The Defect Hunter Syndrome: Deficiency Sulfoiduronate …2136 MedicalSciences: Bachet al. 2400 z 4 4C z 0. U) 0 z 4 2 u-20001 16001 12001 2.0 0 m-q 0 0 0-4 1.0 < z-C w-4 r"-4 / I I

Proc. Nat. Acad. Sci. USAVol. 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; andt 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-fected with the Hunter syndrome are deficient in the ac-tivity of a protein, named the "Hunter corrective factor,"that is required for degradation of dermatan and hep-aran sulfates. We now show that this factor, purified fromhuman urine, removes about 2% of the sulfate residuesfrom [3aS]mucopolysaccharide accumulated within Hunterfibroblasts; these groups are derived from "oversulfated"regions of the polymer. Acetone-powder extracts of fibro-blasts derived from patients with the Hunter syndrome aredeficient in this sulfatase, in contrast to similar extractsfrom fibroblasts of individuals of other genotype. Huntercorrective factor coupled to a-L-iduronidase (or alterna-tively, mixed extracts from Hurler and Hunter fibroblasts)release iduronic acid from 4-0-a-L-sulfoiduronosyl-D-sulfoanhydromannose. We conclude that the Huntercorrective factor is a sulfatase for sulfated iduronic acidresidues.

The Hunter syndrome is a genetic disorder associated withfailure to degrade dermatan sulfate and heparan sulfate;lysosomal storage of these polymers leads to numerous clinicalproblems, including skeletal abnormalities, limitation ofjoint motion, hepatosplenomegaly, deafness, and cardiovas-cular disease (1, 2). Of the known mucopolysaccharidoses, theHunter syndrome is the only one transmitted as an X-linkedrecessive trait.

Fibroblasts cultured from the skin of Hunter patients do notadequately degrade sulfated mucopolysaccharide because ofa deficiency of a specific protein that is present in cell secre-tions, cells, and urine of individuals who do not have theHunter syndrome (3-5). Because this protein, when addedexogenously to Hunter cells, accelerates the degradation ofsulfated mucopolysaccharide, it has been named the "Huntercorrective factor," and abbreviated simply as "Hunterfactor." Purified Hunter factor has no effect on the muco-polysaccharide metabolism of cells derived from normal in-dividuals or from patients with mucopolysaccharide storagedisorders other than the Hunter syndrome (5).

Several analogous corrective factors have recently beenidentified as the "missing enzyme" in the corresponding dis-order. Thus, the Hurler corrective factor has been identifiedas the enzyme a-iiduronidase, and the Hurler and Scheiesyndromes as a-riduronidase deficiency diseases (6, 7); theSanfilippo A corrective factor has been identified as heparansulfate sulfatase (probably an N-sulfatase) (8), and the San-filippo B factor as N-acetyl-a-glucosaminidase (9, 10). In amucopolysaccharidosis due to #3-glucuronidase (EC 3.2.1.31)deficiency, jB-glucuronidase serves as corrective factor (11).We have now identified the Hunter corrective factor as a

sulfatase for sulfated iduronic acid residues, which occur in

$ Address reprint requests to this author.

several mucopolysaccharides (12-15). A preliminary report ofthis work has been presented (16).

MATERIALS AND METHODS

Reagents. H285SO4, - [6-3H]glucosamine, and D-[1-'H ]-galactose were purchased from New England Nuclear Corp.;Sephadex G-200 and DEAE-Sephadex A-50, from Phar-macia; Biogel P-2 from Bio-Rad; chondroitinase ABC, andthe reference unsaturated disaccharides A Di4S, ADi-6S, andA Di-OS from Miles Laboratories; chondroitin 4-sulfate fromSeikagaku Kogyo Co. The unsaturated disulfated disac-charides, A DidiSD and A DidiSE, were gifts from Dr. SakaruSuzuki, and 4-O-a--sulfoiduronosyl-D-sulfoanhydromannose,from Dr. Ulf Lindahl.

Cell Culture. Fibroblasts derived from skin of normal oraffected individuals were maintained as described (17); ace-tone powders of the cells were made by the procedure of Halletal. (11).

Corrective Factors. Pools 1-5 of the Hunter corrective factorpreparation of Cantz et al. (5) were used in the present experi-ments. Their corrective activity, assayed as described (5, 17),had diminished by no more than one half after storage at-15° for 18 months. The a-r-iduronidase (Hurler correctivefactor) was a fraction eluted from hydroxylapatite (18).

