3′-phosphoadenosine 5′-phosphosulfate biosynthesis and the sulfation of cholecystokinin by the tyrosylprotein-sulfotransferase in rat brain tissue

Chemico-Bioiogical Interactions ELSEVIER

Chemico-Biological Interactions 92 (1994) 281-291

3 '-Phosphoadenosine 5'-phosphosulfate biosynthesis and the sulfation of cholecystokinin by the tyrosylprotein-sulfotransferase in rat brain tissue Froylan Vargas*~f a'b, Olivier Frerot a, Francoise Brion a, My Dam Trung Tuong a, Andree Lafitte a, Christiane G u l a t - M a r n a y a aLaboratoire de Neurobiologie et Pharmacologie, Unite 109, Centre Paul Broca de l'lnserm, Paris, France bOklahoma Medical Research Foundation, Noble Center for Biomedical Research, Carcinogenesis Group, 825 NE 13th Street, Oklahoma City, OK 73104, USA

Received 16 August 1993; revision received 14 February 1994; accepted 14 February 1994

Abstract

This article resumes the work we have accomplished in the past few years. Cholecystokinin sulfation is an important post-translational modification necessary for the biological activity of this peptide hormone. The tyrosyl protein sulfotransferase (TPST) activity from rat cerebral cortex was characterized. TPST activity is most probably responsible for the endogenous sulfation of CCK. TPST reaction kinetic properties were studied using radiolabeled 3'-phosphoadenosine 5'ophosphosulfate (PAPS) and the non-sulfated peptide acceptor terbutyloxycarbonyl-cholecystokinin octapeptide (BocCCK-8(ns)) as substrates, and brain microsomes as the enzyme source. The BocCCK-8 sulfating reaction data is consistent with the idea that TPST forward reaction follows an ordered Bi Bi mechanism. PAPS biosynthesis

* Corresponding author, Oklahoma Medical Research Foundation, Noble Center for Biomedical Research, Carcinogenesis Group, 825 NE 13th Street, Oklahoma City, OK 73104, USA. Abbreviations: PAPS, 3 '-phosphoadenosine 5'-phosphosulfate; TPST, tyrosyl protein sulfotransferase;2NP, 2-naphtol; [35S]-2-NS, 2-naphtyl-[35S]sulfate; DCNP, 2,6-dichloro-4-nitrophenol; DIDS, 4,4'diisothiocyanostilbene-2,2'-disulfonic acid; BocCCK-8(ns), terbutyloxycarbonyl-cholecystokinin octapeptide non-sulfated. tl dedicate this article to the memory of my mother who died while I was accomplishing my duties. 0009-2797/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0009-2797(94)03306-S

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and availability was studied in slices from rat cerebral cortex incubated in the presence of [35S]sulfate. There is a rapid and dynamic turnover of the steady-state level of PAPS in brain cells which is decreased by depolarizing agents such as potassium, veratridine and glutamate. Furthermore, the presence of a membrane-bound PAPS biosynthesis inhibitor was observed. These results are discussed in view of the biological importance that the cell sulfating pathways might play in nerve cell activity.

Keywords: PAPS biosynthesis; Tyrosyl protein sulfotransferase; Cholecystokinin sulfation; Sulfation in brain

