Developmental profiles of arylsulfatases A and B in rat cerebral cortex and spinal cord

Iliochtmica et Bioph)~ica Acta. 1081 (19911 315-320 ,1991 ElsevierSciencePublishers n,v. tBiomedical Divisionl009~-2769/91/S03.50 A D O N I S 000527609100093F

315

D e v e l o p m e n t a l p r o f i l e s o f a r y l s u l f a t a s e s A a n d B in r a t c e r e b r a l cortex and spinal cord Ron H.M. van der Pal, Wil Klein, Lambert M.G. van Golde and Matthijs Lopes-Cardozo Labarato O' of Vetermam- B:r~hemtsl~'. Utrecht ~rnH'e~lq'. Utrecht (The Netherlatrd¢)

(Received 6 August lq~l Key words: ArylsulfataseA: Arylsulfataseg: Cerebrosldesultatase: Developmenl:Suffolipid;(Rat brain) Arylsullatases A (EC 3.1.6.1) and B (EC 3.1.6.12) are lysosomal enzymes that can remove sulfate groups from sulfatides and soifo-glycosaminoglycans, respectively. The activities of these enzymes in cerebral cortex and in spinal cord of developing rat pups were measured. The tissues were homogenized "and the arylsul|atases A and B in the soluble fraction were separated from each other by anion exchange chromatography on DE-52 cellulose, Subsequently, the enzyme activities were assayed with p-nilroeatechol sul|ate as substrale at 37"C 'and pH 5.6. We observed a developmental profile o| erydsulfatase A, similar to thai previously reported for cerebroside sullat~se (EC 3.L6.8; (Van tier Pal et al. (1990) Biochim. Biophys. Acta 1043, 91-96)). The activity' of arylsuliatase A increased gradually during development, whereas arylsal|atase B rose more steeldy, peaked arotmd day 15 and declined thereaiter. As a conseqnence the ratio between B and A forms of arylsul|atase dropl~tl from about 4 in 1-week-old imwi to 2.2 (cortex) and 0.7 (cordl in 7-week-old rat pups, Introduction

Sulfur|des are galactosylceramides (cerebrosides) with a sulfate group attached to the 3-position of the sugar moiety [1]. These sulfolipids constitute approx, one fifth of the glyeolipids and 6 mol% of total lipids in the myelin membrane of the adult mammalian central nervous system (CNS; [2]). Thus. salfatides are the major anionic lipids in the specialized extensions of the oligodendrocyte plasma membrane that insulate and stabilize neuronal axons [3]. The physiological function of sulfatides in mammalian tissues is still a matter of specu!ation [a]. l-ln~,. ever. it is intriguing that within the C N S sulfur|des are found ;dmost exclusively in the myelin sheaths: an observation that could indicate a specific role of this lipid class, for instance, as a glue sticking adjacent myelin lamellae together. In fact it was proposed alread',' in the early 1970s that sulfatides stabilize the compact structure of my,:lin through electrostatic inter-

Abbreviations: AS. arylsultatase" CNS. central nervous system; CSase, ee~broside sulfala~; NCS, p-nitr~atcehoI sulfate. Correspondence: M. Lopes-Cardoz~, Labora*ory or V¢~efit,al) iilochemistry, P.O.Box 80.176, 3508 TD Utrecht. The Netherlands.

actions with myelin basic protein [5,6]. This hypothesis is les,s likely since it was established that myelin basic protein is located at the cytoplasmic side (see Rot'. 7), whereas ~sulfolglycolipids generally are found at the external face of plasma membranes (see Re|. 8). However. the possibility still holds that sulfur|des bind to positively charged groups of myelin proteins such as proteolipid protein extending from the opposing membrane or that a minor fraction of the sulfur|des is located in the inner leaflet of the lipid hilayer. Therefore, it is feasible that the enzymes which cataly~ the sulfation of cerebrosides and desulfation of sulfatides control the negative surface charge at the the plasma membrane [91. and that sulfatases possibly destabilize the multi-layered structure of compact myelin. Hence, we considered the further biochemical characterization of enzymes that hydrolyse sulf¢~conjugat~ in the C N S relevant in view of their possible involvement in pathological demyelination. This study focusses on cerebroside-3-sulfate 3-sulfohydrt~lase (CSase; EC 3.1.6.8), a lysosomal enzyme. which removes the sulfate group from sutfatides I10l. CSase became a topic of clinical :nterest as a result of the pioneering work of Austin and colleagues and of the group of Jatzkewitz Ireviewed in Re|. ll). They disi~overed that patients suffering from Metachromatic Leukodystrophy are deficient in CSase. As a conse-

