THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No. 1, Igsue of January 10, pp. 224-232, 1982 Prrnted m U.S.A.

Structure Analysis of the Major Oligosaccharides Canine GM, Gangliosidosis Liver*

Accumulating in

(Received for publication, July 27, 1981)

Thomas G. Warner and John S. O’BrienS From the Department of Neurosciences, University of California, Sun Diego School of Medicine, La Jolla, California 92093

A homologous series of structurally related, high molecular weight oligosaccharides have been isolated and purified from the livers of a mixed breed of Beagle dogs affected with GMI gangliosidosis. Five individual oligosaccharide fractions were purified by charcoal chromatography, preparative silicic acid thin layer chromatography, and gel filtration chromatography. Molecular size determinations revealed that these oligosac- charides contained 6, 9, 11, and 13 sugar residues, respectively. Detailed structure analysis was carried out on the most abundant fractions, oligosaccharides 1,2 and 3 (OS 1,2 and OS 3) using permethylation analysis and 360-MHz proton magnetic resonance spectroscopy coupled with sequential exoglycosidase degradation. OS 1,2 was a mixture of two linear isomeric hexasaccharides and OS 3 was a nonasaccharide containing a bianntenary branched mannosyl core. The proposed structures are:

Oligosaccharide 1,2 Gal B(1-4)GlcNAc B(1-2)Man a(1-3)Man B(1-4)GlcNAc B(1-4)GlcNAc Gal B(1-4)GlcNAc p(1-2)Man a(1-6)Man P(1-4)GlcNAc B(1-4)GlcNAc

Oligosaccharide 3 Gal P(1-4)GlcNAc B(1-2)Man a(1-3)

\

/

These compounds are nearly identical with the oligosaccharides stored in human GMI gangliosidosis liver but they differ from the human compounds uniquely since they contain 2 GlcNAc residues at the reducing terminus instead of 1, suggesting that there may be significant differences in glycoprotein metabolism or structure between mam- malian species.

Man B(1-4)GlcNAc B(1-4)GlcNAc

Gal B(1-4)GlcNAc B(1-2)Man a(1-6)

GMI gangliosidosis is a lysosomal neurovisceral storage disorder which results from a near total deficiency of acid p- galactosidase activity (1). As a result of the severely depressed enzyme levels, two major classes of storage products accu- mulate, the ganglioside GMI and water-soluble galactose-con- taining glycoconjugates (1,2). Although the disorder is rare in humans, it is expressed with varying degrees of onset and phenotype (3) and it has been discovered in other mammals including cats (4-6), cattle (7), and dogs (8).

Rigorous structural analysis of the water-soluble glycocon- jugates accumulating in human GMI gangliosidosis liver was initially reported by Wolfe et al. (2). Subsequently, urinary excretion products have also been analyzed in detail (9) and recent reports of more complex structures in GM1 gangliosi- dosis urine have appeared (10). These galactosyl oligosaccha- rides are of interest and importance since they presumably

* This work was supported by grants from the Gould Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. * Supported by National Institutes of Health Grants GM17702 and NS08682.

reflect the partially hydrolyzed oligosaccharide side chains of a broad spectrum of cellular glycoproteins which are subject to lysosomal catabolism. Moreover, the oligosaccharide com- ponents of many glycoproteins including a-1 protease inhibitor (ll), human ceruloplasm (12), plasma a-1 acid glycoprotein (13), rat lactalbumin (14), and human plasminogen (15) are sialylated or fucosylated derivatives of the major oligosaccha- rides accumulating in human GM1 gangliosidosis tissue.

Recently, a canine form of GM1 gangliosidosis was described (8) and the residual ,&galactosidase activity in tissue of this affected animal has been characterized (16). We report here detailed structural analysis of the major accumulating hepatic oligosaccharides in the canine disorder and compare their structures to those found in human GM1 gangliosidosis, Type 1.

MATERIALS AND METHODS

Reagents-Sialyl lactose, sodium borohydride, /3-galactosidase (from Escherichia coli), grade VIII, ,&hexosaminidase, and a-man- nosidase (from the jack bean) were obtained from Sigma. The latter two glycosidases were dialyzed overnight against 1 liter of 0.01 M phosphate buffer, pH 7.2, a t 4 “C prior to use. Glacial acetic acid and sulfuric acid, employed for oligosaccharide hydrolysis, were Ultrex grade from J. T. Baker, Philipsburg, NJ. Acetic anhydride and iodo-

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Oligosaccharides of Canine GMl Gangliosidosis 225

methane (Alrich) were freshly distilled before use. All other solvents were of reagent grade or better and were used without further purification.

Tissue Source-A breeding colony of Beagle dogs was established at University of California San Diego. Affected animals and normal siblings were obtained by the mating of two adult Beagle dogs previously diagnosed by enzyme assay (16) as heterozygote-carriers for GMI gangliosidosis. Normal and GMI gangliosidosis canine liver, and human GMI gangliosidosis, Type 1, liver were obtained immedi- ately after autopsy and were stored at -20 "C.

Isolation and Purification of GMI Dog Oligosaccharides-01igo- saccharides were isolated from GMI dog liver (alternatively, GMI human liver) by homogenization of frozen tissue (3 g of tissue/ml) in methanol/water, 9:1, followed by centrifugation (2000 X g, 15 min, 23 "C). After removal of the supernatant, the pellet was extracted twice in a similar manner, the supernatants were pooled and the solvent was removed under vacuum. The resulting residue was sus- pended in 30 ml of water and applied to a column of charcoal/Celite, 1:l (4.5 x 3.0 cm), 5 g each. The column was washed with 30 ml of water followed by 30 ml of water/ethanol, 9 1 (v/v), and these eluates were discarded. The crude oligosaccharides were eluted from the column with 150 ml of water/ethanol, 1:l (v/v). Further purification was carried out with preparative thin layer chromatography on four silicic acid (0.25 m m ) thin layer plates (20 X 20 cm) (E. Merck, Darmstadt, Germany). The plates were developed overnight in pro- panol/acetic acid/water, 3:2:2 (v/v), with a wick of several layers of filter paper fixed on the top of each plate (17). The location of the individual oligosaccharides was determined by treating the edges of the plate with orcinol spray (1% orcinol in 50% sulfuric acid) followed by heating at 100 "C for 5-10 min for color development. The appropriate areas were scraped from the plates and the residue was suspended in 10 ml of water. After centrifugation (2000 X g, 15 min) the supernatant was removed and the silicic acid pellet was extracted four additional times with 10 ml of water. The extracts were pooled and the solvent removed under vacuum.

