The appearance of an extracellular arylsulfatase during morphogenesis of the sea urchin Strongylocentrotus purpuratus

DEVELOPMENTAL

BIOLOGY

88.269-278

(1981)

The Appearance of an Extracellular Arylsulfatase Morphogenesis of the Sea Urchin Strongylocen&ofus ALAN C. RAPRAEGER’ Hopkins Marine Station, Department

of

during purpuratus

AND DAVID EPEL

Biological Sciences,

Stnrlford University,

P&c

Grove, CalZfornia 93950

Received February 8, 1981; accepted in revised form June 18, 1981 An increase in arylsulfatase activity occurs after hatching in embryos of the sea urchin Strongylocentrotuspqnwatus. The bulk of this increase can be attributed to the accumulation of an extracellular activity, since it can be removed by an embryo dissociation medium or upon protease treatment of intact embryos. Also, intact embryos can hydrolyze exogenous substrate, displaying activity equivalent to that which can be removed by the dissociation or protease treatment. The data indicate that the activity appears as a newly secreted molecule ionically coupled to a membrane site or to other components in the extracellular matrix. The majority of the activity is a single component of apparently large molecular size (analyzed on polyacrylamide gel electrophoresis), consistent with the suggestion that it may be complexed with other extracellular components. A morphogenetic role for this enzymeis suggested by the appearance of the extracellular sulfatase temporally coincident with the requirement of sulfated proteoglycans and glycoproteins for cell movement and shape changes. INTRODUCTION

A dramatic in&ease in arylsulfatase activity occurs after hatching in the embryos of the sea urchin Strongylocentrotus purpuratus (Fedecka-Bruner et aL, 1971). A closer examination of this enzyme is prompted by the indication that a portion of the activity might be extracellular, in addition to the enzyme’s appearance at a time when the embryos require sulfated macromolecules for normal morphogenesis and differentiation. Early sea urchin embryos develop normally in seawater devoid of sulfate ions, but stop development at the late mesenchyme blastula stage (Immers, 1956; Sugiyama, 1972), coinciding with the time when a pronounced incorporation of sulfate into proteogylcan (Karp and Solursh, 1974; Oguri and Yamagata, 1978) and glycoproteins (Heifetz and Lennarz, 1979) occurs. These sulfated macromolecules possibly play an important role in governing cell adhesion and shape. The appearance of sulfatase at this time suggests that it is also involved in morphogenesis, possibly degrading sulfated proteoglycans, glycoproteins, or sulfatides. Arylsulfatases have been implicated in the lysosomal degradation of such molecules in mammalian cells. For example, arylsulfatase B is required for the lysosomal desulfation of dermatan sulfate, a glycosaminoglycan (Austin, 1973; Fluharty et aL, 1974; Beratis et al, 1975). Arylsulfatase A, also a lysosomal enzyme, takes part in the degradation of cerebroside sulfate (see Dorfman 1 Present address: School of Medicine,

Department of Pediatrics, Stanford, Calif. 94305.

Stanford

University

and Matalon, 1976). Also, arylsulfatases from a number of invertebr,ates have activity against cerebroside sulfate (Mratz ltind Jatzkewitz, 1974). Since these sulfated molecules are typically cell surface constituents and are likely present on sea urchin cells, they may even be remodeled in situ by an extracellular sulfatase, thereby altering cell adhesions or cell shape. The location of the sulfatase in the embryo, particularly if its location is extracellular, would support or rule out this suggestion. We have employed several criteria to define a postulated extracellular location of the arylsulfatase. These include (1) the removal of enzyme activity in a dissociation mediuni which removes the hyaline layer and dissociates the embryo in the apparent absence of cell lysis or the release of other measurable enzyme activities, (2) the rklease of a similar amount of sulfatase activity from the embryo by proteases acting extracellularly, and (3) the display of an equivalent amount of activity by intact embryos in the presence of exogenous substrate. We also briefly examine the possible nature of the enzyme’s binding to the cell and its relationship to the intracellular enzyme(s). MATERIALS

AND

METHODS

Embryo Culture S. purpuratus collected subtidally at Santa Barbara, California, were maintained in running seawater tanks at Hopkins Marine Station, Pacific Grove, California. Gametes were shed by intracoelomic injection of 0.5 M KCl. After fertilization, zygotes were washed threefold

269 0012/1606/81/160269-10$02.00/O Copyright All rights

Q 1981 by Academic Press, Inc. of reproduction in any form reserved.

