Biochemical characterization of arylsulfatase-C isozymes in human fibroblasts

Vol. 128, No. 3, 1985 May 16, 1985

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1388-l 394

BIOCHEMICALCHARACTERIZATION OF ARYLSULFATASE-C ISOZYMESIN HUMANFIBROBLASTS Jean-Pierre

S. Simard, Mohamned&neen and Patricia

L. Chang"

Department of Pediatrics M&laster University Hamilton, Ontario, Canada Received April

9, 1985

SUt+lARY Arylsulfatase-C and sterol sulfatase were thought to be identical enzymes whose X-linked locus escapes inactivation. However, recent evidence shows that they are not identical but that arylsu1fatase-C in hcnnanfibroblasts exists in two isozymic forms, designated as slow and fast. We now report that the two forms are enzymatically different. When assayed with an artificial fluorogenic substrate, the slow form showed a pH optima of 8.00 and a Km of 228 pM. In contrast, the fast form showeda pH optimum of 7.67 and a IGnof 86.7 uM with substrate inhibition occurring above 0.33 n@l. The heat stability of the fast form was slightly below that of the slow form. Polyclonal antibodies raised against the slow form did not cross-react with the fast form. Hence, the two isozymic forms of arylsu1fatase-C are enzymatically and structurally different and the slow form is associated with sterol sulfatase activity. a 1985

Academic

Press,

Inc.

Arylsulfatase-C biochemical identity substrates,

i.e.

(ARC) [EC3.1.6.1]

has not been clarified.

the 3-@hydroxysterol

dehydroepiandrosterone sterol

sulfatase

sulfate

ichthyosis

sameenzyme (3,4). escape X-inactivation not identical

When its activity

sulfates,

and cholesterol

(STS) [EC3.1.6.2].

associated with the X-linked and X-linked

is a microsomal membrane-boundenzyme whose

Deficient

such as estrone sulfate, sulfate,

Therefore,

it is also referred

STS and ARCactivities

recessive conditions,

in man (2).

is assayed with natural

placental

to as

have been

sulfatase deficiency

(1)

ARCand STS have been asslnnedto be the

Their locus was mappedto the humanXp22-pter and appeared to (5,6).

Recent evidence from our laboratory

with STS but exists

as two isozymic forms, designated as slow and fast,

which are separable by electrophoresis *Address corresoondence to:

showed that ARC is

on nitrocellulose

acetate (7).

Dr. P.L. Chang ROCITI 3N18

M&aster University Medical Centre 1200 Main St. W. Hamilton, Ontario, Canada L8N 325 0006-291X/85$1.50 Copyright 0 1985 by Academic Press, Inc. All rights of reproducrion in any form reserved.

1388

This report

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lescribes

128,

No.

3, 1985

the characterization

BIOCHEMICAL

AND

BIOPHYSICAL

of the two ARC isozymes

RESEARCH

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from human fibroblast

membrane

extracts. MATERIALS AND METHODS Cell Culture: Normal human fibroblasts were obtained from skin biopsies of individuals with no known metabolic diseases. X-linked Ichthyosis fibroblast strain 503 was obtained from H. Kihara and GM3227 fran the Camden Mutant Cell Repository Cells were maintained in Ml++HEPES medium supplemented with penicillin (100 (N-J.). (100 pg/ml), L-glutamine (2mM) and lo-20% fetal calf serum under U/ml) , streptomycin the usual culture conditions. Cells were harvested with trqsinization and pellets were rinsed twice with isotonic saline before storing at -70 C until use. Extraction of arylsulfatase C: Cell pellets were resuspended in 0.15 M sodium acetate (50 pl/lOO mn dish) and disrupted with ultrasonic treatment at Power setting 4 with a micro-ultrasonic cell disruptor (Kontes, Vineland, NJ) for 3x10 sec. with 30 s %c. cooling intervals on ice. The cell suspension was centrifuged at 12,000 x g at 4 C for 3 min. The membrane pellets were washed twice in 1.0 ml of 0.04 M Tris pH 8.5 and then extracted with 0.04 M Tris-HCl (pH 8.0) containing 1% (w/v) Miranol H2M (Miranol Chemical Co., Irvington, NJ) and centrifuged at 12,000 x g at 4'C for 5 min. The supernatant was used as the extract for protein and enzyme analyses. Electrophoresis: The Separation electrophoresis on nitrocellulose described by Chang et al (8).