I. Introduction

Endogenous sulfated compounds display a variety of cellular roles such as (1) neurotransmitters and hormones, e.g. CCK [1-3] and lutropin [4]; (2) modulators of the L-type Ca 2÷ channel, e.g. heparin [5]; (3) storage of neurotransmitters and hormones in vesicles and secretory granules, e.g. chromogranins/secretogranins [6], heparan and chondroitin proteoglycans [7]; (4) storage and modulatory activity of growth factors on the cell surface, and extracellular membrane matrix, e.g. heparan sulfate and heparan sulfate proteoglycans [8]; (5) allosteric modulatory effect on serine proteases inhibition by serpins, e.g. glycosaminoglycans and heparin [9], an essential process in regulating blood coagulation; and (6) hirudin [10,11], a tyrosine sulfated peptide from the leech Hirudo medicinalis which is one of the most potent anticoagulants known through its binding with thrombin [12]. Sulfation of these endogenous compounds is essential for their biological activity. The sulfation processing of these endogenous compounds takes place in the Golgi system. Tyrosine sulfated biological compounds are modified by the action of a tyrosyl protein sulfotransferase (TPST) located in the inner side of the Golgi membrane [6,13,14]. 3'-Phosphoadenosine 5'-phosphosulfate (PAPS), the sulfate donor, is synthesized in the cytosol of brain cells as in the peripheral tissues [15], and is made available for the sulfation process in the Golgi system by the nucleotide PAPS carrier [ 16,171. Sulfation reactions like the phosphorylation/dephosphorylation system, e.g. biosynthesis and turnover of endogenous sulfated compounds, may also participate in the regulation of cell responses to hormones and neurotransmitters [18], besides their we!l-known participation in monoamines catabolism [19] and xenobiotic catabolism [20,211. PAPS biosynthesis and turnover is essential for all these biological processes. Sulfate activation takes place in the cytosol by the action of the ATP-sulfurylase and APS-kinase. Previously, we have shown that there is steady-state level of PAPS within the nerve cells of brain slices [18] and that depolarizing agents reduce the available levels of PAPS. This effect is not due to reduced ATP levels [22]. In addition, membrane thermolabile components have been reported [15] which inhibit PAPS biosynthesis. In this report we summarize the properties of the biosynthesis of PAPS by nerve tissue and the sulfation of cholecystokinin octapeptide in vitro.

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283

2. [3SS]PAPS formation by rat brain slices Following the rapid uptake of inorganic [35S]sulfate by rat brain slices, presumably via a saturable transport system [23], and of 2-napthol (2-NP), a different pattern was observed for the formation of [35S]PAPS and 2-naphthyl[35S]sulfate ([35S]-2-NS) with incubation time. Newly synthetized [35S]PAPS reached a plateau within 5 min, whereas the [35S]-2-NS formation, being detectable shortly after the labeled nucleotide appears, was continuously formed by the brain slices throughout the incubation period [18]. [35S]PAPS was separated by ion exchange chromatography and quantitated by the enzymatic transfer reaction as follows: 2-naphtol-OH + [35S]PAPS

-

2-napthyi-O[35S]SO3 + PAP

the transfer reaction being catalysed by the cytosolic phenolsulfotransferase [15]. This assay was validated by comparison of data with those derived from direct quantification of [35S]PAPS isolated by either TLC or anion exchange chromatography [151. Indeed, 2-NP, a lipophilic compound, readily enters cells [24] and represents a high affinity substrate for the cytosolic phenolsulfotransferases [25]. [35S]-2-NS formation in the slice preparation appeared to occur with apparent saturation kinetics (Km = 1 #M) [18] consistent with previous results. Both the phenolsulfotransferase(s) responsible for sulfation ofcatecholamines and various other phenols and the enzymes participating in the PAPS-synthesizing systems are soluble enzymes widely distributed in brain regions and localized in the cytoplasm of nerve endings and brain cells [15,26] (Table I). [35S]PAPS formation and transfer of its activated [35S]sulfate group to the 2-NP acceptor occurring in the brain slice preparation was followed by a continuous efflux of [35S]-2-NS into the medium, where the major fraction of this sulfated product was found [18]. The effect of 2,6-dichloro-4-nitrophenol (DCNP) on [35S]PAPS formation was studied to estimate the amounts of this nucleotide involved in the endogenous sulfation reactions catalysed by phenolsulfotransferases and tyrosyl protein sulfation 3. Indeed, this compound was shown to be, in vitro, a rather potent inhibitor of phenolsulfotransferases [13,27] and a good inhibitor of the microsomal TPST catalysing cholecystokinin sulfation [28-30], as well as in vivo to prevent sulfation of harmol in liver [31] and endogenous cholecystokinin biosynthesis in rat brain [30]. Contrary to our expectations, however, [35S]PAPS formation in brain slices was inhibited in a concentration dependent manner by DCNP with an IC50 = 10 #M. This effect could partially be attributed to a direct inhibition of the PAPS-synthesizing enzyme system, because this was observed to occur when formation of the nucleotide by a cytosolic fraction of rat cerebral cortex [18] was evaluated with an IC50 = 0.25 mM. The development of specific drugs for the different endogenous cell sulfation systems is necessary to understand the biological function of endobiotics sulfated compounds [6-9,25]. Available inhibitors are active at 10-6-10 -2 M, but much higher specificity is required.

