3-hydroxy-3-methylglutaryl-coenzyme a reductase of the

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 34, Issue of December 5, pp. 18411-18418,1988 Printed in U. S. A.

3-Hydroxy-3-methylglutaryl-coenzyme A Reductase of the Sea Urchin Embryo DEDUCED STRUCTURE AND REGULATORY PROPERTIES*

(Received for publication, June 16, 1988)

Harold D. Woodward, Jamie M. C. Allen, and William J. LennarzS From the Department of Biochemistu and Molecular Biology, University of Teras M . D. Anderson Cancer Center, Houston, Te&s 77030

3-Hydroxy-3-methylglutaryl-coenzyme A reductase activity is developmentally regulated in the sea urchin Strongylocentrotuspurpuratue (Woodward, H. D., Al- len, J. M. C., and Lennarz, W. J. (1988) J. Biol. Chern. 263,2513-2517). To study the structural and regulatory properties of this enzyme, we isolated and sequenced a 3-kb cDNA encoding the sea urchin embryo reductase. The deduced amino acid sequence of this cDNA predicted a protein structure consisting of a hydrophobic N-terminal region containing seven potential membrane-spanning domains and a somewhat less hydrophobic C-terminal domain joined by a hydrophilic linker region. Comparison with reductase from mammalian sources revealed that the N-terminal membrane domain and the C-terminal cytoplasmic domain exhibited high sequence similarity, whereas the domain that linked these two showed little or no sequence similarity. We investigated the possibility that sterols or sterol derivatives might be involved in the marked change that occurs in the level of reductase activity over development. Enzyme activity and reductase mRNA levels measured in extracts from embryos cultured in the presence of cholesterol, 25-hydroxycholesterol, dolichol, or mevalonic acid were found to be virtually unchanged as compared to control embryos. Similar experiments with mevinolin, a competitive inhibitor of reductase, failed to show a drug-induced change in enzyme or mRNA level. Thus, despite structural similarities the sea urchin embryo enzyme differs markedly from the mammalian enzyme with respect to regulation, since its level is neither repressed by sterols nor induced by mevinolin. Moreover, it appears unlikely that sterols or sterol derivatives play a role in the striking change in the level of this enzyme that occurs during development.

Mammalian 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA’ reductase) is a 97-kDa transmembrane

* This work was supported by National Institutes of Health Grant GM 33184 (to W. J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertkement” 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 GenBankTM/EMBL Data Bank with accession number($ 504200.

$ A Robert A. Welch Professor of Chemistry, gratefully acknowl- edges the Robert A. Welch Foundation and Welch Grant G-1088. To whom correspondence should be addressed.

The abbreviations used are: HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; TLC, thin layer chromatography; kb, kilobase; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; bp, base pairs; NaDod SO,, sodium dodecyl sulfate.

glycoprotein of the endoplasmic reticulum (1-3). The mammalian enzyme consists of an amino-terminal membrane domain of 339 residues and a carboxyl-terminal domain of 338 residues joined by a linker region of 110 residues (2). The C- terminal domain projects into the cytosol and contains the catalytic site. Modeling studies of the deduced primary structure of mammalian HMG-CoA reductase suggest that the amino-terminal region contains seven distinct membrane- spanning segments. The linker region contains proteolytic sites, which, when acted on by endogenous or exogenous proteases, release a soluble form of catalytically active reductase.

HMG-CoA reductase is the rate-controlling enzyme of polyisoprenoid biosynthesis in mammals (4) and catalyzes the synthesis of mevalonate, the precursor common to cholesterol, dolichol, and coenzyme Q. We have demonstrated that de rwuo synthesis of dolichol is essential for normal development in the sea urchin embryo (5,6). This biosynthetic requirement is a reflection of the need for dolichyl phosphate, which serves as a carrier of oligosaccharide chains that are ultimately N- linked to proteins required for normal gastrulation (6). Com- pactin, a competitive inhibitor of HMG-CoA reductase, inhibits both dolichol and cholesterol synthesis in the embryo; the consequence is a block in N-linked glycoprotein synthesis and concomitant cessation of normal development just prior to gastrulation. This block in development can be overcome by adding dolichol or dolichyl phosphate to the culture medium containing compactin (5).

Although sterol-mediated regulation of HMG-CoA reductase in mammalian cells has received a great deal of attention (4), little is known about control of expression of this enzyme during embryonic development. Because the studies discussed above suggested that regulation of HMG-CoA reductase activity has direct consequences on early development of the sea urchin embryo, earlier we partially characterized sea urchin embryo HMG-CoA reductase and measured its activity and some of its physical properties during embryogenesis (7). Enzyme activity was found to increase approximately 200- fold during development from the unfertilized egg to the pluteus stage embryo. The finding that, unlike the mammalian enzyme, the sea urchin embryo HMG-CoA reductase could not be solubilized by detergents or protease treatment, led us to investigate the possibility that the structure of the sea urchin embryo and mammalian enzymes might be very different. As a first step in characterizing the sea urchin embryo reductase polypeptide, a genomic clone was identified which hybridized to a cDNA-encoding hamster HMG-CoA reductase (7). Analysis of sea urchin embryo reductase mRNA levels using a restriction fragment derived from this clone revealed a 5.5-kb poly(A+) mRNA whose level increased 15-

