Biochimica et BiophysicaActa, 1076(1991)282-288 :'~1991ElsevierSciencePublishersB.V.(Biomedical Division)0167-.4838/91/$03.50 ADONIS 0167483891000860
282
BBAPRO33814
Enzymic synthesis of steroid sulphates XVII. On the structure of bovine estrogen sulphotransferase John B. Adams School of Biochemistr): Universityof New South Wales. Sydney (Australia)
(Received21 May1990) Keywords: Estrogensu[photransferase:Isomorphicform;Transferrin;(Bovine placenta) Estrogen sulphotransferase plays a major role in controlling intracdlular levies of 17~-estradiol in human mammary cancer cells and human endometrium. Bovine estrogen sulphotransferase e.DNA has recently been cloned: the encoded protein having a maximum M r of 35000 (Nash, A.R. et al. (1988) Aust. J. Biol. Sci. 41, 507-516). Enzyme of M r 35000 by SDS-PAGE has now been isolated and cyanogen bromide-deaved peptides sequenced. The latter were identified in the c-DNA-predicted amino acid sequence which confirms that the active enzyme (Mr ~ 70000) exists as a dimer of identical subunits. Sequence data on similar peptides isolated from an enzyme preparation containing a protein of M r 74000 as the major species on SDS-PAGE, which was previously thought to represent the enzyme, suggested that this protein was transferrin. This was confirmed by PAGE, SDS-PAGE, susceptibility to neuraminidase and reaction with bovine transferrin antibody. Isoelectric focusing experiments show that active enzyme exists in two or three polymorphic forms (pl values 5.3, 5.7 and possibly 5.9) having similar physicochemical properties ot polymorphic forms of transferrin so that they overlap on ion-exchange chromatography and PAGE. The enzyme shows some homology to the amino acid sequence close to the Fe-binding site in laetoferrin and the question is raised as to the possible presence of a tightly bound metal in estrogen sulphotransferase involved in the binding of adenosine Y-phosphate 5'-phosphosulphate. Introduction Apart from its conjugating role in ttle liver, estrogen sulphotransferase (EC 2.8.2.4, Y-phosphoadenylylsulfate:estrone 3-sulfotransferase) is atso involved in the regulation of 17fl-estradiol levels in target tissue cells. For example, in both human and porcine endometrium, progesterone induces 17fl-estradiol dehydrogenase and estrogen sulphotcansferase, thus preventing estrogen action by effectively converting most of the 17fl-estradiol to estrone sulphate [1-5]. Again, when estrogen receptor positive human mammary cancer cells are exposed to physiological concentrations of 17fl-estradiol (~ 1 nM), a major route of metabolism is the formation of estrogen sulphates [6,7]. In such cells, estrogen sulphotransferase exhibits high affinity for 17fl-estradioland estrone and cooperativity in their binding [6]. Properties of estrogen sulphotransf¢rase have been examined in puriAbbreviations: PAGE, polyacrylamidegel electrophoresis:SDS, sodiumdodecylsulphate. Correspondence:J.B. Adams,Schoolof Biochemistry.,Universityof New SouthWales,Sydney,N.S.W.2033,Australia.
fled preparations of enzyme isolated from bovine placenta and adrenals. By means of ultracentrifugation on sucrose gradients, enzyme activity was associated with a species of Mr---74000 and in the analytical ultracentrifuge, both in the presenc and ,-bsence of guanidine hydrochloride, the purified enzyme possessed an Mr of 74000 [8]. Enzyme activity was associated with four polymorphic protein species which ran in close proximity on PAGE. Each of these species could be isolated by repeated ion-exchange chromatography when they were found to possess similar amino acid compositions and to exhibit wave-fike kinetics [8,9]. On SDS-PAGE, all purified enzyme preparations showed an Mr 74000 species, often accompanied by bands in the Mr 65-70000 and Mr 35000 regions. These bands were thought to be generated by proteinase action [10]. Recently, enzyme composed almost exclusively of an Mr 35000 species on SDS-PAGE has been isolated from bovine placenta and cloned. The c-DNA sequence indicated a protein of Mr 34600 suggesting that the enzyme exists as a dimer of identical subunits [11]. We have now reinvestigated the nature of the protein showing a Mr of 74000 by SOS-PAGE and have identified it as transferrin, It has been confirmed that estro-
283 gen sulphotraasferase is composed of two identical subunits of M,. 35000. The active enzyme can exist in two or three pc!ymorphic forms with very similar physicochemical properties to the polymorphic forms of bovine transfer, in. The possibility that estrogen sulphotransferase may be genetically related to the transferrin family of ~roteins has been explored by comparison of amino acia sequence data. Materials ana Methods
