THE JOURNAI. OF B~m.ocrcn~ CHEMISTRY Vol. 254. No. 23, Issue of December 10, pp. 12224-12229, 1979 Prrnted in U.S. A.

Isolation and Characterization of Two Isomers of Brain Tetrasialogangliosides*

(Received for publication, April 23,1979)

Susumu Ando+ and Robert K. Yu

From the Department of Neurology, Yale University School of Medicine, New Haven, Connecticut 06510

Two isomers of tetrasialogangliosides were isolated and purified to homogeneity from human, bovine, chicken, and cod fish brains by employing DEAE-Seph- adex and Iatrobeads column chromatographies. The tetrasialogangliosides of human, bovine, and chicken brains appeared to be identical because they had iden- tical mobilities on thin layer plates developed with six different solvent systems. The tetrasialoganglioside of cod fish brain moved slower on thin layer plates than the tetrasialoganglioside from the other species. The ganglioside preparations were subjected to mild acid hydrolysis, neuraminidase treatment, and periodate oxidation followed by borohydride reduction. The structures of the two isomers were differentiated from each other by controlled mild acid hydrolysis in both aqueous and organic solvents. The structure IV3(NeuAc)2,113(NeuAc)z-GgOse4ceramide is assigned to the tetrasialoganglioside of human, bovine, and chicken brains; and the structure IV3NeuAc,I12(NeuAc)3- GgOse4ceram;de is assigned to that of cod fish brain. The possible pathways for the synthesis of the two tetrasialogangliosides are discussed.

Klenk et al. (2) reported in 1967 that the major tetrasialo- ganglioside isolated from human brain contained two sialo- syl(2-8)sialosyl residues attached to a ganglio-N-tetraosylcer- amide backbone. They realized, however, that the ganglioside’ preparation could be either Gqlb or GQ~,, or a mixture of both. Johnson and McCluer suggested an unusual structure for Ggl that contained a sialosyl(2-6)N-acetylgalactosaminosyl resi- due, but no evidence was presented (4). In 1972, Ishizuka and Wiegandt (5) isolated a tetrasialooligosaccharide from the ozonolysate of crude ganglioside mixtures of fish brain, and proposed the structure as desphingosino-Get,+, (desphingo- sino-G&. This was taken as evidence that fish brain tetrasi- aloganglioside possessed a GQ~, structure (5). They further

* This work was supported by United States Public Health Service Grant NS-11853 and a grant from the Kroc Foundation. This work has partially been presented at the Sixth International Meeting of the International Society for Neurochemistry (1). The costs of pub- lication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertise- ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

+ Present address, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan.

’ The ganglioside nomenclature used here is that of Svennerholm (3). Other gangliosides which have not been designated by Svenner- holm include the following: GQ,~, IV”(NeuAc)2,113(NeuAc)2- GgOse&er; GQI~, IV3NeuAc, I13(NeuAc)a-GgOse&er; GTI,, IV”(NeuAc)2,II”NeuAc-GgOse&er; GT~~, IV”NeuAc, I13(NeuAc)a- GgOse.+Cer; G.rl,, I13(NeuAc):s-GgOse&er; GTS, II”(NeuAc)a- GgOse&er; G.1.3, I13(NeuAc)3-LacCer; GM~, 13NeuAc-GalCer. Other abbreviations used are: Cer, ceramide; GalNAc, N-acetylgalactosa- mine; NeuAc, N-acetylneuraminic acid.

speculated that the pig brain tetrasialoganglioside had the

same structure. At present, the structures of intact brain tetrasialogangliosides of various animal species are still re-

garded as tentative. We recently isolated and purified the tetrasialogangliosides

as intact glycolipids from human, bovine, chicken, and cod fish brains by the sequential application of DEAE-Sephadex and Iatrobeads column chromatographies (6, 7). The charac- terization of these gangliosides and evidence for the occur- rence of the two isomers of tetrasialogangliosides, Ggn, and

GBlo are described in the present paper.

