Acta Diabetologica

, Volume 50, Issue 1, pp 61–72 | Cite as

Transglutaminase 2 transamidation activity during first-phase insulin secretion: natural substrates in INS-1E

  • Lucia Russo
  • Claudia Marsella
  • Giovanni Nardo
  • Tania Massignan
  • Massimo Alessio
  • Emanuela Piermarini
  • Stefano La Rosa
  • Giovanna Finzi
  • Valentina Bonetto
  • Federico Bertuzzi
  • Pierre Maechler
  • Ornella MassaEmail author
Original Article


Transglutaminase 2 (TG2) is a multifunctional protein with Ca2+-dependent transamidating and G protein activity. Previously, we reported that tgm2 −/− mice have an impaired insulin secretion and that naturally occurring TG2 mutations associated with familial, early-onset type 2 diabetes, show a defective transamidating activity. Aim of this study was to get a better insight into the role of TG2 in insulin secretion by identifying substrates of TG2 transamidating activity in the pancreatic beta cell line INS-1E. To this end, we labeled INS-1E that are capable of secreting insulin upon glucose stimulation in the physiologic range, with an artificial acyl acceptor (biotinamido-pentylamine) or donor (biotinylated peptide), in basal condition and after stimulus with glucose for 2, 5, and 8 min. Biotinylated proteins were analyzed by two-dimensional electrophoresis and mass spectrometry. In addition, subcellular localization of TG2 in human endocrine pancreas was studied by electron microscopy. Among several TG2’s transamidating substrates in INS-1E, mass spectrometry identified cytoplasmic actin (a result confirmed in human pancreatic islet), tropomyosin, and molecules that participate in insulin granule structure (e.g., GAPDH), glucose metabolism, or [Ca2+] sensing (e.g., calreticulin). Physical interaction between TG2 and cytoplasmic actin during glucose-stimulated first-phase insulin secretion was confirmed by co-immunoprecipitation. Electron microscopy revealed that TG2 is localized close to insulin and glucagon granules in human pancreatic islet. We propose that TG2’s role in insulin secretion may involve cytoplasmic actin remodeling and may have a regulative action on other proteins during granule movement. A similar role of TG2 in glucagon secretion is also suggested.


Transglutaminase 2 Insulin secretion Calcium β-Cell INS-1E Human islet 



Part of this work has been supported by the Telethon grants GGP09147 to Ornella Massa and by Swiss National Science Foundation to Pierre Maechler. Authors are grateful to Prof. F. Barbetti for his continuous support and helpful contribution to discussion, to Prof. C. Capella (Department of Human Morphology and Department of Pathology, Ospedale di Circolo, Varese, Italy), to Prof. M. Piacentini (Department of Biology, University of Rome “Tor Vergata”, Rome, Italy), Dr. G.M. Fimia and Dr. F. Ciccosanti (National Institute for Infectious Diseases, IRCCS “L. Spallanzani”, Rome, Italy) for MALDI TOF experiments on A25 labeled substrates and to Dr. C. Placidi (Department of Human Morphology, University of Insubria and Department of Pathology, Ospedale di Circolo, Varese, Italy) for helping with human pancreas microscopy.

Conflicts of interest

No potential conflicts of interest relevant to this article were reported.

Supplementary material

592_2012_381_MOESM1_ESM.pdf (5.8 mb)
Supplementary material 1 (PDF 5918 kb)


