Characterization of the human oxysterol receptor overexpressed in the baculovirus system.
Oxysterols are potent regulators of enzymes of the de novo cholesterol biosynthetic pathway and do not require the LDL (low density lipoprotein):LDL receptor system for their regulatory actions. The search for an alternate transduction system led to the identification of an oxysterol binding protein. This cytosolic protein has been extensively characterized, purified, and cloned. Although it fulfills the pharmacologic criteria for an oxysterol receptor by binding to oxysterols with affinities corresponding to their regulatory potencies, its function in maintaining cholesterol homeostasis has not been determined. We have overexpressed the human oxysterol receptor in Spodoptera frugiperda cells using the Baculovirus system. The overexpressed protein binds oxysterols, but not cholesterol. The affinity for 25-hydroxycholesterol determined by competitive binding assay was 7.3 +/- 4.4 nM (mean +/- SD), and the relative affinities of several other oxysterols approximately corresponded to their potencies in cell systems. The expressed protein migrated as a single immunoreactive band on denaturing polyacrylamide gels with a molecular mass of 94 kDa. The molecular mass calculated from sucrose gradient centrifugation and gel filtration was 273 kDa for the 9.8S form, 217 kDa for the 7.8S form, and 184 kDa for the 6.6S form. However, velocity gradient centrifugation and heparin-sepharose chromatography each indicated that there were at least two fractions containing specific oxysterol binding. We conclude that we have successfully overexpressed the human oxysterol receptor and that biochemical analysis of the overexpressed protein provides evidence of interactions with other proteins. Further analysis of the overexpressed protein should provide clues regarding its role in maintaining cholesterol homeostasis.
Srinivasan
G
G
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77555.
Patel
N T
NT
Thompson
E B
EB
D000818:Animals; D016367:Baculoviridae / Q000737:chemistry / Q000235:genetics*; D001667:Binding, Competitive; D002478:Cells, Cultured; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D006801:Humans; D008970:Molecular Weight; D009036:Moths / Q000737:chemistry / Q000382:microbiology; D011869:Radioligand Assay; D011987:Receptors, Steroid / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D000098825:Oxysterol Binding Proteins
D011987:Receptors, Steroid; D011994:Recombinant Proteins; D000098825:Oxysterol Binding Proteins