title: Dissecting the interaction between transglutaminase 2 and fibronectin.
abstract: In the extracellular environment, the enzyme transglutaminase 2 (TG2) is involved in cell-matrix interactions through association with the extracellular matrix protein, fibronectin (FN). The 45 kDa gelatin-binding domain of FN (45FN) is responsible for the binding to TG2. Previous studies have demonstrated that the FN-binding site of TG2 is located in the N-terminal domain of the enzyme although with conflicting results regarding the specific residues involved. Here we have mapped the FN interaction site of human TG2 by use of hydrogen/deuterium exchange coupled with mass spectrometry, and we confirm that the FN-binding site is located in the N-terminal domain of TG2. Furthermore, by combination of site-directed mutagenesis and surface plasmon resonance analysis we have identified the TG2 residues K30, R116 and H134 as crucial to maintain the high affinity interaction with FN. Mutation of all three residues simultaneously reduced binding to 45FN by more than 2000-fold. We also identified residues in the catalytic core domain of TG2 that contributed to FN binding, hence extending the binding interface between TG2 and FN. This study provides new insights into the high affinity interaction between TG2 and FN.
authors.affiliation: Centre for Immune Regulation and Department of Immunology, University of Oslo, Oslo University Hospital, Oslo, Norway. i.d.r.cardoso@medisin.uio.no.
authors.forename: Inês
authors.identifier: 0000-0002-2569-2336
authors.initials: I
authors.lastname: Cardoso
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.
authors.forename: Eva Christina
authors.initials: EC
authors.lastname: Østerlund
authors.affiliation: Centre for Immune Regulation and Department of Immunology, University of Oslo, Oslo University Hospital, Oslo, Norway.
authors.forename: Jorunn
authors.initials: J
authors.lastname: Stamnaes
authors.affiliation: Centre for Immune Regulation and Department of Immunology, University of Oslo, Oslo University Hospital, Oslo, Norway.
authors.forename: Rasmus
authors.initials: R
authors.lastname: Iversen
authors.affiliation: Centre for Immune Regulation and Department of Immunology, University of Oslo, Oslo University Hospital, Oslo, Norway.|Department of Biosciences, University of Oslo, Oslo, Norway.
authors.forename: Jan Terje
authors.initials: JT
authors.lastname: Andersen
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.
authors.forename: Thomas J D
authors.initials: TJ
authors.lastname: Jørgensen
authors.affiliation: Centre for Immune Regulation and Department of Immunology, University of Oslo, Oslo University Hospital, Oslo, Norway.
authors.forename: Ludvig M
authors.initials: LM
authors.lastname: Sollid
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000302:isolation & purification; D020134:Catalytic Domain; D003001:Cloning, Molecular; D041961:Deuterium Exchange Measurement; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D011503:Transglutaminases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000906:Antibodies; D005353:Fibronectins; D011994:Recombinant Proteins; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins
keywords: Fibronectin; Hydrogen/deuterium exchange; Site-directed mutagenesis; Surface plasmon resonance; Transglutaminase 2