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J. Biol. Chem., Vol. 277, Issue 4, 2562-2572, January 25, 2002

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Physiological Substrates for Human Lysosomal -Hexosaminidase S^*

Stefan T. Hepbildikler, Roger Sandhoff^§, Melanie Kölzer, Richard L. Proia^¶, and Konrad Sandhoff

From the  Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany, the ^§ Deutsches Krebsforschungszentrum Heidelberg, Abteilung für Zelluläre und Molekulare Pathologie, INF 280, 69120 Heidelberg, Germany, and the ^¶ Genetics of Development and Disease Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, June 13, 2001, and in revised form, October 10, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Human lysosomal -hexosaminidases remove terminal -glycosidically bound N-acetylhexosamine residues from a number of glycoconjugates. Three different isozymes composed of two noncovalently linked subunits  and  exist: Hex A (), Hex B (), and Hex S (). While the role of Hex A and B for the degradation of several anionic and neutral glycoconjugates has been well established, the physiological significance of labile Hex S has remained unclear. However, the striking accumulation of anionic oligosaccharides in double knockout mice totally deficient in hexosaminidase activity but not in mice expressing Hex S (Sango, K., McDonald, M. P., Crawley, J. N., Mack, M. L., Tifft, C.J., Skop, E., Starr, C. M., Hoffmann, A., Sandhoff, K., Suzuki, K., and Proia, R. L., (1996) Nat. Genet. 14, 348-352) prompted us to reinvestigate the substrate specificity of Hex S. To identify physiological substrates of Hex S, anionic and neutral oligosaccharides excreted in the urine of the double knockout mice were isolated and analyzed. Using ESI-MS/MS and glycosidase digestion the anionic glycans were identified as products of incomplete dermatan sulfate degradation whereas the neutral storage oligosaccharides were found to be fragments of N-glycan degradation. In vitro, recombinant Hex S was highly active on water-soluble and amphiphilic glycoconjugates including artificial substrates, sulfated GAG fragments, and the sulfated glycosphingolipid SM2. Hydrolysis of membrane-bound SM2 by the recombinant Hex S was synergistically stimulated by the GM2 activator protein and the lysosomal anionic phospholipid bis(monoacylglycero)phosphate.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Lysosomal hexosaminidases (EC 3.2.1.52) release terminal -glycosidically linked N-acetylglucosamine and N-acetylgalactosamine residues from a number of glycoconjugates (1). They are composed of two subunits,  and , derived from homologous genes HEXA and HEXB. Hexosaminidase A (Hex A,¹ ) and Hex B () were believed to be the major functional isozymes, and Hex S () a minor labile form without significant activity (2). Several attempts to isolate pure Hex S from human tissues or cell homogenates yielded preparations with poor enzymatic activity (3-5).

Each subunit possesses an active site characterized by its own substrate specificity (5). The active site of the -subunit hydrolyzes uncharged substrates, whereas the -subunit, in addition, cleaves negatively charged substrates. Only the -heterodimer Hex A is able to degrade ganglioside GM2 (Fig. 1B) at significant rates in the presence of the GM2 activator protein (GM2AP).

A group of severe neurodegenerative storage diseases, the GM2 gangliosidoses, results from mutations in any of the genes encoding the two hexosaminidase subunits and GM2AP. Tay-Sachs disease is caused by mutations in the HEXA gene resulting in a deficiency in Hex A and Hex S, whereas in Sandhoff disease the lack of Hex A and Hex B activity is observed due to mutations in the HEXB gene. The GM2 gangliosidoses are characterized by a massive accumulation of ganglioside GM2 and related glycolipids in neuronal lysosomes. Depending on the defect and its severity, other tissues may also be affected by lipid and oligosaccharide accumulation (1). The severity of the clinical phenotype is directly related to the amount of residual enzyme activity (6). In addition, the isozyme that is not affected by mutation is able to compensate in part for the loss of the affected hexosaminidase activity (1).

Mouse models have been generated for Tay-Sachs disease (deficient in the -subunit but expressing Hex B), for Sandhoff disease (deficient in the -subunit and expressing Hex S) as well as double knockout mice (totally deficient in hexosaminidase activity) (7, 8) (Table I).

Surprisingly, the double knockout mice show a mucopolysaccharidosis phenotype with an accumulation of anionic oligosaccharides in addition to the pathological and biochemical features of GM2 gangliosidoses (8, 9). In the Hexb / mice no increased accumulation of glycosaminoglycans (GAGs) was observed indicating that Hex S, which is still expressed in the mutant mice, is involved in GAG catabolism. GAGs contain (1-4)- and (1-3)-linked N-acetylhexosamine residues which become sensitive to hexosaminidases when exposed as terminal sugar residues during their degradation (10).

We have purified and characterized recombinant Hex S (rHexS) after expression in insect cells. To study its substrate specificity the storage glycans were isolated from the urine of the hexosaminidase double knockout mice. In addition, other water-soluble and lipid substrates were evaluated. The results demonstrate that Hex S is highly active on a wide range of substrates and support the contention that the enzyme is physiologically important in vivo.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Commercial Products

Phosphatidylcholine (egg yolk) (PC), cholesterol (Chol), the maltose oligosaccharide standard, concanavalin A-Sepharose, chondroitin 6-sulfate (type IV-S), -glucuronidase from bovine liver (type B-10), and hyaluronidase (bovine testes) were purchased from Sigma, Deisenhofen, Germany. BMP was purchased from Avanti Polar Lipids. 2,5-Dihydroxybenzoic acid was supplied by ICN Biochemicals. POROS 20 HQ and HS ion exchange resins were purchased from Roche Molecular Biochemicals, Mannheim, Germany. LichroPrep RP18, TLC, and HPTLC plates were obtained from Merck, Darmstadt, Germany. Carbograph SPE tubes were obtained from Alltech, Unterhaching, Germany. DEAE-Sephacel and Sephadex G-25 fine were purchased from Amersham Biosciences, Inc., München, Germany. TSK-Gel HW40F was kindly provided from TosoHaas, Stuttgart, Germany. The anionic standard saccharides N-acetylgalactosamine 6-sulfate (GalNAc-6-sulfate) and the saturated disaccharide derived from chondroitin 6-sulfate (C6S-2) were obtained from Dextra Laboratories, Reading, UK. The unsaturated GAG disaccharide standards Di2S, Di4S, and Di6S were purchased from Calbiochem, Bad Soden, Germany. The synthetic substrates 4-methylumbelliferyl-2-acetamido-2-deoxy--D-glucopyranoside (MUG) and 4-methylumbelliferyl-2-acetamido-2-deoxy--D-glucopyranoside-6-sulfate (MUGS) used for the hexosaminidase assay were supplied by Toronto Research Chemicals, Downswood, Ontario, Canada. 8-Aminonaphthalene-1,3,6-tris-sulfonic acid, N-acetylgalactosamine, chondroitinase AC, chondroitinase ABC, and methyl--D-mannoside were purchased from Fluka, Neu-Ulm, Germany. Trypsin was purchased from Promega GmbH, chymotrypsin was obtained from Roche Molecular Biochemicals GmbH, both in Mannheim, Germany. All other chemicals were of analytical grade or of the highest purity available.

