title: Monoclonal antibodies detect receptor-induced binding sites in Glu-plasminogen.
abstract: When Glu-plasminogen binds to cells, its activation to plasmin is markedly enhanced compared with the reaction in solution, suggesting that Glu-plasminogen on cell surfaces adopts a conformation distinct from that in solution. However, direct evidence for such conformational changes has not been obtained. Therefore, we developed anti-plasminogen mAbs to test the hypothesis that Glu-plasminogen undergoes conformational changes on its interaction with cells. Six anti-plasminogen mAbs (recognizing 3 distinct epitopes) that preferentially recognized receptor-induced binding sites (RIBS) in Glu-plasminogen were obtained. The mAbs also preferentially recognized Glu-plasminogen bound to the C-terminal peptide of the plasminogen receptor, Plg-R(KT), and to fibrin, plasmin-treated fibrinogen, and Matrigel. We used trypsin proteolysis, immunoaffinity chromatography, and tandem mass spectrometry and identified Glu-plasminogen sequences containing epitopes recognized by the anti-plasminogen-RIBS mAbs: a linear epitope within a domain linking kringles 1 and 2; a nonlinear epitope contained within the kringle 5 domain and the latent protease domain; and a nonlinear epitope contained within the N-terminal peptide of Glu-plasminogen and the latent protease domain. Our results identify neoepitopes latent in soluble Glu-plasminogen that become available when Glu-plasminogen binds to cells and demonstrate that binding of Glu-plasminogen to cells induces a conformational change in Glu-plasminogen distinct from that of Lys-Pg.
authors.affiliation: Departments of Cell Biology, The Scripps Research Institute, La Jolla, CA, USA.
authors.forename: Jaena
authors.initials: J
authors.lastname: Han
authors.forename: Nagyung
authors.initials: N
authors.lastname: Baik
authors.forename: Kee-Hwan
authors.initials: KH
authors.lastname: Kim
authors.forename: Jian-Ming
authors.initials: JM
authors.lastname: Yang
authors.forename: Gye Won
authors.initials: GW
authors.lastname: Han
authors.forename: Yun
authors.initials: Y
authors.lastname: Gong
authors.forename: Mercè
authors.initials: M
authors.lastname: Jardí
authors.forename: Francis J
authors.initials: FJ
authors.lastname: Castellino
authors.forename: Jordi
authors.initials: J
authors.lastname: Felez
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Parmer
authors.forename: Lindsey A
authors.initials: LA
authors.lastname: Miles
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D003094:Collagen / Q000276:immunology / Q000378:metabolism; D004338:Drug Combinations; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology / Q000378:metabolism*; D005337:Fibrin / Q000276:immunology / Q000378:metabolism; D005340:Fibrinogen / Q000276:immunology / Q000378:metabolism; D006801:Humans; D018082:Kringles; D007797:Laminin / Q000276:immunology / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D010958:Plasminogen / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011509:Proteoglycans / Q000276:immunology / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D012995:Solubility; D053719:Tandem Mass Spectrometry; D020298:U937 Cells
chemical_list: D000911:Antibodies, Monoclonal; D004338:Drug Combinations; D000939:Epitopes; D007797:Laminin; C548063:PLGRKT protein, human; D010446:Peptide Fragments; D011509:Proteoglycans; D011956:Receptors, Cell Surface; C059485:matrigel; D005337:Fibrin; D005340:Fibrinogen; D010958:Plasminogen; D003094:Collagen