title: Transglutaminase 2 transamidation activity during first-phase insulin secretion: natural substrates in INS-1E.
abstract: Transglutaminase 2 (TG2) is a multifunctional protein with Ca(2+)-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 [Ca(2+)] 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.
authors.affiliation: Laboratory of Mendelian Diabetes, Bambino Gesù Children's Hospital, Research Institute, Rome, Italy.
authors.forename: Lucia
authors.initials: L
authors.lastname: Russo
authors.forename: Claudia
authors.initials: C
authors.lastname: Marsella
authors.forename: Giovanni
authors.initials: G
authors.lastname: Nardo
authors.forename: Tania
authors.initials: T
authors.lastname: Massignan
authors.forename: Massimo
authors.initials: M
authors.lastname: Alessio
authors.forename: Emanuela
authors.initials: E
authors.lastname: Piermarini
authors.forename: Stefano
authors.initials: S
authors.lastname: La Rosa
authors.forename: Giovanna
authors.initials: G
authors.lastname: Finzi
authors.forename: Valentina
authors.initials: V
authors.lastname: Bonetto
authors.forename: Federico
authors.initials: F
authors.lastname: Bertuzzi
authors.forename: Pierre
authors.initials: P
authors.lastname: Maechler
authors.forename: Ornella
authors.initials: O
authors.lastname: Massa
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D051381:Rats; D013379:Substrate Specificity; D011503:Transglutaminases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D007328:Insulin; C000717608:TGM2 protein, human; C000717609:Tgm2 protein, rat; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins; D005947:Glucose