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Table 1 Subcellular distribution of tyrosyl protein sulfotransferase activity, CCK-8 content and PAPS synthesizing activities in rat cerebral cortex Subcellular fraction

TPST activity RSA

PAPS synthesis (pmol/mg protein/min)

[35S]PAPStransport sites (RSA)

CCK-8(ir) content RSA

Homogenate Nuclear (Pl) Crude particulate (P2) Microsomal (P3) Soluble Mitochondrial (M 0 Vesicular (M2) Synaptoplasm (M 3)

1.00 0.44 0.84 4.30 nd 0.52 4.10 nd

< 0.1 <0.1 <0.1 <0.1 44.3 + 3.0 <0.1 < 0.1 12.7 ± 1.0

1.00 0.64 0.69 1.26 nd ne ne ne

1.00 0.22 0.88 0.56 0.07 0.61 1.48 0.27

± 0.1 + 0.3 ± 0.3 ± 0.2 + 1.0

Subcellular fractions were prepared as described by Brion et al. [15]. In brief, tissues were homogenized in 20 volumes of 0.32 M sucrose with 10 up-and-down strokes at 700 rev./min in a Teflon-glass homogenizer. The homogenate was centrifuged at 1000 x g for 10 min, and the pellet was resuspended in 10 volumes of 0.32 M sucrose and recentrifuged. The resulting pellet (PI) was resuspended in 5 ml of 10 mM Tris-maleate buffer (pH 7.0) and kept at 4°C. The combined supernatants were centrifuged at 20 000 x g for 20 min to give a mitochondrial pellet (P2)- The supernatant was then centrifuged at 100 000 x g for 60 min to give the P3 fraction. TPST activity values and cholecytokinin immunoreactive content (CCK-8(ir)) are from Trung-Tuong et al. [29], and Vargas and Schwartz [3]. PAPS synthesis by the different subcellular fraction values are from Brion et al. [15]. [35SIPAPS transport specific binding values are from Vargas [17]. RSA represents the relative specific activity. Values given in columns 1 and 2 are mean values + S.E.M. from three and two separate experiments, respectively [29,3]. Values in columns 3 and 4 are mean values from two distinct experiments [17,29]. Abbreviations: nd, not detectable; ne, not estimated.