The purified oligosaccharides were desalted by gel filtration chro- matography on a column of Bio-Gel P2 (1.0 X 90 cm) collecting 2-ml fractions. The progress of the column was monitored by TLC' and the appropriate fractions pooled, and the solvent removed under vacuum.

Glycosidase Treatment-Oligosaccharides 1,2 and 3 were sequen- tially degraded by 8-galactosidase, 8-hexosaminidase, and a-mannos- idase. In a typical preparation, the oligosaccharide (1-3 mmol) was suspended in 0.3 ml of a buffer containing 0.1 M phosphate, pH 7.3, 1 m~ MgC12, and 100 m~ 2-mercaptoethanol followed by addition of 30 units of 8-galactosidase (3000 units/ml in 0.01 m~ phosphate, pH 7 .3 ) . After incubation at 37 "C for 2 h, an equivalent amount of enzyme was added and the reaction was allowed to proceed overnight. The progress of the reaction was monitored with TLC and, after complete degradation was observed, the reaction was terminated and the enzyme precipitated by heating the reaction mixture a t 100 "C for 1-2 min. The particulate enzyme was removed by centrifugation (2000 X g, 15 min, 23 "C). An aliquot of the reaction mixture was removed and the products separated by gel fitration chromatography on Bio-Gel P2. The remaining oligosaccharides in the reaction mixture were lyophilized and subsequently treated with /?-hexosaminidase and a-mannosidase using the same protocol except that the reaction was carried out with 20 m~ acetate buffer, pH 5.0, and two additions of 5 units of 8-hexosaminidase, and in 20 mM acetate at pH 4.4 with two additions of 6.5 units of a-mannosidase.

Alditol Acetate Analysis-The carbohydrate content of the puri- fied dog oligosaccharides was determined and quantified by conver- sion of the neutral and amino sugars to their alditol acetate deriva- tives. Oligosaccharides (100-200 pg) were hydrolyzed in sealed am- pules under nitrogen in 0.5 ml of 1 N HCI at 100 "C for 10 h. After cooling to room temperature, the hydrolysis mixture was applied to a small column (1.0 X 1.5 cm) containing the anion exchange resin AG 1-X2, acetate form, 200-400 mesh (Bio-Rad). The column was eluted with 5 ml of methanol, the eluate pooled, and the solvent removed under vacuum. The resulting residue was suspended in 0.3 ml of water and the sugars were reduced with the addition of 5-7 mg of NaBH,. After 3 h a t room temperature the reaction was terminated with acetic acid. Boric acid in the reaction mixture was removed by three successive additions and evaporations of methanol. The result-

' The abbreviations used are: TLC, thin layer chromatography; OS, oligosaccharides; GlcNAc, 2-acetamido-2-deoxy-~-glucose; Man, D- mannose; Gal, D-galactose.

ing alditols were dried overnight under vacuum over phosphorus pentoxide. Acetylation was carried out in 0.3 ml of acetic anhydride and 0.2 ml of pyridine at 80 "C for 30 min. The solvent was removed under vacuum and the excess acetic anhydride removed by three successive additions and evaporations of toluene. The resulting white powder was suspended in 3 ml of chloroform and washed three times with 1 ml of water to remove salts. The solvent was removed under a stream of nitrogen and the residue suspended in 30 pl of chloroform.

The alditol acetates were separated and quantified using a Varian gas chromatograph (Model 3700) fitted with a flame ionization detec- tor using a 6-foot, 2-mm, glass column packed with SP 2340, 3% on Supelcoport, 100-200 mesh, Supelco Co., Bellefonte, PA. The column temperature was maintained at 180 "C for 6 min after injection of the sample, followed by a linear increase in temperature to 250 "C at 6 "C/min. The neutral alditol acetates were identified by comparison of their retention times to those of a commercial reference mixture (Supelco Co.). Authentic hexosamines were derivatized similarly and were employed as reference standards.

Permethylation Analysis-The oligosaccharides were also con- verted to their permethylated alditol acetates in order to determine positional linkage of the individual carbohydrates in the oligosaccha- ride chain. The method of Stellner et al. (18) was employed with the following modifications (2). Samples of oligosaccharides (100-200 pg) were dried overnight over phosphorus pentoxide under vacuum. After suspension in 0.3 ml of dimethyl sulfoxide (silylation grade) (Pierce Chemical Co., Rockford, IL), 50p1 of dimethyl sulfoxide anion solution (prepared from 300 mg of NaH in 3.0 ml of dimethyl sulfoxide) were added. The reaction mixture was sealed under nitrogen and allowed to react at room temperature with agitation for 3-4 h. Freshly distilled iodomethane, 0.5 ml, was slowly added to the reaction mixture main- taining the temperature below 20 "C. After 30 min at room tempera- ture the excess iodomethane was removed under nitrogen. The per- methylated products were suspended in 8 ml of chloroform and washed four times with 8 ml of water. After removal of solvent, the residue was subjected to acetolysis-hydrolysis by heating in 0.5 N sulfuric acid in acetic acid (0.5 ml) at 80 "C for 16 h in a sealed ampule followed by addition of 0.3 ml of water with additional heating for 4 h. The hydrolysis products were neutralized, reduced with borohy- dride, and acetylated as described for the preparation of the alditol acetates.

Gas chromatographic analysis of the resulting permethylated al- ditol acetates was carried out on columns containing (a) OV-17, 3% on chromosorb HP, 80-100 mesh, and ECNSS-M, 3% on Gas-Chrom Q, 100-200 mesh for the amino and neutral sugars and ( 6 ) OV-225, 3% on Gas-Chrom Q, 100-120 mesh, for the neutral sugars (Applied Science, State College, PA). Identification of the resulting products was made based on the reported retention times of the methylated alditol acetates (19) or by employing oligosaccharides of known struc- ture and composition, such as lactose, sialyl lactose, dichitobiose, the ganglioside GM,, and the oligosaccharides from human GMI ganglio- sidosis liver, as reference standards.

Mass Spectral Analysis-The identity of each of the methylated alditol acetates was confmed using gas chromatography-mass spec- tral analysis. Spectra were taken on an LKB spectrometer, Model 7000, using an ionization voltage of 70 eV.

Magnetic Resonance Analysis-In order to complete the structure determination and to confm the results of methylation analysis, proton magnetic resonance spectroscopy was carried out on OS 1,2 and OS 3 using a 360-MHz Oxford superconducting magnet coupled with a Nicolet 1180 E computer, operating in the Fourier transform mode using quadrature phase detection. Spectra were obtained at a sweep width of +.2000 Hz using a near 90 pulse, with a delay time between pulses of 3.0 s. Reported spectra were computer expanded to 1600 Hz and 500 Hz. Spectra were recorded with the samples main- tained at ambient temperature or in some experiments a t 64 "C.

