The Defect in the Hunter Syndrome

Proc. Nat. Acad. Sci. USA Vol. 70, No. 7, pp. 2134-2138, July 1973

The Defect in the Hunter Syndrome: Deficiency of Sulfoiduronate Sulfatase (skin fibroblasts/mucopolysaccharide degradation/mucopolysaccharidosis) GIDEON BACH*, FRANK EISENBERG, JR.*, MICHAEL CANTZt, AND ELIZABETH F. NEUFELD*j

* National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014; and t Department of Pediatrics, University of Kiel, Kiel, Federal Republic of Germany Communicated by C. B. Anfinsen, May 1, 1973

ABSTRACT Skin fibroblasts cultured from patients af- several mucopolysaccharides (12-15). A preliminary report of fected with the Hunter syndrome are deficient in the activity of a protein, named the "Hunter corrective factor," this work has been presented (16). that is required for degradation of dermatan and heparan sulfates. We now show that this factor, purified from MATERIALS AND METHODS human urine, removes about 2% of the sulfate residues from [3aS]mucopolysaccharide accumulated within Hunter Reagents. H285SO4, - [6-3H]glucosamine, and D-[1-'H ]- fibroblasts; these groups are derived from "oversulfated" galactose were purchased from New England Nuclear Corp.; regions of the polymer. Acetone-powder extracts of fibro- Sephadex G-200 and DEAE-Sephadex A-50, from Phar- blasts derived from patients with the Hunter syndrome are macia; Biogel P-2 from Bio-Rad; chondroitinase ABC, and deficient in this sulfatase, in contrast to similar extracts from fibroblasts of individuals of other genotype. Hunter the reference unsaturated disaccharides A Di4S, ADi-6S, and corrective factor coupled to a-L-iduronidase (or alterna- A Di-OS from Miles Laboratories; chondroitin 4-sulfate from tively, mixed extracts from Hurler and Hunter fibroblasts) Seikagaku Kogyo Co. The unsaturated disulfated disac- release iduronic acid from 4-0-a-L-sulfoiduronosyl-D- charides, A DidiSD and A DidiSE, were gifts from Dr. Sakaru sulfoanhydromannose. We conclude that the Hunter Suzuki, corrective factor is a sulfatase for sulfated iduronic acid and 4-O-a--sulfoiduronosyl-D-sulfoanhydromannose, residues. from Dr. Ulf Lindahl. The Hunter syndrome is a genetic disorder associated with Cell Culture. Fibroblasts derived from skin of normal or failure to degrade dermatan sulfate and heparan sulfate; affected individuals were maintained as described (17); ace- lysosomal storage of these polymers leads to numerous clinical tone powders of the cells were made by the procedure of Hall problems, including skeletal abnormalities, limitation of etal. (11). joint motion, hepatosplenomegaly, deafness, and cardiovas- Corrective Factors. Pools 1-5 of the Hunter corrective factor cular disease (1, 2). Of the known mucopolysaccharidoses, the preparation of Cantz et al. (5) were used in the present experi- Hunter syndrome is the only one transmitted as an X-linked ments. Their corrective activity, assayed as described (5, 17), recessive trait. had diminished by no more than one half after storage at Fibroblasts cultured from the skin of Hunter patients do not -15° for 18 months. The a-r-iduronidase (Hurler corrective adequately degrade sulfated mucopolysaccharide because of factor) was a fraction eluted from hydroxylapatite (18). a deficiency of a specific protein that is present in cell secre- tions, cells, and urine of individuals who do not have the Preparation of Radioactive Mucopolysaccharide. Very heavy Hunter syndrome (3-5). Because this protein, when added cultures of Hunter fibrobists (cells maintained for 1 month exogenously to Hunter cells, accelerates the degradation of after transplantation, to a density of 5 mg of protein per 75- sulfated mucopolysaccharide, it has been named the "Hunter cm2 Falcon flask) were labeled for 6 days with E5SOt (20 ml corrective factor," and abbreviated simply as "Hunter per flask of medium containing 3 mCi, 9 jmol of inorganic factor." Purified Hunter factor has no effect on the muco- sulfate). The cells were then harvested by trypsinization, polysaccharide metabolism of cells derived from normal in- washed twice with 0.9% NaCl, suspended in 1.0 ml of 0.9% dividuals or from patients with mucopolysaccharide storage NaCl, and disrupted by 10 cycles of freezing and thawing. disorders other than the Hunter syndrome (5). The insoluble debris was removed by centrifugation at Several analogous corrective factors have recently been 10,000 X g for 20 min. The supernatant fluid, which con- identified as the "missing enzyme" in the corresponding dis- tained 90% of the cell-associated radioactivity, was dialyzed order. Thus, the Hurler corrective factor has been identified against 2 liters of 0.05 M (NH4)2S04, followed by four changes, as the enzyme a-iiduronidase, and the Hurler and Scheie 2 liters each, of water. This dialyzed solution was used as syndromes as a-riduronidase deficiency diseases (6, 7); the "substrate" for the Hunter factor without further purifica- Sanfilippo A corrective factor has been identified as heparan tion, unless otherwise indicated. sulfate sulfatase (probably an N-sulfatase) (8), and the San- Incorporation of [8H]glucosamine or [8H]galactose, which filippo B factor as N-acetyl-a-glucosaminidase (9, 10). In a label the hexosamine or uronic-acid residues of mucopoly- mucopolysaccharidosis due to #3-glucuronidase (EC 3.2.1.31) saccharide, respectively, was performed in fibroblasts grown deficiency, jB-glucuronidase serves as corrective factor (11). to confluence but not later than 2 weeks after transplanta- We have now identified the Hunter corrective factor as a tion. The usual medium was modified to contain only 2 mg sulfatase for sulfated iduronic acid residues, which occur in of glucose and 1 mCi of either radioactive precursor per 10 ml. After 3 days, cells were harvested and mucopolysaccharide $ Address reprint requests to this author. was extracted as above. 2134 Downloaded by guest on September 25, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) Defect in the Hunter Syndrome 2135