270

DEVELOPMENTALBIOLOGY

by settling and cultured at 16°C in Millipore-filtered seawater. Embryo suspensions ranging from 2000 to 10,000 embryos/ml were stirred by paddles attached to 60 rpm Synchron clock motors. The embryos were harvested and washed in hand-cranked centrifuges. Embryo

Dissociation

and Hwrnogenization

Sulfatase is spontaneously released from embryos in media devoid of divalent cations. Therefore, soluble activity was measured after the embryos were dissociated by the method of Kane (1973). An embryo pellet was suspended at room temperature in 1 M glycine (adjusted to pH 8.0) containing 10 mM EDTA-Na, for 20-30 min, then placed on ice. This incubation time was sufficient for maximal enzyme release. During this treatment, the embryos usually dissociated into their component cells which remained motile. Cells were removed from the dissociation medium (DM) by 5 min centrifugation at 12,OOOg, then were homogenized in a medium consisting of DM + 1% Triton X-100 (referred to as HM) at 4°C in a glass homogenizer. Enzyme activity was stable in these homogenates. The sulfatase cannot be removed from embryos contained in fertilization envelopes. Therefore, in order to assay for extracellular sulfatase prior to hatching, fertilization envelope hardening was prevented by fertilizing in the presence of 1 mM 3-amino-1,2,4,-triazole (Foerder and Shapiro, 1977). At 5 min after fertilization, the envelopes were removed by passing the embryos through a 90-pm Nitex mesh followed by several washes in seawater. Sulfa&se

Assays

Arylsulfatase activity was determined by hydrolysis of p-nitrophenylsulfate (Sigma). For the data shown in Figs. 1, 2, 5, and 6, the l-ml assay mix contained sulfatase (50-100 ~1 DM, SW, or HM), 200 mM sodium acetate (pH 6.0), 5 mM EDTA-Naz, and 20 mM substrate. The assay mix was incubated at 37”C, and activity was terminated by the addition of an equal volume of 1 N NaOH. The mix was clarified by centrifugation at 12,000g and pnitrophenol accumulation measured by its absorbance at 420 nm. For K, determinations (Fig. 8) and the hydrolysis of other aryl derivatives (Sigma) shown in Table 1, activity was measured continuously by means of a recording spectrophotometer at room temperature under conditions described in the figure legends. Since these assays were not terminated with NaOH, it was necessary to correct for the reduced absorbance of p-nitrophenol at the pH of the assays by the use of a p-nitrophenol standard. Glucose-gphosphate dehydrogenase activity was mea-

VOLUME 88.1981

sured by the reduction of NADP (measured at 340 nm) in the presence of glucose 6-phosphate at pH 8.0. Activity

Displayed

by Whole Embryos

Substrate hydrolysis by whole embryos (Fig. 4) was determined in 16°C seawater buffered at pH 8.0 with 10 mM Tris-HCl and containing 20 mM pnitrophenylsulfate. Assays were terminated after 20-30 min with 1 N NaOH as described above. Aliquots of DM-derived sulfatase or of homogenized embryos were incubated under identical conditions (by combination with one part of 2X artificial seawater (Hinegardner, 1967) containing 20 mM Tris-HCl (pH 8.0) and 40 mM pnitrophenylsulfate) at 16°C for comparison with the amount of activity displayed by intact embryos. Protease Removal

of Sulfatase

Proteolytic removal of arylsulfatase from embryos was studied using Pronase (Sigma, protease VI) and bovine trypsin (Sigma, type III). Sulfatase activity was stable in the presence of these proteases. Embryos were suspended in 1 mg/ml concentrations of protease in seawater + 10 mMTris-HCl (pH 8.0) at 16°C. After incubation, the embryos were washed three or four times by centrifugation (1OOOg) and resuspension in seawater. Embryos were subsequently treated with dissociation medium as above to determine cell-associated and remaining DM-susceptible activity. Gel Electrophoresis Polyacrylamide gel electrophoresis was performed according to a modification of the Laemmli method (1970) utilizing 4-12% acrylamide gradient gels containing 2.7% bisacrylamide. Enzyme samples were dialyzed against 100 mMTris-HCl (pH 6.8), then brought to 1% sodium dodecyl sulfate (SDS) and 5% glycerol. The discontinuous buffer system consisted of 375 mM Tris-HCl (pH 8.0) + 0.1% SDS in the gradient gel (8 X 15 cm), 125 mMTris-HCl (pH 6.8) + 0.1% SDS in the 4% stacking gel (1 X 15 cm), and a 25 mM Tris-borate (pH 8.0) chamber buffer + 0.1% SDS. Gels were run at constant current (30 mA) at 20°C. Enzyme activity was visualized by placing the gel in 100 ml 100 mM TrisHCl (pH 6.8) containing 0.2 g 6-benzoyl-2-napthyl sulfate (Sigma) at 37°C for an hour, after which 0.1 g Fast Blue B was added for staining. The gels were washed in distilled water and photographed. RESULTS

Increase in Sulfatase Activity

during

Gastrulation

1) Pattern of total activity during development. Analysis of S. purpuratus embryo homogenates during early

RAPRAEGER

AND EPEL

development reveals an arylsulfatase activity which begins to increase at embryo hatching (Fig. 1) confirming prior data (Fedecka-Bruner et aL, 1971). The sulfatase activity increases more than lo-fold during development from the blastula stage (20 hr) to the early pluteus stage (60 hr). Although the present findings indicate a greater amount of activity per embryo than that described previously, this discrepancy is explained in part by different assay conditions (e.g., temperature, buffer). The data presented here are reproducible under the specified conditions and show good agreement over two spawning seasons. 2) The majority of the arylsu~atase is released by embryo dissociation. Embryo dissociation in glycine dissociation medium (DM) by the method of Kane (1973) results in release of sulfatase. The dissociated cells were centrifuged from the medium and homogenized to compare cell-associated and DM-released activities. From posthatching to the early pluteus stage (60 hr), the cellassociated enzyme activity exhibits approximately a 2fold increase while the DM-released form increases more than 20-fold (Fig. 2). Although these data are the results of a single experiment, the pattern and magnitude of arylsulfatase appearance was consistent in other experiments (see also Fig. 6). This comparison indicates that the increase in sulfatase activity during gastrulation can be attributed almost solely to what might be an extracellular sulfatase (Fig. 2). To assay sulfatase activity before hatching, the fertilization envelopes were removed as described and embryos were dissociated in DM. Small amounts of DMreleased sulfatase can be measured just prior to and during the hatching period but the majority of the ent: 2

