Accumulation of Gangliosides with A^-Acetylneuraminosyl(a2-6

(CANCER RESEARCH 50. 1284-1290. February 15. 1990|

Accumulation of Gangliosides with A^-Acetylneuraminosyl(a2-6)lactosamine

Structure in Primary Human HepatomaTakao Taki,1 Riunirne Yamamoto, Miki Takamatsu, Kazuo Ishii, Akio Myoga, Kiyoshi Sekiguchi, Isao Ikeda,

Kunio Kurata, Jun Nakayama, Shizuo Handa, and Makoto MatsumotoDepartment of Biochemistry, School of Pharmaceutical Science, University ofShizuoka, Shizuoka-shi 422 [T. T., K. Y., M. T., K. I., M. M.]; Institute ofDainabot Co.,Ltd., 344 Minorudai Matsudo-shi, Chiba 271 [A. M., K. S., I. I., K. K.J; Central Clinical Laboratories, Shinshu University Hospital, 3-I-I Asahi, Matsumoto-shi 390(J. N.J; and Department of Biochemistry, Faculty of Medicine, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113 [S. H.], Japan

ABSTRACT

Gangliosides of hepatomas have been analyzed by using a monoclonalantibody directed to /V-acetylneuraminosyl(a2-6)lactoneotetraosyl-ceramide (sialyl(a2-6)paragloboside), which was prepared by injectingthe monosialoganglioside fraction of human meconium into BALB/cmice. The monoclonal antibody, named MSG-15, was found to bindsialyl(a2-6)paragloboside, but it failed to react with other gangliosides,including /V-acetylneuraminosyl(a2-3)lactoneotetraosylceramide (sialyl(a2-3)paragloboside) and "IP-type gangliosides. MSG-15 was found torecognize NeuAcÂ«2-6Gal/3structure of the ganglioside. Gangliosidesobtained from human hepatomas were analyzed by immunostaining onhigh-performance thin-layer chromatography plates using the monoclonal antibody MSG-15. All primary hepatoma samples used in thisstudy (nine samples) were found to contain sialyl(a2-6)paragloboside,which accounted for 13-31% of the monosialoganglioside fractions inthe hepatomas. Furthermore, MSG-15 recognized several monosialo-gangliosides in addition to sialyl(a2-6)paragloboside. These gangliosides apparently also contain a terminal NeuAca2-6Gal/J structure.Other ganglioside fractions obtained from hepatoma and meconium wereimmunostained on thin layer chromatography plates with MSG-15. Additionally, another monoclonal antibody (II-11 ), which recognizes terminal lactosamine structure, was used to immunostain these fractions aftersialidase treatment. Bands stained with both monoclonal antibodiesshowed similar mobilities to each other in the di- and trisialogangliosidefractions as well as monosialoganglioside fraction. In control liver, GM,ganglioside accounted for 92% of monosialoganglioside fraction, andsialyl(a2-6)paragloboside accounted for less than 1% of the fraction.Immunohistochemical study by using MSG-15 in tissue sections fromhepatocellular carcinoma and normal liver tissues demonstrated that onlyhepatocellular carcinoma cells gave a positive reaction. These resultssuggest that the biosynthetic pathway of gangliosides containingNeuAca2-6Gal#l-4GIcNAc/3 structure is activated in hepatoma cells.

INTRODUCTION

Glycoconjugates on cell surfaces change their chemical compositions and metabolism in association with oncogenic transformation (1-6) and cell differentiation (7-10). Some gangliosides in tumor tissues or cells have been proposed as tumor-associated antigens (11-16). Glycosphingolipids of meconiumhave been demonstrated to contain many kinds of oligosac-charide structures, including Le" and Les, which are tumorassociated ( 17-20). Based on these results, we tried to preparemonoclonal antibodies directed to tumor-associated antigensby immunizing mice with the ganglioside fraction from meconium.

In the present paper, we describe the preparation of mono-

Received 1/6/89; revised 5/15/89. 10/11/89; accepted 11/10/89.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' Recipient of a grant-in-aid from the Ministry of Education, Science andCulture of Japan. Present address: Department of Biochemistry, Faculty ofMedicine, Tokyo Medical and Dental University. Yushima 1-5-45, Bunkyo-ku.Tokyo 113. Japan. To whom requests for reprints should be addressed.

clonal antibody against sialyl(Â«2-6)paragloboside2 and its ap

plication to the detection of the gangliosides in hepatomasamples. We could find various glycolipids containing theNeuAcÂ«2-6Gal/31-4GlcNAc)3 structure at their nonreducingterminals by a TLC immunostaining technique in hepatomasand meconium gangliosides.

MATERIALS AND METHODS

Glycolipids. Glucosylceramide was prepared from the liver of apatient with Gaucher's disease. Lactosylceramide was prepared fromboviflleerythrocytes. GM,, sialyl(Â«2-3)paragloboside, and "IP-type gan

gliosides were prepared from human placenta (21). Paragloboside,nLc6Cer, and 1-glycolipid were prepared by the hydrolysis of sialyl(Â«2-3)paragloboside, Â¡-ganglioside,and I-ganglioside with Clostridium per-fringens sialidase, respectively. LcjCer was prepared by the hydrolysisof paragloboside with jack bean /3-galactosidase.

Preparation of Monoclonal Antibody Directed to Monosialoganglioside Fraction from Meconium. Immunization was performed by themethod of Young et al. (22). About 100 Â¿tgof monosialosylgangliosideof meconium were suspended in 40 ^g of acid-treated SalmonellaMinnesota in 0.5 ml of PBS (pH 7.4). The monosialoganglioside suspension was injected s.c. into BALB/c mice four times at weeklyintervals. Three days after the last injection, the spleen was removedfor fusion. Preparation of hybridomas and the ELISA method fordetecting antibody were performed as described previously (23). Forthe detection of antibody activity by ELISA, 0.1 Mgof the monosialoganglioside fraction was coated on a microtiter plate (Titertek; FlowLaboratories, Ltd.).

