Abstract
Advanced glycosylation end products of proteins (AGEs) are nonenzymatically glycosylated proteins which accumulate in vascular tissue in aging and at an accelerated rate in diabetes. A approximately 35-kDa polypeptide with a unique NH2-terminal sequence has been isolated from bovine lung and found to be present on the surface of endothelial cells where it mediates the binding of AGEs (receptor for advanced glycosylation end product or RAGE). Using an oligonucleotide probe based on the amino-terminal sequence of RAGE, an apparently full-length cDNA of 1.5 kilobases was isolated from a bovine lung cDNA library. This cDNA encoded a 394 amino acid mature protein comprised of the following putative domains: an extracellular domain of 332 amino acids, a single hydrophobic membrane spanning domain of 19 amino acids, and a carboxyl-terminal domain of 43 amino acids. A partial clone encoding the human counterpart of RAGE, isolated from a human lung library, was found to be approximately 90% homologous to the bovine molecule. Based on computer analysis of the amino acid sequence of RAGE and comparison with databases, RAGE is a new member of the immunoglobulin superfamily of cell surface molecules and shares significant homology with MUC 18, NCAM, and the cytoplasmic domain of CD20. Expression of the RAGE cDNA in 293 cells allowed them to bind 125I-AGE-albumin in a saturable and dose-dependent manner (Kd approximately 100 nM), blocked by antibody to RAGE. Western blots of 293 cells transfected with RAGE cDNA probed with anti-RAGE IgG demonstrated expression of immunoreactive protein compared to its absence in mock-transfected cells. These results suggest that RAGE functions as a cell surface receptor for AGEs, which could potentially mediate cellular effects of this class of glycosylated proteins.
Highlights
From the $Department of Cellular and Molecular Biology, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486, the **Departmentof Biological Data, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey 07065, the §Department of Physiologyand Cellular Biophysics, Columbia University, College of Physicians and Surgeons, New
Using a n oligonucleotide probe basedon cell types, suggests two mechanisms through which they the amino-terminal sequenceof RAGE, an apparently could contribute to diabetic complications: by altering the full-length cDNA of 1.5 kilobases was isolated from a architecture of the extracellular matrix througthhe formation bovine lung cDNA library
Cells were washed three times with Hank's balanced method [18].25 pg of RNA was separated on aformaldehyde gel and salt solution, pH 7.4, binding buffer (minimal essential medium transferred to a nylon membrane (ICN, Irvine, CA) employing meth- containing 1%bovine serum albumin (Sigma), 0.05 ml/well) was ods asdescribed [18].The blot was hybridized with the bovine cDNA added along with the indicated amount of l2'1-Advanced glycosylation end products of proteins (AGEs) albumin alone or for RAGE under the conditions utilized in the cloning of the human in the presence of a t least a 20-fold molar excess of unlabeled AGE
Summary
Be hereby marked "advertisement" in accordance with 18 Isolation of the 35-kDa AGE Receptor, Trypsin Cleavage, and Se-. Cells were washed three times with Hank's balanced method [18].25 pg of RNA was separated on aformaldehyde gel and salt solution, pH 7.4, binding buffer (minimal essential medium transferred to a nylon membrane (ICN, Irvine, CA) employing meth- containing 1%bovine serum albumin (Sigma), 0.05 ml/well) was ods asdescribed [18].The blot was hybridized with the bovine cDNA added along with the indicated amount of l2'1-AGE albumin alone or for RAGE under the conditions utilized in the cloning of the human in the presence of a t least a 20-fold molar excess of unlabeled AGE cDNA below. E x ~ r e s s ~ oSntudies in 293 Cells-The bovine cDNA was released from pUC19using EcoRI and inserted into themammalian expression vector pD5 behind the adenovirus major late promoter [16] at the
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