3. P A P S utilization by rat brain slices Little is k n o w n a b o u t the m e c h a n i s m s c o n t r o l l i n g P A P S f o r m a t i o n a n d u t i l i z a t i o n in b r a i n . O u r studies have s h o w n t h a t d e p o l a r i z a t i o n o f b r a i n slices by p o t a s s i u m , excitatory a m i n o acids, a n d v e r a t r i d i n e r a p i d l y reduces newly synthesized [35S]PAPS levels. D e p o l a r i z a t i o n with high p o t a s s i u m p r o d u c e d the m a x i m a l decrease in P A P S levels while the effect o f v e r a t r i d i n e was i n h i b i t e d by t e t r o d o t o x i n [18]. T h i s a p p a r e n t l y c o r r e s p o n d s to i n h i b i t i o n o f the n u c l e o t i d e synthesis, because the initial rate o f [35S]PAPS a c c u m u l a t i o n was s t r o n g l y r e d u c e d a n d [35S]-2-NS form a t i o n , i n s t e a d o f p r o c e e d i n g linearly, was c o m p l e t e l y b l o c k e d after a few m i n u t e s . I n a d d i t i o n , the i n h i b i t i o n o f [35S]PAPS f o r m a t i o n by e x c i t a t o r y a m i n o acids o c c u r r e d with a limited degree o f stereospecificity n o t b e i n g affected by the a n t a g o nists L - g l u t a m a t e diethylester, c i s - p i p e r i d i n e d i c a r b o x y l i c acid, a n d D-glutamylglycine. F u r t h e r m o r e , the p h a r m a c o l o g i c a l specificity p a t t e r n o f the r e s p o n s e did n o t reflect t h a t o f established a m i n o acid r e c e p t o r subclasses [32]. O t h e r n e u r o t r a n s m i t ters did n o t p r o d u c e , h o w e v e r , significant effect, e.g. acetylcholine, G A B A , kainic acid, a n d , N - m e t h y l - D - a s p a r t i c acid [18]. F u r t h e r w o r k is necessary to establish w h e t h e r the P A P S r e s p o n s e to excitatory a m i n o acids is a r e c e p t o r - m e d i a t e d one.

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4. Sulfate-activating system in rat brain The formation of [35S]PAPS from inorganic [35S]sulfate and Mg2+-ATP was easily detectable using a crude rat brain cytosol as the enzyme source. The formed radiolabeled nucleotide was assayed by the enzymatic transfer reaction described above. This assay was validated by comparison of data with those derived from direct quantification of [35S]PAPS isolated by either TLC or anion exchange chromatography [15]. Saturation kinetics of [35S]PAPS synthesis showed apparent Km values of 3 mM for Mg2+-ATP and 0.2 mM for SO42-, according to those reported for the ATP sulfurylase from rat liver [33,34], a result implying that the step involving APS kinase was not a limiting one. This is consistent with the high affinity of APS kinase for APS, which allows the first step to proceed in a forward direction. [35S]APS was immediately transformed into [35S]PAPS since no significant amount of [35S]APS was detected in the incubation mixture. Other properties of the PAPSforming system in rat brain are requirement for Mg 2+ and the inhibition by various nucleotides, Mn 2+, and PO43-, which presumably act as competitors of the two substrates of the ATPsulfurylase [ 14,31 ]. PAPS formation measured in several rat brain areas did not show any marked regional differences. PAPS formation in the cytosolic fraction from rat brain was 5 times lower than the corresponding fraction from liver tissue. In contrast, PAPS formation could not be detected in any subcellular fraction containing membranes (Table 1), because of the presence of a membrane bound thermolabile inhibitor (Ref. [15] and Vargas and Ringer, unpublished data). Further studies are now in progress to assess whether this modulatory protein is functionally relevant to the control of PAPS synthesis and sulfating reactions in cell function.

5. [35S]PAPS nucleotide carrier We were particularly interested in assessing the presence of a PAPS-synthesizing activity in the microsomal fraction from rat brain, which contains, among other post-translational enzymes, a peptide tyrosyl sulfotransferase activity, presumably responsible for the sulfation of cholecystokinin [3,13]. The fact that PAPS formation was not detectable in this subcellular rat brain fraction suggested that the sulfation reaction in the Golgi system relies on the cytoplasmic synthesis of the sulfate donor. In agreement with this hypothesis, a PAPS transport system has been recently reported in Golgi vesicles from rat liver [16,35]. Accordingly, evidence of this membrane carrier was obtained in rat brain microsomes. Thus, [35S]PAPS specific binding to rat brain microsomes reached equilibrium within 20 min and was readily displaceable by an excess of non-labeled PAPS or its major metabolites 3 '-PAP and APS and also ATP, with a dissociation constant of tl/2 = 80 s [17]. The specificity of the PAPS transport carrier, in brain microsomes, as determined by [35S]PAPS specific binding (Table 1), had properties similar to those of the PAPS uptake process in rat liver microsomes [16]. First, the affinity constant was similar: 0.45 ± 0.06 #M compared with a Km of 0.7 /~M for PAPS uptake in rat