18411

18412 Sea Urchin HMG-CoA Reductase

fold during development from the egg to the gastrula stage. In the present study we have isolated and sequenced a sea

urchin embryo HMG-CoA reductase cDNA. Analysis of the deduced amino acid sequence revealed considerable similarity with hamster reductase. A sequence difference between the urchin and hamster reductases in the region known to be cleaved proteolytically in the hamster reductase may account for the inability of sea urchin embryo reductase activity to be released in soluble, active form by digestion with proteases. Sequence comparisons of the transmembrane domains of the sea urchin embryo and hamster reductases suggested that, as in the hamster, sterols might modulate the levels of sea urchin embryo reductase activity. However, examination of the ef- fects of sterols on reductase activity and mRNA levels, cou- pled with earlier measurements of the sterol content in the developing embryo (6), lead us to conclude that in the developing sea urchin embryo sterols do not play a role in the regulation of HMG-CoA reductase.

EXPERIMENTAL PROCEDURES

Materials-[a-32P]dCTP (3000 Ci/mmol), [(u-~'S]~ATP (3000 Ci/ mmol), and Hybond-N filters were purchased from Amersham Corp. ["CIHMG-CoA (55.5 mCi/mmol), [3H]cholesterol (51.9 Ci/mmol), and [3H]25-hydroxycholesterol (87.0 Ci/mmol) were purchased from Du Pont-New England Nuclear. Cholesterol and 25-hydroxycholesterol were purchased from Steraloids, Inc. and used without purifi- cation. Dolichol, mevinolin, and mevalonic acid lactone were purchased from Sigma. Silica Gel-G polyethylene TLC sheets were purchased from Scientific Products. The oligonucleotide-labeling kit was purchased from Pharmacia LKB Biotechnology Inc. and used according to the supplier's specifications. The Sequenase dideoxy- sequencing kit was purchased from United States Biochemical Corp. The reverse DNA-sequencing primer was purchased from New Eng- land BioLabs. The Erase-A-Base System was purchased from Pro- mega Biotec and used according to the supplier's specifications. Klenow DNA polymerase, pUC18, and RNA molecular weight markers were purchased from Bethesda Research Laboratories. T4 DNA ligase was purchased from IBI. Restriction enzymes were purchased from commercial sources and used according to the supplier's specifications. All other chemicals were purchased from Sigma.

The sea urchin cDNA library (XZAP) was constructed by Strata- gene. The library was made from 14.5-h sea urchin embryo poly(A+) RNA by cloning cDNA into the EcoRI site of the bacteriophage vector XZAP. Escherichia coli strains BB4 and XL-1 Blue were used as hosts to propagate the library. Two cDNAs used for quantitating mRNA levels, SPEC-2 DNA and a mitochondrial rRNA cDNA, were generous gifts of Dr. William Klein, Department of Biochemistry and Molec- ular Biology, University of Texas M. D. Anderson Cancer Center, Houston, TX.

Culturing of Embryos-Sea urchins (Strongylocentrotus purpura- tus) were purchased from Pacific Biomarine Laboratories, Inc. (Ven- ice, CA). Animals were maintained, gametes collected, and embryos cultured as previously described (6). Mevinolin, cholesterol, 25-hydroxycholesterol, dolichol, or mevalonic acid lactone were added to fertilized eggs 1 h after fertilization. Embryos were cultured contin- uously in the presence of these compounds until harvesting. Mevi- nolin was added to cultures from a 20 mM solution in dimethyl sulfoxide; cholesterol, 25-hydroxycholestero1, and dolichol dissolved in ethanol were added so that the final ethanol concentration was 0.25%. Mevalonic acid lactone was added from a 10 mM solution in water. Vitamin E (5 pg/ml) was included in cultures containing sterols to inhibit their autoxidation.

Isolation of Crude Microsomes-Prior to preparation of microsomes, embryos grown to the indicated stages were washed three times with 50 volumes of artificial sea water and then dissociated into single cells by repeated washing with Cap+, Me-f ree sea water containing 25 mM EGTA (8). Microsomes were then prepared as previously described (7). HMG-CoA reductase activity in the microsomes was measured as described previously (7). Protein was determined by the method of Peterson (9).

Incorporation of Sterols into Embryos-The uptake and metabolic conversion of the exogenous sterols, cholesterol, and 25-hydroxycholesterol were assessed by measuring the conversion of 3H-labeled sterols into metabolites exhibiting altered mobilities on TLC. Each

labeled sterol in ethanol (100 $ 3 ; 0.46 pg of [3H]25-hydroxycholesterol or 0.75 pg of [3H]cholesterol) was added to 20 ml of a 2% culture of 23-h embryos in a Petri dish. The cultures were incubated for 16 h at 14 "C, and then the embryos were washed three times with 50 volumes of ice-cold artificial seawater. The washed embryos were then extracted three times with 2 volumes of 2:l chloroform/methanol. The chloroform/methanol extract was analyzed by chromatography on polyethylene TLC sheets in Solvent System A (95:5 chloroform/ethanol) or Solvent System B (9:l benzene/ethylacetate). Standard cholesterol, 25-hydroxycholestero1, and cholesteryl ester were detected by exposure to Ip vapors. Regions of 1 cm were cut out and counted in Liquiscint.

