Vol. 185, No. 2, 1992 June 15, 1992
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 588-595
POLYHORPHISHS OF r3UMAN CHOLESTEROL 7a-IXYDROXYLASE1
Walid
G. Karam
and John
Department of Biochemistry and Molecular Ohio Universities College of Medicine, Received April
Y.L.
Chiang Pathology, Rootstown,
Northeastern OH 44272
21, 1992
Human liver cholesterol 7a-hydroxylase (CYP7) cDNAs were isolated from a human liver cDNA library. A full-length cDNA has 2901 nucleotides which encode a typical P450 polypeptide of 504 Two different sequences of codon 100, TTT amino acid residues. were identified in cDNA clones. In addition, (Phe) and TCT (Ser), codons 347 and 385 are GAT (Asp) and GAC (Asp) in all cDNA CIOneS,y whereas those reported previously (FEBS Lett. 268,137-140, 1990) are AAT,(Asn) and SC (Ser), respectively. Since there is only one 7a-hydroxylase gene in the human genome, it is likely that polymorphisms at the codon 100 of cDNA clones arise from two different alleles in the 7a-hydroxylase gene of this human liver. SumpparV:
Q 1992
Academic
Press,
Inc.
Cholesterol 7a-hydroxylase (CYP7) catalyzes the first and rate-limiting step in bile acid synthesis in the liver. This cytochrome P450 isozyme is thought to be down-regulated by bile acids returning to the liver via enterohepatic recirculation of bile (1). This laboratory has recently accomplished the cloning, purification and heterologous expression of rat liver cholesterol 7a-hydroxylase (2-6). Using purified enzyme, antibody and cDNA as probes, we demonstrated that cholesterol 7a-hydroxylase activity is regulated at the gene transcriptional level by hydrophobic bile salts, thyroid hormone and cholesterol in the rat (2-6). However, not much is known about the structure, function and regulation of 7a-hydroxylase in humans. Since cholesterol 7a-hydroxylase is an essential enzyme in mammals, it is expected that rat and human enzymes should share a high sequence homology. Thus rat cDNA could be used as a hybridization probe to clone human 7a-hydroxylase cDNA. In this communication, We report the cloning and sequencing of several human 7a-hydroxylase cDNAs isolated from a human liver 'Sequence data from this EMBL/GenBank 0006-291X/92
Copyright All rights
Data
Libraries
article have been deposited under Accession No. M93133.
$4.00
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
588
with
the
Vol.
185,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
cDNA library and discovered at least one polymorphism in the coding sequence. Several other discrepancies in DNA and the deduced amino acid sequences were noticed when comparing our sequences with those reported by Noshiro and Okuda (7). Analyses of the deduced amino acid sequences of the human and rat 7a-hydroxylase revealed several highly conserved regions which may be important in determining cholesterol binding and substrate specificity of CYP7. WATERIALS
AND BETHODS
Using a rat 7a-hydroxylase cDNA (3) as a hybridization probe, we screened a Agt 11 human liver cDNA library (Clonetech, Palo Alto, from a female human liver with high CA) constructed stringency (8). One positive clone containing a 1.8 kb insert was plaque purified and subcloned into a Bluescript vector. A 0.43 kb .Eco RI-Pst I fragment containing coding sequence for the C-terminal 100 amino acid residues was used as a hybridization probe to screen an Uni-ZAP XFt male human liver cDNA library (Cat. No. 937200, Stratagene, La Jolla, CA). A total of 23 positive clones were plaque-purified through three rounds of screenings from 1 million pfu of phages under a high stringency condition. Six of these clones were restriction and DNA characterized by mapping sequencing. cDNA inserts in a pBluescript vector were in viva excised and rescued into bacteria XL-1 blue cells according to the manufacturer's instruction (Stratagene). Plasmids were digested with restriction enzymes to generate restriction fragments for subcloning and sequencing. Nucleotide sequences of these cDNA were determined according to the dideoxy chain termination method (9) using the Sequenase system (United State Biochemical Co., Cleveland, OH). Two oligonucleotide primers, 5'-TGTTTGAGTCAAGCAGA3'(nucleotides and 5'-AGTCAAAGGGTCTGGGT1060 to 1076) 3'(complemental sequence to nucleotides 1261-1277), were synthesized by National Biosciences (Plymouth, MN) and used as sequencing primers for nucleotide sequences in the region covering DNASIS (Hitachi both codons 347 and 385 of partial cDNA clones. CA) and MicroGenie (Beckman, Palo Alto, CA) America, Brisbane, softwares were used to analyze DNA and Amino acid sequences.
