by Genentech, Inc. The costs of publication of this article were defrayed in part by ... ll Recipient of a Josiah P. Macy, Jr. predoctoral fellowship. 11 Established ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 264, No. 36, Issue of December 25, PP.21573-21581,1989 Printed in U. S A .
Tissue-specific Expression, Developmental Regulation, and Chromosomal Mapping ofthe Lecithin:Cholesterol Acyltransferase Gene EVIDENCEFOREXPRESSIONINBRAINANDTESTES
AS WELL AS LIVER* (Received for publication, April 18, 1989)
Craig H. Warden$ $9, Carol A. LangnerF, Jeffrey I. Gordon§[[, Benjamin A. Taylor**, John W. McLean$$, and Aldons J. Lusis$ From the $Departmentsof Medicine and Microbiology, University of California, Los Angeles, California 90024, the §Departments School of Medicine, St. Louis, Missouri 63310, of Medicine, Biochemistry and Molecular Biophysics, Washington University **The Jackson Laboratory, Bar Harbor, Maine 04609, and the $$Department of Cardiovascular Research, Genentech Inc., South Sun Francisco, California 94080
Lecithin:cholesterol acyltransferase (LCAT) cata- tral role in transport of excess cholesterol from peripheral lyzes the esterification of cholesterol in high density tissues to liver (1,2), the only organ capable of both catabollipoproteins, thereby facilitating transport of excess izing and exporting cholesterol. LCAT is synthesized in the cholesterol from peripheral tissues to liver. We report liver and secreted into the blood (3, 4). In blood free choleshere studiesof the developmental, dietary, andgenetic terol from cell membranes is first transferred tohigh density control of LCAT geneexpression. Inadultmale lipoprotein (HDL) particles, where it is esterified, in a reacSprague-Dawley rats fed a standard chow diet LCAT tion catalyzed byLCAT and incorporated into lipoprotein the mRNA was most abundant in liver, a major source of core (5). The cholesterol esters are then transported to liver the plasma enzyme, but appreciable levels were also present in brain andtestes. Since bothbrain andtestes by poorly understood mechanisms(6).The physiological funcare isolated from blood by tight cellular barriers, un- tion of LCAT i n vivo is demonstrated by the symptoms of doubtedly greatly reducing the level of plasma-derived patients with familial LCAT deficiency. Such patients accumulate cholesterol intheirperipheraltissuesand develop LCAT in cerebrospinal fluid and testes, the production of LCAT in these tissues may be important for removal premature atherosclerosis and centralnervous system impairments (7, 8). of excesscholesterol.Noteworthychanges inthe There is as yet little information relating to the developexpression of LCAT mRNA were observed during development of both rodents and humans. On the other mental, dietary, orgenetic control of LCAT gene expression. hand, LCAT mRNA levels were relatively resistant to In this reportwe describe the cloning and characterizationof dietary challenge or to drugs affecting cholesterolme- mouse LCAT cDNA and the use of the cDNA to address the tabolism. Since human epidemiological studies have following questions. First, in which tissues is the LCATgene suggested an association betweenLCAT levels and var- expressed, and how do thelevels of expression change during iations of high density lipoprotein cholesterol, we ex- development? Second, is LCAT gene expression affected by amined LCAT gene polymorphisms in amouse animal diet ordrugs affecting cholesterol metabolism? Andthird, are model. Mapping of the LCAT gene (Lcat) to mouse mutations of the LCAT gene responsible for phenotypic difChromosome 8 within 2 centimorgans of the Es-2 locus ferences in the levels and physical characteristics of high indicates thatit does not correspond to any previously mapped loci affecting high density lipoproteinpheno- density lipoproteins among inbred strainsof mice? One surprising finding toemerge from this work is that LCAT gene types in themouse. expression is not restricted to liver, but also occurs in brain and testes. This extrahepatic expression appears to be of physiological significance and may help explain sensory imLecithin:cholesterol acyltransferase (LCAT)’ plays a cen- pairment, such as hearing loss, associated with LCAT deficiencies. * This work was supported in part by Grants HL42488, HL28481, DK 37960, and CA 33093 from the National Institutes of Health and EXPERIMENTALPROCEDURES by Genentech, Inc. The costs of publication of this article were cDNA Library Construction, Screening, and Clone Analysis-Livers defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 were obtained from adult female SWR mice (Simonsen) and frozen immediately in liquid nitrogen. Total RNA was extracted from pulU.S.C. Section 1734 solely to indicate this fact. T h e nucleotide sequence(s)reported in this paper has been submittedverized frozen tissuewith guanidinium thiocyanate (9).Poly(A)+ totheGenBankTM/EMBLDataBankwith accession number(s) RNA was prepared by poly(U)-Sepharose affinity chromatography (10). Double-stranded cDNA was synthesized by priming with 505154. oligo(dT) (11, 12). Size-selected double-stranded cDNAs were ligated ll Recipient of a Josiah P. Macy, Jr. predoctoral fellowship. into X g t l O (13) and screened unamplified. Sixhundred thousand 11 Established Investigator of the American Heart Association. clones were screened in duplicate with a full-length human LCAT § § To whom correspondence should be addressed Dept. of MedicDNA (14). Filters were hybridized in 25% formamide, 5 X SSC (1 X cine, UCLA, Los Angeles, CA 90024. The abbreviations used are: LCAT, 1ecithin:cholesterol acyltransssc = 150 mM NaC1, 15 mM trisodium citrate), 50 mM sodium ferase; HDL, high density lipoprotein; HMG-CoA, 3-hydroxy-3-meth- phosphate (pH 6.8), 5 X Denhardt’s solution, 10% dextran sulfate, ylglutaryl coenzyme A; kb, kilobase pairs; LDL, low density lipopro- and 20 pg/ml single-stranded sheared salmon sperm DNA, at 42‘C tein; VLDL, very low density lipoprotein; SDS, sodium dodecyl sul- overnight. Filters were washed for 30 min in 2 X SSC at room fate. temperature and for 30 min in 2 X SSC at 37 “C. Phage DNA was
21573
21574
Expression of L C A T in Brain and Testes as Well as Liver
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1 CCAGGGCTGGA ATG GGG CCG CCC GGC TCC CCA TGG CAG TGG GTG ACGCTGCTGCTG
"_ "_ "_
HLCAT MLCAT MLCAT
1 77
HLCAT
84
______ ___ ___
---
pro Trp Thr Ala M e tG l yL e uP r oG l y Ser P r o T r p G l n A r q V a l L e u L e u L e u L e u G l y L e u L e u L e u Pro P r o A l a T h r Pro TGTGATG GGG CTG CCT GGC TCC CCA TGG CAG CGG GTG CTGTTGCTGTTG GGG CTA CTGCTCCCTCCT GCC ACCCCC
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CCG CAC ACC ACG CCC AAG GCT GAG CTCAGT
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___ ___ ___ ___ ___ ___ ___ ___ ___ ___ ______ ___ ___ ___ ___ __-___ __-
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HLCAT 1 6 5
GTG
ccc
GGC TGC CTG GGG AAT CAG CTA GAA GCC AAG CTG GAC AAA CCA GAT GTG GTG AAC TGG ATG TGC TACCGC
HLCAT MLCAT 55 MLCAT 2 3 9
-" Leu Met GluAspPhePheThr Ile T r pL e uA s pP h eA s nL e uP h eL e uP r oL e uG l yV a lA s pC y sT r p GAG GAC TTCTTCACCATC TGG CTG GAT TTC AACTTG TTTCTCCCCCTC GGG GTG GAC TGCTGG ttt ttt
tt.
