Separation and properties of five glycosaminoglycan sulfatases from rat skin.

Chondroitin sulfates, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, and oligosaccharides derived from these sulfated glycosaminoglycans have been used for the measurement of sulfatase activity of rat skin extracts. Chromatographic fractionation of the extracts followed by specificity studies demonstrated the existence of five different sulfatases, specific for 1) the nonreducing N-acetylglucosamine 6-sulfate end groups of heparin sulfate and keratan sulfate, 2) the nonreducing N-acetylgalactosamine (or galactose) 6-sulfate end groups of chondroitin sulfate (or keratan sulfate), 3) the nonreducing N-acetylgalactosamine 4-sulfate end groups of chondroitin sulfate and dermatan sulfate, 4) certain suitably located glucosamine N-sulfate groups of heparin and heparan sulfate, or 5) certain suitably located iduronate sulfate groups of heparan sulfate and dermatan sulfate. Two arylsulfatases, one of which was identical in its chromatographic behaviors with the third enzyme described above, were also demonstrated in the extracts. These results taken together with those previously obtained from studies on human fibroblast cultures suggest that normal skin fibroblasts contain at least five specific sulfatases and diminished activity of any one may result in a specific storage disease.


Separation
and Properties of Five Glycosaminoglycan Sulfatases from Rat Skin* (Received for publication, November 27, 1978, andin revised form, March 22, 1979)  sulfate. Two arylsulfatases, one of which was identical in its chromatographic behaviors with the third enzyme described above, were also demonstrated in the extracts. These results taken together with those previously obtained from studies on human fibroblast cultures suggest that normal skin fibroblasts contain at least five specific sulfatases and diminished activity of any one may result in a specific storage disease.
The physiological role of sulfatases in mammalian tissues has long been a subject for speculation (for a review see Ref. 1). In recent years, investigation into the enzymatic defects of mucopolysaccharidoses have resulted in a number of important observations on the role of sulfatases in the metabolism of sulfated glycosaminoglycans.
Thus, the enzymatic deficiencies that have been identified are glucosamine-N-SO3 sulfamidase for the Sanfilippo A syndrome (2-4), iduronate-SO4 sulfatase for the Hunter syndrome (5-7), N-acetylgalactosamine-4-SO4 sulfatase or arylsulfatase B for the Maroteaux-Lamy syndrome (8)(9)(10)(11)(12)(13), and N-acetylhexosamine-6-SOd sulfatase for the Morquio syndrome (14). More recently, evidence has been provided to indicate the existence of two different N-acetylhexosamine-6-SOd sulfatases (15), an N-acetylgalactosamine-6-SO4 (or galactose-6-S04) sulfatase whose deliciency causes inability to degrade chondroitin g-sulfate and keratan sulfate (as is the case of Morquio syndrome) and an N-acetylglucosamine-6-SOd sulfatase relating to a novel mucopolysaccharidosis characterized by inadequate catabolism of keratan sulfate and heparan sulfate. It has also been dem-* This work was supported by grants-in-aid for Cancer Research and for Scientific Research from the Ministry of Education, Science and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC. Section 1734 solely to indicate this fact. on&rated that 6-SO4 hydrolysis by these enzymes occurs only on nonreducing residues (16). Much of the information regarding these sulfatases is obtained using cultured human skin fibroblasts as enzyme source, suggesting that many different types of sulfatase necessary for glycosaminoglycan metabolism reside in normal skin. However, as yet few of the sulfatases have been obtained in a purified state and much further work is required before the specificities of the sulfatases are understood and their separate identities made certain. We report here the results of our systematic study on the desulfation of chondroitin 4/6-sulfate, dermatan sulfate, heparan sulfate, heparin, and keratan sulfate by extracts of rat skin, and the separation and characterization of the enzymes responsible for these reactions.
Generous gifts of the following materials are acknowledged: phenyl cY-L-iduronide, Dr was prepared from a commercial heparin sample in a similar way.

Preparation
of Crude Enzymes from Rat Skin-The fractionation scheme is shown in Scheme 1. Fresh skins, obtained from 10 newborn rats, were placed in ice-cold 0.9% NaCl, freed from adhering tissues, and cut into small pieces (about 5 x 5 mm). All subsequent procedures were carried out at 0-4'C. Skin pieces, 10 g (wet weight), were homogenized in 50 ml of 0.02 M Tris-HCl, pH 7.2, containing 0.25 M sucrose ("Buffer A") with a Polytron (Kinematica CMBH, Luzern-Schweiz) and the homogenate was centrifuged at 800 x g for 15 min. The pellet (P2) was retreated with the homogenizer and the mixture was centrifuged at 800 x g. The two supernatants obtained by Polytron homogenization were combined and centrifuged at 10,000 x ,e for 15 min to yield supernatant "S," and pellet "PI." The pellet, P1(equivalent to about 36mg of Lowry protein), was washed with 20 ml of Buffer A and resusnended in 4 ml of fresh Buffer A. Following disruption by sonication for 3 min at 10,000 cycles in a sonic oscillate< the sample was centrifuged at 10,000 x g for 15 min and the super- Two kinds of control assays, in which either the substrate or the enzyme was omitted, were always performed. One unit of enzyme was defined as the quantity that catalyzed the release of 1 pmol of product/h. Glycosidase activities were assayed with p-nitrophenyl N-acetyl-/?-n-glucosaminide, p-nitrophenyl N-acetyl-P-n-galactosaminide, pnitrophenyl P-n-glucuronide, p-nitrophenyl B-n-galactoside, and phenyl a-L-iduronide as substrate. The incubation mixture contained 0.2 pmol of substrate, 2.5 pmol of sodium acetate/acetic acid buffer, pH 5.0, 7.5 pg of bovine serum albumin, and enzyme in a final volume of 50 ~1. After incubation for 1 h at 37"C, the reaction was stopped by addition of 0.5 ml of 0.   4, AGlcUA-GalNAc-4,6-bis-Sod; 5, GlcUA-GalNAc-6-SO,; 6u, GlcUA-GalNAc-4-SOI; 6b, AGlcUA-GalNAc-B-Sod; 6c, GalNAc-4,6-bis-S04; 7u, AGlcUA-GalNAc-4-Sod; 76, SO,"-; 8, GalNAc-6-SOI; 9, GalNAc-4-S04.
To separate 6u, 66, and 6c from one another, the corresponding zone is cut out and eluted with water. Aliquots of the eluate are examined by paper chromatography in Solvent B (24 h) and by paper electrophoresis (60 min), as indicated. Similarly, 7u and 7b are eluted from the strip of the initial chromatography and separated by paper electrophoresis. The zone corresponding to each degradation product is cut out and counted for radioactivity in a scintillation spectrometer.
by guest on March 24, 2020 http://www.jbc.org/ Downloaded from (in the digestion of dermatan sulfates) at 37°C for 2 h. The digest was chromatographed on paper in Solvent A. By this procedure, some compounds were not separated from one another, as indicated in Scheme 2. To separate these, ""S-labeled materials in the corresponding zones were eluted with water and subjected to paper chromatography in Solvent B or to paper electrophoresis, as indicated in Scheme 2. Radioactive materials on paper strips were localized with a strip scanner. The radioactive zones were cut out, placed in scintillation vials, and counted.

Chromatogruphic and Electrophoretic
Techniques-Ion exchange chromatography of enzymes was done at 4°C on a small column (1 x 5.5 cm) of DEAE-cellulose, equilibrated with 0.02 M Tris-HCI, pH 7.2. Samples (4 ml, 15 to 20 mg as protein) were applied to the column. After washed with 16 ml of the same buffer, the column was developed by linear gradient elution with 30 ml of 0.02 M Tris-HCl, pH 7.2, in the mixing flask and 30 ml of 0.4 M NaCl in the same buffer in the reservoir. Effluent fractions of 1 ml were collected at a rate of 10 ml/ h and analyzed for protein and enzyme activity.
Gel chromatography of enzymes was done at 4°C on a column (1.2 x 85 cm) of Sephadex G-200, equilibrated with 0.2 M NaCl in 0.02 M Tris-HCl, pH 7.2. Samples (1.0 ml, 0.3 to 1.6 mg as protein) were applied to the column, and were then eluted with the same salt solution. Effluent fractions of 1 ml were collected at a rate of 7 ml/h and analyzed for protein and enzyme activity.
