Transglutaminase 2 and Factor XIII catalyze distinct substrates in differentiating osteoblastic cell line: utility of highly reactive substrate peptides

Abstract

Differentiated osteoblastic cell line, MC3T3-E1 expresses transglutaminase 2 (TG2) and Factor XIII (FXIII). In previous studies, we identified isozyme-specific and highly reactive glutamine-donor substrate peptides (pepF11KA and pepT26) for each isozyme. Using these peptides, we compared the reaction products with lysine-donor substrates for each isozyme in differentiating MC3T3-E1 cells. By this analysis, distinct substrates for the activated TG2 and FXIII were detected in cultured cellular extract. Possible substrates that incorporated biotin-labeled peptides were further purified using streptavidin-affinity chromatography. Several isozyme-specific substrates were identified by mass spectrometry analysis of the purified fractions. These analyses also indicate the benefit of the substrate peptides for obtaining distinct substrates in a reaction mixture where two isozymes co-exist.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Abbreviations

Asc-P:

l-ascorbic acid 2-phosphate

CBB:

Coomassie Brilliant Blue

DTT:

Dithiothreitol

TG2:

Transglutaminase 2

FXIII:

Factor XIII

References

  1. Al-Jallad HF, Nakano Y, Chen JL, McMillan E, Lefebvre C, Kaartinen MT (2006) Transglutaminase activity regulates osteoblast differentiation and matrix mineralization in MC3T3-E1 osteoblast cultures. Matrix Biol 25:135–148

    PubMed  Article  CAS  Google Scholar 

  2. Beninati S, Piacentini M (2004) The transglutaminase family: an overview. Amino Acids 26:367–372

    PubMed  CAS  Google Scholar 

  3. Chen JSK, Mehta K (1999) Tissue transglutaminase: an enzyme with a split personality. Int J Biochem Cell Biol 31:817–836

    PubMed  Article  CAS  Google Scholar 

  4. Esposito C, Caputo I (2005) Mammalian transglutaminases: identification of substrates as a key to physiological function and physiological relevance. FEBS J 272:615–631

    PubMed  Article  CAS  Google Scholar 

  5. Fesus L, Piacentini M (2002) Transglutaminase 2: an enigmatic enzyme with diverstic functions. Trends Biochem Sci 27:534–539

    PubMed  Article  CAS  Google Scholar 

  6. Forsprecher J, Wang Z, Nelea V, Kaartinen MT (2009) Enhanced osteoblast adhesion on transglutaminase 2-crosslinked fibronectin. Amino Acids 36:747–753

    PubMed  Article  CAS  Google Scholar 

  7. Griffin M, Casadio R, Bergamini CM (2002) Transglutaminases: nature’s biological glues. Biochem J 368:377–396

    PubMed  Article  CAS  Google Scholar 

  8. Hitomi K, Kitamura M, Sugimura Y (2009a) Preferred substrate sequences for transglutaminase 2: screening using a phage-display peptide library. Amino Acids 36:619–624

    PubMed  Article  CAS  Google Scholar 

  9. Hitomi K, Kitamura M, Perez-Alea M, Ceylan I, Thomas V, El-Alaoui S (2009b) A specific colorimetric assay for measuring transglutaminase 1 and factor XIII activities. Anal Biochem 394:281–283

    PubMed  Article  CAS  Google Scholar 

  10. Itoh M, Kawamoto T, Tatsukawa H, Kojima S, Yamanishi K, Hitomi K (2011) in situ detection of active transglutaminases for keratinocyte (TGase 1) and tissue type (TGase 2) using fluorescence-labeled highly reactive substrate peptides. J Histochem Cytochem 59:180–187

    PubMed  Article  CAS  Google Scholar 

  11. Johnson KA, Rose DM, Terkeltaub RA (2008) Factor XIIIA mobilizes transglutaminases 2 to induce chondrocyte hypertrophic differentiation. J Cell Sci 121:2256–2264

    PubMed  Article  CAS  Google Scholar 

  12. Kaartinen MT, El-Maadawy S, Rasanen NH, Mckee MD (2002) Tissue transglutaminase and its substrate in bone. J Bone Miner Res 17:2161–2173

    PubMed  Article  CAS  Google Scholar 

  13. Lorand L, Graham RM (2003) Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat Rev Mol Cell Biol 4:140–156

    PubMed  Article  CAS  Google Scholar 

  14. Nakano Y, Al-Jallad HF, Mousa A, Kaartinen MT (2007) Expression and localization of plasma transglutaminase factor XIIIA in bone. J Histochem Cytochem 55:675–685

    PubMed  Article  CAS  Google Scholar 

  15. Nakano Y, Forsprecher J, Kaartinen MT (2010) Regulation of ATPase activity of transglutaminase 2 by MT1-MMP: implications for mineralization of MC3T3-E1 osteoblast cultures. J Cell Physiol 223:260–269

    PubMed  CAS  Google Scholar 

  16. Nurminskaya M, Kaartinen MT (2006) Transglutaminase in mineralized tissues. Front Biosci 11:1591–1606

    PubMed  Article  CAS  Google Scholar 

  17. Perez-Alea M, Kitamura M, Martin G, Thomas V, Hitomi K, El Alaoui S (2009) Development of an isoenzyme-specific colorimetric assay for tissue transglutaminase 2 cross-linking activity. Anal Biochem 389:150–156

    PubMed  Article  CAS  Google Scholar 

  18. Quarles LD, Yohay DA, Lever LW, Caton R, Wenstrup RJ (1992) Distinct proliferative and differentiated stages of murine MC-3T3-E1 culture; an in vitro model of osteoblast development. J Bone Miner Res 7:683–692

    PubMed  Article  CAS  Google Scholar 

  19. Ruoppolo M, Orru S, D’Amato A, Francese S, Rovero P, Marino G, Esposito C (2003) Analysis of transglutaminase protein substrates by functional proteomics. Protein Sci 12:1290–1297

    PubMed  Article  CAS  Google Scholar 

  20. Sugimura Y, Hosono M, Wada F, Yoshimura T, Maki M, Hitomi K (2006) Screening for the preferred substrate sequence of transglutaminase using a phage-displayed peptide library: identification of peptide substrates for TGase 2 and Factor XIIIa. J Biol Chem 281:17699–17706

    PubMed  Article  CAS  Google Scholar 

Download references

Acknowledgments

We greatly appreciate Dr. Masatoshi Maki and Dr. Hideki Shibata in our laboratory for providing valuable suggestions. This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas (No. 20200072) (to K. H.) from the Ministry of Education, Sports, Science and Technology (MEXT, Japan).

Author information

Affiliations

Authors

Corresponding author

Correspondence to Kiyotaka Hitomi.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Watanabe, K., Tsunoda, K., Itoh, M. et al. Transglutaminase 2 and Factor XIII catalyze distinct substrates in differentiating osteoblastic cell line: utility of highly reactive substrate peptides. Amino Acids 44, 209–214 (2013). https://doi.org/10.1007/s00726-011-1131-8

Download citation

Keywords

  • Transglutaminase
  • Osteoblast
  • TG2
  • Factor XIII