HLCAT 2 4 6
GAG GAC
TTCTTCACCATC
HLCAT MLCAT 8 2 MLCAT 3 2 0
Arq Leu Tyr V a Tl y r Ser G l A y rV q al Ser A s nA l Pa r oG l yV a Gl l n I l e A r qV a Pl r oG l yP h G e l yL y Ts h G r lu Ser GTC TAC AAC CAC AGC TCT GGG CGC GTG TCC AAT GCC CCT GGT GTC CAG ATC CGT GTCCCT GGC TTT GGC AAG ACC GAA TCT
HLCAT 327
GTCTACAAC
HLCAT MLCAT 1 0 9 MLCAT 4 0 1
L e u --- Ser ser -" --- -" "- -" V a l G l u T y r V a l A s p Asp Asn Lys Leu A l a G l y T y r Leu H i s T h r LeuVal Gln Asn L e u V a l Asn Asn G l y T y r V a l A r q GTT GAG TAC GTG GAT GAC AAC AAG CTA GCA GGC TAC CTG CAC ACA CTG GTG CAG AAT CTG GTT AAC AAC GGA TAT GTG CGG
HLCAT 4 0 8
GTG GAG TAC CTG GAC AGC AGC AAG CTG GCA GGG TAC CTG CAC ACA CTG GTG CAG AAC CTG GTC AAC AAT GGC TAC GTG CGG
ttt
HLCAT MLCAT 2 8 MLCAT 1 5 8
Gln V a l P r o G l yC y s L e uG l yA s nA r q LeuGluAlaLysLeuAspLysProAspValValAsnTrpMetCys GTG CCT GGC TGC TTG GGG AAT CGG CTA GAA GCC AAG CTG GAT AAA CCA GAT GTG GTG AAC TGGATGTGC
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Val Ile A s pA s nT h rA r q Ile ATT GATAATACC AGG ATT
GGG GTA GAC TGCTGGATCGAT
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CTC AAC ATG TTC CTA CCCCTT
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HLCAT MLCAT 1 3 6 MLCAT 4 8 2
Glu Gly Glu Arq P r o T y r A s p T r p A r q L e u A l a P r o His G l n G l n A s p G l u T y r T y r L y s L y s L e u A l a Asp GluThrValArqAlaAla Gly GAT GAG ACA GTG CGG GCC GCA CCC TAT GAC TGG CGT CTG GCA CCC CAC CAG CAG GAT GAA TAC TAC AAG AAG CTG GCT GGC
HLCAT 4 8 9
GAC GAG ACT GTG CGC GCC GCC CCC TAT GAC TGG CGG CTG GAG CCC GGC CAG CAG GAG
HLCAT MLCAT 1 6 3 MLCAT 563
L e uV a lG l uG l uM e tT y rA l aA l aT y rG l yL y sP r oV a lP h eL e u CTG GTA GAG GAG ATGTAT GCC GCT TAT GGG AAG CCTGTCTTCCTCATT
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CTG GTG GAG GAG
HLCAT MLCAT 1 9 0 MLCAT 6 4 4
P h eL e uL e uA r qG l nP r o TTCTTACTG CGG CAGCCT
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GAG TAC TAC CGC AAG CTC GCA GGG
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GGC CAC AGC CTC GGC TGTCTA
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CAC TTGCTCTAT
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Ala Arq G l n Ser T r p L y s A s p H i s P h e Ile AspGlyPhe I l e Ser L e u G l y A l a ProTrpGlyGly Ser CAG TCC TGG AAG GAC CAC TTCATT GAT GGT TTTATCTCTCTC GGG GCT CCG TGG GGT GGT TCC
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TTCCTGCTG
HLCAT MLCAT 2 1 7 MLCAT 7 2 5
Met ser pro L e uV a l I l e L y sA l aM e tA r q I l e L e u A l a Ser G l y A s p A s n G l n G l y I l e P r o I l e L e u Ser A s n I l e L y s L e u L y s G l u G l u G l n ATC AAG GCCATG CGG ATC CTG GCC TCA GGT GAC AAC CAG GGC ATC CCCATCCTGTCC GAG CAG AAC ATA AAG CTG AAA GAG
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HLCAT 7 3 2 HLCAT MLCAT 2 4 4 MLCAT 8 0 6 HLCAT 813 HLCAT MLCAT 2 7 1 MLCAT 8 8 7
CGC CAGCCC
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CAG GCC TGG AAG GAC CGC TTTATT
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TTG GCC TCA GGT GAC AAC CAG GGC ATC CCCATC
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___ ___ _-_
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P h e -Val TTC AAC TACACCGTC ttt
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ATG TCC AGC ATC AAG CTG AAA GAG GAG
phe Ser A r q Met A l a A r q Ile T h r T h r T h r Ser Pro T r p M e t L e u P r o A l a P r o H i s V a l T r p P r o G l u A s p H i s V a l CGC ATA ACC ACG ACTTCCCCC TGG ATG CTC CCA GCC CCT CAC GTG TGG CCT GAA GAC CATGTG It.
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TTC AAC TAC ACA GGC CGT GAC TTC CAA CGC T T CT T T
HLCAT MLCAT 2 9 8 MLCAT 9 6 8
__A l a G l y --- -__ Leu Arq Arq A s p L e u L e u G l u A r q L e u P r o A l a Pro G l y V a l G l u V a l T y r C y s L e u T y r G l y V a l G l y A r q P r o T h r P r o His T h r CGT GAC CTACTG GAG CGC CTCCCC GCA CCT GGT GTA GAA GTA TATTGTCTC TAC GGT GTG GGC AGA CCC ACA CCC CAC ACC
___ ttt
HLCAT 9 7 5
--- --_
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CGT GAC CTCCTG
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GCA GAC CTG CAC T T T GAG GAA GGC
______
__-___
___ ______ ___ ___ ___ --- ___ __-
t t tt t t
ttl
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HLCAT 1 0 5 6 TACATCTAC
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GAC CAC GGC TTC CCCTAC
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GCA GGA CTC CCA GCA CCT GGT GTG GAA GTA TACTGT
_.._ HLCAT MLCAT 3 2 5 T y r Ile T y r A s p H i s PheProTyrLysAspProVal MLCAT 1 0 4 9 TACATCTAT GAC CAC AAC TTC CCCTAC AAA GAC CCCGTG t t t ..t
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TGGTACATG
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CTT TAC GGC GTG GGC CTGCCC
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ACG CCC CGC ACC
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.
