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Unfolding studies of tissue transglutaminase

Amino Acids volume 36, Article number: 633 (2009) Cite this article

Abstract

Activation of tissue transglutaminase by calcium involves a conformational change which allows exposition of the active site to the substrate via movements of domains 3 and 4 that lead to an increase of the inter-domain distance. The inhibitor GTP counteracts these changes. Here we investigate the possible existence of non-native conformational states still compatible with the enzyme activity produced by chemical and thermal perturbations. The results indicate that chemical denaturation is reversible at low guanidine concentrations but irreversible at high concentrations of guanidine. Indeed, at low guanidine concentrations tissue TG-ase exists in a non-native state which is still affected by the ligands as in the native form. In contrast, thermal unfolding is always irreversible, with aggregation and protein self-crosslinkage in the presence of calcium. DSC thermograms of the native protein in the absence of ligands consist of two partly overlapped transitions, which weaken in the presence of calcium and merge together and strengthen in the presence of GTP. Overall, the present work shows, for the first time, the reversible denaturation of a TG-ase isoenzyme and suggests the possibility that also in in vivo, the enzyme may acquire non-native conformations relevant to its patho-physiological functions.

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Abbreviations

TG-ase:

Transglutaminase

DSC:

Differential scanning calorimetry

CD:

Circular dichroism

Tm:

Melting temperature

SAS:

Small-angle-scattering

GdmHCl:

Guanidinium hydrochloride

References

  • Achyuthan KE, Greenberg CS (1987) Identification of a guanosine triphosphate binding site on guinea-pig liver transglutaminase. Role of GTP and calcium in modulating activity. J Biol Chem 262:1901–1906

    PubMed  CAS  Google Scholar 

  • Bai JH, Xu D, Wang HR, Zheng SY, Zhou HM (1999) Evidence for the existence of an unfolding intermediate for aminoacylase during denaturation in guanidine solutions. Biochim Biophys Acta 1430:39–45

    PubMed  CAS  Google Scholar 

  • Baskakov I, Wang A, Bolen DW (1998) Trimethylamine-N-oxide counteracts urea effects on rabbit muscle lactate dehydrogenase function: a test of the counteraction hypothesis. Biophys J 74:2666–2673

    PubMed  Article  CAS  Google Scholar 

  • Bergamini CM (1988) GTP modulates calcium binding and cation-induced conformational changes in erythrocyte transglutaminase. FEBS Lett 239:255–258

    PubMed  Article  CAS  Google Scholar 

  • Bergamini CM (2007) Effects of ligands on the stability of tissue transglutaminase: studies in vitro suggest possible modulation by ligands of protein turn-over in vivo. Amino Acids 33:415–421

    PubMed  Article  CAS  Google Scholar 

  • Bergamini CM, Dean M, Matteucci G, Hanau S, Tanfani F, Ferrari C, Boggian M, Scatturin A (1999) Conformational stability of human erythrocyte transglutaminase: patterns of thermal unfolding at acid and alkaline pH. Eur J Biochem 266:575–582

    PubMed  Article  CAS  Google Scholar 

  • Casadio R, Polverini E, Mariani P, Spinozzi F, Carsughi F, Fontana A, Polverino de Laureto P, Matteucci G, Bergamini CM (1999) The structural basis for regulation of tissular transglutaminase by calcium ions. Eur J Biochem 262:672–679

    PubMed  Article  CAS  Google Scholar 

  • Di Venere A, Rossi A, De Matteis F, Rosato N, Finazzi-Agrò AF, Mei G (2000) Opposite effects of Ca2+ and GTP binding on tissue transglutaminase tertiary structure. J Biol Chem 275:3915–3921

    PubMed  Article  CAS  Google Scholar 

  • Dong A, Kendrick B, Kreilgârd L, Matsuura J, Manning MC, Carpenter JF (1997) Spectroscopic study of secondary structure and thermal denaturation of recombinant human factor XIII in aqueous solution. Arch Biochem Biophys 347:213–220

