title: Cell surface targeting of pregnancy-associated plasma protein A proteolytic activity. Reversible adhesion is mediated by two neighboring short consensus repeats.
abstract: The activities of insulin-like growth factor (IGF)-I and -II are regulated by IGF-binding proteins (IGFBPs). Cleavage of IGFBP-4 by the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) causes release of bound IGF and has been established in several biological systems including the human reproductive system. Using flow cytometry, we first demonstrate that PAPP-A reversibly binds to the cell surface of several cell types analyzed. Heparin and heparan sulfate, but not dermatan or chondroitin sulfate, effectively compete for PAPP-A surface binding, and because incubation of cells with heparinase abrogated PAPP-A adhesion, binding is probably mediated by a cell surface heparan sulfate proteoglycan. Furthermore, the proteolytic activity of PAPP-A is preserved while bound to cells, suggesting that adhesion functions to target its activity to the vicinity of the IGF receptor, decreasing the probability that released IGF is captured by another IGFBP molecule before receptor binding. This mechanism potentially functions in both autocrine and paracrine regulation, as PAPP-A need not be synthesized in a cell to which it adheres. A truncated PAPP-A variant without the five short consensus repeats in the C-terminal third of the 1547-residue PAPP-A subunit, lacked surface binding. We also show that PAPP-A2, a recently discovered IGFBP-5 proteinase with homology to PAPP-A, does not bind cells. This finding allowed further mapping of the PAPP-A adhesion site to short consensus repeat modules 3 and 4 by the expression and analysis of nine PAPP-A/PAPP-A2 chimeras. Interestingly, the proteolytically inactive, disulfide-bound complex of PAPP-A and the proform of eosinophil major basic protein (proMBP), PAPP-A.proMBP, shows only weak surface binding, probably because the adhesion site of PAPP-A is occupied by heparan sulfate, known to be covalently bound to proMBP. This hypothesis was further substantiated by demonstrating that heparinase treatment of PAPP-A.proMBP restores surface binding. We finally propose a model in which IGF bioactivity is regulated by reversible cell surface binding of PAPP-A, which in turn is regulated by proMBP.
authors.affiliation: Department of Molecular Biology, Science Park, University of Aarhus, Aarhus C, Denmark.
authors.forename: Lisbeth S
authors.initials: LS
authors.lastname: Laursen
authors.forename: Michael T
authors.initials: MT
authors.lastname: Overgaard
authors.forename: Kathrin
authors.initials: K
authors.lastname: Weyer
authors.forename: Henning B
authors.initials: HB
authors.lastname: Boldt
authors.forename: Peter
authors.initials: P
authors.lastname: Ebbesen
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: D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry* / Q000378:metabolism; D015153:Blotting, Western; D002448:Cell Adhesion; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D047091:Eosinophil Granule Proteins; D005434:Flow Cytometry; D006025:Glycosaminoglycans / Q000494:pharmacology; D006493:Heparin / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D008954:Models, Biological; D010957:Plasmids / Q000378:metabolism; D011266:Pregnancy-Associated Plasma Protein-A / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012260:Ribonucleases; D014162:Transfection
chemical_list: D001798:Blood Proteins; D003545:Cysteine; D018076:DNA, Complementary; D047091:Eosinophil Granule Proteins; D006025:Glycosaminoglycans; D006493:Heparin; D006497:Heparan Sulfate; D007334:Insulin-Like Growth Factor I; D011266:Pregnancy-Associated Plasma Protein-A; D012260:Ribonucleases