and identification of arylsulfatase acetate (Cellogel, Kalex) was according

C by to the method

Enzyme Assays: All assays were performed under conditions in which enzyme activities were directly proportional to duration of incubation and amount of protein used. ARC was assayed with 4-methyltilliferyl sulfate (4-MUS, Koch-Light Laboratories Ltd., England) as the substrate according to the method of Eto et al (9). Estrone sulfatase was assayed with estrone-sulfate-3H (40-60 Ci/mnole, NEN, Lachine, Quebec) as the labelled substrate according to the method of Iwamori et al (10). Dehvdroepiandrosterone-sulfatase was assayed with dehydroepiandrosterone-sulfate-3H (lo-25 Ci/nole, NEN, Lachine, Quebec) as the labelled substrate according to the method of Gauthier et al (11). Characterization of enzymatic parameters: The pH optima of ARC, estrone-sulfatase - -.-_ and dehydroepiandrosterone sulfatase activities in fibroblasts membrane extracts were measured with 0.2M Tris-maleate buffer (QH 5.2 - 8.6) and 0.15M sodium aceate buffer (DH 3.7 - 5.6). The heat stability of the enzyme activities was measured according to the method of Feinstein et al (12). Cell membrane extracts were inactivated at temperatures from 50 to 75'C for 10 min. each and the residual activities were measured. The temperature at which 50% of the activity remained (T ) was calculated by linear regression analysis of the data points after transf 8O rmation into logarithmic functions. Km. The activity of the enzyme preparations over a range of substrate concexrations was measured for each substrate. The Km was calculated from a linear reqression analysis of the Lineweaver-Burke plot. Imnunoprecipitation and electrophoresis. Polvclonal antibodies to partially purified ARC from human placenta(I3,I6)were raised as described by Stevens et al (14). Five microlitre of rabbit serum was mixed with 2-8 ul of fibroblast extract containing 0.15 units of ARC activity, incubated at 37 C for one hour and the mixture applied for electrophoresis as described for ARC (8). RESULTS Fibroblasts Y-linked

Ichthyosis

representative were disrupted

of normal

individuals

to remove all 1389

and patients

soluble

enzymes,

suffering including

from

Vol.

128,

No. 3, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Figure 1 Cellogel electrophoresis of ARC in fibroblasts from normal individuals and patients with X-linked Ichthyosis. Fibroblasts were disrupted and washed to provide membrane fractions that were extracted with miranol. The detergent extract was applied on Cellogel, subjected to electrophoresis and stained for ARC activity with ~-MUS. + anode - cathode -origin N: normal fibroblasts from individuals 1 and 2 XLI: X-linked Ichthyosis fibroblasts from individuals 1 and 2

arylsulfatase-A

and -B.

electrophoresis

and stained

individuals

The Miranol-extracts

showed either

of membrane pellets

for ARC activity two ancdal

activity

the two bands in both males and females. Ichthyosis

patients

fast-anodic

representative

ARC activity

according single

with

experiments

than that

The optimal

parameters:

and slow,or

extracts

only the slower

of

from two X-linked which migrated

to the

ichthyosis

were cornoared in

4-methylumbelliferyl

optimum,

while

heat stability

the means are calculated

sulfate

(4~~s)

Results

and Km.

from three

above 0.33 pM.

The heat stability

50% of the activity

different

different

the fast

(Fig.2a).

at which

(O.Ol
pM, which was again significantly

concentration

pH

band at 7.67 (p
This was significantly

In addition,

X-linked

and the

pH of the slow band was 8.00 which was significantly

band at 57.8 + 0.8'C

(p
with

substrate,

are shown in Fig.2

of the fast

0.4’C.

the fast

from normal

band alone from normal fibroblasts

of patients

band, as measured by the temperature +

bands,fast

However,

of the slow

the artificial

to the following

experiments.

60.2

Extracts

showed only about 10% of normal activity

band alone from fibroblasts

their

1).

to

position.

Extracts fast

(Fig.

were subjected

at 95% confidence (Fig.2b).

from that of the fast

substrate

form of ARC (Fig.2c). 1390

higher

(TsO), from that

was of

The Km for the slow band was 217

form of ARC demonstrated

Similar

to four

of the slow

was lost level,

from

inhibition

inhibition

band at 91 uM by substrate

was not observed

in the slow

Vol. 128, No. 3, 1985

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

/P

-\

i

-_‘.