Recombinant DNA Methodology-The maintenance and propaga- tion of plasmids and bacteriophages, as well as the isolation of vectors and restriction enzyme mapping, were performed as described by Maniatis et al. (10). The hybridization conditions for screening the cDNA library consisted of prehybridization at 42 "C in 50% formamide, 5 X SSPE (0.75 M NaCl, 50 mM sodium phosphate, pH 6.8, 5 mM Na2EDTA), 5 X Denhardt's solution (0.1% Ficoll (Mr -400,000), 0.1% polyvinylpyrrolidene, and 0.1% bovine serum albumin), 0.5% NaDodSO4, 100 pg/ml sheared salmon sperm DNA, and 1 pg/ml poly(A); hybridization was performed at 42 "C in the same buffer containing 10% (weight/volume) dextran sulfate and 1 X lo' cpm/ml denatured, radiolabeled probe. To remove nonspecific hybridization, washing was performed at 68 "C in 0.5 X SSC (0.075 M NaCl, 0.0075 M sodium citrate, pH 7.0) and 1% NaDodS04.

Library Screening-DNA from 600,000 plaques of the sea urchin XZAP cDNA library was transferred to Hybond N filters. A 0.22-kb Hinff restriction fragment derived from the sea urchin genomic clone described previously (7) was used to screen these plaques. This DNA fragment contains sequence similarity to a hamster reductase cDNA. The restriction fragment was isolated by agarose gel electrophoresis, radiolabeled to 1.1 X lo9 cpm/pg by the oligonucleotide-labeling method (11) and hybridized to the filters under high stringency as described above. Phage that gave a positive signal in the initial screen were plaque-purified twice more. Seven different phages were isolated which cross-hybridized with the 0.22-kb Hinff sea urchin genomic DNA restriction fragment. The cDNA insert sizes in these clones were 0.5, 0.8, 1.3, 1.4, 1.8, 2.0, and 3 kb. All seven of these cDNAs cross-hybridized with the cDNA containing the 3-kb insert. One of these cDNA clones, XZ3, containing the 3-kb cDNA insert, was chosen for further analysis. The excision process that converts the phage XZAP DNA to Bluescript plasmid DNA was performed according to Stratagene's specifications. All seven of the phage DNAs were converted to their corresponding Bluescript plasmid DNAs. The Blue- script form of XZ3, designated pXZ3, was used for DNA sequencing.

Construction of Deletion Clones of pU3 for DNA Sequencing-The pXZ3 cDNA was digested with EcoRI and BglII, and the four restriction fragments generated (1.7,0.7,0.24, and 0.36 kb) were isolated by agarose gel electrophoresis. These restriction fragments were converted to blunt end fragments with Klenow DNA polymerase and ligated into SmaI-digested pUC18. Ligation mixtures were used to transform competent JM109 cells. DNA from cells containing the pUC18 derivatives was isolated by the procedure of Krieg and Melton (12).

The pUC18 DNA clones containing the 1.7- and 0.7-kb pXZ3 restriction fragments were used for construction of deletion clones. The clone containing the 1.7-kb DNA fragment was digested with PstI and XbaII; the clone containing the 0.7-kb DNA fragment was digested with SphI and XbaI. Each of these double-digested DNAs was treated with Exonuclease I11 and S1 nuclease under conditions recommended in the Erase-A-Base System that remove 200-250 nucleotides from the 5' end of each DNA at 30-s intervals. The DNAs at each 30-s time point were subsequently religated and transformed into competent JM109 cells according to the Erase-A-Base System specifications. DNA from these deletion clones was isolated by the procedure of Krieg and Melton (12) and used directly for sequencing.

DNA Sequencing-The pXZ3 cDNA clone was sequenced utilizing two different strategies. Initially, approximately 250 nucleotides of the 5' and 3' ends of the cDNA were sequenced directly in the Bluescript vector using the m13 universal primer and the reverse sequencing primer. Approximately 250-300 nucleotides of the 5' and 3' ends of the pUC18 clones containing the 1.7-kb and 0.7-kb DNA restriction fragments of pXZ3 were also sequenced utilizing these primers. The remainder of the DNA sequence of pXZ3 was determined by sequencing one strand of each of the DNAs from the deletion subclones. Each 200 bp of DNA of pXZ3 was sequenced at least two times from DNA isolated from different deletion clones. In all cases,

Sea Urchin HMG-CoA Reductase 18413

only one DNA strand was sequenced from each deletion clone. TWO oligonucleotide primers (18-mers) were generated by the Oligonucle- otide Synthesis Laboratory, University of Texas M. D. Anderson

approximately 400 bp of DNA in the 3' portion of pXZ3. The DNA Cancer Center, Houston, TX. These primers were used to sequence

was sequenced by the dideoxy chain termination method (13), using a Sequenase DNA sequencing kit and [ c ~ - ~ ~ S ] ~ A T P . The DNA sequence was computer analyzed using MICROGENIE and PC GENE software packages.