    PubMed  Article  CAS  Google Scholar 

  • Fabbi M, Marimpietri D, Martini S, Brancolini C, Amoresano A, Scaloni A, Bargellesi A, Cosulich E (1999) Tissue transglutaminase is a caspase substrate during apoptosis. Cleavage causes loss of transamidating function and is a biochemical marker of caspase 3 activation. Cell Death Differ 6:992–1001

    PubMed  Article  CAS  Google Scholar 

  • Frustaci A, Cuoco L, Chimenti C, Pieroni M, Fioravanti G, Gentiloni N, Maseri A, Gasbarrini G (2002) Celiac disease associated with autoimmune myocarditis. Circulation 105:2611–2618

    PubMed  Article  Google Scholar 

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

    PubMed  Article  CAS  Google Scholar 

  • Haynie DT, Freire E (1993) Structural energetics of the molten globule state. Proteins 16:115–140

    PubMed  Article  CAS  Google Scholar 

  • Kishore BK, Kallay Z, Tulkens PM (1989) Clinico-biochemical aspects of guanidine compounds in uraemic toxicity. Int Urol Nephrol 21:223–232

    PubMed  Article  CAS  Google Scholar 

  • Kurochkin IV, Procyk R, Bishop PD, Yee VC, Teller DC, Ingham KC, Medved LV (1995) Domain structure, stability and domain-domain interactions in recombinant factor XIII. J Mol Biol 248:414–430

    PubMed  CAS  Google Scholar 

  • Lai TS, Bielawska A, Peoples KA, Hannun YA, Greenberg CS (1997) Sphingosyl-phosphocholine reduces the calcium requirement for activating tissue transglutaminase. J Biol Chem 272:16295–16300

    PubMed  Article  CAS  Google Scholar 

  • Lee KN, Birckbichler PJ, Patterson MK Jr (1989) GTP hydrolysis by guinea pig liver transglutaminase. Biochem Biophys Res Commun 162:1370–1375

    PubMed  Article  CAS  Google Scholar 

  • Luft LM, Barr SG, Martin LO, Chan EK, Fritzler MJ (2003) Autoantibodies to tissue transglutaminase in Sjogren’s syndrome and related rheumatic diseases. J Rheumatol 30:2613–2619

    PubMed  CAS  Google Scholar 

  • Mariani P, Carsughi F, Spinozzi F, Romanzetti S, Meier G, Casadio R, Bergamini CM (2000) Ligand induced conformational changes in tissue transglutaminase: Monte Carlo analysis of small angle scattering data. Biophys J 78:3240–3251

    PubMed  Article  CAS  Google Scholar 

  • Mitkevich OV, Shainoff JR, DiBello PM, Yee VC, Teller DC, Smejkal GB, Bishop PD, Kolotushkina IS, Fickenscher K, Samokhin GP (1998) Coagulation factor XIIIa undergoes a conformational change evoked by glutamine substrate. Studies on kinetics of inhibition and binding of XIIIA by a cross-reacting antifibrinogen antibody. J Biol Chem 273:14387–14391

    PubMed  Article  CAS  Google Scholar 

  • Monsonego A, Friedmann I, Shani Y, Eisenstein M, Schwartz M (1998) GTP dependent conformational changes associated with the functional switch between Galpha and crosslinking activities of brain derived tissue transglutaminase. J Mol Biol 282:713–720

    PubMed  Article  CAS  Google Scholar 

  • Nakaoka H, Perez DM, Baek KJ, Das T, Husain A, Misono K, Im MJ, Graham RM (1994) Gh: a GTP-binding protein with transglutaminase activity and receptor signaling function. Science 264:1593–1596

    PubMed  Article  CAS  Google Scholar 

  • Nury S, Meunier J-C, Mouranche A (1989) The kinetics of the thermal deactivation of transglutaminase from guinea-pig liver. Eur J Biochem 180:61–66

    Article  Google Scholar 

  • Perczel A, Park K, Fasman GD (1992) Analysis of the circular dichroism spectrum of proteins using the convex constraints algorithm: a practical guide. Anal Biochem 203:83–93