L e uT y rG l uA s pG l yA s pA s pT h rV a lA l aT h rA r q GCT GCA CTCTAT GAA GAT GGG GAC GAC ACC GTA GCC ACCCGC
ttt
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t
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ACG GAC CCT GTG GGT GTGCTCTAT
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t t. tt
TGG CTG CAG TCA
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t t t ..t
GAG GAT GGT GAT GAC ACG GTG GCG ACC CGC
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--- ---
Leu pro Leu H i s G l v Ile G l n HLCAT MLCAT 3 5 2 Ser T h r G l u L e u C y s G l y G l n T r p G l n G l y A r g G l n Ser G l n P r o V a l His L e uL e uP r oM e tA s nG l GT h rA s p His L e u MLCAT 1 1 3 0 AGCACT GAG CTCTGT GGC CAG TGG CAG GGC CGC CAG TCG CAG CCC GTA CATTTGCTGCCCATG AAC GAG ACA GAT CAC CTC HLCAT 1 1 3 7 AGCACC
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HLCAT 1 2 1 9 AAC ATG GTCTTC
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GGC CTG TGG CAG GGC CGC CAG CCA CAG CCT GTG CAC CTGCTGCCCCTG
"_ "_ "_
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HLCAT MLCAT 3 1 A 9 sM n eVt aPl h e MLCAT 1 2 1 1 AACATG GTCTTC . . t
ttt
GAG CTCTGT
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Leu L eG u lH u is lle A sA n la I l e L e Lu e G u lA y l Ta yAr r q AAG ACA ATG GAG CATATCAAT GCC ATC CTA CTG GGT GCCTACCGC
* **
t tt t t
AGC AAC CTGACCCTG
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GAG CACATCAAT
______ ___
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GCC ATC CTGCTG
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t
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G l n G l y P r o --- A l a ----- --- T h Pr r o LYS Ser --- --- ACTCCT AAG TCG
. ..* *.
GGT GCC TAC CGC CAG GGTCCCCCT
GCA TCC
Thr Glu HLCAT MLCAT 4 0 6 P r o A l a A l a Ser P r o Ser P r o P r o P r o Pro G l u E n d MLCAT 1 2 9 2 CCA GCT GCC AGC CCA AGT CCC CCA CCC CCT GAA TAA AGACCTAGCTGTTATAAATA
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CAC GGG ATA CAG CAT CTC
tt
HLCAT 1 2 9 9 CCG ACT GCC AGC CCA GAG CCC CCG CCT CCT GAA TAA AGACCTTCCTTTGCTA
FIG. 1. Nucleotide sequence of mouse LCAT cDNA and predicted amino acid sequence of mouse and humanLCAT. Nucleotides for mouse LCAT (MLCAT) are numbered from the 5' terminus of cDNAclone MLCAT7. Nucleotides for human LCAT(HLCAT) are numbered from the 5' terminus of clone pL2 (14). Positions a t which mouse LCAT and human LCAT are identical are marked with an asterisk (*). The complete predicted amino acid sequence of mouse LCAT is shown immediately above the mouse nucleotide sequence. Negative amino
pressio ion of LCAT in Bruin and Testes as Well as Liver prepared from 11 strongly hybridizing plaques. Restriction endonuclease digestion revealed that all contained overlapping sequences. Four recombinant phage, containing the largest inserts, were analyzed further. All were sequenced on both strands using the dideoxy chain termination method (15). Sources of Tissues-Timed, pregnant female, neonatal, and young adult male Sprague-Dawleyrats were obtained from Sasco (St. Louis, MO).Weaned animals were maintained on a standard rat chow diet ad libitum and a fixed 12-h light cycle. Animals ( n = 10-40 for each time point representing 2-5 litters) were killed by decapitation between 1200 and 1400 h. Their tissues were quickly harvested and frozen in liquid nitrogen. Human fetal liver samples from first and second trimester aborted fetuses were obtained by Schwartz et al. (16) and stored at -90 "C for 15-20 years. Fetal age wasestimated from measurements of crown rump lengths using the nomogram of Tanimura etal. (17). Additional human liver samples were obtained from a preterm and a full term newborn each of whom died of acute respiratory failure (16). These samples were stored at -90 "C for approximately 15 years. A sample of adult liver was isolated from a male organ donor who had no laboratorv or clinical evidence of hepatic dysfunction at the time of his death"(l8). Dietaw Manidations-Female C3H/HeJ. . , BALBfcJ. and C57BLI 6J mice"of 2-4 months ofagewere obtained from The Jackson Laboratory (Bar Harbor, ME) and fed either (i) a normal chow diet (Purina chow containing 4% fat) or a (ii) ahigh fat diet (prepared by mixing the Thomas-Hartroft diet purchased from Teklad Test Diets (Madison, WI) with Purina breeder chow (5015) in a ratio of 1 part to 3 parts, respectively). The latter diet contains (by weight) 7.5% cocoa butt.er, 1.25%cholesterol, and 0.5%cholic acid (19). Mice were given the high fat diet ad l i b i t u ~for 3 weeks before killing. Another group of mice ( n = 3-4 for each group) were given a normal chow diet supplemented with 5% cholestyramine for 4 days and 0.1% mevinolin for 2 days prior to killing. Isolation of RNA and Quantitation of Steady-state n R N A Levels by Blot Hybridization-Total cellular RNA was extracted from frozen tissues using guanidine thiocyanate and further purified by cesium chloride ultracentrifugation (20). RNA integrity was confirmed by denaturing methylmercury agarose gel electrophoresis (21). For Northern blot analyses, samples of RNA (15 pg) were subjected to electrophoresis in 1%formaldehyde-agarose gels (21), transferred to nylon, and cross-linked by exposure to ultraviolet light. Blots were prehybridized in 0.5 M sodium phosphate buffer, pH 7.0, 1mM EDTA, 7% SDS, and 0.1% bovine serum albumin, as described by Church and Gilbert (22). Blots were then hybridized in the same buffer containing 2 X lo6 cpm/ml of mouse LCAT cDNAlabeled with ["PI dCTP by random oligonucleotide priming (23) to a specific activity of 1 X lo@cpm/pg DNA. The cDNAs encoding rat 3-hydroxy-3methylglutaryl coenzyme A reductase (HMG-CoA reductase) (clone D S l l in Ref. 24f, rat HMG-CoA synthase (clone XLA2a in Ref. 251, and a-tubulin were also labeled as above (23). Stringencies selected for filter washing are listed in Refs. 25 and 26 (final wash = 0.1 X SSC, 0.1% SDS at65 "C). RNA dot blots were prepared using a template manifold as described in Elshourbagy et al. (27). Four different concentrations of given tissues total cellular RNA sample (0.5, 1, 2, and 3 pg) were combined with yeast tRNA prior to formaldehyde denaturation so that the final amount of RNA per sample equaled 3 pg. Blots containing samples of rat tissue RNA were probed with a cloned mouse LCAT cDNA. The human liver RNA dot blot was incubated with a human LCAT cDNA(14). Conditions selected for filter hybridization and washing were similar to those employed for the Northern blot studies (26, 28). The relative abundance of LCAT mRNA in RNA samples prepared from a given tissue was calculated after quantitative scanning laser densitometry of filter autoradiographs. Only signals in the linear range of film sensitivity were utilized. In a previous study with the same mRNA preparations it was shown that there was no significant variation in the amount of RNA used for each blot, as judged by hybridization to a variety of other mRNA species, including several which remained relatively constant in levels during development (29).