Materials [2,4,6,7-3H] 17/~-estradiol (specific activity 110 Ci/mmol) was obtt~ined from Amersham International. Bovine transferrin, l, uman transfcrrin, human transfertin antibody, neuram/nidase (Type X Ctostridium perfringens) and adenosine Y-phosFhate 5'-phosphosulphate, were from Sigma. Rabbit anti bovine transferrin was kindly supplied by Dr. E. Momotani. Adenosine 3'-phosphate 5'-phospho[~'S]sulphate was prepared as described previously [12]. Poly buffer exchanger (PBE94) and poly buffer-74 for d~romatofocusing were obtained from Pharmacia. Methods Purification of estrogen sulpho:ransferase with apparent M, 35000 by SDS-PAGE eP-35K). This was carried out by an adaptation of the method described by Moore et al. [13]. Bovine placenta c~tyledons (600 g) were homogenized in 2 voL of 0.01 M sodium phosphate buffer (pH 7A) in 0.9~ NaCI cow,raining 1 mM EDTA, 1 mM dithiothreitol and 200 KIU/ml aprotinin. After straining through two layers of cheesecloth, the homogenate was centrifuged at 13000 × g for 15 rain, and the supernatant reeentrifuged at 40000 >'~g for I h. An (NH4)2SO4 fraction precipitating between 0.5 and 0.7 saturation was made and dissol;'~ in 50 ml of the homogenization buffer. This was dialysed against 0.02 M sodium phosphate buffer (pH 7.8) containing I mM EDTA and 0.1 mM dithiothreitol (buffer A). The dialysed material was clarified by centrifugation at 100000 × g for 1 h and applied to a column (3 × 35 cm) of DEAE-Sepharose CL-6B (Pharmacia) previously equilibrated with buffer A. After application of one column volume of buffer A to remove unbound proteins, elution was carried out with a linear gradient of 0.02 M sodium phosphate (pH 7.~;, 250 ml) and 0.1 M sodium phosphate (pH 7.8, 250 ml) each containing 0.1 mM dithiothreitol and 200 KIU/ml of aprotinin. The flow rate was 0.5 ml/min and 9 ml fractions collected. The latter were assayed for protein (280 nm absorption) and 0.1 ml aliquots taken for enzyme assay. Active fractions were pooled, concentrated by uhrafiltration and dialysed against 5 mM ammonium bicarbonate (pH 8.1) containing 1 mM EDTA and 0.1 mM dithiothreitol. This was then applied to a column (3 × 35 cm) of
DEAE-Sepharose CL-6B equilibrated with the buffer. Elution was carried out with a linear gradient of 5 mM ammonium bicarbonate (pH 8.1,300 ml) and 500 mM ammonium bicarbonate (pH 8.1, 300 ml), each containing 1 mM EDTA, 0.1 mM dithiothreitol and 200 KIU/ml aprotinin. The flow rate was 0.25 ml/min and 3 ml fractions collected. Aliquots (0.01 ml) were removed for enzyme assay and active fractions pooled. Final purification was achieved on a column (1.5 x 50 cm) of DEAE-Sepharose CL-6B with a linear gradient of 0.02 M sodium phosphate (pH 7.4, 300 ml) and 0.06 M sodium phosphate (pH 7.4, 300 ml), each containing 1 mM EDTA and 0.1 mM dithiothreitol. Aliquots (0.01 m:) of fractions were assayed for activity. The active fractions were pooled, concentrated by ultrafiltration (6 ml) and stored in 1 ml allquc4s at -80°C. The protein concentration was 2.2 mg/ml and the specific activity 21 nmol estrogen sulphate formed/mg protein per min. Purification of estrogen sulphotransferase with apparent Mr 74000 by SD$-PAGE (P-74K). This material was isolated from pooled bovine adrenals by (NH,s)2SO4 fractionation and repeated chromatography on DEAEcellulose columns, as described previously [8]. The specific activity was 10 nmol estrogen sulphate formed/rag protein per min. Enzyme asmy. Unless otherwise stated, this was carried out by incubation (10 rain 37°C), with [3HI 17.8¢stradiol (30/~M, 80000 dpm), adenosine 3'-phosphate 5'-phosphosulphate (0.1 raM), MgCI 2 (15 mM), TrisHCI (pH 7.4, 50 raM), EDTA (1 raM) and dithiothreitol (0.1 raM), in a final vol. of 0.15 ml. Carriers of 17.8estradiol and 17.8-estradiol-3-sulphate (50 ~g of each) were added in acetone (0.6 ml). Protein was removed by centrifugation, the supernatant dried under N 2, and the [3H]estradiol-3-sulphate isolated and counted by liquid scintillation after thin-layer chromatography [3]. Gel electrophoresis. PAGE was carried out with 6% gels, and SDS-PAGE with 10~ gels. For the latter, molecular weight markers were: phosphorylase b, bovine serum albumin, egg albumin, pepsin, trypsinogen and lysozyme. Proteins were stained with Coomassie blue. Immunological studies, immunodiffusion was carried out on microscopy slides using 1~ agar containing 0.15 M NaCI and 20 mM sodium phosphate (pH 7.0). Chromatofocusing chromatography. An (NH4)2SO4 fraction was prepared from bovine placenta cotyledons (400 g) as described above and estrogen sulphotransferase subsequently purified on a column (4 x 40 cm) of DEAE-cellulose (DE52, Whatman), Elution was carried out with 0.02 M sodium phosphate (pH 7.4) before switching to a linear gradient of 0.02-0.1 M phosphate (pH 7.4) containing 0.1 mM dithiothreitol and 200 KIU aprotinin as described previously [10]. Enzymically active fractions were pooled, dialysed against 0.025 M imidazole HCI (pH 6.6) and concentrated by ultrafiltration. Chromatofocusing was carried out on a 20 mi bed
284