PII\ I\ II 60 -

d, II I\ \ P : I ‘I0 t Q

--s ---

‘\\ \ dy\ \\ \\ \ \

b

TEMP.

f

(“C)

C (Sib) (mM)-1 Figure 2a pi profile of ARC activity in crude membrane extracts showing the slow and the fast Each curve represents one experiment in duplicate. forms. mean AX activity* + range fran extracts showing a slow band pattern. ---mean AIEC activity* 7 range fran extracts showing a fast band pattern.

l puol of 4-MUS hydrolysed/ul/hr

at 37OC

Figure 2b Heat stability of AW in crude membrane extracts representing the slow and the fast Membrane extracts were subjected to 10 min. of heating at the various forms. temperatures. me residual ARC activity was determined and compared to the control Each curve represents one experiment in that had not been subjected to heating. duplicate. mean residual ARC activity 2 range in extracts representing the slow band pattern. ---mean residual ARC activity + range in extracts representing the fast band pattern. Figure 2c Lineweaver-Burk nlot of the ARC activity in crude membrane extracts representing the slow and fast band pattern. activity fran extracts representing the slow band pattern. ---activity from extracts representing the fast band pattern. 1391

Vol. 128, No. 3, 1985

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE1: Enzymatic characterization of crude preparations from fibroblast membranes representing the slow and fast forms of ARC Crude extract

Substrate

Characterization T,, (Cl

PN slow ARC

I-Mus DS

8.00+0.10(3) 8.07+0.06(3) 8.0350.06(3)

60.2+0.4(3) 62.2+0.7(4) 61.7zO.6(3)

4-Mus

7.67+0.03(3)

57.8+0.8(3)

ES

fast ARC

IQn(WM) 217+3.2(3) 5.0+0.6(3)

8.9?3.1(4) 91.0254.0(3)

Fibroblasts representing the slow and fast ARCforms were disrupted and washedto provide the membrane pellets which were extracted with Miranol. The solubilized suparnatant wasused as the crude extract for the various characterizations. Each determination was the meanfran (N) experiments + S.D. and each experiment wasperformed in duplicate.

The ARCof the slow band and the fast band were then compared in their T50 and Km towards the natural substrates, dehydroisoandrosterone

sulfate

(DS).

estrone sulfate

QHoptima,

(ES), and

The data are presented in Table 1.

STS activity

was found to be associated only with the slow band; the fast band of ARCactivity no detectable

activity

towards the natural substrate.

showing the slow band were almost identical and DS (8.03) were used. was not significantly (61.7o)'were used. significantly

Similarly,

different

The PH optima of extracts

when the substrates 4-MUS (8.001, ES (8.07)

the heat stability

(T50) of the enzyme activity

when the substrates 4-MDS (60.2'C),

The Kmvalues of the enzyme activity

different

Imnunoprecipitation

from that of 4-MUSat 217 ).IM (p
with normal membraneextracts

polyclonal

that showedeither

fast forms of ARC, and extracts

from oatient

retarded in its electrophoretic

mobility

of either

The antiserum was incubated

fibroblasts

with X-linked

(lanes d and g, Fig.3).

of immunization did not affect

form of ARC (lanes b, e and h, Fig.3),

any serum (lanes c, f and i, Fig. 3). 1392

ichthyosis

that

only the slow form of ARCwas

due to the imnuno-reaction

while the fast form was not affected

the samerabbit before initiation

antibodies were raised in the

the slow form or both the slow and

showed only the fast form of ARC. As shown in Fig.3,

mobility

FS (62.2'C) and DS

towards ES and DS were

rabbit against the slow form of ARCfrom humanplacenta.

Fig.3)

had

(lanes a and g, Control serum fran

the electrophoretic

nor did incubation without

Vol.

128,

BIOCHEMICAL

No. 3, 1985

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Figure 3 Semxn obtained from rabbit before (control) Imnunoprecipitation and electrophoresis. and after (test) imnunization with the slow form of ARCwas incubated with 2-8 ul of fibroblast extracts for I hr. at 37'C. Similar incubation of fibroblast extract with 0.04M Tris-El, pH 8.0, instead of rabbit serum was also performed. The mixture was applied to nitrocellulose acetate and electrophoresed as described for ARC in Waterials and Methods". a : test rabbit serum slow-banded ARC vs b : control rabbit serum c : tris-HCl buffer fast-banded ARC vs d : test rabbit serm e : control rabbit serum f : tris-HCl buffer slow + fast-ban&d ARC-vs g : test rabbit serum h : control rabbit sermn i : tris-HCl )ISCUSSION

Two anionic iibroblasts. fast

forms of ARC (slow

Normal fibroblasts Fibroblasts

forms.

and fast)

are recognized

show either

from three

the slow

out of six

in human cultured

form alone OK both the slow

individuals

with

X-linked

lave been found to demonstrate

the fast

band alone while

:how either

The fast

form of ARC in these patient

of the two forms.

lot associated optimal Iheir

catalytic

functions

raised

the slow

isozymic

significant

against

zhe possibility zhe substrate s-p-hydroxysterol

immunologically

the artificial

sulfatase

sulfates.

heat stability

itself

may

4-EVIUS, ES and

identical

(Fig.

