(Received for publication, July 24, 1995; and in revised form, September 12, 1995)
From the
hct-1 (hippocampal transcript) was detected in a
differential screen of a rat hippocampal cDNA library. Expression of hct-1 was enriched in the formation but was also detected in
rat liver and kidney, though at much lower levels; expression was
barely detectable in testis, ovary, and adrenal. In liver, unlike
brain, expression was sexually dimorphic; hepatic expression was
greatly reduced in female rats. In mouse, brain expression was
widespread, with the highest levels being detected in corpus callosum;
only low levels were detected in liver. Sequence analysis of rat and
mouse hct-1 cDNAs revealed extensive homologies with
cytochrome P450s (CYPs), a diverse family of heme-binding
monooxygenases that metabolize a range of substrates including
steroids, fatty acids, and xenobiotics. Among the CYPs, hct-1 is most similar (39% at the amino acid sequence) to cholesterol
7-hydroxylase (CYP7) and contains a postulated steroidogenic
domain present in other steroid-metabolizing CYPs but clearly
represents a type of CYP not previously reported. Genomic Southern
analysis suggests that a single gene corresponding to hct-1 is
present in mouse, rat, and human. hct-1 is unusual in that,
unlike all other CYPs described, the primary site of expression is in
the brain. Similarity to CYP7 and other steroid-metabolizing CYPs may
argue that hct-1 (CYP7B) plays a role in steroid metabolism in
brain, notable because of the documented ability of brain-derived
steroids (neurosteroids) to modulate cognitive function in
vivo.
Cytochromes P450, a diverse group of heme-containing
monooxygenases (termed CYPs) ()(for nomenclature see Nelson et al.(1) ), catalyze a variety of oxidative
conversions, notably of steroids but also of fatty acids and
xenobiotics. Though most abundantly expressed in the testis, ovary,
placenta, adrenal, and liver, the brain is a further site of CYP
expression(2, 3, 4, 5, 6) .
Several CYP activities or mRNAs have been reported in the nervous
system, predominantly of types metabolizing fatty acids and xenobiotics
(subclasses CYP2C, -2D, -2E, and -4(6, 7) ). However,
primary rat brain-derived glial cells can synthesize pregnenolone and
progesterone in vitro(8) . Mellon and Deschepper (9) provided molecular evidence for the presence, in brain, of
key steroidogenic enzymes CYP11A1 (scc) and CYP11B1 (11
) but
failed to detect CYP17 (c17) or CYP11B2 (AS). Though CYP21A1 (c21)
activity is reported to be present in brain (10) authentic
CYP21A1 transcripts were not detected(11) .
Interest in brain steroid metabolism has been fueled by the finding that adrenal- and brain-derived steroids (neurosteroids) can modulate cognitive function and synaptic plasticity (reviewed in (12, 13, 14, 15, 16, 17) ). For instance, pregnenolone and steroids derived from it are reported to have memory-enhancing effects in mice(18, 19) . However, the full spectrum of brain CYPs and the biological roles of their metabolites in vivo have not been established.
To
investigate such regulation of brain function our studies have focused
on the hippocampus, a brain region important in learning and memory.
Patients with lesions that include the hippocampus display pronounced
deficits in the acquisition of new explicit memories; in rat,
neurotoxic lesions to the hippocampus lead to a pronounced inability to
learn a spatial navigation task, such as the water maze (20) .
Hippocampal synapses, notably those in region CA1, display a
particularly robust form of activity-dependent plasticity known as long
term potentiation (LTP) (21) that satisfies some of the
requirements for a molecular mechanism underlying memory processes,
persistence, synapse specificity, and associativity. LTP is thought to
be initiated by calcium influx through the NMDA (N-methyl-D-aspartate) subclass of receptor activated
by the excitatory neurotransmitter, L-glutamate (reviewed in (22) ); occlusion of NMDA receptors with AP5 both blocks LTP
and the acquisition of the spatial navigation task(23) . In
vivo, simultaneous release of -aminobutyric acid from
inhibitory interneurons inhibits NMDA channel opening and LTP
induction(22) . It is of note that some naturally occurring
steroids, such as pregnenolone sulfate, act as agonists of the
-aminobutyric acid receptor (e.g. see (24) and (25) ) and may also directly modulate NMDA
currents(26, 27) . Though brain steroids principally
appear to exert their effects via the
-aminobutyric acid and NMDA
receptors, there are indications that neurosteroids may also interact
with
and progesterone receptors (28, 29) .
However, the pathways of CYP-mediated steroid metabolism in the central
nervous system have not been fully elucidated.
In addition, non-steroid CYP metabolites also play important roles in brain; CYP-mediated metabolism of psychoactive agents (30) and CYP metabolites of arachidonic acid such as prostanoids and eicosanoids (31) clearly contribute to the regulation of brain function.
As part of a study into the molecular biology of the hippocampal formation and the mechanisms underlying synaptic plasticity, we have sought molecular clones corresponding to mRNA's expressed selectively in the formation. One such cDNA, hct-1 (for hippocampal transcript), was isolated from a cDNA library prepared from adult rat hippocampus. Sequence analysis revealed that hct-1 is a novel cytochrome P450 most closely related to cholesterol- and steroid-metabolizing CYPs but, unlike other CYPs, is predominantly expressed in brain. We present molecular characterization of hct-1 coding sequences from rat and mouse and their expression patterns and discuss the possible role of hct-1 in the central nervous system.
Clones 12 and 7 were then fully sequenced and
compared with the data base. Homology was detected between clone 12 and
the human (36) and rat (37, 38) cDNA's
encoding cholesterol 7-hydroxylase (CYP7), though the sequences
are clearly distinct. At the nucleic acid level, the 1428-nt cDNA clone
for rat hct-1 shared 55% identity over an 1100-nt overlap with
human CYP7 and 54% identity over a 1117-nt overlap with rat CYP7 (not
presented). Fig. 1gives the partial cDNA sequences of rat hct-1 and the encoded polypeptide.
Figure 1: Sequence of partial rat hct-1 cDNA and the encoded polypeptide. The nucleotide sequence and translation product of the 1.4-kb cDNA clone 12 including additional sequence data derived from clone 7 (lower case). The two putative polyadenylation signals are underlined.
Figure 2:
Northern analysis of hct-1 expression in adult rat and mouse brain. A, expression in
rat brain and other tissues; B, sexually dimorphic expression
in rat liver; C, expression in mouse tissues.
Poly(A) (A) or total (B and C) RNA from organs of adult animals was resolved by gel
electrophoresis; the hybridization probe was rat hct-1 cDNA
clone 12 (1.4 kb); the probe for the loading control (below)
corresponds to ribosomal protein S26. Tissues analyzed were: Hi, hippocampus; RB, remainder of brain lacking
hippocampus; Cx, cortex; Cb, cerebellum; Ob;
olfactory bulb; Li, liver; He, heart; Th,
thymus; Ki, kidney; Ov, ovary; Te, testis; Lu, lung.
The sequences obtained were identical throughout the region of overlap. The mouse hct-1 open reading frame commences with a methionine at nucleotide 81 (numbering from clone 40) and terminates with a TGA codon at nucleotide 1600, encoding a protein of 507 amino acids (Fig. 3). At the 5` end the ATG initiation codon leading the open reading frame does not correspond to the translation initiation consensus sequence (YYAYYATGR(40) ). However, the 5`-untranslated region cloned is devoid of other possible initiation codons, and an in-frame termination triplet (TAA) lies 20 codons upstream of the ATG. The encoded polypeptide sequence aligns well with other cytochrome P450 sequences (see below); we surmise that the ATG at position 81 represents the correct start site for translation. At the 3` end the truncation of clone 40 lies entirely in the non-coding region downstream of the stop codon. Neither clone contained a poly(A) tail, but both contained a potential polyadenylation sequence (AATAAA) at a position corresponding precisely to that seen in the rat cDNA.
Figure 3: Mouse hct-1 cDNA and the sequence of the encoded polypeptide. The restriction map of the cDNA (top) corresponds to the compilation of two independent clones sequenced; the cross-hatched box indicates the coding region. The nucleotide sequence and translation product (bottom) are derived from this compilation. Lower case sequences indicate the 59 additional 5` nucleotides in clone 40 and the 99 additional 3` nucleotides in clone 35. The putative polyadenylation site is underlined.
Figure 4:
Alignment of mouse hct-1 with
human CYP7 (cholesterol 7-hydroxylase) and steroidogenic P450 s. A, identical amino acids are indicated by a bar; hyphens in the amino acid sequences indicate gaps introduced
during alignment. The N-terminal hydrophobic leader sequences are underlined. The position of the conserved Thr residue within
the O
-binding pocket of other CYPs(43) , but
replaced by Asn in hct-1 (position 294) and CYP7, is indicated
by an asterisk. B and C, conserved residues
in the heme-binding (residues 440-453, B) and postulated
steroidogenic (residues 348-362, C) domains conserved
between hct-1 and other similar CYPs (overlined in A). Sequences are human CYP7
(7
-hydroxylase(36) ), bovine CYP17 (17
-hydroxylase (42) ), human CYP11B1 (steroid
-hydroxylase(43) ),
bovine CYP21B (21-hydroxylase(11) ), human CYP11A1 (P450scc;
cholesterol side chain cleavage(44) ), and rabbit CYP27
(27-hydroxylase(45) ).
The N terminus of the hct-1 polypeptide is hydrophobic, a feature shared by microsomal CYPs. This portion of the polypeptide is thought to insert into the membrane of the endoplasmic reticulum. Consistent with microsomal CYPs, the N terminus lacks basic amino acids prior to the hydrophobic core (amino acids 9-34). Previous alignment studies have highlighted conserved regions within CYP polypeptides (e.g.(46) ). CYPs contain a highly conserved motif, FXXGXXXCXG(XXXA), present in 202 of the 205 compiled sequences(1) , thought to represent the heme binding site with the arrangement of amino acids around the cysteine residue postulated to preserve the three-dimensional structure of this region for ligand binding(41) . This motif is fully conserved in hct-1 (Fig. 4B). A second domain, which may be conserved in CYPs responsible for steroid interconversions(47, 36) , also is featured in hct-1 though an invariant Pro residue is replaced, in hct-1, by Val (Fig. 4C).
To refine this analysis, a 42-mer oligonucleotide was designed according to the DNA sequence of the 3`-untranslated region of the cDNA clone upstream of the first polyadenylation site to minimize cross-hybridization with other CYP mRNAs. Coronal sections of mouse brain were hybridized, emulsion dipped, and exposed for autoradiography (Fig. 5). Transcripts were detected throughout mouse brain and were not restricted to the hippocampus (Fig. 5, A and B). Strongest expression was observed in the corpus callosum, the anterior commissure, and fornix while, as in rat, hippocampal expression was particularly prominent in the dentate gyrus (Fig. 5C). Moderate expression levels, comparable with those observed in hippocampus, were observed in cerebellum, cortex, and olfactory bulb.
Figure 5: Analysis of hct-1 expression in adult mouse brain. The hybridization probe was a synthetic oligonucleotide corresponding to the 3`-untranslated region of mouse hct-1 cDNA. A, coronal section; B, coronal section, rostral to A, showing hybridization in corpus callosum (cc), fornix (f), and anterior commissure (ac); C, enlargement of section through the hippocampus (DG, dentate gyrus); D, section adjacent to the section in A hybridized with an oligonucleotide specific for opsin (negative control).
Figure 6: Southern analysis of hct-1 coding sequences in mouse (Mo), rat (Ra), and human (Hu). Total DNA was cleaved as indicated with restriction endonucleases (B, BamHI; E, EcoRI; H, HindIII; X, XbaI), resolved by agarose gel electrophoresis, and probed with rat hct-1 cDNA clone 12 before exposure to autoradiography.