Isolation of Gangliosides from Human Hepatomas and Meconium.Nine samples of primary hepatomas were donated from Shikoku CancerCenter, Japan. The wet weight of each sample was about 1-5 g.Hepatoma tissue was homogenized with 20 volumes of a solventmixture of chloroform/methanol (2:1, v/v). The homogenate was filtered. The filtrate was evaporated under reduced pressure in a rotaryevaporator. The residue was dissolved in 20 ml of a mixture ofchloro-form/methanol/water (30:60:8, v/v/v) and applied to a DEAE-Sepha-dex A-25 column (1.2 x 10 cm). Neutral lipid, phospholipid, and neutralglycolipid were eluted with the same solvent mixture. Monosialogan-gliosides were eluted with a mixture of chloroform/methanol/0.05 Msodium acetate (30:60:8, v/v/v), disialogangliosides with a mixture ofchloroform/methanol/0.2 M sodium acetate (30:60:8, v/v/v), trisialo-gangliosides with a mixture of chloroform/methanol/0.4 M sodiumacetate (30:60:8, v/v/v), and tetra- or polysialogangliosides with amixture of chloroform/methanol/0.8 M sodium acetate (30:60:8, v/v/v).

2The abbreviations used are: sialyl(ir2-6)paragloboside (sialyl(o2-6)PG),NeuACrt2-6Galfn-4GlcNAcdl-3Galf)l-4Glcfn-lCer; GlcCer, Glcfil-lCer;LacCer, Gal/Jl-4Glc(il-lCer; Lc,Cer, GlcNAcfil-3Galf)l-4Glcfil-lCer; nLc.Cer(PG), Gal/31-4GlcNAct(i-3GaIfM-4Glcdl-lCer; nLc6Cer (i-glycolipid), Gal/Jl-4GlcNAc/31-3Galdl-4GlcNAcfll-3GaltÂ¡l-4GlcOl-lCer; I-glycolipid, Galtfl-4GlcN Ac/il -3(Gal/31 -4GlcNAcfl 1-6)Gal/31 -4GlcN Ac/J1-3Galff 1-4Glcd 1-1Cer;sialyl(o2-3)paragloboside (sialyl(Â«2-3)PG). NeuAcÂ«2-3Gali)l-4GlcNAc/31-3Galdl-4GlcfÃ¼-lCer; i-ganglioside. NeuAcn2-3GaltÃl-4GlcNAcfÃl-3Gal/31-4GlcN Acd1 -3Gal/31-4Glcrf 1-1Cer; I-ganglioside. NeuAcÂ«2-3GalfÂ¡1-4GlcNAcd 1-3(NeuAcrt2-3Gal/Ãl-4GlcNAcdl-6)GalÃn-4GlcNAcf)l-SGalfil-4G Icrfl-ICer(other abbreviations for ganglio-serios gangliosides are according to Svennerholm(39); TLC, thin-layer chromatography; HPTLC. high-performance thin-layerchromatography; HPLC, high-performance liquid chromatography; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay: NMR. nuclear magnetic resonance.

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HEPATOMA GANGLIOSIDES

Meconium (720 g) was homogenized with 7.2 liters of the solventmixture of chloroform/methanol (2:1, v/v). The lipid extract was evaporated to dryness under reduced pressure, suspended in distilled water,dialyzed against distilled water, and lyophilized. The dry weight of theextract was 27 g. Ten g of the crude extract were dissolved in 100 mlof the mixture of chloroform/methanol/water (30:60:8, v/v/v). Insoluble material was removed by centrifugal ion and washed with the solventmixture several times. The supernatants were combined and applied toa DEAE-Sephadex A-25 column (3.2 x 45 cm). Neutral glycolipid waseluted with the solvent mixture of chloroform/methanol/water(30:60:8, v/v/v), and each ganglioside fraction was eluted with thesolvent mixtures mentioned above, increasing the concentration ofsodium acetate in a stepwise manner. By this procedure, 2.53 g ofneutral glycolipids were obtained, and 0.87 g of monosialogangliosides.0.22 g of disialogangliosides (mostly sulfatide), 0.12 g of trisialogan-gliosides. and 0.16 g of tetra- and polysialoganglioside fractions wererecovered.

High-Performance Liquid Chromatograph}'. About 10 mg of mono

sialoganglioside fraction was applied to an HPLC apparatus (ShimadzuLC-4A) equipped with an latrobeads column (6RS 8010, 3 x 180 mm;donated by latron Co., Ltd.) which was equilibrated with a mixture ofisopropanol/hexane/water (55:40:5, v/v/v). Gangliosides were dissolved with the solvent mixture of chloroform/methanol (2:1, v/v) andsubjected to HPLC. Gangliosides were eluted with a gradient of isopropanol/hexane/water (55:40:5-55:25:20, v/v/v). The flow rate was 1ml/min, and each fraction contained 1 ml. An aliquot was taken fromeach fraction and analyzed by TLC.

Thin-Layer Chromatograph}'. Gangliosides were separated by TLCon precoated silica gel 60 plates (HPTLC plates; E. Merck, Darmstadt,Federal Republic of Germany). Solvent systems for developing were asfollows: chloroform/methanol/water (65:35:8, v/v/v), chloroform/methanol/0.2% CaCl2 (60:40:9, v/v/v), and chloroform/methanol/0.2% CaCl2 (55:45:10, v/v/v). Gangliosides were visualized by sprayingthe plate with resorcinol/HCl reagent. GM3, sialyl(Â«2-3)paragloboside,and Ii-type gangliosides were used as standards.

MG-1-

MG-2-MG-3-

40 60 80 100 120Fig. 1. HPLC elution profile of monosialogangliosides from human mcco-

nium. The fractions eluted from HPLC were monitored by TLC. The solventsystem was chloroform/methanol/0.2DÂ£ CaCI2 (60:40:9, v/v/v). Gangliosides were

visualized by spraying the plate with resorcinol/HCl reagent. Lane S, standardganglioside <.M,.

