Abstract
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.
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Data availability
All data generated or analyzed in this study are available from the corresponding author upon reasonable request.
Abbreviations
- FRET:
-
Förster resonance energy transfer
- FITC:
-
Fluorescein isothiocyanate
- PBS:
-
Phosphate buffered saline
- RhoB:
-
Rhodamine B
- TG:
-
Transglutaminase
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Acknowledgements
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, Nagoya University—Amano Enzyme Research Grant, Aichi Kidney Foundation, Tatematsu Foundation, Takeda Science Foundation, and Terumo Life Science Foundation. Authors are gratefully thankful to the CARE (The Center for Animal Research and Education) at Nagoya University for technical supports for animal experiments.
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HT designed the study and drafted the manuscript. RA performed the development and characterization of biosensor probes. KH 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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726_2023_3272_MOESM1_ESM.tif
Supplementary Figure 1. Emission spectrums when FITC was excited in the developed biosensors. Schematic diagram of the FRET probe detecting TG activity. Cross-linking of reactive TG substrate peptides, including Gln or Lys, closes the distance between FITC and RhoB fluorescent molecules, leading to the decrease of FITC emission intensity (red arrow) and the increase of RhoB emission intensity (blue arrow) (A). FITC-K5/RhoB-Kpep and FITC-T26/RhoB-Kpep were incubated with TG1 and TG2, respectively, and the emission spectrums of 500–600 nm, when the FITC was excited at 480 nm, were measured at each indicated time (B and C) (TIF 919 KB)
726_2023_3272_MOESM2_ESM.tif
Supplementary Figure 2. Emission spectrums when RhoB was excited in the biosensors. FITC-K5/RhoB-Kpep and FITC-T26/RhoB-Kpep were incubated with TG1 and TG2, respectively. The emission spectrums of 575–635 nm, when the RhoB was excited at 555 nm, were measured at each indicated time (A and B) (TIF 772 KB)
726_2023_3272_MOESM3_ESM.tif
Supplementary Figure 3. Mass spectrometry analyses in the biosensors. FITC-K5/RhoB-Kpep and FITC-T26/RhoB-Kpep were incubated with TG1 and TG2, respectively. These samples were dissolved with 5% acetonitrile/0.1% formic acid and desalted using GL-tip SDB (GL Sciences Inc.). Mass spectrometry was performed on a Q Exactive mass spectrometer (Thermo Fisher Scientific) and the spectrums in FITC-K5/RhoB-Kpep (A) and FITC-T26/RhoB-Kpep (B and C) were indicated after peak deconvolution using the Xtract algorithm within FreeStyle software (Themo Fisher Scientific). Cal. Mass, Calculated Mass (TIF 717 KB)
726_2023_3272_MOESM4_ESM.tif
Supplementary Figure 4. Dose response to TG inhibitor treatments in the biosensor. FITC-T26/RhoB-Kpep was incubated with TG2 in the presence or absence of each TG inhibitor, Z-DON (A), Boc-DON (B), and cystamine (C) at the indicated dose. The emission intensities of FITC were measured at each indicated time (TIF 1096 KB)
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Tatsukawa, H., Aoyama, R. & Hitomi, K. Development of peptide-based biosensors for detecting cross-linking and deamidation activities of transglutaminases. Amino Acids 55, 807–819 (2023). https://doi.org/10.1007/s00726-023-03272-7
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DOI: https://doi.org/10.1007/s00726-023-03272-7