RESULTS Two full-length and four partial-length cDNAs isolated from an Uni-ZAP XR human liver cDNA library were characterized by restriction mapping and DNA sequencing. All clones share the same restriction sites in the overlapping region (Figure 1). The nucleotide sequences of clone PHC7F was determined (Figure 2). It has 2901 bp nucleotides and contains an open reading frame encoding for a typical cytochrome P450 polypeptide containing 504 amino acid sequence which is 20 residues. This clone has a 59 bp 5 '-upstream bp longer than the one reported by Noshiro and Okuda (7). A of this upstream sequence with the gene sequence comparison (10) reveals that this cDNA recently reported by Molowa et al. 589
Vol.
185,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
PHC7F/PHC7R
E
DD
S
SP
X
X
1211
EDD LIII
SP 1 1
X I
x
Fiqure 1. Restriction Maps of Human Cholesterol 7a-Hydroxylase cDNAs and the Sites of Polymorphisms. PHC7F and PIiC7R are two full-length clones. Hill, Bill, I211 and Llll are identical, partial-length clones. The heavy lines represent the amino acid coding regions in these clones. Triangles indicate the polymorphic codon numbers and amino acid residues. Amino acids, D, Asp: F, Phe; S, Ser;. Restriction enzyme sites, E, Eco RI; X, Xho I; P, Pst 1; and S, Sma I.
sequence starts two bases However, nucleotides 5 to TCCTTCC in the gene and a G C in the gene. The reasons 3'-flanking region has 1471 translatable region is 1 kb rat
cDNA
(3).
Clone
PHC7R
after the start site of the gene. 8 in the cDNA are substituted with at nucleotide 35 is substituted with a for this variation are not known. The bp including a poly A-tail. This nonshorter than the Type I clone of the has
an identical
restriction
map and
sequences in the 5' and 3 '-flanking regions to clone PHC7F. These two clones are apparently identical and are of full-length. The 5'- and 3'- regions of about 200 to 300 bp of four other cDNA clones were determined. To our surprise, codon 100 in clone Hlll is TCT (Figure 3), this codon is TTT in both full-length clones. Other three clones are short of codon 100. The codon 100 in the human clone reported by Noshiro and Okuda (7) is TTT. This single change of T to c converts the corresponding amino acid residue from a Phe to a Ser. We also identified discrepancies of three bases in codons 347 and 385 when comparing our sequences to those of Noshiro and Okuda (7). All six clones we sequenced have GAT (Asp) and GAC (Asp) at these two codons, respectively. The corresponding sequences reported by Noshiro and Okuda are UT (Asn) and MC (Ser), respectively. All these base conversions are A/G or T/C and resulted in non-conserved changes of amino acid residues. 590
Vol.
185,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Figure 2. Nucleotide and Deduced Amino Acid Sequences of the FullLength cholesterol 7a-Hydroxylase cDNA clone PHC7F or PHC7R. Arrows and numbers indicate the locations of intron/exon junctions and exon numbers, respectively, according to the rat gene structure. The highly conserved, putative steroid-binding site and highly conserved aromatic amino acid region are indicated by brackets. The highly conserved heme-binding site is indicated by underline of amino acid residues. The cysteine heme ligand is indicated by a star. The polyadenylation sugnal AATAAA is underlined near the 3' -end. Vindicates the site of polymorphisms. 591
Vol.
185, No. 2, 1992
AG
BIOCHEMICAL
C T
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
AGCT
T C T DA A A A A A G G T T A G T T
Phe
iLiE l-
PHC7F/PHC7R
H 111
Figure 3. Nucleotide Sequences Surrounding Polymorphic Codon 100 of the Human Cholesterol 7a-Hydroxylase cDNAs. The boxes indicate codons 100 in full-length clones PHC7F and PHC7R and a partial clone Hill.