Proton NMR Spectroscopy. The NMR spectrum of the intact glycolipid (MG-3) was obtained with a JEOL JNM-GX500 spectrometer(500 MHz). The operation was performed at 45T in Me2SO-D6solution and then in a Me2SO-D6 solution containing 2c,i D;O at the

same temperature. Chemical shifts are given as ppm from the peak ofthe internal standard, tetramcthylsilane.

Immunostaining on TLC Plate. Immunostaining of glycolipid on theTLC plate was performed as reported by Saito et al. (24) with thefollowing modification: the substrate, 3,3'-diaminobenzidine, for per-

oxidase was replaced with 4-chloro-l-naphthol reagent. The substratesolution was prepared as follows: 1 mlof methanol solution of 4-chloro-l-naphthol (3 mg/ml) was mixed with 5 ml of 100 m\i NaCl/0. l Mtrisaminomethane/HCl buffer (pH 7.2), and 10 /il of hydrogen peroxidewere added. As the first antibody, MSG-15 [directed to sialyl(Â«2-6)paragloboside. IgM type] or H-ll [directed to paragloboside. IgMtype (23)1 was used. When the monoclonal antibody H-l I was used forimmunostaining, gangliosides separated on the TLC plate were hydro-lyzed with sialidase as follows: 5 ml of a solution of C. perfringenssialidase (0.5 unit) in 0.2 M acetate buffer, pH 5.4, containing 0.1%bovine serum albumin were placed on the HPTLC plate and incubatedat 37Â°Cfor 10 h. After washing the plate with distilled water, immuno

staining with the H-l 1 antibody was carried out as described above.Methylation Analysis. About 1 mgof MG-3 ganglioside was dissolved

in 0.5 ml of dimethyl sulfoxide, and methylation of the glycolipid wasperformed by the method of MÃ¡nssonet al. (25). Methylated ganglioside was subjected to acetolysis in 0.7 N HCl/80% acetic acid at 80Â°C

for 18 h. After the extraction of fatty acids with Â«-hexane,the methylated sugars were reduced with 1 mg of NaBHj in 300 /jl of distilledwater at room temperature for 10 min. The methylated alditols wereacetylated in a mixture of pyridine/acetic anhydride (1:1) at IOOÂ°Cfor

10 min (26). The products were dried under reduced pressure andanalyzed by gas-liquid chromatography (Shimadzu GC-7A equippedwith a 30-m capillary column packed with 1% OV-1). An aliquot of themethylated sugars was applied to the column, which was maintained at160Â°Cfor the first 16 min and then programmed at the rate of 5Â°C/min from 160Â°to 230Â°C.The partially methylated alditol acetates were

analyzed by gas chromatography-mass spectrometry on a DB-1 capillary' column (0.25 mm x 30 m).

Treatment of MG-3 Ganglioside with Exoglycosidases. About 20 n%of MG-3 ganglioside was incubated at 37Â°Covernight with C. perfrin-

gens sialidase (0.1 unit; Sigma Chemical Co., St. Louis, MO) in 200 /ilof 5 mM acetate buffer (pH 5.0) (27). Neutral glycolipid was incubatedat 37Â°Covernight with various exoglycosidases in 200 Â¿ilof 25 imi

citrate buffer (pH 4.0) containing 50 ng of taurodeoxycholate (28. 29).The following exoglycosidases were used: 0.25 unit of /j-galactosidasefrom jack bean (Sigma) and 0.1 unit of fi-jV-acetylhexosaminidase fromjack bean (Sigma). Incubation was stopped by putting the incubationtube in a boiling water bath for 1 min. The hydrolysate was extractedwith 5 volumes of chloroform/methanol (2:1), evaporated to drynessunder reduced pressure, and analyzed by TLC.

Immunohistochemical Staining. An indirect immunoperoxidasemethod (30) was performed to determine the distribution of sialyl(Â«2-6)paragloboside in hepatocellular carcinoma tissues. Tissue blocks ofhepatocellular carcinoma (three cases) as well as normal liver tissues

Fig. 2. Immunostaining of gangliosides on TLC withmonoclonal antibody MSG-15 directed to the monosialo-ganglioside fraction of nicconium. I. stained with resorcinol/HCl reagent: B. immunoslaincd with MSG-15. LaneS, standard gangliosides, <Â¡M,. sialyl(<>2-3)paragloboside(SÂ¡alyI(u2-3)PG),i-ganglioside. and I-ganglioside from lopto bottom: Lane I. the monosialoganglioside fraction: Lane2. MG-1; Lane 3. MG-2; Lane 4. MG-3. The solventsystem was chloroform/methanol/water (65:35:8. v/v/v).

GM3Sialyl(tÃt2-3)PG- ~

Â¡-ganglioside- â€¢¿�*.

I-ganglioside-S 1 2 3 4 234

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Fig. 3. Stepwise degradation of the gangliosideMG-3 by exoglycosidases. Glycolipids treated withexoglycosidases were analyzed by TLC. Lane I,the standard glycolipids, LacCer. nLc4Cer (PG),nLc6Cer (i-glycolipid), and l-glycolipid from top tobottom; Lane 2, MG-3; Lane 3, MG-3 + C. per-fringem sialidase: Lane 4, lipid in lane 3 + jackbean /j-galactosidase; Lane 5, lipid in Lane 4 +jack bean /3-/V-acetylhexosaminidase; Lane 6, lipidin Lane 5 + jack bean /^-galactosidase; Lane 7, thestandard glycolipids. GlcCer and Lc3Cer, and tanrodeoxycholate (TDC). The solvent system was thesame as in Fig. 2. Glycolipids were visualized byspraying the plate with orcinol/H2SO4 reagent.