volume column of polybuffer exchange resin-94 (pH 6.6) and elution made with polybuffer-74 (pH 5.0) containing 1 mM dithiothreitol. Fractions (2 ml) were collected and assayed for protein (280 nm absorption) and enzyme activity. lsoelectric focusing. Estrogen sulphotransferase was purified from bovine placenta cotyledons as described 18,9|. On PAGE is showed 3-4 bands in the usual position and was free of other proteins as judged by this technique. Electrofocusing of this preparation was carfled out on an LKBSI02 (440 ml) apparatus, employing 6 ml of wide-range carrier ampholyte (pH 3-10). Enzyme (4 mg protein) in 6.7 ml of 0.1 M Tris-HCl (pH 7.5) was introduced into the mixing vessel of the gradient mixer when about one quarter of the gradient bad been formed. This prevented the enzyme coming into contact with the bottom electrode and subjecting it to extremes of pH. Voltage was set at 500 V and the column run for 3 days at 0°C. Fractions (4 ml) were collected at a flow rate of 32 ml/h and the pH and enzyme activity determined. The latter was assayed by the method given in the legend to Fig. 8. Amino acid sequencing. This was carried out on peptides isolated by cyanogen bromide cleavage. Briefly, protein (200/~g) was dried and reacted with 100 #l of cyanogen bromide solution (10 mg/m170% formic acid) by standing overnight at room temperature. Following iyophilisation, /~-mercaptocthanol (20 /~l) and buffer (200 #l, 6 M guanidine hydrochloride in 0.5 M Tris, pH 7.5) were added and the mixture stood at 370C for 1 h. Peptides were resolved on a reverse-phase column (15 x 3.2 mm Brownlee C4 cartridge). A gradient (A: 0.1% trifluoroacetic acid in water, B: 0.1% trifluoracetic acid in acetonitdle) was applied of 0-35% B in 30 rain. Monitoring was carried out at 210 and 254 nm. Cyanogen bromide cleavage was performed directly on P-74K but P-35K was purified on the same column and buffer system; the gradient in this case was 0-100% B in 40 rain. The major protein component (M r 35000 on SDS-PAGE} was used for cleavage. Sequencing was carried out on an Applied Biosystems Pulsed Liquid 477A sequencer with online 120A PTH analyser, using standard (normal -1) cycles. The SWISS-PROT protein database was employed for comparison of sequence data obtained with P-74K. Scq,,ence homology of estrogen sulphotransferase with lactoferrin was carried out with the SEQHP programme. Resulls
PAGE and SDS-PAGE of the two enzyme preparations P-35K and P.74K The four band (two major and two minor) pattern, previously exhibited by either bovine adrenal or placen-
hal preparations of estrogen sulphotransferase [9], was again evident by PAGE of P-74K (Fig. 1). Preparation P-35K exhibited a band in the same area. No other proteins were present in either preparation. In repeated runs, it was not possible to determine whether a single band or two poorly resolved bands were present in P-351C Mixtures of the two preparations were not resolvable (Fig. 1). Upon SDS-PAGE, preparation P-74K contained a major protein of Mr 74000, together with minor proteins of lower molecular weight, including an Mf 35 000 species. Preparation P-351L on the other hand, contained a protein of M, 35000 as the major species proteins of Mr 74000 and Mr 50000 being the minor components (Fig. 2). Amino acid sequence data Preparation P-74K was cleaved directly with cyanogen bromide and the resulting polypeptides separated by reverse-phase HPLC. Some twelve pepfides were obtained and two of these (fractions 4 and 6 numbered in sequence of elut~on) subjected to amino acid sequence analysis. Neither of these polypepfides showed any homology to the amino acid sequence predicted from the c-DNA data obtained for cloned estrogen sulphotransferase [111. However, data base searches revealed that fractions 4 and 6 possessed > 70% and > 80% homology, respectively, with porcine transferrin (data on bovine transferrin is unavailable) (Fig. 3). Bovine transferfin has been reported to have an M, of 74000 (single chain) and to exist in a number of polymorphic forms, as shown by starch gel electrophoresis [14]. The behaviour of a pure commercial preparation of
dye-- : :::~Y~ P35K P74K "[" P"/4K "IT
R35K + tR74K T
Fig. l. PAGE of estrogen sulphou'ansferase preparations P-74K and P-35K. Also shown is bovine transferrin ('i') and gel electrophoresis patterns obtained with mixtures of the individual protein preparations.
285 145 .... MVTAIPDPDgFQDFVE ....
Kr"MDGEVPYGSWFEHT~WWEKSKN
PQVLFLF
.:: . . . . . .
74K--1 2 3 ~4 •
R74K R35K T Fig. 2. SDS-PAGE of estrogen sulphotransferase preparations P-74K and P-35K. Bovine transferrin (T) is also shown for comparison. The main protein component of preparation P-351C when stored at -20°C and thawed twice, irreversibly precipitated from solution as demonstrated in lane 3 compared to unthawed material stored at -80°C shown in lane 2 (equal vols. of clear supernatants were applied).
Y E D M K E N I R K E V M K L L E F L G R K A S D E L V ....
Fig. 4. Sequence identity of two peptides, isolated from the main protein component of P-35K, witk the sequence deduced from the c.DNA of estrogen sulpbotransfera.- [lll. The sequence of the latter is shown from residue 145 onwards and the sequences o[ the two peptides as a solid line. An additional peptide (Arg-Lys-G|y),corresponding to residues 25"1-2~9 of the deduced enzyme sequence, was also isolated (data not shown).
N-glycosidic linkages [15]. in bovine transferrin, the sialic acid residues are susceptible to neuraminidase generating a new set of polymorphic forms with lowered electrophoretic mobility [14]. The influence of neuraminidase action on P-35K and P-74K is shown in Fig. 5. Whilst the electrophoretic mobility of P-74K on P A G E was reduced, that of P-35K was unaffected. The behaviour of bovine transferrin is shown for comparison. Such data support the conclusion reached from amino acid sequence studies, viz. that P-74K contains a high proportion of transfertin.