RNA Isolation and Analysis of Sea Urchin HMG-CoA Reductase mRNA Leuels-RNA was isolated from sea urchin eggs and embryos at the indicated stages of development and electrophoresed and transferred to Hybond-N filters as described by Woodward et al. (7). RNA from different developmental stages of the sea urchin embryo or RNA from mevinolin-, mevalonic acid lactone-, or polyisoprenoid- supplemented cultures was hybridized with the 3-kb restriction fragment isolated from pXZ3 and radiolabeled to 9 X 10' cpmlpg by the oligonucleotide-labeling method (11). The hybridization conditions consisted of prehybridization at 42 "C in 50% formamide, 5 X SSPE, pH 6.8,5 X Denhardt's, 0.5% NaDodS04, 100 pg/ml sheared salmon sperm DNA, and 1 pg/ml poly(A) followed by hybridization in the same buffer containing 10% dextran sulfate and denatured, radiolabeled probe. To remove nonspecific hybridization the filter was washed at 68 "C in 0.4 X SSC, 1% NaDodS04. RNA levels were quantitated by densitometric scanning. Reductase RNA levels from the developmental time course were normalized to the levels of mitochondrial rRNA (which remains constant during development). Reductase mRNA levels in polyisoprenoid-supplemented embryos were normalized to SPEC-2 1.9-kb mRNA. In these experiments, the RNA was initially hybridized with the pXZ3 3-kb cDNA fragment. After autoradiography at -80 "C for 10 days, the RNA was rehybrid- ized with the SPEC-2 DNA restriction fragment, and the results with the former probe were normalized against the latter.

RESULTS

Isolation of a Sea Urchin Embryo HMG-CoA Reductase cDNA Clone-In a previous report, a sea urchin embryo genomic DNA fragment was sequenced and shown to exhibit 70% deduced amino acid sequence identity to a 47-amino acid segment of the C-terminal region of hamster HMG-CoA reductase that contains the catalytic site (7). To obtain a more detailed understanding of the structure of sea urchin embryo reductase, we undertook the isolation of a cDNA clone. A sea urchin embryo cDNA library prepared from poly(A+) RNA from 14.5-h embryos (midblastula stage) was hybridized with the previously described 0.22-kb HinfI genomic DNA restriction fragment (7). Fifteen positive plaques were initially identified after screening under high stringency. These phage were plaque purified two additional times and then converted to their corresponding Bluescript plasmid derivatives. Seven different cDNA clones that cross-hybridized with the genomic restriction fragment were identified. The BglII restriction map of one of these clones, pXZ3, con-

1000 2000 m '

3ooo I , I

"

5' I I 1

k- - *", - FIG. 1. Restriction map of pXZ3 and strategy used to se-

quence the DNA. Scale on the top designates the nucleotide posi- tions in base pairs (Bp) . The BgnI (B) restriction map below this is indicated with 5' to 3' direction from left to right. The protein coding region is indicated by a heauy bar. The arrows above and below the map indicate the direction and extent of sequence determination using deletion clones (arrows above the map), by sequencing 5' and/ or 3' ends of pXZ3 cDNA or its BgZII fragments (arrows below the map), or by using synthetic oligonucleotide primers (dashed arrows below the map).

3' - ""4

taining a 3-kb cDNA insert is shown in Fig. 1. The 0.7-kb BglII restriction fragment in pXZ3 cross-hybridized with the genomic DNA restriction fragment. Partial restriction mapping of the other six cDNA clones suggested that they were 5"truncated derivates of pXZ3.

DNA Sequence Analysis-The nucleotide sequence of pXZ3 was determined utilizing the strategy outlined in Fig. 1; this strategy involved sequencing DNA deletion clones and EcoRI/ BglII subclones of pXZ3. In Fig. 2 is shown the complete nucleotide sequence and the deduced amino acid sequence of pXZ3. The nucleotide sequence of 2966 bp contained a single open reading frame of 2796 bp (+1 to +2,796) encoding a 932- amino acid protein (Mr 101,049). This open reading frame was flanked by 27 and 143 bp, respectively, of 5'- and 3'- untranslated sequences. The 5"untranslated sequence contained stop codons in all three reading frames. The 3'-untranslated region lacked a consensus polyadenylation signal (AATAAA) and a poly(A) tail. One of the other cDNA clones that cross-hybridized with pXZ3 was found by nucleotide sequence analysis to contain approximately 900 bp of 3'- coding sequence as well as 400 bp of 3"untranslated sequence containing the poly(A) tail (data not shown). Comparison of the complete nucleotide sequence of pXZ3 with hamster (3, 14) and human (15) reductases indicated 56 and 57% identity, respectively. No significant sequence similarity was found between the limited amount of either of the untranslated regions of pXZ3 and the corresponding hamster and human sequences. The hamster reductase contains a 3"untranslated region of approximately 2 kb (3). Since the cDNA clone for sea urchin embryo reductase contains all of the coding sequence and is 3 kb versus 5.5 kb for the mRNA, it is probably missing additional 3"untranslated sequence.