LacCer-

PG-

i-glycolipid~

l-glycolipid-

GlcCer

m,'Lc3Cer'TDC

6

GlcNAc/3

Glc/S

G.il Gal/3

sphingosine>c=c<

fatty acidÂ»cÂ«c<

JU V5.6 6.5 54 5.3 4.7 4.6 4.5 4.4 4.3 4.2 4.1 2.7 2.6 2.5

-ppm

Fig. 4. NMR spectrum of MG-3. MG-3 was dissolved in 0.5 ml of Me2SO-D6 containing 2% D2O. The NMR spectrum was obtained with a JEOL JNM-GX500 (500 MHz) at 45'C.

Ill

I 0-6j:

_Â§5 MIÂ«U

IODO 100 10 1

Ganglioside (ng)

Fig. 5. Assay of binding activity of MSG-15 to the sialyl(n2-6)paraglobosideby immunostaining on TLC. a, various amounts of the sialyl(i>2-6)paraglobosidewere subjected to TLC, and the binding activity of MSG-15 to the gangliosidewas tested by immunostaining. b, titration curve obtained by the immunostainingmethod. Color intensity was measured with a TLC chromatoscanner (ShimadzuCS 910). â€¢¿�,sialyl(Â«2-6)paragloboside; D, sialyl(a2-3)paragloboside; A, GM3.

(three cases) were selected from the pathology files of Central ClinicalLaboratories of Shinshu University Hospital. After blocking the endogenous peroxidase activity with 0.3% H2O2, tissue sections were incubated with MSG-15 (1:200 dilution, 5 Mg/ml) at 4Â°Covernight. After

rinsing with PBS, horseradish-conjugated anti-mouse IgM anti-mouseIgM antibody (1:100 dilution; Medical Biological Laboratories, Na-goya, Japan) was overlayered. After 2 h, the section was rinsed withPBS, and the peroxidase activity was visualized with diaminobenzidine/H2O2 solution. Counterstaining was carried out with methyl green.

RESULTS AND DISCUSSION

Monoclonal Antibody against Monosialoganglioside of Meco-nium. Seven hybridomas secreting monoclonal antibodies whichbound to the monosialoganglioside fraction of meconium wereselected by ELISA. One of the seven monoclonal antibodies,MSG-15, was selected on the basis of its reactivity and specificity to a monosialoganglioside of meconium by HPTLC immunostaining. In order to determine the ganglioside structurerecognized by MSG-15, the monosialoganglioside fraction wasseparated by HPLC, and each fraction eluted from the columnwas analyzed by TLC (Fig. 1). Three gangliosides (MG-1, MG-2, and MG-3) were separated as shown in Fig. 1. Gangliosideseluted after fraction 100 contained several components. MG-1was identified as GM3 by TLC. MG-2 showed similar mobilityto sialyl(a2-3)paragloboside on TLC. According to Iwamori etal. (31), MG-2 seems to be sialyl(a2-3)lactotetraosylceramide,and MG-3 has been suggested to be sialyl(a2-6)paraglobosideby Nilsson et al. (20). Purified MG-1, MG-2, and MG-3 weresubjected to TLC and immunostained with the MSG-15 antibody (Fig. 2). MG-3 was found to be a ganglioside which MSG-15 recognized. In addition to the ganglioside MG-3, two othergangliosides in the monosialoganglioside fraction were stainedwith MSG-15 (Fig. IB, Lane I). Therefore, we attempted toconfirm the structure of MG-3 ganglioside in order to determinethe epitope which MSG-15 recognizes.

Structure Analysis of MG-3 Ganglioside. MG-3 gangliosidewas subjected to stepwise degradation with various glycosidasesand each product was analyzed by TLC (Fig. 3). When MG-3was treated with C. perfringens sialidase, the product was converted to a lipid showing similar mobility to that of paraglo-boside. Asialo-MG-3 was converted to a lipid having similarmobility to that of Lc3Cer by jack bean /j-galactosidase treatment, and the product was further converted to LacCer by jackbean ÃŸ-hexosaminidase treatment. Finally the product was hy-drolyzed to GlcCer by 0-galactosidase treatment. Each productshows a slightly slower mobility compared with the standardglycolipid, as shown in Fig. 3. This is probably due to thedifference in fatty acid composition of the ceramide moiety.MG-3 was found to contain moderate amounts of hydroxy fattyacids (data not shown). This result indicates that MG-3 has asialosyllactotetraosylceramide or sialosyllactoneotetraosylcer-amide structure.

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â€¢¿�â€¢Â»ftm*M> MUÃ•~- K

Fig. 6. Immunostaining of monosialoganglioside fractionsobtained from nine hepatoma samples. I. immunostaining ofhepatoma monosialoganglioside fractions by MSG-1S. B, staining of the hepatoma monosialoganglioside fractions with resor-cinol/HCI reagent. Lanes 1-9. the monosialogangliosides obtained from hepatoma tissues. Lane S, standard gangliosides,GMj, sialyl(Â«2-3)paragloboside (Sialyl(a2-3)PG). and sialyl(Â«2-6)paragloboside (Sialyl(a2-6)PG). The plate was developed oncewith a mixture of chloroform/methanol (8:2, v/v) and then witha mixture of chloroform/methanol/0.2% CaCl2 (55:45:10,v/v/v).

12345678S 9B

GM3

3lalyl(ci2-3)PQ

SIÂ«lyl(d2-6)Pa

12345678S 9

Fig. 7. Composition of monosialogangliosides fromhepatoma samples. The gangliosides separated on TLCwere visualized by spraying with resorcinol/HCI reagent.Color intensity was measured by the TLC chromatos-canner. Percentages were expressed on the basis of sialicacid. Bars in panel of control, S.D. Five control livertissues were used for the analysis.

