0.5% bis-acrylamide, 1M Tris-HCl pH 8.45, 13.3% glycerol, 0.05% APS, 0.1% SDS, 0.05% TEMED), spacer gel (10% acrylamide, 0.3% bisacrylamide, 1M Tris-HCl pH 8.45, 0.05% APS, 0.1% SDS, 0.05% TEMED), and a concentrated gel (3.8% acrylamide, 0.12% bisacrylamide, 0.75M Tris-HCl pH 8.45, 0.08% APS, 0.074% SDS, 0.083% TEMED). The mobility of fluorescent probe bands was evaluated by observing the fluorescence at 565 nm when excited at 480 nm using the high sensitivity reading (FUSION; Vilber, France).

Development of peptide-based biosensors for measuring TG activity

Peptide-based biosensors for TG activity were constructed as a donor fluorescein-linked Gln substrate peptide and an acceptor fluorescein-linked Lys substrate peptide. TG induced Gln–Lys cross-linking of substrate peptides causes the dimerization of probes. This allows donor and acceptor probes to be close together, resulting in the FRET phenomenon (Fig. 1A). The emission wavelength of the fluorescent donor and excitation wavelength of the acceptor must be close for the fluorescent molecules to cause FRET. Due to this, FITC and RhoB are commonly used together. In addition, TAMRA, which has fluorescence properties comparable to RhoB, was also examined. These fluorescent molecules were labeled with Gln substrate peptides for TG1 and TG2 designated as K5 and T26 or with Lys substrate peptide named Kpep for detecting cross-linking activities (Hitomi et al. 2009b). Several FRET pairs were firstly tested for the ability to detect TG1 and TG2 activities. Representative results showed that probe pairs of FITC-K5/RhoB-Kpep and FITC-K5/TAMRA-Kpep significantly decreased the emission intensity of 520 nm in the cross-linking reaction by TG1 compared to the probe pair of FITC-Kpep/TAMRA-K5 with fluorescent labeling replaced (Fig. 1B). In the case of TG2, the emission intensity of FITC-T26/RhoB-Kpep showed a marked reduction compared to those of FITC-T26/TAMRA-Kpep and FITC-Kpep/TAMRA-T26 (Fig. 1C). Based on these results, we determined the FITC-K5/RhoB-Kpep and FITC-T26/RhoB-Kpep as prominent probe pairs for monitoring the cross-linking activity of TG1 and TG2, respectively. Next, we analyze the specificity of the probe pair against cross-linking reaction by TG1 and TG2. The point mutants of each reactive Gln residue of the substrate peptide (change of Gln to Asn), named K5QN and T26QN, significantly reduced the emission intensity in the FRET probe (Figs. 1D and 1E).

Specificity and sensitivity of the selected biosensors

To investigate whether the decrease in emission intensity of FITC-K5 and FITC-T26 due to the cross-linking reaction by TG1 and TG2 is dependent on the presence of RhoB-Kpep, the reduced emission intensity was measured using Kpep without fluorescent labeling, biotinylated pentylamine (BPA), and no probe instead of RhoB-Kpep. As a result, no emission intensity reductions were detected (Figs. 2A and 2B). These results indicated that the proximity of RhoB to FITC by cross-linking reaction is essential for the reduced intensity of FITC emission. Moreover, this decrease in emission intensity of FITC may be the result of enhanced excitation and emission of RhoB due to FRET induction (Suppl. Figure 1A). However, the emission spectrum of 500–600 nm when excited at 480 nm was checked, and no enhancement of emission intensity of RhoB was observed at 580 nm, the emission peak of RhoB (Suppl. Figures 1B and 1C). Interestingly, these emission peaks of RhoB when excited at 555 nm (excitation wavelength for RhoB), also decreased markedly in a time-dependent manner (Suppl. Figures 2A and 2B). The sensitivities of these probe pairs were analyzed using various enzyme concentrations, resulting in evident concentration-dependent changes (Figs. 2C and 2D). In terms of enzyme concentration, detection limits of FITC-K5/RhoB-Kpep and FITC-T26/RhoB-Kpep were approximately 1.25 µg/mL (31.25 ng/well) of TG1 and TG2, respectively.