Prior to recording spectra, the oligosaccharides were applied to ion exchange columns containing Chelex resin (Bio-Rad), 200-400 mesh, OH form, 1.0 X 1.3 cm (20), followed by four successive lyophilizations from HO'H (99.98% HO'H, low paramagnetic ion grade from Aldrich). t-Butanol was included as an internal standard (8 = 1.253 ppm) and chemical shifts were reported relative to external [3-(trimethylsily1)- tetradeuterio]sodium propionate. Coupling constants were taken from the directly observed signal spacing.

Signal assignments were made by comparison to assignments of oligosaccharides of similar structure previously reported elsewhere (20-22) and by acquiring spectra on the products obtained by sequen- tial exoglycosidase degradation of each of the respective oligosaccha- rides.

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226 Oligosaccharides of Canine GM, Gangliosidosis

RESULTS

Thin Layer Chromatography and Molecular Size Deter- mination-Tissue extracts of GMl gangliosidosis dog liver revealed a series of high molecular weight orcinol-positive oligosaccharides when analyzed with thin layer chromatog- raphy (Fig. 1). In general, the pattern of the dog oligosaccha- rides resembled the pattern obtained with the extract from human GMI gangliosidosis liver, with the second compound in the series being the most abundant. However, each of the oligosaccharides in the dog GMI gangliosidosis extract had a slightly slower relative mobility ( RF) on the plate, indicating that they were less polar or of higher molecular weight than the corresponding compounds in the human GMI gangliosi- dosis liver extract. Extracts of normal dog liver contained no detectable levels of storage material.

The first four oligosaccharide fractions in the dog GMI gangliosidosis liver extract were clearly resolved from one another with the thin layer chromatography system em- ployed, and each fraction appeared as a single spot after purification. The oligosaccharide fraction with greatest rela- tive mobility appeared as a diffuse spot on the plate after purification indicating that it may be composed of at least two components of similar molecular weight, for this reason, this fraction was designated OS 1,2. This material and fraction OS 3 comprised the major portion of the isolated dog oligosac- charides (Table I). Although some losses of material occurred during purification, the amounts of the oligosaccharides iso- lated indicate the massive amounts of material stored in the liver of the affected animal. The crude Oligosaccharide extract accounts for about 2-3% of the wet tissue weight.

Recently, Holmes and O’Brien (17) demonstrated that an approximation of the molecular size of oligosaccharides can be determined from semilogarithmic plots of the mobility of oligosaccharides on thin layer plates uersus the number of hexose residues in oligosaccharide standards of known molec- ular weight and similar carbohydrate content. We employed

A B C D E F G FIG. 1. Silicic acid thin layer chromatogram. Lane A, normal

dog liver extract; lane B, human GMI gangliosidosis liver extract; lane C, canine GM, gangliosidosis liver extract; lanes D, E, F, and G, purified GM, gangliosidosis dog oligosaccharides OS 1,2, OS 3, OS 4, and OS 5, respectively. The plate was developed overnight in pro- panol/acetic acid/water, 3:22, and oligosaccharides detected with

TABLE I Yields of purified oligosaccharides from GMI gangliosidosis dog

liver Oligosaccharides were isolated from frozen liver tissue (4 g) as

described under “Materials and Methods” and purified by preparative thin layer chromatography followed by gel filtration chromatography on Bio-Gel P-2.

Oligosaccharide Yield mg/g tissue

os 1,2 0.6 OS 3 1.5 OS 4 0.3 OS 5 0.4

4 6 8 IO 12 14

NUMBER OF HEXOSE RESIDUES

6

FIG. 2. Molecular size determination of purified GMI gan- gliosidosis dog oligosaccharides. The relative mobilities of the dog oligosaccharides (X) on silicic acid thin layer chromatography (see Fig. 1) were compared to the human GM, gangliosidosis (0) oligosaccharides. The mobilities of each oligosaccharide are calculated relative to the mobility of the human pentasaccharide fraction.

this method of analysis (Fig. 2) for determining the number of hexose residues in the puriiied GM1 dog oligosaccharides. The oligosaccharides in the human GMI liver extract were em- ployed as reference standards. These compounds have been well characterized and the first four compounds in the series contain 5, 8, 10, and 12 sugar residues, respectively (2,9, 10). These results indicated that each of the purified dog oligosac- charides contained one additional hexose residue, relative to the corresponding compounds in the human GMI gangliosi- dosis liver extract, having 6, 9, 11, and 13 residues in OS 1,2, OS 3, OS 4, and OS 5, respectively.

Composition and Methylation Analysis-The major dog oligosaccharide fractions, OS 1.2 and OS 3, were subjected to compositional analysis as their alditol acetates and as their methylated alditol acetate derivatives (Tables 11,111, and IV). Both oligosaccharides were sequentially degraded by a series of exoglycosidase treatments and the resulting oligosaccharide products were analyzed similarly. Each enzyme treatment exposed a new, nonreducing terminus of the oligosaccharide chain and, after permethylation analysis, the position of link- age of the enzyme-released carbohydrate residue on the chain was revealed. In addition, the anomeric configuration of each glycoside linkage can be ascertained since each of the enzymes employed was highly specific for a particular glycoside con- former. The penta- and octasaccharides accumulating in the liver of human GMl gangliosidosis patients were utilized as reference standards (Fig. 3).

The analysis revealed that, in general, the dog and human GMI gangliosidosis oligosaccharides were nearly identical in carbohydrate structures and composition (Table 11) except

orcinol spray reagent followed by heating for 5 min at 100 “C. that the dog OS 1,2 and OS 3 both contained one additional

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Oligosaccharides of Canine GMI Gangliosidosis 227

disubstituted N-acetylglucosamine residue (Tables I11 and IV) and this additional hexosamine residue accounts for their greater molecular size on TLC analysis.

Both the dog and human oligosaccharides contained a GlcNAc residue at the reducing terminus. Sodium borohy- dride reduction of OS 3 resulted in the formation of an N- acetylglucosaminitol residue which was identified as 4-O-ace- tyl-2-deoxy-2-(N-methylacetamido)-1,3,5,6-tetra-O-methyl- gluctitol after methylation analysis (Table 111).

