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Section on Steroid Regulation, Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892-4510
Address all correspondence and requests for reprints to: Dr. Charles A. Strott, Building 49, Room 6A36, National Institutes of Health, Bethesda, Maryland 20892-4510.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cholesterol sulfonate is widely distributed (12, 13, 14, 15, 16, 17, 18) and
circulates in plasma at concentrations ranging from 328–924 µg/100
ml, with a blood production rate of 35–163 mg/day (19).
Cholesterol sulfonate has been implicated in a wide variety of
biological processes, e.g. regulation of cholesterol
synthesis (20), sperm capacitation (21),
thrombin and plasmin activities (22), and activation of
protein kinase C isozymes (23), particularly the 
isoform (24). Furthermore, cholesterol sulfonate can serve
as a substrate for adrenal (25) and ovarian
(26) steroidogenesis. Cholesterol sulfonate plays an
important, but unclear, role in the normal development and physiology
of skin, where an epidermal cholesterol sulfate cycle has been
described (27). What is clear, however, is the pathogenic
role of excessive cholesterol sulfonate deposition in the stratum
corneum of the epidermis in the development of recessive X-linked
ichthyosis (28). Although the sulfonates of cholesterol
and its hydroxylated metabolites are naturally occurring, the
sulfotransferase enzyme(s) that produces them has not been clearly
identified. In human liver, hydroxysteroid sulfotransferase (HST),
commonly referred to as DHEA sulfotransferase, was
suggested as the major enzyme to catalyze the sulfonation of
cholesterol (29). The complementary DNA (cDNA) for human
DHEA sulfotransferase (HST1) was originally cloned from
the liver (30, 31, 32) and adrenal (33). More
recently, cDNAs of two novel human HST isoenzymes (HST2a and HST2b)
were cloned from a placental cDNA library (34). As part of
a program to identify the specific sulfotransferase(s) that catalyzes
the sulfonation of cholesterol and C27
oxysterols, we evaluated the specificity of cloned human HST1,
HST2a, and HST2b using novel techniques that permit an assessment of
substrate preference. Our findings clearly indicate that the HST2
isoforms sulfonate cholesterol and other C27
sterols, in preference to DHEA, and, whereas HST1 is
distinguishable as a DHEA sulfotransferase.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression and purification of human HST1, HST2a, and
HST2b
To overexpress HST1 (GenBank accession no. U08024), HST2a
(GenBank accession no. U92314), and HST2b (GenBank accession no.
U92315) in Escherichia coli, the open reading frames
of cDNAs were amplified by PCR using the templates SULT2A1-p91023,
SULT2B1a-pCR3.1, and SULT2B1b-pCR 3.1, generously supplied by Dr.
Richard M. Weinshilboum at the Department of Pharmacology, Mayo Medical
School/Mayo Clinic/Mayo Foundation, Rochester, MN. HST1 sense and
antisense primers were, respectively,
5'-ATGTCGGACGATTTCTTATGGTTTG-3'
and 5'-TTATTCCCATGGGAACAGCTC-3'. HST2a and HST2b sense primers
were, respectively, 5'-GTCGACATGGCGTCTCCCCCACCTTTCCAC-3' and
5'-GTCACAATGGACGGGCCCGCCGAGCCCCAGATC-3'; both sense
primers were paired with the common antisense primer
5'-GCGGCCGCTTATTATGAGGGTCGTGGGTGCGG-3'. PCR products were
isolated by the agarose gel purification method (QIAGEN,
Santa Clarita, CA) and subcloned into a bacterial expression vector
pCRT7/NT-TOPO (Invitrogen, San Diego, CA). Subclones
containing the insert cDNA in the correct orientation were selected by
PCR using T7 sense primer and gene-specific antisense primer. The
bacterial expression constructs were used to transform BL21(DE3)pLysS
cells (Stratagene). Transformed bacteria were grown in 50
ml LB broth with ampicillin, for 3 h at 37 C, and then
overnight at 26 C. The overnight seed cultures were added to 1 l
LB broth with ampicillin and were incubated at room temperature until
A600 reached 0.4–0.8, at which time IPTG
was added to a final concentration of 0.1 mM.
Incubations were continued overnight at room temperature. Bacteria were
harvested and pellets suspended in 5 x (wt/vol) of chilled lysis
buffer (50 mM sodium phosphate, 300
mM NaCl, 10 mM imidazole,
pH 8.0) containing proteolytic enzyme inhibitors (1 pellet/25 ml,
Boehringer Mannheim) and 1 mg/ml lysozyme. The bacterial
suspensions were sonicated and centrifuged at 45,000 rpm for 1 h.
