Abstract
Nonenzymatic glycosylation of proteins, as occurs at an accelerated rate in diabetes, can lead to the formation of advanced glycosylation end products of proteins (AGEs), which can bind to endothelial cells, thereby altering cellular function in a manner which could contribute to the pathogenesis of diabetic angiopathy. In this report, we describe the isolation of two endothelial cell surface-associated proteins which mediate, at least in part, the interaction of AGEs with endothelium. Based on pilot studies demonstrating AGE binding activity with comparable characteristics in bovine endothelial cell and lung extracts, the material from lung was sequentially subjected to chromatography on hydroxylapatite, fast protein liquid chromatography Mono S, and gel filtration. Two distinct polypeptides, approximately 35 and approximately 80 kDa, were purified to homogeneity, each of which bound AGEs as demonstrated by competitive binding assays using cellular binding proteins immobilized on a plastic surface. NH2-terminal sequence analysis indicated that the approximately 35-kDa protein was novel, whereas the NH2-terminal sequence of the approximately 80-kDa protein was identical to that of lactoferrin. Immunocytologic studies using polyclonal antibody prepared to each of the purified polypeptides demonstrated the presence of immunoreactive material on the surface of bovine endothelial cells maintained under serum-free conditions. Furthermore, immunoelectron microscopic studies with antibodies to the approximately 35- and approximately 80-kDa AGE-binding proteins conjugated to different size colloidal gold particles confirmed the presence of the target antigens on the cell surface and suggested that they were closely associated. IgG purified from polyclonal antisera to either the 35- or 80-kDa AGE-binding proteins blocked the binding of 125I-AGE-albumin to the cell surface. These results indicate that endothelial cells express specific cell surface molecules which mediate AGE-endothelial interaction. These polypeptides represent a novel class of cell surface acceptor molecules for glucose-modified proteins which may promote degradation and/or transcytosis of the ligand, and modulation of cellular function.
Highlights
In this report, we describe the isolation of two endothelial cell surface-associated proteins which mediate, at least in part, the interactioonf advanced glycosylation end products (AGEs) with endothe- Interaction of aldoses with proteinsinitiatesachain of lium
Albumin glycosylated after shorterincubations with glucose, before AGEs become detectable, did not compete with '251-AGE-albumin for binding to endothelium [4]
Since binding of AGE-albumin to thendothelium could be blocked by either excess unlabeled AGE-albumin or AGE-hemoglobin, but not by the same unmodified proteins, it was evident that theadvanced glucose derivative on the protein was necessary for recognition of this cellular binding site [4]
Summary
We describe the isolation of two endothelial cell surface-associated proteins which mediate, at least in part, the interactioonf AGEs with endothe- Interaction of aldoses with proteinsinitiatesachain of lium. Endothelial cell monolayers were preincubated with antibody to purified AGE-binding proteins(2h a t 4"C), excess unlabeled AGE-albumin or trypsin (0.05 unit/ml) for 30 min at 37 "C.
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