/?-Galactosidase treatment of OS 3 resulted in the release of 2 galactose residues and the concomitant formation of a heptasaccharide which contained 4 GlcNAc residues. Two of these residues were identified as 1,5-di-O-acetyl-2-deoxy-2- (N-methylacetamido)-3,4,6-tri-O-methylgluctitol in the meth- ylated heptasaccharide and thus they constitute the newly generated nonreducing termini after enzyme treatment. Pre- viously, all GlcNAc residues appeared as 1,4,5-tri-O-acetyl-2- deoxy-2-( N-methylacetamido)-3,6-di-O-methylgluctitol in the

TABLE I1 Alditol acetate analysis of OS 1,2, OS 3, and exoglycosidase

degradation products The oligosaccharides were converted to their alditol acetate deriv-

atives described under "Materials and Methods." The derivatives were separated and quantified by gas chromatography on a column containing SP 2340 maintained at 160 "C for 6 min followed by a linear increase in temDerature to 220 "C at 6 "C/min.

Alditol acetate No. of

Oligosaccharide hexose Gal- mido-2- 2-Aceta-

residues" %ti actitol deoxyglucti- tolb

mol ratio os 1,2 6 2.27 1.01 2.72 OS 3 9 3.10 2.15 3.76 OS 3 P-galactosidase-treated 7 3.05 3.95 OS 3 P-galactosidase- and P- 5 2.85 2.14

OS 3 P-galactosidase, P-hex- 3 0.93 2.07 hexosaminidase treated

osaminidase, and a-mannos- idase treated a The number of hexose residues in each oligosaccharide was de-

termined by TLC analysis using the oligosaccharides in human GM, gangliosidosis liver as standards (see Fig. 2).

bDegradation products of the hexosamine residue which form during acid hydrolysis were included here in order to accurately determine the amounts of this sugar residue.

~~~

methylated OS 3 product. On this basis, it can be concluded that the 2 galactose residues occupied the terminal position in the intact OS 3 chain and that they were linked Dl-4 to 2 penultimate GlcNAc residues.

Subsequently, degradation of the resulting heptasaccharide with P-hexosaminidase released 2 of the 4 N-acetylhexosamine residues with the formation of a pentasaccharide. Methylation analysis of this product indicated the presence of two 1,5-di- O-acety1-2,3,4,6-tetra-O-methylmannitol residues located at the nonreducing terminus after enzyme degradation. These residues were previously disubstituted in the heptasaccharide precursor and were identified as 1,2,5-tri-O-acety1-3,4,6-tri-O- methylmannitol. This demonstrates that the enzyme-released GlcNAc residues were linked Pl-2 to these 2 mannose resi- dues. The pentasaccharide also contained 1 additional man- nose residue which was trisubstituted at positions 1, 3, and 6 and was identified as 1,3,5,6-tetra-O-acety1-2,4-di-O-methyl- mannitol in the methylated product.

Degradation of the pentasaccharide with a-mannosidase released 2 of the 3 mannose residues and resulted in the formation of a trisaccharide which contained one terminal mannose residue. This was converted to 1,5-di-O-acetyl- 2,3,4,6-0-methylmannitol with methylation analysis. It can be concluded that a-mannosidase-released mannose residues had been linked al-3 and a1-6 to the trisubstituted mannose residue.

Two 1,4,5-tri-0-acetyl-2-deoxy-2-(N-methylacetamido)- 3,6-di-O-methylgluctitol residues were identified in the a-man- nosidase-generated trisaccharide. Since it was previously determined that 1 GlcNAc residue occupied the reducing terminus, the terminal nonreducing mannose residue must be linked 1-4 to the remaining GlcNAc residue (probably in P-linkage) which in turn is linked 1-4 to the reducing GlcNAc terminus. However, since this trisaccharide was not degraded by additional P-hexosaminidase or a-mannosidase treatment, the anomeric configuration of the two remaining glycosidic linkages cannot be determined unequivocally. These results indicate the following structure for the P-hexosaminidase- generated pentasaccharide:

Man a(1-3) \

/ Man 1-4 GlcNAc 1-4 GlcNAc

Man a(1-6)

TABLE I11 Methylation analysis of OS 3 and exoglycosidase degradation products

The nonasaccharide OS 3 and the reaction products derived by gxoglycosidase treatment were permethylated in dimethylsulfoxide and iodomethane and, following acid hydrolysis, they were converted to their alditol acetates as described under "Materials and Methods." The permethylated alditol acetates were separated by gas chromatography on columns containing OV-17, maintained at 160 "C for 6 min followed by an increase in temperature to 220 "C at 2 "C/min, and OV-225, maintained at 170 "C.

Permethylated alditol acetate'

mol ratio mol ratio mol ratio GM1 human octasaccharide OS 3

2.95 3.84

OS 3 borohydride reduced 1.83 2.03 1.50 0.566 3.02 OS 3 P-galactosidase treated 1.76 1.27 1.50 1.60 OS 3 P-galactosidase and P-hexosaminidase 1.80 1.29 1.81

OS 3 P-galactosidase, P-hexosaminidase, and 0.98 2.02

1.76 1.84 1.44 1.73 2.06 1.35

treated

a-mannosidase treated a It is implicit that the hydroxyl positions which are not methylated contain acetyl groups. * Borohydride-reduced dichitobiose and GM1 human octasaccharide were employed as reference standards for this methylated alditol

acetate.

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228 Oligosaccharides of Canine GMl Gangliosidosis

TABLE IV Methylation analysis of OS 1,2 and exoglycosidase degradation products

The GM1 dog hexasaccharide fraction OS 1,2 and its exoglycosidase derived products were converted to their permethylated alditol acetate derivatives as described under "Materials and Methods." The carbohydrate derivatives were separated by gas chromatography on columns containing OV-17,OV-225, and ECNSS-M. Chromatography on ECNSS-M was carried out at 160 "C for 6 min followed by an increase to 200 "C at 3 "C min.

Oligosaccharide

Permethylated alditol acetate"

Galactitol (7 R d 6-

2-N-Methylacetamido- Mannitol 2-deoxygluctitol

tetra-OMe) 2,3,4,6- 3,4,6-Tri- 2,4,6-Tri- 2,3,4-Tri- 3,4,6-Tri- 3,6-Di- x-,-, -,-

Tetra-OMe OMe OMe OMe OMe OMe mol ratio mol ratio mol ratio

GMI human pentasaccharide 0.79 1.32 0.56 0.48 1.86 os 1,2 0.81 1.21 0.41 0.61 2.91 OS 1,2 P-galactosidase treated 1.25 0.35 0.89 0.76 1.80 OS 1,2 P-galactosidase and P-hexosaminidase 1.00 0.41 0.54 2.05

OS 1,2 P-galactosidase, P-hexosaminidase, and 1.02 1.98 treated

a-mannosidase treated a All hydroxyl positions that are not methylated contain acetyl groups.

1. Gal P(1-4)GlcNAc p(l-2)Man (~(1-3) \

/ Man P(1-4)GlcNAc

Gal P(1-4)GlcNAc p(l-2)Man a(1-6) 2. Gal P(1-4)GlcNAc P(1-2)Man a(1-3)Man P(1-4)GlcNAc

Gal P(l-4)GlcNAc P(1-2)Man a(1-6)Man P(1-4)GlcNAc FIG. 3. Human GM1 gangliosidosis hepatic oligosaccharides

employed as standards for permethylation analysis.

Methylation analysis of intact OS 1,2 revealed that it con- tained no trisubstituted carbohydrate residues indicating a linear, unbranched, hexasaccharide chain (Table IV). A single 1,5-di-0-acetyl-2,3,4,6-tetra-O-methylgalactitol residue was identified as the nonreducing terminus of the methylated alditol acetate derivative. Consistent with this was the release of the galactose residue after treatment of OS 1,2 with /?- galactosidase. The pentasaccharide which resulted from the enzyme treatment contained a GlcNAc residue as its non- reducing terminus, giving rise to 1,5-di-O-acetyl-2-deoxy-2- (N-methylacetamido)-3,4,6-tri-O-methylgluctitol after meth- ylation analysis. Since methylation analysis of OS 1,2 had previously demonstrated that all the GlcNAc residues were substituted at position 4, the terminal galactose must be linked /?l-4 to a penultimate GlcNAc residue.

Subsequent degradation of the pentasaccharide mixture with /?-hexosaminidase resulted in the loss of the terminal GlcNAc residue and the generation of a mannose residue at the nonreducing terminus in the resulting tetrasaccharide. In the pentasaccharide precursor, this mannose residue was sub- stituted at position 2 by the terminal GlcNAc and was iden- tified as 1,2,5-tri-O-acetyl-3,4,6-tri-O-methyhannitol in the methylated product. The methylation data indicated that OS 1,2 contained the trisaccharide unit, Gal P(1-4)GlcNAc p(1- 2)Man, which is identical with one of the outer branches of the nonasaccharide OS 3. Both 1,3,5-tri-O-acetyl-2,4,6-tri-O- methylmannitol (0.41 mol residues) and 1,5,6-tri-O-acetyl- 2,3,4-tri-O-methylmannitol (0.61 mol residues) were detected in the methylation products of OS 1,2 and its /?-galactosidase and /?-hexosaminidase degradation products (Table IV). The presence of less than whole molar ratios of these 2 residues suggested that OS 1,2 is a mixture of two structural isomers with identical carbohydrate compositions. One isomer con- tained the trisaccharide unit, Gal P(1-4)GlcNAc /?(l-B)Man, linked to position 3 of a mannose residue and the other isomer contained the same trisaccharide unit linked to position 6 of a mannose residue. Supporting this conclusion is the obser-