150 ,

I

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t

20 30 40 50 HOURS OF DEVELOPMENT FIG.

I

60 (16OC)

1. Total arylsulfatase activity during early development of S. Embryos were homogenized in glycine dissociation medium + 1% Triton X-100 and enzyme activity was determined by p nitrophenylsulfate hydrolysis (see Materials and Methods). Activity is expressed as quantity of substrate hydrolyzed (mol X 10-‘3/embryo/min).

purpratus.

Extracellular

271

Arylsulfatase

I

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I

o DISSOCIATION MEDIUM l CELL HOMOGENATE

g

/

8 8

/

Q

/

20 30 40 50 HOURS OF DEVELOPMENT

60 (16’C)

FIG. 2. Arylsulfatase release from S purpuratvs embryos by treatment with dissociation medium. Enzyme activity removed by the medium (0) or remaining with the cells (0) was determined by measuring pnitrophenyl sulfate hydrolysis, as in legend to Fig. 1.

zyme activity remains cell associated (data not shown). The presence of relatively minor amounts of soluble activity at hatching (compared to later stages) and the lack of a marked peak of sulfatase release at this time suggests that the enzyme is not being released as a hatching activity. Ver$icution of the Extracellular Location of the Sulfatase by Other Biochemical Criteria 1) Intracellular enzymes are not released by the dissociation treatment. Release of sulfatase by dissociation medium (DM) did not result in visible cell lysis despite the report by Harkey and Whiteley (1980) that some of the primary mesenchyme cells lyse after even short exposures to DM. Microscopic observation (Figs. 3A, B) indicated that these cells were intact and that ciliated cells even remained motile for well over 30 min. However, this does not rule out the possible exocytosis of a vesicle-contained enzyme in response to osmotic or ionic changes; for example, prolonged exposure to dissociation medium does cause the release of some echinochrome from these cells, a process that has been reported to occur spontaneously under a variety of conditions (see Harvey, 1956). The enzyme is probably not contained in the echinochrome chromatophores, however, since short dissociation treatments can achieve almost total sulfatase release with very little pigment loss. Also, dissociation apparently did not release glucose-6-phosphate dehydrogenase, a cytoplasmic enzyme; no detectable activity was present in the medium

272

FIG. 3. Treatment of 8. purpuratus 48-hr were suspended in DM for 20 min at room terminate cell motility and photographed. for 30 min as in A. (C) Normal prism embryos 50 mM Tris-HCl (pH 8) at 16°C. Embryos swimming.

DEVELOPMENTAL

BIOLOGY

VOLUME

88, 1981

prism embryos with dissociation medium (DM) or Pronase (magnification = 250X). (A) Embryos temperature without agitation, then placed on a slide including a drop of 2% glutaraldehyde to The arrow indicates the archenteron which is slow to dissociate. (B) Embryos suspended in DM in seawater. (D) Embryos treated for 1 hr in Pronase (1 mg/ml) in artificial seawater containing looked very similar to the controls but did shrink slightly when fixative was added to stop their

from dissociated embryos despite considerable activity in the homogenates of prism-stage embryos. The sulfatase might be expected to be contained in lysosomes by analogy to mammalian arylsulfatases which are commonly lysosomal enzymes (see review by

Nicholls and Roy, 1971). If lysosomal arylsulfatase is being released by the dissociation treatment, other lysosomal enzymes might be expected to be released as well. Other p-nitrophenyl-linked derivatives were used to assay for possible lysosomal-type enzymes released

RAPRAEGER

AND EPEL

by DM treatment of prism larvae (Table 1). Assays were conducted at pH 5 or 7.5, comparing enzyme activity released into DM with the total activity of the embryos (which were homogenized in DM + 1% Triton X-100). In addition to sulfatase activity, significant phosphatase, galactosidase, glucosidase, and glucuronidase activities were present in the total embryo homogenates. However, the dissociation medium, into which 70% of the total arylsulfatase activity has been released, did not contain measurable amounts of these enzyme activities. 2) Intact embryos hydrolyze substrate. Another criterion for the apparent extracellular location of the sulfatase is the display of activity by intact embryos. Comparisons were made between (1) substrate hydrolysis by intact embryos in seawater containing 20 mM p-nitrophenylsulfate, and (2) the amount of activity released into the DM. As a control for the possible contribution of intracellular activity to the measurement, the activity in the DM still containing the intact cells is also shown. As seen (Fig. 4), intact embryos in seawater hydrolyze exogenous p-nitrophenylsulfate to an extent comparable to the activity released into dissociation medium. In addition, there is only a minimal contribution to the activity resulting from the presence TABLE 1 ASSAY FOR POSSIBLE LYSOSOMAL ENZYME ACTIVITIES BY DISSOCIATION MEDIUM