Immunological sludies with transferrin antibodies bovine transferrin on PAGE, run alone, or mixed with P-74K or P*35K, is shown in Fig,. 1. Since P-35K contained a considerable amount of protein contaminants (Fig. 2), a sample was purified on a micro scale by reverse.phase HPLC and the major proZein component, showing only a single band (M, 35 000) on SDS-PAGE, was subsequently cleaved with cyanogen bromide. Polypeptides were separated by reverse-phase HPLC as above and three fractions (fractions 4, 7 and 8 numbered in sequeace of elution) subjected to amino acid sequence analysis. All three fractions were homologons to the data reported for cloned estrogen sulphotransferase (Fig, 4).
Results of immunodiffusion experiments employing a polyclonal antibody to pure bovine transferrin are shown in Fig. 6. Both P-74K and P-35K contained protein reacting against the antibody. When a polyclonal antibody to human transferrin was employed, no reaction was observed with bovine transferrin or the two enzyme preparations (data not shown).
:
!
Treatment with neuraminidase Sialic acid occurs as part of two glycans which are attached to the polypepfide chain of transferrin via V A Q K I ' V R W C I ' i S N Q E A N KCS5 FRENMS K A V K N G P L V S C V K K S S Y L I ~ I K A I R D K - - S . N
EAD^VrU)^GLVRAGLAPV~,'LKPVWJ~'V~sPomYYAVAVVK KGSS~ - - - - V - Fr6 QW~QL~KgsCm~LGRSAGWtWMCCL~Rr~ZEKAVASt.~SSSCVPC ADIArNFPKLCQQCAGKGAEKCACSh*HEPYFGVAGAFNCLKEDAGDVAFVVA-I -(3 E. SWI=ENLPDKXDRD" " " --
~
-
~
N
~
Fr4
Fig. 3. Amino acid sequences of two peptides (fractions 4 and 6) isolated from P-74K compared to the data base sequence of pig transferrin (residues 1-225 only). The solid line represents a peptide sequence identical to the sequence of transferrin. A dot indicates an uncertainty in the identification of a single amino acid. The sequence of peptide 6 appears first.
74K ~K 74kT 74K "~k 35k T 4+
35K T neuraminidase
Fig. 5. Influence of neuraminidasc on P.74K, P-35K and bovine transfemn. Samples (30/~g protein) were treated with 0,1 unii of
neuraminidase for 3El at 37°C in 0.05 M sodium acetate buffer (pH 5.0). Controls were incubated under identical conditions in the absence of neuraminidase. Aliquots (,=!5 #g protein) were then examined by PAGE. (T = bovine transferrin).
28~
T
P-74K
T
P-35K
Fig. 6. Demonstration of the presence of transferrin in both P-74K and P-35K by tic Ouchlerlony technique employing a commercial polyclonal aafibody (centre well) to pure bovine transferrin ('1"). In studies on the cloning of estrogen sulpbotransferase, it was found that a polyclonal antibody, purified by absorption to enzyme conjugated to Sepharose 4B, reacted against bovine serum albumin (Cohn Fraction V), However, this antibody did not react with crystallized serum albumin [23]. This result was very likely due to antibodies raised against transferrin present in the enzyme preparation.
lsoelectric focusing experiments As menfioaed in the Introduction, previous studies established that estrogen sulphotransferase activity was always associated with protein which was comprised of four polymorphic forms; this association persisting when these polymorphic forms were individually purified [8,9]. From the data presented here, it appears that transferrin co-purifies with estrogen sulphotransferase and in some preparations can represent the major protein component. Evidence for the existence of polymorphic forms of estrogen sulphotransferase itself was then sought by the use of isoelectric focusing techniques. Chromatofocusing of a partially purified preparation of enzyme from bovine placenta (see Materials and Methods) is shown in Fig. 7. The presence of three species of enzyme with differing isolectric points is suggest~.~ from the enzyme activity profile. Although
3[
the latter was coincident with the protein profile, examination of fractions by PAGE revealed protein species having high electrophoretic mobilities in addition tobands in the normal region (see Figs. 1 and 5). Protein ,.~luting in fractions 50-70 (Fig. 7), and lacking enzynle activity, showed four bands on PAGE in the positions occupied by isomorphic forms of bovine transferrin (data not shown). lso:lectric focusing of bovine transferrin has been reported to yield a complex picture of 6-8 peaks - the most dearly defined having a pl of 5.2 [14]. This is then consistent with elution of transferrin in fractions 50-70. Enzyme which had been purified by (NH4)2SO4 fractionation and repeated ion-exchange chromatography (see Materials and Methods), and exhibiting 4 bands in the normal position and free of other proteins as judged by PAGE, was then subjected to isolectric focus:,ng. In this instance, two definite peaks of enzyme activity with p l values of 5.3 and 5.7 were obtained. The presence of a possible third species was indicated by an inflection near pH 6. Material in the two peak fractions when examined by PAGE gave a single proteir, band in each instance (Fig. 8). Discussion The results have demonstrated that the protein of M~ 74000 (SDS-PAGE), present in P-74K and P-35K, is ~,ransferrin. This was established by amino acid sequence data, susceptibility to neuraminidase, behaviour on PAGE and SDS-PAGE and reaction with bovine transferrin antibody. Amino acid sequence data on the major protein component of P-35K are in complete agreement with the deduced amino acid sequence data from the c-DNA of estrogen sulphotransferase [11]. The latter would then normally exist as a dimer (M r 70000) of two identical subunits. 10 6-O
g-. !
