Synthesis and biochemical properties of a new photoactivatable cholesterol analog 7,7-azocholestanol and its linoleate ester in Chinese hamster ovary cell lines.
We report the chemical synthesis of a new photoactivatable cholesterol analog 7,7-azocholestanol (AC) and its linoleate ester (ACL). We also examined the biochemical properties of the sterol and its ester by employing several different mutant Chinese hamster ovary (CHO) cell lines with defined abnormalities in cholesterol metabolism as tools. AC mimics cholesterol in supporting the growth of a mutant cell line (M19) that requires cholesterol for growth. In normal cells, tritiated ACL present in low-density lipoprotein (LDL) was hydrolyzed and reesterified in a manner similar to tritiated cholesteryl linoleate (CL) in LDL. Also, in the mutant cell line (AC29) lacking the enzyme acyl-coenzyme A:cholesterol acyltransferase or in the mutant cell line (CT60) defective in the Niemann-Pick type C1 protein, the hydrolysis of ACL in LDL was normal, but the reesterification of the liberated AC was defective. Therefore, the metabolism of ACL in LDL is very similar to that of CL in LDL. Tritium-labeled AC delivered to intact CHO cells as a cyclodextrin complex was shown to photoaffinity label several discrete polypeptides, including caveolin-1. These results demonstrate AC as an effective reagent for studying cholesterol-protein interactions involved in intracellular cholesterol trafficking.
Cruz
Jonathan C
JC
Department of Biochemistry, Medical School, Dartmouth College, Hanover, NH, USA.
Thomas
Matthew
M
Wong
Edmund
E
Ohgami
Nobutaka
N
Sugii
Shigeki
S
Curphey
Thomas
T
Chang
Catherine C Y
CC
Chang
Ta-Yuan
TY
D000818:Animals; D001391:Azo Compounds / Q000138:chemical synthesis* / Q000494:pharmacology*; D016466:CHO Cells; D004083:Cholestanol / Q000031:analogs & derivatives / Q000138:chemical synthesis* / Q000494:pharmacology*; D006224:Cricetinae; D003470:Culture Media; D003505:Cyclodextrins / Q000737:chemistry; D004952:Esters; D019787:Linoleic Acid / Q000737:chemistry*; D008077:Lipoproteins, LDL / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D010777:Photochemistry; D013055:Spectrophotometry, Infrared
C472706:7,7-azocholestanol; D001391:Azo Compounds; D003470:Culture Media; D003505:Cyclodextrins; D004952:Esters; D008077:Lipoproteins, LDL; D004083:Cholestanol; D019787:Linoleic Acid