Enzyme Preparation and Identification

Recombinant Hex S was expressed in insect cells using the baculovirus expression system. Sf21 cells were infected with a recombinant virus containing the cDNA encoding for the human hexosaminidase -subunit. The baculovirus construct was prepared as described for the expression of recombinant Hex B (11). The expression was terminated 96 h after infection. After centrifugation of the serum-free culture medium of infected cells the supernatant was subjected to lectin affinity chromatography with concanavalin A. The column was eluted with 15% (w/v) methyl--D-mannoside in equilibration buffer (25 mM sodium phosphate, pH 7.0, 200 mM sodium chloride) and the enzyme activity was monitored with MUG as substrate. The eluate of the concanavalin A column was concentrated and passed through a fast-flow cation exchange resin (POROS 20 HS) equilibrated in 50 mM sodium phosphate buffer, pH 6.5. rHexS was eluted without binding, in contrast to contaminating glycoproteins in the ConA eluate. Subsequently the fraction containing Hex S activity was applied to a weak anion-exchange material (POROS 20 HQ) equilibrated in 50 mM sodium phosphate buffer, pH 7.0. The column was eluted by increasing the concentration of sodium chloride in the equilibration buffer (by 10 mM/column volume), at a flow rate of 4 ml/min. Purification to 90% homogeneity was shown by SDS-PAGE. The purified preparation of rHexS was identified by Western blotting and N-terminal sequencing using Edman degradation and ESI-MS/MS sequencing of tryptic and chymotryptic peptides after narrow-bore reverse phase HPLC separation as described below. Western blotting was performed according to Towbin et al. (12) using an antiserum raised against the denatured hexosaminidase -subunit (13). Hex A and Hex B were purified from human liver to apparent homogeneity according to Liessem et al. (14). Their purity and identity were ensured by SDS-PAGE and Western blotting (12, 13).

Characterization of Post-translational Modifications of rHexS

After proteolytic cleavage and reverse phase separation of the resulting peptides the post-translational modifications of rHexS were analyzed by mass spectrometry: N-glycosylation of rHexS glycopeptides was demonstrated by treatment with N-glycosidase F (15) and by ESI-MS/MS fragment analysis as described below. Disulfide linkages were analyzed by comparing the masses of the proteolytic cleavage products of unmodified active rHexS with those of active rHexS alkylated by iodoacetamide, and those of reduced and alkylated rHexS as established in our laboratory for the analysis of the hexosaminidase -subunit (15). In addition, chymotryptic cleavage of rHexS was used under the conditions described for tryptic degradation (15) and proteolytic digestion products were also analyzed by ESI-MS/MS as described below, in addition to MALDI-MS.

Activator Preparation

Recombinant GM2AP was expressed in insect cells using the baculovirus expression system and purified as described previously (16).

Determination of K_m and V_max

The K_m and V_max values for recombinant -hexosaminidase S were determined by the method of Lineweaver and Burk (17). Enzyme activities were measured as described (5) using various concentrations of the synthetic substrates MUG (final concentrations of 0.25-2.5 mM in 50 mM citrate buffer, pH 4.4) and MUGS (0.1-1.0 mM in 50 mM citrate buffer, pH 4.0), respectively. Enzyme kinetics with these substrates followed Michaelis-Menten theory.

Determination of pH Optimum with the Artificial Substrates MUG and MUGS

Enzyme activities were measured as described (5) using MUG (final concentrations of 1 mM in 50 mM citrate buffer) and MUGS (0.5 mM in 25 mM citrate buffer), respectively, and varying the pH of the solution in the range from 3.0 to 6.0.

Presentation of Data

The data obtained with the artificial substrates are means of at least duplicate determinations. Deviations did not exceed ± 5% of the mean.

Animals

Generation of hexosaminidase-deficient mice through targeted gene disruption and cross-breeding of the knockout animals to obtain the double knockout mice has been described previously (7, 8). The urine and kidneys of knockout mice and wild type littermates of 10-20 weeks of age were analyzed.

Isolation of the Sulfoglycolipids SM2 and SB2 from Rat Kidney

SM2 and SB2 were isolated according to Jennemann et al. (18) with some modifications. Kidneys were homogenized with an Ultra-Turrax, freeze-dried, and extracted twice with acetone using an ultrasonic bath. The residual pellet then was extracted for GSLs two times with chloroform/methanol/water (10/10/1, v/v), once with chloroform/methanol/water (30/60/8, v/v) using an ultrasonic bath, and the combined chloroform/methanol/water extracts were dried in a rotary evaporator. To remove most phospholipids the chloroform/methanol/water extract was treated with 0.1 M methanolic KOH for 2 h at 37 °C and neutralized with acetic acid. To remove salts the extract was dialyzed 5 times against water and subsequently freeze dried. Neutral and acidic GSLs were separated on a DEAE column and acidic GSLs were split into fractions by eluting with a stepwise gradient of 20, 80, 200, 500, and 1000 mM potassium acetate in methanol. Again salts were removed by dialyzing 5 times against water and subsequently freeze drying the micellar lipid solution. SM2 then was isolated from the 80 mM potassium acetate fraction and SB2 from the 500 mM potassium acetate fraction. Each fraction was further purified by silica gel flash chromatography using a stepwise gradient of propanol-2/n-hexane/water, with changes in the ratio of n-hexane and water (55/44/1, 55/43/2, etc. until 55/37/8, v/v). Final purification of SM2 was achieved by repeated silica gel flash chromatography of the combined SM2-containing fractions using the solvent systems chloroform/methanol/water (62/30/1.8, v/v/v) and then chloroform/methanol/water (70/30/2, v/v/v). Final purification of SB2 was achieved by silica gel flash chromatography of the combined SB2-containing fractions using the solvent system chloroform/methanol/water (65/30/2.5, v/v/v).

Vesicle Preparation

Large unilamellar vesicles (LUVs) were prepared by the following procedure: PC (50 mM, toluol/ethanol, 2/1, v/v), BMP (5 mM, chloroform/methanol, 2/1, v/v), Chol (25.6 mM, chloroform/methanol, 2/1, v/v), and SM2 (740 µM, chloroform/methanol/water, 60/35/8, v/v/v) were dissolved in organic solvents. Appropriate aliquots of the lipid solutions were mixed and dried under nitrogen. The lipid mixture was hydrated to a total lipid concentration of 2 mM in Tris/HCl buffer (1 mM, pH 7.4) and freeze-thawed 10 times in liquid nitrogen to ensure solute equilibration between trapped and bulk solutions. The standard lipid composition was 50 mol % PC, 20 mol % BMP, 20 mol % Chol, and 10 mol % SM2.

Unilamellar vesicles were prepared by the passage of liposomes through 2 polycarbonate filters (100-nm pore size, Avestin) mounted in tandem in a mini-extruder (Liposo-Fast, Avestin). Samples were subjected to 19 passages as recommended (19).

