Isolation of surface binding protein specific for advanced glycosylation end products from mouse macrophage-derived cell line RAW 264.7.
Macrophages internalize and degrade proteins modified by advanced glycosylation end products (AGEs) via a specific receptor (AGE-R). Chemical cross-linking studies with AGE-bovine serum albumin have demonstrated that the molecular weight of this receptor is approximately 90,000. We previously established that the binding constant (Ka) of this receptor site for the chemically synthesized model AGE, 2-(2-furoyl)-4(5)-(2-furanyl)-1H- imidazole-butyric acid (FFI-BA), on cells of the mouse macrophagelike cell line RAW 264.7 is identical to that for AGE proteins. Therefore, FFI was used as an affinity matrix in the first purification step of the AGE-R. The membranes of RAW 264.7 cells were solubilized in octyl-beta-glucoside and subjected to affinity chromatography on FFI-sepharose and gel permeation on Superose 6 fast protein liquid chromatography. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis of this material revealed a high enrichment of a 90,000-Mr protein that had AGE binding activity. Approximately 25% of the protein at this step was the 90,000-Mr protein. The 90,000-Mr membrane protein was purified to homogeneity by rechromatographing the material on Superose 12 in the presence of SDS before and after reduction with 2-mercaptoethanol. After these harsh conditions, the 90,000-Mr protein lost AGE binding activity. Additional cross-linking studies on human peripheral monocytes revealed an AGE-R protein of identical size to that on RAW 264.7 cells, suggesting the relatively highly conserved nature of this molecule.(ABSTRACT TRUNCATED AT 250 WORDS)
Radoff
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S
Laboratory of Medical Biochemistry, Rockefeller University, New York, New York 10021.
Cerami
A
A
Vlassara
H
H
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology; D002460:Cell Line; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D008264:Macrophages / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D008562:Membrane Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology; D051379:Mice; D009000:Monocytes / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface