Binding of high density lipoprotein to cultured fibroblasts after chemical alteration of apoprotein amino acid residues.
Cultured extrahepatic cells possess a specific high affinity binding site (receptor) for high density lipoprotein (HDL) that is induced by cholesterol delivery to cells. To characterize the binding recognition site(s) on HDL, the ability of HDL to interact with cultured human fibroblasts was assayed after chemical alteration of specific apoprotein amino acid residues. Reduction and alkylation, acetylation, and cyclohexanedione treatment of HDL3 had little or no effect on its cellular binding. Treatment of HDL3 with tetranitromethane (TNM), however, caused a large dose-dependent decrease in binding, with maximum inhibition at 3 mM. Amino acid analysis of the TNM-treated particles showed specific alteration of tyrosine residues, but sodium dodecyl sulfate-gel electrophoresis demonstrated apoprotein cross-linking coincident with decreased binding. These results suggest that modification of HDL tyrosine residues and/or cross-linking of HDL apoproteins alters the ligand site recognized by the HDL receptor. Gradient gel electrophoresis, molecular sieve chromatography, and electron microscopy showed only minor changes in size distribution and shape of HDL3 particles after treatment with 3 mM TNM, but at higher TNM concentrations, coalescence and aggregation of particles was evident. Treatment of HDL3 with 3 mM TNM affected neither its promotion of the low affinity (receptor-independent) cholesterol efflux from cells nor its ability to accept cholesterol from an albumin suspension, yet promotion of high affinity (receptor-dependent) cholesterol efflux from cells was abolished. The finding that TNM treatment of HDL3 decreases both its receptor binding and its promotion of cholesterol efflux from cells without substantial alteration of its physical properties supports the hypothesis that the HDL receptor functions to facilitate cholesterol transport from cells.
D000596:Amino Acids / Q000378:metabolism; D001053:Apolipoproteins / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism; D002788:Cholesterol Esters / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004587:Electrophoresis, Agar Gel; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D008075:Lipoproteins, HDL / Q000378:metabolism*; D053439:Lipoproteins, HDL2; D053440:Lipoproteins, HDL3; D008854:Microscopy, Electron; D013329:Structure-Activity Relationship; D013774:Tetranitromethane / Q000494:pharmacology
D000596:Amino Acids; D001053:Apolipoproteins; D002788:Cholesterol Esters; D008075:Lipoproteins, HDL; D053439:Lipoproteins, HDL2; D053440:Lipoproteins, HDL3; D002784:Cholesterol; D013774:Tetranitromethane