REMOVED

Enzyme activity

Activity released by DM (%)

Homogenized embryos (pH 5.0) Dissociation medium (pH 5.0)

6.27 N.D.”

-

Homogenized embryos (pH 7.5) Dissociation medium (pH 1.5)

0.54 N.D.

-

pNitrophenyl+o-galaetoside

Homogenized embryos (pH 5.0)’ Dissociation medium (pH 5.0)

6.45 N.D.

-

pNitrophenyl-8-D-glueoside

Homogenized embryos (pH 5.0)’ Dissociation medium (pH 5.0)

6.45 N.D.

-

pNitrophenyl-8-wglucuronide

Homogenized embryos (pH 5.0) Dissociation medium (pH 5.0)

6.52 N.D.

-

Homogenized embryos (pH 7.5) Dissociation medium (pH 7.5)

0.82 N.D.

-

Homogenized embryos (pH 5.0) Homogenized embryos (pH 7.5) Dissociation medium (pH 7.5)

10.61 5.90 4.13

70

Substrate (26 mnn) pNitrophenylphosphate

pNitrophenylsulfate

Test

Note S pwpu7atur 54-hr prism larvae were placed in dissociation medium (DM) for 30 min at 2O’C. The medium ~88 centrifuged at 12,ooOg for 5 min to remwe the cells, then the supernatant ~88 brought to 1% Trikm X-100 and B final volume of 3 ml. Another aliquot of embryos ~88 directly homogenized in 3 ml of dissociation medium + 1% Triton X-100. Equal aliquote of dissociation medium or homogenized embryos were assayed in 30 mMTris/ HCl (pH 7.5) or 400 mMsodium acetate (pH 5.0) at 2O’C with B final substrate concentration of 20 mM. A,., was measured continuously and converted to moles of p-nitmphenol released using the extinction ceefficients of 94 m&‘cm-’ and 9500 m&km-’ at pH 5.0 and 7.5 respectively. Activity is expressed aa moles of pnitro~henol released x lo~‘/aliquot/min. ’ N.D., not detectable. b No measurable activity at pH 1.5.

Extradular

Arylsulfatase

q q q

273

WHOLE EMBRYOS DISSOCIATION MEDIUM + CELLS DISSOCIATION MEDIUM ALONE

28 HOUR 35 HOUR 45 HOUR 52 HOUR (Blastula) (Gastrula)(Early Prism)(Late Prism) FIG. 4. Comparison of arylsulfatase activity displayed by embryos before and after treatment with dissociation medium. pNitrophenylsulfate hydrolysis was measured at 16°C using aliquots of intact, untreated embryos in seawater, dissociated embryos in dissociation medium, or dissociation medium from which the cells were removed via a 12,OOOgcentrifugation. Enzyme activity is expressed as in legend to Fig. 1.

of intact cells, as removal of these cells by centrifugation only slightly reduces the amount of active enzyme measured. These intact cells do contain sulfatase activity; for example, at the 2%hr stage, the cells contain one-half the total activity, yet this activity is not displayed in the DM + cell mixture. Therefore, cell-associated activity cannot be measured unless the cells are homogenized. These data suggest that the substrate is unable to enter the cells and therefore its hydrolysis by intact cells or embryos is an accurate measure of extracellular activity. Since the activity displayed by the intact embryos is so similar to that extracted by the DM, the DM apparently removes the extracellular enzyme quite efficiently. 3) Proteolytic removal of arylsulfatase. Experiments with the arylsulfatase were aided by the remarkable stability of the enzyme, even in the presence of proteases. Sulfatase activity in embryo homogenates was stable to incubation at 37”C, was stable in the presence of 1% Triton X-100 or 1% SDS, and was also completely stable in 1 mg/ml Pronase or bovine trypsin at 37”C, even after several hours. Protease treatment of intact embryos was therefore

274

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BIOLOGY

used as an additional probe to assess the extracellular location of the sulfatase. Treatment of prism embryos with either 1 mg/ml bovine trypsin or 1 mg/ml Pronase results in arylsulfatase release into the seawater and a corresponding decrease in the amount of activity that can be released by a subsequent treatment in dissociation medium (Fig. 5). Enzyme release into the seawater by untreated embryos is negligible. A 60-min Pronase treatment releases over 95% of the sulfatase ordinarily susceptible to DM release. Trypsin-mediated release is less rapid, reaching approximately 60% of the enzyme susceptible to DM release after 3 hr. Boiled trypsin or Pronase do not remove sulfatase, nor does trypsin in the presence of 5 mM benzamidine, 1 mM phenylmeth-

100

80

I

c

SEAWAiER ALON;