To characterize transcripts enriched in the hippocampal formation we isolated clones corresponding to hct-1 from a rat hippocampus cDNA library. In rat, expression appeared to be most abundant in hippocampus with some expression in cortex and substantially less expression in other brain regions. Elsewhere in the body transcripts were only detected in liver and, to a lesser extent, in kidney; expression was barely detectable in ovary, testis, and adrenal, also sites of steroid transformations and CYP activity. Hepatic expression was sexually dimorphic with hct-1 mRNA barely detectable in female liver. In rat brain and liver, two hct-1 transcripts of 1.8 and 2.1 kb appear to be generated by alternative polyadenylation; a 5.0-kb transcript was weakly detected in rat brain but was not observed in mouse.
Sequence analysis of hct-1 cDNA clones revealed that hct-1 encodes a novel
cytochrome P450 (CYP). Although the mouse cDNA coding region appears
complete, the absence of a consensus translation initiation site (40) flanking the presumed initiation codon could indicate that hct-1 polypeptide synthesis is subject to regulation at the
level of translation initiation. Homology was highest with rat and
human cholesterol
7-hydroxylase(36, 37, 38) , known as
CYP7. While related, hct-1 is distinct from CYP7, sharing only
39% homology over the full length of the protein. CYP polypeptides
sharing greater than 40% sequence identity are generally regarded as
belonging to the same family(1) ; hct-1 and CYP7 (39%
similarity) are borderline. The conservation of other unique features
between hct-1 and CYP7 (see below), however, argues for a
close relationship, and hct-1 has been designated
``CYP7B'' (Cyp7b in mouse) by the Committee on Standardized
Cytochrome P450 Nomenclature. (
)
From the hct-1 leader sequence we surmise that the hct-1 polypeptide
resides, like CYP7, in the endoplasmic reticulum and not in
mitochondria, the other principal cellular site of CYP activity. The
strictly conserved heme binding site motif
FXXGXXXCXG(XXXA) (1) is
present in hct-1 (residues 440-453) while the postulated
``steroidogenic domain'' (e.g.(36) ) shared
by CYPs responsible for steroid interconversions is also present in hct-1 (amino acids 348-362), except that a consensus Pro
residue is replaced by Val in both the mouse and rat hct-1 polypeptides. Of 34 CYP sequences compiled by Nelson and
Strobel(46) , only 4 contain an amino acid residue other than
Pro at this position. Whereas 2 of these harbor an unrelated amino acid
(Glu; CYP3A1, CYP3A3), interestingly, a Val residue is present in
bovine CYP17 (steroid 17-hydroxylase(42) ) at a position
equivalent to that in hct-1 while human CYP17 harbors a
conservative substitution at this site (Leu(47) ). Despite this
similarity, however, the overall extent of homology between hct-1 and CYP17 (22.5%, not shown) is lower than with CYP7 (39%).
Neither hct-1 nor CYP7 appears to contain a conserved
O binding pocket (equivalent to residues 285-301 in hct-1) as highlighted by Poulos(41) . Crystallographic
studies on the bacterial CYP101 indicated that a Thr residue
(corresponding to position 294 in hct-1) disrupts helix
formation in that region and is important in providing a structural
pocket for an oxygen molecule(41) . Site-directed mutagenesis
of this Thr residue in both CYP4A1 and CYP2C11 demonstrated that this
region can influence substrate specificity and affinity(48) .
In both hct-1 and CYP7 the conserved Thr residue is replaced
by Asn. This modification suggests that hct-1 and CYP7 are
both structurally distinct from other CYPs in this region; this may be
reflected both in modified oxygen interaction and substrate choice.