Analysis of the Deduced Amino Acid Sequence-A comparison of the deduced amino acid sequence of pXZ3 and hamster HMG-CoA reductase is shown in Fig. 3. The deduced amino acid sequence of pXZ3 shares 56% identity with hamster HMG-CoA reductase. When conservative substitutions are included, the overall amino acid similarity with hamster reductase increases to 69%. We conclude from this comparison that pXZ3 encodes sea urchin embryo HMG-CoA reductase.

The method of Kyte and Doolittle (16) was applied to the deduced amino acid sequence to predict the hydropathy plot shown in Fig. 4. The primary structure of sea urchin embryo HMG-CoA reductase was predicted to consist of a hydrophobic N-terminal region (amino acid residues 1-340) connected to a C-terminal region (amino acid residues 537-932) by a 196-residue hydrophilic linker region (amino acid residues 341-536). The N-terminal and C-terminal regions share 61 and 65% identity, respectively, with hamster HMG-CoA reductase. The most divergent region of the sea urchin embryo reductase is contained in the linker region, which shares only 30% identity to the corresponding region of hamster reductase. The hydrophobic N-terminal region contains seven potential membrane spanning domains (Fig. 4). The location of each of these domains in the primary structure of the polypeptide (Fig. 3) and their similarity to hamster reductase are shown in Table I. It is remarkable that if conservative substitutions are allowed, the sequence similarity ranges from a low of 60% to a high of 100%. The N-terminal region of the sea urchin enzyme has one additional stretch of 16 hydrophobic amino acids (residues 262-277) between membrane-spanning domains 6 and 7. However, the length of this segment is probably insufficient for it to span a membrane bilayer (17).

It is evident from Fig. 4 that the C-terminal region (amino acid residues 537-932) of sea urchin HMG-CoA reductase is nearly as hydrophobic as the N-terminal region. The most

18414

FIG. 2. Nucleotide sequence of pXZ3 corresponding to sea urchin embryo HMG-CoA reductase and the predicted amino acid sequence of the protein. Position 1 corresponds to the first nucleotide of the ATG codon for the initiator methionine. The nucleotides on the 5' side of position 1 are indicated by negative numbers. The predicted amino acid sequence is shown below the nucleotide sequence. The amino acids are numbered beginning with the initiator methionine. The four potential N-glycosylation sites are indicated in boxes.

63 21

147 49

231 77

315 105

399 133

483 161

561 189

651 217

735 245

819 273

903 301

987 329

1071 357

1155 385

1239 413

1323 4 4 1

1407 469

1 4 9 1 497

I575 525

1659 553

1743 581

1827 609

1911 637

1995 665

2079 693

2163 721

2247 749

2331 777

2415 805

2499 833

2583 861

2667 889

2 7 5 1 917

2846 932

2939

hydrophilic region is the linker domain, encompassed in residues 341-536. A segment of this domain (residues 365-385 in Fig. 3) is enriched in a PEST sequence that is common to many proteins with rapid turnover times (18). However, this sequence does not correspond either in location or amino acid residues to that found in hamster reductase (18). The linker region contains a long stretch of acidic amino acids at posi- tions 436-449 (EDEEEEVIKEEEVE) that is absent from the hamster reductase. Sea urchin embryo reductase contains three potential N-glycosylation sites in the C-terminal region at residues 850, 886, and 930, and one potential N-glycosylation site in the N-terminal region at residue 279.

Analysis of Sea Urchin Embryo HMG-CoA Reductase mRNA during Development-The 0.7-kb BglII DNA fragment from pXZ3 was hybridized with RNA from four different developmental stages of the embryo. In agreement with earlier

work using a genomic fragment from the C-terminal domain (7), the 0.7-kb BglII DNA fragment detected a 5.5-kb mRNA that increased approximately 15-fold from the egg to the gastrula stage (Fig. 5).

Effect of a Competitive Inhibitor on Sea Urchin HMG-CoA Reductase-Previous studies have revealed that the mammalian enzyme is competitively inhibited by compactin (19) and the more potent substrate analog mevinolin (20). This inhibition results in hyperinduction of enzyme levels as a result of a marked increase in the level of reductase mRNA. As previously reported using compactin (5, 6), mevinolin was found to block normal development of the sea urchin embryo and cause exogastrulation. Both the morphological effect on development and the inhibition of acetate incorporation into cholesterol were dependent on the concentration of mevinolin (data not shown). Microsomes isolated from embryos cultured

H a m s t e r V E;I;E S A ; i I d A i F V L G A U T S P P V A A - -;R;T Q M . C i A - j ! j - [ L P - 5 E D N E i C l Q a E 5 A E O A K 4 5 9 U r c h i n S H ~ S ~ V K P , A , R F T I G S S G S G S E D E E E E V I ~ K ~ E E E ~ ~ E U ' ~ ~ L E T E L K A P R P M P E L L E I L N V G - K G P N 4 7 6