Preparation of a Trisaccharide from Chondroitin 6-Sulfate

The model substrate for Hex S was obtained by enzymatic degradation of chondroitin 6-sulfate according to Kresse et al. (20). Briefly, after exhaustive digestion of chondroitin 6-sulfate with testicular hyaluronidase (EC 3.2.1.35) in 100 mM NaAc buffer, pH 5.0, containing 150 mM NaCl, for 45 h at 37 °C the ethanol-soluble degradation products were chromatographed on a column of Sephadex G-25 fine (24 × 850 mm) equilibrated and eluted with 1.0 M NaCl, at a flow rate of 0.3 ml/min. The fractions were monitored by FACE profiling. Tetrasaccharide peak fractions were desalted using the same column equilibrated with water. The tetrasaccharide was then treated with -glucuronidase (EC 3.2.1.31) in 100 mM sodium citrate buffer, pH 5.2, for 48 h at 37 °C. The trisaccharide product C6S-3 (Fig. 1C) was chromatographed and desalted as described for the tetrasaccharide.

Isolation of Urinary Glycans in the Hexosaminidase-deficient Mice

The urine was collected in a metabolism cage for mice (Scanbur A/S) in which the mice stayed 8 h per day. Neutral urinary oligosaccharides were prepared by solid phase extraction on graphitized carbon as described by Klein et al. (21). Briefly, the columns (0.5 ml) equilibrated with 3 ml of water were loaded with 150-300 µl of urine sample and washed with 2 ml of water again. Neutral oligosaccharides were eluted with 2 ml of 25% (v/v) acetonitrile in water. Anionic urinary oligosaccharides were prepared according to Lindahl et al. (22) with some modifications. A 2.6-ml DEAE-Sephacel column was equilibrated first with 50 mM Tris/HCl, pH 7.2, and then with washing buffer (50 mM sodium acetate buffer, pH 4.0) at a flow-rate of 1.2 ml/min. After loading of 150-300 µl of urine and 15 ml of washing buffer the column was eluted with either 150 mM lithium chloride in 50 mM sodium acetate buffer, pH 4.0, for the isolation of DS-5 (A) or 2.0 M lithium chloride in the same buffer for the preparation of GAG OS (B). The eluates A and B were separately desalted by size exclusion chromatography on a TSK-Gel HW40F column (16 × 390 mm) with a flow rate of 1.0 ml/min.

Enzyme Assays

Hex S activity was assayed with the artificial substrates MUG or MUGS as described (5). Incubation mixtures using MUG as substrate contained the following components in a final volume of 200 µl: sodium citrate buffer (50 mM, pH 4.4), 20-µl aliquots of enzyme solution, 10 µg of bovine serum albumin, and 1 mM MUG. When using MUGS the final substrate concentration was 0.5 mM and the buffer concentration was 25 mM, pH 4.0. The incubation was stopped after 30 min at 37 °C by adding 1 ml of sodium carbonate/glycine buffer (200 mM each, pH 9.5). Then, the amount of 4-methylumbelliferone released was measured fluorimetrically. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the hydrolysis of 1 µmol of MUG/min at 37 °C.

Oligosaccharide degradation by hexosaminidases was assayed as follows: 1-5 nmol of purified N-glycan fragment H₃N₃ (Fig. 1D), 0.5-2 nmol/oligosaccharide DS-5 (Fig. 1C) or GAG OS, and 4 nmol of C6S-3 (Fig. 1C), respectively, were dried in a centrifugal vacuum evaporator (CVE, Savant). Standard assay mixtures contained 200 milliunits of (MUG) -hexosaminidase activity and 4 µg of bovine serum albumin in a final volume of 40 µl. For subsequent mass spectrometric analysis sample solutions were buffered by ammonium acetate (5 mM, pH 4.0). By addition of 40 µl of methanol the incubation was stopped after 5 h unless indicated otherwise. Concomitantly, samples lacking enzyme activity as well as samples devoid of substrate were incubated as control experiments under identical conditions.

The GAG OS mixture isolated from the urine of double knockout mice was incubated with chondroitinase AC (EC 4.2.2.5) and chondroitinase ABC (EC 4.2.2.4), respectively, under the same conditions as with the hexosaminidases except that 175 milliunits of enzyme activity and ammonium acetate buffer (5 mM, pH 8.0) were used; the samples were incubated for 9 h at 37 °C and the reaction products were analyzed by FACE. Concomitantly, control assays lacking either enzymes or substrates were run under identical conditions, none of them indicating that any digestion took place. The degradation of glycolipid substrates by the hexosaminidase isozymes was tested in a micellar assay (A) and the hydrolysis of SM2 by Hex S was also examined in a liposomal assay (B).

Micellar Assay-- Glycolipids were incubated with the A, S, and B isozymes under the same conditions as described in the oligosaccharide assay with the following alterations: 8 nmol of glycolipid per assay were used and sodium citrate buffer (5 mM, pH 4.3) replaced the ammonium acetate buffer in a final volume of 20 µl. 1.5 µg of GM2AP was added and incubation times ranged from 15 min to 17 h, as indicated in the legends to the figures. After the incubations were stopped with 20 µl of methanol the assay mixtures were dried in a CVE and redissolved in 40 µl of ammonium acetate buffer (0.3 M, pH 7.0). Prior to TLC analysis the samples were desalted by reverse phase chromatography. RP18 columns (0.5 ml) equilibrated with a solution of chloroform, methanol, 0.1 M KCl (3/48/47, v/v/v) were loaded with sample, washed with water, and eluted with 2 ml of chloroform/methanol 1/1 (v/v). The eluted lipids were applied to a TLC plate.

Liposomal Assay-- LUVs containing SM2 as substrate were incubated with rHexS and GM2AP in a liposomal assay. The micellar assay described above was modified as follows. Standard incubation mixtures contained sodium citrate buffer (10 mM, pH 4.3), 4 µg of bovine serum albumin, 1.0 µg of GM2AP, and 30 milliunits of rHexS in a final volume of 40 µl. A total volume of 20 µl of protein mixture in citrate buffer was added to the same volume of unilamellar vesicles dissolved in Tris/Cl buffer (1 mM, pH 7.2). The liposomes (1 mM, amount of total liposomal lipids) were composed of 50 mol % PC, 20 mol % Chol, 20 mol % BMP and 10 mol % SM2 or 70 mol % PC, 20 mol % Chol, and 10 mol % SM2. After incubation stop with 40 µl of methanol the mixtures were concentrated to dryness by a stream of nitrogen and then subjected to alkaline methanolysis with 0.1 M NaOH in 200 µl of methanol for 4.5 h at 37 °C to remove the phospholipids. After neutralization with 1.2 µl of glacial acetic acid, the samples were desalted by reverse phase chromatography on RP18 and applied to HPTLC plates.