The clear supernatants were applied to 2 ml Ni-NTA Agarose columns
(QIAGEN) equilibrated with lysis buffer. Columns were
washed with 10 column vol of wash buffer (50 mM
sodium phosphate, 300 mM NaCl, 20
mM imidazole, pH 8.0), and the bound proteins
were eluted with 5 column vol of elution buffer (50
mM sodium phosphate, 300 mM
NaCl, 250 mM imidazole, pH 8.0). Eluted proteins
were concentrated using Amicon Centricon filters (Amicon, Beverly,
MA).
Enzyme assays
For determination of substrate specificity, an equimolar mixture
of DHEA, cholesterol, and oxysterol was prepared by
solubilization in 3 mM (final concentration)
hydroxypropyl-ß-cyclodextrin (38). The incubation
mixture consisted of 20 µM each of DHEA,
cholesterol, and an oxysterol [24(S)-, 25-, or
27-hydroxycholesterol], 100 µM
[35S]PAPS, and either 27 µg HST1 or 13.6 µg
of an HST2a or 2b isoform in a total vol of 0.1 ml 100 µM
Tris-HCl (pH 7.1) and 5 mM magnesium acetate. Reactions
were stopped at 30 min, by the addition of 1 ml chloroform, and the
free alcohols and sulfoconjugates were extracted into chloroform by
complexing with methylene blue (39). The chloroform layer
was backwashed 4 times with 1 ml water to remove unreacted
[35S]PAPS. The chloroform was taken to dryness
and redissolved in 0.1 ml methanol, for analysis by HPLC and gradient
elution.
HPLC analysis
A model no. 1100 instrument (Hewlett-Packard Co.), equipped with a diode array detector, set to absorb
at 202 ± 20 nm, was used. Samples were injected onto an Aqua
3_C18 reverse phase column, 150 x 4.6 cm (Phenomenex, Torrance,
CA). An initial trinary solvent system of acetonitrile, methanol, and
water (5:55:45), each containing 1.0 ml of 7.4 M ammonium
acetate/liter (40), was used, with an initial flow rate of
0.5 ml/min. During a 25-min period, the flow rate and solvent system
changed linearly to 1.5 ml/min and 5:95:0, respectively, and this was
followed by an additional isocratic period of 15 min, for a total
duration of 40 min. Fractions were collected at intervals of either 0.5
or 1.0 min, and the total counts-per-minute in each fraction was
determined by liquid scintillation spectrometry at constant
efficiency.
RT-PCR analysis
RT was performed using the ThermoScript RT-PCR system, according
to the manual provided by the company (Life Technologies, Inc.). Briefly, in one reaction tube, the first cDNA strand was
synthesized at 50 C for 60 min using 3 µg of total RNA as template
and the gene-specific antisense primers,
5'-TTCTCCTCTCTCATGGGCATCCAGCCATG-3' for HST1 and
5'-TCCAGGATGGATCCCCTTCCTTCAGGATTAAG-3' for both HST2a and HST2b. cDNAs
were amplified by PCR using 2 µl cDNA reaction mix as template and
gene-specific primer pairs: HST1, 5'-CGTGATGAGTTCGTGATAAGGGATGAAGATG-3'
(sense primer) and 5'-TGTGGTCAAACCATGACCCATATAGCACAG-3'
(antisense primer); HST2a, 5'-GTGTCACCACTTTACAGAAGAGGGACTGAG-3' (sense
primer) and 5'-AGATGATCTCGATCATCCAGGTCGTGCCGT-3' (antisense primer);
and HST2b, 5'-GGCTTGTGGGACACCTATGAAGATGACATC-3' (sense primer),
5'-AGATGATCTCGATCA-TCCAGGTCGTGCCTG-3' (antisense
primer). PCR conditions were: initial denaturation at 94 C for 2 min,
followed by 40 cycles of 30 sec at 94 C, 30 sec at 60 C, and 30 sec at
72 C.
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Results |
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, the sulfonates
of DHEA, 27-hydroxycholesterol, and cholesterol
were clearly resolved by HPLC; and although individual oxysterol
monosulfonates could not be adequately resolved from each other, they
were completely resolved as monosulfonate/monoacetate derivatives.
Table 1 gives a listing of the retention
times for all compounds analyzed.