~-2000- -800 - 200- '500- 600- 1000- -400 - 100 500.~ ~ ~ ~ -200- 30 40 50 60 70 80 30 35 50 60 70 80 90 100 FRACTION NO. 90100 10 152025 FIG. 1. Low molecular weight3040fragment released by the Hunter corrective factor from [*Slmucopolysaccharide of Hunter fibroblasts. Dermatan sulfate fraction (Panel A) or heparan sulfate fraction (Panel B) was incubated for 72 hr, either with 150 units of Hunter factor in 0.5 ml of 0.9% NaCI-0.01 M sodium phosphate, pH 6.0 (0) or without factor but with an equal amount of buffered saline (0) in a final volume of 0.65 ml. The reaction mixture also contained 0.1 M sodium acetate (pH 4.4) and 5 mM NaN3. Radioactive products of the incubation were separated on a column (1.5 X 80 cm) of Sephadex G-200, eluted in fractions of 2 ml with 0.9% NaCl. The small peak of totally retarded material was collected, concentrated by lyophilization, and applied to a column of BioGel P-2 (1 X 55 cm) in 1 M NaCl (Panel C); fractions of 1 ml were collected. Identical elution patterns on BioGel P-2 were obtained for the retarded material released from the dermatan sulfate and from the heparan sulfate fractions, although only the former is plotted here.