286

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liver microsomes. Second, 3'-PAP was a strong inhibitor of [35S]PAPS specific binding with an IC50 of 320 ± 60 ~M. Third, APS and ATP, the biosynthetic precursors of PAPS, were more active in inhibiting [35S]PAPS specific binding than affecting PAPS uptake as reported in chick embryo microsomes [36]. Fourth, DIDS, the specific inhibitor of chloride transport in erythrocytes [37], inhibited in a similar manner [35S]PAPS specific binding in brain microsomes [17] and PAPS uptake by rat liver microsomes [35]. Because sulfation is important for cholecystokinin activity in the CNS [1,2,41] and the biochemical expression of glycoproteins and proteoglycan properties in receptor function [8], membrane permeability [5] and transmitter release [38,39], PAPS transport system in the Golgi apparatus is an important pharmacological target for the design of new inhibitors with potential pharmacological applications. 6. Tyrosine sulfation of cholecystokinin The sulfate group on CCK-8 (the most abundant form of the known biologically active CCK molecules) is essential for its recognition by receptors mediating its various hormonal or neuronal actions [1,40,41]. Recently, TPST activities associated with the microsomal fractions from rat brain [3,13,28,29] and Golgi membranes from bovine adrenal medulla [12,13,42] were characterized. This enzyme activity is responsible, most probably, for the endogenous sulfation of CCK [13], and for the sulfation of the secretory proteins [42]. The TPST properties from the rat brain microsomal fraction are: first, the enzyme is located in the membrane [13], being solubilized only with high detergent concentration [3]. Second, CCK-8 sulfation was not detected in the cytosolic fractions from rat brain, where the phenolsulfotransferases [19,20,26] and arylsulfotransferase IV [25] are located. Third, the optimal pH of 6.0 is consistent with the observation that the secretory granules is the major site of maturation for several prohormones [43,44]. Fourth, TPST sulfates tyrosine residues within the peptide chains providing one, two, or more, Asp or Glu residues in the vicinity of the amino terminal side of the tyrosine. Fifth, TPST is located in the microsomal, synaptic vesicular fraction [29] and Golgi fractions from rat cerebral cortex (unpublished data). Its localization in the synaptic vesicular fraction is in agreement with the CCK content (Table 1). Sixth, the peripheral distribution of TPST activity showed high levels of peptide sulfation in brain, pituitary, liver, spleen, duodenum, and lung, in accord with the exocrine function of these organs [29,42]. Seventh, TPST activity is higher in neonatal rat brain tissue than the mature values (Vmax decreased from 0.83 ± 0.05 to 0.31 ± 0.02 pmol of sulfated peptide/rag protein per min) [29], in agreement with the idea of higher rate of biosynthesis of cellular components in neonatal and developing tissue. Eighth, the solubilized enzyme preparation showed similar properties to sulfate the cholecystokinin peptide and the TPST synthetic polymer substrates poly(Glu, Ala, Tyr) [3,14]. Ninth, and finally, the TPST initial reaction velocity plots with [35S]PAPS and non-sulfated BocCCK-8(ns) as substrates suggests the possibility that the peptide sulfation reaction follows a sequential ordered mechanism [28]. These properties suggest that the TPST sulfating BocCCK-8 corresponds to the adrenal medulla TPST enzyme purified and characterized by Huttner and coworkers [12,42], or an isoenzyme of the sulfotransferase. Purification of this brain TPST is necessary to establish its identity.

F. Vargas et al./ Chem.-Biol. Interact. 92 (1994) 281-291

287

APS lOoM 40

*7

JM

~t r E. . .