H O ~ H

H-2

N -Acetyl r -J-7

e /h

L , , , , , , , , , , , ~ , , , , , , l , , , l , , , l / / ~

5.2 5.0 4.8 4.6 4.4 4.2 4.0 2.2 2.0

PPM

FIG. 4. The 360-MHz 'H magnetic resonance spectra of OS 3. Signals for ring protons have been omitted. Shown are signals for anomeric protons and the acetamido methyl region. Spectra were acquired on 2 m~ solution with t-butanol as internal standard using 64 acquisitions at room temperature.

vation that further degradation with a-mannosidase resulted in the loss of both the Man 1-3 residue and the Man 1-6 residues with the formation of the trisaccharide, Man 1-4

This compound is identical with the trisaccharide obtained by the complete enzyme degradation of OS 3. The anomeric configuration of the last 3 residues could not be determined from this data.

Proton Spectra of OS 3-Spectra obtained from OS 3 (Fig. 4 and Table V) contained three multiplet signals near the anomeric proton region at S = 4.270, 4.207, and 4.126 ppm. These signals, which are assigned to Man H-2 protons were suggestive of a bianntenary mannosyl core structure of the

GlcNAc 1-4 GlcNAc.

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Oligosaccharides of Canine GMl Gangliosidosis 229

TABLE V Magnetic resonance signal assignments for GMl gangliosidosis dog oligosaccharides

The 360-MHZ proton spectra were recorded at a spectral window o f f 2000 Hz using quandrature phase detection. t-Butanol was included as an internal standard and chemical shifts are reported relative to external [3-(trimethylsily1)-tetradeuterio]sodium propionate taken from computer expanded spectra at 1600 Hz sweep width. Spectra were acquired at 22 "C for OS 3 and its glycosidase degradation products. Additional spectra were taken at 64 "C shifting the HOZH signal upfield and no additional signals were revealed. OS 1,2 spectra were recorded at 64 "C.

dase treated reduced

H-1 proton GlcNAc, a

GlcNAc, b Man, c Man, c' Man, d Man, g GlcNAc, e GlcNAc, h Gal, f Gal, i

H-2 proton Man, c Man, c' Man, d

N-Acetyl Man, g

GlcNAc, a GlcNAc, b GlcNAc, e GlcNAc, h

a5.200 p4.722

4.777

5.129 4.939 4.584 4.584 4.480' 4.477'

4.270

4.207 4.126

2.047 2.090 2.053' 2.058'

a5.196 p4.717

4.617 4.780

5.126 4.930 4.561 4.561

4.265

4.200 4.126

2.050 2.088 2.063 2.063

a5.200 p4.704

4.619 4.755

5.117 4.935

4.269

4.070 3.978

2.048 2.090

a5.196 p4.716

4.617 4.774

4.070

2.045 2.067

4.637 4.779

4.074

a5.200 p4.722

4.780 4.755 5.142 4.922 4.600 4.600

4.492' 4.488'

4.236 4.222 4.195 4.100

2.051 2.052 2.061 2.072

2.052 2.052

" Not resolved. Assignments are interchangeable.

type, Man al-3 (Man a1-6) Man ,B. Oligosaccharides derived from various glycoprotein sources that are known to contain a bianntenary mannosyl core with this structure have been reported to give signals in this region of the spectra with nearly identical chemical shifts (20, 22). This was also con- sistent with the results obtained with methylation analysis which indicated that one of the Man residues in OS 3 was trisubstituted at positions 1, 3, and 6 and served as a branch point for two outer oligosaccharide chains.

Further inspection of the OS 3 spectrum revealed a pair of closely overlapping doublets directly downfield from the Man H-2 signals at 4.480 and 4.477 ppm, J1,2 P 8 Hz; these were assigned to two terminal Gal residues in the oligosaccharide chain. The asialo-oligosaccharide side chain of human sero- transferrin (22) is similar to the anticipated structure of OS 3 and gave signals a t 4.470 ppm with a nearly identical pair of overlapping doublets as observed here. Also, the product obtained by ,B-galactosidase degradation of OS 3 did not contain these signals. The observed coupling constant of about 8 Hz was consistent with Gal residues in the p configuration.

The acetamido methyl region of OS 3 was complex; four overlapping singlets were detected. The signals at 2.053 and 2.058 ppm were the most intense and were assigned to 2 N- acetylhexosamine residues in nearly similar environments in the oligosaccharide chain. After treatment of OS 3 with ,B- galactosidase, these two singlets coalesced to a single reso- nance at 2.063 ppm, suggesting that the GlcNAc residues were probably closely linked to the terminal Gal residues. Removal of the Gal residues from the oligosaccharide chain reduced the magnetic nonequivalence of these GlcNAc residues giving rise to a single resonance. After ,&galactosidase treatment three singlet signals were clearly apparent in the acetamido region at 2.050, 2.063, and 2.088 ppm with an approximate ratio of signal intensities of 1:1.7:1, respectively. Based on these observations, the signals at 2.053 and 2.058 ppm in the

OS 3 spectrum were assigned to two penultimate GlcNAc residues, substituted by 2 terminal Gal residues. The signals for the anomeric protons for these GlcNAc residues were assigned to the distorted doublet at 4.585 ppm, J1.2 8 Hz. This assignment was based on a similar assignment for the hexosamine residues in the asialo-oligosaccharide of human serotransferrin (22) which also contains penultimate, galac- tosyl-linked GlcNAc residues. Subsequent degradation of ,B- galactosidase-treated OS 3 with ,B-hexosaminidase resulted in the loss of these signals. Also lacking was the acetamido methyl singlet a t 2.063 ppm, confirming the previous assign- ment for this resonance.

As discussed above, exoglycosidase degradation of OS 3 was completed with a-mannosidase treatment and this resulted in the formation of the trisaccharide, Man (1-4)GlcNAc (1- 4)GlcNAc. Assignment of the remaining signals in the OS 3 spectrum was facilitated by inspection of the spectra obtained with this material (Fig. 5). The furthest downfield signal in OS 3 (5.200 ppm) was present in the trisaccharide spectrum (5.196 pprn). Integration of the peak area of this signal, in both spectra, indicated that it was equivalent to less than a whole proton residue. Due to its downfield resonance and its relative peak intensity, this signal was assigned to the H-1 proton of the a anomer of the GlcNAc residue at the reducing terminus. The H-1 proton resonance of the corresponding ,B anomer was assigned to the doublet at 4.722 in OS 3 and a t 4.716 ppm, J1.2 E 8 Hz, in the trisaccharide. a-Mannosidase treatment led to a loss of the anomeric proton signals in the OS 3 spectra a t 4.939 and 5.129 ppm and the H-2 resonances a t 4.207 and 4.126 ppm. The resonance at 4.939 ppm was assigned to a Man residue a-linked 1-6 to an additional Man residue, and the signal at 5.129 ppm was attributed to a Man residue a-linked 1-3 to an additional Man residue. These assignments were made based on comparison to the assign- ments for the oligosaccharide side chain of serotransferrin

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230 Oligosaccharides of Canine GMI Gangliosidosis

H O ~ H N -Acetyl *

I I C

, 1 1 1 ~ 1 1 1 ( 1 1 1 , 1 1 1 , 1 1 1 1 , , , 1 f/"n 5.2 5.0 4.8 4.6 4.4 4.2 4.0 2.2 2 0

PPM

FIG. 5. The 360-MHz 'H magnetic resonance spectra of the trisaccharide derived from OS 3 after 8-galactosidase, 8-hex- osaminidase, and a-mannosidase treatment. Spectra were ac- quired on a 1.4-mm solution using 128 acquisitions at room tempera- ture. Ring protons have been omitted for clarity. Upper inset, ano- meric proton region after sodium borohydride reduction of the trisac- charide. The sample was 0.3 mM and 512 acquisitions were taken.