GMjSialyl(a2-3)PG

Other gangliosidesSialyl(Â«2-6)PG

GangliosideswithNeuAca2-6Gal/3CM,Sialyl(o2-3)PG

Other gangliosidesSialyl(d2-6)PG

Gangliosides withNeuAcd2-6Gal|3H)}

Control120

40 60 80*|]

5â€¢ZI

1 120

40 60BO*D

]6I

20 40 M 80* 40 60 80* 20 40 60 80'

I20 40 60 80* 20 Â«060 BO% 20 40 60 BOÂ« 2040 60 80X 20 40 60 BO%

In order to determine the anomeric configuration and molarratios of sugar components, an NMR spectrum of MG-3 wasobtained (Fig. 4). Assignments of signals of anomeric protonsbetween 4 and 5 ppm are indicated in Fig. 4. The peak at 4.7ppm was assigned to the /3-anomeric proton of GlcNAc fromthe data reported by Vliegenthart et al. (32) and us (21). Signalsat 4.267 and 4.215 ppm were assigned to the 0-anomericprotons of Gal. The anomeric proton of ÃŸ-Glcshowed splitsignals at 4.20 and 4.23 ppm (Fig. 4). This suggests the presenceof hydroxy fatty acids in the ceramide moiety of the MG-3 (33).Quartet peaks at 2.61, 2.62, 2.63, and 2.64 ppm were assignedto equatorial 3H of A'-acetylneuraminic acid, which is attachedto the 6C position of galactose or jV-acetylglucosamine (32, 34).From the peak areas of anomeric protons and equatorial 3H of

NeuAc, the molar ratios of /3Glc:0Gal:/3GlcNAc:Â«NeuAc weredetermined to be 1:2:1:1. Methylation analysis of MG-3 indicated the presence of 4-substituted glucose, 3-substituted galactose, 4-substituted yV-acetylglucosamine, and 6-substituted galactose. From these analytical data, the structure of MG-3was confirmed to be NeuAca2-6Gal/il-4GlcNAc/31-3Gal/31-4Glc01-lceramide (sialyl(a2-6)paragloboside).

Immunostaining of Sialyl(a2-6)paragloboside with MSG-15.Purified MG-3 was found to be a ganglioside recognized by theMSG-15 antibody as shown in Fig. 2. In addition to the MG-3ganglioside, two other bands were stained with this antibody.We purified these gangliosides and checked the terminal structures by NMR spectroscopy. NMR spectra of these gangliosidesclearly indicated the presence of characteristic signals derived

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GM3Sialyl(d2-3)PG

OthergangliosidesSialyl(d2-6)PG

Gangliosides withNeuAcet2-6Gal^3GMjSÂ¡alyl(i2-3)PG

Other gangliosidesSlalyl(*2-6)PG

Gangliosides withNeuAcd2-6Galf31='20

40 6080*|1

1rziâ€¢20

40 BO80"1b]C20 40 BO 80"5|C

20 40 BO80% 20 40 60 80% 20 40 60 80'.

Fig. 8. Composition of monosialogangliosides from hepatoma tissues (A-C)and their adjacent cirrhotic livers (a-c). Assay procedure was the same as in Fig.7. Other gangliosides in A accounted for 48% of monosialoganglioside and themajority of them were GM2.

from the equatorial 3H of NeuAc linked to 6C of galactose.3Therefore, MSG-15 seems to recognize the terminal NeuAcÂ«2-6Gal/3 structure of the ganglioside. To confirm the epitope ofMSG-15, various gangliosides were subjected to immunostain-ing on TLC plates. MSG-15 failed to react with other gangliosides, including GM3, GM2, GMla, GDU, GD)b, GTjb, sialyl(a2-3)paragloboside, and Ii-type gangliosides. The results indicatethat the antibody specifically recognizes NeuAca2-6Galj3 structure. A titration curve of MSG-15 obtained by using sialyl(a2-6)paragloboside as the antigen is shown in Fig. 5. One ng ofsialyl(cv2-6)paragloboside could be visualized by the immuno-staining technique using MSG-15 (0.5 ÃŸg/m\in PBS).

Accumulation of Sialyl(a2-6)paragloboside in Monosialoganglioside Fraction of Primary Hepatomas. Monosialogangliosidefractions from nine primary hepatoma samples were developedon TLC and immunostained with MSG-15 (Fig. 6). All hepatoma samples were found to contain bands stained with theantibody, and these bands showed the same mobilities as thatof sialyl(Â«2-6)paragloboside. Furthermore, five samples (samples 3 and 6 to 9) were found to contain other gangliosidesstained with MSG-15, and they showed much slower mobilitiesthan that of sialyl(Â«2-6)paragloboside. These bands are apparently NeuAca2-6Gal0 structure-containing gangliosides withmore complicated carbohydrate structures than sialyl(a2-6)paragloboside. In Fig. dA, gangliosides corresponding to sia-Iyl(tt2-6)paragloboside and other immunostained gangliosidesare split into two or three bands. In this chromatography, theplate was developed with a solvent mixture of chloroform/methanol (8:2, v/v) and then developed again with the solventmixture of chloroform/methanol/0.2% CaCl2 (55:45:10, v/v/v)to get better separation. Gangliosides that consisted of threebands seem to have the same carbohydrate structure but different ceramide species, because these three bands were combinedto form a single, rather broad, band as shown in Fig. 10 whenthe solvent system for developing was changed.

The concentration of sialyl(a2-6)paragloboside and gangliosides with NeuAca2-6Gal|S structure in each hepatoma monosialoganglioside fraction was determined by densitometricanalysis, and the results are summarized in Fig. 7. Sialyl(a2-6)paragloboside accounted for 13-31% of monosialoganglioside fractions, but that of normal control liver accounted foronly about 1% of the fraction (Fig. 7). If the gangliosides stainedwith MSG-15 were combined, NeuAcÂ«2-6Gal/3-containing gly-colipid accounted for about 50% in one case (sample 9). Fivecontrol liver tissues were used for monosialoganglioside assay.

>T. Taki. K. Ishii. S. Ando, K. Kon, C. Rokukawa, T. Abe. S. Manda,

manuscript in preparation.