Preparation of Radioactive Mucopolysaccharide. Very heavycultures of Hunter fibrobists (cells maintained for 1 monthafter transplantation, to a density of 5 mg of protein per 75-cm2 Falcon flask) were labeled for 6 days with E5SOt (20 mlper flask of medium containing 3 mCi, 9 jmol of inorganicsulfate). The cells were then harvested by trypsinization,washed twice with 0.9% NaCl, suspended in 1.0 ml of 0.9%NaCl, and disrupted by 10 cycles of freezing and thawing.The insoluble debris was removed by centrifugation at10,000 X g for 20 min. The supernatant fluid, which con-tained 90% of the cell-associated radioactivity, was dialyzedagainst 2 liters of 0.05M (NH4)2S04, followed by four changes,2 liters each, of water. This dialyzed solution was used as"substrate" for the Hunter factor without further purifica-tion, unless otherwise indicated.

Incorporation of [8H ]glucosamine or [8H ]galactose, whichlabel the hexosamine or uronic-acid residues of mucopoly-saccharide, respectively, was performed in fibroblasts grownto confluence but not later than 2 weeks after transplanta-tion. The usual medium was modified to contain only 2 mgof glucose and 1 mCi of either radioactive precursor per 10ml. After 3 days, cells were harvested and mucopolysaccharidewas extracted as above.

2134

Defect in the Hunter Syndrome 2135

~-2000- -800 - 200-

'500- 600-

1000- -400 - 100

500.~ ~ ~ ~ -200-

3040 50 60 70 80 90 10030 40 50 60 70 80 90100 10 152025

30 35FRACTION NO.

FIG. 1. Low molecular weight fragment 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 in0.5 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 finalvolume 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 in-cubation 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 oftotally 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 fromthe dermatan sulfate and from the heparan sulfate fractions, although only the former is plotted here.

Labeled mucopolysaccharides were separated on SephadexG-200. A major peak, appearing immediately after the voidvolume, contained 90% dermatan sulfate, as previouslydocumented (5), whereas a minor, greatly retarded peak con-tained 75% heparan sulfate and 25% dermatan sulfate.Further purification was achieved by adsorption to DEAE-Sephadex A-50 in 5mM phosphate buffer (pH 7.0) and elutionby discontinuous increments of NaCl in the same buffer.The heparan sulfate was eluted with 0.6 M NaCl, and thedermatan sulfate with 0.8-1.0M NaCl.

Radioactivity. Except for corrective factor assays, for whicha scintillation fluid has been stipulated (17), samples werecounted in a mixture of Liquifluor (New England NuclearCorp.), ]3io-Solv BBS-3 (Beckman Instrument Co.), andtoluene, 1:2.7:24 (v/v).

Digestion with Chondroitinase ABC. [15S]Mucopolysac-charide was treated with chondroitinase ABC by the procedureof Saito et al. (19); presumably, only the dermatan sulfatecomponent was digested. Mucopolysaccharide (about 100,000cpm) was mixed with 10 ,l of "enriched Tris buffer" (19)and 0.05 units of chondroitinase ABC in a total volume of 0.05ml. After 45 min at 370, another 0.05 unit of enzyme in 5 ,dIwas added, and the incubation was continued for 45 minlonger; the reaction was stopped by immersing the tubes in aboiling-water bath for 2 min. An aliquot was applied toWhatman 1 and subjected to descending chromatography inn-butanol-acetic acid-0.1 N NH40H (2:3: 1, v/v) for 22 hr.The paper was cut into 3-mm strips, each of which wascounted in 0. 5 ml H20 and 9.5 ml of scintillation fluid.

Paper Electrophoresis. To identify inorganic sulfate, elec-trophoresis was performed in 0.2 M ammonium acetatebuffer (pH 5.0) at 25 V/cm. Authentic ;5SO04 was appliedin the same salt mixture as the unknown sample, to correct forthe interference of chloride with the migration of sulfate ions.