Labeled mucopolysaccharides were separated on Sephadex idonolactone butaneboronate by the procedure of Eisenberg G-200. A major peak, appearing immediately after the void [(20) and manuscript in preparation]. A tube containing a volume, contained 90% dermatan sulfate, as previously known amount of barium iduronate was prepared as standard, documented (5), whereas a minor, greatly retarded peak con- and to this tube as well as to each unknown sample a constant tained 75% heparan sulfate and 25% dermatan sulfate. amount of mannitol was added as internal standard. The Further purification was achieved by adsorption to DEAE- samples were reduced with sodium borohydride, freed of Sephadex A-50 in 5 mM phosphate buffer (pH 7.0) and elution boric acid, lactonized, and derivatized. They were then by discontinuous increments of NaCl in the same buffer. analyzed with a Beckman GC65 gas chromatograph on a The heparan sulfate was eluted with 0.6 M NaCl, and the column of OV17 on GasChrom Q. From the ratio of peak dermatan sulfate with 0.8-1.0 M NaCl. areas of idonolactone butaneboronate to mannitol butaneboronate, the amount of iduronic acid in each unknown sample Radioactivity. Except for corrective factor assays, for which was calculated. a scintillation fluid has been stipulated (17), samples were counted in a mixture of Liquifluor (New England Nuclear RESULTS Corp.), ]3io-Solv BBS-3 (Beckman Instrument Co.), and Sulfatase activity of Hunter corrective factor toluene, 1:2.7:24 (v/v). Incubation of the Hunter corrective factor with [15S]muco- Digestion with Chondroitinase ABC. [15S]Mucopolysac- polysaccharide isolated from Hunter fibroblasts resulted in charide was treated with chondroitinase ABC by the procedure the release of a small amount of material of low molecular of Saito et al. (19); presumably, only the dermatan sulfate weight (Fig. 1). About 1.5% of the radioactivity was released component was digested. Mucopolysaccharide (about 100,000 from the fraction that contained primarily dermatan sulfate cpm) was mixed with 10 ,l of "enriched Tris buffer" (19) and 4.5% from the fraction that contained primarily heparan and 0.05 units of chondroitinase ABC in a total volume of 0.05 sulfate. In both cases, the radioactive product was shown to ml. After 45 min at 370, another 0.05 unit of enzyme in 5 ,dI correspond to inorganic sulfate by chromatography on Biogel was added, and the incubation was continued for 45 min P-2. longer; the reaction was stopped by immersing the tubes in a Similar experiments were done with analogous dermatan boiling-water bath for 2 min. An aliquot was applied to sulfate and heparan sulfate fractions that had been labeled Whatman 1 and subjected to descending chromatography in with 8H in the hexosamine or uronic acid moieties. In contrast n-butanol-acetic acid-0.1 N NH40H (2:3: 1, v/v) for 22 hr. to results obtained with [5JS]mucopolysaccharide, there was The paper was cut into 3-mm strips, each of which was no factor-dependent release of low molecular weight material counted in 0. 5 ml H20 and 9.5 ml of scintillation fluid. from the tritium-labeled polymers§. A routine assay for the sulfatase activity of the Hunter cor- Paper Electrophoresis. To identify inorganic sulfate, elec- rective factor was developed, taking advantage of the solu- trophoresis was performed in 0.2 M ammonium acetate bility of sulfate and insolubility of residual mucopolysaccha- buffer (pH 5.0) at 25 V/cm. Authentic ;5SO04 was applied ride in 80% ethanol. Unfractionated [a5S]mucopolysaccharide in the same salt mixture as the unknown sample, to correct for the interference of chloride with the migration of sulfate ions. § In the experiments with tritium-labeled substrates, a small amount of radioactive material was found in the fractions fully Gas-Liquid Chromatography of Iduronic Acid. Iduronic retarded on Sephadex G-200; this amount was identical in incu- acid was measured by gas-liquid chromatography as 1,4- bations with and without Hunter factor. Downloaded by guest on September 25, 2021 2136 Medical Sciences: Bach et al. Proc. Nat. Acad. Sci. USA 70 (1973) TABLE 2. Absence of inhibitor in extracts from 2400 2.0 Hunter fibroblasts

0 z 20001 m 4 -q Ethanol-soluble 4C 0 z released 0

0. 16001 0 Sample (cpm per sample) -4 U) / I I 1. Extract, normal fibroblasts 2800 0 12001 < z 1.0 2. Extract, Hunter fibroblasts 140 4 z- 3. Mixture, 1 + 2 2200 C 4. Hunter factor Soo0 w 2600 2 -4 5. Mixture, 2 + 4 2700