Thin-layer Chromatography

Desalted assay samples were applied to thin layer Silica Gel 60 or HPTLC plates (Merck Darmstadt, Germany). The chromatograms were developed with chloroform, methanol, 0.22% (w/v) CaCl₂ in water (60/35/8, v/v/v). After development, plates were air-dried, sprayed with 8% (w/v) H₃PO₄ containing 10% (w/v) CuSO₄, and charred at 180 °C for 10 min, and lipids were quantitated by photodensitometry (Shimadzu Kyoto, Japan).

Analysis of Glycan Samples by Fluorophore-assisted Carbohydrate Electrophoresis (FACE)

This method is based on the fluorescence labeling of glycans by reductive amination with 8-aminonaphthalene-1,3,6-tris-sulfonic acid (23). 5 µl of a 0.15 M solution of the fluorophore in 15% (v/v) glacial acetic acid and 5 µl of 1.0 M sodium cyanoborohydride in dimethyl sulfoxide were added to the glycan samples which had previously been dried in a CVE. After incubation for 15 h at 37 °C the samples were dried in a CVE and dissolved in 8 µl of HPLC water. 4 µl of this solution were mixed with the same volume of 25% (v/v) glycerol in HPLC water and applied to a 32% polyacrylamide gel with a 4% stacking gel. The buffer solutions and gel components were the same as for SDS-PAGE according to Laemmli (24) except that SDS was omitted. The fluorescently labeled glycans resolved in the gel were visualized by illumination with UV light of 366 nm wavelength. The gel image was photographed by a ccd camera. The migration distance depends on molecular weight and charge, i.e. the m/z values of the labeled glycans. An anionic oligosaccharide carrying intrinsic negative charges has a higher electrophoretic mobility than a neutral oligosaccharide of the same size. Thus, in the FACE gels it is only possible to compare migration distances of identically charged analytes. From our experiments using FACE we conclude for the GAG fragments with high negative net charge that the labeling efficiency decreases with increasing molecular weight of the saccharide. Therefore, band intensities are only comparable between analytes of the same molecular weight and charge. Despite these limitations the FACE display technique is one of the most powerful methods for glycan profiling currently at hand, combining high sensitivity down to the picomole range, high resolution, and relatively high tolerance of buffers and salt concentrations in the samples.

MALDI Mass Spectrometry

MALDI-MS analysis was performed on a TofSpec E (Micromass, Manchester, UK) mass spectrometer operating at an acceleration voltage of 20 kV with a 337-nm nitrogen laser. Mass spectra were externally calibrated using a maltooligosaccharide standard (4 to 10 glucose units) for glycan samples and commercially available peptides for peptide samples (Sigma). 1 µl of matrix solution were mixed on-target with the same volume of glycan sample. Dihydroxybenzoic acid (10 mg/ml of acetonitrile/water, 7/3, v/v, freshly prepared) was used as matrix for glycan analysis, -hydroxycinnamic acid was used for small peptides (10 mg/ml acetonitrile, 0.1% trifluoroacetic acid in water, 6:4, v/v, freshly prepared) and sinapinic acid was used for high molecular weight peptides (10 mg/ml acetonitrile, 0.1% trifluoroacetic acid in water, 1/1, v/v).

ESI Mass Spectrometry

ESI mass spectra were acquired on a Q-Tof2 (Micromass, Manchester, UK) instrument operating at capillary voltages around 1000 V. It uses a quadrupole mass analyzer and an orthogonal acceleration time of flight mass spectrometer with a hexapole collision cell situated between the two mass analyzers. In MS/MS mode, this collision cell was flooded with argon for fragmentation analysis and collision energy was varied in the range from 20 to 60 eV. Peptides were dissolved in acetonitrile/water, 1/1 (v/v) for positive mode analysis. According to Zaia and Costello (25) anionic glycans were dissolved in acetonitrile, 10 mM hydrochloric acid (1/1, v/v) and analyzed in negative mode. In addition, parent and daughter ion scans of DS-5 were performed on a Quattro II ES-MS/MS instrument (Micromass, Manchester, UK) similar to the Q-Tof2 except that the masses were detected by a quadrupole analyzer instead of a time-of-flight mass spectrometer.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Structural Analysis of Recombinant Hex S-- Recombinant human Hex S was partially purified from the serum-free culture medium of transfected insect cells by affinity chromatography on a concanavalin A-Sepharose column. This preparation contained precursor -subunit and the processed -subunit in a 1:1 ratio. By Edman sequencing the N terminus of the precursor -subunit was identified as L²³WPWPQNFQT and that of the polypeptide r_m in the processed -subunit as G⁸⁵KRHTLKNV which starts five amino acid residues upstream of the N terminus of the native _m-chain (26, 27). After further purification by cation and anion exchange chromatography the preparation contained >90% processed rHexS according to SDS-PAGE. Rechromatography on a POROS 20 HQ resin yielded small amounts of apparently homogeneous processed rHexS and precursor rHexS. This purified rHexS was 40 times more active against the neutral substrate MUG (52.9 units/mg of protein) than the purified pro-rHexS (1.3 units/mg). Both enzymes, precursor rHexS and rHexS, hydrolyzed the sulfated substrate MUGS at the same rate as the neutral substrate MUG. Since the complete separation of the precursor and the processed rHexS by rechromatography caused considerable loss of material and activity we used the purified rHexS preparation still containing up to 10% precursor rHexS for the following studies.

The -subunit of native Hex S carries three potential N-linked glycosylation sites, Asn-115, -157, and -295, each of which has been found to be modified by an oligosaccharide chain in a previous study (28). We analyzed the N-glycosylation of the rHexS using proteolytic cleavage and mass spectrometric analysis of the glycopeptides separated by reverse phase HPLC. N-Glycans were linked to all three N-glycosylation sites. Asn-115 carried an N-linked GlcNAc₂Man₃ oligosaccharide as found for the processed -subunit in Hex A (29), or was not glycosylated; Asn-157 carried either GlcNAc or GlcNAc₂Man_7-9; Asn-295 carried GlcNAc, GlcNAcFuc, or GlcNAc₂Man₄ (data not shown).

To further characterize the structure of the rHexS the disulfide linkage pattern was examined using mass spectrometric analysis of proteolytic peptides as established before for the analysis of Hex B (15). We found that the postulated cystine linkage between the peptides _p and r_m (27) is formed between Cys-58 and Cys-104 (data not shown). Another disulfide bridge of the -subunit is formed by Cys-277 and Cys-328 (data not shown) as it was postulated after analysis of the native -subunit (30). The third disulfide bond was detected between Cys-505 and Cys-522 (data not shown). This disulfide linkage pattern corresponds to that of the homologous -subunit in Hex B (15). The -subunit contains an additional cysteine residue in position 125. Both cystein residues, Cys-125 and Cys-458 of the unreduced and active enzyme protein could easily be alkylated by iodoacetamide, indicating that they are not involved in any disulfide bridge (data not shown). Taken together, the glycosylation and disulfide pattern of the recombinant -subunit in rHexS are in good agreement with data obtained for the hexosaminidase subunits in other studies (15, 27-30).