A major monosialoganglioside in normal liver was GM, (92%),and the rest was mainly sialyl(a2-3)paragloboside. Sialyl(a2-6)paragloboside was hardly detected by resorcinol reagent andwas visualized only by immunostaining. The concentration ofthe ganglioside was less than 1% in control tissues. In the otherthree cases, gangliosides from adjacent cirrhotic livers were alsostudied and compared with those from corresponding carcinoma tissues. These results are shown in Fig. 8, indicating thatpatterns of monosialoganglioside composition in cirrhotic liverswere very similar to those of normal liver. In one case ofcarcinoma tissue, a tremendous increase of GM2 (accountingfor about 48%; column of other gangliosides in Fig. 8/1) wasobserved. In these three cases, sialyl(a2-6)paragloboside accounted for 8-13% in hepatoma tissue, but less than 1% in thecirrhotic liver.

An immunohistochemical study was performed using threehepatocellular carcinomas and three normal control liver tissues. The carcinoma cells showed positive reactions with MSG-15 monoclonal antibody in two cases, and weak positive reaction in one case. Among the positive two cases, positive densebrown stainings are observed along cell membranes of thecarcinoma cells (Fig. 9a). In the other case, cytoplasmic stain-ings of the carcinoma cells are demonstrated (Fig. 9b). Nopositive staining was observed in the normal liver tissues,including hepatocytes and bile ducts (Fig. 9c). In cirrhotic liver,positive staining could not be observed. Immunohistochemicalstudy using MSG-15 in tissue sections from hepatocellularcarcinoma, liver cirrhosis, and biliary and gastrointestinal tractmalignancies revealed that only hepatocellular carcinoma sections gave positive dense brown granules.4 These results indicate

that the activity of sialyltransferase catalyzing the formation ofNeuAca2-6Gal/3 structure at nonreducing terminals of glyco-

lipids is enhanced in hepatoma tissue. The results of the analysisof monosialoganglioside fractions with MSG-15 suggested theoccurrence of gangliosides with the NeuAcÂ«2-6Gal0 structurein disialo- or trisialoganglioside fractions. Next, we examinedthe appearance of NeuAcÂ«2-6Gal/3linkage in other gangliosidefractions from a hepatoma sample (sample 8 in Fig. 6) andmeconium by using MSG-15.

Comparison of Sialyl(a2-6)lactosamine Structure-containingGangliosides in Hepatoma and Meconium. Gangliosides from ahepatoma sample (Fig. 6, Lane 8) and meconium were separatedby HPTLC. One plate (Fig. IOA) was sprayed with the resor-cinol/HCl reagent to visualize all gangliosides. A second plate(Fig. IOA) was immunostained with the MSG-15 antibody, anda third plate (Fig. IOC) was treated with C. perfringens sialidaseand then immunostained with a monoclonal antibody, H-ll,which recognizes terminal lactosamine structure of glycolipids(23). Three bands stained with MSG-15 were detected in bothmonosialoganglioside fractions. Bands split into three bands inFig. 6 are compacted into one band in Fig. 10 because of thedifferent solvent system used for developing; these gangliosidesof hepatoma and meconium show very similar mobilities toeach other on the TLC plate. In the disialoganglioside fractions,one major band in meconium and two bands in the hepatomawere observed. The upper band of hepatoma showed similarmobility to that of the band of meconium. In the trisialoganglioside fractions, two bands in the case of hepatoma and oneband in the meconium were visualized with the antibody MSG-15. These gangliosides, stained with MSG-15, show very similarmobilities to each other. When the ganglioside fractions weretreated with sialidase and stained with H-ll, multiple bands

4 H. Yamada. personal communication.

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Fig. 9. Immunohistochemical staining of hepatocellular carcinoma and normal liver tissues, a, hepatocellular carcinoma (trabecular type). Positive reaction withMSG-15 antibody along the cell membranes of the carcinoma cells, o, hepatocellular carcinoma (solid type). Positive reaction with MSG-15 antibody in the cytoplasmof the carcinoma cells, c, normal liver tissue. Hepatocytes and bile ducts showed no reaction, p, portal area.

Sialyl(d2-3)PGâ€” -

i-gangliosÂ¡de > â€¢¿�

l-ganglioside Â»-â€”¿�

A1234B567 8

â€¢¿�

1234B5678

* * "**#_-

1 2 3 4 B5 6 7 8Fig. 10. Immunostaining of the ganglioside fractions from meconium and a hepatoma sample, a, the mono-, di-, tri-, and polysialoganglioside fractions were

developed by TLC and visualized with resorcinol/HCI reagent. A. the ganglioside fractions developed by TLC were immunostained by MSG-15. c. the gangliosidesseparated by TLC were treated with C perfringens sialidase and immunostained with a monoclonal antibody, H-l I, which recognizes the terminal lactosamine residueof glycolipid (23). The bands marked with asterisks corresponded to those stained by MSG-15 antibody. The band marked with a star shows ganglioside notimmunostained with MSG-15. Lane A. standard gangliosides. CM,, sialyl(Â«2-3)paragloboside, i- and l-type ganglioside from top to bottom; Lane B, sialyl(Â«2-6)paragloboside; Lanes 1-4, mono-, di-, tri-, and polysialoganglioside fractions of meconium, respectively. Lanes 5-8 show mono-, di-, tri-, and polysialogangliosidefractions of hepatoma tissue, respectively. The solvent system was a mixture of chloroform/methanol/0.2% CaCI2 (60:35:8, v/v/v).

appeared in both samples, indicating that these samples contained a variety of lactosamine-containing gangliosides. Thebands marked with asterisks in Fig. 10 show glycolipids corresponding to those visualized on the second plate. All the bandsin Fig. 10, B and C, comigrated and probably represent thesame structures except the one marked with a star. The gangliosides from the two different sources have similar profilesin terms of NeuAcÂ«2-6Gal/31-4-GlcNAc/3-containing gangliosides, indicating that the biosynthetic pathway is activated inhepatoma cells.

