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

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Abstract

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.

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Acknowledgments

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.

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Authors and Affiliations

  1. Laboratory of Mendelian Diabetes, Bambino Gesù Children’s Hospital, Research Institute, c/o Padiglione Giovanni Paolo II (-2), Piazza Sant’Onofrio 4, 00165, Rome, Italy

    Lucia Russo, Claudia Marsella, Emanuela Piermarini & Ornella Massa

  2. Dulbecco Telethon Institute, Mario Negri Institute for Pharmacological Research, Milan, Italy

    Giovanni Nardo, Tania Massignan & Valentina Bonetto

  3. DIBIT, San Raffaele Scientific Institute, Milan, Italy

    Massimo Alessio

  4. Department of Human Morphology, University of Insubria, Varese, Italy

    Stefano La Rosa & Giovanna Finzi

  5. Department of Pathology, Ospedale di Circolo, Varese, Italy

    Stefano La Rosa & Giovanna Finzi

  6. S.S.D. Diabetologia, Ca’ Granda Niguarda Hospital, Milan, Italy

    Federico Bertuzzi

  7. Department of Cell Physiology and Metabolism, Geneva University Medical Centre, Geneva 4, Switzerland

    Pierre Maechler

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  1. Lucia Russo
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  2. Claudia Marsella
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  3. Giovanni Nardo
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  4. Tania Massignan
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  12. Ornella Massa
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Correspondence to Ornella Massa.

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Communicated by Massimo Federici.

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Russo, L., Marsella, C., Nardo, G. et al. Transglutaminase 2 transamidation activity during first-phase insulin secretion: natural substrates in INS-1E. Acta Diabetol 50, 61–72 (2013). https://doi.org/10.1007/s00592-012-0381-6

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