A comparison of amino acid sequences between the rat and human 7a-hydroxylase revealed that there is an overall sequence identity of 85 % which is much higher than the identity between orthologues of other cytochrome P450 gene families. The gene structure of the rat cholesterol 7a-hydroxylase has been reported (11). We have recently cloned a hamster cholesterol 7e-hydroxylase gene, the sequence of which revealed an identical intron/exon boundary structure in exon 1 to 3 so far determined (M. Crestani and J. Chiang, unpublished results). It is likely that the human cholesterol 7a-hydroxylase gene also has the same gene structure as do the rat and hamster genes. The first exon contains the coding sequence for the membrane-binding domain of cytochrome P450 enzyme. Based on the heterologous expression of a catalytically active cholesterol 7a-hydroxylase lacking the N-terminus 24 amino acid residues, we suggested that this highly hydrophobic membranebinding segment is not necessary for the catalytic activity (4). The sequence identity of the exon 1 is 70 % between the rat and human sequences. The exon 2, in which the polymorphic codon 100 is
er
Vol.
185, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
located, has a sequence identity of 90 % between the rat and human. In both the rat and hamster, codon 100 is TTT (data not shown) which is the same as one of the two codons we identified in human cDNAs. The exon 3 has a sequence identity of 81 % between the rat and human. The exon 4 has a rather low sequence identity of 62 %. The exon 5 is the most conserved region with a sequence identity of 97 %. There are only two conserved amino acid substitutions in the exon 5 between the rat and human. The polymorphic codon 347 (Asp) is located in the highly conserved, putative steroid-binding site (Figure 2) which was identified by us previously (3). Codon 347 in the rat is the same as in all human sequences reported here, but it is an Asn in the human sequence of Noshiro and Okuda (7). In addition, all six clones we sequenced have the same codon 385 which is conserved in the rat, but is a Ser in the human (Asp), sequence of Noshiro and Okuda (7). The highly conserved amino acid sequences in the exon 5 must play an important role in the binding of cholesterol and determining substrate specificity. A highly conserved aromatic amino acid region (Figure 2) is splitted and located in both exons 5 and 6. This region is completely identical in the rat and human sequences. The close localization of this aromatic amino acid region to both steroid-binding site and hemebinding site also indicates its importance in cholesterol-binding and catalysis. The coding sequence in the exon 6 has a sequence conserved cytochrome P450 heme-binding identity of 84 %. A highly site (Figure 2) is completely conserved in the rat and human. DISCUSSION
Several evidences suggested that there is only one cholesterol 7a-hydroxylase gene in the rat. Genomic Southern blot hybridization of human genomic DNA and the isolation of a human cholesterol 7a-hydroxylase gene in this laboratory (unpublished results) also provided some evidences that there is only one cholesterol 7a-hydroxylase gene in the human genome. A Phe (TTT) at codon 100 is conserved in both rat and human cDNAs as well as in the hamster gene. Therefore, a Ser (TCT) at codon 100 may be a in codon 100 may be due to the variant and the polymorphisms Amino acid presence of two different alleles in this male. residues located in the exon 2 have been implicated to play an important role in the substrate specificity and catalytic activity The E. coli expression system of cytochrome P450 isozyme (12). recently developed by us for a high level expression of an active 593
Vol.
185,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
rat cholesterol 7a-hydroxylase could be used to study the effect of mutations in codon 100 in the catalytic activity of the human The observed discrepancies in cholesterol 7a-hydroxylase (4). codons 347 and 385 may be also true polymorphisms in the human. However, these two residues are highly conserved in all six human clones reported here as well as in the rat cDNA. Thus nucleotide sequencing errors in the previous report (7) could not be ruled out completely. Sequencing of these polymorphic sites in the human genomic DNAs of individuals should be done to verify these polymorphisms. Restriction site polymorphisms analysis could be applied to detect and confirm these polymorphisms in the human genomes. The conversion of AGC to GAC in codon 385 destroys a Sfa NI site in our sequence. The conversion of AAT to GAT in codon 347 destroys a Mse I site in our sequence. Tsp EI will cut a sequence surrounding TTT at codon 100, but will not cut sequence containing TCT at codon 100. Site-directed mutagenesis study of amino acid residues in these polymorphic sites may be useful for the elucidation of structure and function relationship of this important regulatory enzyme in bile acid synthesis. ACKNOWLEDGMEN!l’S
This research is supported by a NIH grant GM31584 and a grantin-aid from American Heart Association Ohio Affiliates (A91-01). Excellent technical assistance of Mr. Richard Verne11 is greatly acknowledged. REFHRENCES 1. 2.
