(Received for publication, January 24, 1995; and in revised form, April 13, 1995)
From the
Three aryl sulfotransferases (ASTs) isolated from rat liver catalyze the sulfuric acid esterification of the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-2AAF). These three ASTs were separated by high resolution anion exchange chromatography and were designated Q1, Q2, and Q3. Q1 and Q2 had high N-OH-2AAF sulfonation activity, whereas Q3 showed low activity. Reversed phase high performance liquid chromatography/mass spectrometry analysis showed Q1-Q3 to be comprised of 33,945- and 35,675-Da protein subunits. Q1 contained only the 35,675-Da protein subunit, Q2 contained equal quantities of 33,945- and 35,675-Da subunits, and Q3 contained only the 33,945-Da subunit. The subunit compositions of Q1-Q3 were confirmed by immunochemical analysis. Size exclusion high performance liquid chromatography confirmed that the active quaternary structure of the three isoenzymes was dimeric. Analysis of liver cytosols for the relative contributions of Q1-Q3 to total cytosolic N-OH-2AAF sulfotransferase activity indicated that Q1, Q2, and Q3 accounted for 44, 46, and 10% of the activity, respectively. These results demonstrate the existence of both homodimeric and heterodimeric aryl sulfotransferases and show that two ASTs, a homodimer of 35,675-Da subunits and a heterodimer of a 33,945- and a 35,675-Da subunit, are primarily responsible for hepatic N-OH-2AAF sulfotransferase activity.
Cytosolic sulfotransferases have been shown to catalyze the
PAPS()-dependent sulfonation of a wide variety of
hydroxylated endobiotic and xenobiotic compounds (1, 2, 3, 4) . Whereas the primary
function of xenobiotic sulfoconjugation is to permit detoxication of
the compound, it occasionally results in the production of highly
reactive intermediates capable of causing genotoxic and cytotoxic
damage to cells. One sulfotransferase activity that has been studied
with great interest is that responsible for the sulfoconjugation of N-hydroxyarylamic acids and N-hydroxyarylamines(5, 6, 7) . The
sulfuric acid esterification of these compounds results in bioactivated
forms reported to cause liver cancer in
rodents(6, 7, 8, 9, 10) . A
model compound for this class of carcinogens that has been extensively
investigated is N-OH-2AAF. Sulfuric acid esters of N-OH-2AAF have been implicated in the in vivo production of the N-(deoxyguanosin-8-yl)- and
3-(deoxyguanosin-N
-yl)-2AAF DNA adducts in rat
liver (11, 12) and the N-(deoxyguanosin-8-yl)-2-aminofluorene DNA adduct in mouse
liver(10) . In addition, carcinogen-mediated loss of this
activity among rat liver cells, putatively initiated for
carcinogenesis, has been suggested to contribute to the development of
a resistance phenotype to carcinogen toxicity(9, 13) .
This phenotype contributes to the selective clonal proliferation of
initiated cells into preneoplastic nodules during the promotion stage
of carcinogenesis (14) .
Rapid advances in the knowledge of the molecular structure of sulfotransferases (see (15) , and references therein) have stimulated efforts to associate the individual catalytic activities with specific sulfotransferase amino acid sequences(4) . It has been suggested previously that a principal source for liver cytosolic N-OH-2AAF sulfotransferase activity is aryl sulfotransferase IV (AST IV)(1, 16, 17, 18) . This enzyme was reported to be a dimeric molecule comprised of two identical subunits of known amino acid sequence and a subunit mass of 33,906 Da(19, 20) . The primary sequence for the AST IV subunit is identical to the deduced amino acid sequences reported for cDNAs of phenol sulfotransferase (PST-I; (21) ) and for minoxidil sulfotransferase(22) . Recently, two additional sources of liver cytosolic N-OH-2AAF activity have been reported, HAST-I and HAST-II(23) . HAST-II was shown to have a higher enzymatic activity toward N-OH-2AAF than HAST-I and to be cross-reactive with a polyclonal antibody raised against HAST-I. Recently, the HAST-I polyclonal antibody was used to isolate an HAST-I cDNA clone, ST1C1(24) . This cDNA was found to code for a 35,768-Da protein that displayed high N-OH-2AAF sulfoconjugation activity when transfected and expressed in COS-1 cells. The 36-kDa HAST-I protein was found to have a 51% amino acid sequence homology with the 34-kDa PST-1/AST IV protein.