-4 u- 400 r" Sulfatase activity was assayed as described in Methods, with 2 X 105 cpm of [IS] mucopolysaccharide, and amounts of Hunter 4 8 12 16 20 24 28 factor or normal acetone-powder extract selected tobe in the linear HUNTER. CORRECTIVE FACTOR, UNITS range. Extract from Hunter fibroblasts contained 0.1 mg of protein; higher amounts gave an apparent FIG. 2. Ethanol-soluble material released from [3S]muco- inhibition, perhaps be- polysaccharide of Hunter fibroblasts by increasing amounts of cause of dilution of the radioactive substrate with endogenous mucopolysacchoride. Hunter corrective factor. The assay is as described in Methods, except that the incubation volume was increased to 0.29 ml. The ethanol-soluble reaction product behaves as inorganic extracted from Hunter fibroblasts (about 100,000 cpm) is sulfate by chromatography on Biogel P-2 and by paper incubated with Hunter corrective factor, usually 5-20 units, in electrophoresis at pH 5.0 (Rpicrate = 3.0). 0.12 M sodium acetate (pH 4.0) and 5 mM NaN3. The in- Up to a point, there is a linear relationship between the cubation mixture, 0.07-0.11 ml, contains, in addition, NaCl amount of Hunter factor added and the amount of '5SO4= re- (<0.07 M) and sodium phosphate (pH 6.0) (<5 mM) that are leased (Fig. 2). Maximal release corresponds to about 2% of introduced with the Hunter factor. After 20 hr at 370, 0.1 ml the added radioactivity (the amount varies somewhat be- of chondroitin 4-sulfate (10 mg/ml in 1 M NaHCO3) and 4 tween different preparations of [15S ]mucopolysaccharide). volumes of absolute ethanol are added. The mixture is This means that only 2% of the sulfate residues of the muco- thoroughly agitated on a Vortex mixer, chilled in ice for 30 polysaccharide substrate are susceptible to the action of the mim; and centrifuged at 10,000 X g for 20 min at 20. The Hunter corrective factor. supernatant solution is collected and again centrifuged. An No sulfate was released at all when the [B5S]mucopoly- aliquot of the supernatant solution and of the precipitate saccharide derived from Hunter fibroblasts was replaced by (redissolved in water) are counted. A control incubation an analogous preparation derived from Hurler fibroblasts. without Hunter factor is carried through the same steps, and all values are corrected for this blank (usually about 300 cpm). TABLE 3. Decrease of "oversulfated" areas of [33S]muco- The assay is based on preliminary experiments that showed an polysaccharide upon incubation with Hunter factor optimal pH at 4.0, with half maximal activity at pH 3.5 and 4.7. Citrate-phosphate buffer is inhibitory. The rate of release Addition of Hunter factor of ethanol-soluble is for hr diminishes material linear 10 but Units of markedly thereafter. corrective Fraction* activity ADidiS (%)t TABLE 1. Sulfate releasedfrom [35S]mucopolysaccha ide None (3 samples) 7.6 by acetone-powder extracts offibroblasts Pool 1 60 5.1 Pool 2 50 5.3 Num- Ethanol-soluble released Pool 3 46 5.4 ber of (cpm/mg of protein) Pool 4 16 6.1 cell Pool 5 10 6.0 Genotype lines Mean Range Hunter 6 1,400 200-3,000 * The fractions "pools 1-5" are described in Figs. 2 and 3 of Hurler 3 25,000 15,000-31,000 ref. 5. Normal 2 15,000 t The % of ADidiS is calculated on the basis of moles of disul- Maroteaux-Lamy 1 33,000 fated disaccharide obtained relative to total disaccharide released Sanfilippo A 1 20,000 by chondroitinase ABC. Because the specific activity of disulfated Bl-Glucuronidase disaccharide is twice that of monosulfated, the percentage is cal- deficiency 1 24,000 culated as follows: I-cell 1 6,000 0.5 (cpm, ADidiS) X 100 % _ 0.5 (cpm, ADidiS) + (cpm, ADi4S) + (cpm, ADi-6S) Sulfatase activity of acetone-powder extracts was assayed as described in Methods, with 105 cpm of [33S]mucopolysaccharide Less than half of the mucopolysaccharide was converted to un- substrate, about 0.1 mg of protein for extracts of Hunter fibro- saturated disaccharides by chondroitinase ABC. The proportion blasts, and about 0.05 mg of protein for extracts of fibroblasts of degraded was the same in samples that had been incubated with other genotype. the Hunter factor and controls. Downloaded by guest on September 25, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) Defect in the Hunter Syndrome 2137

TABLE 4. Iduronic acid released from 4-O-c-L-sulfo- TABLE 5. Iduronic acid released from 4-0-a-L-sulfoiduronosyl-D-sulfoanhydromannose by the combined iduronosyl-D-sulfoanrhydromannose by acetone-powder action of Hunter factor and a-iiduronidase extracts offibroblasts