Myrant, N. B., and Mitropoulos, K. A. (1979) J. Lipid 135-153. W. F. and Lin, G.-M. (1990) Chiang, J. Y. L., Miller, Chem. 265, 3889-3897.
3.
Li, Y. C., Chem. 265,
Wang, D. P. 12012-12019.
and Chiang,
4.
Li, Y. C. 19186-19191.
and
J.
5.
Pandak, Gurley, B.(1991)
Chiang,
Y. L.
J. (1991)
Y. L. J.
(1990) Biol.
Res.
18,
J.
Biol.
J.
Biol.
Chem.
266,
W. M., Li, Y. C., Chiang, J. Y. L., Studer, E. J., E. C., Heuman, D. M., Vlahcevic, Z. R. and Hylemon, P. J. Biol. Chem. 266, 3416-3421. G.C., Pendleton, L.C., Li, Y.C. and Chiang, J.Y.L. Biochem. Biophys. Res. commun. 172, 1150-1156.
6.
Ness, (1990)
7.
Noshiro,
M. and Okuda,
8.
Sambrook, Cloning, Press.
J., Fritsch, A Laboratory
K (1990) FEBS Lett. 268, 137-140. E. F. and Maniatis, T. (1989) Molecular Manual, Cold Spring Harbor Laboratories, 594
Vol.
185, No. 2, 1992
BIOCHEMICAL
9.
Sanger, F. Nicklen, Acad. Sci. U.S.A.
10.
Molowa, Biochem.
11.
Jelinek,
D.
T., 31,
AND BIOPHYSICAL
S., and CoUlSOn, 74, 5463-5467.
Chen,
W. S.,
Cimis,
RESEARCH COMMUNICATIONS
A. R. (1977) G. M. and Tan,
Proc.
Natl.
C. P. (1992)
2539-2544.
D. F. and Russell,
D. W. (1990)
Biochem.
29,
7781-
7785. 12.
Gotoh, 0. and Biotransformation Francis, N. Y.
Fujii-Kuriyama, (Ruckpau1,K.C.
595
Y. ed.)
(1989) in Frontier pp. 195-243, Taylor
in and
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 588-595
POLYHORPHISHS OF r3UMAN CHOLESTEROL 7a-IXYDROXYLASE1
Walid
G. Karam
and John
Department of Biochemistry and Molecular Ohio Universities College of Medicine, Received April
Y.L.
Chiang Pathology, Rootstown,
Northeastern OH 44272
21, 1992
Human liver cholesterol 7a-hydroxylase (CYP7) cDNAs were isolated from a human liver cDNA library. A full-length cDNA has 2901 nucleotides which encode a typical P450 polypeptide of 504 Two different sequences of codon 100, TTT amino acid residues. were identified in cDNA clones. In addition, (Phe) and TCT (Ser), codons 347 and 385 are GAT (Asp) and GAC (Asp) in all cDNA CIOneS,y whereas those reported previously (FEBS Lett. 268,137-140, 1990) are AAT,(Asn) and SC (Ser), respectively. Since there is only one 7a-hydroxylase gene in the human genome, it is likely that polymorphisms at the codon 100 of cDNA clones arise from two different alleles in the 7a-hydroxylase gene of this human liver. SumpparV:
Q 1992
Academic
Press,
Inc.
Cholesterol 7a-hydroxylase (CYP7) catalyzes the first and rate-limiting step in bile acid synthesis in the liver. This cytochrome P450 isozyme is thought to be down-regulated by bile acids returning to the liver via enterohepatic recirculation of bile (1). This laboratory has recently accomplished the cloning, purification and heterologous expression of rat liver cholesterol 7a-hydroxylase (2-6). Using purified enzyme, antibody and cDNA as probes, we demonstrated that cholesterol 7a-hydroxylase activity is regulated at the gene transcriptional level by hydrophobic bile salts, thyroid hormone and cholesterol in the rat (2-6). However, not much is known about the structure, function and regulation of 7a-hydroxylase in humans. Since cholesterol 7a-hydroxylase is an essential enzyme in mammals, it is expected that rat and human enzymes should share a high sequence homology. Thus rat cDNA could be used as a hybridization probe to clone human 7a-hydroxylase cDNA. In this communication, We report the cloning and sequencing of several human 7a-hydroxylase cDNAs isolated from a human liver 'Sequence data from this EMBL/GenBank 0006-291X/92
Copyright All rights
Data
Libraries
article have been deposited under Accession No. M93133.