cI.
g v
282
BBAPRO33814
Enzymic synthesis of steroid sulphates XVII. On the structure of bovine estrogen sulphotransferase John B. Adams School of Biochemistr): Universityof New South Wales. Sydney (Australia)
(Received21 May1990) Keywords: Estrogensu[photransferase:Isomorphicform;Transferrin;(Bovine placenta) Estrogen sulphotransferase plays a major role in controlling intracdlular levies of 17~-estradiol in human mammary cancer cells and human endometrium. Bovine estrogen sulphotransferase e.DNA has recently been cloned: the encoded protein having a maximum M r of 35000 (Nash, A.R. et al. (1988) Aust. J. Biol. Sci. 41, 507-516). Enzyme of M r 35000 by SDS-PAGE has now been isolated and cyanogen bromide-deaved peptides sequenced. The latter were identified in the c-DNA-predicted amino acid sequence which confirms that the active enzyme (Mr ~ 70000) exists as a dimer of identical subunits. Sequence data on similar peptides isolated from an enzyme preparation containing a protein of M r 74000 as the major species on SDS-PAGE, which was previously thought to represent the enzyme, suggested that this protein was transferrin. This was confirmed by PAGE, SDS-PAGE, susceptibility to neuraminidase and reaction with bovine transferrin antibody. Isoelectric focusing experiments show that active enzyme exists in two or three polymorphic forms (pl values 5.3, 5.7 and possibly 5.9) having similar physicochemical properties ot polymorphic forms of transferrin so that they overlap on ion-exchange chromatography and PAGE. The enzyme shows some homology to the amino acid sequence close to the Fe-binding site in laetoferrin and the question is raised as to the possible presence of a tightly bound metal in estrogen sulphotransferase involved in the binding of adenosine Y-phosphate 5'-phosphosulphate. Introduction Apart from its conjugating role in ttle liver, estrogen sulphotransferase (EC 2.8.2.4, Y-phosphoadenylylsulfate:estrone 3-sulfotransferase) is atso involved in the regulation of 17fl-estradiol levels in target tissue cells. For example, in both human and porcine endometrium, progesterone induces 17fl-estradiol dehydrogenase and estrogen sulphotcansferase, thus preventing estrogen action by effectively converting most of the 17fl-estradiol to estrone sulphate [1-5]. Again, when estrogen receptor positive human mammary cancer cells are exposed to physiological concentrations of 17fl-estradiol (~ 1 nM), a major route of metabolism is the formation of estrogen sulphates [6,7]. In such cells, estrogen sulphotransferase exhibits high affinity for 17fl-estradioland estrone and cooperativity in their binding [6]. Properties of estrogen sulphotransf¢rase have been examined in puriAbbreviations: PAGE, polyacrylamidegel electrophoresis:SDS, sodiumdodecylsulphate. Correspondence:J.B. Adams,Schoolof Biochemistry.,Universityof New SouthWales,Sydney,N.S.W.2033,Australia.
fled preparations of enzyme isolated from bovine placenta and adrenals. By means of ultracentrifugation on sucrose gradients, enzyme activity was associated with a species of Mr---74000 and in the analytical ultracentrifuge, both in the presenc and ,-bsence of guanidine hydrochloride, the purified enzyme possessed an Mr of 74000 [8]. Enzyme activity was associated with four polymorphic protein species which ran in close proximity on PAGE. Each of these species could be isolated by repeated ion-exchange chromatography when they were found to possess similar amino acid compositions and to exhibit wave-fike kinetics [8,9]. On SDS-PAGE, all purified enzyme preparations showed an Mr 74000 species, often accompanied by bands in the Mr 65-70000 and Mr 35000 regions. These bands were thought to be generated by proteinase action [10]. Recently, enzyme composed almost exclusively of an Mr 35000 species on SDS-PAGE has been isolated from bovine placenta and cloned. The c-DNA sequence indicated a protein of Mr 34600 suggesting that the enzyme exists as a dimer of identical subunits [11]. We have now reinvestigated the nature of the protein showing a Mr of 74000 by SOS-PAGE and have identified it as transferrin, It has been confirmed that estro-
283 gen sulphotraasferase is composed of two identical subunits of M,. 35000. The active enzyme can exist in two or three pc!ymorphic forms with very similar physicochemical properties to the polymorphic forms of bovine transfer, in. The possibility that estrogen sulphotransferase may be genetically related to the transferrin family of ~roteins has been explored by comparison of amino acia sequence data. Materials ana Methods