Gas-Liquid Chromatography of Iduronic Acid. Iduronicacid was measured by gas-liquid chromatography as 1,4-

idonolactone butaneboronate by the procedure of Eisenberg[(20) and manuscript in preparation]. A tube containing aknown amount of barium iduronate was prepared as standard,and to this tube as well as to each unknown sample a constantamount of mannitol was added as internal standard. Thesamples were reduced with sodium borohydride, freed ofboric acid, lactonized, and derivatized. They were thenanalyzed with a Beckman GC65 gas chromatograph on acolumn of OV17 on GasChrom Q. From the ratio of peakareas of idonolactone butaneboronate to mannitol butane-boronate, the amount of iduronic acid in each unknown samplewas calculated.

RESULTSSulfatase activity of Hunter corrective factor

Incubation of the Hunter corrective factor with [15S]muco-polysaccharide isolated from Hunter fibroblasts resulted inthe release of a small amount of material of low molecularweight (Fig. 1). About 1.5% of the radioactivity was releasedfrom the fraction that contained primarily dermatan sulfateand 4.5% from the fraction that contained primarily heparansulfate. In both cases, the radioactive product was shown tocorrespond to inorganic sulfate by chromatography on BiogelP-2.

Similar experiments were done with analogous dermatansulfate and heparan sulfate fractions that had been labeledwith 8H in the hexosamine or uronic acid moieties. In contrastto results obtained with [5JS]mucopolysaccharide, there wasno factor-dependent release of low molecular weight materialfrom the tritium-labeled polymers§.A routine assay for the sulfatase activity of the Hunter cor-

rective factor was developed, taking advantage of the solu-bility of sulfate and insolubility of residual mucopolysaccha-ride in 80% ethanol. Unfractionated [a5S]mucopolysaccharide§ In the experiments with tritium-labeled substrates, a smallamount of radioactive material was found in the fractions fullyretarded on Sephadex G-200; this amount was identical in incu-bations with and without Hunter factor.

Proc. Nat. Acad. Sci. USA 70 (1973)

2136 Medical Sciences: Bach et al.

2400

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0.

U)

0

z

4

2

u-

20001

16001

12001

2.0

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m-q0

0

0

-4

1.0 <z-

Cw

-4

-4

r"

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4 8 12 16 20 24 28HUNTER. CORRECTIVE FACTOR, UNITS

FIG. 2. Ethanol-soluble material released from [3S]muco-polysaccharide of Hunter fibroblasts by increasing amounts ofHunter corrective factor. The assay is as described in Methods,except that the incubation volume was increased to 0.29 ml.

extracted from Hunter fibroblasts (about 100,000 cpm) isincubated with Hunter corrective factor, usually 5-20 units, in0.12 M sodium acetate (pH 4.0) and 5 mM NaN3. The in-cubation mixture, 0.07-0.11 ml, contains, in addition, NaCl(<0.07 M) and sodium phosphate (pH 6.0) (<5 mM) that are

introduced with the Hunter factor. After 20 hr at 370, 0.1 mlof chondroitin 4-sulfate (10 mg/ml in 1 M NaHCO3) and 4volumes of absolute ethanol are added. The mixture isthoroughly agitated on a Vortex mixer, chilled in ice for 30mim; and centrifuged at 10,000 X g for 20 min at 20. Thesupernatant solution is collected and again centrifuged. Analiquot of the supernatant solution and of the precipitate(redissolved in water) are counted. A control incubationwithout Hunter factor is carried through the same steps, andall values are corrected for this blank (usually about 300 cpm).The assay is based on preliminary experiments that showed an

optimal pH at 4.0, with half maximal activity at pH 3.5 and4.7. Citrate-phosphate buffer is inhibitory. The rate of releaseof ethanol-soluble material is linear for 10 hr but diminishesmarkedly thereafter.

TABLE 1. Sulfate releasedfrom [35S]mucopolysaccha ideby acetone-powder extracts of fibroblasts

Num- Ethanol-soluble released

ber of (cpm/mg of protein)

cell

Genotype lines Mean Range

Hunter 6 1,400 200-3,000Hurler 3 25,000 15,000-31,000Normal 2 15,000Maroteaux-Lamy 1 33,000Sanfilippo A 1 20,000Bl-Glucuronidase

deficiency 1 24,000I-cell 1 6,000

Sulfatase activity of acetone-powder extracts was assayed as

described in Methods, with 105 cpm of [33S]mucopolysaccharidesubstrate, about 0.1 mg of protein for extracts of Hunter fibro-blasts, and about 0.05 mg of protein for extracts of fibroblasts ofother genotype.

