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The Biotransformation of
Endobiotics by Sulfonation
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Charles A. Strott,
MD, Head, Section on Steroid Regulation Young C. Lee, PhD, Staff Scientist Hirotoshi Fuda, PhD, Research Fellow Atsushi Kojitani, DDS, PhD, Postdoctoral
Fellow Norman B. Javitt,
MD, PhD, Adjunct Investigator |
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The hydroxysteroid sulfotransferases SULT2A1,
SULT2B1a, and SULT2B1b Fuda, Lee, Yanai,
Strott; in collaboration with Javitt,
Pedersen Often referred to as hydroxysteroid
sulfotransferases, the enzymes that sulfoconjugate
neutral steroids constitute the SULT2 family, which, in turn, comprises two
subfamilies, SULT2A1 and SULT2B1. Whereas the SULT2A1 subfamily consists of a
single form, the SULT2B1 subfamily consists of the two isoforms SULT2B1a and
SULT2B1b, which derive from an alternative exon 1. SULT2A1 has a broad
substrate predilection, acting on alpha- and beta-hydroxylated compounds,
whereas the SULT2B1 isoforms have narrower substrate preferences that are
limited to selective 3-beta-hydroxylated steroids. For example, SULT2B1a avidly
sulfonates pregnenolone, whereas it less efficiently sulfoconjugates
cholesterol; on the other hand, SULT2B1b functions as the physiologic
cholesterol sulfotransferase. The fact that the SULT2A1 and SULT2B1 isozymes
are differentially expressed and display dissimilar substrate propensities
strongly suggests that they play distinct biologic roles. Importantly, the
SULT2B1 isoforms are structurally unique when compared with SULT2A1 and all
other known cognate cytosolic sulfotransferases. For instance, the SULT2B1
isoforms have extended amino- and carboxy-termini. Moreover,
the carboxy termini are enriched in prolines as
well as in glutamate, serine, and threonine, suggesting polypeptide sequences
that target proteins for rapid degradation. The crystal structure of human
SULT2B1b reveals a catalytic binding orientation for the substrate that
differs from the previously determined structure for the SULT2A1
substrate-binding pocket. In addition, an amino terminal helix, comprising
residues D19 to K26, determines the specificity difference between the
SULT2B1 isoforms, which becomes ordered upon substrate binding covering the
binding pocket. Mutational analysis suggests that the specificity difference
for cholesterol and pregnenolone between the SULT2B1a and SULT2B1b isoforms
can be traced to the unique amino-terminal residues 19-DISEI-23. Alanine
scanning of this region reveals that only the I20A and I23A mutants are
associated with a complete loss of cholesterol sulfonating activity.
Furthermore, the activity can be partially restored by replacement with
conservative substitutions such as leucine and methionine. I20 and I23 lie on
the same side of the helix facing on the inside of the hydrophobic pocket,
whereas residues S21 and E22 are exposed to solvent. The structure of the SULT2B1 gene is
highly conserved in that the human and mouse genes are essentially identical.
Differential expression of mouse SULT2B1a, SULT2B1b, and SULT2A1, along with
their distinct substrate preferences, has physiologic relevance. For instance,
the exclusive expression of SULT2B1a in the central nervous system, and its
tendency for greater activity with pregnenolone as a substrate, would be in
keeping with the importance of pregnenolone sulfate as a neurosteroid
involved in learning and memory processes. The sole expression of SULT2B1b in
skin, and the fact that it clearly has a predilection for cholesterol as a
substrate, is consistent with the importance of cholesterol sulfate as a
regulatory molecule in keratinocyte differentiation and development of the
epidermal barrier. On the other hand, the overwhelming expression of SULT2A1
in the liver is in accord with its role in general metabolism involving both
xenobiotics and endobiotics. Importantly, the mouse
SULT2B1 and SULT2A1 genes are differentially expressed during
embryonic development, with the former gene expressed at all stages from E8.5
to E19 and the latter gene not expressed until E19. The results suggest that
expression of the mouse SULT2B1 gene is more critical during early
development than that of the SULT2A1 gene, especially the expression
of SULT2B1a during the early stages of brain development and the
expression of SULT2B1b during growth of epithelia. Lee KA, Fuda H, Lee
YC, Negishi M, Strott CA, Pedersen LC. Shimizu C, Fuda H, Yanai H, Strott CA. Conservation of the hydroxysteroid
sulfotransferase SULT2B1 gene structure in the mouse: pre- and postnatal
expression, kinetic analysis of isoforms, and comparison with prototypical SULT2A1. Endocrinology 2003;144:1186-1193.
Strott CA.
Hydroxysteroid sulfotransferases SULT2A1, SULT2B1a and SULT2B1b. In: Coughtrie MWH, Pacifici GM, eds. Human Cytosolic Sulfotransferases. Cholesterol sulfate Higashi,
Yanai, Fuda, Lee, Strott; in collaboration with Javitt,
Kanzaki Cholesterol sulfate is widely distributed in
human plasma and is quantitatively more significant than other known sterol
sulfates. While our knowledge of the physiologic role(s) of cholesterol
sulfate is still evolving, the steroid has been implicated in several
biologic systems. For example, it is a component of cell membranes, where it
has a stabilizing effect, such as protecting erythrocytes from osmotic lysis.
Cholesterol sulfate can regulate serine proteases such as those involved in
blood clotting, fibrinolysis, and epidermal cell adhesion. Cholesterol
sulfate has also been implicated in adrenal and gonadal steroid synthesis, in
the regulation of cholesterol and fatty acid synthesis, and in controlling
sperm capacitation. Given its ability to regulate the activity of selective
protein kinase C isoforms and modulate the specificity of
phosphatidylinositol 3-kinase, cholesterol sulfate is involved in signal
transduction. In keratinocyte differentiation, cholesterol sulfate can induce
genes that encode key components involved in development of the barrier. In
addition, cholesterol sulfate can induce platelet aggregation. That
cholesterol sulfate circulates in blood and demonstrates an emerging role as
a regulatory molecule suggests a similarity to compounds categorized as
hormones. Quantitative expression of the gene encoding
SULT2B1b is consistent with the involvement of cholesterol sulfate in
keratinocyte development, i.e., the progressive expression of SULT2B1b mRNA
in primary cultures of normal human epidermal keratinocytes (NHEK) during Ca2+-induced
differentiation. The rise in SULT2B1b mRNA expression parallels increases in
protein and enzymatic activity. SULT2B1b is the only SULT2 enzyme expressed
in normal human skin as well as in cultured NHEK, which would be the expected
finding if the sole purpose of SULT2 expression in skin were the production
of cholesterol sulfate. Nonetheless, the question remains as to the principal
physiologic role of cholesterol sulfate in the epidermis. The answer can be
found, in part, by knowing where SULT2B1b is expressed during keratinocyte
development. In this regard, immunocytochemical analysis has revealed that
expression of SULT2B1b is confined to the granular layer of the epidermis,
suggesting that it is a late marker of keratinocyte differentiation, i.e.,
the expression pattern is the same as that of the barrier protein filaggrin,
an acknowledged late marker of differentiation. On the other hand, involucrin, another barrier protein, is clearly expressed
by cells in the immediate suprabasal region or
spinous layer of the epidermis, the earliest-appearing cells in the pathway
toward keratinocyte differentiation. The confinement of cholesterol
sulfotransferase to the granular layer of the living epidermis beneath the
stratum corneum, coupled with the fact that this region of the epidermis
contains the highest content of cholesterol sulfate, strongly suggests that
the principal function of this sulfolipid is carried out primarily in the
region of the granular-stratum corneum junction. The epidermis is a
perpetually renewing tissue whereby keratinocytes arise from stem cells in
the basal layer and move through a series of cellular differentiation events
until, as dead squames, they are finally sloughed off from the outer stratum
corneum. Cholesterol sulfate could play a significant role in the proper
functioning of the stratum corneum and the normal sloughing of dead cells, a
process termed desquamation. Cholesterol sulfate can retard desquamation by
inhibiting serine proteases in the stratum corneum that are responsible for
the degradation of cell adhesion structures known as desmosomes. In addition,
cholesterol sulfate can influence cell cohesiveness by affecting the
stability of corneocyte lipid bilayers. We therefore hypothesized that the
inhibitory influence of cholesterol sulfate on normal desquamation would
subsequently be removed by the presence of a cholesterol sulfate
sulfohydrolase in the outer stratum corneum, thus allowing normal
desquamation to occur. Our hypothesis is supported by the finding of markedly
elevated cholesterol sulfate in the stratum corneum of patients with
recessive X-linked ichthyosis, a scaling disorder that is a form of abnormal
desquamation resulting from a deficiency in steroid sulfatase activity.