EXPERIMENTAL PROCEDURES

Isolation of Tetrasialogangliosides-The total gangliosides of a human whole brain (57-year-old man) were prepared as described previously (8). The gangliosides were applied to a DEAE-Sephadex A-25 (acetate form, Pharmacia Fine Chemicals) column (1.4 cm, inner diameter, X 110 cm) packed in methanol. The gangliosides were separated according to their differences in acidity by continuous gradient elution with 2100 ml of ammonium acetate in methanol (0 to 0.5 M) (Fig. 1). The gangliosides eluted were monitored with the resorcinol-HCl reagent (9). The fourth peak fraction which corre- sponded to the tetrasialoganglioside was collected and evaporated to near dryness. The residue, dissolved in water, was first dialyzed against distilled water, then 0.2 M sodium acetate, and finally against distilled water. This dialysis procedure converted gangliosides from ammonium salts to sodium salts. The dialyzed material obtained after lyophilization was dissolved in 3 ml of chloroform/methanol/3.5 N ammonium hydroxide (50:47:3, v/v), and then applied to an Iatro- beads column (6). The column (1.6 cm, inner diameter, x 80 cm) was packed with 80 g of Iatrobeads 6RS-8060 (Iatron Laboratories, Inc., Tokyo, Japan) in the same solvent, and the gangliosides were eluted with a linear gradient system prepared from a total of 750 ml of chloroform/methanol/3.5 N ammonium hydroxide (45:52:3 and 30:67: 3). The purity of the tetrasialoganglioside in each fraction was ex- amined by thin layer chromatography. The fractions showing a single band were combined to give 34 mg (1.8% of the total gangliosides) of white powder.

The total chicken brain gangliosides (177 mg of sialic acid) were prepared from 308 g of white matter from brains of 6- to 16-week-old chicken by the procedure described previously (8). The gangliosides were separated into four major peaks by a DEAE-Sephadex column. The material in the fourth peak was purified by Iatrobeads column chromatography in a similar manner as described above to yield 5.0 mg of the pure tetrasialoganglioside (0.94% of the total ganglioside).

A bovine brain polysialoganglioside fraction, which was a generous gift of Dr. Herbert Yohe in our laboratory, was treated in a similar manner as above to give the pure tetrasialoganglioside.

The total fish brain gangliosides (16 mg of sialic acid) were prepared from 126 g of whole cod fish brains by the method described previously (8). The gangliosides were separated into six major peaks by a DEAE- Sephadex column (1.4 cm, inner diameter, X 110 cm) using gradient elution with ammonium acetate in methanol (0 to 0.6 M, total volume, 3600 ml). The fifth peak corresponded to the tetrasialoganglioside fraction. The fraction was evaporated to near dryness. The residue was suspended in water. It was dialyzed against distilled water, 0.2 M sodium acetate, and then distilled water. The retentate was lyophi- lized and the residue applied to a column (1.4 cm, inner diameter, x

80 cm) that was packed with 60 g of Iatrobeads 6RS-8060 in chloro-

12224

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Brain Tetrasialogangliosides 12225

form/methanol/35 N ammonium hydroxide (50:47:3). The ganglio- sides were eluted with a similar gradient system as described for the human tetrasialoganglioside. In the tetrasialoganglioside fraction, 5.6 mg of sialic acid were recovered (yield: 35% of the total ganglioside sialic acid).

Thin Layer Chromatography-Precoated analytical and high per- formance thin layer chromatographic plates of Silica Gel 60 (E. Merck, Darmstadt, West Germany) were used with the following developing solvent systems: chloroform/methanol/water (50:45:10) containing 0.02% CaCls.2HaO; chloroform/methanol/2.5 N ammonia (50:45:10); 1-propanol/water (7520); 1-propanol/2.5 N ammonia (75: 20); I-propanol/pyridine/water (70:10:20); 1-butanol/pyridine/water (9:6:4) containing 0.1% KCl. Gangliosides were visualized by spraying with resorcinol-HCl reagent (9) followed by heating the covered plate at 95°C on an aluminum block heater (IO).