The rHexS Cleaves the Synthetic Substrates MUG and MUGS at High Rates-- The purified rHexS showed higher specific activities toward the artificial substrates MUG and MUGS (Fig. 1A) than described previously for partially purified Hex S preparations obtained from liver and an apparently homogeneous preparation from human fibroblasts (4, 5). As given in Table II the corresponding V_max values were of the same order of magnitude as for Hex A. Interestingly, rHexS catalyzed the hydrolysis of the anionic MUGS substrate even at a higher rate than the other isozymes (Table II). rHexS hydrolyzed the neutral substrate MUG optimally at pH 4.5 (data not shown) as did Hex S and Hex A prepared from human liver (3). The pH optimum of MUGS degradation by rHexS was at pH 3.8 (data not shown).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Structures susceptible to Hex S. A, synthetic substrates. MUG and MUGS were used for the analysis of the kinetic properties of rHexS. B, anionic glycolipids. C, anionic oligosaccharides. C6S-3 is a trisaccharide derived from chondroitin 6-sulfate. DS-5 is the pentasaccharide accumulated in the urine of hexosaminidase double knockout mice derived from partial dermatan sulfate degradation. According to MS data it carries two sulfate ester groups in position 4 of the GalNAc residues (Fig. 5B, see also Fig. 6). As shown by enzymatic digestion the GalNAc residue at the nonreducing end is not sulfated in this molecule (Fig. 7). Digestion by chondroitinases ABC and AC indicated that the anionic storage oligosaccharides contained both iduronic and glucuronic acid residues. However, their position in the oligosaccharide chain has not been determined. Thus, the DS-5 structure shown here is one of four constitutional isomers. D, neutral oligosaccharide. The N-glycan fragment structure H3N3 is the main component of the neutral storage glycans in the urine of hexosaminidase double knockout mice and Hexb / mice (Fig. 2).

Neutral Oligosaccharides Are Excreted in the Urine of Hexosaminidase Knockout Mice-- Uncharged glycans were found in the urine of both, the Hexb / mice and the double knockout mice (Hexa /; Hexb /) (Fig. 2). The most abundant neutral storage oligosaccharide was isolated and analyzed by ESI-MS/MS (data not shown) and enzymatic degradation by hexosaminidases (Fig. 3). Based on the data obtained from this and additional monosaccharide and methylation analysis (31) we assign the following structure to the major neutral storage glycan: GlcNAc(1-2)Man(1-3)[GlcNAc(1-2)Man(1-6)]Man(1-4)GlcNAc (H₃N₃, Fig. 1D) which appears to be a product of partial N-glycan degradation (data not shown). Other neutral glycans were detected by MALDI-MS that contained 3 hexose (H) and 3-6 N-acetylhexosamine residues (N) and 4 hexose and 4-7 N-acetylhexosamine residues. In the urine of Hexb / mice, ions that could be assigned to a saccharide exclusively composed of N-acetylhexosamine and hexose residues were observed up to m/z = 2841.4 and corresponded to the sodium adduct of H₆N₉ (calculated average mass: 2841.6 Da). In the urine of double knockout mice, H_xN_y compounds were detected up to a signal at m/z 3816.4 corresponding to H₇N₁₃ (calculated average mass: 3816.5 Da; data not shown). In the urine of human patients deficient in Hex A and B activity, H₃N₃, two structural isomers of H₃N₄, and smaller fragments have been identified by NMR spectroscopy (32) carrying only N-acetylglucosamine residues at their non reducing ends. The structure of H₃N₃ obtained from human urine is identical to the N-glycan fragment H₃N₃ that we characterized in the knockout mice (Fig. 1D).

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Excretion of neutral glycans in the urine of the hexosaminidase deficient mice. The urine of mice deficient in Hex A and S (Hexa /) and deficient in Hex A and B (Hexb /) as well as the urine of mice totally deficient in hexosaminidase activity (double knockout) and of wild type littermates was collected for 8 h/day in a metabolism cage. The neutral oligosaccharides were prepared by solid phase extraction on Carbograph columns. The neutral glycans were eluted with 25% (v/v) acetonitrile in water. Aliquots of desalted glycan fractions corresponding to equal amounts of urine were evaporated to dryness and, together with a maltose oligosaccharide standard (4 to 10 glucose units), were profiled by FACE. This included fluorescence labeling with 8-aminonaphthalene-1,3,6-tris-sulfonic acid and subsequent electrophoresis on a 32% polyacrylamide gel.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Enzymic degradation of the neutral glycan H3N3 by rHexS and Hex B. Equal amounts of the neutral glycan H3N3 isolated from urine of Hexb / mice corresponding to ~1 to 5 nmol of oligosaccharide were incubated for 17 h at pH 4.5 without enzyme (upper panel) and with rHexS (middle panel), respectively. In the bottom panel the glycan H3N3 was incubated for 15 h with Hex B at pH 5.2. Reaction products were analyzed by MALDI-MS. In the enzyme blank incubated under these conditions no glycan degradation was observed. H, hexose; N, N-acetylhexosamine.

Neutral Storage Oligosaccharides Are Preferentially Degraded by Hex B-- The N-glycan fragment H₃N₃ (Fig. 1D) was isolated from the urine of Hex B-deficient mice. Long-term incubation with rHexS yielded only minor amounts of degradation products as detected in MALDI-MS (Fig. 3). The bottom panel in Fig. 3 shows that this substrate was most efficiently degraded by Hex B.

The Anionic Urinary Oligosaccharides Are Fragments of Dermatan Sulfate Degradation-- The anionic storage glycans were prepared from the urine of the double knockout mice by anion exchange chromatography (Fig. 4). Elution with a buffer containing 150 mM LiCl (A) yielded a single, low molecular weight oligosaccharide termed DS-5 in fraction A4 (Fig. 4) whereas elution with 2.0 M LiCl (B) yielded a complex mixture of anionic oligosaccharides including DS-5 in fraction B4 (Fig. 4). This anionic oligosaccharide mixture was named GAG OS. Both, DS-5 and the GAG OS mixture, were analyzed structurally by ESI-MS/MS as well as by enzymatic degradation (Figs. 5-7). Fig. 5A shows a negative mode ESI-TOF-MS spectrum of the GAG OS mixture. Within the series of multiply charged molecular ions the negative charge increases with decreasing m/z values. The masses calculated from these m/z values each differ by 459 Da, by the expected mass of a disaccharide composed of a sulfated N-acetylhexosamine and a uronic acid residue as it occurs in galactosaminoglycans but not in keratan sulfate. The molecular mass of the free acid DS-5 was determined to be 1139.21 Da, which is identical to the molecular mass of a bis-sulfated GAG pentasaccharide composed of three N-acetylhexosamine and two uronic acid residues (1139.23 Da). Using a triple quadrupole ESI-MS/MS in the negative precursor ion mode, the pure DS-5 sample was scanned for molecular ions that should contain sulfate groups. Only molecular ions were scanned that upon fragmentation in the collision chamber gave rise to the fragment m/z 97, indicating the presence of sulfate or phosphate groups. By this, we detected the singly charged alkali metal ion adducts of DS-5 that corresponded by the loss of one Li⁺ to the doubly charged ions found by ESI-TOF-MS previously (Fig. 5B). To confirm that DS-5 contains sulfate but not phosphate we performed an ESI-MS/MS product ion scan of the molecular ion [M  4H⁺ + Li⁺ + Na⁺]² (m/z 582.6, M = DS-5) (Fig. 5C). By increasing the collision energy 4.7-fold (inserted spectrum) a new fragment of m/z 80 (due to [·SO₃]) but not m/z 79 (due to [PO₃]) appeared (33). Further fragments of this product ion scan confirmed the existence of a sulfated N-acetylhexosamine (HexNAc) residue [HexNAc-sulfate  H₃O⁺] at m/z 282 and a monosulfated trisaccharide containing one uronic acid (HexUA) and two HexNAc residues in complex with either a Li⁺ or Na⁺ (m/z 685 or 701: [sulfated (HexNAc)₂HexUA  2 H⁺ +(Li⁺ or Na⁺)] and m/z 667 or 683 due to a further loss of H₂O (18 Da), respectively).