H a m s t e r F U , i I A B ! j I QONP K H I P A V K L E[TLL-M-J;T H [ R [ V [ S - ! I R - C - L ; ] T K U E P S L S j J Q V D R O M N U 5 1 9 U r c h i n A L - 7 0 0 P Q L L V G K H I P A V K L E N:i-L"O:N P E R G V 3 - i R R Q i-I 5 K L L P I T D'A'L E K L P V A S Y 0 V 5 5 3 6

H a m s t e r L u M l G A C C E N V I G V M P l - I i m C L 1 K u ; Q V P M A T T E G C L V A S T N R G C R A[!:G L G [ E - E I A S 5 7 9 U r c h i n F V S G A C C E N V I G V M P ~ ~ 7 P V G V A G P L L L D G Q E F , Q V P M A T T E G C L V A S T N R G C R A i ~ R S A G G I H 5 9 6

U r c h i n ~ i 4 ~ D V K G i : N I H G S G L N A S L A R 1 V C A T V H A G E L S L H S : A L A A G H L V K S H M K H N R S A L N I A S P L 8 9 6 H a m s t e r ; ~ ~ G ~ Q ~ A ~ ~ K D N P ~ E ~ R ~ l G ~ V M A G E L S L ~ ~ A ~ ~ ~ ~ ~ ~ V ~ t - - K ~ N - - ~ 8 7 5

T C T A N I A " S I 9 3 2 K l K K g _ A : 8 8 7

FIG. 3. Comparison of the deduced amino acid sequences of sea urchin embryo and hamster reductases. Solid boxes indicate regions of amino acid sequence identity. Dashed boxes indicate conservative amino acid substitutions. Dashes indicate absent residues.

4011 2 3 4 5 6 7

-40

1 100 200 300 400 500 600 700 SO0 900 Amino Acid Residue

FIG. 4. Hydropathy plot of the deduced amino acid sequence of sea urchin embryo HMG-CoA reductase. The method of Kyte and Doolittle (16) was used to generate this plot using an interval of 15 amino acids. The vertical axis indicates the units of hydropathicity; the horizontal line indicates the amino acid residues. The horizontal line at the hydropathy value of -5 divides hydrophobic regions above from hydrophilic regions below. The seven predicted membrane spanning domains are numbered 1-7.

in the presence of mevinolin expressed only 16% of the HMG- CoA reductase activity found in control microsomes. Because mevinolin is only sparingly soluble in seawater, we reasoned that it might remain associated with the embryos and contam- inate the subsequently isolated microsomes. In fact, mixing experiments with control microsomes and microsomes from mevinolin-treated embryos suggested that this was the case. Inhibition by contamination with mevinolin should be overcome by competition with excess substrate. Therefore, micro-

TABLE I Predicted N-terminal membrane-spanning domains of sea urchin

embryo HMG-CoA reductase % Similarity with

hamster Membrane Residues Length reductase membrane

domains domain in residues Absolute Conservative

substitutions

1 12-48 37 76 86 2 61-80 20 40 75 3 89-119 31 77 84 4 125-148 24 aa 100 5 162-185 24 71 83 6 193-222 30 77 93 7 304-343 40 35 60

somes from nonsupplemented and mevinolin-supplemented embryos were incubated with increasing concentrations of HMG-CoA prior to measuring in vitro enzyme activity. The results in Fig. 6 indicate that microsomal reductase from control and mevinolin-supplemented embryos, when incubated with increasing amounts of HMG-CoA, reached the same maximal level of reductase, i.e. no more (or less) activity was present in microsomes from mevinolin-cultured embryos than in control microsomes at saturating levels of HMG-CoA (-4 mM). These results suggested that although mevinolin was a competitive inhibitor of the sea urchin reductase, it had no effect on the level of HMG-CoA reductase; the enzyme was neither repressed nor, as in the case of the hamster (2, 19), induced by cultivation in the presence of this drug. Similarly, when the level of reductase mRNA in control and mevinolin-treated embryos was measured as described in Ta-

18416 Sea Urchin HMG-CoA Reductase

9.5 - 7.5 - 4.4 - 2.4 -

- 1.3 -

FIG. 5. Analysis of the level of sea urchin embryo HMG- CoA reductase mRNA during development. RNA from eggs, 16- cell embryos (5.5 h after fertilization), blastula stage embryos (23 h after fertilization), and gastrula stage embryos (48 h after fertilization) was electrophoresed and transferred to Hybond N filters as described under “Experimental Procedures.” The RNA was hybridized as described under “Experimental Procedures.” The washed filter was exposed to Kodak X-omat AR at -80 “C for 12 h. The arrow indicates the location of the 5.5-kb mRNA. The sizes of the markers are expressed in kb. Tot = Total RNA; PA+ = poly(A+) RNA.