‘A

-I

DM

A...........A.................-...--

60 40

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C

+ TRYPSIN

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+ PRONASE

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VOLUME

88,198l

ylsulfonylfluoride (PMSF) and 10 mM tosylarginine methyl ester (TAME). During the protease treatment, the embryos remain intact (Figs. 3C, D) suggesting that the blastocoelic contents are not released. However, the hyaline layer becomes very indistinct and the apical surfaces of the cells become rounded. Neither enzyme treatment alters the amount of cell-associated sulfatase (i.e., sulfatase remaining with the cells following dissociation), suggesting that this portion is inaccessible to protease. 4) Extracellular activity increases via enzyme secretion It would be expected that the extracellular arylsulfatase would appear during early morphogenesis by secretion of an intracellular precursor. However, the possibility exists that the enzyme is extracellular initially and becomes activated in situ. This can be tested by treating embryos with Pronase to remove the extracellular enzyme, and then determining if extracellular sulfatase accumulates after this treatment, indicating that new sulfatase is being secreted. Mesenchyme blastulae (24 hr) returned to seawater following removal of the extracellular sulfatase by Pronase continue to accumulate and secrete the enzyme (Fig. 6). However, the majority of the replenished sulfatase now appears in the seawater, implying that it continues to be secreted but is not trapped or bound at the embryo’s surface. The total amount of extracellular enzyme secreted by the Pronase-treated embryos by 47 hr of development (i.e., the activity accumulating in the seawater in addition to the DM-susceptible activity) is similar to the amount of active enzyme appearing on the controls during the same period (reaching the prism stage). Following protease treatment, the treated blastulae fail to undergo normal gastrulation, but do synthesize pigment (a postgastrula event) and appear to replace their hyaline layer. Morphologically, they range from animalized-type embryos with large blastocoels to condensed balls of cells.

40 Partial

20 0

0

1 HOURS

2

3

OF TREATMENT

FIG. 5. Protease removal of arylsulfatase from intact S purpurat~~ embryos. Prism-stage (42-hr) embryos were incubated at 16°C in artificial seawater alone (A), or in artificial seawater containing 1 mg/ ml bovine trypsin (B), or 1 mg/ml Pronase (C). Zero-hour time points for B and C were obtained by adding boiled protease to the embryos and processing them immediately. Processing involved washing the embryos extensively in seawater, then placing them in dissociation medium (DM) for 30 min. The dissociated cells were removed from the DM via centrifugation at 12,990 g and were homogenized. Arylsulfatase activity was measured in the seawater (0) DM (A) and homogenized cells (0) and is expressed as a percentage of the total enzyme activity.

Enzyme

Characterkation

1) Sensitivity to su&atase inhibitors. Arylsulfatases are generally classified according to their sensitivity to various inhibitors, particularly sulfate, sulfite and phosphate ions (see reviews by Roy and Trudinger, 1970; Nicholls and Roy, 1971). In an attempt to characterize the enzyme, the effects of pH, temperature and various ions on pnitrophenylsulfate hydrolysis was studied. The enzyme hydrolyzes this sulfate ester over a pH range of 4.5 to 10.0, with an optimum at pH 6 (Fig. 7). The enzyme activity increases with temperature to 3’7°C and exhibits maximal activity between 37 and 50°C. The most potent inhibitor found was sulfite ion (Table 2). Sulfate, fluoride and phosphate are mildly inhibi-

RAPRAEGER

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AND EPEL

Extracelhlar

275

Arglsulfatase

II 0.6

CELLS

I

B

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BRIEF PRONASE

4

5

6

7

8

9

10

PH 24 44

28

32 36 40 44 HOURS OF DEVELOPMENT(lG°C)

48

FIG. 6. Arylsulfatase accumulation following a 1-hr treatment with Pronase. S. pqwuratus mesenchyme blastulae were placed (first arrow) in seawater (A) or in seawater containing 1 mg/ml Pronase (B) for an hour at 16°C. The embryos were then washed (second arrow) and suspended in seawater. Aliquots were withdrawn immediately or at subsequent intervals and the embryos were dissociated. Arylsulfa&e activity in the seawater, dissociation medium and homogenized cells was determined. Enzyme activity is expressed as substrate hydrolyzed (mol X lo-‘/min/aliquot).

tory, requiring concentrations substantially greater than the inhibitory concentrations of sulfite. Thus, the sea urchin arylsulfatase does not readily fall into the classification scheme for mammalian arylsulfatases, which are either sensitive or insensitive to sulfite, sulfate and phosphate. 2) Cmpatim

of cell-associated and extracellular sul-

fatase. The fractionation of sulfatase into activities resistant or susceptible to release by DM raises the question of the number of sulfatases involved and their relationship to one another. The cell-associated form may be lysosomal, or may be a precursor to the extracellular form. The latter possibility is supported by a common Michaelis-Menten constant of 3.9 mM pnitrophenylsulfate (Fig. 8) and an identical pH optimum (comparison not shown) for the two activities. The ability of the sulfatase to retain full activity in SDS allows the use of SDS-polyacrylamide gels (Weber and Osborn, 1969) in an attempt to separate sulfatase species, although as the enzyme is probably not totally denatured, the inverse proportionality of its mobility to size on an SDS gel must be qualified. Sulfatase was removed from prism-stage embryos by