Materials [2,4,6,7-3H] 17/~-estradiol (specific activity 110 Ci/mmol) was obtt~ined from Amersham International. Bovine transferrin, l, uman transfcrrin, human transfertin antibody, neuram/nidase (Type X Ctostridium perfringens) and adenosine Y-phosFhate 5'-phosphosulphate, were from Sigma. Rabbit anti bovine transferrin was kindly supplied by Dr. E. Momotani. Adenosine 3'-phosphate 5'-phospho[~'S]sulphate was prepared as described previously [12]. Poly buffer exchanger (PBE94) and poly buffer-74 for d~romatofocusing were obtained from Pharmacia. Methods Purification of estrogen sulpho:ransferase with apparent M, 35000 by SDS-PAGE eP-35K). This was carried out by an adaptation of the method described by Moore et al. [13]. Bovine placenta c~tyledons (600 g) were homogenized in 2 voL of 0.01 M sodium phosphate buffer (pH 7A) in 0.9~ NaCI cow,raining 1 mM EDTA, 1 mM dithiothreitol and 200 KIU/ml aprotinin. After straining through two layers of cheesecloth, the homogenate was centrifuged at 13000 × g for 15 rain, and the supernatant reeentrifuged at 40000 >'~g for I h. An (NH4)2SO4 fraction precipitating between 0.5 and 0.7 saturation was made and dissol;'~ in 50 ml of the homogenization buffer. This was dialysed against 0.02 M sodium phosphate buffer (pH 7.8) containing I mM EDTA and 0.1 mM dithiothreitol (buffer A). The dialysed material was clarified by centrifugation at 100000 × g for 1 h and applied to a column (3 × 35 cm) of DEAE-Sepharose CL-6B (Pharmacia) previously equilibrated with buffer A. After application of one column volume of buffer A to remove unbound proteins, elution was carried out with a linear gradient of 0.02 M sodium phosphate (pH 7.~;, 250 ml) and 0.1 M sodium phosphate (pH 7.8, 250 ml) each containing 0.1 mM dithiothreitol and 200 KIU/ml of aprotinin. The flow rate was 0.5 ml/min and 9 ml fractions collected. The latter were assayed for protein (280 nm absorption) and 0.1 ml aliquots taken for enzyme assay. Active fractions were pooled, concentrated by uhrafiltration and dialysed against 5 mM ammonium bicarbonate (pH 8.1) containing 1 mM EDTA and 0.1 mM dithiothreitol. This was then applied to a column (3 × 35 cm) of
DEAE-Sepharose CL-6B equilibrated with the buffer. Elution was carried out with a linear gradient of 5 mM ammonium bicarbonate (pH 8.1,300 ml) and 500 mM ammonium bicarbonate (pH 8.1, 300 ml), each containing 1 mM EDTA, 0.1 mM dithiothreitol and 200 KIU/ml aprotinin. The flow rate was 0.25 ml/min and 3 ml fractions collected. Aliquots (0.01 ml) were removed for enzyme assay and active fractions pooled. Final purification was achieved on a column (1.5 x 50 cm) of DEAE-Sepharose CL-6B with a linear gradient of 0.02 M sodium phosphate (pH 7.4, 300 ml) and 0.06 M sodium phosphate (pH 7.4, 300 ml), each containing 1 mM EDTA and 0.1 mM dithiothreitol. Aliquots (0.01 m:) of fractions were assayed for activity. The active fractions were pooled, concentrated by ultrafiltration (6 ml) and stored in 1 ml allquc4s at -80°C. The protein concentration was 2.2 mg/ml and the specific activity 21 nmol estrogen sulphate formed/mg protein per min. Purification of estrogen sulphotransferase with apparent Mr 74000 by SD$-PAGE (P-74K). This material was isolated from pooled bovine adrenals by (NH,s)2SO4 fractionation and repeated chromatography on DEAEcellulose columns, as described previously [8]. The specific activity was 10 nmol estrogen sulphate formed/rag protein per min. Enzyme asmy. Unless otherwise stated, this was carried out by incubation (10 rain 37°C), with [3HI 17.8¢stradiol (30/~M, 80000 dpm), adenosine 3'-phosphate 5'-phosphosulphate (0.1 raM), MgCI 2 (15 mM), TrisHCI (pH 7.4, 50 raM), EDTA (1 raM) and dithiothreitol (0.1 raM), in a final vol. of 0.15 ml. Carriers of 17.8estradiol and 17.8-estradiol-3-sulphate (50 ~g of each) were added in acetone (0.6 ml). Protein was removed by centrifugation, the supernatant dried under N 2, and the [3H]estradiol-3-sulphate isolated and counted by liquid scintillation after thin-layer chromatography [3]. Gel electrophoresis. PAGE was carried out with 6% gels, and SDS-PAGE with 10~ gels. For the latter, molecular weight markers were: phosphorylase b, bovine serum albumin, egg albumin, pepsin, trypsinogen and lysozyme. Proteins were stained with Coomassie blue. Immunological studies, immunodiffusion was carried out on microscopy slides using 1~ agar containing 0.15 M NaCI and 20 mM sodium phosphate (pH 7.0). Chromatofocusing chromatography. An (NH4)2SO4 fraction was prepared from bovine placenta cotyledons (400 g) as described above and estrogen sulphotransferase subsequently purified on a column (4 x 40 cm) of DEAE-cellulose (DE52, Whatman), Elution was carried out with 0.02 M sodium phosphate (pH 7.4) before switching to a linear gradient of 0.02-0.1 M phosphate (pH 7.4) containing 0.1 mM dithiothreitol and 200 KIU aprotinin as described previously [10]. Enzymically active fractions were pooled, dialysed against 0.025 M imidazole HCI (pH 6.6) and concentrated by ultrafiltration. Chromatofocusing was carried out on a 20 mi bed
284