Cotnpositional Analysis-The molar ratios of carbohydrate and long chain base constituents were determined by gas-liquid chroma- tography as their iV,O-trifluoroacetyl derivatives (11). Samples con- taining 15 to 40 pg of sialic acid were methanolyzed for 3 h at 100°C with 200 ~1 of anhydrous methanolicHC1(3%, w/v) in l-ml Reactivials capped with Tufbond (Applied Science Laboratories, State College, Pa.). After the fatty acid methyl esters were removed by extraction with n-hexane, the solvent and HCl were evaporated under a stream of nitrogen and the sample was further dried in UCZCUO. The residue was then dissolved in 80 pl of trifluoroacetic anhydride/ethyl acetate mixture (1:l) and heated for 10 min at 60°C. Aliquots of the solution were injected into a column (6 feet x 2 mm, inner diameter) filled with Gas-chrom Q that was coated with a mixture of 6% (w/w) SP- 2401 and 0.5% (w/w) OV-225 (8). The oven temperature was pro- grammed at P’C/min from 140-23O’C. N-Acetyl- and N-glycolylneu- raminic acids were analyzed by gas-liquid chromatography as their trimethylsilyl derivatives (12). The fatty acid methyl esters were analyzed on a 10% SP-222PS column (6 feet X 2 mm, inner diameter, Supelco, Inc., Bellefonte, Pa.) by gas-liquid chromatography. Long chain base composition was determined by a modified procedure of Carter and Gaver (13). A ganglioside sample, containing 60 pg of sialic acid, was hydrolyzed for 3 h at 75°C with 300 ~1 of 2 N HCl in 82% methanol. After the fatty acids were extracted with n-hexane, the hydrolysate was evaporated under a stream of nitrogen. The residue was dissolved in 0.3 ml of a mixture of methanol/aqueous 0.1 N NaOH (4/3) with vortexing, and then partitioned with 0.36 ml of chloroform. The lower phase was washed twice with Folch’s theoretical upper phase, evaporated, and dried over NaOH pellets in U~CUO overnight. The long chain bases present in the residue were converted to their trimethylsilyl ether derivatives by treatment with 40 ~1 of a mixture of hexamethyldisilazane/trimethylchlorosilane/pyridine (2.6/1.6/2, by volume) at 60’ for 10 min. Aliquots were injected into a gas chromatographic column of 3% OV-1 maintained at 210°C.

Mild Acid Hydrolysis-A ganglioside sample, containing 40 ,sg of sialic acid, was dissolved in 200 ~1 of aqueous 5.6 mM formic acid (pH 3.0) and heated at 80°C (5) for 30 min. The solution was then neutralized bv adding 200 ~1 of 0.2 N NaOH followed bv incubation at 37°C for 1 hr. The solution was then desalted by passing through a Sephadex G-50 (fine) column (bed volume, 40 ml) packed in distilled water (14). Water was used as the eluant. The fist 12.5 ml of eluate were discarded and the next 2.5 ml, containing glycolipid products, were collected and lyophilized. The residue was dissolved in 40 ~1 of chloroform/methanol (1:2) and the glycolipid products were analyzed by thin layer chromatography using the following developing solvent systems: chloroform/methanol/water (50:45:10) containing 0.02% CaCb. 2H20, and chloroform/methanol/2.5 N ammonia (50:45:10). In a separate experiment, the ganglioside sample was hydrolyzed for 90 min at 80°C with 200 ~1 of 50 mM formic acid in chloroform/methanol (1:2). The reaction mixture was evaporated, neutralized, desalted, and then analyzed by thin layer chromatography in the same manner as described above.

Neuraminidase Treatment-A ganglioside sample, containing 45 pg of sialic acid, was dissolved in 140 ~1 of 0.1 M sodium acetate buffer ‘(pH 5.0, containing 0.1% (w/v) CaC12.2HzO). The sample was then mixed with 15 ~1 of a neuraminidase solution (EC 3.2.1.18. Clostridium perfringens, type VI, Sigma Chemical Co., St. Louis, Mo., 1 unit in 1 ml of the same buffer). The solution was fnst incubated for 150 min at 20°C. One-third of the solution was removed and set aside at the end of the reaction. To the rest of the sample, an additional 15 ~1 of the enzyme solution were added and the second incubation was carried out at 37°C for 16 hr. At this point, one-half of the reaction mixture was set aside, and the other half was further incubated for 24 h at 37°C with 15 ~1 of 2% sodium taurocholate (ox bile, Sigma) and

100 ~1 of fresh enzyme solution. Each of the three portions, obtained under the above different hydrolytic conditions,-was applied to a Sephadex G-50 (fine) column (bed volume, 40 ml) in order to remove inorganic salts and most of the free sialic acid (14). The glycolipid products were then analyzed by thin layer chromatography as de- scribed above.