FIG. 7. Activity of arylsulfatase versus pH. Arylsulfatase activity released into dissociation medium from prism-stage embryos was measured at various pH values in 200 m&f Tris-acetate + 0.1% SDS (3’7°C) containing 20 mM pnitrophenylsulfate. Assays were terminated with an equal volume of 1 N NaOH. Enzyme activity is expressed as the hydrolysis of pnitrophenylsulfate as measured by the absorbance of free pnitrophenol at 420 nm.

digestion with 1 mg/ml Pronase or treatment with dissociation medium and compared to sulfatase activity remaining with the cells or that present on whole embryos on gradient gels (4-12s). These activities were visualized by the hydrolysis of 6-benzoyl-2-napthyl sulfate and subsequent staining of the insoluble cleavage product with Fast Blue B. SENSITIVITY

Test

(m&f)

No additive (control) 200 KH,PO, 50 10 10 NapSO 1 0.1

TABLE 2 TO SULFATASE

INHIBITORS Percentage of control activity 100 15 43 88 1 10 37

100 Na2S04 10 2

60 81 84

10 NaF 1

34 78

Note. p-Nitrophenylsulfate hydrolysis by aliquots of homogenates in 0.2 MTris-acetate (pH 6.0) + 0.1% SDS at 37°C. Solutions were adjusted to pH 6.0 with NaOH necessary. Activity in the presence of added inhibitors sured and expressed as a percentage of the control.

prism embryo was measured or HCl where was also mea-

276

DEVELOPMENTAL

4

-l/K,,,

0

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0.2

0.4

0.6

0.8

p-NITROPHENYLSULFATE

BIOLOGY

I 1.0

(ITIM-‘)

FIG. 8. Lineweaver-Burke plot of arylsulfatase from 8. purpur&m prism embryos. Embryos were dissociated in DM, then the cells were pelleted and homogenized in DM + 1% Triton (HM). The volumes were adjusted so that sulfatase activity/ml was identical in DM and HM. Enzyme activity in DM + boiled HM (0) and HM + boiled DM (0) was determined at pH 8.0 against various concentrations of pnitrophenylsulfate. The initial rate of pnitrophenol released (measured at A&.& was expressed as moles of substrate hydrolyzed X lo-“/min using the extinction coefficient (pH 8) of 14,206 mol-‘cm-‘. Activity is plotted as a reciprocal of the rate (l/V) versus the reciprocal of the substrate concentration. K,,, equals 3.9 mAf.

The bulk of the sulfatase activity of embryo homogenates runs as a single band with relatively low mobility, possibly indicating large molecular size (Fig. 9). Duplicate gels stained with Coomassie blue demonstrate that almost all the protein bands run ahead of this sulfatase band, which itself coincides with a Coomassie blue band (data not shown). The majority of the major sulfatase band can be removed by incubation of the embryos in dissociation medium prior to homogenization suggesting that a large portion of it is the extracellular activity. Coomassie blue staining again demonstrates the coincidence of a significant amount of protein with this activity. No other Coomassie bluestaining bands were detectable in the quantity of DM applied to these gels. The whole embryo and cell homogenates (enzyme remaining with the cells after dissociation) contain two minor sulfatase bands of greater mobility than the major DM-susceptible band. These bands are not diminished by the DM treatment (compare lanes 1,2, and 3) nor do they appear in the DM. Thus, these bands represent cell-associated activity and may be intracellular. In addition, a portion of the major band is resistant to release by dissociation medium and would therefore be defined as cell-associated activity. This may be intracellular, or may be associated with a portion of the extracellular matrix which is not susceptible to release by Ca2+-free media. As expected, Pronase treatment of embryos releases

VOLUME

881981

a significant amount of sulfatase activity into the seawater (Fig. 9, lane 5). This activity is comprised of the major sulfatase band, with a slightly increased mobility, and two novel moieties of apparent smaller size generated by the proteolytic treatment. This introduces the possibility that the slowly migrating activity may be a complex, possibly linked to other extracellular components, and that the protease partially cleaves the complex without substantial digestion of the sulfatase itself. Dissociation of the Pronase-treated embryos in DM releases more enzyme, again comprising the single major band, but with the slightly increased mobility (lane 6); homogenization of the cells following dissociation again reveals the major band, although with slightly increased mobility, as well as one of the cell-associated bands. However, one of the cell-associated bands is no longer apparent after Pronase digestion. It is likely that some digestion of cell-associated activity can be attributed to residual Pronase following cell homogenization, although the embryos were washed four to five times. DISCUSSION

Gastrulation of the S. purpuratus embryo is accompanied by the accumulation of an arylsulfatase which

r embryos ,

cells

PRONASE TREATED

DM

SW

DM

4

5

6

-

1 cells

1

1

2

3

7

FIG. 9. SDS-polyacrylamide gel electrophoresis of sulfatase from prism-stage embryos. Gels were run and stained as described under Materials and Methods. Lane 1, one-fifth of an aliquot of embryos homogenized in HM; lane 2, one aliquot of embryos homogenized in HM; lane 3, homogenized cells from an aliquot of dissociated embryos; lane 4, DM (no cells) from embryos in lane 3; lane 5, sulfatase released into seawater from treatment of an aliquot of embryos for 1 hr with Pronase; lane 6, DM (no cells) after dissociation of the proteasetreated aliquot; lane 7, homogenized cells after dissociation of the protease-treated aliquot. (DM = glycine dissociation medium; HM = DM + 1% Triton X-100.)