21575
RESULTS
Murine LCAT Cloning and Structure-We isolated and characterized a mouse hepatic LCAT cDNA to compare the structure of murine LCAT with the human enzyme and to derive a probe for studies of murine LCAT expression and polymorphisms. A full-length human LCAT cDNA (14) was used to identify LCAT cDNA in a SWR mouse liver library. Sequence analysis of four overlapping cDNA clones yielded the DNA sequence shown in Fig. 1. One mouse clone, designated MLCAT7, contained aninsert of 1340 base pairs, including the entire coding sequence. The first ATG of the cDNA clone initiates anopen reading frame specifying a 438amino acid protein with a calculated molecular mass of 47,194 daltons. This includes a signal peptide of 24 residues. The site of co-translational processing of mouse LCAT was inferred based on the remarkable degree of homology of the human and murine preproteins. Overall, human and mouse LCAT show about 85% identity of both the nucleotide and amino acid sequences (Fig. 1).In fact several interesting and important structural features of human LCAT are strongly conserved in mouse LCAT. These include a region from amino acid 158 to 170 in which 7 of 13 amino acids are identical to human apolipoprotein E (30). Amino acids 150-173 of human and mouse LCAT can also be fit to a helical wheel, suggesting an amphipathic helical structure in this region. Serine 181, which has been identified by Farooqui et al. (31) as important for the catalytic activity of LCAT, is conserved, as are free cysteines 31 and 184, which have also been reported to be necessary for LCAT activity (32). There are four potential asparagine-linked glycosylation sequences in LCAT at amino acids 20, 84, 272, and 384 (note that the estimated mass of human LCAT purified from serum is 60,000-67,000 daltons). The only notable sequence similarity to otherproteins occurs at amino acids 178-183 ofmature LCAT, where six contiguous residues are identical with the interfacial binding site of pancreatic lipase (33). Clone MLCAT7 extends four nucleotides upstreamof the initiator ATG and ends ninenucleotides short of the polyadenylation site. The mouse polyadenylation signal appears to overlap with carboxyl-terminal glutamic acid and stopcodons, as is thecase for human LCAT (34). Tissue Distribution of LCAT mRNA-A large body of data has been produced over the pastseveral years using SpragueDawley rats concerning the tissue-specific expression and developmenta~regulation of a number of genes encoding proteins involved in theintracellular and extracellular transport of lipids as well as their biosynthesis and metabolic processing. Thesestudies have provided insightsnot only about the factors which modulate specific gene expression but also about the function of these proteins. Given this extensive base of information, we used the murine LCAT cDNA to initially catalog steady-state levels of its mRNA in a variety of adult Sprague-Dawley rat tissues. Total cellular RNA was prepared from 13 different tissues: brain,stomach,small intestine, colon, liver, kidney, heart, lung, psoas muscle, soleus muscle, spleen, testes, and adrenals. Each tissue was harvested and pooled from 10 250-g male rats representing two litters. The distribution of LCAT mRNA among the tissue RNA samples was examined by probing Northern blots with the 3*P-labeled1340-base pair insert from MLCAT7. The results are shown in Fig. 2. Liver RNA contained the highest concen-
acid numbers refer to thepredicted leader prepeptide; positive amino acids refer to themature protein. The mature amino terminus (tripleasterisk, ***) is positioned by agreement with the amino terminus residues of human LCAT. The predicted N-linked glycosylation sites are underlined, and amino acids in common with the interfacial binding site of porcine pancreatic lipase are double underlined. Those amino acids of human LCAT which differ from mouse LCAT are shown above the mouse LCAT protein sequence.
Expression of LCATBrain in
21576
and Testes as Well as Liver
r: c .-c 0
P
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0 0
L Q)
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3
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0
c
0
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0
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FIG. 3. Expression of LCAT mRNA in rat brain and mouse liver. Total cellular RNA was prepared from adult male Sprague Dawley rats ( n = 2-4). The brains were divided into four sections: cortex, cerebellum, hippocampus,and brain stem. Total cellular RNA was also prepared from adult female BALB/cJ and C57BL/6J mouse livers. Ten micrograms of total RNA were electrophoresed through a 1% agarose formaldehyde gel, transferred to nylon, and probed with a 32P-labeled MLCAT7. Blots were washed to a final stringency of 0.1 X SSC, 0.1% SDS at 65 "C. Relative levels were estimated by quantitative scanning laser densitometry.