$4.00
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
588
with
the
Vol.
185,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
cDNA library and discovered at least one polymorphism in the coding sequence. Several other discrepancies in DNA and the deduced amino acid sequences were noticed when comparing our sequences with those reported by Noshiro and Okuda (7). Analyses of the deduced amino acid sequences of the human and rat 7a-hydroxylase revealed several highly conserved regions which may be important in determining cholesterol binding and substrate specificity of CYP7. WATERIALS
AND BETHODS
Using a rat 7a-hydroxylase cDNA (3) as a hybridization probe, we screened a Agt 11 human liver cDNA library (Clonetech, Palo Alto, from a female human liver with high CA) constructed stringency (8). One positive clone containing a 1.8 kb insert was plaque purified and subcloned into a Bluescript vector. A 0.43 kb .Eco RI-Pst I fragment containing coding sequence for the C-terminal 100 amino acid residues was used as a hybridization probe to screen an Uni-ZAP XFt male human liver cDNA library (Cat. No. 937200, Stratagene, La Jolla, CA). A total of 23 positive clones were plaque-purified through three rounds of screenings from 1 million pfu of phages under a high stringency condition. Six of these clones were restriction and DNA characterized by mapping sequencing. cDNA inserts in a pBluescript vector were in viva excised and rescued into bacteria XL-1 blue cells according to the manufacturer's instruction (Stratagene). Plasmids were digested with restriction enzymes to generate restriction fragments for subcloning and sequencing. Nucleotide sequences of these cDNA were determined according to the dideoxy chain termination method (9) using the Sequenase system (United State Biochemical Co., Cleveland, OH). Two oligonucleotide primers, 5'-TGTTTGAGTCAAGCAGA3'(nucleotides and 5'-AGTCAAAGGGTCTGGGT1060 to 1076) 3'(complemental sequence to nucleotides 1261-1277), were synthesized by National Biosciences (Plymouth, MN) and used as sequencing primers for nucleotide sequences in the region covering DNASIS (Hitachi both codons 347 and 385 of partial cDNA clones. CA) and MicroGenie (Beckman, Palo Alto, CA) America, Brisbane, softwares were used to analyze DNA and Amino acid sequences.
RESULTS Two full-length and four partial-length cDNAs isolated from an Uni-ZAP XR human liver cDNA library were characterized by restriction mapping and DNA sequencing. All clones share the same restriction sites in the overlapping region (Figure 1). The nucleotide sequences of clone PHC7F was determined (Figure 2). It has 2901 bp nucleotides and contains an open reading frame encoding for a typical cytochrome P450 polypeptide containing 504 amino acid sequence which is 20 residues. This clone has a 59 bp 5 '-upstream bp longer than the one reported by Noshiro and Okuda (7). A of this upstream sequence with the gene sequence comparison (10) reveals that this cDNA recently reported by Molowa et al. 589
Vol.
185,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
PHC7F/PHC7R
E
DD
S
SP
X
X
1211
EDD LIII
SP 1 1
X I
x
Fiqure 1. Restriction Maps of Human Cholesterol 7a-Hydroxylase cDNAs and the Sites of Polymorphisms. PHC7F and PIiC7R are two full-length clones. Hill, Bill, I211 and Llll are identical, partial-length clones. The heavy lines represent the amino acid coding regions in these clones. Triangles indicate the polymorphic codon numbers and amino acid residues. Amino acids, D, Asp: F, Phe; S, Ser;. Restriction enzyme sites, E, Eco RI; X, Xho I; P, Pst 1; and S, Sma I.
sequence starts two bases However, nucleotides 5 to TCCTTCC in the gene and a G C in the gene. The reasons 3'-flanking region has 1471 translatable region is 1 kb rat
cDNA
(3).