RAPRAEGERANDEPEL

can be classified as extracellular on the bases of its release from the embryos by dissociation medium (DM), its release from intact embryos by proteases, and the hydrolysis of exogenous substrate by whole embryos. It is unlikely that methods such as embryo dissociation result in the release of intracellular sulfatase since the DM does not contain measurable quantities of cytoplasmic or lysosomal enzymes. The presence of other lysosomal activities in the DM is an important consideration since mammalian arylsulfatases are common lysosomal enzymes. Possible extracellular locations of the sulfatase include sites in the blastocoel, between cells of the epithelium, or on the outer surface of the embryo. The embryo surface site is consistent with the finding that exogenous p-nitrophenylsulfate is hydrolyzed by intact embryos; it is unlikely that p-nitrophenylsulfate penetrates into the blastocoel or into the intracellular space since the septate desmosomes at the cell apices are effective barriers to the penetration of small molecules, such as sucrose or amino acids (Moore, 1940; Rapraeger, unpublished results). It is also unlikely that the protease treatment readily removes sulfatase from the blastocoel. Although the proteases dissolved the hyaline layer (see also Vacquier and Mazia, 1968;. Citkowitz, 1971) and the apical cell surfaces became rounded, the embryos did not dissociate. In addition, sulfatase can be fully released by short treatments with DM under conditions which leave most embryos still intact. Finally, preliminary histochemical staining suggests that the enzyme is uniformly distributed on the outer surface, probably associated with the hyaline or related layers (Rapraeger and Epel, 1980). These studies provide some insights into the nature of sulfatase attachment to the embryo. Release will occur in media devoid of divalent cations or by the action of proteases in seawater. Thus, it would not appear to be an integral part of the plasma membrane nor covalently bound to an integral component, yet is attached sufficiently well so that it is not normally released into seawater. The proteolytic release could ensue from digestion of part of the enzyme or from digestion of an anchoring component. Both possibilities are consistent with the limited effects of Pronase on the electrophoretic mobility of the sulfatase. However, the apparent high molecular weight of the activity suggests it is a macromolecular complex, more consistent with the concept of an associated anchoring molecule perhaps a hyaline layer constituent. The continued appearance of extracellular sulfatase following a brief protease treatment suggests that the enzyme appears via secretion of a newly synthesized or newly activated intracellular precursor. Furthermore, its spontaneous release into the seawater supports the

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concept that it is not an integral part of the membrane but is probably associated with membrane binding sites or matrix components which were removed by the prior proteolytic digestion. The partial characterization of the sulfatase indicates that it does not readily fit the classification scheme devised for the mammalian enzymes based on its major sensitivity only to sulfite and a minor inhibition by sulfate and phosphate. In this regard, it bears more resemblance to an invertebrate arylsulfatase derived from the snail Helix powcatia (Dodgson and Powell, 1959a,b). An arylsulfatase present in the sperm and semen of sea urchins has also been described (Moriya and Hoshi, 1980; Hoshi and Moriya, 1980) and is reported to be capable of dissolving the jelly and viteline layers around eggs. This enzyme does not resemble the embryonic sulfatase based on the requirements of the sperm enzyme for calcium, its greater sensitivity to inhibition by sulfate and its greater affinity for pnitrophenysulfate. It is tempting to postulate that the extracellular arylsulfatase plays a critical role in sea urchin embryonic morphogenesis. Sulfated glycoproteins and sulfated proteoglycans are synthesized to an increased extent beginning at early gastrulation. Posthatching development is disrupted in the presence of tunicamycin (Schneider et al., 1978) which blocks the attachment of N-linked oligosaccharides on glycoproteins, and in the presence of xylosides (Kinoshita and Saiga, 1979) which compete with the initiation of O-linked glycosaminoglycan chains on the protein core of proteoglycans. Also, studies with animalizing treatments such as sulfatefree seawater (Immers, 1956; Sugiyama 1972) thiocyanate (Kinoshita, 1974), or selenate ions (Sugiyama, 1972) point to the requirement for newly synthesized sulfated macromolecules for development beginning at early gastrulation. A plausible explanation for this is that these typically extracellular molecules are necessary for morphogenesis. One possible model is that the sulfatase is involved in processing these sulfated materials prior to or at the time of their export, and then is exported with them. In this case, the extracellular enzyme activity may no longer be performing a useful role. An alternate possibility is that the extracellular sulfatase actually has a cell-surface role, such as participating in the assembly of the extracellular matrix or, more likely, by releasing sulfate from these materials-possibly to reduce cell adhesions and allow changes in cell shape. Many questions remain to be answered about the sea urchin embryonic sulfatase. Among these are its distribution on the embryo surface, the identity of its natural substrate, the universality of its occurrence among sea urchin species, as well as other phyla, and the elu-