Periodate Oxidation-Borohydride Reduction-The experiment was carried out according to the procedure reported previously (8). A ganglioside sample, containing 40 pg of sialic acid, was dissolved in 270 ~1 of 0.2 M sodium acetate buffer (pH 4.4). A solution of 0.5 M

sodium metaperiodate (30 ~1) was added at 0°C. After 48 h at 4°C the reaction was stopped by adding 50 ~1 of 10 M ethylene glycol. After 3 h the solution was neutralized with 90 ~1 of 0.2 N NaOH, and then mixed at 0°C with 60 ~1 of 9% sodium borohydride (final pH 10 to 11). The solution was allowed to stand at 4°C overnight. Excess borohydride was decomposed by the addition of 70 ~1 of 2 M acetic acid (final pH 6.5). The reaction product was desalted by the Sepha- dex G-50 column described above. The ganglioside fraction was then subjected to methanolysis and the resulting carbohydrate components were analyzed by gas-liquid chromatography as their trifluoroacetyl derivatives (11).

RESULTS AND DISCUSSION

It is well known that the brains of most higher vertebrates contain at least four major gangliosides, GM~, GoI,, Gnlh, and GT~,,, which comprise roughly 75% of the total brain ganglio- side sialic acids (10, 15). All these gangliosides contain one or more sialic acid moieties attached to a common ganglio-N- tetraosylceramide backbone whose structure was established by Kuhn and Wiegandt in 1963 (16, 17). However, the struc- ture of the mammalian tetrasialoganglioside, which generally constitutes a minor component of most mammalian total brain ganglioside mixtures, has not been established. In 1967, Klenk et al. isolated small amounts of tetrasialoganglioside (termed ganglioside C4) from human brain (2). Periodate oxidation experiments on the isolated ganglioside fraction suggested the presence of two sialosyl(2-8)sialosyl groupings attached to a ganglio-N-tetraosylceramide backbone. However, the exact arrangement of the sialic acid residues was not determined.

In contrast to the brains of higher vertebrates, the brains of lower vertebrates such as fish contain a preponderance of polysialogangliosides and relatively small amounts of mono- sialogangliosides (5, 18-20). The tetrasialoganglioside, which is particularly abundant, accounts for nearly 40% of the total brain ganglioside in certain fish species. The structure of the carbohydrate moiety (desphingosinooligosaccharide) of cod fish brain tetrasialoganglioside was subsequently elucidated by Ishizuka and Wiegandt (5). They showed that it had a trisialosyl group attached to the inner galactosyl moiety with the remaining sialosyl residue linked to the terminal galactosyl moiety of the ganglio-N-tetraose backbone. This backbone was identical with that found in the major mammalian brain gangliosides. The structure was therefore assigned as GoLet or Go,,, although the intact ganglioside was never isolated. They further assumed that the mammalian brain tetrasialoganglio- side might have the same structure (5, 21).

In the present investigation, we have successfully achieved the isolation of intact tetrasialogangliosides from human, bo- vine, chicken, and cod fish brains. The total ganglioside mix- ture from each species was subjected to DEAE-Sephadex column chromatography which effecti .Tely separated the var- ious gangliosides based on their sialic acid contents (Fig. 1). The tetrasialogangliosides (Goi), eluted from the column with a solution of ammonium acetate in methanol, were recovered in the fourth major peak (yield: 3% of the total gangliosides) in the case of human brain; and in the fifth major fraction (yield: 35% of the total gangliosides) in the fish brain prepa- ration. The Go, preparations of bovii:e and chicken brains were obtained in the fourth peak on DEA.?Sephadex columns similar to the human brain tetrasialoganglioside. All the Go,

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12226 Brain Tetrasialogangliosides

J 20 40 60 80 100 120

Fraction number

FIG. 1. Ganglioside elution profiles from DEAE-Sephadex columns. A, gangliosides from human brain;peahs I to ZVcorrespond to the mono-, di-, tri-, and tetrasialo- fractions, respectively; B, gangliosides from cod fish brain: peaks Z, ZI, V, and VI correspond to the mono-, di-, tetra-, and pentasialo- fractions. IZZ and IV were both assigned the trisialogangliosides. Gangliosides were eluted with a linear gradient with increasing concentration of ammonium acetate in methanol.