Interestingly, about 90 percent of the cholesterol sulfate formed during Ca2+
induction of keratinocyte differentiation is found in the cellular membrane
fraction, with only 10 percent present in the soluble cell fraction. The
membrane localization of cholesterol sulfate suggests that such localization
is an important determinant of cholesterol sulfate’s physiologic
effect. Cholesterol sulfate supports platelet
aggregation: it potentiates arachidonic acid–, ADP-, and
thrombin-induced platelet aggregation as well as serotonin secretion, effects
specific for cholesterol sulfate that require both the sterol ring structure
and the sulfate moiety. The content of cholesterol sulfate in platelets is
566 ± 62 pmol/109 platelets. The presence of SUL2B1b confirmed
that platelet cholesterol sulfate can be produced locally and not just taken
up from the circulation. In fact, as in skin, platelet SULT2B1b is the only
SULT2 enzyme expressed by these discoid anucleate
particles. Although platelets lack a nucleus, they do contain rough
endoplasmic reticulum and polysomes and are known to engage in protein
synthesis. It is recognized that human platelets and plasma lipoproteins
interact and are intimately involved in the pathogenesis of atherosclerosis,
thrombosis, and coronary artery disease. Based on real-time PCR, the level of
SULT2B1b mRNA in platelets is stably maintained at 4°C but markedly
diminished over a period of 4 h at 37°C. The presence of HDL but not of LDL,
however, significantly attenuates the loss of SULT2B1b mRNA. Furthermore, the
stabilizing influence of HDL is attributable specifically to its
apolipoprotein A-I component, whereas apolipoprotein A-II and apolipoprotein
E are without effect. Importantly, we observed a direct correlation between
platelet SULT2B1b mRNA and protein levels in the presence or absence of
lipoprotein as reflected in enzymatic activity and cholesterol sulfate
production. Higashi Y, Fuda H, Yanai H, Lee Y, Fukushige T, Kanzaki T, Strott CA.
Expression of cholesterol sulfotransferase (SULT2B1b) in human skin and
primary cultures of human epidermal keratinocytes. J Invest Dermatol 2004;122:1207-1213. Yanai H, Javitt NB, Higashi Y, Fuda H, Strott CA. Expression of cholesterol sulfotransferase
(SULT2B1b) in human platelets. Circulation 2004;109:92-96. Transcriptional regulation of cholesterol
sulfotransferase (SULT2B1b) Lee,
Higashi, Fuda, Strott Studies involving RT-PCR reveal that human
SULT2B1a and SULT2B1b are differentially expressed. Real-time PCR
demonstrates that expression of SULT2B1b is high in the prostate, placenta,
and skin; weak in the kidney, lung, small intestine, and colon; and with
little or no expression in other tissues. Information is lacking as to how
expression of the two human SULT2B1 isoforms is regulated. Given the
importance of cholesterol sulfate in the epidermis of human skin, we have
focused on the expression of SUT2B1b by using primary cultures of normal
human epidermal keratinocytes (NHEK) as well as immortalized but highly
differentiated human keratinocytes (HaCaT) cells. The gene encoding SULT2B1 contains neither a
canonical TATAAA nor a CCAAT motif in the upstream region flanking exon 1B,
and there is no initiator motif. An RLM-RACE investigation of the start of
transcription yielded multiple transcription start sites (TSS) that tended to
cluster in two areas. Some TATA-less promoters retain the ability to direct
transcription initiation from a specific nucleotide, whereas others appear to
direct transcription initiation from several sites, ranging from a few
tightly clustered start sites to dozens of sites spanning hundreds of
nucleotides. Although inconsistency with the RLM-RACE procedure might be
attributable to the existence of multiple TSS, we cannot exclude the
possibility of premature termination of RT as a consequence of the high GC
content in the 5´-UTR. In fact, experiments employing RT-PCR placed the TSS
upstream of the sites determined by RLM-RACE, suggesting that premature
termination of RT might have occurred. Many TATA-less promoters are characterized by
the presence of multiple GC boxes, which bind to the Sp1 transcription
activator and thus play a central role in the assembly of the transcription
complex of these promoters. Regulation of the gene encoding SULT2B1 appears
to be similarly under the influence, at least part, of the Sp1 family of
transcription factors; that is, the area upstream of the coding region of
SULT2B1b contains several GC/GT boxes, and mutational analyses suggest
involvement of specific motifs in transcriptional regulation. In addition,
deletion analyses correctly confirmed the mutational analyses. Importantly,
nuclear extracts from HaCaT cells contain proteins
that bind to probes incorporating Sp1 motifs implicated in gene regulation by
the mutational and deletion analyses; furthermore, supershift
analyses confirmed the presence of Sp1 and Sp2 proteins in the HaCaT cell nuclear extracts. Co-transfection experiments