FIG. 2. Tissue-specific expression of the rat liver LCAT gene. Total cellular RNA was prepared from adult male Sprague
entering puberty (day 35). In addition, testicular RNA was isolated during days 4,8, 14, and 35 of postnatal life. Material was collected from 10-40 rats representing 2-5 litters at each developmental stage analyzed. Stringencies selected for dot blot hybridization and washing were identical to those used for the Northern analyses. LCAT mRNA is present in liver at theearliest time point tration of a 1600-nucleotide-long mRNA which reacted with surveyed in fetal life (day 16 of the 21-day gestation, see upper the probe. In addition, an identical sized mRNA was also noted in brain and testes. Scanning laser densitometry indi- panel of Fig. 4). A progressive 3-fold rise in mRNA levels cated that LCAT mRNA levels in these latter two tissues occurs during the 4 final days of gestation. Following parturition, there is an abrupt increase in hepatic LCAT mRNA were 17 and 6%,respectively, of that noted in liver. Steady-state levels of LCAT mRNA in different regions of concentration, reaching apeak by the 4th postnatal day. the brain were documented in cerebral cortex, cerebellum, Thereafter levels decline through the suckling weaning tranhippocampus, and brain stem( n = 2-4 adult males). Northern sition, rise transiently during midweaning and fall through blots indicated that all four brain regions expressed apprecia- early puberty. Although the cellular patterns of expression of ble levels of LCAT mRNA (Fig. 3). Cerebellum had approxi- the LCAT gene cannot be inferred from this type of analysis, mately twice the level of LCAT mRNA as cerebral cortex and it is important tonote that thedevelopmental changes in rat hippocampus. The presence of LCAT mRNA in brain has hepatic LCAT mRNA concentration do not simply parallel also been observed by Kathy Smith, Josiah Wilcox, and alterationsin its cellular populationduring the perinatal Richard Lawn at Genentech, Inc., using in situ hybridization period. Morphometric studies (35) have shown that approxito Rhesus monkey tissues.' In these studies,the LCAT mRNA mately 50% of late gestation fetalliver cells are hematopoietic was found to be present in neuronsand neuroglia throughout and that thisnumber rapidly declines during the early suckthe brain, with levels highest in the cerebellum. The distri- ling phase. The developmental profiles of LCAT mRNA accumulation bution differed from that of apoE and apoD. LCAT mRNA Accumulation in the Developing Rat Liver, in brain and in liver are not precisely coincident, but there Brain, and Testes-Themouse LCAT cDNAwas subse- are some similarities duringlate fetal and early postnatal life quently used to probe dot blotsof total cellular RNA prepared (compare the upper and middlepanels of Fig. 4). LCAT mRNA from rat liver and brain harvested during late gestation (fetal is present in both organs priorto birth, thelevels in liver rise days 16-21), the suckling period (postnatal days 1-13), the sharply at birth through the early suckling phase, while levels weaning phase (postnatal days 14-28), and from animals just in brain begin to rise on day 4 after birth.Unlike in theliver, LCAT mRNA levels continue to rise in brain through the remainder of the suckling and weaning periods reaching a R. Lawn, personal communication. Dawley rats ( n = 10 littermates). Twenty micrograms of RNA were applied to a denaturing formaldehyde-agarose gel. Northern blots were prepared and probed with LCAT cDNA. The mRNA visualized in liver, brain, and testicular RNA has acalculated size of 1600 nucleotides.
21577
Expression of LCAT in Brain and Testesas Well as Liver
/Liver
rn 8.5 weeks
A 16.5
18.5
, 8.5
10-12 13-14 16-18
21
I
25
, New-
Adult
I Born
Fetal age(weeks)
14
24
=I
35
FIG. 6. Accumulation of LCAT mRNA in human fetal liver. A human LCATcDNA was used to probe dot blots containing samples of human liver RNA prepared at various time points itemized in the inset. Data are expressed in arbitrary densitometric units.
We next compared these findings with the developmental pattern of expression of the gene which encodes the principal activator of LCAT,apolipoprotein A-I. Fig. 5providesa 3 summary of our survey of rat apoA-I mRNA accumulation in 2 the two tissues of the rat which have been shown to support 1 its transcription, liver and intestine (see Ref. 27). The same liver RNA preparations which were used to document LCAT 35 mRNA concentrations were alsousedfor these analyses. Day of Development Thereis a slight correspondence of expression of LCAT FIG. 4. Developmental patterns of accumulation of LCAT mRNA in liver with apoA-I mRNA in intestine since levels mRNA in rat liver, brain, and testes. Dot blot hybridization of both rise sharply in the lastfew days of development and analyses were used to determine the relative levels of this mRNA in Figs. 4 and 5). each tissue at various stages of fetal and postnatal life. As noted in peak just after birth (compare Expression of the Human LCAT Gene in the Developing the text, tissues were pooled from 10-40 animals at every time point Liver-A humanLCAT cDNA was used tocomparethe surveyed. Data are expressed in arbitrary densitometric units. developmental profile of LCAT mRNA accumulation in human liver (Fig. 6) with that observed in the rat. As early as the 8th week of human fetal life, this mRNA had achieved a level which exceeds that present intwo newborn and a single adult liver sample. A progressive rise in mRNA concentration takes place during thesecond trimester. Remarkably, thereis little variation among individuals in those cases where multiple samples were obtained at similar stages of fetal development. Dietary and Genetic Regulation-Since LCAT is a central enzyme in cholesterol transport, it was of interest to determine whether LCAT gene expression is affected by dietary influences or by drugs affecting cholesterol metabolism. We therefore quantitated the levels of hepatic LCAT mRNA in mice maintained on a low fat chow diet, or for 3 weeks on a high fat diet containing 1.25% cholesterol and 7.5% cocoa butter. The latter diet has been shown to produce atheroscleroticlesions and decreased HDL levels incertaininbred strains of mice (including C57BL/6J) but not others (includFold ing BALB/cJ and C3H/HeJ) (36).3 Since genetic variations Day of Development FIG. 5. Expression of the rat preproapoA-I gene in devel- could affect the response to diet,we examined LCAT mRNA oping liver and intestine. The same liver RNA samples used to in both susceptible and resistant strains. The results,shown in Fig. 7, indicate that the high fat diet results in a small quantitate LCAT mRNA concentrations were used formeasuring apoA-I mRNA levels by dot blot hybridization. Data are expressed in increase in the levels of LCAT mRNA in strain C3H/HeJ arbitrary densitometric units. and slight decreases in strains BALB/cJ and C57BL/6J. In contrast to this very slight effect, the expression of various peak at postnatal day 24. Although the levels of LCAT mRNA sterol responsivegenes, such as HMG-CoA reductase and HMG-CoA synthase, was greatly altered by the high fat diet in brain arelow, the patternwas reproducible. Our survey of testicular RNA was less complete than the in the same mice (Fig. 7). These results demonstrate that ones conducted for brain and liver largely as a consequence LCAT mRNA levels are not significantly affected by diet or of the small size of this organ during the fetal and perinatal by genetic variation between mouse strains. periods. A sharp rise in LCAT mRNA levels coincides with sexual maturation (see lower panel of Fig. 4). R. LeBoeuf and A. J. Lusis, unpublished data.
4l
Expression of LCAT in Brain and Testes
21578
C3HkJ
BALBW
CS7BL/6J
as Well as Liver
C57BL/61
Lecithin cholesterol acyl transferase
C57BU6J
HMG-CoA Reductase
HMG-CoA Synthase
FIG. 7. Levels of LCAT, HMG-CoA reductase, and HMG-CoA synthase mRNA among inbred mouse strains maintained on chow ( ), high fat (a),or cholestyramine plus mevinolin (B) diets. Four separate preparations of total RNA were isolated from the livers of four each C3H/HeJ, BALB/cJ, or C57BL/6J mice. The RNAs were electrophoresed through a 1%agarose formaldehyde gel, transferred to nylon, and probed with a 32Plabeled MLCAT7, HMG-CoA reductase probe DS11, HMG-CoA synthase probe XLA2a, or an a-tubulin cDNA probe. Blots were washed to a final stringencyof 0.1 X SSC, 0.1% SDS a t 65 “C. Values were decided by quantitative scanning laser densitometry of the Northern blots. Data have been expressed in arbitrary unitsnormalized for the intensity of a-tubulin bands.
