The endothelial cell binding site for advanced glycation end products consists of a complex: an integral membrane protein and a lactoferrin-like polypeptide.
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.
Schmidt
A M
AM
Department of Physiology, Columbia University, College of Physicians and Surgeons, New York, New York 10032.
Mora
R
R
Cao
R
R
Yan
S D
SD
Brett
J
J
Ramakrishnan
R
R
Tsang
T C
TC
Simionescu
M
M
Stern
D
D
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*
D017127:Glycation End Products, Advanced; D008565:Membrane Proteins; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; D007781:Lactoferrin