GDlb

GTlb

GQI

WM H B C F WM H 0 C F

FIG. 2. Thin layer chromatograms of Ggl preparations. WIt4, total gangliosides from human brain white matter. H, B, C, and Fare GQ, preparations from human, bovine, chicken, and cod fish brains, respectively. The left plate was developed with chloroform/methanol/ water (50:45:10) containing 0.02% CaC12.2Hr0, and the rzght plate with 1-propanol/pyridine/water (70:10:20). Bands were visualized by heating at 95°C with resorcinol-HCl reagent (8).

fractions were further purified by chromatography on Iatro- beads columns (6). The purified GQ~ samples from human, bovine, and chicken brains showed identical RF values on thin layer plates employing six different developing solvent sys-

terns. The fish brain GQ~ appeared to have a slight but dis- tinctly slower mobility than the other preparations on thin layer plates developed with the two solvent systems shown in Fig. 2. It showed an apparently identical RF as the other GQ1 preparations in the other four solvent systems.

TABLE I Chemical compositions of human and cod fish brain GQ,

Human Ga, Cod fish Go,

Molar ratios of carbohydrate and long chain base

Glucose Galactose N-Acetylgalactosamine N-Acetylneuraminic acid Long chain base

1.0 1.0 2.00 1.86 0.99 0.98 3.74 3.82 0.75 1.26

Fatty acid composition (%) CM1 CIR:O CM GO:0 C22:O f&l c&4:0 cm 1 C261

0.9 5.7 90.5 22.0

0.6 8.2 1.3 0.3 2.0

3.2 0.7

60.7 3.8

Long chain base composition (%) d16:l’” 1.2 2.8 d18:l’ 60.8 94.1 d18:O” 2.3 3.0 d20:14 31.7 0 d20:0” 4.0 0

(1 1, (4E)-hexadecasphingenine; 2, (4E)-sphingenine; 3, sphinganine, 4, (4E)-icosasphingenine; 5, icosasphinganine.

G~I-GalNAc-Gal-Glc-cer d”AC t&c

G~1-GalNAc-Gpl-Glc-cet N&AC Ne”AC

GDIO

GDlb

GTlb

123456789

FIG. 3. Degradation patterns of Ggl by mild acid hydrolysis. The upper scheme shows the expected degradative pathways. The lower chromatogram represents the products of the hydrolysis. 1, total gangliosides from human brain white matter; 2, human GQ] before hydrolysis; 3, human GoI after hydrolysis with aqueous 5.6 mM formic acid; 4, human GQ~ after hydrolysis with 50 mM formic acid in chloroform/methanol (1:2); 5, G,l la (8); 6, cod fish GQ, before hydrol- ysis; 7, cod fish GQI after hydrolysis with aqueous 5.6 mM formic acid; 8, cod fish GQ~ after hydrolysis with 50 mM formic acid in chloroform/ methanol (1:2); 9, a synthetic mixture of GM,, Gm,, Go,h, and G,r,h. The plate was developed with chloroform/methanol/water (50:45:10) containing 0.02% CaClz. 2H90, and bands were visualized by heating with resorcinol-HCl reagent.

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Brain Tetrasialogangliosides 12227

GMI

Gora

GDlb 0

GTlb m GQI am-

-

I 2 3 4 5 6 7 8 9 IO

GAI

GMI

FIG. 4. Thin layer chromatogram of hydrolysis products by neuraminidase. 1, total gangliosides from human brain white matter; 2, human Go, before hydrolysis; 3, human GoI after incubation for 150 min at 2O’C; 4, human Go, after incubation for 16 h at 37’C; 5, human GoI after incubation for 24 h at 37’C in the presence of taurocholate; 6, cod fish GoI before hydrolysis; 7, cod fish GQI after incubation for 150 min at 2O’C; 8, cod fish GQ~ after incubation for 16 h at 37’C; 9, cod fish GoI after incubation for 24 h at 37’C in the presence of taurocholate; 10, asialo GM (GA,). The plate was devel- oped with chloroform/methanol/water (50:45:10) containing 0.02% CaC12.2Hz0, and bands were visualized by heating with resorcinol- HCl reagent. All spots were purple except GAI, which was brownish yellow.