(22), a urinary hexasaccharide isolated from a patient with Sandhoff s disease (21), and the mannosyl core tetrasaccharide of human IgM protein (20), each of which contain Man residues with similar linkages and give signals in this region of their spectra.

It has been established that the a and /I anomers of mannose and mannosyl glycosides cannot be distinguished by the mag- nitude of the H-1, H-2 coupling constant (20). However, since the /I anomers resonate at higher fields than the a anomers, we conclude that the doublet at 4.774 ppm, J1,2 P 3 Hz, can be assigned to the terminal Man residue in the trisaccharide and that it is P-linked to the adjacent GlcNAc residue. This accounts for the observation that although this Man residue is in a terminal nonreducing position of the trisaccharide chain, it is not released by extensive treatment with a-man- nosidase, since the enzyme is highly specific and does not degrade P-linked mannosyl glycosides. The apparent doublet at 4.070 ppm is assigned to the H-2 proton of this residue.

The remaining unassigned signals in the anomeric proton region of the trisaccharide were two overlapping doublets at about 4.617 ppm, J1.2 8 Hz. These resonances were partially obscured in the original OS 3 spectra, being situated under the envelope of the H-1 resonance of the 2 outer chain GlcNAc residues. The presence of two sets of doublets with nearly identical chemical shifts could suggest: (a) the presence of structural isomers, each giving rise to a separate resonance for this residue or ( b ) a conformational induced nonequivalence for the H-1 proton of this residue. The former possibility is inconsistent with the methylation results which clearly indi- cated that the Man residue was linked solely to position 4 of the interior GlcNAc and this residue was also exclusively linked to position 4 of the reducing GlcNAc terminus, no other linkages were observed. In order to test the later possibility, the trisaccharide was reduced with sodium borohydride and spectra were acquired on the resulting product (Fig. 5). After distruction of the anomeric center at the reducing terminus

and elimination of the presence of the a and /I conformers, the two doublets coalesced into a single, sharply resolved doublet at 4.637 ppm, J1,2 z 8 Hz. We conclude that the presence of each anomer in the trisaccharide creates a slightly different magnetic environment for the H-1 proton of the interior GlcNAc residue in the chain and thus accounts for the presence of two sets of doublet signals for this resonance.

The borohydride-reduced trisaccharide did not contain sig- nals at 5.196 and 4.716 ppm, supporting our earlier assign- ments as the a and /I H-1 resonances, respectively, for the terminal GlcNAc residue. The Man H-1 and H-2 resonances were not affected by chemical reduction. The magnetic reso- nance data supported the following structure for the OS 3 derived trisaccharide:

Man P(1-4)GlcNAc P(1-4)GlcNAc

Proton Spectra of OS 1,2-1n general, the proton spectra of OS 1,2 (Fig. 6 and Table V) resembled that obtained with OS 3, however, several notable differences were observed. One of the major differences was the presence of the two signals in the OS 1,2 spectrum at 4.780 and 4.755 ppm. Methylation analysis previously indicated that this oligosaccharide fraction was a mixture of two linear isomeric hexasaccharides. These two signals suggested that each hexasaccharide contained a P-linked Man residue. The bianntenary branched /I-linked Man residue in the OS 3 spectrum gave a single signal in this spectral region with an intermediate chemical shift at 4.777 ppm. The downfield signal at 5.142 ppm was consistent with a Man residue in a-linkage to position 3 of the P-linked Man residue in one of the hexasaccharide isomers. The other structural isomer contained a Man residue (4.922 ppm) a- linked to position 6 of the P-Man residue in the oligosaccharide chain. The relative ratios of the two isomers was estimated by integration of the peak areas of these signals (Fig. 6, upper inset). This indicated that the OS 1,2 mixture contained 38% of the isomer with a Man a(1-3) Man /I unit in the hexasac-

N-Acetyl A

/h

L l , . , [ , I I [ 1 V l ~ ~ ~ I T ~ ~ ~ ~ ~ ' l ' ~ + " - r -

5.2 5.0 4.8 4.6 4.4 4.2 4.0 2 2 2.0

PPM

FIG. 6. The 360-MHz 'H magnetic resonance spectra of 0s 1,2. Spectra were acquired on a 2 m~ solution at 65 "C with 64 acquisitions. Upper inset, anomeric proton signals for Man 41-3) and Man (~(1-6) residues at 500 Hz sweep width for dog OS 1,2 and the GM, human pentasaccharide fraction.

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Oligosaccharides of Canine GMl Gangliosidosis 23 1

charide chain and about 62% of the isomer with a Man 41-6) Man p unit, in good agreement with the methylation data. Interestingly, the human GM1 gangliosidosis pentasaccharide mixture (which was employed as a reference standard for the methylation analysis of OS 1,2) contained different ratios, giving 55% and 45% for the two isomeric pentasaccharides (Fig. 6, inset).

The proton spectrum of the trisaccharide resulting from enzyme treatment of OS 1,2 was similar to the spectrum of the OS 3-derived trisaccharide. A pair of overlapping doublets at 4.630 ppm, J1,2 z 8 Hz (data not shown) was assigned to the P-linked, interior GlcNAc residue. The anomeric proton signal for the terminal Man residue, 4.068 ppm, was also consistent with a P-linked glycoside. The remaining OS 1,2 signals were assigned by comparison to the assignments of the OS 3 spec- trum.

Based on the results of methylation and composition anal- ysis, sequential exoglycosidase degradation, and nuclear mag- netic resonance spectroscopy, the structures proposed for canine GM1 gangliosidosis OS 1,2 and OS 3 are:

os 1.2

f e d C b a ~ a l p(1-4)GlcNAc P(1-2)Man a(l-3)Man P(1-4)GlcNAc P(1-4)GlcNAc

I h L? C’ b a ~ a l fl(1-4)GlcNAc P(1-2)Man a(l-6)Man P(1-4)GlcNAc P(1-4)GlcNAc

OS 3

f e d Gal P(1-4)GlcNAc P(1-2)Man 41-3)

\ C b a Man P(1-4)GlcNAc P(1-4)GlcNAc

i h g / Gal p(1-4)GlcNAc B(1-2)Man 41-6)

DISCUSSION

A large number of urinary and hepatic oligosaccharides and glycoconjugates have been described in human disorders which result from deficiencies of lysosomal exoglycosidases (10, 23). Many of these stored or excreted products are struc- turally homologous, appearing to be remnants of the incom- plete catabolism of the N-acetyl lactosaminic-asparagine- linked oligosaccharide side chains of glycoproteins. Although the precise sequence of events involved in degradation of glycoproteins and their oligosaccharide components is unclear at the present time, it has been assumed that one of the earliest steps in this process is the liberation of the oligosac- charide side chain from the glycoprotein by the action of an endo-N-acetyl-p-D-ghcosaminidase (24). This enzyme hydro- lyzes the dichitobiose bond that links the oligosaccharide to the polypeptide resulting in an oligosaccharide with a single N-acetylglycosamine residue at its reducing terminus and a glycopeptide containing an N-acetylglucosamine-asparagine unit. The latter glycoconjugate is hydrolyzed by an asparatyl- N-acetyl-P-glucosaminidase (EC 3.5.2.26) (25) and the enzyme released oligosaccharide side chain is further degraded in a stepwise manner by lysosomal exoglycosidases.

