title: The endothelial cell binding site for advanced glycation end products consists of a complex: an integral membrane protein and a lactoferrin-like polypeptide.
abstract: Advanced glycation end products (AGEs), formed as the result of the extended interaction of proteins with ketoses, modulate central properties of endothelial cells and mononuclear phagocytes by interacting with a cell surface binding site comprised of a novel integral membrane protein (receptor for AGE = RAGE) and a lactoferrin-like polypeptide (LF-L), the latter having sequence identity to lactoferrin (LF). To further understand this cellular binding site, the interaction of RAGE with LF-L and LF was characterized. By ligand blotting and a solid state competitive binding assay, 125I-LF-L and 125I-LF bound to RAGE immobilized on nitrocellulose membranes or polypropylene tubes in a time-dependent and reversible manner, demonstrating a high affinity component with Kd approximately 100 pM. The interaction of 125I-LF-L and 125I-LF with RAGE was independent of iron in LF and was competed by addition of an excess of unlabeled carboxyl-terminal portion of LF. Cross-linking studies with purified 125I-LF-L and RAGE, in the presence of disuccinimidyl suberate, showed a new, slowly migrating band, corresponding to a complex of RAGE and LF-L, and cross-linking on mouse aortic endothelial cells showed two new slowly migrating bands on immunoblotting visualized with both anti-RAGE IgG and anti-LF-L IgG. These data lead us to propose that the endothelial cell surface binding site for AGEs consists of LF-L bound noncovalently to RAGE anchored in the cell membrane.
authors.affiliation: Department of Physiology, Columbia University, College of Physicians and Surgeons, New York, New York 10032.
authors.forename: A M
authors.initials: AM
authors.lastname: Schmidt
authors.forename: R
authors.initials: R
authors.lastname: Mora
authors.forename: R
authors.initials: R
authors.lastname: Cao
authors.forename: S D
authors.initials: SD
authors.lastname: Yan
authors.forename: J
authors.initials: J
authors.lastname: Brett
authors.forename: R
authors.initials: R
authors.lastname: Ramakrishnan
authors.forename: T C
authors.initials: TC
authors.lastname: Tsang
authors.forename: M
authors.initials: M
authors.lastname: Simionescu
authors.forename: D
authors.initials: D
authors.lastname: Stern
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000502:physiology; D017127:Glycation End Products, Advanced / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D007781:Lactoferrin / Q000302:isolation & purification / Q000378:metabolism*; D008168:Lung / Q000378:metabolism; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010586:Phagocytes / Q000502:physiology; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D017127:Glycation End Products, Advanced; D008565:Membrane Proteins; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; D007781:Lactoferrin