Gal Glc Gal G GalNAc !J? Gal 2 Glc-cer I4

L-l A /2,3 /2,3

NeuAc NeuAc NeuAc

Gal ‘2 GolNAc@Gal !LT Glc -cer

/2,3 / 2,3

Gal!!? GalNAc@ Gal E Glc - cer

/ 2,3 NeuAc

/2,3 NeuAc

/2,8 NeuAc

/ 2.8 NeuAc

/ I 1

IO 15 20 r --7 25 Retentvm Time (mm)

FIG. 5. Gas-liquid chromatograms of ganglioside compo- nents after periodate oxidation-borohydride reduction fol- lowed by methanolysis and trifluoroacetylation. Upper, GT it,; center, human GoI; lower, cod fish Gol. CT, N-acetylheptulosaminic acid, C,, intact N-acetylneuraminic acid. A column packed with a mixture of SP-2401 and OV-225 was operated under variable temper- ature from 140-23O’C at 2”C/min.

The chemical compositions of human and fish Go1 prepa- rations were analyzed by gas-liquid chromatography. The results, expressed as molar ratios, are shown in Table I. Both Go, preparations contained glucose, galactose, N-acetylgalac- tosamine, N-acetylneuraminic acid, and long chain base in the molar ratios of 1:2:1:4:1. The data are consistent with a tetra- sialoganglio-N-tetraosylceramide structure. However, the li- pophilic constituents of the two Go1 preparations differed considerably. Human Go1 contained stearic acid as the major fatty acid, and (4E)-sphingenine and (4E)-icosasphingenine as the predominant long chain bases (Table I). On the other hand, fish Ggr contained large amounts of nervonic acid in addition to stearic acid. In addition, fish Go1 apparently con- tained (4E)sphingenine as the predominant long chain base, with no detectable amounts of (4E)-icosaphingenine (Table I).

Human GoI was subjected to mild acid hydrolysis employ- ing aqueous formic acid at pH 3. This produced a series of partially desialylated glycolipid products, i.e. Grla as well as Grrb, Gnrb, Gm,, and GM~ (Fig. 3, lane 3). These products were identified by thin layer chromatography with at least two different developing solvent systems. Interestingly, mild acid hydrolysis of human Go1 in an organic medium under the conditions described produced Grla as the major partially desialylated product (Fig. 3, lane 4). We have previously isolated Grrs from human brain gangliosides as a minor com- ponent and established its structure (8). The mild acid hy- drolysis experiment suggests that human GoI contained the

m-CERAMIDE

GQlb

o-CERAMIDE

GQIC

FIG. 6. Structures of tetrasialogangliosides. GQ]~ is from hu- man brain and Ggl, is from cod fish brain.

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12228 Brain Tetrasialogangliosides

Glc-cer

Gal-Glc-cer

1 Gal-Gic-cer

Neu/Ac GalNAc-GalGlc-cer

Gal-GalNAc-Gal-Glc-cer

1

GM~ tm> NehAc

6~2 - Neu/Ac

1

GMI

Gal-Glc-cer

Ne:Ac Gal-GalNAc-Gpl-Glc-cer

GDS Ne6Ac

Ne:Ac Ne”Ac GDla

1

\ 1

GalNAc-Gal-Glc-cer Gal-GalNAc-Gal-Glc-cer

Ne:Ac Nei Ac NeiAc

Gal-Glc-cer Neu/Ac GDZ Ne/uAc GTla

A/ ,$fc GalNAc-G/al-Glc-cer

Neh Ac

ti NNee:uAAcc GTZ GalNAc--T;;Glc-cer N&Ac

N&AC GTlc

N&AC

1

Gal-GalNAc-Gal-Glc-cer

Neu/Ac NedAc Neu/Ac GQIC

Neu/Ac

1

GPI

GTS

Gal-GalNAc-Gal-Glc-cer

Ne:Ac Neu/Ac

GDlb

4 Gal-GalNAc-Gal-Glc-cer

Neu/Ac Ne:Ac Ne&Ac

G-fib

4 Gal-GalNAc-Gal-Glc-cer

Neh Ac Ne:Ac

Neh Ac Neu/Ac Galb

FIG. 7. Proposed pathways for ganglioside synthesis. The +, +, and =+ represent the transfer of galactose, N-acetylneuraminic acid, and N-acetylgalactosamine from their nucleotides catalyzed by specific glycosyltransferases, respectively.

above partial structures. Because human Goi yielded both GTla and Gr,b, it could only be assigned as GQI~. On the other hand, fish GoI produced GTU,, Gnlb, GD~,, and GM~, but no Gria under identical hydrolytic conditions (Fig. 3, lanes 7 and 8). Its structure is therefore different from that of Golb. A possible structure for fish GoI that appeared to be compatible with these data has to be that of Go,, (5). The hydrolysis products of fish Goi generally had slightly higher RF values than those of the corresponding human gangliosides. This was probably attributable to the differences in their fatty acid compositions (Table I).

