Cholesterol Biosynthesis from Lanosterol

doi:10.1074/jbc.274.21.14624

Cholesterol Biosynthesis from Lanosterol

MOLECULAR CLONING, TISSUE DISTRIBUTION, EXPRESSION, CHROMOSOMAL LOCALIZATION, AND REGULATION OF RAT 7-DEHYDROCHOLESTEROL REDUCTASE, A SMITH-LEMLI-OPITZ SYNDROME-RELATED PROTEIN*

From the ^‡Department of Biochemistry and Bioproducts Research Center and ^§Institut fur Biochemische Pharmakologie A-6020 Innsbruck, Peter Mayr-Strasse 1, Austria and ^¶Medical Research Center, Yonsei University, 134 Shinchon-dong, Sudaemoon-ku, Seoul 120-749, Korea

Abstract

The cDNA encoding the 471-amino acid rat 7-dehydrocholesterol reductase (DHCR), an enzyme that has been implicated in both cholesterol biosynthesis and developmental abnormalities (e.g. Smith-Lemli-Opitz syndrome) in mammals, has been cloned and sequenced, and the primary structure of the enzyme has been deduced. The DHCR gene was mapped to chromosome 8q2.1 by fluorescence in situ hybridization. Rat DHCR, calculated molecular mass of 54.15-kDa polypeptide, shares a close amino acid identity with mouse and human DHCRs (96 and 87%, respectively) as compared with its other related proteins (e.g. fungal sterol Δ¹⁴-reductase) and exhibits high hydrophobicity (>68%) with 9 transmembrane domains. Five putative sterol-sensing domains were predicted to be localized in transmembrane domains 4–8, which are highly homologous to those found in 3-hydroxymethylglutaryl-CoA reductase, sterol regulatory element-binding protein cleavage-activating protein, and patched protein. The polypeptide encoded by DHCR cDNA was expressed in yeast as a 55.45-kDa myc-tagged fusion protein, which was recognized with anti-myc monoclonal antibody 9E10 and shown to possess full DHCR activity with respect to dependence on NADPH and sensitivity to DHCR inhibitors. Northern blot analysis indicates that the highest expression of DHCR mRNA was detected in liver, followed by kidney and brain. In rat brains, the highest level of mRNA encoding DHCR was detected in the midbrain, followed by the spinal cord and medulla. Feeding rats 5% cholestyramine plus 0.1% lovastatin in chow resulted in both approximately a 3-fold induction of DHCR mRNA and a 5-fold increase of the enzymic activity in the liver. When rats were fed 0.1% (w/w) AY-9944 (in chow) for 14-days, a complete inhibition of DHCR activity and a significant reduction in serum total cholesterol level were observed. However, the level of hepatic DHCR mRNA fell only slightly, suggesting that AY-9944 may act more rapidly at the protein level than at the level of transcription of the DHCR gene under these conditions.

Footnotes

↵* This work was supported by grants from the Korean Ministry of Science and Technology Molecular Medicine Project 98MM-0204A03 (to Y.-K. P.) and Korea Science and Engineering Foundation through the Bioproducts Research Center at Yonsei University (9514-0401-00-12-3 (to Y.-K. P.)).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AF071500.
↵‖ To whom correspondence should be addressed: Yonsei University, Dept. of Biochemistry, 134 Shinchon-dong, Sudaemoon-ku, Seoul, 120–749, Korea. Tel.: 82-2-361-2702; Fax: 82-2-362-9897; E-mail:paikyk{at}bubble.yonsei.ac.kr.
↵2 J. N. Lee and Paik, Y.-K., unpublished data.
↵3 S-H. Bae and Y.-K. Paik, unpublished observation.
Abbreviations:

SLOS

Smith-Lemli-Opitz syndrome

DHCR

7-dehydrocholesterol reductase

kb

kilobase(s)

ORF

open reading frame

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid

SSD

sterol-sensing domain

AY-9944

trans1,4-bis(2-chlorobenzylaminomethyl)cyclohexane dihydrochloride

BM15. 766

4(2-(1-(4-chlorocinnamyl)-piperazin-4-ylethyl)-benzoic acid

CL-diet

5% cholestyramine plus 0.1% lovastatin in normal chow

IC₅₀

a concentration of inhibitor required for 50% inhibition

lanosterol

4,4′,14α-trimethyl-5α-cholesta-8,24-dien-3β-ol

lathosterol

5α-cholesta-7-en-3β-ol

PTC

patched protein

tamoxifen

trans-2-[4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethylamine

U18666A

3-β-[2-(diethylamino)ethoxy]androst-5-en-17-one
- Received December 22, 1998.
- Revision received March 4, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.