316 quence, sulfatides accumulate in lysosomes of the brain, normal myelination is disturbed and the patients suffer from severe neurological symptoms. For clinical sc,-eening, CSase activity can be measured conveniently with the artificial, chromogenic substrate p-nitrocatechol sulfate (NCS; 2-hydroxy-5nitrophenyl sulfate) as arylsulfatase A (AS-A; EC 3.1.6A). This is based on the observation that CSaae and AS-A reactions are catalysed by the same protein [12]. The AS-A assay is less laborious than the determination of the CSase activity but it is also less specific. Sulfatases other than CSase ( = AS-A) can hydrolyse NCS as well; for instance, arylsulfatase B (ASB), a lysosomal enzyme that splits sulfate off from -N-acctyl-galactosamine-4-sulfate moieties of sulfated glycosamlnoglycans [13]. For diagnostic purposes in human medicine, reaction conditions have been worked out that allow the measurement of AS-A independcndy of AS-B (with N C S as substrate). This was realized by including salts in the medium [14] or by performing the assay at a low temperature (e.g., see Ref. 15). A complication arose when the activity of AS-A was determined in tissues of experimental animals. For instance, Worwood and colleagues [16,17] found that the assay conditions that discriminate between AS-A and AS-B in human tissues are not appropriate for specific assays of these enzymes in tissues from the rat. The most likely explanation for this phenomenon is that, as AS-A and AS-B are glycoproteins like most lysosomal enzymes, variable processing of the glycoconjugate induces heterogeneity in the catalytic properties of AS-A and AS-B among species and even among tissues of the same animal. We now report on the independent assays of AS-A and AS-B in rat cerebral cortex and spinal cord. We show that the developmental profile of AS-A differs significantly from that of AS-B. Also, we provide evidence that the discrepancy between CSase and AS-A activities previously observed by us [18] can be ex. plained by the contribution of AS-B in the assay titat was used to estimate the AS-A activity. Finally. we make a note of caution with regard to the use of N C S for the specific assay of AS-A in animal tissues. Materials and Methods

Animals and chemicals Female rats 16-18 days pregnant were obtained from Harlan CPB (Zeist, The Netherlands). The pups were separated from their mothers 28 days after birth. The animals were killed at various time points of their development by decapitation. Cerebral hemispheres were dissected out of the brain discarding the cerebellum and the brain stem. The cord was flushed out of the ~pine with a syringe [19]. Chemicals were purchased from Sigma (St.Lonis,

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M NaCI (I and II) and with O.2S M NaCI (Ill mid IV). The total clnate of 40-ml was collected in 2-ml fractions of which 100 pl was used to assay the activity o[ AS, measured spectrophotometricallyat a wa'~elengthof 515 nra. M O , U.S.A) and the celluloseanion exchange material, DE-52, from Serva (Heidelberg, Germany). 3SS-radiolabelled sulfatideswere prepared by intracranialinjection of rat pups with ~SO4:" as described [18].

Enzyme preparations and assays Spinal cords and cerebral tissue were homogenized (20~, w / v ) in 20 m M Tris-HCl (pH 7.4), containing 0.1% (v/ v) Triton X-100, at 0 ° C with an UItra-Turrax (IKA. T-25; 20 s, 1aaximal speed). The homoganates were centrifuged in the SS34 rotor o f a Sorvall centrifuge (30 rain, 4 8 0 0 0 X g at 4°C). About 80% of thc total arylsulfatase activity of the homogenate was secovered in the supernataut and this percentage did not vary with the age of the pups. Arylsulfatascs A and B were separated from each other by anion exchange chromatography (essentially as described in Ref. 17). An aliquot of the sapcmalant (0.5-1 ml) was applied onto a column (10 × 0,8 cm: bed volume of 5 ml) of DE.52 cellulose, equilibrated with 20 mM Tris-HCl (pH 7.4). The column was washed with 5 ml of the same buffer. This eluate did not contain AS activity. Subsequently, the column was eluted stepwise with 10 ml portions of this buffer containing NaCI. Four fractions were collec!ed: fractions 1 and 11 were eluted with 0.125 M NaCI and fractions III and IV with 0.25 M NaCI (see Fig. 1). The arylsulfatase activity of th~ various fractions was assayed with N C S as substrate. Fraction I contained much protein and it was necessary to dilme it with four volumes of 0.125 M NaCI (20 m M Tris, p H 7.4) to assay in a linear range of protein eoncentratiolt. The