Iduronate Genotype Iduronate released (jhg) Sample no. Hunter factor a-L-Iduronidase released (ug) Normal 2.4 1 - - 0.4 Hunter 0.6 2 - + 0.9 Hurler 0.09 3 + _ 0.6 Hunter + Hurler, mixed 2.9 4 + + 9 5 + + 16 Incubation mixtures contained 0.2 jumol of disaccharide; acetone- powder extract in 0.9% NaCl containing about 0.1 mg of protein; The complete incubation mixture, no. 4, contained 0.2 jumol of a final concentration of 0.12 M sodium acetate buffer (pH 4.0) disaccharide; Hunter factor, 60 corrective units, in 0.25 ml of and 5 mM NaNs, in a total volume of 70 ul. After 24 hr at 370, 0.9% NaCl-0.01 M Na phosphate (pH 6.0) enriched with 0.25 the iduronate released was measured by gas-liquid chromatog- mg of albumin; a final concentration of 0.12 M sodium acetate raphy, as described in Methods. Higher amounts of acetone-powder buffer (pH 4.0) and 5 mM NaN3, in a total volume of 0.5 ml. extracts resulted in apparent inhibition in the mixed sample, After 24 hr at 370, a-Liduronidase was added [sufficient to hy- perhaps because of its high content of endogenous mucopoly- drolyze 0.3 jumol of phenyliduronide per 17 hr under standardized saccharide. conditions (6)] in phosphate-NaCl-albumin buffer as above. The buffer was adjusted to remain as 0.12 M sodium acetate (pH 4.0) and 5 mM NaN3. After another 24 hr at 370, iduronate disulfated5. After treatment of the [a5S]mucopolysaccharide released was measured by gas-liquid chromatography as de- with Hunter factor, the proportion of disulfated disaccharides scribed in Methods. Samples 1-3, from which one or both enzymes produced by chondroitinase ABC was reduced to a limiting were omitted, were incubated with the equivalent amount of value of about 5% (Table 3). Apparently, one-third of the albumin in buffered saline. Sample 5 differed in that both Hunter "oversulfated" areas of mucopolysaccharide isolated from factor and a-Liduronidase were incubated together for 48 hr. Hunter fibroblasts was removed by the Hunter factor. By contrast, mucopolysaccharide isolated from Hurler fibroblasts and similarly treated with chondroitinase ABC, showed Sulfatase activity of acetone powders of fibroblasts the presence of about 4% oversulfated areas, none of which Acetone-powder extracts prepared from Hunter fibroblasts was removed by the Hunter factor (4.1 or 4.5%, for control are profoundly deficient in the sulfatase associated with sample or sample treated with Hunter factor, respectively). Hunter corrective factor, when compared to similar extracts These results are consistent with the hypothesis that the of fibroblasts from normal individuals or from patients with Hunter corrective factor is a sulfatase for oversulfated areas other mucopolysaccharidoses (Table 1). The deficit is not due of the mucopolysaccharide, provided these are suitably ex- to the presence of inhibitors in extracts of Hunter fibroblasts, posed. Since the sulfate residue that is unique to such areas as shown by mixing experiments (Table 2). The Hunter is that linked to L-iduronic acid, the results further suggest genotype, shown to be associated with deficiency of Hunter that the Hunter factor is a sulfatase for sulfated iduronic factor, is now correlated with a deficiency of sulfatase ac- acid. A direct demonstration thereof was obtained by using tivity. for substrate the disaccharide 4-0-a-L-sulfoiduronosyl-D- An interesting exception to that correlation is the marked sulfoanhydromannose, and measuring iduronic acid re- deficiency of sulfatase in extracts of fibroblasts from I-cell leased by the combined action of Hunter factor and a-L- disease patients (Table 1). These cells have markedly re- iduronidase. Whereas either enzyme alone released only traces duced activity of several lysosomal enzymes (e.g., ref. 21) of iduronic acid, as determined by gas-liquid chromatog- and are unable to correct the defect of Hunter fibroblasts (4). raphy, the two enzymes together released as much as 40% Homogenates prepared by freeze-thawing of fibroblasts, of the available iduronic acid in the best experiment pre- were found unsatisfactory for studying the sulfatase because sented in Table 4. of the presence of apparent inhibitors, particularly in homog- Release of iduronic acid from 4-0-ca-L-sulfoiduronosyl-D- enates of normal cells. The interfering substances are pre- sulfoanhydromannose was also catalyzed by acetone-powder sumably removed in the preparation of acetone powders. extracts of normal fibroblasts. Extracts of Hurler fibroblasts (which have Hunter factor but not a-i-iduronidase) or of

