title: Transglutaminase 2-Catalyzed Intramolecular Cross-Linking of Osteopontin.
abstract: Osteopontin (OPN) is a multifunctional integrin-binding protein present in several tissues and body fluids. OPN is a substrate for the enzyme transglutaminase 2 (TG2), which catalyzes inter- and intramolecular cross-linking affecting the biological activity of the protein. Polymerization of OPN by intermolecular cross-linking has mostly been studied using relatively high TG2 concentrations, whereas the effect of lower concentrations of TG2 has remained unexplored. Here we show that TG2 at physiologically relevant concentrations predominantly catalyzes the formation of intramolecular cross-links in OPN. By site-directed mutagenesis and mass spectrometry, we demonstrate that Gln(42) and Gln(193) serve as the primary amine acceptor sites for isopeptide bond formation. We find that Gln(42) predominantly is linked to Lys(4) and that Gln(193) participates in a cross-link with Lys(154), Lys(157), or Lys(231). The formation of specific isopeptide bonds was not dependent on OPN phosphorylation, and similar patterns of cross-linking were observed in human and mouse OPN. Furthermore, we find that OPN purified from human urine contains the Lys(154)-Gln(193) isopeptide bond, indicating that intramolecular cross-linking of OPN occurs in vivo. Collectively, these data suggest that specific intramolecular cross-linking in the N- and C-terminal parts of OPN is most likely the dominant step in TG2-catalyzed modification of OPN.
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University , 8000 Aarhus C, Denmark.
authors.forename: Brian
authors.initials: B
authors.lastname: Christensen
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University , 8000 Aarhus C, Denmark.
authors.forename: Elias D
authors.initials: ED
authors.lastname: Zachariae
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University , 8000 Aarhus C, Denmark.
authors.forename: Carsten
authors.initials: C
authors.lastname: Scavenius
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University , 8000 Aarhus C, Denmark.
authors.forename: Søren
authors.initials: S
authors.lastname: Kløverpris
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University , 8000 Aarhus C, Denmark.
authors.forename: Claus
authors.initials: C
authors.lastname: Oxvig
authors.affiliation: Department of Biomedicine, Aarhus University , 8000 Aarhus C, Denmark.
authors.forename: Steen V
authors.initials: SV
authors.lastname: Petersen
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University , 8000 Aarhus C, Denmark.|Interdisciplinary Nanoscience Center, Aarhus University , 8000 Aarhus C, Denmark.
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Enghild
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University , 8000 Aarhus C, Denmark.|Interdisciplinary Nanoscience Center, Aarhus University , 8000 Aarhus C, Denmark.
authors.forename: Esben S
authors.initials: ES
authors.lastname: Sørensen
mesh_terms: D000595:Amino Acid Sequence; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D053495:Osteopontin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000652:urine; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D011503:Transglutaminases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D053495:Osteopontin; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins