THE JOURNAL OF BIOLOGICAL CHEM1S”RY Q 1986 by The American Society of Biological Chemists, Inc.

Vol. 261, No. 12, Issue of April 25, pp. 5625-5630,1986 Printed in U.S.A.

Gangliosides of Bovine Buttermilk ISOLATION AND CHARACTERIZATION OF A NOVEL MONOSIALOGANGLIOSIDE WITH A NEW BRANCHING STRUCTURE*

(Received for publication, November 20, 1985)

Kotaro TakamizawaSq, Masao IwamoriS, Masahiko Mutaig, and Yoshitaka NagaiS From the $Department of Biochemistry, Faculty of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, and SYakuEt Central Institute for Microbiological Research, Yaho, Kunitachi, Tokyo 186, Japan

Bovine buttermilk was found to contain 0.92 pmol of lipid-bound sialic acid/g dry weight. On ganglioside mapping, at least seven gangliosides were detected, and the structures of the five major molecules were determined by degradation with exoglycosidases and methylation analysis. Gw3, G D ~ , and GT3 were found to comprise 80% of the total gangliosides. The other two gangliosides had a new core structure with a branched oligosaccharide chain. One was a novel monosialogan- glioside with a 2 + 6 linked sialic acid residue with the following structure:

Gala1 -). 4GlcNAcSl L 6

3 G a u l - 4GlcSl -+ Cer

/ NeuAca2 + 6 G a 1 + 4GlcNAcPl

There were 41 nmol of this ganglioside/g of buttermilk (4.5% of total gangliosides). The other was a trisialo- ganglioside with the above new core structure.

Gangliosides in bovine milk are mainly present in the buttermilk, which primarily consists of the milk fat globule membrane (MFGM’) derived from the apical plasma mem- brane of the secretory cells of the lactating bovine mammary gland (1; for reviews see Refs. 2-4). Until now, only a few reports on the ganglioside composition of bovine milk have appeared. Keenan (5 ) , Huang (6) and Ito et al. (7) reported the ganglioside compositions of MFGM, buttermilk, and whole milk, respectively, and they found the major ganglio- sides in bovine milk were GD3 and G M ~ . In addition, Bushway and Keenan (8) reported the existence of G M ~ , Guz, Gib, and GDz in the bovine mammary gland on the basis of their mobilities on TLC. However, detailed analysis of the ganglio-

* This work was supported in part by a grant from the Ministry of Education, Science, and Culture of Japan, a Grant for Specific Diseases from the Ministry of Health and Welfare of Japan, and Special Coordination Funds for Promoting Science and Technology from the Science and Technology Agency of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: MFGM, milk fat globule membrane; Cer, ceramide; CDH, ceramide dihexoside; GLC, gas-liquid chroma- tography; G M ~ , I13NeuAc-LacCer; G D ~ , I13(NeuAc)2-LacCer; GM2, I13NeuAc-GgOse~Cer; G M ~ , I13NeuAc-GgOse4Cer; GDIb, 1I3(NeuAc), GgOse4Cer; GD~,, IV3NeuAc,I13NeuAc-GgOse~Cer; G D ~ , I13(NeuAc)*- GpOseaCer; GTlb, I13(NeuAc)21V3NeuAc-GgOse4Cer; GT3, 1I3(NeuAc),- LacCer.

~ ~”

side composition, particularly of the minor components in bovine milk, has not yet been performed systematically. Re- cently, monoclonal antibodies prepared by immunization with human MFGM were found to react with human mammary carcinoma cells, and the antigens recognized by the monoclo- nal antibodies were postulated to be glycoconjugates including glycolipids (9-17). Also, since glycolipids are thought to be important as recognition molecules, they may participate in the formation of MFGM. To elucidate the possible function of gangliosides in bovine milk, we determined the composition of the gangliosides in buttermilk in detail by the ganglioside- mapping procedure and found a ganglioside with a new core structure with a branched oligosaccharide chain.

MATERIALS AND METHODS

Preparation of Gangliosides-Buttermilk powder (1.1 kg) prepared from about 230 kg of raw bovine milk was extracted twice with 3 volumes of acetone to remove neutral lipids and then successively extracted with 10 volumes of chloroform/methanol/water, 2010:1 and 10201, and chloroform/methanol, 1:l (by volume), a t 45 “C. Gangliosides were recovered in the upper phase by Folch’s partition- ing and then dialyzed against water to remove free oligosaccharides and salts. Gangliosides were also recovered from the lower phase by DEAE-Sephadex A-25 column chromatography and combined with the fraction from the upper phase.