317 TABLE I

Comparison o/~a:; condltto,s for AS-A and A S.B An aliquot of supernatant derived from the cerebral eorlex of a 19-day-old ~at was fraction°ted by anion ~change chromatography (Fig. l). The aedvity of arylsulfatase in fractions I ',toni°ins AS-B) and Ill (oontai,~ AS-A) was measu~d: (I) according Io Wor~'ood el aL 1i61. 60 min al ]5°C in 25 mM sodluma acetate. 25 RIM NaCI. 10 ram NCS (pH 5.3); (2) 10 or 60 rain at 37°C in 025 M sodium acetate. 20 mM NCS (pH 5.6) as desctihcd in Materials and Methods A linear time-course was observed for both fraclions assayed at 15°C (reaction conditions (1)). The data were ohtained v,ath the enzyme preparations of the experiment shown in Fig 1. Sinular results were obtained with samples derived from spinal ~ r d and Irom pups of different age (n 7). Arylsulfatas¢ acti~ily with NCS l/final.h- t g wet walght i) Reaction ¢ondition~ (ll Temperature: I5°C

(2} 37°C

Incubation - time (rain): 60 Fraction l (AS-B) Fraction III (AS-A)

7.8 9.2

other fractions could b e a s s a y e d undiluted. Samples of the fractions (300 ttl) were incubated d u r i n g 10 rain at 37°C with 100 FI of an a s s a y mix containing 80 m M N C S a n d 1 M sodium acetate ( p H 5.6). T h e reaction was s t o p p e d with 0.5 ml 2 M N a O H a n d A(515) was m e a s u r e d (~ - 12000 L - real -* - cm*). Dialysis of the fractions prior to the assay did not e n h a n c e the observed e n z y m e activity showing that neither A S - A nor AS-B were inhibited b y the salts in the ehition buffer. T h e total A S activity ( A + B) of the s u p e r n a t a n t was d e t e r m i n e d similarly after diluting a n aliquot with 49 vol of 20 m M T r i s - H C l ( p H 7.4). T h e total A S activity was recovered quantitatively (96 + 4,%; , ~ 10) in the fractions (1 + 111). Cerebroside sulfatase activity was m e a s u r e d (as described in Ref. 18). Resdls

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Fi8, 2. Developmental profiles ef total arylsulfat;~s¢,AS-A and AS-B in rat CNS. The activity of total AS liP) derived from rat spinal cord (panel A) or ¢©zebralcortex ( p ~ a B) was ~ T e d . Aliquots of me same supematams were fraetionated on DE-52 cellulose ~Fig~ II. AS was measured in fractions l ( = AS-B; 4) and Ill ( = AS-A: 0). In panels C (spinal ~,'d) and D (cerebral carte.x) !he ratio AS-B / AS-A is plotted against the age of the red pups. Values for adult rats were: (i) cerebral cori~rx, AS-A ffi 41 _+ 5; AS-B ffi 71_+8 : lotal AS - 124~11: AS-B / AS-A L74_+0.05; ([i) spinal cord: AS-A - 55_+6; AS-B = 39+_3 ; total AS ffi 94_+14; AS-B / AS-A ffi 0.7O _+ 0.03 (Means+S.D.). Differences observed between data from male and female rats were statistically insignificant.

318 arylsulfatases can be separated from each other by anion exchange chromatography on small DE-52 cellulose columns I17], as AS-A is bound more strongly than AS-B at neutral pH. Fig. 1 illustrates that this method worked well for samples derived from rat brain or spinal cord. After a stepwise elution with 125 nqd 250 raM NaCI, two peaks of AS activity, assayed with NCS, were observed. Using [35Slsulfatide as substrate. CSase activity was found only in the second peak. This strongly indicates that the first peak (fraction 1) indeed corresponds to AS-B and the seegnd peak (fraction [II) to AS-A, Additiona! evidence for this was obtained in experiments in which our present assay (NCS; 10 rain; pH 5.6: 37°C) was compared with the conditions (NCS; 60 min; pH 5.3; 15°C) described by Worwood et al. [16] to be selective for the assay of AS-A (Table I). The timecourse of AS in fraction I was linear during 60 min both at 15 and at 37°C and the temperature coefficient was relatively high (Vm~(37°C)/Vm~(15°C) = 13). On the other hand, at 37°C the AS activity in fraction Ill was constant only during the initial 10 rain and decreased abruptly thereafter. The temperature coSfficient was about 3-times lower than that of AS in fraction L These results fit well with the kinetic properties described (in Ref. 16) for AS-B (linear time- courses; high temperature coefficient) and for AS-A (linear with time at 15 but not at 37°C; low temperature c~fficient). Profiles of A S-A and A S-B in developing rat CNS Fig. 2 shows how the activities of the arylsulfatases change during development in rat spinal cord and cerebral cortex. The contribution of AS-A and AS-B to the total AS activity, which was assayed independently, was estimated. Furthermore, the ratios of AS-B/AS-A are plotted in panels C (spinal cord) and D (cortex). Total AS peaked after the second postnatal week and declined gradually thereafter. On the other hand. the activity of AS-A increased continuously, reaching a constant level 3 weeks after birth, whereas the developmental profile of AS-B mimicked that of total AS. It should be emphasized that the AS-A activities in Fig. 2 are almost an order of magnitude higher than reported previously by us [18]. This can he explained by the different assay conditions used. In Ref. 18 we followed the method of Worwood et al. [16] to assay AS-A (60 vain. 15°C), whereas in the present experiments we measmed fetal AS, AS-A and AS-B with NCS as substrafe during 10 rain at 37°C. The fact that total AS (Fig. 2) equals (AS-A + AS-B} indicates that the enzymes are recovered quantitatively from the anion exchange columns. In other words, the ratio AS-B/AS-A truly reflects this ratio in the supernatant, Comparing spinal cord and cortex it is interesting that AS-B decreased much more in spinal cord than in cortex during development. Furthermore, AS-A reached