  1. 1.
    Lorand L, Graham RM (2003) Transglutaminases: crosslinking enzymes with pleiotropic functions. Natl Rev Mol Cell Biol 4:140–156CrossRefGoogle Scholar
  2. 2.
    Fesus L, Piacentini M (2002) Transglutaminase 2: an enigmatic enzyme with diverse functions. Trends Biochem Sci 27:534–539PubMedCrossRefGoogle Scholar
  3. 3.
    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 264:1593–1596PubMedCrossRefGoogle Scholar
  4. 4.
    Hasegawa G, Suwa M, Ichikawa Y, Ohtsuka T, Kumagai S, Kikuchi M, Sato Y, Sato Y (2003) A novel function of tissue—type transglutaminase: protein disulfide isomerase. Biochem J 373:793–803PubMedCrossRefGoogle Scholar
  5. 5.
    Mishra S, Murphy LJ (2004) Tissue transglutaminase has intrinsic kinase activity: identification of transglutaminase 2 as an insulin-like growth factor-binding protein-3 kinase. J Biol Chem 279:23863–23868PubMedCrossRefGoogle Scholar
  6. 6.
    Griffin M, Casadio R, Bergamini CM (2002) Transglutaminases: nature’s biological glues. Biochem J 368:377–396PubMedCrossRefGoogle Scholar
  7. 7.
    De Laurenzi V, Melino G (2001) Gene disruption of tissue transglutaminase. Mol Cell Biol 21:148–155PubMedCrossRefGoogle Scholar
  8. 8.
    Nanda N, Iismaa SE, Owens WA, Husain A, Mackay F, Graham RM (2001) Targeted inactivation Gh/tissue transglutaminase II. J Biol Chem 276:20673–20678PubMedCrossRefGoogle Scholar
  9. 9.
    Bernassola F, Federici M, Corazzari M, Terrinoni A, Hribal ML, De Laurenzi V, Ranalli M, Massa O, Sesti G, McLean WHI, Citro G, Barbetti F, Melino G (2002) Role of transglutaminase 2 in glucose tolerance: knockout mice studies and a putative mutation in a MODY patient. FASEB J 16:1278–1371CrossRefGoogle Scholar
  10. 10.
    Weyer C, Bogardus C, Mott DM, Pratley RE (1999) The natural history of insulin secretory dysfunction and insulin resistance in the pathogenesis of type 2 diabetes mellitus. J Clin Invest 104:787–794PubMedCrossRefGoogle Scholar
  11. 11.
    Gallwitz B, Kazda C, Kraus P, Nicolay C, Schernthaner G (2011) Contribution of insulin deficiency and insulin resistance to the development of type 2 diabetes: nature of early stage diabetes. Acta Diabetol. doi: 10.1007/s00592-011-0319-4
  12. 12.
    Porzio O, Massa O, Cunsolo V, Colombo C, Malaponti M, Bertuzzi F, Hansen T, Johansen A, Pedersen O, Meschi F, Terrinoni A, Melino G, Federici M, Decarlo N, Menicagli M, Campani D, Marchetti P, Ferdaoussi M, Froguel P, Federici G, Vaxillaire M, Barbetti F (2007) Missense mutations in the TGM2 gene encoding transglutaminase 2 are found in patients with early-onset type 2 diabetes. Hum Mutat 28:1150PubMedCrossRefGoogle Scholar
  13. 13.
    Pinkas DM, Strop P, Brunger AT, Khosla C (2007) Transglutaminase 2 undergoes a large conformational change upon activation. PLoS Biol 5:e327PubMedCrossRefGoogle Scholar
  14. 14.
    Bungay PJ, Owen RA, Coutts IC, Griffin M (1986) A role for transglutaminase in glucose-stimulated insulin release from the pancreatic beta-cell. Biochem J 235:269–278PubMedGoogle Scholar
  15. 15.
    Sener A, Dunlop ME, Gomis R, Mathias PC, Malaisse-Lagae F, Malaisse WJ (1985) Role of transglutaminase in insulin release. Study with glycine and sarcosine methylesters. Endocrinology 117:237–242PubMedCrossRefGoogle Scholar
  16. 16.
    Kibbey RG, Pongratz RL, Romanelli AJ, Wollheim CB, Cline GW, Shulman GI (2007) Mitochondrial GTP regulates glucose-stimulated insulin secretion. Cell Metab 5:253–264PubMedCrossRefGoogle Scholar
  17. 17.
    Merglen A, Theander S, Rubi B, Chaffard G, Wollheim CB, Maechler P (2004) Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells. Endocrinology 145:667–678PubMedCrossRefGoogle Scholar
  18. 18.
    Ricordi C, Lacy PE, Finke EH, Olack BJ, Scharp DW (1988) Automated method for isolation of human pancreatic islets. Diabetes 37:413–420PubMedCrossRefGoogle Scholar
  19. 19.
    Vargas F, Vives-Pi M, Somoza N, Alcalde L, Armengol P, Marti M, Serradell L, Costa M, Fernandez-Llamazares J, Sanmarti A, Pujol-Borrell R (1996) Advantages of using a cell separator and metrizamide gradients for human islet purification. Transplantation 61:1562–1566PubMedCrossRefGoogle Scholar