TABLE 2. Absence of inhibitor in extracts fromHunter fibroblasts

Ethanol-solublereleased

Sample (cpm per sample)

1. Extract, normal fibroblasts 28002. Extract, Hunter fibroblasts 1403. Mixture, 1 + 2 22004. Hunter factor 26005. Mixture, 2 + 4 2700

Sulfatase activity was assayed as described in Methods, with2 X 105 cpm of [IS] mucopolysaccharide, and amounts of Hunterfactor or normal acetone-powder extract selected tobe in the linearrange. Extract from Hunter fibroblasts contained 0.1 mg of pro-tein; higher amounts gave an apparent inhibition, perhaps be-cause of dilution of the radioactive substrate with endogenousmucopolysacchoride.

The ethanol-soluble reaction product behaves as inorganicsulfate by chromatography on Biogel P-2 and by paperelectrophoresis at pH 5.0 (Rpicrate = 3.0).Up to a point, there is a linear relationship between the

amount of Hunter factor added and the amount of '5SO4= re-leased (Fig. 2). Maximal release corresponds to about 2% ofthe added radioactivity (the amount varies somewhat be-tween different preparations of [15S ]mucopolysaccharide).This means that only 2% of the sulfate residues of the muco-polysaccharide substrate are susceptible to the action of theHunter corrective factor.No sulfate was released at all when the [B5S]mucopoly-

saccharide derived from Hunter fibroblasts was replaced byan analogous preparation derived from Hurler fibroblasts.

TABLE 3. Decrease of "oversulfated" areas of [33S]muco-polysaccharide upon incubation with Hunter factor

Addition of Hunter factor

Units ofcorrective

Fraction* activity ADidiS (%)t

None (3 samples) 7.6Pool 1 60 5.1Pool 2 50 5.3Pool 3 46 5.4Pool 4 16 6.1Pool 5 10 6.0

* The fractions "pools 1-5" are described in Figs. 2 and 3 ofref. 5.

t The % of ADidiS is calculated on the basis of moles of disul-fated disaccharide obtained relative to total disaccharide releasedby chondroitinase ABC. Because the specific activity of disulfateddisaccharide is twice that of monosulfated, the percentage is cal-culated as follows:

% _0.5 (cpm, ADidiS) X 100

0.5 (cpm, ADidiS) + (cpm, ADi4S) + (cpm, ADi-6S)

Less than half of the mucopolysaccharide was converted to un-saturated disaccharides by chondroitinase ABC. The proportiondegraded was the same in samples that had been incubated withthe Hunter factor and controls.

Proc. Nat. Acad. Sci. USA 70 (1973)

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Defect in the Hunter Syndrome 2137

TABLE 4. Iduronic acid released from 4-O-c-L-sulfo-iduronosyl-D-sulfoanhydromannose by the combined

action of Hunter factor and a-iiduronidase

IduronateSample no. Hunter factor a-L-Iduronidase released (ug)

1 - - 0.42 - + 0.93 + _ 0.64 + + 95 + + 16

The complete incubation mixture, no. 4, contained 0.2 jumol ofdisaccharide; Hunter factor, 60 corrective units, in 0.25 ml of0.9% NaCl-0.01 M Na phosphate (pH 6.0) enriched with 0.25mg of albumin; a final concentration of 0.12 M sodium acetatebuffer (pH 4.0) and 5 mM NaN3, in a total volume of 0.5 ml.After 24 hr at 370, a-Liduronidase was added [sufficient to hy-drolyze 0.3 jumol of phenyliduronide per 17 hr under standardizedconditions (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, iduronatereleased was measured by gas-liquid chromatography as de-scribed in Methods. Samples 1-3, from which one or both enzymeswere omitted, were incubated with the equivalent amount ofalbumin in buffered saline. Sample 5 differed in that both Hunterfactor and a-Liduronidase were incubated together for 48 hr.