Ganglioside-mapping and Isolation of Individual Gangliosides-The crude ganglioside fraction (about 80 mg of lipid-bound sialic acid) was applied on a column (2 cm inner diameter X 15 cm) of DEAE- Sepharose CL-GB and the ganglioside map was obtained as described previously (18). TLC for ganglioside mapping was developed with either solvent a, chloroform/methanol/2.5 N ammonia, 60409 (by volume), or solvent b, chloroform/methanol/0.5% CaClz. 2H20, 554510 (by volume). On the basis of the ganglioside map, the fractions to be combined were decided, and the contaminating am- monium acetate in each fraction was removed by dialysis. Individual gangliosides were purified on a column (1.8 cm inner diameter X 55 cm, or 1 cm inner diameter X 95 cm) packed with Iatrobeads (6RS- 8060) with a gradient formed from either chloroform/methanol/ water, 55:45:4 and 1080:4, or 55:45:4 and 10:904 (by volume). The homogeneity of the isolated gangliosides was examined by TLC with solvents a and b.

Methylation Analysis-Permethylation of the isolated gangliosides was carried out with methyl sulfonyl carbanion and methyl iodide in dimethyl sulfoxide (19), and the permethylatedproducts were purified by solvent partitioning and Iatrobeads column chromatography (18). The purified permethylated gangliosides thus purified were used for determination of the sialic acid composition and for linkage analysis as partially methylated aldohexitol acetates according to the method described previously (20, 21). Standard aldohexitol acetates were prepared from IV30tGal-nLc4Cer, N-acetylneuraminosyl lactose, glo- boside, Gg4Cer, and gum arabic. Identification of each peak was performed by comparing the retention time on GLC and the mass spectrum obtained with a mass spectrometer (QP-1000, Shimadzu, Kyoto).

Enzyme Treatment-Neuraminidase (Vibrio cholerae, Grade B, Calbiochem-Behring), &galactosidase (jackbean, Sigma), and N-ace-

5625

5626 A Novel Monosialoganglioside from Bovine Buttermilk tyl-P-D-glucosaminidases (bovine kidney, Boehringer Mannheim, and jack bean, Sigma) were used for structural determination of the isolated gangliosides. About 20 pg of a ganglioside was hydrolyzed with 0.5 units of P-galactosidase in 500 p1 of 50 mM citrate phosphate buffer, pH 4.0, a t 37°C for 18 h, or with 5 units of N-acetyl-0-D- glucosaminidase in 200 11 of 50 mM sodium citrate buffer, pH 5.0, a t 37 "C for 18 h. The incubation medium contained sodium taurocho- late at a concentration of 1 mg/ml. For neuraminidase treatment, a ganglioside, containing about 10 pg of lipid-bound sialic acid, was dissolved in 100 p1 of distilled water, mixed with 5 units of neuramin- idase, and then incubated at 37 "C for 2 h or a t 20 "C for 20 min. The products after exoglycosidase treatment were examined by TLC with solvent b and the spots were located with orcinol-HpSO1.

Analytical Procedures-After locating the spots with resorcinol- HC1, the per cent distribution of lipid-bound sialic acid in each ganglioside was determined densitometrically with a TLC densitom- eter (CS-910, Shimadzu) a t an analytical wavelength of 570 nm and a control wavelength of 710 nm (22). The fatty acid and long chain base compositions were determined by GLC with fatty acid methyl esters and long chain aldehydes, respectively. For quantitation of lipid components in bovine buttermilk, total lipids were extracted from 10 g of buttermilk powder, and lipid-bound phosphorus in the extracts was determined by Bartlett's method after wet digestion with H,O, and HC104 (23). The extracts were further fractionated into neutral and acidic lipids by DEAE-Sephadex column chromatogra- phy, and lipid-bound sialic acid in the acidic lipid fraction was measured by the resorcinol-HC1 method (24).

RESULTS

Isolation of Gangliosides

Lipids comprised about 13% of bovine buttermilk powder, and the amounts of lipid-bound phosphorus and sialic acid were 11.88 and 0.92 pmol/g dry weight, respectively. Fig. 1 shows the ganglioside map for bovine buttermilk. At least

tG """ ."""