Paper electrophoresis was carried out on 60-cm strips of Toyo No. 51A paper in the apparatus described by Markham and Smith (32) at a potential gradient of 30 V/cm for the periods indicated in individual experiments. The buffer used was 0.05 M ammonium acetate/acetic acid, pH 5.0.
Paper chromatography was carried on 60-cm strips of Toyo No. 51A paper at room temperature by the descending method. The solvent systems used were: Solvent A, butyric acid/O.5 M ammonia (5:3, by volume) and Solvent B, l-butanol/acetic acid/water (10:3:5, by volume).

Other
Methods-Hexuronate was determined by the orcinol method (33), and protein by the method of Lowry et al. (34).

RESULTS
In initial experiments, the 10,000 X g supernatant (S1) and pellet (PI) fractions from rat skin, prepared as shown in Scheme 1, were assayed at pH 5. by Si. Since the data indicated that P1 is a main locus of both of the sulfatase activities, this fraction has been used as a first source of enzyme.
Solubilization and Fractionation of Sulfatases from 10,000 x g Pellet (P&When the 10,000 X g pellet (PI) was treated in a sonic oscillator and the supernatant of the sonicate (PJ was assayed using chondroitin 4/6-[35S]sulfate as substrate, it was shown that essentially ah of the sulfatase activity could be solubilized by sonication for 3 min.
In separate experiments, P1 was exposed to 2% Tween 20 or 0.1% Triton X-100 for 30 min at O"C, after which it was sedimented at 10,000 x g for 10 min. When the resulting supernatant solutions were assayed as above, more than 95% of the sulfatase activity in P1 could be found in the Tween 20 extract whereas little activity was detected in the Triton X-100 extract.
DEAE  substrates, the activity may be due to the occurrence of an Nacetylglucosamine 6-SO4 sulfatase. With keratan [35S]sulfate, a second peak appeared just after and overlapping the first peak (Fig. le) (Fig. lc). The specificity can be accounted for by the occurrence of a sulfatase specific for the 6-SO4 linked to sugars with the galactose configuration.
Peak III (Fractions 29 to 37) differed from Peak II in that it had little activity toward keratan [""Slsulfate.
Thus, the enzyme in this peak must be an N-acetylgalactosamine-4-SOb sulfatase.
Peak Also shown in Fig. 1, g and  as substrates. With the 4-sulfated heptasaccharide only one peak was observed corresponding to Peak III (Fig. lg), whereas with the 4/6-sulfated heptasaccharide an additional peak was obtained corresponding to Peak II (Fig. lh). The results are consistent with the suggestions that Peak II and III represent a sulfatase specific for the 6-SO4 linked to sugars with the galactose configuration and an N-acetylgalactosamine-4-SO1 sulfatase, respectively.
When the fractionation by DEAE-cellulose was monitored with a keratan sulfate tetrasaccharide trisulfate, GlcNAc-6-[35S]S04-Gal-6-[35S]SO~-GlcNA~-6-[35S]SO~-Gal, only one peak was observed corresponding to Peak I (Fig. li). In this assay system, incubation was carried out for 16 h, since the velocity obtained with the tetrasaccharide was much lower than that obtained with keratan [35S]sulfate. Regardless of this difference in assay condition, it is apparent from the results in Fig. 1, e and  but also indicate that desulfation by this enzyme occurs at the nonreducing terminal. Fig. lj indicates that the skin extract contains arylsulfatases which can be resolved into two fractions. It is noteworthy that one of the activities is closely associated with the Peak III activity for N-acetylgalactosamine-4-SOd linkages. For further purification, several runs of DEAE-cellulose chromatography of P, samples were carried out, and peak fractions were separately pooled, concentrated, and subjected to gel filtration on a Sephadex G-200 column that had been calibrated using chymotrypsinogen (Mr = 25,000), hen's egg albumin (MI = 45,000), bovine serum albumin (Mr = 67,000), and aldolase (Mr = 158,000) (Fig. 2). Significant differences in the elution patterns of sulfatases were noted with Peaks I, II, III, IV, and V, as monitored with each most specific substrate. The apparent molecular weights estimated from these data (see Ref. 35 for the molecular weight determination method) were 70,000 for Peak I (N-acetylglucosamine-6-SOd sulfatase), 110,000 for Peak II (N-acetylgalactosamine or galactose-6-SO4 sulfatase), 65,000 for Peak III (N-acetylgalactosamine-4-SOd sulfatase), 130,000 for Peak IV (N-SOS-sulfamidase), and 80,000 for Peak V (iduronate-SO4 sulfatase). Also to be noted is the fact that the peak of the activity toward N-acetylgalactosamine-4-SO, was coincident with a single peak of arylsulfatase (Fig. 2~). The result is consistent with the view that these reactions are catalyzed by the same enzyme (11). Table I shows the degree of purification and the recovery of enzymatic activities achieved. When the most purified enzyme preparations were examined for glycosidase activities toward p?nitrophenyl P-n-glucuronide, p-nitrophenyl P-n-N-acetylglucosaminide, phenyl a+iduronide, and p-nitrophenyl B-Dgalactoside, none of the enzyme preparations catalyzed the release of phenolate ion from these glycosides.