In order to further evaluate the role of AST IV as an N-OH-2AAF sulfotransferase, studies were conducted to determine the possible contribution of the 34-kDa and 36-kDa sulfotransferase subunits to AST IV activity. The use of improved purification techniques and protein characterization methodologies have revealed the presence of three sulfotransferase forms. The first form is a homodimer of two 33.9-kDa subunits, which shows low N-OH-2AAF sulfotransferase activity. A second form is a homodimer comprised of two 35.7-kDa subunits and has the highest level of N-OH-2AAF sulfotransferase activity. The third form is a heterodimer comprised of a 33.9-kDa and a 35.7-kDa protein subunit, possessing high catalytic activity for N-OH-2AAF sulfonation. The existence of heterodimeric sulfotransferases constitutes a new, previously unreported level of sulfotransferase organization.
Figure 1:
Elution profile of AST IV and
characterization of sulfotransferase activity following hydroxylapatite
chromatography. AST IV purification was performed through the
hydroxylapatite purification step as described under
``Experimental Procedures.'' Panel a, eluted
fractions were analyzed for absorbance at 280 nm(- - -) and 2-naphthol
sulfoconjugation activity at pH 5.5 (). Pooled fractions
representing HA1 and HA2 are shown with longdashedverticallines. Panel b,
characterization of HA1 and HA2 pools following ATP-agarose
purification. 2-Naphthol activity at pH 5.5 (hatchedbox) and methionine-agarose bead assay of N-OH-2AAF sulfoconjugation activity (stripedbox) were determined as described under
``Experimental Procedures.'' The relative intensity following
image analysis of Western blots of HA1- and HA2-purified pools stained
with a polyclonal antiserum to AST IV is also shown (filledbox).
Figure 2:
MonoQ FPLC resolution of three
sulfotransferase activity peaks in the HA1 and HA2 sulfotransferase
fractions. Sulfotransferase activity for N-OH-2AAF ()
and 2-naphthol (
) sulfoconjugation was measured following MonoQ
FPLC fractionation of HA1 and HA2 fractions as described under
``Experimental Procedures.'' Labels indicating the elution
positions of Q1, Q2, and Q3 are shown above for
reference.
Figure 3:
Size exclusion HPLC analysis of Q1, Q2,
and Q3 molecular masses under non-denaturing conditions. Profiles were
generated using 0.5-ml fractions from size exclusion chromatography of
Q1-Q3 as described under ``Experimental Procedures.''
The first profile (standards) shows the elution positions as
determined by absorbance at 280 nm for bovine serum albumin (66 kDa)
() and carbonic anhydrase (29 kDa) (
). The enzyme
activity profiles for Q1-Q3 are shown for the sulfoconjugations
of 2-naphthol (
) and N-OH-2AAF
(
).
To further evaluate the basis for the Q1, Q2, and Q3 dimer heterogeneity, subunit composition was assessed by subjecting the dimeric forms to C18 reversed phase chromatography. Elution profiles showed (Fig.4) that Q1 and Q3 were each comprised of a single protein peak with distinct elution positions. In contrast, the Q2 profile contained two protein peaks of equivalent size, which eluted at positions corresponding to the Q1 and Q3 peaks. Subsequent reversed phase liquid chromatography and mass spectrometry of protein peaks found in Q1, Q2, and Q3 (Fig.5) indicated that Q1 contained a single component with a mass of 35,675 ± 3 Da and that Q3 was comprised of a single component with a mass of 33,946 ± 4 Da. Mass spectrometry of the two protein peaks observed in Q2 were found to have masses identical to the masses for Q1 and Q3. The mass for the Q3 protein approximated the theoretical mass of an N-terminally acetylated form of the deduced amino acid sequence from the PST-1 cDNA(21) , i.e. 33,951 Da. The mass of the protein comprising Q1 did not closely correspond with any reported sulfotransferase amino acid sequence. However, since it displayed high N-OH-2AAF activity, the possibility existed that it was a modified form of the ST1C1 subunit comprising HAST-I(24) . N-Acetylation following the removal of the N-terminal methionyl residue of the ST1C1 subunit would result in a mass of 35,679 Da.
Figure 4: C18 reversed phase HPLC analysis of Q1, Q2, and Q3. Purified Q fractions were analyzed by reversed phase HPLC as described under ``Experimental Procedures.'' The subunit elution positions of the Q fractions are shown, as monitored by absorbance at 215 nm. The first eluting peak, observed in Q1 and Q2, was labeled A, as shown at the top of the figure. The later eluting peak, labeled B, was observed in both Q2 and Q3.