  20. 20.
    Orrù S, Caputo I, D’Amato A, Ruoppolo M, Esposito C (2003) Proteomics identification of acyl-acceptor and acyl-donor substrates for transglutaminase in a human intestinal epithelial cell line. Implication for celiac desease. J Biol Chem 278:31766–31773PubMedCrossRefGoogle Scholar
  21. 21.
    Shigeto M, Katsura M, Matsuda M, Ohkuma S, Kaku K (2006) First phase of glucose-stimulated insulin secretion from MIN 6 cells does not always require extracellular calcium influx. J Pharmacol Sci 101:293–302PubMedCrossRefGoogle Scholar
  22. 22.
    Conti A, Ricchiuto P, Iannaccone S, Sferrazza B, Cattaneo A, Bachi A, Reggiani A, Beltramo M, Alessio M (2005) Pigment epithelium-derived factor is differentially expressed in peripheral neuropathies. Proteomics 5:4558–4567PubMedCrossRefGoogle Scholar
  23. 23.
    Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri GM, Caremolla B, Orecchia P, Zardi L, Righetti PG (2004) Blue silver: a very sensitive colloidal coomassie G-250 staining for proteome analysis. Electrophoresis 25:1327–1333PubMedCrossRefGoogle Scholar
  24. 24.
    Casoni F, Basso M, Massignan T, Gianazza E, Cheroni C, Salmona M, Bendotti C, Bonetto V (2005) Protein nitration in a mouse model of familial amyotrophic lateral sclerosis: possible multifunctional role in the pathogenesis. J Biol Chem 280:16295–16304PubMedCrossRefGoogle Scholar
  25. 25.
    Pappin DJ, Hojrup P, Bleasby AJ (1993) Rapid identification of proteins by peptide-mass fingerprinting. Curr Biol 3:327–332PubMedCrossRefGoogle Scholar
  26. 26.
    Kiraly R, Csosz E, Kurtan T, Antis S, Szigeti K, Simon-Vecsei Z, Korponay-Szabo IR, Keresztessy Z, Fesus L (2009) Functional significance of five noncanonical Ca2+-binding sites of human transglutaminase 2 characterized by site-directed mutagenesis. FEBS J 276:7083–7096PubMedCrossRefGoogle Scholar
  27. 27.
    Lai TS, Slaughter TS, Peoples KA, Hettasch JM, Greenberg CS (1998) Regulation of human tissue transglutaminase function by magnesium-nucleotide complexes. Identification of distinct binding sites for Mg-GTP and Mg-ATP. J Biol Chem 273:1776–1781PubMedCrossRefGoogle Scholar
  28. 28.
    Selway JL, Moore CE, Mistry R, R. A. John Challiss, Herbert TP (2011) Molecular mechanisms of muscarinic acetylcholine receptor–stimulated increase in cytosolic free Ca2+ concentration and ERK1/2 activation in the MIN6 pancreatic β-cell line. Acta Diabetol. doi: 10.1007/s00592-011-0314-9
  29. 29.
    Brunner Y, Couté Y, Iezzi M, Foti M, Fukuda M, Hochstrasser DF, Wollheim CB, Sanchez JC (2007) Proteomics analysis of insulin secretory granules. Mol Cell Proteom 6:1007–1017CrossRefGoogle Scholar
  30. 30.
    Suckale J, Solimena M (2010) The insulin secretory granule as a signaling hub. Trends Endocrinol Metab 21:599–609PubMedCrossRefGoogle Scholar
  31. 31.
    D’Hertog W, Overbergh L, Lage K, Ferriera GB, Maris M, Gysemans C, Flamez D, Kupper Cardozo A, Van den Bergh G, Schoofs L, Lut Arckens L, Moreau Y, Hansen D, Eizirik DL, Waelkens E, Mathieu C (2007) Proteomics analysis of cytokine-induced dysfunction and death in insulin-producing INS-1E cells. New insights into the pathways involved. Mol Cell Proteom 6:2180–2199CrossRefGoogle Scholar
  32. 32.
    La Rosa S, Lloyd RV, Capella C (2005) Expression and role of myosins in pancreatic endocrine cells and related tumors. In: Trends in Pancreatic Cancer Research. Maxwell A. Loft, ed. Nova Science Publishers, Inc., New York, p 23–39Google Scholar
  33. 33.
    Tomas A, Yermen B, Min L, Pessin JE, Halban PA (2006) Regulation of pancreatic beta-cell insulin secretion by actin cytoskeleton remodelling: role of gelsolin and cooperation with the MAPK signalling pathway. J Cell Sci 119(Pt 10):2156–2167PubMedCrossRefGoogle Scholar
  34. 34.
    Hegyi G, Michel H, Shabanowitz J, Hunt DF, Chatterjie N, Healy-Louie G, Elzinga M (1992) Gln-41 is intermolecularly cross-linked to Lys-113 in F-actin by N-(4-azidobenzoyl)-putrescine. Protein Sci 1:132–144PubMedCrossRefGoogle Scholar