Sulfatase activity of acetone powders of fibroblasts

Acetone-powder extracts prepared from Hunter fibroblastsare profoundly deficient in the sulfatase associated withHunter corrective factor, when compared to similar extractsof fibroblasts from normal individuals or from patients withother mucopolysaccharidoses (Table 1). The deficit is not dueto the presence of inhibitors in extracts of Hunter fibroblasts,as shown by mixing experiments (Table 2). The Huntergenotype, shown to be associated with deficiency of Hunterfactor, is now correlated with a deficiency of sulfatase ac-tivity.An interesting exception to that correlation is the marked

deficiency of sulfatase in extracts of fibroblasts from I-celldisease patients (Table 1). These cells have markedly re-duced activity of several lysosomal enzymes (e.g., ref. 21)and are unable to correct the defect of Hunter fibroblasts (4).Homogenates prepared by freeze-thawing of fibroblasts,

were found unsatisfactory for studying the sulfatase becauseof the presence of apparent inhibitors, particularly in homog-enates of normal cells. The interfering substances are pre-sumably removed in the preparation of acetone powders.

TABLE 5. Iduronic acid released from 4-0-a-L-sulfo-iduronosyl-D-sulfoanrhydromannose by acetone-powder

extracts offibroblasts

Genotype Iduronate released (jhg)

Normal 2.4Hunter 0.6Hurler 0.09Hunter + Hurler, mixed 2.9

Incubation mixtures contained 0.2 jumol of disaccharide; acetone-powder extract in 0.9% NaCl containing about 0.1 mg of protein;a final concentration of 0.12 M sodium acetate buffer (pH 4.0)and 5 mM NaNs, in a total volume of 70 ul. After 24 hr at 370,the iduronate released was measured by gas-liquid chromatog-raphy, as described in Methods. Higher amounts of acetone-powderextracts resulted in apparent inhibition in the mixed sample,perhaps because of its high content of endogenous mucopoly-saccharide.

disulfated5. After treatment of the [a5S]mucopolysaccharidewith Hunter factor, the proportion of disulfated disaccharidesproduced by chondroitinase ABC was reduced to a limitingvalue of about 5% (Table 3). Apparently, one-third of the"oversulfated" areas of mucopolysaccharide isolated fromHunter fibroblasts was removed by the Hunter factor. Bycontrast, mucopolysaccharide isolated from Hurler fibro-blasts and similarly treated with chondroitinase ABC, showedthe presence of about 4% oversulfated areas, none of whichwas removed by the Hunter factor (4.1 or 4.5%, for controlsample or sample treated with Hunter factor, respectively).These results are consistent with the hypothesis that the

Hunter corrective factor is a sulfatase for oversulfated areasof the mucopolysaccharide, provided these are suitably ex-posed. Since the sulfate residue that is unique to such areasis that linked to L-iduronic acid, the results further suggestthat the Hunter factor is a sulfatase for sulfated iduronicacid. A direct demonstration thereof was obtained by usingfor substrate the disaccharide 4-0-a-L-sulfoiduronosyl-D-sulfoanhydromannose, and measuring iduronic acid re-leased by the combined action of Hunter factor and a-L-iduronidase. Whereas either enzyme alone released only tracesof iduronic acid, as determined by gas-liquid chromatog-raphy, the two enzymes together released as much as 40%of the available iduronic acid in the best experiment pre-sented in Table 4.

Release of iduronic acid from 4-0-ca-L-sulfoiduronosyl-D-sulfoanhydromannose was also catalyzed by acetone-powderextracts of normal fibroblasts. Extracts of Hurler fibroblasts(which have Hunter factor but not a-i-iduronidase) or of

Identification of Hunter corrective factor as asulfoiduronate sulfatase

Action of the Hunter factor on [3S]mucopolysaccharide re-sulted in the disappearance of some "oversulfated" areas-i.e., areas with two sulfate groups per disaccharide consistingof uronic acid and hexosamine. This was determined by di-gesting with chrondroitinase ABC the substrate and theproduct of Hunter factor activity. The bacterial enzyme de-graded a portion of the [E5S]mucopolysaccharide used assubstrate (presumably, the dermatan sulfate componentonly) into unsaturated disaccharides of which 7.5% were

I The radioactive disulfated disaccharide released by chondro-itinase ABC had a chromatographic position similar to that ofADidiSE [2-acetamido-2-deoxy-3-O-(j-D-gluco-4-enepyranosyl-uronic acid)-4,6-di-O-sulfo-i)-galactose] and that of ADidiSD[2-acetamido-2-deoxy-3-O-(2 or 3-0-sulfo-j#-D-gluco4-enepyran-osyluronic acid)-6-O-sulfo-D-galactose]. On prolonged chromatog-raphy (40 hr) the radioactive disulfated disaccharide was partiallyseparated from both reference compounds. Its chromatographicmobility, intermediate between ADidiSD and ADidiSE, was con-sistent with that of ADidiSB [2-acetamido-2-deoxy-3-O-(2 or3-O-sulfo-3-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galac-tose], which was unfortunately not available for comparison.