10 20 30 40

TUBE NUMBER FIG. 1. Ganglioside map of bovine buttermilk gangliosides.

The ganglioside map was obtained as described previously in detail (16). The developing solvent was solvent b and the spots were located with resorcinol-HC1 reagent. Seven gangliosides, designated as GI- G7, were recognized on the map.

TABLE I Per cent distribution of sialic acids in gangliosides from bovine

buttermilk

Ganglioside distribution Sialic acid

seven molecules, which were designated as G1-G7, can be detected on the map. The concentration of each ganglioside expressed as the per cent distribution of ganglioside sialic acid in buttermilk is shown in Table I. G5 was the most abundant ganglioside, accounting for about 70% of the total lipid-bound sialic acid, and, due to the large quantity, it was collected in three fractions in the order of elution. Using the ganglioside map as an indicator of the purification, ganglio- sides G1-G7 were purified by silica gel (Iatrobeads) column chromatography (Fig. 2).

Structures of Gangliosides On the basis of the results of permethylation analysis,

exoglycosidase treatment, mild acid hydrolysis, and alkaline treatment, the structures of the gangliosides were elucidated as follows.

Ganglioside GI-G1 had a mobility identical to that of GM3 on TLC (Fig. 2). After treatment with neuraminidase, G1 was converted to a compound identical with lactosyl ceramide (Fig. 3), and permethylation analysis of G1 showed 1,4,5-tri- O-acetyl-2,3,6-tri-O-methyl glucitol and 1,3,5-tri-O-acetyl- 2,4,6-tri-O-methyl galactitol in a ratio of 1:l (Table 11). Thus, ganglioside G1 was concluded to be G M ~ .

Ganglioside GZ"G2 was resistant to neuraminidase (Fig. 3). CDH, but not asialo-GM2, was produced by mild acid hydrolysis with 5.6 mM formic acid at 80 "C for 30 min. Also, the mobility on TLC of G2 treated with mild alkali coincided with that of G D ~ . Thus, the structure of G2 was suggested to be the lactone of GD~.

GM3

GM 2 GM1

GDlc

. ,-. w

GDlt GTlt

- S T G1 G 2 G3 G4 G5 G5 G5 G6 G7

GM GM

GD1c GM 1

S T GI G2 G3 G4 G5 G5 G5 G6 G7

FIG. 2. TLC of gangliosides isolated from bovine buttermik. The developing solvents used were: top, solvent b, and bottom, solvent a. Gangliosides Gl-G7 correspond to the designations on the ganglio- side map in Fig. 1. S, standard gangliosides; T, total bovine buttermilk gangliosides.

% G1 G2

4.8

G3 3.2

G4 4.5

G5 3.9

G6 68.5

G7 5.2 2.1

Others 7.8

C D H G M l P G O N O N O N O N O N O N O N - - - - - - - 0 1 G2 03 G4 0 5 G6 G7

FIG. 3. Neuraminidase treatment of gangliosides isolated from bovine buttermilk. 0 and N represent the original ganglio- sides and the products after neuraminidase treatment, respectively. The developing solvent was solvent b, and the spots were located with orcinol-HzSO4 reagent. PC, nLc4Cer.

A Novel Monosialoganglioside from Bovine Buttermilk 5627

TABLE I1 Partially methylated aldohexitol acetates from gangliosides of bovine

buttermilk The values are the molar ratios to 1,4,5-tri-O-acetyl-2,3,6-tri-O-

methyl glucitol (=LO).

Hexitol and hexosaminitol acetates identified by GLC

and GC-MS"

Molar ratio of the compo- nents liberated from:

G1 G3 G5 G6

1,4,5-tri-O-acety1-2,3,6-tri- 1.0 1.0 1.0 1.0

1,3,5-tri-O-acetyl-2,4,6-tri- 0.7 0 0.8 0.7

1,5-di-O-acetyl-2,3,4,6-tetra- 0 0.9 0 0

1,5,6-tri-O-acety1-2,3,4-tri- 0 0.9 0 0

1,3,5,6-tetra-O-acetyl-2,4-di- 0 0.8 0 0

1,4,5-tri-O-acetyl-2-deoxy-2- 0 1.7 0 0

0-methyl glucitol

0-methyl galactitol

0-methyl galactitol

0-methyl galactitol

0-methyl galactitol

N-methylacetamido-3,6- di-0-methyl glucitol

GC-MS, gas chromatography-mass spectrometry

Ganglioside G3-G3 was located in the monosialoganglio- side region on the ganglioside map (Fig. 1) and had a mobility close to that of V13NeuGc-nLc&er. On methylation analysis, G3 gave 1,5-di-O-acety1-2,3,4,6-tetra-O-methyl galactitol, 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl glucitol, 1,5,6-tri-O-ace- tyl-2,3,4-tri-O-methyl galactitol, 1,3,5,6-tetra-O-acetyl-2,4-di- 0-methyl galactitol, and 1,4,5-tri-O-acetyl-2-deoxy-2-N- methylacetamido-3,6-di-O-methyl glucitol in the ratio of 0.9:1.0:0.9:0.8:1.7 (Fig. 4), suggesting that G3 is a sialosyl hexahexaosyl ceramide with a branched oligosaccharide with galactose and N-acetylneuraminic acid at the nonreducing terminals. The permethylated methyl glycoside methyl ester of NeuAc was the sole sialic acid derivative derived from G3, and 1,5,6-tri-O-acety1-2,3,4-tri-O-methyl galactitol was absent from asialo-G3, indicating that NeuAc is linked to the ter- minal galactose through the 2 + 6 linkage.

FIG. 4. GLC of partially methyl- ated aldohexitol acetates obtained from ganglioside G3. The partially methylated aldohexitol acetates were analyzed on 3% OV-275 (A) and 2% OV- 17 ( B ) as described under "Materials and Methods." a, 1,5-di-O-acety1-2,3,4,6- tetra-0-methyl galactitol; b, 1,4,5-tri-O- acetyl-2,3,6-tri-O-methyl glucitol; c, 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl gal- actitol; d, 1,3,5,6-tetra-O-acety1-2,4-&- 0-methyl galactitol; e, 1,4,5-di-O-acetyl- 2-deoxy-2-N-methylacetamido-3,6-di-O- methyl glucitol.

w u) z 2 u) w a K w P a

a 00 w

P

As shown in Fig. 5, G3 was hydrolyzed sequentially with exoglycosidases in the following order: 8-galactosidase, neur- aminidase, and N-acetyl-P-D-glucosaminidase, and the final product (C in Fig. 5) had a mobility identical to that of nLc4Cer, whose structure was further confirmed by permethy- lation analysis, 1,5-di-O-acety1-2,3,4,6-tetra-O-methyl galac- titol, 1,4,5-tri-O-acety1-2,3,6-tri-O-methyl glucitol, 1,3,5-tri- O-acetyI-2,4,6-tri-O-methyl galactitol and 1,4,5-tri-O-acetyl- 2-deoxy-2-~-methylacetamido-3,6-di-O-methyl glucitol being detected in the ratio of 1:l:l:l. This indicates that the NeuAc residue is linked to the terminal galactose of the P l - 3 linked lactosamine branch, and not that of the P l -+ 6 linked lactosamine branch. Thus, a branched oligosaccharide with nLclCer was proposed as follows:

GalPl -+ 4GlcNAcP1 I

6

3 GalPl --f 4GlcPl "z Cer

7 NeuAccu2 --f 6GalPl "+ 4GlcNAcP1

In accord with the above structure, when hydrolysis with exoglycosidases was carried out in the order of neuraminidase, &-galactosidase, and N-acetyl-P-D-glucosaminidase, the prod- ucts were found to be hexahexaosyl, tetrahexaosyl, and dihex- osyl ceramides, respectively (Fig. 6). Thus, G3 was concluded to have the above structure.

Ganglioside G4-G4 showed two major and several minor bands (Fig. 2) , and its mobility on TLC was close to that of GM~. The major product on neuraminidase treatment was CDH, some of which had a mobility lower than CDH from human erythrocytes (Fig. 3), suggesting that G4 is also a sialylated lactosyl ceramide derivative. However, due to the small amount and heterogeneity of G4, further analysis of the structure could not be performed.

