title: Cell surface adhesion of pregnancy-associated plasma protein-A is mediated by four clusters of basic residues located in its third and fourth CCP module.
abstract: The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) cleaves a subset of insulin-like growth factor binding proteins (IGFBP), which inhibit the activities of insulin-like growth factor (IGF). Through this proteolytic activity, PAPP-A is believed to regulate IGF bioavailability in several biological systems, including the human reproductive system and the cardiovascular system. PAPP-A adheres to mammalian cells by interactions with glycosaminoglycan (GAG), thus targeting the proteolytic activity of PAPP-A to the cell surface. Based on site-directed mutagenesis, we here delineate the PAPP-A GAG-binding site in the C-terminal modules CCP3 and CCP4. Using heparin affinity chromatography, commonly employed in such studies, we define three clusters of arginines and lysines of CCP3, which are important for the interaction of PAPP-A with heparin. In a model of PAPP-A CCP3-CCP4, basic residues of these sequence clusters form a contiguous patch located on one side of the structure. Binding to the unknown, natural cell surface receptor of PAPP-A, assessed by flow cytometry, also depends on residues of these three basic clusters. However, single or double residue substitutions generally have a modest effect on PAPP-A heparin binding assessed by chromatography, but cell surface adhesion was critically reduced by several of these substitutions, emphasizing the relevance of analysis by flow cytometry. The contributions of positively charged residues located in CCP4 were all minor when analyzed by heparin affinity chromatography. However, the mutation of CCP4 residues Arg1459 and Lys1460 to Ala almost abrogated cell surface adhesion. Furthermore, when acidic residues of the homologous proteinase PAPP-A2 (Asp1547, Glu1555 and Glu1567) were introduced into the corresponding positions in the sequence of PAPP-A, located in each of the three basic clusters of CCP3, binding to heparin was strongly impaired and cell surface binding was abrogated. This explains, at least in part, why PAPP-A2 lacks the ability of cell surface adhesion, and further emphasizes the role of the basic clusters defined in PAPP-A.
authors.affiliation: Department of Molecular Biology, Science Park, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Weyer
authors.forename: Michael T
authors.initials: MT
authors.lastname: Overgaard
authors.forename: Lisbeth S
authors.initials: LS
authors.lastname: Laursen
authors.forename: Claus G
authors.initials: CG
authors.lastname: Nielsen
authors.forename: Alexander
authors.initials: A
authors.lastname: Schmitz
authors.forename: Michael
authors.initials: M
authors.lastname: Christiansen
authors.forename: Lars
authors.initials: L
authors.lastname: Sottrup-Jensen
authors.forename: Linda C
authors.initials: LC
authors.lastname: Giudice
authors.forename: Claus
authors.initials: C
authors.lastname: Oxvig
mesh_terms: D000595:Amino Acid Sequence; D024342:Amino Acids, Acidic / Q000235:genetics / Q000378:metabolism; D024361:Amino Acids, Basic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002448:Cell Adhesion / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D005434:Flow Cytometry; D006025:Glycosaminoglycans / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011266:Pregnancy-Associated Plasma Protein-A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D024342:Amino Acids, Acidic; D024361:Amino Acids, Basic; D006025:Glycosaminoglycans; D021122:Protein Subunits; D011994:Recombinant Proteins; D006493:Heparin; D011266:Pregnancy-Associated Plasma Protein-A