ARCHIVES

OF BIOCHEMISTRY

Vol. 294, No. 2, May

AND

BIOPHYSICS

1, pp. 427-433,

1992

Generation of Murine Monoclonal Antibodies Specific for A/-Glycolylneuraminic Acid-Containing Gangliosides’ Hideki

Ozawa, Ikuo

Kawashima,

and Tadashi

Department of Tumor Immunology, Tokyo Metropolitan Honkomugome, Bunkyo-ku, Tokyo 113, Japan

Received

November

12, 1991, and in revised

form

December

Tai2 Institute

Medical

Science,

13, 1991

We generated two murine monoclonal antibodies (MAbs) specific for mono- and disialylgangliosides having N-glycolylneuraminic acid (NeuGc) as their sialic acid moiety, respectively, by immunizing C3H/HeN mice with these purified gangliosides adsorbed to Salmonella minnesota followed by fusion with mouse myeloma cells. By use of a wide variety of glycolipids, including NeuGccontaining gangliosides, the precise structures recognized by these two antibodies were elucidated through enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatography. One MAb, GMRS, which was generated by immunizing the mice with purified GMS(NeuGc), reacted specifically with gangliosides having NeuGca2+3Galterminal structures, such as GM3(NeuGc), IV’NeuGca-Gg&er, IVSNeuGca-nLc&er, V’NeuGca-Gb&er, and GDla(NeuGc, NeuGc). None of the other gangliosides having internal NeuGca2+3Galsequences, such as GMfL(NeuGc) and GMl(NeuGc), nor corresponding gangliosides having NeuAcaS+SGal- sequences, nor neutral glycolipids were recognized. Thus, the epitope structures recognized by the MAb were found to be strictly NeuGca2+3Galterminal structures. In contrast, the other MAb, GMRS, which was generated by immunizing the mice with purified GDS(NeuGc-NeuGc-) adsorbed to the bacteria, reacted specifically with gangliosides having NeuGca2+8NeuGco2+3Galterminal sequences, such as GDS(NeuGc-NeuGc-), IV3NeuGcazGg&er, IV’NeuGccuz-nLc&er, and VSNeuGcaz-Gb&er, but did not react with corresponding gangliosides having NeuAc as their sialic acid moiety or with the neutral glycolipids tested. The epitope structures recognized by the MAb were suggested to be NeuGca2+8NeuGca2*3Gali This work was supported in part by a Grant-in-Aid for Scientific Research (02660147) and a Grant-in-Aid for Scientific Research on Priority Areas (03255103) from the Ministry of Education, Science and Culture of Japan and by a grant from the Naito Foundation. * To whom correspondence should be addressed. 0003-9S61/92 $3.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form

of

terminal structures. Using these MAbs, we determined the distribution of such gangliosides in the spleen, kidney, and liver of several mice strains. Novel gangliosides reactive with these MAbs were detected in these tissues. 0 1992

Academic

Press,

Inc.

Gangliosides are glycosphingolipids containing sialic acids, of which structural diversity exists (1). Only Nacetylneuraminic acid (NeuAc)3 and its derivatives are found in humans, whereas some other animal speciesalso synthesize N-glycolylneuraminic acid (NeuGc). A number of murine monoclonal antibodies (MAbs) against NeuAccontaining gangliosides have been generated previously (2-4), whereas few murine MAbs to gangliosides containing NeuGc have been reported yet (5-7). It would be preferable to establish hybridomas by immunizing mice directly with purified gangliosides. It is, however, still difficult to routinely generate MAbs to these gangliosides, in particular to those containing NeuGc as their sialic acid moiety. In the past decade, MAbs to gangliosides have been established by immunizing BALB/c or C57BL/6 mice with neuroectoderm-derived tumor cells (8). To develop an improved method for generating murine MAbs to gangliosides with high efficiency, we recently compared antibody responsesto various gangliosides containing NeuAc and NeuGc as their sialic acid moiety in different strains of inbred mice (9). We have found that (i) C3H/HeN and A/J mice demonstrate maximum antibody responses ’ Abbreviations used: NeuAc, N-acetylneuraminic acid, NeuGc, Nglycolylneuraminic acid; MAb, monoclonal antibody; ELISA, enzymelinked immunosorbent assay; PBS, phosphate-buffered saline; The sialic acid moiety of gangliosides is NeuAc unless otherwise noted. Gangliosides are named according to the nomenclature of Svennerholm (29); otherwise, the nomenclature used follows IUPAC-IUB recommendations (30). 427

Inc. reserved.

