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Journal of Lipid Research, Vol. 43, 1341-1347, August 2002
Copyright © 2002 by Lipid Research, Inc.

Methods

Synthesis and biochemical properties of a new photoactivatable cholesterol analog 7,7-azocholestanol and its linoleate ester in Chinese hamster ovary cell lines

Jonathan C. Cruz^1,*, Matthew Thomas^1,*, Edmund Wong^*, Nobutaka Ohgami^*, Shigeki Sugii^*, Thomas Curphey, Catherine C. Y. Chang^* and Ta-Yuan Chang^2,*

^* Department of Biochemistry, Darmouth Medical School, and Department of Chemistry, Dartmouth College, Hanover, NH
Department of Pathology, Darmouth Medical School, and Department of Chemistry, Dartmouth College, Hanover, NH

² To whom correspondence should be addressed. e-mail: ta.yuan.chang{at}dartmouth.edu

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.

Abbreviations: AC, 7,7-azocholestanol; ACL, 7,7-azocholestanol linoleate; ACO, 7,7-azo-5-cholestan-3ß-ol oleate; BHT, 2,6-di-tert-butyl-p-cresol; CHO, Chinese hamster ovary; CL, cholesteryl linoleate; ER, endoplasmic reticulum; MßCD, methyl ß-cyclodextrin; NPC1, Niemann-Pick Type C1; PCC, pyridinium chlorochromate

Supplementary key words UV cross-linking • photoactivatable cholesterol analog • cholesterol-protein interaction • cholesterol mutants • Niemann-Pick type C disease • acyl-coenzyme A:cholesterol acyltransferase • caveolin-1

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