Identification of Hunter corrective factor as a I sulfoiduronate sulfatase The radioactive disulfated disaccharide released by chondroitinase ABC had a chromatographic position similar to that of Action of the Hunter factor on [3S]mucopolysaccharide re- ADidiSE [2-acetamido-2-deoxy-3-O-(j-D-gluco-4-enepyranosyl- sulted in the disappearance of some "oversulfated" areas- uronic acid)-4,6-di-O-sulfo-i)-galactose] and that of ADidiSD i.e., areas with two sulfate groups per disaccharide consisting [2-acetamido-2-deoxy-3-O-(2 or 3-0-sulfo-j#-D-gluco4-enepyran- of uronic acid and hexosamine. This was determined by di- osyluronic acid)-6-O-sulfo-D-galactose]. On prolonged chromatog- with raphy (40 hr) the radioactive disulfated disaccharide was partially gesting chrondroitinase ABC the substrate and the separated from both reference compounds. Its chromatographic product of Hunter factor activity. The bacterial enzyme de- mobility, intermediate between ADidiSD and ADidiSE, was con- graded a portion of the [E5S]mucopolysaccharide used as sistent with that of ADidiSB [2-acetamido-2-deoxy-3-O-(2 or substrate (presumably, the dermatan sulfate component 3-O-sulfo-3-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galac- only) into unsaturated disaccharides of which 7.5% were tose], which was unfortunately not available for comparison. Downloaded by guest on September 25, 2021 2138 Medical Sciences: Bach et al. Proc. Nat. Acad. Sci. USA 70 (1973)