Clone
PHC7R
after the start site of the gene. 8 in the cDNA are substituted with at nucleotide 35 is substituted with a for this variation are not known. The bp including a poly A-tail. This nonshorter than the Type I clone of the has
an identical
restriction
map and
sequences in the 5' and 3 '-flanking regions to clone PHC7F. These two clones are apparently identical and are of full-length. The 5'- and 3'- regions of about 200 to 300 bp of four other cDNA clones were determined. To our surprise, codon 100 in clone Hlll is TCT (Figure 3), this codon is TTT in both full-length clones. Other three clones are short of codon 100. The codon 100 in the human clone reported by Noshiro and Okuda (7) is TTT. This single change of T to c converts the corresponding amino acid residue from a Phe to a Ser. We also identified discrepancies of three bases in codons 347 and 385 when comparing our sequences to those of Noshiro and Okuda (7). All six clones we sequenced have GAT (Asp) and GAC (Asp) at these two codons, respectively. The corresponding sequences reported by Noshiro and Okuda are UT (Asn) and MC (Ser), respectively. All these base conversions are A/G or T/C and resulted in non-conserved changes of amino acid residues. 590
Vol.
185,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Figure 2. Nucleotide and Deduced Amino Acid Sequences of the FullLength cholesterol 7a-Hydroxylase cDNA clone PHC7F or PHC7R. Arrows and numbers indicate the locations of intron/exon junctions and exon numbers, respectively, according to the rat gene structure. The highly conserved, putative steroid-binding site and highly conserved aromatic amino acid region are indicated by brackets. The highly conserved heme-binding site is indicated by underline of amino acid residues. The cysteine heme ligand is indicated by a star. The polyadenylation sugnal AATAAA is underlined near the 3' -end. Vindicates the site of polymorphisms. 591
Vol.
185, No. 2, 1992
AG
BIOCHEMICAL
C T
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
AGCT
T C T DA A A A A A G G T T A G T T
Phe
iLiE l-
PHC7F/PHC7R
H 111
Figure 3. Nucleotide Sequences Surrounding Polymorphic Codon 100 of the Human Cholesterol 7a-Hydroxylase cDNAs. The boxes indicate codons 100 in full-length clones PHC7F and PHC7R and a partial clone Hill.
A comparison of amino acid sequences between the rat and human 7a-hydroxylase revealed that there is an overall sequence identity of 85 % which is much higher than the identity between orthologues of other cytochrome P450 gene families. The gene structure of the rat cholesterol 7a-hydroxylase has been reported (11). We have recently cloned a hamster cholesterol 7e-hydroxylase gene, the sequence of which revealed an identical intron/exon boundary structure in exon 1 to 3 so far determined (M. Crestani and J. Chiang, unpublished results). It is likely that the human cholesterol 7a-hydroxylase gene also has the same gene structure as do the rat and hamster genes. The first exon contains the coding sequence for the membrane-binding domain of cytochrome P450 enzyme. Based on the heterologous expression of a catalytically active cholesterol 7a-hydroxylase lacking the N-terminus 24 amino acid residues, we suggested that this highly hydrophobic membranebinding segment is not necessary for the catalytic activity (4). The sequence identity of the exon 1 is 70 % between the rat and human sequences. The exon 2, in which the polymorphic codon 100 is
er
Vol.
185, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
located, has a sequence identity of 90 % between the rat and human. In both the rat and hamster, codon 100 is TTT (data not shown) which is the same as one of the two codons we identified in human cDNAs. The exon 3 has a sequence identity of 81 % between the rat and human. The exon 4 has a rather low sequence identity of 62 %. The exon 5 is the most conserved region with a sequence identity of 97 %. There are only two conserved amino acid substitutions in the exon 5 between the rat and human. The polymorphic codon 347 (Asp) is located in the highly conserved, putative steroid-binding site (Figure 2) which was identified by us previously (3). Codon 347 in the rat is the same as in all human sequences reported here, but it is an Asn in the human sequence of Noshiro and Okuda (7). In addition, all six clones we sequenced have the same codon 385 which is conserved in the rat, but is a Ser in the human (Asp), sequence of Noshiro and Okuda (7). The highly conserved amino acid sequences in the exon 5 must play an important role in the binding of cholesterol and determining substrate specificity. A highly conserved aromatic amino acid region (Figure 2) is splitted and located in both exons 5 and 6. This region is completely identical in the rat and human sequences. The close localization of this aromatic amino acid region to both steroid-binding site and hemebinding site also indicates its importance in cholesterol-binding and catalysis. The coding sequence in the exon 6 has a sequence conserved cytochrome P450 heme-binding identity of 84 %. A highly site (Figure 2) is completely conserved in the rat and human. DISCUSSION
Several evidences suggested that there is only one cholesterol 7a-hydroxylase gene in the rat. Genomic Southern blot hybridization of human genomic DNA and the isolation of a human cholesterol 7a-hydroxylase gene in this laboratory (unpublished results) also provided some evidences that there is only one cholesterol 7a-hydroxylase gene in the human genome. A Phe (TTT) at codon 100 is conserved in both rat and human cDNAs as well as in the hamster gene. Therefore, a Ser (TCT) at codon 100 may be a in codon 100 may be due to the variant and the polymorphisms Amino acid presence of two different alleles in this male. residues located in the exon 2 have been implicated to play an important role in the substrate specificity and catalytic activity The E. coli expression system of cytochrome P450 isozyme (12). recently developed by us for a high level expression of an active 593
Vol.