Ganglioside G5-G5 was the most abundant ganglioside, amounting to about 70% of the total lipid-bound sialic acid,

n 8 1 I I

0 10 20 30 40 50 0 10 20

RETENTION TIME ( m i d

5628 A Novel Monosialoganglioside from Bovine Buttermilk

1 G 3 A B C P G

FIG. 5. Enzymatic hydrolysis of ganglioside G3. The plate was developed with solvent b and the spots were located with orcinol- HzS04 reagent. Lane 1, V13NeuGc-nLc&er; lane (product) A , G3 treated with @-galactosidase; lune (product) B, product A treated with neuraminidase; lune (product) C, product B treated with N-acetyl-@- D-glucosaminidase (bovine kidney); PG, nLc4Cer.

1 2 G3 A B C CDH FIG. 6. Enzymatic hydrolysis of ganglioside G3. The plate

was developed with solvent b and the spots were located with orcinol- HzSO, reagent. Lune 1, V13NeuGc-nLc&er; lune 2, 1 treated with neuraminidase; lane (product) A, G3 treated with neuraminidase; lane (product) B, product A treated with @-galactosidase; lane (product) C, product B treated with N-acetyl-@-D-glucosaminidase (jack bean). Arrows indicate the orcinol-negative spots.

and its mobility on TLC was identical with that of G D ~ . Permethylation analysis of G5 showed 1,4,5-tri-O-acetyl- 2,3,6-tri-O-methyl glucitol and 1,3,5-tri-O-acetyl-2,4,6-tri-O- methyl galactitol in a ratio of 1:l (Table 11), and the 8-0- trimethylsilyl-4,7,9-tri-O-methyl derivative of the methyl gly- coside methyl ester of NeuAc and the permethylated methyl glycoside methyl ester of NeuAc in a ratio of 1:l. Moreover, CDH and GM3 were produced from G5 on neuraminidase treatment at 37 "C (Fig. 3) and at 20 "C. Thus, the structure of G5 was concluded to be GD3, NeuAca2 + 8NeuAca2 + 3GalP1 + 4GalP1 + Cer. Three fractions of G5 collected in the order of elution from a DEAE-Sepharose column (Fig. 2) were identical in its fatty acid, long chain base, and sialic acid compositions.

Ganglioside G6-G6 was located between GDla and GDlb on

TLC with solvents a and b. Permethylation analysis of G6 showed 1,4,5-tri-O-acety1-2,3,6-tri-O-methyl glucitol and 1,3,5-tri-O-acety1-2,4,6-tri-O-methyl galactitol in a ratio of 1:l (Table 11), and the 8-0-trimethylsilyl-4,7,9-tri-O-methyl de- rivative of the methyl glycoside methyl ester of NeuAc and the permethylated methyl glycoside methyl ester of NeuAc in a ratio of 2:l. Also, as shown in Fig. 3, G D ~ , G M ~ and CDH were produced on neuraminidase treatment at 37 "C (Fig. 3) and at 20 "C. Thus, the structure of G6 was concluded to be GT3, NeuAca2 + 8NeuAca2 + 8NeuAca2 + 3GalP1 + 4Glc@1+ Cer.

Ganglioside G7-Due to its small quantity, complete struc- tural elucidation could not be performed. However, as shown in Fig. 3, asialo-G7 prepared by neuraminidase treatment was identical with asialo-G3. And judging from its position on the map (Fig. 1) and the results of analysis of the permethylated sialic acid, G7 was proposed to be a trisialoganglioside with a new oligosaccharide structure similar to that of G3.

Fatty Acid, Long Chain Base, and Sialic Acid Compositions of Gangliosides from Bovine Buttermilk

On GLC of the permethylated sialic acids, N-acetylneura- minic acid was found to be the sole sialic acid of Gl-G7. And sphingosine was the major long chain base of all gangliosides isolated from bovine buttermilk (Table 111). Thus, a clear difference was found in the fatty acid composition. Palmitic, oleic, and stearic acids were the major acids of GM3, but those of GD3, GT3 and the new monosialoganglioside G3 were be- henic, tricosanoic, and lignoceric acids. The fatty acid com- position of GD3 was quite different from that reported by Keenan (5), but similar to that reported by Ito et al. (7).