428

OZAWA,

KAWASHIMA, TABLE

Structure

of Gangliosides

Ganglioside

AND

TAI

I Used

in This

Study

Structure

NeuGc-containing gangliosides GMB(NeuGc) GMB(NeuGc) GMl(NeuGc) IV3NeuGca-Gg&er IV3NeuGca-nLc&er V3NeuGca-Gb&er GDla(NeuGc, NeuGc) GDB(NeuGc-NeuGc-) IV3NeuGccu2-Gg,Cer IV3NeuGcaZ-nLc&er V3NeuGcaz-GbsCer

NeuGcaZ GalNAc@l GalPl + NeuGca2 NeuGca2 NeuGca2 NeuGccu2 NeuGca2 NeuGcaQ NeuGca2 NeuGca2

NeuAc-containing gangliosides GM4 GM3 GM2 GM1 IV3NeuAca-Gg,Cer IV3NeuAccY-nLc,Cer GDla GDB(NeuAc-Net&-) 0-AC-GD3 GD2 GDlb IV3NeuAca,-nLc,Cer GTla GTlb GT3 GT2 GQlb

NeuAca2 + BGal-Cer NeuAccu2 + 3Galpl + 4GlcCer GalNAc@l + 4(NeuAccu2 + 3)Gal/31+ 4Glc-Cer Gala1 + BGalNA@l + 4(NeuAca2 + 3)Galj31+ 4Glc-Cer NeuAca2 + 3Galj31+ 3GalNAcj31--* 4Gal/31+ 4GlcCer NeuAccu2 + 3Galj31+ 4GlcNAcfll + 3Gal/31 --t 4Glc-Cer NeuAca2 + 3Gal/31 --t 3GalNAc/31+ 4(NeuAca2 + 3)GalSl+ 4Glc-Cer NeuAca2 + 8NeuAcaP + 3Galal+ 4Glc-Cer 0-Ac-NeuAca2 + 8NeuAccu2 + 3Galal+ 4Glc-Cer GalNAcfll + 4(NeuAca2 + 8NeuAca2 + 3)Gal@l+ 4Glc-Cer Gal/31 + 3GalNAc$l+ 4(NeuAccY2 --+ SNeuAca2 + 3)Gal@l+ 4Glc-Cer NeuAccY2 + 8NeuAca2 + 3Galal+ 4GlcNAc@l+ 3GalPl+ 4Glc-Cer NeuAcaX + 8NeuAccu2 + 3Galj31+ 3GalNAcfll+ ri(NeuAca2 + 3)Gal/31+ NeuAca2 + 3GalSl-+ 3GalNAcfil -+ 4(NeuAca2 + SNeuAca2 + 3)Gal/31+ NeuAccu2 + 8NeuAca2 + 8NeuAca2 + 3Gal/31+ 4Glc-Cer GalNAc@l + 4(NeuAca2 + 8NeuAca2 + 8NeuAca2 + 3)Gal@+ 4Glc-Cer NeuAca2 + 8NeuAca2 + 3Gal/31* 3GalNAcPl + 4(NeuAccu2 + SNeuAccuL

+ 3Gal/31+ --* 4(NeuGccu2 3GalNAq31 --t + 3Gal/31+ + 3Galj31+ + 3Galj31+ + 3Gal/31+ + 8NeuGccY2 + 8NeuGccr2 + SNeuGca2 --t 8NeuGca2

4GlcCer + 3)Galfll+ 4Glc-Cer 4(NeuGccu2 + 3)Gal pl+ QGlc-Cer 3GalNAc/31+ 4Gal j31 + 4Glc-Cer lGlcNAc@l --* 3Gal/31+ 4Glc-Cer 3GalNAc@l+ 3Galal + 4Gal/31--* 4Glc-Cer 3GalNAcfll+ 4(NeuGca2 + 3)Gal/31+ 4Glc-Cer + 3Galfil--* QGlc-Cer + 3Gal/31+ 3GalNAc@l+ 4Gal fll + 4Glc-Cer + 3GalPl+ 4GlcNAc/31+ 3Gal j31+ 4Glc-Cer + 3GalPl + 3GalNAc@l+ 3Galal+ 4Gal @l+

against these gangliosides among these mice; (ii) gangliosides having NeuAc induce relatively high-titer antibody responses, whereas those having NeuGc induce relatively low-titer antibody responses; (iii) the pattern of reactivity to the various gangliosides is similar in all the strains tested. These results suggest that C3H/HeN and A/J mice are more suitable than other strains of mice for raising MAbs to gangliosides. The different antibody responses among various mice strains may reflect the sensitivity to lipopolysaccharide. In the present paper, we describe the production of murine MAbs specific for NeuGc-containing gangliosides. These MAbs were useful for detecting gangliosides having NeuGc as their sialic acid residue in some tissues of mice. Also, they would be useful for analyzing the biological functions of these gangliosides on the cell surface. MATERIALS