185,
No.
BIOCHEMICAL
2, 1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
rat cholesterol 7a-hydroxylase could be used to study the effect of mutations in codon 100 in the catalytic activity of the human The observed discrepancies in cholesterol 7a-hydroxylase (4). codons 347 and 385 may be also true polymorphisms in the human. However, these two residues are highly conserved in all six human clones reported here as well as in the rat cDNA. Thus nucleotide sequencing errors in the previous report (7) could not be ruled out completely. Sequencing of these polymorphic sites in the human genomic DNAs of individuals should be done to verify these polymorphisms. Restriction site polymorphisms analysis could be applied to detect and confirm these polymorphisms in the human genomes. The conversion of AGC to GAC in codon 385 destroys a Sfa NI site in our sequence. The conversion of AAT to GAT in codon 347 destroys a Mse I site in our sequence. Tsp EI will cut a sequence surrounding TTT at codon 100, but will not cut sequence containing TCT at codon 100. Site-directed mutagenesis study of amino acid residues in these polymorphic sites may be useful for the elucidation of structure and function relationship of this important regulatory enzyme in bile acid synthesis. ACKNOWLEDGMEN!l’S
This research is supported by a NIH grant GM31584 and a grantin-aid from American Heart Association Ohio Affiliates (A91-01). Excellent technical assistance of Mr. Richard Verne11 is greatly acknowledged. REFHRENCES 1. 2.
Myrant, N. B., and Mitropoulos, K. A. (1979) J. Lipid 135-153. W. F. and Lin, G.-M. (1990) Chiang, J. Y. L., Miller, Chem. 265, 3889-3897.
3.
Li, Y. C., Chem. 265,
Wang, D. P. 12012-12019.
and Chiang,
4.
Li, Y. C. 19186-19191.
and
J.
5.
Pandak, Gurley, B.(1991)
Chiang,
Y. L.
J. (1991)
Y. L. J.
(1990) Biol.
Res.
18,
J.
Biol.
J.
Biol.
Chem.
266,
W. M., Li, Y. C., Chiang, J. Y. L., Studer, E. J., E. C., Heuman, D. M., Vlahcevic, Z. R. and Hylemon, P. J. Biol. Chem. 266, 3416-3421. G.C., Pendleton, L.C., Li, Y.C. and Chiang, J.Y.L. Biochem. Biophys. Res. commun. 172, 1150-1156.
6.
Ness, (1990)
7.
Noshiro,
M. and Okuda,
8.
Sambrook, Cloning, Press.
J., Fritsch, A Laboratory
K (1990) FEBS Lett. 268, 137-140. E. F. and Maniatis, T. (1989) Molecular Manual, Cold Spring Harbor Laboratories, 594
Vol.
185, No. 2, 1992
BIOCHEMICAL
9.
Sanger, F. Nicklen, Acad. Sci. U.S.A.
10.
Molowa, Biochem.
11.
Jelinek,
D.
T., 31,
AND BIOPHYSICAL
S., and CoUlSOn, 74, 5463-5467.
Chen,
W. S.,
Cimis,
RESEARCH COMMUNICATIONS
A. R. (1977) G. M. and Tan,
Proc.
Natl.
C. P. (1992)
2539-2544.
D. F. and Russell,
D. W. (1990)
Biochem.
29,
7781-
7785. 12.
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