Abstract
Advanced glycosylation end products (AGEs) arise from glucose-derived Amadori products and have been implicated in the pathogenesis of diabetic vascular disease. We recently reported the presence of an AGE-modified form of low density lipoprotein (LDL) that circulates in high amounts in patients with diabetes or renal insufficiency and that exhibits impaired plasma clearance kinetics. We utilized AGE-specific antibodies to identify the major sites of AGE modification within protease-digested preparations of apolipoprotein B that impair the binding of the AGE-modified form of LDL by human fibroblast LDL receptors. The predominant site of AGE immunoreactivity was found to lie within a single, 67-amino acid region located 1791 residues NH2-terminal of the putative LDL receptor binding domain. These data point to the high reactivity and specificity of this site for AGE formation and provide further evidence for important structural interactions between the LDL receptor binding domain and remote regions of the apolipoprotein B polypeptide.
Highlights
From the Picower Institute for Medical Research, Manhasset, New York 11030 and the §Gladstone Institute of Cardiovascular Diseases, San Francisco, California 94141
We recently reported the presence of an Advanced glycosylation end products (AGEs)-modified form of low density lipoprotein (LDL) that circulates in high amounts in patients with diabetes or renal insufficiency and that exhibits impaired plasma clearance kinetics
Patients with diabetes or renal insufficiency suffer a high incidence of atherosclerotic vascular disease, which has been attributed in part to persistent elevation in the plasma level of the lipoprotein components very low density lipoprotein and LDL 1 [1,2,3,4]
Summary
AGE-modified LDL was prepared by incubating LDL (2.5 mg) with glucose (200 mra) at 37°C for 4-14 days in phosphate-buffered saline containing 1 mM EDTAand 20 /LM BHT. The LDL was dialyzed against phosphate-buffered saline containing 1 mM EDTAand 10 !LM BHT, and aliquots were subjected to analysis by an AGE-specific enzyme-linked immunosorbent assay [12, 16]. To validate the specificity of the anti-AGE antibodies, control experiments were performed in which AGE-BSA was added to the primary antibody incubation at a final concentration of 1 mglml. LDL Epitope Analysis by Enzyme-linked Immunosorbent AssayNative and AGE-modified LDLs were analyzed for reactivity to antibodies directed against synthetic apoB pep tides whose sequences are located in close proximity to the LDL receptor binding domain [22]. The bound antibodies were detected with an alkaline phosphatase-linked secondary antibody and p-nitrophenol as substrate
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