AND

METHODS

Glycosphingolipids. GMl, GDla, GDlb, and GTlb were prepared from bovine brain. GM3 and GQlb (bovine brain) and GD3 (bovine milk) were purchased from Iatron (Tokyo, Japan). GM2 and GD2 were prepared from GM1 and GDlb, respectively, by treatment of bovine testis @galactosidase (Boehringer-Mannheim-Yamanouchi, Tokyo,

4Glc-Cer

4Glc-Cer 4Glc-Cer

+

3)Galbl+

QGlc-Cer

Japan) as described previously (10). 9-0-Acetylated GD3 (0-AC-GD3) was isolated from a human melanoma cell line (9). Codfish brain gangliosides (mixtures of GT3, GT2, and other gangliosides) and GTla (monkey brain) were gifts from S. Ando (Tokyo Metropolitan Institute of Gerontology). GM4 (human myelin) was provided by T. Ariga (Virginia Commonwealth University). IV3NeuAccu-nLc&er and IVsNeuGcanLc,Cer were isolated from human and bear erythrocytes, respectively. GM3(NeuGc), GM2(NeuGc), GMl(NeuGc), and GDla(NeuGc, NeuGc) of mouse (ICR) liver and four GD3 isomers [GDB(NeuAc-NeuAc-), GDB(NeuAc-NeuGc-), GD3(NeuGc-NeuAc-), and (NeuGc-NeuGc-)] of bear erythrocytes were donated by Y. Hashimoto and A. Suzuki (Tokyo Metropolitan Institute of Medical Science). GD3 (NeuGc-NeuGc-) of cat erythrocytes was a gift from S. Handa (Tokyo Medical and Dental College). IV3NeuAcaz-nLc,Cer of human kidney was donated by I. Ishixuka (Teikyo University School of Medicine). IV3NeuGca,-nLc,Cer was prepared from sheep erythrocytes. IV3NeuGca,-Gg&er (mouse thymoma) and V3NeuGcaz-GbsCer of mouse (DBA/Z) kidney were donated by K. Nakamura and M. Sekine (Tokyo Metropolitan Institute of Medical Science), respectively. IV3NeuGccu-Gg&er and V3NeuGccY-Gb,Cer were isolated from mouse spleen and kidney, respectively. GalCer (bovine brain) was purchased from Sigma (St. Louis, MO). LacCer, Gg,Cer, and Gg&er were prepared from GM3, GM2, and GMl, respectively, by treatment with sialidase as previously described (10). Gb,Cer, Gb,Cer, and IV3GalNAcol-Gb&er were purchased from Iatron. The structures of gangliosides used in this study are shown in Table I. Cells and tissues. PA1 (BALB/c-derived supplied by the Japanese Cancer Research