    PubMed  Article  CAS  Google Scholar 

  • Pinkas DM, Strop P, Brunger AT, Khosla C (2007) Transglutaminase 2 undergoes a large conformational change upon activation. PLoS Biol 5(e327):2788–2796

    CAS  Google Scholar 

  • Privalov PL, Dragan AI (2007) Microcalorimetry of biological macromolecules. Biophys Chem 126:16–24

    PubMed  Article  CAS  Google Scholar 

  • Rinas U, Risse B, Jaenicke R, Abel KJ, Zettlmeissl GB (1990) Denaturation-renaturation of the fibrin stabilizing factor XIII a-chain isolated from human placenta. Biol Chem Hoppe Seyler 371:49–56

    PubMed  CAS  Google Scholar 

  • Schuppan D, Hahn EG (2001) Celiac disease and its link to type 1 diabetes mellitus. J Pediatr Endocrinol Metab Suppl 1:597–605

    Google Scholar 

  • Smethurst PA, Griffin M (1996) Measurement of tissue transglutaminase activity in a permeabilized cell system: its regulation by Ca2+ and nucleotides. Biochem J 313:803–808

    PubMed  CAS  Google Scholar 

  • Sollid LM (2000) Molecular basis of celiac disease. Ann Rev Immunol 18:53–81

    Article  CAS  Google Scholar 

  • Strucksberg KH, Rosenkranz T, Fitter J (2007) Reversible and irreversible unfolding of multi-domain proteins. Biochim Biophys Acta 1774:1591–1603

    PubMed  CAS  Google Scholar 

  • Tanfani F, Bertoli E, Signorini M, Bergamini CM (1993) Structural investigation of transglutaminase by Fourier transform infrared spectroscopy. Eur J Biochem 218:499–505

    PubMed  Article  CAS  Google Scholar 

  • Tsou C-L (1995) Inactivation precedes overall molecular conformational changes during enzyme denaturation. Biochim Biophys Acta 1253:151–162

    PubMed  Google Scholar 

  • Zhang J, Lesort M, Guttmann RP, Johnson GV (1998) Modulation of the in situ activity of tissue transglutaminase by calcium and GTP. J Biol Chem 273:2288–2295

    PubMed  Article  CAS  Google Scholar 

Download references

Acknowledgments

Authors are grateful to Prof. Franco Dallocchio for fruitful discussions, to Mr. Marco Signorini and to Ms Alessia Dondi, for help in initial experiments and in thermal inactivation studies. This research was supported by grants from MIUR A.S., C.M.B. and CNR (A.S.)

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Affiliations

  1. Department of Biochemistry and Molecular Biology, Interdisciplinary Centre for Study of Inflammation, University of Ferrara, Via L. Borsari 46, 44100, Ferrara, Italy

    Carlo Cervellati, Monica Squerzanti, Carlo M. Bergamini & Vincenzo Lanzara

  2. Department of Experimental Medicine, Section of Chemistry and Structural Biochemistry, University of Parma, Parma, Italy

    Lorella Franzoni & Alberto Spisni

  3. Department of Applied Science for Complex Systems, Marche Polytechnic University, Ancona, Italy

    Francesco Spinozzi & Paolo Mariani

Authors
  1. Carlo Cervellati
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  2. Lorella Franzoni
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  3. Monica Squerzanti
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  4. Carlo M. Bergamini
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  5. Francesco Spinozzi
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  6. Paolo Mariani
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  7. Vincenzo Lanzara
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  8. Alberto Spisni
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Correspondence to Carlo M. Bergamini.

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Cervellati, C., Franzoni, L., Squerzanti, M. et al. Unfolding studies of tissue transglutaminase. Amino Acids 36, 633 (2009). https://doi.org/10.1007/s00726-008-0161-3

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  • DOI: https://doi.org/10.1007/s00726-008-0161-3

Keywords

  • Tissue transglutaminase
  • Denaturation
  • Guanidine hydrochloride
  • Thermal unfolding
  • Differential scanning calorimetry
  • Ligands