title: Identification of transglutaminase reactive residues in human osteopontin and their role in polymerization.
abstract: Osteopontin (OPN) is a highly posttranslationally modified protein present in several tissues where it is implicated in numerous physiological processes. OPN primarily exerts its functions through interaction with integrins via the Arg-Gly-Asp and Ser-Val-Val-Tyr-Gly-Leu-Arg sequences located in the N-terminal part of the protein. OPN can be polymerized by the cross-linking enzyme transglutaminase 2 (TG2), and polymerization has been shown to enhance the biological activity of OPN. However, little is known about the reactivity and location of the glutamine and lysine residues involved in the TG2-mediated modification of OPN. Here we show that TG2 catalyses the incorporation of 5-(Biotinamido)pentylamine at glutamines in both the N- and C-terminal parts of OPN, whereas TG2 primarily incorporated the glutamine-donor peptide biotinyl-TVQQEL-OH into the C-terminal part of OPN. By mass spectrometric analyses we identified Gln34, Gln42, Gln193 and Gln248 as the major TG2 reactive glutamines in OPN. The distribution of reactive Gln and Lys residues in OPN proved to be important, as the full-length protein but not the physiologically highly active integrin-binding N-terminal part of OPN were able to polymerize in a TG2-mediated reaction. Collectively, these data provide important new molecular knowledge about the mechanism of OPN polymerization.
authors.affiliation: Department of Molecular Biology and Genetics, Science Park, Aarhus University, Aarhus, Denmark.
authors.forename: Brian
authors.initials: B
authors.lastname: Christensen
authors.affiliation: Department of Molecular Biology and Genetics, Science Park, Aarhus University, Aarhus, Denmark.
authors.forename: Elias D
authors.initials: ED
authors.lastname: Zachariae
authors.affiliation: Department of Molecular Biology and Genetics, Science Park, Aarhus University, Aarhus, Denmark.
authors.forename: Carsten
authors.initials: C
authors.lastname: Scavenius
authors.affiliation: Department of Molecular Biology and Genetics, Science Park, Aarhus University, Aarhus, Denmark.
authors.forename: Morten
authors.initials: M
authors.lastname: Thybo
authors.affiliation: Department of Molecular Biology and Genetics, Science Park, Aarhus University, Aarhus, Denmark.
authors.forename: Morten M
authors.initials: MM
authors.lastname: Callesen
authors.affiliation: Department of Molecular Biology and Genetics, Science Park, Aarhus University, Aarhus, Denmark.
authors.forename: Søren
authors.initials: S
authors.lastname: Kløverpris
authors.affiliation: Department of Molecular Biology and Genetics, Science Park, Aarhus University, Aarhus, Denmark.
authors.forename: Claus
authors.initials: C
authors.lastname: Oxvig
authors.affiliation: Department of Molecular Biology and Genetics, Science Park, Aarhus University, Aarhus, Denmark; Interdisciplinary Nanoscience Center, Aarhus University, Aarhus, Denmark.
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Enghild
authors.affiliation: Department of Molecular Biology and Genetics, Science Park, Aarhus University, Aarhus, Denmark; Interdisciplinary Nanoscience Center, Aarhus University, Aarhus, Denmark.
authors.forename: Esben S
authors.initials: ES
authors.lastname: Sørensen
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005973:Glutamine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism*; D058105:Polymerization; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D011503:Transglutaminases / Q000378:metabolism*
chemical_list: D009842:Oligopeptides; D005973:Glutamine; D053495:Osteopontin; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins; D008239:Lysine