Figure 5: Mass spectrometry analysis of the protein subunits comprising the sulfotransferase isoforms Q1, Q2, and Q3. Molecular mass determination by mass spectrometry of the subunit peaks A and B, isolated by C18 reversed phase HPLC of Q1, Q2, and Q3, was performed as described under ``Experimental Procedures.'' A representative mass spectra, spectral data, and corresponding masses are shown for peak A (a) and B (b).
To further assess the identity of the
sulfotransferase subunits found in Q1, Q2, and Q3, N-terminal amino
acid sequencing was attempted on purified subunit proteins. Both
subunits were found to be N-terminally blocked and thus were subjected
to tryptic digestion followed by chromatographic isolation of peptides
and subsequent analysis of peptides for amino acid sequence. As shown
in Table1, amino acid sequencing of peptides showed that the
subunit comprising Q1 was homologous to ST1C1 (24) and the
subunit for Q3 was homologous to PST-1(21) . These findings
indicate that cytosolic N-OH-2AAF sulfotransferase activity is
a sum of the activities of three different dimer forms in which Q1 is
an ST1C1ST1C1 homodimer, Q2 is an ST1C1
PST-1 heterodimer,
and Q3 is a PST-1
PST-1 homodimer.
Figure 6:
DEAE-HPLC sulfotransferase elution profile
of male cytosol. Male rat liver cytosol (5 ml) was prepared as
described previously, applied to a DEAE-HPLC column, and eluted with a
gradient as described under ``Experimental Procedures.''
Fractions (3 ml/fraction) were collected and analyzed for N-OH-2AAF () and 2-naphthol (
) sulfonation
activities. Fractions were also analyzed for absorbance at 280 nm(- -
-) and are shown for reference. The elution positions for Q1, Q2, and
Q3 are indicated by labels.
Figure 7: Immunochemical analysis of sulfotransferase subunit compositions of fractions from DEAE-HPLC of male cytosol. The apex fractions from the sulfotransferase peaks labeled Q1, Q2, and Q3 in Fig.6were evaluated by Western blot immunohistochemical analysis for the presence of the 33.9-kDa and 35.7-kDa subunits. Panel a demonstrates the ability of the polyclonal antiserum to be used for detection of the two subunits: lanes1 and 2, 0.1 µg of 33.9-kDa subunit; lanes 3-5, 0.1 µg of 35.7-kDa subunit; lanes6 and 7, 2 µg of cytosol protein. Immunochemical staining was performed with polyclonal antibody pretreated as follows: lanes2 and 4, preabsorbed with purified 33.9-kDa subunit; lanes5 and 7, preabsorbed with both 33.9-kDa and 35.7-kDa subunits. In panels b and c, lanes1-3 contained 0.1 µg of Q1, Q2, and Q3, respectively. The immunochemical staining was performed with sulfotransferase polyclonal antiserum preabsorbed with 33.9-kDa subunit in b and 35.7-kDa subunit in c. See ``Experimental Procedures'' for additional details.
The N-OH-2AAF sulfotransferase activity of rat liver AST IV has been shown to be comprised of the activities of three different dimers with distinct structural and functional features. The three dimers, Q1, Q2, and Q3, were elucidated by subjecting purified AST IV to high performance liquid chromatography with an anion exchange resin. Each dimer was shown to have N-OH-2AAF sulfotransferase activity, and upon SDS-PAGE generated a single 34-kDa band that stained positively upon Western immunochemical staining with polyclonal antiserum to AST IV. Denaturation of the individual native AST IV dimers to monomers by reversed phase HPLC showed that each dimer had a unique subunit composition, and established reversed phase HPLC as an important tool for examining heterogeneity of dimer composition. Subsequent analysis of the subunits by mass spectrometry and amino acid sequencing provided a basis for the structural identification of the three dimers. This approach has allowed a definitive determination of sulfotransferase subunit composition. Q1 was shown to be a homodimer comprised of a 35,675 ± 3-Da protein subunit, which, based upon partial amino acid sequence analysis, was found to be homologous to the ST1C1 sulfotransferase subunit ((24) ; deduced amino acid sequence molecular mass of 35,768 Da). Q1 showed high N-OH-2AAF sulfotransferase activity and may correspond to the HAST I sulfotransferase whose composition also included the ST1C1 subunit (24) . The Q3 dimer was shown to be a homodimer comprised of a protein subunit with a molecular mass of 33,946 ± 4 Da and amino acid sequence homologies that identified it as the PST-1 sulfotransferase subunit ((21) ; deduced amino acid sequence molecular mass of 33,906 Da). It showed significant but much lower N-OH-2AAF sulfotransferase capacity than the Q1 homodimer and had also been implicated previously as a source of N-OH-2AAF sulfotransferase activity(16, 19) . The Q2 dimer was found to be a heterodimer comprised of one 35.7-kDa subunit (ST1C1) and one 33.9-kDa subunit (PST-1). It possessed a high N-OH-2AAF sulfotransferase capacity, which was approximately 50% that of the ST1C1 homodimer (Q1). Q2 appeared to have an anion exchange chromatography elution position similar to that of a partially characterized sulfotransferase termed HAST II, which had been previously characterized as having strong N-OH-2AAF sulfotransferase activity and reacting positively to an antiserum raised against ST1C1(23) .