myeloma cells) was kindly Resources Bank (Tokyo,

MONOCLONAL

ANTIBODIES

Japan). Ml4 (Human melanoma cell line) cells were donated by Irie (UCLA School of Medicine), and maintained in serum-free medium. Sheep and bovine erythrocytes were obtained commercially. Female mice of the inbred strains BALB/c, C57BLf6, DBAj2, and CBA/N were purchased from Japan SLC Inc. (Shizuoka, Japan). The spleen, kidney, and liver of these mice aged 7 to 12 weeks were stored at -20°C until glycolipid extraction. Total glycolipids were extracted from cells or tissues with chloroform:methanol:water, and gangliosides were separated from neutral glycolipids using DEAE-Sephadex chromatography and Iatrobead column chromatography as described previously (11). Monoclonal antibodies. Hybridomas producing MAbs were established as previously described (12). Briefly, female mice of the C3H/ HeN inbred strain (7 weeks of age), which were obtained from Japan Clea (Tokyo, Japan), were immunized with purified ganglioside (10 pg, total amount per mouse) adsorbed to acid-treated Salmonella minnesota (250 pg, total amount per mouse) by the procedure of Young et al. (13). Each mouse received intravenous injections of the antigen-bacteria complex on Days 0, 4, 7, 11, and 21. The spleen cells were obtained 3 days after the last injection and were fused with a myeloma cell line, PAI, by a routine cell hybridization procedure. The hybridoma fusions were screened against the immunizing ganglioside. The antibody titers in the supernatant of hybridoma cells were assayed by enzyme-linked immunosorbent assay (ELISA). The positive hybrid was cloned twice by limiting dilution. The isotype of antibody was determined by a mouse monoclonal antibody isotyping kit (Amersham, UK). MAb GMR24 specific for GA1 was prepared in our laboratory. The specificity of the MAb will be described in detail elsewhere. Enzyme-linked immunosorbent assay. Solid-phase ELISA was performed as previously reported (14). A 96-well polystyrene microtiter plate (Dynatech, Alexandria, VA) was used. Glycolipid (0.5 nmol) was serially diluted in ethanol and applied to the plate in a volume of 50 al/ well. The supernatant of cloning hybridoma cells was diluted in PBS containing 1% human serum albumin. The plates were incubated for 1 h at room temperature. After washing, the second antibody, horseradish peroxidase-conjugated goat anti-mouse IgG or IgM (Cappel, Malvern, PA), diluted 1:200, was added to the plates and incubated for another 1 h at room temperature. The wells were washed again. This was followed

A-l

A405

A-2

1

A405 1

:--@ 0.5

‘. \

- \ .

0.5

-

% \ ;

A\.

OLor? 2” Antigen

\

9 \.‘.

‘\ \.:~s+:lb.-.

2-3

2-5 dilution

‘\ 2-7

o2-o

(n mol/well)

2-71

,

2-z

Antibody

24

2-6

2-8

.p

dilution

FIG. 1. Reactivity of MAb GMR6 with various authentic glycolipids in ELISA. (A-l) Wells were coated with each glycolipid at different concentrations and incubated with MAb GMR3. The supernatant of hybridoma was used as MAb. (A-2) Wells were coated with each ganglioside (0.5 nmol/well) and incubated with different concentrations of the MAb. 0, GMB(NeuGc); n , IV3NeuGca-Gg,Cer, IV3NeuGca-nLc,Cer, VsNeuGca-Gg&er, and GDla(NeuGc, NeuGc); A, GM2(NeuGc), GDB(NeuGc-NeuGc-), and GDS(NeuGc-NeuAc-); 0, GMl(NeuGc), GDB(NeuAc-NeuGc-), IV3NeuGcoc2-Gg,Cer, IV3NeuGcal-nLc,Cer, and V3NeuGcaZ-Gb&er; 0, gangliosides having NeuAc (GM4, GM3, GM2, GMl, IVsNeuAca-Gg,Cer, IVsNeuAca-nLc&er, GDla, GTla, GD3, GD2, GDlb, GTlb, G&lb, and IV3NeuAccuz-nLc,Cer); A, neutral glycolipids (LacCer, Gg,Cer, GgCer, Gb&er, Gb,Cer, and IV3GalNAcaGb&er).

TO

429

GANGLIOSIDES A-l

A-2

A405

A405

2-l

Antigen

2-3

2-s

dilution

2-7

2-9

(n mokwell)

2.11

,

*2

Antibody

2-4

2-6

2-8

2‘lo

dilution

FIG. 2. Reactivity of MAb GMR3 with various authentic glycolipids in ELISA. (A-l) Wells were coated with each glycolipid at different concentrations and incubated with MAb GMR3. (A-2) Wells were coated with each ganglioside (0.5 nmol/well) and incubated with different concentrations of the MAb. 0, GD3(NeuGc-NeuGc-); n , IV3NeuGca2Gg,Cer and IVsNeuGccu,-nLc,Cer; A, GD3(NeuAc-NeuGc-), GD3(NeuGc-NeuAc-), and V3NeuGca,-GbsCer; 0, gangliosides having NeuGccuP+BGal structures (GM3(NeuGc), GMB(NeuGc), GMl(NeuGc), IV’NeuGccr-Gg,&er, IVsNeuGcot-nLc,Cer, PNeuGca-Gb&er, and GDla(NeuGc, NeuGc)); Cl, gangliosides having NeuAc (GM4, GM3, GM2, GMl, IV3NeuAccu-Gg&er, IV3NeuAca-nLc,Cer, GDla, GTla, GD3, GD2, GDlb, GTlb, GQlb, and IV3NeuAca,-nLc&er); A, neutral glycolipids (LacCer, Gg,Cer, Gg,Cer, Gb&er, Gbrcer, and IVsGalNAccuGb,Cer).