Proc. Nat. Acad. Sci. USA 70 (1973)

2138 Medical Sciences: Bach et al.

Hunter fibroblasts (which have a--iduronidase but notHunter factor) had only slight activity, whereas a mixture ofthe two caused the release of as much iduronic acid as did thenormal extract (Table 5).

DISCUSSION

The results demonstrate that the Hunter corrective factor hassulfoiduronate sulfatase activity. It is unlikely that the sul-fatase is a chance contaminant of the admittedly impurefactor preparation, since it is specifically absent from extractsof Hunter fibroblasts.We were surprised to find that acetone-powder extracts of

normal fibroblasts did not release more 35SO4t from [n5S]-mucopolysaccharide than did the purified Hunter factor.These extracts contain several of the enzymes necessary formucopolysaccharide degradation, such as a-L-iduronidase and,3-glucuronidase (6, 11), as well as the sulfoiduronate sulfatase.Presumably, one or more of the unknown degradative en-zymes (e.g., hydrolases for the 0-sulfated N-acetylgalactosa-mine residues of dermatan sulfate) is inactivated during prep-aration or assay of the acetone-powder extract.Hunter patients store and secrete both dermatan sulfate

and heparan sulfate. Sulfoiduronate residues have been wellcharacterized in mammalian dermatan sulfate and heparin(12-15). They have not been reported in heparan sulfate.That, however, is probably because heparan sulfate is poorlystudied; it is generally assumed to be similar to heparin (22,23), the difference being primarily in molecular size and shape(larger and more branched molecule in heparan sulfate) andratio of N-acetylated to N-sulfated glucosamine (higher inheparan sulfate 11) .Not all sulfated iduronic-acid residues are available to the

sulfatase. We speculate that the unavailable ones are buriedwithin the molecule. This hypothesis is consistent with thefinding of a disulfated disaccharide at the nonreducing endof dermatan sulfate isolated from Hunter fibroblasts but not

0

from Hurler fibroblasts (Sj6berg, I., Fransson, L. A., Matalon,R. & Dorfman, A., manuscript in preparation).A disulfated disaccharide containing iduronic acid has

been found in the urine of a Hunter patient (27). Such afragment could result in vivo from the action of an endogly-cosidase (e.g., hyaluronidase) on dermatan sulfate.The clinical consequences of sulfoiduronate sulfatase

deficiency are, on the whole, similar to those of a-iiduroni-dase deficiency. The severe and mild forms of the Huntersyndrome have as their counterpart the Hurler and Scheiesyndromes, respectively (1, 28). There is, however, one con-sistent phenotypic difference between the two enzyme de-

1I Because of the large proportion of sulfated iduronic-acid residuesin heparin, it is puzzling that this polymer should not be storedand excreted in the Hunter syndrome. Although "heparin-like" fractions have been reported among the mucopolysac-charides of Hurler and Sanfilippo patients these are not considered"true" heparin but rather degradation products of heparan sul-fate (24, 25). Perhaps man, in contrast to other mammals,does not synthesize heparin. Experimental data on the subjectare sparse (e.g., ref. 26).

ficiencies. a-IIduronidase deficiency is always associatedwith cloudy corneas, whereas corneas remain clear in theHunter syndrome. One must presume that corneal muco-polysaccharides do not require Hunter factor for degradation,and, therefore, that they are devoid of sulfoiduronate residues.

We thank Dr. Ulf Lindahl (University of Uppsala, Sweden) andDr. Sakaru Suzuki (University of Nagoya, Japan) for generousgifts of disulfated disaccharides. This work was supported in partby a grant from the Deutsche Forschungsgemeinschaft.

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2. Dorfman, A. & Matalon, R. (1972) in The Metabolic Basis ofInherited Disease, eds. Stanbury, J. B., Wyngaarden, J. B. &Fredrickson, D. S. (McGraw Hill, New York), pp. 1218-1272.

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Proc. Nat. Acad. Sci. USA 70 (1973)