Comparison of Some Properties of Five Sulfatases from Rat Skin- Fig.  3 shows the effect of pH on the rates of [35S]S042-release from 35S-labeled substrates by the most purified enzyme preparations.
It is noteworthy that the pH optimum for N-acetylglucosamine-6-SOh sulfatase activity on the keratan sulfate tetrasaccharide is approximately pH 3.7, while that on heparan sulfate is about 5.0 (Fig. 3a). The difference in pH optima for the two substrates might possibly be due to the maintenance of the substrates in the proper  Table II shows the sensitivity of each enzyme to various inorganic salts which added to the standard incubation medium at optimal pH for each enzyme. The five sulfatases can be distinguished from one another by their sensitivities toward the inorganic ions: e.g. N-acetylgalactosamine-6-SOd sulfatase, N-SO3 sulfamidase, and iduronate-SO4 sulfatase were not affected by 10 mM NaCl or KC1 whereas the other sulfatases were inhibited to 50 to 60% by this concentration of NaCl or KCl; a stimulation of N-SO3 sulfamidase was seen with 10 mM MnC12 or MgC12, although moderate inhibition was noted on the activities of N-acetylglucosamine-6-SOa sulfatase and Nacetylgalactosamine-4-SOd sulfatase; Na2S04 was a potent inhibitor of N-acetylglucosamine-6-SOd sulfatase and N-acetylgalactosamine-4-SOd sulfatase at 0.5 to 2 mM, although it had much less effects on the other three enzymes.
The substrates and pH used for the assay were as in Table I. ml, in 0.05 mM acetate buffer at optimal pH for each enzyme, was preincubated at 50°C for 15 min, it was observed that Nacetylglucosamine-6-SOd sulfatase, N-acetylgalactosamine-6-SO1 sulfatase, and N-acetylgalactosamine-4-SOd sulfatase had lost approximately 30% of their initial activity while the other two enzymes had lost only 5 to 10% of the activity. When stored frozen (with occasional thawing and refreezing), Nacetylgalactosamine-6-SOd sulfatase was found to be relatively more unstable than the other enzymes; i.e. about 80% of its initial activity was lost in 2 months while the other four enzymes retained more than 70% of the activities. Under the standard conditions for enzyme assay (3 h incubation), there was a linear relationship between the amount of each sulfatase added and the amount of [35S]S042-released up to an enzyme amount equivalent to   was digested with the most purified Peak V preparation, the compound was quantitatively converted to two radioactive products which behaved as inorganic sulfate and IdUA-Anhydroman(SOJ, respectively, on paper electrophoresis and on paper chromatography in Solvent A (Fig. 4). The results are consistent with the findings by Bach et al. (5) and Lim et al. (7) of the Hunter corrective factor that has been shown to act as an iduronate-SO4 sulfatase on "abnormal" glycosaminoglycans synthesized by Hunter fibroblasts. The fact that the rat skin enzyme failed to react with the glycosaminoglycan samples prepared from normal rat skin is probably due to the absence of iduronate [35S]S04 residues at the nonreducing termini of these "normal" glycosaminoglycans (cf. Refs. 5 and 31).