Optimization and isozyme cross-reactivity of the biosensors

Next, the reaction ratios between donor and acceptor probes in the FITC-K5/RhoB-Kpep or FITC-T26/RhoB-Kpep were evaluated. TG1 was in the incubation of each concentration pair of FITC-K5 (10–50 µM) and 50 µM RhoB-Kpep; the emission intensity of FITC decreased the most when the reaction ratio of FITC-K5:RhoB-Kpep was 1:5 (Fig. 3A). In the reverse order, incubation of 10 µM FITC-K5 and 10–50 µM RhoB-Kpep with TG1 also showed the highest decrease in the emission intensity at a FITC-K5:RhoB-Kpep ratio of 1:5 (Fig. 3B). Similar results were obtained with TG2 (Figs. 3C and 3D). The specific reactivity of these probe pairs was demonstrated, and FITC-K5/RhoB-Kpep and FITC-T26/RhoB-Kpep specifically recognized the cross-linking activity of TG1 and TG2, respectively (Figs. 3E and 3F). The biochemical properties of Ca²⁺ dependency in these biosensors were evaluated; a decrease in emission intensity responded in a stepwise manner dependent on the concentration of Ca²⁺ (Fig. 3G).

Evaluation of the pH-dependent reaction of TG1 and TG2 in the biosensors

Protein cross-linking reactions by TGs are affected by pH (Fleckenstein et al. 2002; Sivadó et al. 2019). Here, we investigated cross-linking activities of TG1 and TG2 at a pH of 6–10 using selected probe pairs. The emission intensity of FITC strongly decreased at pH 7–10, and this alteration was most pronounced at pH 9; however, this was suppressed at pH 6 (Figs. 4A and 4C). After the reaction at each pH, these samples were then collected and examined to detect cross-linking products by Tricine-SDS-PAGE, as described in the Materials and Methods. Consistent with the decrease in emission intensity of FITC, the cross-linking products were increased in the reaction sample at pH 7–10 (Figs. 4B and 4D). While these bands were faint at pH 6, a band corresponding to deamidation obviously increased.

Evaluation of biosensors’ response to TG inhibitor treatments

Next, we tested whether the decrease in the emission intensity of FITC was attenuated by the addition of TG inhibitors in this assay system. The TG activities were evaluated using inhibitors Z006 and B003, which show relatively high specificity for TG2, and cystamine, a competitive inhibitor with no isozyme specificity. As expected, the decrease in emission intensity of FITC in FITC-T26/RhoB-Kpep was dose-dependently suppressed by inhibitor treatments (Suppl. Figure 3). The IC₅₀ of Z006 is known to be 7.3 µM and 0.02 µM against TG1 and TG2, respectively. The reduced emission intensity of FITC by TG1 in FITC-K5/RhoB-Kpep was slightly suppressed by treatment of 7.5 µM Z006 while the reduction in emission intensity by TG2 in FITC-T26/RhoB-Kpep was expectedly suppressed by about 50% with treatment of 0.02 µM Z006 (Figs. 5A and 5C). Similar results from these samples were obtained in Tricine-SDS-PAGE. The cross-linked products by TG1 in FITC-K5/RhoB-Kpep were insignificantly inhibited after 40 min of incubation, while those by TG2 in FITC-T26/RhoB-Kpep were markedly inhibited after 40 and 120 min of incubation (Figs. 5B and 5D).

TG activity has been directly implicated in various diseases, including skin diseases, celiac disease, fibrosis, atherosclerosis, diabetes, and cancer progression. TG1 and TG2 have been well-investigated for their structures, tissue distribution patterns, substrates, regulation of activities, and associated diseases. However, few reports have analyzed the relationship between the enhanced cross-linking activity or altered distribution of each TG isozyme and disease exacerbation. This is because few methods have been developed to analyze isozyme-specific activity and subcellular localization in living cells. To examine the cross-linking activity of TG2 in living cells, a FRET probe has already been developed for monitoring the conformational change of TG2 (Caron et al. 2012; Pavlyukov et al. 2012). However, this probe cannot detect endogenous TG2 activity because it requires introducing a plasmid encoding TG2 fused with YFP and CFP at its N- and C-termini, respectively, into live cells.