TABLEI Linkage of Lcat with Es-2 and Emu-26 on Chromosome 8 Alleles inherited from the hybrid parent in (CAST/Ei X MEV)Fl X BXD-32 testcross are denoted as either M or C to indicate their origin in either MEV or CAST/Ei, respectively. Genotype Es-2
M C C M
6.1 -
C M
2.6 -
*
1.9 FIG. 8. Restriction fragment length polymorphism of the LCAT gene in MEV and Castaneous mice. Southern blots of MEV, Castaneous, and B X D-32 mouse DNA with32P-labeled MLCAT7 were prepared as described under “Experimental Procedures” using the restriction enzyme BamHI.
We also tested whether the combination of cholestyramine (an inhibitor of intestinal cholesterol absorption) and mevinolin (an inhibitor of cholesterol synthesis) would affect LCAT gene expression. Mice were fed the drugs in a chow diet for 2 days, and hepatic LCAT mRNA levels were determined. As shown in Fig. 7, the drugs had no significant effect on LCAT mRNA levels in any of the strains tested. Under these conditions, HMG-CoA reductase mRNA was induced 17-fold compared to chow fed mice (Fig. 7). LCAT Gene Polymorphisms and Chromosomal Mapping-
5
M C M C M C M C
hat
M C
No. of
Emu-26
M C C M C M M C
progeny
25 17 1 0 1 0 0 Total 49
Human epidemiological studies have revealed associations between LCAT activity and HDLcholesterol (37). Therefore, we were interested in examining whether genetic variations of the LCAT gene contribute to phenotypic variations of HDL among inbred strains of mice. In particular, the Ath-1 gene on mouse Chromosome 1 controls HDL levels in mice fed a high fat diet (36), while the size and cholesterol content of HDL aredetermined by the Hdl-1 gene that is either identical with or tightly linked to the apoA-I1 (Apoa-2) gene on Chromosome 1 (38). Given the association between LCAT mRNA levels and HDLlevels among three mouse strains (see above), it was possible that theLCAT gene corresponded to Hdl-1 or Ath-1. We approached this by searching for LCAT gene restriction fragment lengthpolymorphisms to allow mapping of the LCAT gene. We initially screened DNA from nine inbred strains that are progenitors of recombinant inbred strains. These strains were chosen because of the utility of recombinant inbred strains for genetic analysis (39). Genomic DNAs isolated from these strains of micewere cut with one of 23 restriction enzymes, subjected to electrophoresis and Southern transfer, and hybridized to a mouse LCAT cDNA probe. Twenty-one of the enzymes (BclI, DraI, BglII, PuuII, SacI, XbaI, ScaI, StuI, HaeIII, AluI, Asp700, BasII, EcoRI, HindIII, MspI, PstI, RsaI, EcoRV, NciI, HinfI, and BamHI) yielded identical bands for each strain examined. The enzyme KpnI yielded two different patterns.StrainsAKR/J, BALB/cJ, C3H/HeJ, C57BL/6J, DBA/J, A/J, NZB/BlNJ, and SWR/J showed a
Expression of LCAT in Brain and Testes as Well as Liver Mouse
Human
8
16
12 11.2 11.1
11.2 12.1 12.2 13 21
U
22
LCAT
23 7
Mt-1 Mt-2
FIG. 9. Loci containing theLCAT gene in mice and humans. Shown are mouse Chromosome 8 and human Chromosome 16 containing thek a t locus as well as selected flanking markers. The mouse genechromosomallocations were determined by recombinational analysis. Distances separating genes are given in centimorgans. The human chromosomal mapping and regional mapping was performed using somatic cell hybrids, in situ hybridization, and family studies. The positions of genes which have been localized to chromosome bands are indicatedby brackets.
21579
gene, and the observed distribution of alleles was compared with that of 44 previously typed loci located on 15 of the 19 autosomes. Close linkage was found between Lcat and serum esterase-2 (Es-2) on Chromosome 8, with only a single-crossover detected (TableI). Six recombinants were found between Lcat and Emu-26 (previously known as C58v-1), an ecotropic provirus known to be distal to Es-2 on Chromosome 8 (40, 41). On the basis of these 49 progeny the gene order and estimated map distances (in centimorgans) are Es-2 - 2.0 f 2.0 - Lcat - 12.2 f 4.7 - Emu-26. The location of Lcat relative to other Chromosome 8 markers is shown in Fig. 9. Several genes, including Lcat, that have been mapped to this region of mouse Chromosome 8 have human homologs on Chromosome16q (Fig. 9, Ref. 42). Other members of this conserved segment are the genes encoding mitochondrial glutamic-oxaloacetic transaminase, haptoglobin, chymotrypsinogen B, tyrosine aminotransferase, metallothionein-1 and-2, andadenine phosphoribosyltransferase. Theproximity of Lcat to the clusterof esterases (including Es-2) that hasbeen mapped to the central part of Chromosome 8 (reviewed in Ref. 43) is intriguing because LCAT catalyzes the transesterification of acylgroupsfrom lecithinto cholesterol. It is conceivable that LCAT and the esterases have evolved from a common protein. DISCUSSION
We have examined thedevelopmental, dietary, andgenetic control of LCAT gene expression. The most striking finding to emerge from the studyis that LCATgene expression is not restricted to liver, but also occurs in brain and testes. Given the physical separation, by tight cellular junctions, of brain finding may be of considsingle hybridizingband 6 kb insize, while strain 129/Jshowed and testesfrom the circulation, this erable physiological significance. We have also shown that three bands 6, 5.1, and 4.1 kb in size (data not shown). The there are significant changes in the accumulation of LCAT enzyme TaqI yielded two different patterns. Strains AKR/J, mRNA in developing tissues, in both the rat and in humans. BALB/cJ, C3H/HeJ, C57BL/6J, DBA/J, A/J, NZB/BlNJ, and SWR/J showed a single hybridizing band 6.4 kb in size, On the other hand, the expression of the gene appears to be while strain 129/J showed three bands 6.4, 4.1, and 3.9 kb in relatively resistant to dietary or drugchallenge, even though role incholesterolhomeostasis. size (data not shown). Unfortunately,only a relatively small the enzymeplaysamajor Finally, we have examined the genetic control of LCAT number of recombinant inbred strains involving the 129/J strain are available, and it was not possible to assign the expression in mice. Although some differences in expression LCAT gene (proposed locus symbol Lcat) on thebasis of the were observed among inbred strains of mice, gene mapping studies indicated that mutations of the LCAT gene are not observed polymorphismsusing theserecombinantinbred strains. It is noteworthy that digestion with several enzymes responsible for certain phenotypic variationsof the levels and yielded a single hybridizing band on Southernblots, suggest- structure of HDL that have been previously characterized. ing that there is a single copy of the LCAT gene (data not Each of these points isdiscussed in turn below. liver Extrahepatic Expression of LCAT-Prior to this study shown). We