Hunter fibroblasts (which have a--iduronidase but not ficiencies. a-IIduronidase deficiency is always associated Hunter factor) had only slight activity, whereas a mixture of with cloudy corneas, whereas corneas remain clear in the the two caused the release of as much iduronic acid as did the Hunter syndrome. One must presume that corneal muco- normal extract (Table 5). polysaccharides do not require Hunter factor for degradation, and, therefore, that they are devoid of sulfoiduronate residues. DISCUSSION We thank Dr. Ulf Lindahl (University of Uppsala, Sweden) and The results demonstrate that the Hunter corrective factor has Dr. Sakaru Suzuki (University of Nagoya, Japan) for generous sulfoiduronate sulfatase activity. It is unlikely that the sul- gifts of disulfated disaccharides. This work was supported in part fatase is a chance contaminant of the admittedly impure by a grant from the Deutsche Forschungsgemeinschaft. factor preparation, since it is specifically absent from extracts 1. McKusick, V. A. (1972) in Heritable Disorders of Connective of Hunter fibroblasts. Tissue (C. V. Mosby, St. Louis), pp. 521-687. We were surprised to find that acetone-powder extracts of 2. Dorfman, A. & Matalon, R. (1972) in The Metabolic Basis of normal fibroblasts did not release more 35SO4t from [n5S]- Inherited Disease, eds. Stanbury, J. B., Wyngaarden, J. B. & Fredrickson, D. S. (McGraw Hill, New York), pp. 1218- mucopolysaccharide than did the purified Hunter factor. 1272. These extracts contain several of the enzymes necessary for 3. Fratantoni, J. C., Hall, C. W. & Neufeld, E. F. (1969) Proc. mucopolysaccharide degradation, such as a-L-iduronidase and Nat. Acad. Sci. USA 64, 360-366. ,3-glucuronidase (6, 11), as well as the sulfoiduronate sulfatase. 4. Neufeld, E. F. & Cantz, M. J. (1971) Ann. N.Y. Acad. Sci. Presumably, one or more of the unknown degradative en- 179, 580-587. 5. Cantz, M., Chrambach, A., Bach, G. & Neufeld, E. F. zymes (e.g., hydrolases for the 0-sulfated N-acetylgalactosa- (1972) J. Biol. Chem. 247, 5456-5462. mine residues of dermatan sulfate) is inactivated during prep- 6. Bach, G., Friedman, R., Weissmann, B. & Neufeld, E. F. aration or assay of the acetone-powder extract. (1972) Proc. Nat. Acad. Sci. USA 69, 2048-2051. Hunter patients store and secrete both dermatan sulfate 7. Matalon, R. & Dorfman, A. (1972) Biochem. Biophys. Res. Commun. 47, 959-964. and heparan sulfate. Sulfoiduronate residues have been well 8. Kresse, H. & Neufeld, E. F. (1972) J. Biol. Chem. 247, 2164- characterized in mammalian dermatan sulfate and heparin 2170. (12-15). They have not been reported in heparan sulfate. 9. O'Brien, J. S. (1972) Proc. Nat. Acad. Sci. USA 69, 1720- That, however, is probably because heparan sulfate is poorly 1722. studied; it is generally assumed to be similar to heparin (22, 10. Von Figura, K. & Kresse, H. (1972) Biochem. Biophys. Res. Commun. 48, 262-269. 23), the difference being primarily in molecular size and shape 11. Hall, C. W., Cantz, M. & Neufeld, E. F. (1973) Arch. (larger and more branched molecule in heparan sulfate) and Biochem. Biophys. 155, 32-38. ratio of N-acetylated to N-sulfated glucosamine (higher in 12. Suzuki, S. (1960) J. Biol. Chem. 235, 3580-3588. heparan sulfate 11). 13. Suzuki, S., Saito, H., Yamagata, T., Anno, K., Seno, N., Kawai, Y. & Furuhashi, T. (1968) J. Biol. Chem. 243, 1543- Not all sulfated iduronic-acid residues are available to the 1550. sulfatase. We speculate that the unavailable ones are buried 14. Lindahl, U. & Axelsson, 0. (1971) J. Biol. Chem. 246, 74-82. within the molecule. This hypothesis is consistent with the 15. Malstrom, A. & Fransson, L. A. (1971) Eur. J. Biochem. 18, finding of a disulfated disaccharide at the nonreducing end 431-435. of dermatan sulfate isolated from Hunter fibroblasts but not 16. Bach, G., Cantz, M., Okada, S. & Neufeld, E. F. (1973) 0 Fed. Proc. 32, 483. from Hurler fibroblasts (Sj6berg, I., Fransson, L. A., Matalon, 17. Cantz, M., Kresse, H., Barton, R. W. & Neufeld, E. F. R. & Dorfman, A., manuscript in preparation). (1972) in Methods in Enzymology, ed. Ginsburg, V. (Aca- A disulfated disaccharide containing iduronic acid has demic Press, New York), Vol. 28, pp. 884-896. been found in the urine of a Hunter patient (27). Such a 18. Barton, R. W. & Neufeld, E. F. (1971) J. Biol. Chem. 246, of an 7773-7779. fragment could result in vivo from the action endogly- 19. Saito, H., Yamagata, T. & Suzuki, S. (1968) J. Biol. Chem. cosidase (e.g., hyaluronidase) on dermatan sulfate. 243, 1536-1542. The clinical consequences of sulfoiduronate sulfatase 20. Eisenberg, F., Jr. (1972) in Methods in Enzymology, ed. deficiency are, on the whole, similar to those of a-iiduroni- Ginsburg, V. (Academic Press, New York), Vol. 28, pp. dase deficiency. The severe and mild forms of the Hunter 168-178. 21. Hickman, S. & Neufeld, E. F. (1972) Biochem. Biophys. Res. syndrome have as their counterpart the Hurler and Scheie Commun. 49, 992-999. syndromes, respectively (1, 28). There is, however, one con- 22. Lindahl, U. (1970) in Chemistry and Biology of the Inter- sistent phenotypic difference between the two enzyme de- cellular Matrix, ed. Balasz, E. A. (Academic Press, London), Vol. 2, pp. 943-960. 23. Cifonelli, J. A. & King, J. (1972) Carbohyd. Res. 21, 173-186. 1I Because of the large proportion of sulfated iduronic-acid residues 24. Knecht, J., Cifonelli, J. A. & Dorfman, A. (1967) J. Biol. in heparin, it is puzzling that this polymer should not be stored Chem. 242, 4652-4661. 25. Stone, A. L., Constantopoulos, G., Sotsky, S. M. & Dekaban, and excreted in the Hunter syndrome. Although "heparin- A. (1970) Biochim. Biophys. Acta 222, 79-89. like" fractions have been reported among the mucopolysac- 26. Selye, S. (1965) in The Mast Cells (Butterworth's, Wash- charides of Hurler and Sanfilippo patients these are not considered ington), pp. 304-305. "true" heparin but rather degradation products of heparan sul- 27. Coppa, G. V., Singh, J., Nichols, B. L. & DiFerrante, N. fate (24, 25). Perhaps man, in contrast to other mammals, (1973) Anal. Let. 6, 225-233. does not synthesize heparin. Experimental data on the subject 28. Spranger, J. (1972) Ergeb. Inn. Med. Kinderheilk. 32, 165- are sparse (e.g., ref. 26). 265. Downloaded by guest on September 25, 2021