View larger version (49K): [in this window] [in a new window]   Fig. 4.   Preparation of the anionic storage oligosaccharides DS-5 and GAG OS from the hexosaminidase double knockout mice. The urine of mice totally deficient in hexosaminidase activity was applied to an anion exchange column which was eluted by 150 mM LiCl (A) or 2.0 M LiCl (B). The eluates A and B were separately desalted by size exclusion chromatography. The fractions of the desalting step were subjected to FACE analysis.

View larger version (36K): [in this window] [in a new window]   Fig. 5.   ESI-MS/MS analysis of the GAG OS and DS-5. The GAG pentasaccharide DS-5 was separated from the urine of hexosaminidase double knockout mice on a DEAE-Sephacel column by elution with 150 mM LiCl. The fraction named GAG OS was eluted from the column by 2.0 M LiCl. Prior to negative mode mass spectrometry the samples were desalted by size exclusion chromatography on TSK-Gel HW40. A, ESI-TOF-MS of the GAG OS. The m/z values of the multiply charged molecular ions point to the following molecular masses 1139.20 Da (DS-5), 1598.28 Da (DS-7), 2057.31 Da (DS-9), and 2516.39 Da (DS-11). The theoretical molecular mass of the structure assigned to DS-5 in Fig. 1C is 1139.23 Da. The higher polymers DS-7, DS-9, and DS-11 produce signals at lower m/z values due to additional negative charge. Signals derived from alkali metal ion adducts are labeled in italics. B, precursor ion scan of m/z 97 in negative mode ES-MS/MS of DS-5. Each ion appearing in this spectrum contains a 97-Da fragment corresponding to HSO. C, daughter ion scan of [M - 4H+ + Li+ + Na+]2, M = DS-5. Among the singly charged fragment ions derived from DS-5 are HSO (97 Da), a monosulfated HexNAc residue (282 Da) and a lithium as well as a sodium adduct of a sulfated trisaccharide containing one uronic acid and two HexNAc residues (Li+-complex: m/z 685 and 667 (-H2O) and Na+ complex: m/z 701 and 683 (-H2O). This confirms the structure we propose for DS-5 in Fig. 1C. Inset, to detect the sulfate specific fragment [·SO3] (m/z 80) the collision energy was increased 4.7-fold. HexNAc, N-acetylhexosamine; HexUA, hexuronic acid.

To determine whether DS-5 and the GAG OS are derived from either chondroitin or dermatan sulfate the GAG OS were incubated with the bacterial endolyases chondroitinase AC (EC 4.2.2.5) and chondroitinase ABC (EC 4.2.2.4). Whereas chondroitinase AC specifically hydrolyzes chondroitin sulfate and chondroitin sulfate-like units in polysaccharide chains, chondroitinase ABC additionally hydrolyzes dermatan sulfate (34). Both enzymes produce galactosaminoglycan disaccharides unsaturated between C4 and C5 of the hexuronic acid residues when incubated with appropriate substrates (34). By FACE analysis chondroitinase AC activity caused a shift of the complete ladder of oligosaccharides with the formation of low molecular weight products. However, degradation was not complete as it was after incubation with chondroitinase ABC (data not shown). The products of chondroitinase ABC digestion were analyzed by negative mode ESI-MS/MS. The main signal at m/z = 458.07 was identified as a deprotonated monosulfated, unsaturated disaccharide (Fig. 6, calculated molecular mass 458.07 Da). Therefore, the origin of the GAG OS including the pentasaccharide DS-5 is dermatan sulfate.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   ESI-MS/MS of disaccharides derived from chondroitinase ABC digestion of the GAG OS isolated from the urine of the hexosaminidase double knockout mice. Panels A-C show the characteristic fragmentation patterns for the standard disaccharides Di-2-sulfate (A), Di-4-sulfate (B), and Di-6-sulfate (C), respectively. Panel D shows the fragmentation analysis of the disaccharide product formed by chondroitinase ABC digestion of the GAG OS. HexNAc, N-acetylhexosamine; HexUA, hexuronic acid.

The Repeating Disaccharide Unit in the Urinary GAG OS Is Sulfated in Position 4 of the GalNAc Residue-- In ESI-MS/MS analysis the unsaturated disaccharide formed by chondroitinase ABC digestion of the GAG OS showed the same fragmentation pattern (Fig. 6D) as the unsaturated standard disaccharide Di-4-sulfate which is sulfated in position 4 of the GalNAc residue (Fig. 6B). We conclude that the dermatan sulfate fragments GAG OS and, thus DS-5, are sulfated in position 4 of the GalNAc residues.

rHexS and Hex A Degrade the Dermatan Sulfate Fragment Mixture GAG OS and the Dermatan Sulfate Pentasaccharide DS-5-- The anionic GAG OS fragments isolated from the urine of the hexosaminidase double knockout mice were degraded in vitro by rHexS and Hex A (Fig. 7A, lanes 2 and 3), but not by Hex B (Fig. 7A, lane 1). Incubation of the GAG OS or DS-5 with rHexS and Hex A, respectively, gave rise to two products (Fig. 7A, lanes 2 and 3, B, lanes 3 and 4), one of them comigrating with GalNAc. The second degradation product, derived from the pentasaccharide DS-5 by loss of one monosaccharide unit is termed DS-4. As indicated by the FACE gel in Fig. 7C, N-acetylgalactosamine (lane 3) but not -glucosamine (lane 2) or -galactosamine 6-sulfate (lane 4) was the monosaccharide released from DS-5 (lane 1). This indicates that an exo-sulfatase acted on the nonreducing sugar residue in vivo before DS-5 was excreted with the urine and confirms that the pentasaccharide DS-5 is derived from a galactosaminoglycan. Together with the structural information obtained from mass spectrometry and chondroitinase digestion this suggests the structure of the dermatan sulfate pentasaccharide DS-5 suggested in Fig. 1C.