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. In the presence of an equimolar amount
of DHEA, the C27 sterols accounted
for 88 and 97% of the sulfonates formed with HST2a and HST2b,
respectively (Fig. 2). On the other hand, DHEA was
primarily sulfonated by HST1. It is also noteworthy that HST1
sulfonated cholesterol only minimally (
2%), whereas cholesterol
sulfonation rose to 13% with HST2a, and 48% with HST2b. To obtain an
easily detectable rate for cholesterol sulfonation, the protein
concentration used for HST1 was generally double that of the HST2
isoforms. The striking difference in cholesterol sulfonation is
reflected in the HST-specific activities: HST1, 0.01 pmol/min·mg
protein; HST 2a, 0.62 pmol/min·mg protein; and HST2b, 0.83
pmol/min·mg protein.
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It should also be noted that standards for the 3-sulfonate 27-acetate
and 3-acetate 27-sulfonate derivatives of 27-hydroxycholesterol were
clearly resolved by HPLC (Fig. 3). In other studies, it was found that
no sulfonates were formed with the 3-acetates of 24(S)-, 25-, or
27-hydroxycholesterol using either of the HST2 isoforms (data not
shown).
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). Expression
of HST1 was barely detectable in the bone marrow and spleen, and no
expression was noted for lung and skin. HST2a was clearly expressed in
the placenta, prostate, and skin and (to a lesser extent) in the
kidney, lung, ovary, and small intestine (Fig. 4). In contrast to
HST2a, expression of HST2b was ubiquitous, with expression being
most prominent in the colon, kidney, lung, placenta, prostate,
skin, small intestine, stomach, thymus, and thyroid; expression was
clearly less in the adrenal, ovary, spleen, and liver and barely
detectable in the bone marrow and brain (Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because human HST2a and HST2b are structurally unique vis-à-vis other soluble C18, C19, and C21 steroid sulfotransferases, we were interested in examining their reactivity against the larger C27 sterols, i.e. cholesterol and its hydroxylated metabolites, particularly because a specific sulfotransferase had not been identified for either cholesterol or oxysterols. For this purpose, it was necessary to develop a chromatographic system that could separate the different products generated by an enzyme when using multiple substrates in combination. Based on our previous studies (38) and those of others (45), we used the capacity for water-soluble hydroxypropyl-ß-cyclodextrin to include sterols within the torus and thus maintain nonpolar compounds in solution. Because several studies have documented the usefulness of hydroxypropyl-ß-cyclodextrin in enhancing catalytic rates with cholesterol as a substrate (46, 47), it can be concluded that the differences observed in these studies properly reflect the relative affinity of a substrate for a specific HST, rather than access to the enzyme because of a difference in solubility.
We are not aware of previous studies of oxysterol sulfonate formation.
Although our studies do not exclude the possibility that other
naturally occurring sulfonate derivatives of oxysterols may exist, we
can be certain that the human HST2 enzymes generated 3-sulfonate
metabolites. Previous studies had established the existence of
endogenously occurring sulfonates of 27-hydroxycholesterol
(9) and 24S-hydroxycholesterol (10, 11). In
the latter reports, however, the methods used were based on derivative
analysis after cleavage of the sulfonate moiety. Therefore, no direct
structural information was possible, although a 3-sulfonate structure
could be deduced. In our studies, the direct determination of the
sulfonate product formed by the HST enzymes permitted a definitive
assignment of the sulfonate moiety to only the 3ß-hydroxyl group of
the sterol. Furthermore, stereospecificity involving the 3-hydroxyl
group was maintained by the inability of the HST2 isoforms to sulfonate
lithocholic acid whose 3-hydroxyl group is 
-oriented (data not
presented).
All previously cloned members of the soluble sulfotransferase
superfamily, i.e. estrogen and phenol sulfotransferases and
HSTs, have sizes that range from 282–295 amino acids
(48, 49, 50). Therefore, a principal difference between the
HST1 and HST2 isoforms relates to their respective sizes. HST1 contains
285 amino acids, whereas HST2a and HST2b consist of 350 and 365 amino
acids, respectively (see Fig. 5).
Interestingly, the sizes of the HST2 isoforms are more closely aligned
with members of the larger Golgi membrane-associated class of
sulfotransferases (51, 52, 53, 54). Although it is assumed
that the HST2 isoforms are soluble and not membrane-associated,
this has not been definitively demonstrated. The increase in size of
the human HST2 isoforms is attributable primarily to their extended
amino- and carboxyterminal ends; otherwise, a significant structural
similarity between the HST1 and HST2 proteins in their core regions is
present (see Fig. 5). Furthermore, P-loop motifs (55, 56, 57)
and specific amino acid residues important in protein-cofactor
interaction of soluble sulfotransferases (57) are
completely conserved.