One of the characteristic features of the Peak V sulfatase (iduronate-SO1 sulfatase) is its difficulty in releasing from the particulate fraction of rat skin. This was noted when the relative sulfatase activities recovered in Fractions S1, PI, and P, were compared (Table I). Thus, after treatment with Polytron, the activity of iduronate-SO4 sulfatase was found in P, (pellet) and S, (supernatant) in an approximate ratio of 1:l.Z while the other sulfatase activities were in ratios of 1:2.3 to 1: 11.5. Moreover, only a part of the iduronate-SO4 sulfatase activity in PI could be solubilized by a 3-min sonication while essentially all of the other activities were solubilized.
The following compounds were tested at two to four concentrations in the ranges indicated in parentheses and found to be inactive: p-nitrophenyl sulfate ( to only 1.6% of the added radioactivity (see above), suggesting that only the 4-sulfated end groups may be attacked by this enzyme. Compatible with this suggestion was the observation that, upon preincubation of GalNAc-4-["'S]-S04-(GlcUA-GalNAc-4-[""ElISO& with the sulfatase, the percentage of N-acetylgalactosamine-4-E"5S]S04 released by chondroitinase-AC digestion was reduced from 20.5 to 3.5%, with a corresponding increase of [35S]S042-from 0 to 17%. Further evidence for the specificity of sulfate hydrolysis was obtained by examination of GalNAc-4/6-[""S]S04-(GlcUA-G~~NAc-~/~-[~~S]SO& before and after treatment with the sulfatase; following incubation of 1 x lo4 cpm of the substrate with 0.013 unit of the enzyme for 3 h, there was a reduction of N-acetylgalactosamine-4-35S (from 19 to 15%) and a corresponding appearance of ["5S]S042-(from 0 to 4%) with no detectable change in the amount of N-acetylgalactosamine-6-[35S]S04 and unsaturated disaccharides. The V,,, and K,,, calculated from the rates of S04'-release with varying concentrations of tri-, penta-, hepta-, and nonasaccharide 4-SO4 (Table III) showed that, if the calculation is based on the content of nonreducing N-acetylgalactosamine-4-SO4 end group, the V,,, values increase with increasing chain length while the K,,, values do not differ greatly.
The most purified enzyme preparation was tested for its ability to release ["5S]S042-from various sulfate esters (concentration range, 1 -4. Among the sulfate esters, only the first three compounds gave positive results. As shown in Figs. 1 and 2, there was a close parallelism between the activity for chondroitin-4-SO4 nonasaccharide and that for p-nitrophenyl sulfate. Moreover, the purified enzyme was shown to hydrolyze 4-nitrocatechol sulfate at a rate (150 pmol/h/mg of protein) higher than that for p-nitrophenyl sulfate (6 pmol/h/mg of protein). The results are consistent with the notion that arylsulfatase B and the Nacetylgalactosamine-4-SOd sulfatase are one and the same (8, 10, 11). Comparison of N-Acetylgalactosamine-l-SO4 and -6-SO4 Sulfatases Localized in Soluble and Particulate Fractions-As described above, considerable proportions of the sulfatases in rat skin were released into the soluble fraction (S1) by the The incubation conditions for each substrate were those for enzyme assay described under "Methods," except that varying amounts of the indicated substrate were used in each case. The radioactive substrates, 2 X lo7 cpm/pol, were added to the incubation mixtures containing 0.1 units (determined with heptasaccharide as substrate) of enzyme. The assays measured the radioactivities released as S04'-. Values for V,,, are expressed relative to the maximal rate of trisaccharide hydrolysis, which has been assigned a value of unity. The results suggest that some of the readily solubilized sulfatases might differ from their particulate counterparts. To test this possibility, the soluble fraction (S,) obtained by the initial Polytron treatment was chromatographed on a DEAE-cellulose column under the same conditions used for P, (CL Fig. 1). Fig. 5 illustrates the elution patterns of sulfatase activities towardsGalNAc-4/6-[35S]S04-(GlcUA-GalNAc-4/6-["%]S0J3 and GalNAc-4-E"5S]S04-(GlcUA-GalNAc-4-[""S]-SOJ3 together with those of arylsulfatase activities and proteins. As can be seen, the sulfatase patterns of S, and P, were essentially identical, although their protein patterns were quite different from each other.