DS all

optimal

(Table 1). 1393

was of their

4-MUS, indicated and Table 1).

3),

that

Because mobility

it was clear

that

of

the two

distinct.

with

sterol

be heterogeneous

by characterizing

fibroblasts

the electroohoretic

and hence, structurally

It was thus shown that

towards

(Fig.2

form of ARC retarded form unchanged

three did not

Characterization

substrate,

different

ichthyosis

the remaininq

activity.

form of ARC is associated

sterol

as shown by their identical

the fast

used, was investigated

ionn of ARC, activity

their

with

the slow

only the slow that

sulfatase

are significantly

form but left

forms were

Although

similar,

sterol

Km and heat stability

pH,

antibodies ,nly

with

and

its

(15),

activities

in fibroblasts appeared

sulfatase

to

pH, and structurally

activity,

depending with

with

on

various

only the slow

be catalytically related,

as shown by

Vol.

128,

No. 3, 1985

In conclusion, fast.

properties. sulfatase

AND

ARC in human fibroblasts

They have been shown to differ

structural sterol

BIOCHEMICAL

while

the natural

exists

in their

It is possible

BIOPHYSICAL

in two isozymic

electrophoretic,

that only

substrate

RESEARCH

for

COMMUNICATIONS

forms,

catalytic

slow

and

and

the slow

form of ARC is related

the fast

form remains

to

to be

elucidated. ACKNOWLEDGEMENT A supply of placental ARC by Dr. R. Shankaran, encouragement and advice from Dr. R.G. Davidson, support from the Ontario Mental Health Foundation (P.L.C.) and Medical Research Council of Canada (MA-5789 to P.L.C. and R.G.D.), and award of an Ontario Mental Health Scholarship to P.L.C. are gratefully acknowledged. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

France, J.T. and Liggins, G.C. (1969) J. Clin. Endocrinol. Metab. 29, 138-141. Shapiro, L.J., Weiss, R., Webster, D., and France, J.T. (1978). Lancet i, 70-72. Dolly, J.O., Dodgson, K.S., and Rose, F.A. (1972). Biochem. J. 128, 337-345. Thomas, S.B., and Rose, F.A. (1976). Biochem. J. 158, 631-633. Mohandas, T., Shapiro, L.J., Sparkes, R.S., and Sparkes, M.C. (1979). Proc. Natl. Acad. Sci. (USA) 76, 5779-5783. Muller, C.R., Wahlstram, J., and Ropers, H.H. (1981). Hum. Genet. 58, 446-447. Chang, P.L., Varey, P.A., Rosa, M.G. and Davidson, R.G. (1985). Manuscript submitted. Chanq, P.L., Ameen, M., Lafferty, K.I., Varey, P.A., Davidson, A-R., and Davidson, R.G. (1985). Analyt. Biochem.144,362-370, Eto, Y., Rampini, S., Weismann, U., and Herschkowitz, N.N. (1974). J. Neurochen. 23, 1161-1170. Iwamori, M., Moser, H.W., and Kishimoto, Y. (1976). Arch. Biochem. Biophys. 174, 199-208. Gauthier, R., Vigneault, N., Bleau, G., Chapdelaine, A., and Roberts, K.D. (1978). Steroids 31, 783-798. Feinstein, R.N., Sacher, G.A., Howard, J.B., and Braun, J.T. (1967). Arch. Biochem. Biophys. 122, 338-343. Shankaran, R., Ameen, M., Davidson, R.G., and Chang, P.L. (1985). Purification and properties of the arylsu1fatase-C isozymes from human liver and placenta. Manuscript in preparation. Stevens, R.L., Fluharty, A.L., Skokut, M.H., and Kihara, H. (1975). J. Biol. Chem. 250, 2495-2501. Zuckennan, N.G. and Hagerman, D.D. (1969) Arch. Biochem. Biophys. 135, 440-445. Noel,H., Plante. L., Bleau. G.. Chapdelaine, A., and Roberts, K.D. (1983). J. Steroid Biochem. 19, 1591-1598.

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