TABLE I1 Effect of sterols, polyisoprenols, and mevalonic acid on the level of

HMG-CoA reductase activity and mRNA

Supplementation HMG-CoA reductase Relativeb specific activity” mRNA level

cpm rneualonatel minlmg protein

None 15,300 1.0 Cholesterol (20 Fg/ml) 19,900 1.1 25-Hydroxycholesterol 15,200 1.0

Dolichol (2 pg/ml) 17,500 1.1 Mevalonic acid lactone 20,600 1.1

(10 rg/ml)

(1 mM) Enzyme activity represents the average of duplicate assays from

two different preparations of sea urchin embryo microsomes. *The relative mRNA levels are expressed as the ratio of 5.5-kb

reductase mRNA to 1.9 kb SPEC-2 mRNA as determined by densitometric scanning of two different preparations of RNA from supplemented embryos. The value obtained in the control (none) was set equal to 1.0.

’O h Cholesterol

Cholesterol

p 40

5 m 5 1 / 4 U P

0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2 3.6 4.0

m M HMGCoA

FIG. 6. Effect of HMG-CoA on HMG-CoA reductase activity in microsomes isolated from control (U) and mevinolin- treated (o“-o) embryos. Embryos were grown in the presence or absence of 20 p~ mevinolin for 48 h and microsomes then isolated as described under “Experimental Procedures.” Microsomes were incubated with increasing concentrations of HMG-CoA for 15 min a t 37 “C prior to assaying reductase activity. The results represent the specific activities from duplicate assays in two different preparations of microsomes from control and mevinolin-treated embryos.

ble I1 (see below), it was found that mevinolin did not cause any change.

Effect of Sterol Supplementation on Sea Urchin HMG-CoA Reductase-Sterol regulates mammalian HMG-CoA reductase at the level of transcription (19) as well as post-translationally (21-23). Since studies with the hamster reductase have provided evidence that the N-terminal region of hamster reductase is essential for post-translational regulation by sterols (21-23), and because we found marked similarity in sequence between urchin and hamster in this region, we investigated the possibility of sterol regulation of sea urchin reductase. Embryos were cultured in the presence of cholesterol, 25-hydroxycholesterol, dolichol, or mevalonic acid lactone for 48 h. None of the compounds added had any effect

2 4 6 8 10 12 14

cm from Origin

FIG. 7. Thin layer chromatography of the chloroform/methanol extract of embryos incubated for 16 h with r3H]25- hydroxycholesterol. The thin layer Silica Gel-G polyethylene sheet was chromatographed in solvent B (benzene/ethylacetate, 9:l) until the solvent front migrated 15 cm. Standards of cholesterol and 25- hydroxycholesterol (25-OH cholesterol) were visualized by exposure to I I vapors. One-cm segments were cut from the chromatograms and counted in Liquiscint. The arrows denote the position of migration of the indicated standards.

on development. After 48 h, microsomes were isolated and HMG-CoA reductase activity and mRNA levels were measured.

The results shown in Table I1 reveal that cultivation of embryos in the presence of cholesterol, 25-hydroxycholestero1, dolichol, or mevalonate had no effect on either the level of microsomal HMG-CoA reductase activity or the level of reductase mRNA. Although it was previous:y shown that mevalonate is readily taken up by embryos and that it inhibits reductase activity in cultured cells (24, 25), little was known about uptake of the hydrophobic lipids. Radiolabeling experiments using [3H]cholesterol or [3H]25-hydr~xycholesterol indicated that these labeled sterols could be taken up by embryos and converted to metabolites having different mobilities than the starting lipids, as analyzed on thin layer chromatograms. A representative chromatogram of [3H]25- hydroxycholesterol-labeled embryos is shown in Fig. 7; approximately 30% of the added [3H]25-hydroxycholestero1 was recovered in the chloroform/methanol extract. Upon TLC of this extract using Solvent System B, 63% of the radioactivity

Sea Urchin HMG-CoA Reductase 18417

remained at the origin, 7% migrated with 25-hydroxycholesterol, and 25% migrated with a mobility comparable to that of cholesterol. Similar results were obtained with Solvent System A (data not shown). TLC of the labeled lipid recovered from [3H]cholesterol-labeled embryos demonstrated that this lipid was also taken up and converted to products having mobilities distinct from that of cholesterol (data not shown). Thus, the lack of an effect on reductase activity could not be attributed to a failure in uptake of these lipids by the sea urchin embryo.