The sexual dimorphism of hct-1 expression observed in rat resembles that observed with a number of other CYPs. CYP2C12 is expressed preferentially in liver of the female rat while, like hct-1, CYP2C11 is highly expressed in male liver but only at low levels in the female tissue(49) . This dimorphic expression pattern of CYP2C family members is thought to be determined by the dimorphism of pulsatility of growth hormone secretion(39) . Brain expression of hct-1 is not subject to this control, suggesting that regulatory elements determining hct-1 expression in brain differ from those utilized in liver. However, we have not examined species other than rat; it cannot be assumed that the same regulation will exist in other species. Indeed, sexually dimorphic gene expression is not necessarily conserved between different strains of mouse (e.g.(50) ).
Expression of hct-1 was widespread in mouse brain. Here the expression pattern was most consistent with glial expression, but further experiments will be required to compare neuronal and non-neuronal levels of expression. In mouse brain only the 1.8-kb transcript was detected. However, cDNAs were obtained corresponding to transcripts extending beyond the first polyadenylation site; such extended transcripts are thought to give rise to the 2.1-kb transcript in rat. This suggests the downstream polyadenylation site seen in rat hct-1 is underutilized in mouse hct-1 or absent. While in situ hybridization studies of hct-1 in rat brain were inconclusive, a difference in expression pattern between mouse and rat appears likely; further work will be required to confirm this. Such a difference would be unsurprising because cytochromes P450 are well known to vary widely in their level and pattern of expression in different species; for instance, hepatic testosterone 16-hydroxylation levels differ by more than 100-fold between guinea pig and rat(51) .
Our data indicate that the hct-1 gene is present in rat, mouse, and human and does not appear to be duplicated in the mammalian genome. While CYP genes are scattered over the mouse and human genomes, CYP subfamilies can cluster on the same chromosome. For instance, the human CYP2A and -2B subfamily genes are linked to chromosome 19, CYP2C and -2E subfamilies are located on human chromosome 10, and the mouse Cyp2a, -2b, and -2e subfamilies are present on mouse chromosome 7 (reviewed by Paine(52) ). The gene encoding human CYP7 is located on chromosome 8q11-q12(53) . It will be of future interest to determine the chromosomal location of the human hct-1 homolog. Experiments are in progress to address this question.
What role might hct-1 play in the brain? In the adult CYPs are generally expressed
abundantly in liver, adrenal, and gonads, while the level of CYP
activity in brain is estimated to be 0.3-3% of that found in
liver (see (54) ). Unusually, levels of hct-1 mRNA
expression in rat and mouse brain far exceed those in liver, and it
could be argued that the primary function of hct-1 lies in the
central nervous system. Our data argue that hct-1 is related
to the steroid-metabolizing CYPs, and most similar to CYP7, this may
suggest that the substrate for hct-1, so far unknown, is
likely to be related to cholesterol or one of its steroid metabolites.
This interpretation is borne out by the presence, in hct-1, of
a postulated steroidogenic domain (37) that appears to be
conserved in steroid-metabolizing CYPs. However, the functional
significance of this domain in steroidogenic CYPs has not yet been
demonstrated; it remains a possibility that hct-1 metabolizes
substrates other than cholesterol or steroids. While experiments are
presently under way to determine the substrate specificity of hct-1, the possibility that hct-1 might act on
cholesterol or its steroid metabolites in brain is of some interest.
CYP7 (cholesterol 7-hydroxylase) is responsible for the first step
in the metabolic degradation of cholesterol. This is of note in view of
the association of particular alleles of the APOE gene
encoding the cholesterol transporter protein apolipoprotein E with the
onset of Alzheimer's disease(55, 56) , a
neurodegenerative condition whose cognitive impairments are associated
with early dysfunction of the hippocampus. The documented ability of
cholesterol-derived steroids to interact with neurotransmitter
receptors and modulate both synaptic plasticity and cognitive
function(12, 13, 14, 15, 16, 17, 18, 19) may
suggest that hct-1 and its metabolic product(s) regulate
neuronal function in vivo.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U36992 [GenBank]and U36993[GenBank].