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Degradation of the dermatan sulfate fragments GAG OS and DS-5 by the hexosaminidase isozymes. A, equal amounts of the anionic GAG OS corresponding to about 0.5-2 nmol/oligosaccharide were incubated for 9 h with the following enzymes: Hex B (lane 1), rHexS (lane 2), and Hex A (lane 3). A standard mixture containing GalNAc-6-sulfate and a maltooligosaccharide mixture ranging from 4 to 10 glucose units was applied to lane 4. The assays were run and analyzed by FACE profiling as described under "Experimental Procedures." B, hydrolysis of DS-5 by rHexS and Hex A. Equal amounts of DS-5 corresponding to 0.5-2 nmol of oligosaccharide were incubated for 5 h with Hex A (lane 3) and rHexS (lane 4). Lane 1 contains neutral standard glycans and lane 2 contains an enzyme blank as negative control. The arrows with a question mark point to bands that could not be identified. However, since they are observed in all incubated samples including the enzyme blank in lane 2 they are not formed by hexosaminidase action. C, comparison of DS-5 degradation products with neutral monosaccharides. The products of DS-5 degradation by Hex A (DS-5 + Hex A, lane 1) were compared with standards of GlcNAc (lane 2), GalNAc (lane 3), and GalNAc-6-sulfate (lane 4) in FACE analysis.

rHexS and Hex A Degrade the Anionic Trisaccharide C6S-3-- To study the degradation of chondroitin 6-sulfate by hexosaminidases we prepared the bis-sulfated trisaccharide C6S-3 (Fig. 1C) from commercial chondroitin 6-sulfate using testicular hyaluronidase and -glucuronidase. After incubation of C6S-3 with the hexosaminidase isozymes the reaction products were separated and visualized by FACE profiling as shown in Fig. 8. In the presence of either rHexS or Hex A two new product bands appeared (lanes 3 and 4) which comigrated with a GalNAc-6-sulfate standard (lane 5) and a commercially available saturated chondroitin 6-sulfate disaccharide standard (C6S-2, lanes 1 and 9), respectively. Hex B exhibited no detectable hydrolyzing activity on C6S-3 (Fig. 8, lane 2).

View larger version (31K): [in this window] [in a new window]   Fig. 8.   Degradation of the trisaccharide C6S-3 by hexosaminidases A, B, and S. 2 nmol of C6S-3 dissolved in water were incubated for 5 h at pH 4.0 with Hex B (lane 2), rHexS (lane 3), and Hex A (lane 4), respectively, as described under "Experimental Procedures." Blanks devoid of substrate (lanes 6 and 7) and devoid of enzyme (lane 8) were run under identical conditions. After incubation, the assay samples were subjected to FACE analysis. GalNAc-6-sulfate (lane 5) and the commercially available chondroitin 6-sulfate disaccharide bearing a glucuronic acid residue at the nonreducing terminus (lanes 1 and 9) served as standards for the digestion products.

rHexS and Hex A Degrade the Sulfated Glycolipid SM2 but Not SB2-- We examined the enzymatic degradation of the monosulfated glycolipid SM2 and the bis-sulfated glycolipid SB2 by hexosaminidases. SM2 is an analogue of ganglioside GM2, and the glycolipid SB2 carries an additional sulfate ester bound to position 3 of the terminal GalNAc residue. The purified sulfated GSLs were incubated with the different hexosaminidase isozymes in a micellar assay (Fig. 9). rHexS was more active than Hex A and Hex B and catalyzed the degradation of SM2 to SM3 at significant rates even in the absence of GM2AP. Addition of GM2AP stimulated the SM2 hydrolysis by both, rHexS and Hex A. Hex B showed no significant activity in hydrolyzing SM2 in the presence or absence of GM2AP (Fig. 9A). None of the three hexosaminidases showed significant activity in degrading SB2 (Fig. 9B).

View larger version (66K): [in this window] [in a new window]   Fig. 9.   Hydrolysis of the sulfated glycosphingolipids SM2 and SB2 by hexosaminidases in the presence/absence of the GM2AP. A, SM2 micelles were incubated for 15 min with the three hexosaminidase isoenzymes Hex A, rHexS, and Hex B, respectively, in the presence and absence of GM2AP. The assays were performed as described under "Experimental Procedures." B, the same degradation experiment was performed using SB2 instead of SM2 and incubating for 17 h instead of 15 min.

To reproduce the lysosomal conditions as closely as possible, additional degradation experiments were performed using a liposomal assay system. In this system, the lipid substrate was presented as a component of a unilamellar lipid bilayer to the water-soluble enzyme. Standard liposomes contained phosphatidylcholine and cholesterol as carrier lipids, and BMP, which is a characteristic component of lysosomal and intraendosomal membranes (35, 36). Under these conditions rHexS degraded membrane-bound SM2. Significant degradation rates were achieved only in the presence of both, BMP and the GM2AP (Fig. 10A). The optimum for the reaction was pH 4.3 (Fig. 10B).

View larger version (21K): [in this window] [in a new window]   Fig. 10.   Hydrolysis of liposomal SM2 by rHexS. LUVs containing 10 mol % SM2, 20 mol % BMP, 20 mol % Chol, and 50 mol % PC were incubated with rHexS in the presence/absence of GM2AP. The assays were performed as described under "Experimental Procedures." After mild alkaline methanolysis the samples were desalted by reverse phase chromatography and applied to a HPTLC plate. The resolved bands were stained with cuprous sulfate reagent in phosphoric acid. The relative intensities of the product band, corresponding to SM3 and of the substrate band corresponding to SM2 were determined densitometrically. Blanks containing denatured enzyme were run for each assay under identical conditions and subtracted from the experimental values. A, time dependence. LUVs containing SM2 and BMP were incubated with rHexS in the presence of GM2AP in 10 mM citrate buffer () and in 50 mM citrate buffer (). LUVs composed of 10 mol % SM2, 20 mol % Chol, and 70 mol % PC but devoid of BMP were incubated with rHexS in 50 mM citrate buffer in the presence of GM2AP (). LUVs containing both SM2 and BMP were incubated with rHexS in 50 mM citrate buffer in the absence of GM2AP (). B, pH dependence. LUVs containing both SM2 and BMP were incubated for 30 min with rHexS in the presence () or absence () of GM2AP. The pH ranged from 3.6 to 5.7.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hexosaminidase S (Hex S, ) is a labile lysosomal enzyme that occurs in much smaller quantities in normal human tissues than the major isoenzymes Hex A () and Hex B (). Since rather low specific activities have been reported for purified Hex S (3-5) its physiological significance has been questioned (2). The striking accumulation of anionic oligosaccharides in the urine of mice deficient in all hexosaminidases compared with those still expressing Hex S (Hexb /) (8) prompted us to reinvestigate the substrate specificity of Hex S.

For these studies we expressed human Hex S cDNA in cultured insect cells and purified the recombinant Hex S from the secretions. All three N-glycosylation sites were utilized in the recombinant -subunit consistent with previous studies on the enzyme (28). Its disulfide linkage pattern, as analyzed by mass spectrometry after proteolytic cleavage, corresponded to that of the homologous hexosaminidase -subunit (15). rHexS hydrolyzed the synthetic anionic substrate MUGS at even higher rates than the other isozymes and was as active as Hex A on the neutral substrate MUG (Table II). Interestingly, the precursor form of rHexS showed only 2-3% of the specific molecular activity of processed, mature rHexS.