that used NHEK and HaCaT cells provided additional
support for the involvement of Sp1 and Sp2 in transcriptional regulation. While the case for Sp1 and Sp2 involvement in
regulating expression of SULT2B1b is appealing, the picture is undoubtedly
more complicated. Activation of a given promoter requires several
transcription factors that bind cooperatively to their cognate sites or
possibly act synergistically through other mechanisms. In this regard, a unique
feature of Sp1 as a transcription factor is its synergistic activation and
interaction with other transcription factors. Interestingly, in the case of
the SULT2B21b promoter, we observed, in addition to Sp1 and Sp2, two
unidentified proteins that specifically bind to probes containing the Sp1
motifs; we are currently trying to identify these proteins. Notably, Sp1 and
related family members are not the only proteins to recognize GC/GT boxes. We
have found several other zinc-finger proteins that have a binding specificity
similar to Sp1. Assuming that the location of the TSS for
SULT2B1b is correct, then a major Sp1 regulatory element is located in the 5´
UTR. Reports, however, support transcriptional regulation by the 5´ UTR. The
presence of transcriptional regulatory activity within exon 1 represents a
positive internal promoter regulatory sequence. Interestingly, promoter
activity within the 5´ UTR is usually attributable to the presence of
functional Sp1 binding elements. Furthermore, CpG
dinucleotides, common within the promoter and 5´ UTR of SULT2B1b, may be
subject to hypermethylation, a process that can be
associated with loss of expression. Importantly, Sp1 has been implicated in
the protection of CpG islands from methylation.
Most CpG islands encompass the promoter and first
exon of most housekeeping genes and more than 50 percent of tissue-restricted
genes. We are currently investigating the role of CpG
methylation in transcriptional regulation of human SULT2B1b expression. Isolation, identification, and
characterization of human platelet cholesterol sulfate-binding protein Yanai, Fuda,
Higashi, Lee, Strott Based on preliminary experiments demonstrating
saturable and specific cholesterol sulfate-binding activity in human platelet
membrane extracts, we initiated experiments to identify the binding factor.
We used anion-exchange chromatography to fractionate platelet membrane
extracts. Analysis of the fractions demonstrated a protein of about 55,000 MW
with an N-terminal sequence EPAVYFKEQFLDG, which is identical to the
N-terminus (minus the signal sequence) of calreticulin, a calcium-dependent chaperone protein first detected in the endoplasmic
reticulum but now also known to be present in the plasma membrane. As a
confirmatory experiment, we examined the effect of anticalreticulin
antibodies on cholesterol sulfate binding to human platelets. The results
supported the notion that calreticulin is capable of binding to cholesterol
sulfate and is responsible for the binding of cholesterol sulfate to human
platelets. As human calreticulin was not initially
available, we used the bovine ortholog (93 percent
identical to human calreticulin) for binding analyses, competition studies,
and the effect of calcium on the binding activity. In summary, cholesterol sulfate
binding to bovine calreticulin is saturable with a Kd
of 193 pmol/mg; furthermore, the binding activity is significantly enhanced
by calcium at low concentration, whereas binding is inhibited at high calcium
concentration. Competition studies reveal that binding of cholesterol sulfate
to bovine calreticulin is highly specific, i.e., other steroid sulfates as
well as cholesterol and other cholesterol derivatives do not compete
effectively. With the recent acquisition of the cDNA for human calreticulin,
we will now proceed to overexpress and purify the
human protein for further characterization and cholesterol sulfate-binding
analyses. Characterization of unknown cytosolic
sulfotransferases Fuda, Hanyu,
Javitt, Strott Recently, we cloned two structurally unique
cytosolic sulfotransferases for which substrates have not yet been
identified. Nonetheless, it is interesting that one sulfotransferase
(SULT4A1) appears to be exclusively expressed in the central nervous system,
whereas expression of the other sulfotransferase (SULT6B1) appears to be
restricted to the testes. Although it is possible that the proteins are
products of pseudogenes and are thus inactive enzymes, the specific tissue
expression patterns would suggest otherwise; that is, the fact that the
proteins are expressed in selective tissues suggests that they have a
specific function in those tissues. To explore such possibility, we cloned, overexpressed, and purified both SULT4A1 and SULT6B1 and
have initiated a search to identify active substrates for them. COLLABORATORS Norman B. Javitt,
MD, PhD, Lars C.
Pedersen, PhD, Laboratory of Structural Biology, NIEHS, For
further information, contact chastro@mail.nih.gov |
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