a level that was almost 3-fold higher in spinal cord than in cortex. As a result the ratio of AS g/AS-A in spinal cord decreased durin~ development to approx. 0.7, whereas in cerebral cortex this ratio remained relatively high (>__2). Discussion In a previous paper [18] we reported that the developmental profile of CSase in rat brain and spinal cord, measured with [3~g]sulfatide as substrate, differed significantly from that of AS-A, measured with NCS as substrate. This was a puzzling observation because Mehl and Jatzkewitz [12] have shown that both CSase and AS-A activities are eatalysed by the same enzyme. The aim of the present investigation was to resolve the apparent discrepancy between the activities ~f AS-A and CSase in rat CNS. Our working hypothesis was that not only AS-A but also AS-B was active in the assay [16] that we had used. Therefore. we separated AS-A from AS-B by anion exchange chromatography and assayed the two sulfatase activities independently. Fig. 2 shows that we now find a developmental profile of AS-A which is very similar to that of CSase [18] but different from that of AS-B. These results show that the developmental profile of sulfatase activity [18] measured in supernatants of rat brain according to [16] reflects the profile of AS-B rather than that of AS-A. In other words, it is not possible to distinguish unambiguously between the two types of sulfatase in homogenates of rat CNS by using the artificial, ehromogenie substrate NCS. Pertinent to this are two points, First, the assays with NCS were develol~d clinically to estimate AS-A in human tissues [14] and do not discriminate between AS-A and AS-B when used for tissues of the rat I161. Secondly, it has been reported [171 that the activity of AS-B is an order of magnitude higher than that of AS-A in tissues of the rat. Worwood et al. [16] investigated arylsulfatases in rat liver and reported that AS-B was inhibited for 95% under the assay conditions for AS-A (60 rain, pH 5.3, 15°C). The data in Table I indicate t~at in case of rat brain. AS-B was inhibited also for more than 90% under these conditions. However, the remaining activity of AS-B, measured at 15°C, was of the same order as the activity of AS-A. It is noteworthy that a contribution of AS-B in the assay of AS-A can be traced back if one scrutinizes data in the literature. For instance, Farooqui and Bachhawat [21] estimated AS-A and AS-B in rat brain as a function of age following the assay procedures of Baum i14] and found profiles different from those in Fig. 2. Furthermore, Sarli~ve et al. [22] and Burkart et al. [23] independently reported on the activity of AS-A in developing mouse brain using the method of Baum [14]. They found a symmetrical profile around a maximum (day