next attempted to map Lcat using DNA samples from was the only known site of LCAT synthesis (4), and thus it mRNA is present at apprea recently described (40) interspecific testcross. A newly de- was surprising to find that LCAT veloped strain (designated MEV) that possesses multiple cop- ciable levels in brain and testes. LCATproduced by the liver aconsequence anytissue ies of the endogenous ecotropic murine leukemia virus was is secreted intoplasma,andas exposed to plasma is exposed to LCAT. However, brain and crossed to the CAST/Ei strain. The latter had been inbred from Mus musculus castaneus mice trapped in Thailand. The testes haveblood-tissue barriers which would prevent the (CAST/Ei x MEV)Fl mice were mated to the BXD-32 recom- movement of macromolecules, such as LCAT, from plasma into brain or testes. All tissues, includingbrain, cansynthesize binant inbred strain to produce a testcross generation. Because of the large evolutionary divergence between M. mus- cholesterol (44); thus, we were motivated to examine LCAT could play a role in culus castaneusandlaboratorystrains, which are derived expression in brain to test whether it principally from the western European house mouse, Mus cholesterol homeostasis in the brain and to ascertain whether musculus domesticus, this testcross could be classified with some of the symptomsof LCAT-deficient patients could result respect to numerous biochemical and DNA variants. Using from impaired LCAT activity in the central nervous system the LCAT cDNA probe, a restriction variant was found that (see below). distinguished the CAST/Ei strainfrom both MEV and BXDSeveral recent papers have provided evidence that the brain 32. The probe detected 2.3- and 1.8-kb fragments in BamHI- has a lipid transport system for maintaining cholesterol hodigested CAST/Ei DNA, while 6.0- and 2.3-kb fragments were meostasis which is separate from that of plasma. Maintenance found in MEV and BXD-32 DNAs (Fig. 8). DNAs from 49 of brain cholesterol homeostasis must include both routes for testcross mice were scored for the presence or absence of the cholesterol uptake andremoval. Pitas etal. (45) have reported 1.8-kb BamHI fragment associated with the CAST/Ei Lcat that canine cerebrospinal fluid has separate populations of
2 1580
Expression of LCAT in Brain and Testes as Well as Liver
apoE- and apoA-I-containing lipoproteins, in approximately equal concentrations. They also suggested that these lipoproteins may each perform distinct roles in brain cholesterol homeostasis. Astrocytes in the brain synthesize apoE and secrete lipoprotein particles. Pitas et al. (45) suggested that the apoE-containing lipoproteins transport and redistribute cholesterol from astrocytes to the neurons and glia which express LDL receptors (46). The apoA-I-containing lipoproteins in cerebrospinal fluid have HDL-like density (45). Pitas et al. (45) suggested that the apoA-I-containing lipoproteins may function in the removal of excess cholesterol from brain. Our present findings, indicating that appreciable LCAT gene expression occurs throughout the brain,suggest that thebrain cholesterol transport system is capable of esterifying excess cholesterol for its removal from brain. Illingworth and Glover (47) have reported that cerebrospinal fluid contains low levels of active LCAT, but they assumed that thisLCAT was made in the liver and had crossed the blood brain barrier. However, the production of LCAT by cells of the central nervous system would provide a straightforward explanationfor the presence of LCAT in cerebrospinal fluid. Clinical observation of patients with familial LCAT deficiency provides support for the hypothesis that theexpression of LCAT inbrain maybe important for central nervous system function. Six out of twenty-four patients reported in a reviewof the literature on familial LCAT deficiency had central nervous system impairments, including hearing loss and sensory impairment (7). Such impairments may not occur in all patients with familial LCAT deficiency since familial LCAT deficiency is a genetically heterogeneous disease with considerable variation in LCAT activity and serum cholesteryl ester concentrationsbetween patients (7, 8, 48). DevelopmentalRegulation-The accumulation of LCAT mRNA in developing liver is coincident with the appearance of HDL in newborn rat serum and with the development of LCAT activity in human cord blood. LCAT mRNA levels in liver peak at birth and remain at high levels forapproximately 1week, after which they decrease by 3-4-fold (Figs. 4 and 6). Argiles and Herrera (49) reported that there is no HDL in the serum of 19-day-old rat fetuses and that HDLappears on the 1stday after birth, suggesting a correlation between LCAT mRNA levels and HDL production. LCAT activity in human cord blood also coincides with LCAT mRNA expression since cord blood LCAT activity is increased 2-fold in full-term infants compared with 24-30-week-old premature infants (50). In contrast with the results obtained in fetuses and newborns, the correspondence between adult levels of hepatic LCAT mRNA, LCAT activity, HDL, or apoA-I was not apparent. In particular, adults had higher levels of LCAT activity but lower levels of LCAT mRNA as compared with newborns. It is important to note that, while the increases in relative LCAT mRNA concentrations during fetal liver development/ differentiation are modest, they differ from the almost totally flat profiles observed with mRNAs encoding three cholesterogenic enzymes (HMG-CoA reductase, HMG-CoA synthase, prenyltransferase) and liver fatty acid binding protein (51). Theyare also distinct from the profiles exhibited by afetoprotein mRNA which undergoes a precipitous drop between the 18th fetal week and birth (51) and those of E - , y-, and &globin mRNAs as well as ceruloplasmin mRNA (52). Regulation in Response to Diet and Drugs-Previous data pertaining to the expression of serum LCAT activity have generally reported negative results or only veryslight changes of LCAT activity in response to drug or dietary challenge (53-