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). The aminoterminal
ends do not seem to contain a signal sequence (58), and
their amino acid sequences do not suggest an integral membrane protein
(59). The aminoterminus of the HST2a isoform contains a
proline-dominated motif (SPPPF) suggestive of a motif that serves as a
binding site for proteins containing WW domains
(60). The prominence of proline residues is most striking
in the carboxyterminal ends of the HST2 proteins, which are
proline-enriched (see Fig. 5). This is significant because proline-rich
regions of proteins, which occur widely in prokaryotes and eukaryotes
and are commonly located in the C-termini of proteins, are involved in
protein-protein interactions (61). Additionally, the
carboxyterminal ends of the HST2 proteins are enriched not only in
proline but also glutamic acid, serine, and threonine suggestive of
polypeptide sequences that target proteins for rapid degradation by the
26S proteasome (62). It is to be noted that a structurally
related novel HST has also been cloned in the mouse
(63). Another significant difference among HST1, HST2a, and HST2b was that their messenger RNA (mRNA) expression patterns were clearly distinct. HST1 was relatively highly expressed in steroidogenic organs (adrenal and ovary), androgen-dependent tissue (prostate), tissues of the alimentary tract (stomach, small intestine, and colon), and the liver. Presumably, reflecting a greater sensitivity, expression of HST1 by RT-PCR was more extensive than previous studies employing Northern analysis would suggest, i.e. the latter demonstrated HST1 expression in only the adrenal (64), liver (64, 65), and small intestine (65). Additionally, HST1 protein and mRNA have been localized to parietal cells in human gastric mucosa (66). Similar to HST1, Northern analysis of HST2 revealed only limited expression involving the placenta, prostate, trachea, and small intestine (34). Furthermore, the latter study did not distinguish between HST2a and HST2b (34). The RT-PCR expression patterns of HST2a and HST2b were distinct from each other, in that HST2b was clearly more widely expressed than the HST2a isoform. A notable distinction among the RT-PCR expression patterns of HST1, HST2a, and HST2b was the lack of HST1 expression in skin, an organ where the HST2 isoforms were highly expressed.
Based on the substrate specificity results, we would suggest that the HST2 isoforms, in contrast to HST1, are the sulfotransferases that normally sulfonate cholesterol and its hydroxylated metabolites. Tissue expression patterns also support this judgment, in that skin expresses only the HST2 isoforms and not HST1. Sulfonation of cholesterol, an important metabolic step during normal skin development and creation of the barrier, has been subject to extensive investigation. Normally, cholesterol is sulfonated in the living epidermis; the cholesterol sulfonate then accumulates in the stratum corneum, where it undergoes sulfohydrolysis, creating a phenomenon that has been termed the epidermal cholesterol sulfate cycle (27). The fact that the normal cholesterol/cholesterol sulfonate ratio in blood and gastrointestinal epithelia is approximately 500:1, whereas in the normal stratum corneum the ratio is 5–10:1 suggests that regulation of this ratio may be critical to normal desquamation (67). Differentiation of normal human epidermal keratinocytes is accompanied by an accumulation of cholesterol sulfonate, which is accounted for by an increase in cholesterol sulfotransferase activity (68). Epidermal cornification involves the cross-linking of precursor proteins, a process dependent on the activity of transglutaminase 1, which in turn is dependent on the accumulation of cholesterol sulfonate, a transcriptional activator of the transglutaminase 1 gene (69).
Abnormalities in the metabolism of 24(S)-hydroxycholesterol and 27-hydroxycholesterol have been associated with different diseases. Accelerated atherosclerosis is a well- recognized sequela in some individuals lacking 27-hydroxycholesterol on a genetic basis (70). In others, only neurological abnormalities occur, which range from severe mental retardation to peripheral neuropathies (70). A disturbance in 24(S)-hydroxycholesterol metabolism has recently been reported in Alzheimer’s disease (71). It is of particular interest that the genes coding for the P450 hydroxylase enzymes that convert cholesterol to these oxysterols are expressed in the central nervous system (7, 72) and that a sulfonated ester of 24(S)-hydroxycholesterol is a constituent of normal brain tissue (20). The protean manifestations that occur with disturbances in these metabolic pathways indicate that further insights into disease mechanisms can only be obtained from detailed knowledge of the interrelationships of the biological effects of these oxysterols and their sulfonated esters.
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Footnotes |
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2 Guest Investigator, current address: New York University Medical
Center, 550 First Avenue, New York, New York 10016.
Received January 9, 2001.
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