Ganglioside Composition of Bovine Buttermilk

The molar ratio of lipid-bound sialic acid to lipid-bound phosphorus in bovine buttermilk was 1:13, which was rela- tively higher than those of various tissues (25). G D ~ was the

TABLE I11 Fatty acid and long chain base compositions of gangliosides isolated

from bovine buttermilk Gangliosides

G1 G3 G5 G6 %

Fatty acid 140 1.3 0.7 Tr a Tr 161 1.0 1.0 Tr Tr 160 14.5 10.3 8.3 4.9 18:l 37.9 5.9 1.7 1.9 180 36.2 9.7 3.7 3.3 20:o 1.0 1.6 1.6 1.7 21:o 0.6 1.1 1.5 1.4 22:o 2.8 22.8 31.0 31.1 23:O 2.1 25.5 31.2 32.0 241 0.4 3.6 3.3 4.5 240 2.1 16.3 17.6 19.2 251 Tr 0.6 Tr Tr 25:O Tr 0.9 Tr Tr

14db:1 9.7 13.6 12.0 7.3 16d0 0.2 0.5 0.3 NDc 16d:l 5.0 6.0 5.5 4.4

18td:0 4.7 3.7 6.0 4.1 18d0 13.4 20.5 14.2 17.1 18d1 67.0 55.7 62.0 67.1

Long chain base

a Tr, trace amount. d, dihydroxy.

e t, trihydroxy. ND, not detected.

A Novel Monosialoganglioside from Bovine Buttermilk 5629

TABLE IV Gangliosides of bovine battermilk

Structure Concentration nmollg dry wt

G1 NeuAc~~2+3Gal~l+4Glc~l+Cer 44.16 G2 14.72 G3 Galfil-AGlcNAcfil

\6 3

2 Galpl-AGlc@l+Cer 41.40

NeuAca2-+6Gal~1+=4GlcNAc~l G4 35.88 G5 NeuAca24NeuAca2+3Gal/31+4Glc~14er 315.10 G6 NeuAca24NeuAca24NeuAca2+=3Gal~l+4Glc~l+Cer 15.95 G7 6.44

major ganglioside and comprised 70% of the total gangliosides. Lactose and a new branched hexaose were found to be the major asialo-carbohydrates, and in contrast to the report of Bushway and Keenan (8), gangliosides with lacto-N-mote- traose and ganglio-N-tetraose were not detected. About 90% of the gangliosides had a lactose core (Table IV).

DISCUSSION

As in the previous studies (5-7), we found that G D ~ was the major ganglioside in bovine buttermilk. However, none of the gangliosides proposed by Bushway and Keenan (8) was de- tected on ganglioside mapping. About 90% of the gangliosides contained lactose as the asialo-carbohydrate, such as GM3, GD3, and GT3. And the other major asialo-carbohydrate was a new branching structure, as shown below.

GalPl + 4GlcNAcfi1 I

6

3 Gal61 + 4GlcPl + Cer

7 Gal61 -+ 4GlcNAc@l

At least two molecules, a monosialo- and a trisialoganglioside, having the new structure were detected in buttermilk. They contained 4.5 and 2.1% of the total lipid-bound sialic acid, respectively. Up to now, two core structures with a branching oligosaccharide of gangliosides have been reported in the literature (26-32). A structure, of which two lactosamines are attached to the terminal galactose of nLc4Cer, is widely dis- tributed in various tissues and cells, and a unique structure, of which GalNAc is linked to the internal galactose of nLc&er, is found in human erythrocytes. This is the first report of the occurrence of a branching structure, of which two lactosamines are linked to the galactose residue of cer- amide lactoside. The branching structure at the terminal galactose residue of nLc4Cer has been shown to be essential for I-antigenicity (33). The complete expression of I-antigen- icity a t 18 months after birth is closely associated with an enzyme responsible for the formation of a branching struc- ture. In addition, sialic acid in the new monosialoganglioside is linked at the terminal galactose residue through a 2 + 6 linkage instead of the usual 2 3 linkage. Since GM3, GD3, and GT3 have a NeuAcol2 3 3Gal linkage, sialylation of the 2 -+ 6 substituted ganglioside might be performed by a different linkage-specific sialyltransferase, which has already been pu- rified from rabbit liver (34). Therefore, analysis of the gan- glioside metabolism with attention to the sialyltransferase should be useful for distinguishing the sites of biosynthesis of the 2 + 3 and 2 --., 6 substituted.gangliosides throughout the

MFGM formation. On the other hand, some particular gan- gliosides have been reported to exhibit biological activities as to the promotion of neurite outgrowth of neuroblastomas (35) and the induction of differentiation of human leukemia cell lines, HL-60 and U937 cells (36). Further studies on the localization and metabolism of the gangliosides in the bovine mammary gland are now in progress in our laboratory.

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