volume column of polybuffer exchange resin-94 (pH 6.6) and elution made with polybuffer-74 (pH 5.0) containing 1 mM dithiothreitol. Fractions (2 ml) were collected and assayed for protein (280 nm absorption) and enzyme activity. lsoelectric focusing. Estrogen sulphotransferase was purified from bovine placenta cotyledons as described 18,9|. On PAGE is showed 3-4 bands in the usual position and was free of other proteins as judged by this technique. Electrofocusing of this preparation was carfled out on an LKBSI02 (440 ml) apparatus, employing 6 ml of wide-range carrier ampholyte (pH 3-10). Enzyme (4 mg protein) in 6.7 ml of 0.1 M Tris-HCl (pH 7.5) was introduced into the mixing vessel of the gradient mixer when about one quarter of the gradient bad been formed. This prevented the enzyme coming into contact with the bottom electrode and subjecting it to extremes of pH. Voltage was set at 500 V and the column run for 3 days at 0°C. Fractions (4 ml) were collected at a flow rate of 32 ml/h and the pH and enzyme activity determined. The latter was assayed by the method given in the legend to Fig. 8. Amino acid sequencing. This was carried out on peptides isolated by cyanogen bromide cleavage. Briefly, protein (200/~g) was dried and reacted with 100 #l of cyanogen bromide solution (10 mg/m170% formic acid) by standing overnight at room temperature. Following iyophilisation, /~-mercaptocthanol (20 /~l) and buffer (200 #l, 6 M guanidine hydrochloride in 0.5 M Tris, pH 7.5) were added and the mixture stood at 370C for 1 h. Peptides were resolved on a reverse-phase column (15 x 3.2 mm Brownlee C4 cartridge). A gradient (A: 0.1% trifluoroacetic acid in water, B: 0.1% trifluoracetic acid in acetonitdle) was applied of 0-35% B in 30 rain. Monitoring was carried out at 210 and 254 nm. Cyanogen bromide cleavage was performed directly on P-74K but P-35K was purified on the same column and buffer system; the gradient in this case was 0-100% B in 40 rain. The major protein component (M r 35000 on SDS-PAGE} was used for cleavage. Sequencing was carried out on an Applied Biosystems Pulsed Liquid 477A sequencer with online 120A PTH analyser, using standard (normal -1) cycles. The SWISS-PROT protein database was employed for comparison of sequence data obtained with P-74K. Scq,,ence homology of estrogen sulphotransferase with lactoferrin was carried out with the SEQHP programme. Resulls
PAGE and SDS-PAGE of the two enzyme preparations P-35K and P.74K The four band (two major and two minor) pattern, previously exhibited by either bovine adrenal or placen-
hal preparations of estrogen sulphotransferase [9], was again evident by PAGE of P-74K (Fig. 1). Preparation P-35K exhibited a band in the same area. No other proteins were present in either preparation. In repeated runs, it was not possible to determine whether a single band or two poorly resolved bands were present in P-351C Mixtures of the two preparations were not resolvable (Fig. 1). Upon SDS-PAGE, preparation P-74K contained a major protein of Mr 74000, together with minor proteins of lower molecular weight, including an Mf 35 000 species. Preparation P-351L on the other hand, contained a protein of M, 35000 as the major species proteins of Mr 74000 and Mr 50000 being the minor components (Fig. 2). Amino acid sequence data Preparation P-74K was cleaved directly with cyanogen bromide and the resulting polypeptides separated by reverse-phase HPLC. Some twelve pepfides were obtained and two of these (fractions 4 and 6 numbered in sequence of elut~on) subjected to amino acid sequence analysis. Neither of these polypepfides showed any homology to the amino acid sequence predicted from the c-DNA data obtained for cloned estrogen sulphotransferase [111. However, data base searches revealed that fractions 4 and 6 possessed > 70% and > 80% homology, respectively, with porcine transferrin (data on bovine transferrin is unavailable) (Fig. 3). Bovine transferfin has been reported to have an M, of 74000 (single chain) and to exist in a number of polymorphic forms, as shown by starch gel electrophoresis [14]. The behaviour of a pure commercial preparation of
dye-- : :::~Y~ P35K P74K "[" P"/4K "IT
R35K + tR74K T
Fig. l. PAGE of estrogen sulphou'ansferase preparations P-74K and P-35K. Also shown is bovine transferrin ('i') and gel electrophoresis patterns obtained with mixtures of the individual protein preparations.
285 145 .... MVTAIPDPDgFQDFVE ....
Kr"MDGEVPYGSWFEHT~WWEKSKN
PQVLFLF
.:: . . . . . .
74K--1 2 3 ~4 •
R74K R35K T Fig. 2. SDS-PAGE of estrogen sulphotransferase preparations P-74K and P-35K. Bovine transferrin (T) is also shown for comparison. The main protein component of preparation P-351C when stored at -20°C and thawed twice, irreversibly precipitated from solution as demonstrated in lane 3 compared to unthawed material stored at -80°C shown in lane 2 (equal vols. of clear supernatants were applied).
Y E D M K E N I R K E V M K L L E F L G R K A S D E L V ....
Fig. 4. Sequence identity of two peptides, isolated from the main protein component of P-35K, witk the sequence deduced from the c.DNA of estrogen sulpbotransfera.- [lll. The sequence of the latter is shown from residue 145 onwards and the sequences o[ the two peptides as a solid line. An additional peptide (Arg-Lys-G|y),corresponding to residues 25"1-2~9 of the deduced enzyme sequence, was also isolated (data not shown).
N-glycosidic linkages [15]. in bovine transferrin, the sialic acid residues are susceptible to neuraminidase generating a new set of polymorphic forms with lowered electrophoretic mobility [14]. The influence of neuraminidase action on P-35K and P-74K is shown in Fig. 5. Whilst the electrophoretic mobility of P-74K on P A G E was reduced, that of P-35K was unaffected. The behaviour of bovine transferrin is shown for comparison. Such data support the conclusion reached from amino acid sequence studies, viz. that P-74K contains a high proportion of transfertin.