  35. 35.
    Hegyi G, Belagyi J (2006) Intermonomer cross-linking of F-actin alters the dynamics of its interaction with H-meromyosin in the weak-binding state. FEBS J 873:1896–1905CrossRefGoogle Scholar
  36. 36.
    Eligula L, Chuang L, Phillips ML, Motoki M, Seguro K, Muhlrad A (1998) Transglutaminase-induced cross-linking between subdomain 2 of G-Actin and the 636–642 Lysine-rich loop of myosin subfragment 1. Biophys J 74:953–963PubMedCrossRefGoogle Scholar
  37. 37.
    Nemes Z Jr, Adány R, Balázs M, Boross P, Fésüs L (1997) Identification of cytoplasmic actin as an abundant glutaminyl substrate for tissue transglutaminase in HL-60 and U937 cells undergoing apoptosis. J Biol Chem 272:20577–20583PubMedCrossRefGoogle Scholar
  38. 38.
    Feng JF, Readon M, Yadav SP, Im MJ (1999) Calreticulin down-regulates both GTP binding and transglutaminase activities of transglutaminase II. Biochemistry 38:10743–10749PubMedCrossRefGoogle Scholar
  39. 39.
    Frickel EM, Riek R, Jelesaroy I, Helenius A, Wuthrich K, Ellgaard L (2002) TROSY-NMR reveals interaction between ERp57 and the tip of the calreticulin P-domain. Proc Natl Acad Sci USA 99:1954–1959PubMedCrossRefGoogle Scholar
  40. 40.
    Orrù S, Ruoppolo M, Francese S, Vitagliano L, Marino G, Esposito C (2002) Identification of tissue transglutaminase-reactive lesine residues in glyceraldehyde-3-phosphate dehydrogenase. Protein Sci 11:137–146PubMedCrossRefGoogle Scholar
  41. 41.
    Li C, Ullrich B, Zhang JZ, Anderson RGW, Brose N, Sudhof TC (1995) Ca2+ -dependent and -independent activities of neural and non-neural synaptotagmins. Nature 375:594–599PubMedCrossRefGoogle Scholar
  42. 42.
    Zhang L, Lai E, Teodoro T, Volchuk A (2009) GRP78, but not protein-disulfide isomerase, partially reverses hyperglycemia-induced inhibition of insulin synthesis and secretion in pancreatic β-cells. J Biol Chem 284:5289–5298PubMedCrossRefGoogle Scholar
  43. 43.
    Wilhelmus MMM, Verhaar R, Andringa G, Bol JGJ, Cras P, Shan L, Hoozemans JJM, Drukarch B (2011) Presence of tissue transglutaminase in granular endoplasmic reticulum is characteristic of melanized neurons in Parkinson’s disease brain. Brain Pathol 21:130–139PubMedCrossRefGoogle Scholar
  44. 44.
    Fraij BM, Gonzales RA (1996) A third human tissue transglutaminase homologue as a result of alternative gene transcripts. Biochim Biophys Acta 1306:63–74PubMedCrossRefGoogle Scholar
  45. 45.
    Monsonego A, Shani Y, Friedmann I, Paas Y, Eizenberg O, Schwartz M (1997) Expression of GTP-independent tissue type transglutaminase in cytokine—treated rat brain astrocytes. J Biol Chem 272:3724–3732PubMedCrossRefGoogle Scholar
  46. 46.
    Bowling P, O’Driscoll L, O’Sullivan F, Dowd A, Henry M, Jeppesen PB, Meleady P, Clynes M (2006) Proteomic screening of glucose-responsive and glucose non-responsive MIN-6 beta cells reveals differential expression of proteins involved in protein folding, secretion and oxidative stress. Proteomics 6:6578–6587CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Lucia Russo
    • 1
  • Claudia Marsella
    • 1
  • Giovanni Nardo
    • 2
  • Tania Massignan
    • 2
  • Massimo Alessio
    • 3
  • Emanuela Piermarini
    • 1
  • Stefano La Rosa
    • 4
    • 5
  • Giovanna Finzi
    • 4
    • 5
  • Valentina Bonetto
    • 2
  • Federico Bertuzzi
    • 6
  • Pierre Maechler
    • 7
  • Ornella Massa
    • 1
    Email author
  1. 1.Laboratory of Mendelian DiabetesBambino Gesù Children’s Hospital, Research InstituteRomeItaly
  2. 2.Dulbecco Telethon Institute, Mario Negri Institute for Pharmacological ResearchMilanItaly
  3. 3.DIBIT, San Raffaele Scientific InstituteMilanItaly
  4. 4.Department of Human MorphologyUniversity of InsubriaVareseItaly
  5. 5.Department of PathologyOspedale di CircoloVareseItaly
  6. 6.S.S.D. Diabetologia, Ca’ Granda Niguarda HospitalMilanItaly
  7. 7.Department of Cell Physiology and MetabolismGeneva University Medical CentreGeneva 4Switzerland

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