To identify physiological substrates of Hex S anionic and neutral glycans were isolated from the hexosaminidase double knockout mice (Hexa /, Hexb /) and characterized. Analysis by ESI-MS/MS combined with glycosidase digestion indicated that the isolated GAGs were derived from dermatan sulfate. In addition, elevated levels of neutral N-glycan fragments were found in the urine of the double knockout mice. This was expected from their occurrence in the urine of Hexb / mice. An anionic (DS-5) and a neutral oligosaccharide (H₃N₃) were isolated from the urine of the double knockout mice, and used as physiological substrates for rHexS. As an additional putative substrate the sulfated sphingolipid SM2 was isolated from rat kidney.

rHexS was as active as Hex A on the anionic bis-sulfated glycans, the trisaccharide C6S-3, and the pentasaccharide DS-5, and the sulfated glycolipid SM2. Each of these substrates was refractory to the action of Hex B (Table III). From these anionic substrates rHexS released N-acetylgalactosamine from the nonreducing end and, in the case of C6S-3, N-acetylgalactosamine 6-sulfate, thereby bypassing the exo-sulfatase step. The release of GalNAc-6-sulfate from keratan sulfate oligosaccharides by Hex A has been reported (37) and it was postulated that hexosaminidases are able to release sulfated hexosamines in vivo from the observation that 6- and 4-sulfated hexosamines are excreted with the urine of patients deficient in GAG specific sulfatase activity (38). However, if the terminal N-acetylgalactosamine residue is sulfated in the 3-position, as in the glycolipid SB2, it is resistant against the action of all three hexosaminidases A, B, and S. As with the degradation of other anionic and neutral sphingolipids (39-43), the hydrolysis of the membrane-bound sulfated glycosphingolipid SM2 by rHexS is strongly dependent on both, an activator protein, in this case GM2AP, and the presence of an additional anionic phospholipid such as BMP in the substrate carrying membrane.

As observed by quantitative ESI-MS/MS analysis elevated levels of SM2 were found in the kidney tissues of both Hexa / mice and double knockout mice, both lacking Hex S and Hex A activities.² This suggests an important role of Hex S in the catabolism of SM2, a potentially important glycosphingolipid. High levels of SM2 have been observed in the human renal cancer cell line SMKT-R3 (44) as well as in a uterine endometrial adenocarcinoma (45). Moreover, SM2 and the bis-sulfated SB2 have been identified as the most potent glycosphingolipid ligands for NKR-P1, a membrane protein on natural killer cells that contains an extracellular C type lectin domain, and their contribution to the activation of natural killer cells has been proposed (46).

Comparing the hexosaminidase concentrations and incubation times used in vitro, anionic glycolipids were degraded much faster than anionic oligosaccharides. Rough calculations suggest that the sulfated GSL SM2 is degraded 500 times faster than the sulfated pentasaccharide DS-5 (Fig. 7B and 9A). Slow degradation rates have also been described for digestion of oligosaccharides by other glycosidases, e.g. hyaluronidase and -glucuronidase (34). Presumably, the suitable substrate conformation and the accessibility of the sensitive linkages for the enzyme is less often achieved in a freely movable, water-soluble oligosaccharide than in a glycolipid which may be conformatively fixed in an activator-lipid complex.

The results obtained with Hex A and rHexS expand the array of physiological glycoconjugate substrates acted upon by the lysosomal hexosaminidases. They also add support to the concept that Hex S catalyzes an important set of degradative reactions in vivo. Finally, the unique substrate specificity of Hex S make the enzyme an interesting and valuable research tool in glycobiology.

	ACKNOWLEDGEMENTS

We thank Olaf Wilke for expert assistance in breeding the mice and the group of Rudolf Geyer, Giessen, Germany, for monosaccharide and linkage analysis of the neutral storage glycan H₃N₃ purified from the urine of the Hex B knockout mice. We thank Dr. Christina Schütte, Dr. Bernd Liessem, Michaela Wendeler, and Norbert Werth for providing the hexosaminidase A and B isozymes and GM2AP. We also thank Prof. F. T. Wieland (BZH/University of Heidelberg) for providing the triple Quadrupole ESI-MS/MS.

	FOOTNOTES

^* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 284 and the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Kekulé-Institut für Organische Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany. Tel.: 49-228-735834; Fax: 49-228-737778; E-mail: sandhoff@uni-bonn.de.

Published, JBC Papers in Press, November 13, 2001, DOI 10.1074/jbc.M105457200

² R. Sandhoff, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: Hex, -hexosaminidase; BMP, bis(monoacylglycero)phosphate; Chol, cholesterol; C6S-2, chondroitin-6-sulfate disaccharide; C6S-3, GalNAc-6-sulfate(1-4)GlcUA(1-3)GalNAc-6-sulfate; CVE, centrifugal vacuum evaporator; Di-2-sulfate, _4,5-GlcUA-2-sulfate-(1-3)GalNAc; Di-4-sulfate, _4,5-GlcUA(1-3)GalNAc-4-sulfate; Di-6-sulfate, _4,5-GlcUA(1-3)GalNAc-6-sulfate; DS-4, dermatan sulfate tetrasaccharide; DS-5, dermatan sulfate pentasaccharide; ESI-MS, electrospray ionization mass spectrometry; FACE, fluorophore-assisted carbohydrate electrophoresis; GAG, glycosaminoglycan; GAG OS, glycosaminoglycan oligosaccharide mixture; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; GlcUA, glucuronic acid; GM2, GalNAc (1-4)Gal[(2-3)NeuAc](1-4)Glc(1-1)Cer; GM2AP, GM2 activator protein; GSLs, glycosphingolipids; HexNAc, N-acetylhexosamine; HexUA, hexuronic acid; HPAEC, high pH anion exchange chromatography; LUV, large unilamellar vesicles; MALDI, matrix-assisted laser desorption mass spectrometry; Man, mannose; MUG, 4-methylumbelliferyl-2-acetamido-2-deoxy--D-glucopyranoside; MUGS, 4-methylumbelliferyl-2-acetamido-2-deoxy--D-glucopyranoside-6-sulfate; m/z, mass to charge ratio; PC, phosphatidylcholine (egg yolk); rHexS, recombinant Hex S; r_m, one of two polypeptides in the proteolytically processed recombinant -subunit (Gly⁸⁵-Thr⁵²⁹); SB2, GalNAc-3-sulfate(1-4)Gal-3-sulfate(1-4)Glc(1-1)Cer; SM2, GalNAc(1-4)Gal-3-sulfate(1-4)Glc(1-1)Cer; SM3, Gal-3-sulfate(1-4)Glc(1-1) Cer; SM4, Gal-3-sulfate(1-1)Cer; HPTLC, high performance thin layer chromatography; HPLC, high performance liquid chromatography.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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