30

0

"~" ¢L

OE

20

o 1.5 pM

I

~ ,--I>

OpM 10

I

10

I

1

I

1

20 30 40 50 1/[BocCCK-B Ins)] (rnM) -1

Fig. 1. Dead-end inhibition patterns of the tyrosyl protein sulfotransferase (TPST) from rat brain microsomes by APS. Reciprocal velocity versus l/BocCCK-8(ns) at 0.1 #M of [35S]PAPS with varying APS concentrations. The lines were fit to all the data points, using the equation for mixed non-competitive inhibition [28].

Furthermore, product inhibition of TPST activity was not consistent with a random pathway, since BocCCK-8(s) was an uncompetitive inhibitor with respect to PAPS, at a fixed peptide substrate concentration, and 3'-PAP was a competitive inhibitor versus PAPS and displayed non-competitive inhibition with respect to BocCCK-8(ns) [28]. In addition, APS, a known dead-end inhibitor of sulfotransferases [25], was a competitive inhibitor of the TPST at the PAPS binding site, and non-competitive versus peptide substrate (Fig. 1). This supports the idea that the TPST reaction follows an ordered Bi Bi reaction mechanism [28] as shown in the following scheme:

EEI

A

B

P

Q

k, l~ k_,

k2 11. k,

k_, 11. k,

k_4 1L k4

EA

(EAB ~ EPQ)

EQ

E

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where A and B represent PAPS and BocCCK-8(ns), and P and Q the products, BocCCK-8(s) and 3'-PAP, respectively. APS, the dead-end inhibitor, ATP and the peptide substrate at over-saturating concentrations are represented by I. 7. Conclusion The role of sulfation reactions in cell function is beginning to be more keenly considered. Known for decades, it is only during the last 10 years that a number of highly interesting data have been gathered [12,19-21]. Thus, the enzymatic process of protein and peptide tyrosine sulfation was characterized [13,14], and the responsible enzyme, TPST, was purified [12,421, although the presence of tyrosine-O-sulfate was discovered in mammalian tissues by Bettelheim in 1954 [45]. TPST has been suggested to be responsible for the sulfation of endogenous cholecystokinin [3,13,29] in rat brain. Sulfation of endogenous cellular components also has other physiological roles such as in membrane permeability, hormone recognition and cellular effects and neurotransmitter release. The PAPS biosynthetic pathway will be studied in more detail to characterize and purify the endogenous regulatory components. The lack of specific pharmacological tools has hampered the recognition of the role of sulfation reactions in cell function. Available inhibitors [46-49] are poorly active at 10-6-10 -2 M. Thus, the sulfation system in organisms has come to be recognized as an important cellular metabolic pathway with a marked potential for the development of new specific inhibitors of PAPS synthesis [50,51], sulfotransferases [25,42,52], and sulfatases [53,54] with potential therapeutical applications. 8. Acknowledgements We thank Professor Jean Charles Schwartz for his continuing support throughout this work and Professor David P. Ringer and Professor John Meerman for the invitation to the 2nd International Workshop on Sulfation of Xenobiotics and Endogenous Compounds, 3-6 June 1993, hosted by The Samuel Roberts Noble Foundation, Inc., Ardmore, Oklahoma, USA. We thank Mrs Laura Smith for the typing of the manuscript. 9. References 1 J.G. Dockray, The physiology of cholecystokinin in brain and gut, Br. Med. Bull., 38 (1982) 253-258. 2 A.H. Johnsen and J.F, Rehfeld, A disulfotyrosyl hybrid ofcholecystokinin and gastrin from the neural ganglion of the protochordate Ciona intestinalis, J. Biol. Chem., 265 (1990) 3054-3058. 3 F. Vargas and J.C. Schwartz, Apparent identity of cerebral tyrosylsulfotransferase activities using either a cholecystokinin derivative or an amino acid polymer as substrate, FEBS Lett., 211 (1987) 234-238. 4 E.D. Green, J.U. Baenziger and 1. Boime, Cell-free sulfation of human and bovine pituitary hormones. Comparison of the sulfated oligosaccharides of lutropin, follitropin and thyrotropin, J. Biol. Chem., 260 (1985) 15631-15638.

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