319 15 18) declining to about 50% of the maximal activity with increasing age of the mice. In a later paper Burkart et al. [24] showed another profile of A.q-A in mouse brain, now measured according to Worwood et al. [16], in which the enzyme activity also reaches a maximum around day 20, but remains at about 70% of the maximal activity in older pups. Our present results strongly indicate that the observed decrease of the apparent AS-A activity with age in these experiments is due to a quantatively important contribution of AS-B in their assay, because - at least in rats - AS-B dropped markedly during this developmental period whereas AS-A did not (Fig. 2). Moreover (as discussed in Ref. 16), the contribution of AS-B in the AS-A assay according to Baum [14] is greater than it is in the assay method of Wocwood et al. [16] and this could explain the differences between AS-A profiles (reported in Refs. 23 and 24). In conclusion: (1) the use of N C S as a substrate for the assay of AS-A can lead to erroneous results, doe to the variable contribution of AS-B; and (2) the two forms of arylsulfatases can be measured accurately with N C S if they are separated from each other prior to their assay. The A S - B / A S - A ratio of 2.5, c h a r r e d by us for cerebral cortex of 6-week-old rats (Fig. 2D). is much lower than the ratio of 12 reported by Hook et al. [171 for adult rat brain. This can be explained, least partly, by different assay conditions used. In Ref. 17, AS-A was measured at 15°C and AS-B at 37°C, whereas we assayed both enzymes at 37°C. As indicated in Table L AS-A is more than 4-times as active at the higher temperature. Moreover. Hook et el. [17] state that their AS-A fractions were still contaminated with AS-B and introduce a correction formula. Our method to separate AS-A from AS-B was a slight modification (from that in Ref. 17). For this reason we evaluated our method carefully. The following arguments make us confident that there was no or very tittle cross-contamination of arylsulfatases in the fractions I and Ill obtained from the anion exchange columns, containing AS-A and ASB, respectively (Fig. 1). ( l ) The columns were washed with a large volume of 125 mM NaC1 (fraction ll) after fraction (lI) was discarded; (2) Elation with a gradient rather than by stcpwise elution with 0.125 and 0.25 M NaCI resulted in similar t~coveries of AS-B and AS-A; (3) Rechromatography of fraction I11 on DE-52 yielded one peak at the position of AS-A; (4) CSase activity, assayed with [3SS]sulfatides, was present in fraction 111 but not at all in fraction 1; (5) Silver ions, known to inhibit AS-A selectively [25], blocked the AS activity in fraction Ill but not that in fraction I (results not shown); (6) The time-course of the AS reaction with N C S at 37°C was linear with fraction I but not with fraction Ill and (7) ~tS in fraction I had a much higher temperature co~ffficient than AS in fraction I11 (Table 1). The last two poiats are in agrecment with the kinetic

properties (described in Ref. 16) for AS-A and AS-B of rat liver. Finally. it is interesting to note that the ratio of A S - B / A S - A was about 5-fold higher in the cerebral cortex (Fig. 113') than it was in the spinal cord of a ~.-month-old rat pup. One would expect a ~elatively bdg.h activity of AS-A irt the spinal cord because the metabolism of sulfatides is closely associated with myelin which is enriched in the white matter of the cord, On the other hand, an exclusive localization of CSase in lysosomes of myelin-producing cells in the CNS, the oligr~dcndlocytes, is contra-indicated by observations that the specific activity of CSase in vitro is about equal in the two types of macroglla, astrocytes and oligodendrocytes IVan der Pal et al., submitted for publication). However. in view of the low activity of AS-A in the cortex, one would surmise that the activity of CSase is lower in the neuronal than in the glial compartment of the brain. This contrasts with the suggestion in the literature (see Ref, 26 for refs.) that AS-A is enriched in neuronal cells rather than in glial cells. It should be emphasized, however, that in these reports arylsulfatases were always assayed according to Baum [14]: a method that is unreliable when applied to rodent tissues ([16.17]: Table I). The early rise and fall of AS-B during the development of rat cerebral cortex and especially of the spinal cord (Fig. 2) could well he related to an active phase of desulfation of glycosaminoglycans such as keratan sulfate, chondroitin sulfate and heparan sulfate 127]; components of the extracelhilar matrix associated with the migration of neural cells before myelination starts (see Refs. 28.29 for recent reports). The developmental pattern of AS-B is intriguing but the elucidation of the role of AS-B in brain maturation awaits further investigations. We are currently studying the cellular Iocalizalion of AS-A and AS-B in rat brain. Acknowledgements These investigations were supported in part by the Prinses Beatrix fronds and by the Dutch Foundation for Chemical R~earch (S.O.N.) with financial aid from the Netherlands Organization for Scicniifh: Re~alx.h (N.W.O.I. References ' R:~dirl.N ~. {1982) in Handbook uf Neurochemlstry (Lajtha. A.. ed.). 2nd cdn.. Vol. 3. pp. 163 177. Plenum Press. New York. 2 Norton. W.T. and Cataract. W. (1984l in Myelin(Morell. P.. L~I.). 2nd edn. pp. 147-196. Plenum Press. New York. 3 Rainc. L.~. 11984.~in Mygilni~.~f~][../>..I !. 2nd edm. pp. 1-50. Plenum Pr~,s. New York. 4 Farooqui. A.A. and Horrccks. L.A. 0085) Mok Cell. Biochem.66. 87-95. 5 London. Y. and Vo~senberg. F.G.A 0973) Bi~him. Bioohys. Acta 307. 478-490.

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