title: Functional analyses of two cellular binding domains of bovine lactadherin.
abstract: The glycoprotein bovine lactadherin (formerly known as PAS-6/7) comprises two EGF-like domains and two C-like domains found in blood clotting factors V and VIII. Bovine lactadherin binds to alpha(v)beta(5) integrin in an RGD-dependent manner and also to phospholipids, especially phosphatidyl serine. To define and characterize these bindings the interactions between lactadherin and different mammalian cell types were investigated. Using recombinant forms of bovine lactadherin, the human breast carcinomas MCF-7 cells expressing the alpha(v)beta(5) integrin receptor were shown to bind specifically to RGD containing lactadherin but not to a mutated RGE lactadherin. A monoclonal antibody against the alpha(v)beta(5) integrin receptor and a synthetic RGD-containing peptide inhibited the adhesion of MCF-7 cells to lactadherin. Green monkey kidney MA-104 cells, also expressing the alpha(v)beta(3) together with the alpha(v)beta(5) integrin, showed binding to bovine lactadherin via both integrins. To investigate the interaction of lipid with lactadherin two fragments were expressed corresponding to the C1C2 domains and the C2 domain. Both fragments bound to phosphatidyl serine in a concentration-dependent manner with an affinity similar to native lactadherin (K(d) = 1.8 nM). A peptide corresponding to the C-terminal part of the C2 domain inhibited the binding of lactadherin to phospholipid in a concentration-dependent manner, and finally it was shown that lactadherin mediates binding between artificial phosphatidyl serine membranes and MCF-7 cells. Taken together these results show that lactadherin can act as link between two surfaces by binding to integrin receptors through its N-terminus and to phospholipids through its C-terminus.
authors.affiliation: Protein Chemistry Laboratory, Department of Molecular and Structural Biology, University of Aarhus, Science Park, Gustav Wieds Vej 10C, 8000 Aarhus C, Denmark.
authors.forename: M H
authors.initials: MH
authors.lastname: Andersen
authors.forename: H
authors.initials: H
authors.lastname: Graversen
authors.forename: S N
authors.initials: SN
authors.lastname: Fedosov
authors.forename: T E
authors.initials: TE
authors.lastname: Petersen
authors.forename: J T
authors.initials: JT
authors.lastname: Rasmussen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000954:Antigens, Surface / Q000235:genetics / Q000378:metabolism*; D001666:Binding Sites, Antibody; D001667:Binding, Competitive / Q000276:immunology; D001943:Breast Neoplasms; D002417:Cattle; D002448:Cell Adhesion / Q000235:genetics / Q000276:immunology; D006801:Humans; D016023:Integrins / Q000276:immunology; D008894:Milk Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D010446:Peptide Fragments / Q000502:physiology; D010718:Phosphatidylserines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology; D017434:Protein Structure, Tertiary / Q000235:genetics; D019030:Receptors, Vitronectin / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D016023:Integrins; C098942:MFGE8 protein, human; D008894:Milk Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D010718:Phosphatidylserines; D010743:Phospholipids; D019030:Receptors, Vitronectin; D011994:Recombinant Proteins; C064660:integrin alphaVbeta5; C047981:arginyl-glycyl-aspartic acid