Kinetic analysis of the N-OH-2AAF sulfotransferase activities for the three dimers indicated that the ST1C1 homodimer (Q1) was approximately 40 times more catalytically efficient than the PST-1 homodimer (Q3) and approximately 1.5-fold more efficient than the heterodimer (Q2). Kinetic analysis of p-nitrophenol sulfotransferase activities among the three dimers indicated a different pattern of catalytic efficiencies. The PST-1 homodimer (Q3), was approximately 3-fold more efficient than the ST1C1 homodimer (Q1) and 6-fold more efficient than the heterodimer (Q2). These results indicated that subunit-subunit interactions may have a role in determining dimer catalytic properties. Although these studies indicated that sulfoconjugation of N-OH-2AAF to its highly reactive sulfuric acid ester form was most efficiently catalyzed by a ST1C1-containing sulfotransferase dimer, the actual in vivo contribution of the various dimer forms was less predictable since other factors, including relative dimer abundance and tissue localization, must be taken into account. Studies reported here involving analysis of cytosol suggested that the heterodimer (Q2) was a principal source of N-OH-2AAF sulfotransferase activity in vivo.
Identification of Q2 as an ST1C1PST-1 heterodimer
constitutes the first report of a functional heterodimeric
sulfotransferase. Heterodimeric proteins have been shown to be
important in cellular functions ranging from the regulation of gene
expression by heterodimeric nuclear transcription factors (31, 32) to the metabolism of xenobiotics by
heterodimeric detoxication enzymes(33) . For nuclear
transcription factors, heterodimer interactions change the availability
or specific activity of factors for modulating gene expression. In the
case of xenobiotic metabolizing enzymes such as glutathione S-transferases, heterodimeric interactions produce new dimers
that increase the detoxifying functions of cells(34) . The
xenobiotic detoxifying or bioactivating properties of glutathione S-transferases in rats have been shown to be a result of
tissue-specific (35) and/or sex-specific (36) patterns
of subunit expression, resulting in formation of catalytic homodimers
and permitted heterodimers. Similarly, the existence of heterodimeric
sulfotransferases introduces a new level of functional diversity for
this family of detoxication enzymes. Furthermore, there is considerable
evidence that expression of sulfotransferases in rat liver are also
regulated in a sex-specific
manner(8, 17, 23, 24, 37) .
Interestingly, the rat liver N-hydroxyarylamine
sulfotransferases HAST I and HAST II have been reported to be
male-dominant and male-specific, respectively(23) . If these
sulfotransferases, as suggested above, correspond to Q1 (ST1C1
homodimer) and Q2 (PST-1
ST1C1 heterodimer), respectively, then it
may be concluded that the higher N-OH-2AAF sulfotransferase
activity observed in male liver cytosols (8, 17, 23) arises from the presence of the
male-dominant (homodimer) and male-specific (heterodimer) forms. Future
studies to assess the regulatory basis for sex-specific
sulfotransferase subunit expression and the extent to which
homodimer/heterodimer formation occurs are needed to provide further
insight into the role(s) that sulfotransferases have in xenobiotic
metabolism.
The existence of heterodimeric sulfotransferases also introduces a new level of structural complexity as an enzyme family. Heterodimer formation among glutathione S-transferases is typically restricted to subunits with homologies of 70-90%(33, 34) . In contrast, the amino acid sequence homology between the subunits for the PST-1 and ST1C1 is reported to be only 51%(24) . To date, there have been no investigations of the requirements for sulfotransferase dimer association and dissociation, and little is known about the sulfotransferase domains responsible for dimerization. Questions such as whether sulfotransferase heterodimer formation is restricted to specific subclasses of sulfotransferases or is a common property of all sulfotransferases are subjects for future investigations.