56). Ridgeway and Dolphin (57) found that serum LCAT activity in hypercholesterolemic rats was increased about 60% and that thisresulted primarily from decreased LCAT clearance rather thanincreased production. Our data indicate that LCAT gene expression is relatively resistant toextreme dietary or drug challenge. A high fat diet (1.25% cholesterol, 7.5%cocoa butter) resulted in less than a 25% change in hepatic LCAT mRNA levels in three strains of mice examined. Also, treatment of mice with cholestyramine and mevinolin for 2 days resulted in dramatic modulation of hepatic HMG-CoA reductase and HMG-CoA synthase mRNA but had no significant affect on hepatic LCAT mRNA. Thus, at the level of mRNA, LCAT expression appears to resemble the expression of the apolipoprotein B and E genes in itsrelative resistance to a high fat diet (58). Genetic Control and LCAT Gene Mapping-The mouse is being developed as a model for the genetic control of lipoprotein metabolism and atherosclerosis. The various inbred strains of mice exhibit genetic variations affecting the levels and structures of HDL, LDL, and VLDL (59). Since LCAT plays a central role in cholesterol metabolism, mutations of the gene could be responsible in part for the observed phenotypic variations in mice. Moreover, human epidemiological studies have revealed correlations between LCAT activity and both LDL/VLDL cholesterol levels (60) and HDLcholesterol levels (37). One genetic variation that is of particular interest is the Ath-I gene, located on mouse Chromosome 1, that controls HDL levels in response to a high fat diet as well as susceptibility to atherosclerosis (36). Othergenes controlling lipoprotein parameters in mice have also been characterized (38). To test whether LCAT gene expression may be responsible for these genetic variations, we examined LCAT mRNA levels in several strains ofmice and determined the chromosomal location of the mouse LCAT gene. Interestingly, the C57BL/ 6J strain, which is susceptible to atherosclerosis and has low HDL levels on ahigh fat diet, had lower hepatic LCAT mRNA levels than strainsC3H/HeJ or BALB/cJ, which are resistant and have high HDL levels. Chromosomal mapping of the LCAT gene using restriction fragmentlength polymorphisms, however, indicated that the LCAT gene is on mouse Chromosome 8, and, therefore, it is not identical to the Ath-Igene located on mouse Chromosome 1. These results strongly suggest that theLCAT gene is not responsible for the variations of HDL cholesterol between C57BL/6J and BALB/cJ and C3H/HeJ mice. The present results demonstrate close linkage of Lcat and mouse Chromosome 8 markers. Lcat is tightly linked to Lpl, Got-2, Hp, Es-2, Um, and Emu-26. The placement of these and other markers along Chromosome 8 and their approximate map distances are illustrated inFig. 9. Lcat is linked to loci which have homologs in man mapping to human Chromosomes 8 and 16. The Chromosome 8 genes include lipoprotein lipase (61) and glutathione reductase (62). LCAT, the human homolog of Lcat, is located on human Chromosome 16 (Fig. 9) (63). A large conserved linkage group exists on mouse Chromosome 8 and human Chromosome 16q (Fig. 9) (64). The conserved loci include mitochondrial glutamic oxaloacetic transaminase (human gene symbol GOT-2) (65), haptoglobin (human gene symbol HP) (651, uvomorulin (human gene symbol UVO) (66), tyrosine aminotransferase (human genesymbol TAT) (64), adenine phosphoribosyltransferase (human gene symbol APRT) (671, chymotrypsinogen B (human gene symbol CTRB) (64), and metallothionine-1 and -2 (human gene symbols MT-1, MT-2) (64). The human genefor cholesteryl ester transfer protein (human
Expression of LCAT in Brain and Testesas Well as Liver
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Arch. Biochem. Biophys. 261, 330-335 gene symbol CETP) hasbeen localized to chromosome 16q21 Jauhiainen, M., Stevenson, K. J., and Dolphin, P. J. (1988) J . (68). To date there is no known mouse homolog for CETP, 32. Biol. Chem. 263,6525-6533 but it ispossible that if one exists it will also map to the distal 33. Guidoni, A., Benkouka, F., de Caro, J., and Rovery, M. (1981) part of Chromosome 8. Biochim. Biophys. Acta 660, 148-150 34. McLean, J., Wion, K., Drayna, D., Fielding, C., and Lawn, R. Acknowledgments-We wish to thank Susan Zollman and Dianna Quon for excellent technical assistance. REFERENCES 1. Glomset, J. A. (1962) Biochim. Biophys. Acta 65, 128-135 2. Aron, L., Jones, S., andFielding, C. J. (1978) J. Biol. Chem. 253, 7220-7226 3. Osuga, T., and Portman, 0. W. (1971) Am. J. Physiol. 220, 735741 4. Simon, J. B., andBoyer, J. L. (1970) Biochim. Biophys. Acta218, 549-551 5. Fielding, C. J., and Fielding, P. E. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 3911-3914 6. Fielding, C. J., and Fielding, P. E. (1982) Med. Clin. North. Am. 66,363-373 7. Shojania, A. M., Jain, S. K., and Shohet,S. B. (1983) Clin. Znuest. Med. 6,49-55 8. Teisberg, P., and Gjone, E. (1981) Acta Med. S c a d . 210, 1-2 9. MacDonald, R. J., Swift, G. H., Przybyla, A. E., and Chirgwin, J. M. (1987) Methods Enzymol. 152, 219-227 10. Ricca, G. A,, Hamilton, R. W., McLean, J. W., Conn, A., Kalinyak, J . E., andTaylor, J. M. (1981) J. Biol. Chem. 256, 10362-10368 11. Okayama, H., and Berg, P. (1982) Mol. Cell. Biol. 2, 161-170 12. Gubler, U., and Hoffman, B. J. (1983) Gene (Amst.)25, 263-269 13, Hyunh, T. V., Young, R. A., and Davis, R. W. (1985) in D N A Cloning Techniques, A Practical Approach (Glover, D. M., ed) Vol. I, pp. 49-78, IRL Press, Oxford 14. McLean, J., Fielding, C., Drayna, D., Dieplinger, H., Bear, B., Kohr, W., Henzel, W., and Lawn, R. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2335-2339 15. McLean, J . W., Tomlinson, J. E., Kuang, W.-J., Eaton, D. L., Chen, E. Y., Fless, G.M., Scanu, A.M., and Lawn, R. M. (1987) Nature 330, 132-137 16. Schwartz, A. L., Raiha, N. C. R., and Rall, T. W. (1975) Diabetes 24,1101-1112 17. Tanimura, T., Nelson, R., Hollingsworth, T. T., and Shepard, T. H. (1971) Anat. Rec. 171, 227-236 18. Schwartz, A. L., Steer, C. J., and Kempner, E. S. (1984) J . Biol. Chem. 259,12025-12029 19. Paigen, B., Marrow, A., Brandon, C., Mitchell, D., and Holmes, P. (1985) Atherosclerosis 57, 65-73 20. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18, 5294-5299 21. Bailey, J. M., and Davidson, N. (1976) Anal. Biochem. 70, 75-85 22. Church, G. M., and Gilbert,W. (1984) Proc.Natl.Acad.Sci. U. S. A. 81, 1991-1995 23. Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13 24. Clarke, C. F., Fogelman, A. M., and Edwards, P. A. (1985) J . Biol. Chem. 260, 14363-14367 25. Mehrabian, M., Callaway, K. A., Clarke, C.F., Tanaka, R.D., Greenspan, M., Lusis, A. J., Sparkes, R. S., Mohandas, T., Edmond, J., Fogelman, A.M., and Edwards, P. A. (1986) J . Bid. Chem. 261,16249-16255 26. Demmer, L. A., Levin, M. S., Elovson, J., Reuben, M. A,, Lusis, A. J., and Gordon, J. I. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,8102-8106 27. Elshourbagy, J. A., Boguski, M. S., Liao, W. S. L., Jefferson, L. S., Gordon, J. I., and Taylor,J. M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,8242-8246 28. Thomas, P. S. (1980) Proc. Natl. Acad. Sci. U. S. A . 77, 52015205 29. Ruhin, D. C., Ong, D. E., and Gordon, J. I. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1278-1282 30. McLean, J. W., Fukazawa, C., and Taylor, J. M. (1983) J. Biol. Chem. 258,8993-9000 31. Farooqui, J. Z., Wohl, R. C., KBzdy, F. J., and Scanu,A. M. (1988)
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