Hakomori et al. (35) reported the preparation of a monoclonal antibody directed to NeuAcÂ«2-6Gal/3.The antibody, namedIB9, seems to have similar specificity to the monoclonal antibody MSG-15. They showed the occurrence of NeuAcÂ«2-6Gal/3-containing gangliosides in a human liver adenocarci-noma. Nilsson et al. (36) prepared a monoclonal antibody, LM-4, directed to sialyl(Â«2-6)paragloboside and demonstrated thatgangliosides with a NeuAcÂ«2-6GalÃŸsubstitution were enriched

in many carcinomas, particularly in most colorectal carcinomasand in lung carcinomas. In the present study, we also found theaccumulation of sialyl(Â«2-6)paragloboside and a variety of gan

gliosides containing the terminal NeuAcÂ«2-6Gal/31-4GlcNAc/;istructure in hepatoma and in meconium. Glycolipids containingthe NeuAca2-6GalÃŸl-4GlcNAc0 structure are rarely foundin gangliosides. Two types of gangliosides, sialyl(Â«2-6)paragloboside and sialyl(Â«2-6)lactoneohexaosylceramidehave been isolated from human erythrocytes (37), and one noveltype of ganglioside with this structure, NeuAcÂ«2-6GalÂ£fl-4GlcNAc/n-3(GaI01-4GlcNAc#l-6)Gal#l-4GlcÃÂ¡l-lCer in bovine buttermilk was reported by Takamizawa et al. (38). However, the quantity of these gangliosides was very low (less than2% of total gangliosides of human erythrocytes) (37). Recently,we isolated gangliosides with the structure NeuAcÂ«2-6Gal/31-4GlcNAcÂ£ifrom meconium. The results of a detailed structureanalysis of the gangliosides will be reported elsewhere.

ACKNOWLEDGMENTS

We wish to thank Dr. F. Inagaki (The Tokyo Metropolitan Instituteof Medical Science) for the 500-MHz NMR spectrometric analysis.

REFERENCES1. Hakomori. S., and Kannagi, R. Glycosphingolipids as tumor-associated and

differentiation markers. J. Nati. Cancer Inst.. 71: 231-251. 1983.

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2. Yogeeswaran, G. Cell surface glycolipids and glycoproteins in malignanttransformation. Adv. Cancer Res., 38: 289-350, 1983.

3. Hakomori. S. Tumor-associated carbohydrate antigens. Annu. Rev. Immu-nol.. 2: 103-126, 1984.

4. Magnani, J. I . Carbohydrate differentiation and cancer-associated antigensdetected by monoclonal antibodies. Biochem. Soc. Trans., 12:543-545,1984.

5. Ito, M., Suzuki. E.. Naiki. M.. Sendo, F., and Arai, S. Carbohydrates asantigenic determinants of tumor-associated antigens recognized by monoclonal anti-tumor antibodies produced in a syngeneic system. Int. J. Cancer, 34:689-697, 1984.

6. Hakomori, S. Aberrant glycosylation in cancer cell membranes as focused onglycolipids: overview and perspectives. Cancer Res., 45: 2405-2414, 1985.

7. Hakomori, S. Glycosphingolipids in cellular interaction, differentiation andoncogenesis. Annu. Rev. Biochem.. 50: 733-764. 1981.

8. Akagawa, K. S., Momoi, T., Nagai, Y.. and Tokunaga. T. Appearance ofasialo-GM 1glycosphingolipid on the cell surface during lymphokine-induceddifferentiation of Ml cells. FEBS Lett., 130: 80-84, 1981.

9. Taki, T.. Kawamoto, M., Seto, H., Noro, N., Masuda. T., Kannagi. R., andMatsumoto, M. Differentiation-associated changes of glycolipid compositionand metabolism in mouse leukemia cells. Induction of globotriaosylceramideand a galactosyltransferase. J. Biochem., 94: 633-644, 1983.

10. Kannagi. R., Levery, S. B.. and Hakomori. S. Sequential change of carbohydrate antigen associated with the differentiation of murine leukemia cells,Ml: il-Antigenic conversion and shifting of glycolipid synthesis. Proc. Nati.Acad. Sci. USA, 80: 2844-2848. 1983.

11. Magnani, J. L., Nilsson, B., Brockhaus. M., Zopf, D.. Steplewski, Z.,Koprowski. H., and Ginsburg. V. A monoclonal antibody-defined antigenassociated with gastrointestinal cancer is a ganglioside containing sialylatedlacto-/V-fucopentaose II. J. Biol. Chem., 257: 14365-14369, 1982.

12. Nudelman, E., Hakomori, S., Kannagi. R., Levery, S., Yeh, M.-Y., Hellstrom,K. E., and Hellstrom, I. Characterization of a human melanoma-associatedganglioside antigen defined by a monoclonal antibody. J. Biol. Chem., 257:12752-12756, 1982.

13. Hakomori. S., Nudelman. E., Levery, S. B.. and Patterson. C. M. Humancancer-associated gangliosides defined by a monoclonal antibody (IB9) directed to sialylÂ»2-6galactosyl residue: a preliminary note. Biochem. Biophys.Res. Commun.. 113: 791-798. 1983.

14. Nilsson, O., Mansson. J.-E., Brezicka, T.. Holmgren. J.. Lindholm. L.,Sorenson, S., Yngvason. F.. and Svennerholm, L. Fucosyl-GMlâ€”a ganglioside associated with small cell lung carcinomas. Glycoconjugates J., /: 43-49, 1984.

15. Fukushima, K., Hirota. M., Terasaki, P. I., Wakisaka, A., Togashi, H., Chia,D., Suyama, N., Fukushi, Y., Nudelman, E., and Hakomori, S. Characterization of sialosylated Lewis* as a new tumor-associated antigen. CancerRes., 44: 5279-5285, 1984.

16. Nakakuma. H., Sanai. Y., Shiroki, K., and Nagai, Y. Gene-regulated expression of glycolipids: appearance of GD3 ganglioside in rat cells on transformation with transforming gene El of human adcnovirus type 12 DNA andits transcriptional subunits. J. Biochem.. 96: 1471-1480, 1984.