Immunological sludies with transferrin antibodies bovine transferrin on PAGE, run alone, or mixed with P-74K or P*35K, is shown in Fig,. 1. Since P-35K contained a considerable amount of protein contaminants (Fig. 2), a sample was purified on a micro scale by reverse.phase HPLC and the major proZein component, showing only a single band (M, 35 000) on SDS-PAGE, was subsequently cleaved with cyanogen bromide. Polypeptides were separated by reverse-phase HPLC as above and three fractions (fractions 4, 7 and 8 numbered in sequeace of elution) subjected to amino acid sequence analysis. All three fractions were homologons to the data reported for cloned estrogen sulphotransferase (Fig, 4).
Results of immunodiffusion experiments employing a polyclonal antibody to pure bovine transferrin are shown in Fig. 6. Both P-74K and P-35K contained protein reacting against the antibody. When a polyclonal antibody to human transferrin was employed, no reaction was observed with bovine transferrin or the two enzyme preparations (data not shown).
:
!
Treatment with neuraminidase Sialic acid occurs as part of two glycans which are attached to the polypepfide chain of transferrin via V A Q K I ' V R W C I ' i S N Q E A N KCS5 FRENMS K A V K N G P L V S C V K K S S Y L I ~ I K A I R D K - - S . N
EAD^VrU)^GLVRAGLAPV~,'LKPVWJ~'V~sPomYYAVAVVK KGSS~ - - - - V - Fr6 QW~QL~KgsCm~LGRSAGWtWMCCL~Rr~ZEKAVASt.~SSSCVPC ADIArNFPKLCQQCAGKGAEKCACSh*HEPYFGVAGAFNCLKEDAGDVAFVVA-I -(3 E. SWI=ENLPDKXDRD" " " --
~
-
~
N
~
Fr4
Fig. 3. Amino acid sequences of two peptides (fractions 4 and 6) isolated from P-74K compared to the data base sequence of pig transferrin (residues 1-225 only). The solid line represents a peptide sequence identical to the sequence of transferrin. A dot indicates an uncertainty in the identification of a single amino acid. The sequence of peptide 6 appears first.
74K ~K 74kT 74K "~k 35k T 4+
35K T neuraminidase
Fig. 5. Influence of neuraminidasc on P.74K, P-35K and bovine transfemn. Samples (30/~g protein) were treated with 0,1 unii of
neuraminidase for 3El at 37°C in 0.05 M sodium acetate buffer (pH 5.0). Controls were incubated under identical conditions in the absence of neuraminidase. Aliquots (,=!5 #g protein) were then examined by PAGE. (T = bovine transferrin).
28~
T
P-74K
T
P-35K
Fig. 6. Demonstration of the presence of transferrin in both P-74K and P-35K by tic Ouchlerlony technique employing a commercial polyclonal aafibody (centre well) to pure bovine transferrin ('1"). In studies on the cloning of estrogen sulpbotransferase, it was found that a polyclonal antibody, purified by absorption to enzyme conjugated to Sepharose 4B, reacted against bovine serum albumin (Cohn Fraction V), However, this antibody did not react with crystallized serum albumin [23]. This result was very likely due to antibodies raised against transferrin present in the enzyme preparation.
lsoelectric focusing experiments As menfioaed in the Introduction, previous studies established that estrogen sulphotransferase activity was always associated with protein which was comprised of four polymorphic forms; this association persisting when these polymorphic forms were individually purified [8,9]. From the data presented here, it appears that transferrin co-purifies with estrogen sulphotransferase and in some preparations can represent the major protein component. Evidence for the existence of polymorphic forms of estrogen sulphotransferase itself was then sought by the use of isoelectric focusing techniques. Chromatofocusing of a partially purified preparation of enzyme from bovine placenta (see Materials and Methods) is shown in Fig. 7. The presence of three species of enzyme with differing isolectric points is suggest~.~ from the enzyme activity profile. Although
3[
the latter was coincident with the protein profile, examination of fractions by PAGE revealed protein species having high electrophoretic mobilities in addition tobands in the normal region (see Figs. 1 and 5). Protein ,.~luting in fractions 50-70 (Fig. 7), and lacking enzynle activity, showed four bands on PAGE in the positions occupied by isomorphic forms of bovine transferrin (data not shown). lso:lectric focusing of bovine transferrin has been reported to yield a complex picture of 6-8 peaks - the most dearly defined having a pl of 5.2 [14]. This is then consistent with elution of transferrin in fractions 50-70. Enzyme which had been purified by (NH4)2SO4 fractionation and repeated ion-exchange chromatography (see Materials and Methods), and exhibiting 4 bands in the normal position and free of other proteins as judged by PAGE, was then subjected to isolectric focus:,ng. In this instance, two definite peaks of enzyme activity with p l values of 5.3 and 5.7 were obtained. The presence of a possible third species was indicated by an inflection near pH 6. Material in the two peak fractions when examined by PAGE gave a single proteir, band in each instance (Fig. 8). Discussion The results have demonstrated that the protein of M~ 74000 (SDS-PAGE), present in P-74K and P-35K, is ~,ransferrin. This was established by amino acid sequence data, susceptibility to neuraminidase, behaviour on PAGE and SDS-PAGE and reaction with bovine transferrin antibody. Amino acid sequence data on the major protein component of P-35K are in complete agreement with the deduced amino acid sequence data from the c-DNA of estrogen sulphotransferase [11]. The latter would then normally exist as a dimer (M r 70000) of two identical subunits. 10 6-O
g-. !
cI.
g v