In the cross-linking reaction by TGs, a reaction intermediate is initially formed as a disulfide bond between the cysteine at the active center of TGs and the Gln substrate residue, followed by a nucleophilic reaction with the Lys residue of another substrate. Therefore, to examine the TG activity in isozyme-specific manner, it was essential to determine the peptide sequence containing the Gln residue recognized by each TG isozyme, which we have done in our previous studies. Using each peptide substrate specific for TG1 and TG2, we developed novel peptide-based biosensors for monitoring TG1 and TG2 isozymes’ activities. These are helpful probes that address the disadvantages of previous protein-based FRET probes, such as the complexity of recombinant probe preparation, stability, and reaction specificity issues.

In this study, various probes were synthesized and examined to explore the optimal probe pairs where FRET occurs between two fluorescent molecules linked substrate peptide. FRET is a phenomenon in which energy is transferred from the fluorescent donor molecule to the acceptor fluorescent molecule when the two molecules are close (1–10 nm). The energy of donor fluorescence is not released as emission energy; it excites the acceptor. Then, the emission fluorescence from the acceptor can be observed. The probe pairs of FITC-K5 or FITC-T26 and TAMRA-Kpep showed a marked decrease in emission intensity of FITC. However, even in the probe pair labeled with the same fluorescence molecules, the swapped fluorescent probe pairs of TAMRA-K5 or TAMRA-T26 and FITC-Kpep did not show a decrease in emission intensity of FITC (Figs. 1B and 1C). These results corroborated that the modality of substrate recognition and cross-linking reaction by TG1 and TG2 are similar and that the spatial positioning of fluorescent molecules in the cross-linked products is important for FRET. In the selected probe pairs, such as FITC-K5/RhoB-Kpep and FITC-T26/RhoB-Kpep, their cross-linking reaction by TG1 and TG2 did not increase the emission intensity of RhoB via FRET phenomenon when excited at 480 nm. However, the emission intensities of FITC excited at 480 nm and RhoB excited at 555 nm were significantly decreased in the cross-linking reaction (Suppl. Figures 1 and 2). In these decreased emission intensities of FITC and RhoB after cross-linking reaction, the importance of association between FITC and RhoB was confirmed using non-fluorescent-labeled Kpep and BPA instead of RhoB-Kpep. The decrease in emission intensity of FITC was attributed to the transfer of emission energy from FITC to RhoB, but the release of this excited RhoB energy was not observed as the increase of its emission intensity. A possible reason for this is that RhoB acts as a quencher in this reaction system, and the energy of RhoB is converted into other energy, such as heat and light, and released. Such quenching phenomenon has been reported in some cases. TAMRA, which has a structure similar to RhoB, is used as a quencher dye in real-time PCR, because it interacts with the guanine base in the target DNA, resulting in the quenching of its fluorescence. The other example was reported between rhodamine derivatives and the amino acid tryptophan (Trp) (Vaiana et al. 2003; Doose et al. 2005). This excited singlet state of rhodamine is efficiently and almost exclusively quenched by the Trp via photo-induced electron transfer and released into the solvent as luminescence and heat, and also used for photochemical reactions. The substrate peptides, K5 and T26, used in this study also contain tryptophan at similar positions, suggesting that the proximity of RhoB and Trp by cross-linking absorbed and quenched the fluorescence emission of FITC. Another possible cause of fluorescence quenching is contact quenching. This is the most common quenching mechanism, which occurs when the donor and acceptor dye molecules are in physical contact, and the distance between them is less than 1 nm (Acikgoz et al. 2010). The contact quenching might occur predominantly over FRET due to the closer distance between fluorescence molecules caused by TG induced cross-linking of probe pairs. The fluorescence intensity of RhoB is weaker than that of FITC (there is a tenfold difference in fluorescence intensity between FITC and RhoB at the maximum fluorescence wavelength). This may have resulted in considerable leakage of emission fluorescence from FITC, so that any increase in RhoB fluorescence due to FRET was not observed as a detectable signal change.