17. Karlsson. K.-A., and Larson. G. Structural characterization of lactotetrao-sylceramide. a novel glycosphingolipid isolated from human meconium. J.Biol. Chem., 254: 9311-9316, 1979.

18. Karlsson. K.-A.. and Larson. G. Molecular characterization of cell surfaceantigens of fetal tissue. Detailed analysis of glycosphingolipids of meconiumof a human O Le(a-b+) secretor. J. Biol. Chem.. 25Â«:3512-3524. 1981.

19. Prieto. P. A., and Smith, D. F. A new ganglioside in human meconiumdetected by antiserum against the human milk sialyloligosaccharide. LS-tetrasaccharide b. Arch. Biochem. Biophys., 241: 281-289. 1985.

20. Nilsson, O., Mansson, J.-E.. Tibblin. E., and Svennerholm, L. Gangliosides

of tumor meconium. possible detection of fetal antigen. FEBS Lett., 133:197-200, 1981.

21. Taki, T., Matsuo, K.. Yamamoto, K., Matsubara. T., Hayashi, A., Abe, T.,and Matusmoto, M. Human placenta gangliosides. Lipids, 23: 192-198,1988.

22. Young, W. W.. Jr., MacDonald, E. M. S.. Nowinski, R. C., and Hakomori.S. Production of monoclonal antibodies specific for distinct portions of theglycolipid asialoGM2 (gangliotriaosylceramide) J. Exp. Med., 750: 1008-1019, 1979.

23. Myoga, A., Taki, T., Arai. K., Sekiguchi, K.. Ikeda. I., Kurata, K., andMatsumoto, M. Detection of patients with cancer by monoclonal antibodydirected to lactoneotetraosylceramide (paragloboside). Cancer Res., 48:1512-1516, 1988.

24. Saito, M., Kasai, N., and Yu, R. K. In situ immunological determination ofbasic carbohydrate structures of gangliosides on thin-layer plates. Anal.Biochem., 148: 54-58, 1985.

25. Mansson, J.-E., Egge, H. M. E., and Svennerholm, L. Trisialosyllactosylcer-amide is a ganglioside of human lung. FEBS Lett., 196: 259-262. 1986.

26. Ohashi, M., Uchida, K., and Yamakawa Proc. Jap. Conf. Biochem. Lipids.26:75-77. 1984.

27. Barton. N. W.. Lipovac. V.. and Rosenberg, A. Effect of strong electrolyteupon the activity of Closlridium perfringens sialidase toward sialyllactoneand sialoglycolipids. J. Biol. Chem.. 250: 8462-8466. 1975.

28. Li, S.-C., Mazzotta, M. Y., Chien, S. F., and Li. Y.-T. Isolation andcharacterization of jack bean /3-galactosidase. J. Biol. Chem., 250: 6786-6791, 1975.

29. Li. Y.-T., and Li, S.-C. a-Mannosidase, /J-iV-acetylhexosaminidase and g-galactosidase from jack bean meal. In: V. Ginsburg (ed.). Methods in Enzy-mology. Vol. 28, pp. 702-713. New York: Academic Press, 1975.

30. Nakane, P. K. Recent progress in the peroxidase-labeled antibody method.Ann. NY Acad. Sci., 254: 203-211, 1975.

31. Iwamori, N., Noguchi, M., Yamamoto, T., Yago, M., Nozawa, S., and Nagai,Y. Selective terminal Â«2-3and Â«2-6sialylation of glycosphingolipids withlacto-series type 1 and 2 chains in human meconium. FEBS Lett., 313: 134-138, 1988.

32. Vliegenthart. J. F. G., Dorland. L.. and van Halbcek. H. High-resolution.'H-nuclear magnetic resonance spectroscopy as a tool in the structural

analysis of carbohydrates related to glycoproteins. Adv. Carbohydr. Chem.Biochem.. 41: 209-374. 1983.

33. Kulm. H., Irie, A., Inagaki, F., and Hoshi. M. Melibiosylceramide as the soleceramide dihexoside from the eggs of the sea urchin, Anihocidaris crassispia.J. Biochem.. 104: 755-760. 1988.

34. Taki, T., Hirabayashi, Y., Ishikawa, H., Ando, S., Kon, K., Tanaka, Y., andMatsumoto, M. A ganglioside of rat ascites hepatoma AH 7974F cells.Occurrence of a novel disialoganglioside (GDIÂ«)with a unique W-acetylneu-raminosyl(Â«2-6)-A'-acetylgalactosamine structure. J. Biol. Chem., 267:3075-

3078, 1986.35. Hakomori. S., Patterson. C. M., Nudelman. E., and Sekiguchi. K. A mono

clonal antibody directed to A'-acetylneuraminosylÂ«2-6galactosyl residue ingangliosides and glycoproteins. J. Biol. Chem.. 25Ã„:11819-11822. 1983.

36. Nilsson, O., Lindholm, L.. Holmgren, J.. and Svennerholm, L. Monoclonalantibodies raised against NeuAcÂ«2-6neolactotetraosylceramide detect carcinoma-associated gangliosides. Biochim. Biophys. Acta, 835: 577-583, 1985.

37. Watanabe. K., Powell, M. E.. and Hakomori. S. Isolation and characterization of gangliosides with a new sialyl linkage and core structures. II. Gangliosides of human erythrocyte membranes. J. Biol. Chem., 254:8223-8229,1979.

38. Takamizawa. K., Iwamori, M., Mutai. M., and Nagai, Y. Gangliosides ofbovine buttermilk. J. Biol. Chem., 261: 5625-5630, 1986.

39. Svennerholm. L. The gangliosides. J. Lipid Res., 5: 145-155. 1964.

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1990;50:1284-1290. Cancer Res Takao Taki, Kaname Yamamoto, Miki Takamatsu, et al. 6)lactosamine Structure in Primary Human Hepatoma

−2α-Acetylneuraminosyl(NAccumulation of Gangliosides with

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Accumulation of Gangliosides with A^-Acetylneuraminosyl(a2-6