DISCUSSION

Our interest in the role of dolichyl phosphate in glycoprotein synthesis in the developing sea urchin embryo has led us to the finding that a key enzyme in the synthesis of this polyisoprenoid, HMG-CoA reductase, is developmentally regulated (7). As measured by enzyme activity, reductase increases at least 200-fold between egg and gastrula stage, where activity is maximal. Measurement of the level of reductase mRNA using a genomic fragment in an earlier study (7) or a full-length cDNA in the current study revealed a change of

A great deal has been learned about regulation of HMG- CoA reductase in mammalian cells. Regulation is complex, and sterols can control both expression of the reductase gene and the rate of catabolism of the protein. As a first step in studying factors that might regulate this enzyme in the developing sea urchin embryo, we earlier undertook to elucidate its properties (7). The current studies extend this work and provide information on 1) the deduced primary structure of the enzyme and 2) the effect of exogenous compounds on the level of enzyme activity and mRNA. A number of the previously described properties of the enzyme (7) can now be better understood in terms of the three domains of the protein, the N- and C-terminal domains and the linker domain that joins these two regions. The N-terminal domain of sea urchin embryo reductase contains one potential N-glycosylation site, which is glycosylated in hamster reductase. As in the hamster, the N-terminal domain (amino acid residues 1-340) of the sea urchin reductase is hydrophobic, containing seven putative membrane spanning domains. This region shares 61% amino acid sequence identity with the corresponding region in hamster reductase. Sterols and mevalonate suppress HMG-CoA reductase activity and mRNA levels in cultured mammalian cells by inhibiting transcription (19). Mammalian reductase is also regulated post-translationally by sterols, which appar- ently enhance the degradation of reductase protein (22, 23). Gil et al. (22) and Jingami et al. (23) have provided evidence that the membrane domains of reductase are required for this sterol-enhanced degradation. Experiments by Skalnik et al. (26) using constructs consisting of @-galactosidase fused to the transmembrane domain of HMG-CoA reductase strongly support this conclusion.

The high degree of similarity in the membrane spanning domains of sea urchin embryo and hamster reductases suggested that in the sea urchin enzyme sterols might function in an analogous manner. To test this idea embryos were cultivated in the presence of cholesterol, 25-hydroxycholesterol, dolichol, or mevalonate, and the level of reductase activity in microsomes was measured. Changes in the level of activity could result from an alteration in the reductase degradation rate, whereas changes in the level of reductase mRNA would reflect transcriptional regulation. Surprisingly, none of the compounds tested affected either enzyme activity or mRNA levels, even though their concentrations were comparable to those in experiments with cultured cells (24-29).

15-fold.

Similarly, although mevinolin was a competitive inhibitor of the sea urchin reductase, its addition to embryos did not cause the induction of enzyme and mRNA levels that has been reported with the mammalian enzyme (19).

Gertler et al. (30) have made similar observations on the lack of sterol regulation of reductase in cultured Drosophila cells, despite the fact that Drosophila HMG-CoA shares 32- 61% sequence identity with the seven transmembrane domains of hamster. These authors have argued that since Drosophila cannot synthesize sterol, it is unlikely that structural similarity in the transmembrane regions has been maintained solely to provide sensitivity to sterol-mediated degradation. They suggest that conservation of these domains in hamster (3, 14), human (15), and Drosophila may reflect structural features for targeting the enzymes in the endoplasmic reticulum or be essential for recognizing specific mevalonate derivatives or their binding proteins. Unlike Dro- sophila, the sea urchin embryo synthesizes cholesterol (5, 6), but the endogenous level declines relatively little over development (6) and therefore is unlikely to be a factor in the marked induction in reductase activity. Furthermore, regulation by exogenous cholesterol would not be a useful control mechanism in the sea urchin embryo since it does not eat until the larval stage (90 h after fertilization), when it is first exposed to exogenous sources of sterols.

Amino acid sequence analysis of the linker region (amino acid residues 341-536) in sea urchin embryo reductase showed this domain to be the most divergent from hamster reductase. Indeed, this region shows high divergence even when compar- ing two strains of hamsters (14). However, of partial interest with respect to the sea urchin are the sub-domains including residues 354-368 and 379-393. In hamster reductase, these domains are recognized by proteases that act to release a catalytically active soluble fragment of this enzyme. The low sequence similarity between sea urchin embryo and hamster reductases within these two regions (7 and 20%, respectively) may account for the previously reported finding that the sea urchin embryo reductase cannot be solubilized by proteases (7).

The C-terminal domain of sea urchin embryo reductase (amino acid residues 537-932) shares extensive amino acid sequence similarity (76% if conservative amino acid substitutions are allowed) with hamster HMG-CoA reductase. This finding of conservation within this region is not surprising, given the essential function of this domain as the site of catalytic activity. However, in contrast to hamster reductase, this domain in the sea urchin embryo protein is nearly as hydrophobic as the N-terminal domain. This overall hydrophobic character of the two domains in the sea urchin reductase potentially explains this protein’s refractoriness to solu- bilization by detergents.

These studies have provided insight into the relationship between the properties of the sea urchin embryo reductase and its structure. It is clear that expression of the sea urchin reductase gene is regulated over the course of development. It also seems clear that, unlike the mammalian system, in the sea urchin sterols do not play a role in regulation of expression of this gene. Future studies will be directed toward understanding the molecular basis for this difference in the mode of regulation.

Acknowledgments-We gratefully acknowledge Dr. Paul Hardin, Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, for generously providing us with the SPEC-2 and mitochondrial rRNA cDNA clones. We are most grateful to Dr. Daniel Chin, Department of Pharmacology and Cell Biology, University of California, San Francisco, for providing us

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