When the probe pair ratios of donor and acceptor were examined, the detection sensitivity increased in the probe with less FITC-K5 or FITC-T26 and more RhoB-Kpep (Figs. 3A–D). As described above, in the cross-linking reaction by TGs, a reaction intermediate is initially formed between the active center cysteine of TGs and the Gln residue of the substrate. In this probe, the intermediate is formed between TGs and FITC-Gln substrate peptide, followed by a nucleophilic reaction with the Lys residue of RhoB-Kpep or H₂O, resulting in the cross-linking or deamidation reaction, respectively. Therefore, deamidation reactions are more likely to occur when the amount of amine donor is relatively less. If the ratio of RhoB-Kpep to FITC-Gln substrate peptide is large, deamidation reactions are unlikely to occur. The FITC-Gln substrate peptide and RhoB-Kpep could interact quickly, resulting in the cross-linking reaction and the decreased emission intensity of FITC. These modulations of TG activity between cross-linking and deamidation were also observed at the pH difference in the reaction conditions. Cross-linking reaction by TGs was predominantly induced in the reaction buffer at more than pH 7, while deamidation was induced in that at pH 6 (Figs. 4B and 4D). Interestingly, the results indicate that the rate of the cross-linking reaction increases at alkaline pH, suggesting that it is essential that the amine be deprotonated (Fleckenstein et al. 2002). Notably, the deamidation activity of TG1 was tightly regulated at pH 6, whereas that of TG2 was gradually decreased from pH 6 to pH 9, suggesting that TG2 exerted both cross-linking and deamidation reactions in a wide range of pH compared to TG1. This could be why the FITC-K5/RhoB-Kpep activity detection probe for TG1 indicated a more significant decrease in fluorescence intensity of FITC compared to probe FITC-T26/RhoB-Kpep for TG2.

The probe pairs FITC-K5/RhoB-Kpep and FITC-T26/RhoB-Kpep developed in this study indicated the specific decrease in fluorescence intensity of FITC upon incubation with TG1 and TG2, respectively. In addition, experiments with TG inhibitors also showed that these biosensors could correctly quantify each TG isozyme activity. Further studies are currently underway in our laboratory. These biosensors will allow us to investigate endogenous TG activity in living cells. This will be a valuable tool for elucidating the underlying mechanisms of TG-related diseases and for evaluating the efficacy of TG inhibitors.

Acknowledgments

This work was supported by Grant-in-Aid for Scientiﬁc Research (26292192 and 18H02134 awarded to K.H. and 19K08675 awarded to H. Tatsukawa) from the

Ministry of Education, Sports, Science and Technology (JSPS, KAKENHI, Japan) and by grants from the Japan Foundation for Applied Enzymology as well as the Nagoya University–Amano Enzyme Research Grant. The authors would like to thank Enago for the English language review.

Compliance with Ethical Standards

The authors declare that they have no conflict of interests. This article does not contain any studies with human participants or animals performed by any of the authors.

Author Contribution

H.T. designed the study and drafted the manuscript. R.A. performed the development and characterization of biosensor probes. K.H. helped to design the study and provided the technical and material supports. All authors have read and approved the final manuscript.

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published 10 May, 2023

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Editorial decision: Major revision
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Development of peptide-based biosensors for measuring cross-linking and deamidation activities of transglutaminases

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Journal Publication

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Abstract

Figures

Introduction

Materials And Methods

Results

Development of peptide-based biosensors for measuring TG activity

Specificity and sensitivity of the selected biosensors

Optimization and isozyme cross-reactivity of the biosensors

Evaluation of the pH-dependent reaction of TG1 and TG2 in the biosensors

Evaluation of biosensors’ response to TG inhibitor treatments

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1