All of the oligosaccharides which accumulate or are excreted in human GM, gangliosidosis terminate in a single reducing N-acetylglucosamine residue. The three oligosaccharides ac- cumulating in the GMI gangliosidosis dog, characterized here, differ from the corresponding human compounds since they terminate with 2 N-acetylglucosamine residues instead of 1. One possibility which could account for the presence of this additional GlcNAc residue is that the endoglucosaminidase enzyme present in humans, is not active in dogs and that the initial step in canine glycoprotein catabolism is protease deg-

radation of the polypeptide core. The subsequent action of the asparatyl-N-acetyl-P-glucosaminidase would give rise to an oligosaccharide with 2 GlcNAc residues at the reducing terminus. This presumes, of course, that human and dog N- acetyl-lactosaminic glycoproteins have the same type of di- chitobiosyl linkage between the polypeptide and the oligosac- charide chain. Although this scheme is theoretically possible, endo-P-D-N-acetylglucosaminidase activity has been found in rat (24), pig (26,27), and human (29) and it would be surprising if the enzyme was not also present in dogs.

A second possibility is that the endoglucosaminidase en- zyme is present in dog but the oligosaccharide side chains of dog glycoproteins are linked with 3 N-acetylglucosamine res- idues (a trichitobiosyl unit) to the asparagine residue of the protein. Endoglucosaminidase hydrolysis on this type of link- age could result in an oligosaccharide containing a dichitobio- syl unit at the reducing terminus and a polypeptide containing an N-acetylglucosamine-asparagine unit. This possibility seems unlikely since there has been no precedent for this type of structure in glycoproteins and all N-acetyl lactosaminic type oligosaccharides of glycoproteins analyzed thus far, contain only a dichitobiose linkage unit. However, the unusual storage product, Mana(l-6)Man~(l-4)GlcNAc~(1-4)GlcNAc P( 1-4)GlcNAc, has been described in bovine a-mannosidosis (28). Also a recent preliminary report (29) describes the pres- ence of the trisaccharide, Man p(1-4)GlcNAc P(1-4)GlcNAc in some tissues of goats with P-mannosidosis. Together these observations suggest, indirectly, that other types of oligosac- charide linkages in glycoproteins are plausible and that the additional N-acetylglucosaminyl residues in the dog oligosac- charides are not unique to this animal.

A third possibility is that an N-acetyl-~-D-ghcosamine transferase enzyme acts on the oligosaccharide chain after it is released from the glycoprotein by the endo-N-acetylglucos- aminidase. This biosynthetic scheme would result in the ad- dition of an extra GlcNAc residue to the oligosaccharide chain. This possibility is unlikely, however, since some unreacted oligosaccharide precursors with a single terminal GlcNAc residue would probably be expected to be present in the tissue and none were observed. Also, all transferase enzymes known, transfer sugars from sugar-nucleotide substrates to the non- reducing end of the growing oligosaccharide chain and this scheme would require the addition of the GlcNAc residue at the reducing terminus of the stored oligosaccharide.

Although neither of these three possibilities is complete, whatever mechanism accounts for the origin of these unusual compounds in canine GM1 gangliosidosis, it is apparent that there are significant differences in glycoprotein and glycocon- jugate metabolism or structure between closely related mam- malian species.

Acknowledgments-We wish to thank Dr. John Wright with the Chemistry Department at the University of California, San Diego for his help in acquiring the magnetic resonance spectra and Dr. Chris- toph Peters during his visit here from the Physiologisch-Chemisches Institute, Westfalische Wilhelms University of Munster, West Ger- many, for his helpful suggestions concerning this manuscript.

REFERENCES

1. Okada, S., and O’Brien, J. S. (1968) Science 160, 1002-1004 2. Wolfe, L. S., Senior, R. G., and Ng Ying Kin, N. M. K. (1974) J.

Bwl. Chem. 249, 1828-1838 3. O’Brien, J. S. (1978) in The Metabolic Basis of Inherited Disease

(Stanbury, J. B., Wyngaarden, J. B., and Fredrickson, D. S., eds) pp. 841-867, McGraw-Hill, New York

4. Baker, H. J., Lindsey, J. R., McKann, G. M., and Farrell, D. F. (1971) Science 174,838-839

5. Blakemore, W. F. (1972) J. Comp. Pathol. 82, 179-185

by guest on June 30, 2020http://w

ww

.jbc.org/D

ownloaded from

232 Oligosaccharides of Canine GMI Gangliosidosis

6. Handa, S., and Yamakawa, T. (1971) J. Neurochem. 18, 1275-

7. Donnelly, W. I. C., Sheahan, B. J., and Kelly, M. (1973) Res. Vet.

8. Read, D. H., Harrington, D. D., Keenan, T. W., and Hinsman, E.

9. Lundblad, A,, Sjoblad, S., and Svensson, S. (1978) Arch. Biochem.

10. Yamashita, K., Ohkura, T., Okada, S., Yabuuchi, H., and Kobata,

11. Mega, T., Lujan, E., and Yoshida, A. (1980) J. Biol. Chem. 255,

12. Yamashita, K., Liang, C.-J., Funakoshi, S., and Kobata, A. (1981) J . Biol. Chem. 256, 1283-1289

13. Fournet, B., Montreuil, J., Strecker, G., Dorland, L., Haverkamp, J., Vliegenthart, J. F. G., Binette, J. P., and Schmid, K. (1978) Biochemistry 17, 5206-5214

14. Prasad, R., Hudson, B. G., Strickland, D. K., and Ebner, K. E. (1980) J. Biol. Chem. 255, 1248-1251

15. Hayes, M. L., and Castellino, F. J . (1979) J. Biol. Chem. 254,

16. Rittmann, L. S., Tennant, L. L., and OBrien, J. S. (1980) Am. J.

17. Holmes, E. W., and O’Brien, J. S. (1979) Anal. Biochem. 93,167-

1280

Sei. 15, 139-141

J. (1976) Science 194,442-445

Biophys. 188,130-136

A. (1981) J. Biol. Chem. 256,4789-4798

4057-4061

8772-8776

Hum. Genet. 32,880-889

170

Biophys. 155,464-472 18. Stellner, K., Saito, H., and Hakomori, S. (1973) Arch. Biochem.

19. Lindberg, B. (1972) Methods Enzymol. 28, 178-185 20. Ballou, C. E., and Cohen, R. E. (1980) Biochemistry 19, 4345-

4358 21. Strecker, G., Herlant-Peers, M.-C., Fournet, B., Montreuil, J.,

Dorland, L., Haverkamp, J., Vliegenthart, J . F. G., and Farriaux, J.-P. (1977) Eur. J. Biochem. 81, 165-171

22. Dorland, L., Haverkamp, J., Schut, B. L., Vliegenthart, J. F. G., Spik, G., Strecker, G., Fournet, B., and Montreuil, J . (1977) FEBS Lett. 77, 15-20

23. Strecker, G., and Montreuil, J . (1979) Biochimie 61, 1199-1246 24. Pierce, R. J., Spik, G., and Montreuil, J . (1980) Biochem. J . 185,

25. Dugal, B., and Stramme, J . (1977) Biochem. J. 165,497-502 26. Nishigaki, M., Muramatsu, T., and Kobata, A. (1974) Biochem.

Biophys. Res. Commun. 59, 638-645 27. Boersma, A,, Lamblin, G., Roussel, P., Degand, P., Biserte, G.,

and Roche, J. (1975) C. R. Hebd. Seances Acud. Sci. Ser. D

28. Lundblad, A., Nilsson, B., Norden, N. E., Svensson, S., Ockerman,

29. Jones, M. Z., and Laine, R. A. (1980) Fed. Proc. 39,2082

261-264

Sci. Nut. 281, 1269-1272

P.-A., and Jolly, R. D. (1975) Eur. J. Biochem. 59, 601-605

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ww

.jbc.org/D

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T G Warner and J S O'Briengangliosidosis liver.

Structure analysis of the major oligosaccharides accumulating in canine GM1

1982, 257:224-232.J. Biol. Chem. 

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