Go, from human and fish was subjected to neuraminidase treatment using a C. perfringens preparation. Human GoI produced Grlb, Gnlb, and GM, at 20°C as shown in Fig. 4. After standing at 37°C overnight, the Go, was completely converted to GM]. The GM1 was further partially degraded to asialo- GMI(GAI) in the presence of sodium taurocholate. Fish Go1 showed a similar degradation pattern. Slightly higher mobili- ties of the products from fish Go1 compared to those of human Go1 might be due to the fatty acid differences mentioned above.

Periodiate oxidation-borohydride reduction experiments were carried out in order to delineate the sialosyl-sialosyl linkages. The carbohydrate compositions of the partially de- graded products were determined by gas-liquid chromatogra- phy. Fig. 5 presents the gas-liquid chromatograms and the

supposed structures of the products from Grlb (used as a control sample), human Golb and fish Golc. Any sialic acid with no substitutions on its glyceryl side chain should give rise to a C7 analogue (N-acetylheptulosaminic acid), and the sialic acid with a substitution at position 8 should remain intact. It is clear from Fig. 5 that authentic GT1b yielded 2 mol of the C7 derivative of sialic acid and 1 mol of intact sialic acid. This is in agreement with the expected results that there is only one sialosyl(243)sialosyl grouping in Grlb. Both human and fish Go, yielded identical products under the same experimental conditions: 2 mol of sialic acid remained intact, whereas 2 mol of sialic acid were degraded to the C7 analogue. These results indicate that both Go1 structures contain two sialosyl(2- 8)sialosyl linkages and two sialosyl moieties that remained attached to the terminal positions. It is clear that either structure shown in Fig. 5 would be compatible with these results, as was also pointed out earlier by Klenk et al. (2). However, these results together with the data obtained from the mild acid and neuraminidase hydrolysis experiments, sug- gest that the mammalian and avian brain tetrasialoganglioside has the structure of Ggib (Fig. 6, upper), whereas the fish brain tetrasialoganglioside has the structure of Golc (Fig. 6, lower).

In conclusion, we have shown that the major tetrasialogan- glioside, Gqlb, of the brains of higher vertebrates (human, bovine, and chicken) is structurally different from the major

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tetrasialoganglioside, Goi,, of fish brain. We have not been able to detect Gg,b in fish brain, nor Go,, in the brains of higher vertebrates. It is possible that such a structural diver- gence could be the result of different pathways leading to their biosynthesis. It is known that all brain gangliosides, with the exception of GM4 (22), can be synthesized from GM~ by sequential additions of appropriate carbohydrate units cata- lyzed by appropriate glycosyltransferases (Fig. 7) (23,24). One pathway is through GMS, GAD, GN, Gm, (25, 26), and finally GTla (27). The other is through GMS, Gn3, Grit, and Gnlh (28, 29). Mestrallet et al. (30) further provided evidence that Grlb but not Grrc was also derived from this pathway. Presumably Golb may be synthesized from GTI~ (31). However, neither of these two pathways could adequately explain the formation of Golc. In analogy to the latter pathway, we would like to speculate that Golc could be derived from another route, i.e. GM3, Gna, Gr3, Grz, Grlc, and GQ~,. We have recently isolated and characterized Grs, Grz, and GT~~ in cod fish brain (20,32), thus providing the necessary intermediates for this pathway. Because of the large concentrations of GQ~~ in fish brain, it is possible that this may be a major pathway for ganglioside synthesis in fish brain. Further studies on the proposed path- way are now in progress.

Acknoculeclgment-The technical assistance provided by Mrs. No- buko Miyazawa is greatly appreciated.

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S Ando and R K YuIsolation and characterization of two isomers of brain tetrasialogangliosides.

1979, 254:12224-12229.J. Biol. Chem. 

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