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cidation of its overall role in development. Work is presently proceeding along these lines of investigation. The authors thank Dr. Merton R. Bernfield for his helpful suggestions and criticism of the manuscript. The technical assistance of Chris Patton is also gratefully acknowledged. This work was supported by an NSF Grant to David Epel and a postdoctoral fellowship award (GM 07494) to Alan Rapraeger from the National Institute of General Medical Sciences. REFERENCES AUSTIN, J. H. (1973). Studies in metachromatic leukodystrophy. XII. Multiple sulfatase deficiency. Arch iVeurol. 28,258-264. BERATIS, N. G., TURNER, B. M., WEISS, R., and HIRSCHHORN, K. (1975). Aryl sulfatase B deficiency in Maroteaux-Lamy syndrome: Cellular studies and carrier identification. Pediatr. Res. 9,475-480. CITKOWITZ, E. (1971). The hyaline layer: Its isolation and role in Echinoderm development. Devebp. Biol. 24,348-362. DODGSON, K. S., and POWELL, G. M. (1959a). Studies on sulfatases: Aryl sulfatase activity in the digestive juices and digestive gland of Helix pomatia. B&hem. J. 73, 666-671. DODGSON, K. S., and POWELL, G. M. (1959b). Studies on sulfatases: The partial purification and properties of the aryl sulfatase of the digestive gland of Helix pomatia. B&hem J. 73. 672-679. DORFMAN, A., and MATALON, R. (1976). The mucopolysaccharidoses (a review). Proc. Nat. AccuL Sci. USA 73, 630-637. FEDECKA-BRUNER, B., ANDERSON, M., and EPEL, D. (1971). Control of enzyme synthesis in early sea urchin development: Aryl sulfatase in normal and hybrid embryos. Develop. Biol. 25,655-671. FLUHARTY, A. L., STEVENS, R. L., SANDERS, D. L., and KIHARA, H. (1974). Aryl sulfatase B deficiency in Maroteaux-Lamy syndrome cultured fibroblasts. B&hem. Biqphys. Res. Commun 59,455-461. FOERDER, C. A., and SHAPIRO, B. M. (1977). Release of ovoperoxidase from sea urchin eggs hardens the fertilization membrane with tyrosine crosslinks. Proc. Nat. Acad. Sci. USA 74.4214-4218. GUSTAFSON, T., and WOLPERT, L. (1961). Cellular mechanisms in the morpbogenesis of the sea urchin larva: The formation of arms. Exp. Cell Res. 22, 509-520. HARKEY, M. A., and WHITELEY, A. H. (1980). Isolation, culture, and differentiation of Eehinoid primary mesenchyme cells. Wilhelm RVUX Arch. Dev. Bill. 189, 111-122. HARVEY, E. B. (1956). “The American Arbacia and Other Sea Urchins,” pp. 158-159. Princeton Univ. Press, Princeton, N. J. HEIFETZ, A., and LENNARZ, W. (1979). Biosynthesis of N-glycosidically linked glycoproteins during gastrulation of sea urchin embryos. J. Biol. Chem 254,6119-6127. HINEGARDNER, R. T. (1967). Echinoderms. In “Methods in Developmental Biology” (F. H. Wilt and N. K. Wessells, eds.), p. 139. T. Y. Crowell, New York.

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HOSHI, M., and MORIYA, T. (1980). Arylsulfatase of sea urchin sperm. 2. Arylsulfatase as a lysin of sea urchins. Develop. Biol 74,343-350. IMMERS, J. (1956). Cytological features of the development of the eggs of Paracentrotus lividus reared in artificial sea water devoid of sulphate ions. Exp. Cell Res. 10.546-548. KANE, R. E. (1973). Hyaline release during normal sea urchin development and its replacement after removal at fertilization. Exp. Cell Res. 81, 301-311. KARP, G. C., and SOLURSH, M. (1974). Acid mucopolysaccharide metabolism, the cell surface and primary mesenchyme cell activity in the sea urchin embryo. Develop. BioL 41,110-123. KINOSHITA, S. (1974). Synthesis of sulfate donor in developing sea urchin embryos. Exp. Cell Res., 87,382-385. KINOSHITA, S., and SAIGA, H. (1979). The role of proteoglycan in the development of sea urchins. I. Abnormal development of sea urchin embryos caused by the disturbance of proteoglycan synthesis. Exp. Cell Res. 123, 229-236. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage TI. Nature (Londonj 227, 680-685. MOORE, A. R. (1940). Osmotic and structural properties of the blastular wall in Dewkaster excentricus. J. Ezp. Zoo1 84, 73-79. MORIYA, T., and HOSHI, M. (1980). Characterization and partial purification of arylsulfatase from the seminal plasma of the sea urchin Strongylocatrotus intermedius. Arch. Biochem Biophys. 201, 216223. MRATZ, W., and JATZKEWITZ, H. (1974). Cerebroside sulfatase activity of aryl sulphatases from various invertebrates. Hoppe Seyler 2. Physiol Chem. 355,33-44. NICHOLLS, R. G., and ROY, A. B. (1971). Arylsulfatases. In “The Enzymes” (P. D. Boyer, ed.), Vol. 5, p. 21. Academic Press, New York/ London. OGURI, K., and YAMAGATA, T. (1978). Appearance of a proteoglycan in developing sea urchin embryos. Biochim Biophys. Acta 541.385393.

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