by the addition of a solution of 400 ag/ml o-phenylenediamine (Sigma) in 80 mM citrate phosphate buffer, pH 5.0, containing 0.12% HxO,. After incubation for 15 min, the plate was read at 405 nm. Three samples of each experiment were tested. The standard deviation was less than 10% for all values. Background values of absorbance at 405 nm were less than 0.05. Thin-layer chromatography. Merck precoated TLC plastic sheets (Merck, Darmstadt, Germany) were used for fractionation of glycolipids. The solvent system used for developing chromatograms was chlorofornmethanol-O.22% CaC& in water (5545~10, v/v). Gangliosides and neutral glycolipids were visualized with resorcinol and orcinol stain, respectively. Enzyme immunostaining on TLC plates. Immunostaining on TLC was performed as previously described (11). In brief, after chromatography of the gangliosides, the plates were soaked for 1 h in freshly made PBS containing 1% bovine serum albumin. After being dried, the plates were incubated with MAb solution (1:lO diluted) for 2 h at room temperature. The chromatogram was washed by dipping it in five successive changes of PBS containing 0.05% Tween 20 and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG or IgM (Cappel) for 2 h at room temperature. After the chromatogram was washed again, a solution of 400 pg/ml o-phenylenediamine (Sigma) in 80 mM citratephosphate buffer, pH 5.0, containing 0.12% H,Ox was added. Color development was stopped after 15 min by washing the plate by dipping it in PBS.

RESULTS

Establishment of two MAbs reacting with NeuGc-containing gangliosides. We finally established two hybridoma clones producing MAbs specific for mono- and disialylgangliosides having NeuGc as their sialic acid residue, respectively. Both MAbs (GMRB and GMR3) were of the IgM(K) isotype. ELISA with various authentic glycolipids. ELISA was performed with authentic glycolipids bound to mi-

430

OZAWA,

KAWASHIMA,

AND

TAI

GD3 * GD2Origin

t 1

234567

1

234567

FIG. 3. TLC immunostaining of standard gangliosides with MAb GMR6. (A) Ganglioside fraction (10 nmol as sialic acid content) from bovine brain and from human melanoma cell line Ml4 and the same amount of standard gangliosides were chromatographed with chloroform-methanol-0.22% CaClz in water (554510, v/v) and visualized with resorcinol. (1) gangliosides of Ml4 cells, (2) gangliosides of bovine brain, (3) GMB(NeuAc), (4) GM3(NeuGc), (5) GM2(NeuGc), (6) GMl(NeuGc), (7) GDla(NeuGc, NeuGc). (B) The same ganglioside fractions (total 1 nmol/lane) were chromatographed similarly and immunostained with the MAb. The hybridoma supernatant (diluted 1:lO) was used as MAb.

crotiter wells. The reactivities of MAbs GMR8 and GMR3 with various glycolipids determined by ELISA are shown in Fig. 1 and Fig. 2, respectively. MAb GMRS reacted strongly with several gangliosides having NeuGccu2+3Galsequences, such as GM3(NeuGc), GDla(NeuGc, NeuGc), IV3NeuGca-Gg&er, IV3NeuGcanLc&er, and V3NeuGca-Gb&er. The MAb reacted weakly with GMB(NeuGc), but not with GMl(NeuGc). Both GMB(NeuGc) and GMl(NeuGc) are gangliosides having the same disaccharide structures in internal position, suggesting that the epitope of the MAb may be covered by attachment of the outer saccharides. None of gangliosides having NeuAc as their sialic acid moiety nor the neutral glycolipids tested were recognized at all. Thus, it was suggested that the epitope detected by MAb GMR8 is the disaccharide (NeuGccu2+3Gal-) sequence, which must be in a terminal position. On the other hand, MAb GMR3 reacted specifically with several gangliosides having NeuGca2+8NeuGccu2+3Galsequences, such as GD3(NeuGc-NeuGc-), IV3NeuGcazGg,Cer, IV3NeuGclu2-nLc&er, and V3NeuGcaz-Gb&er, but not with other NeuGc-containing gangliosides. Di- and monosialylgangliosides having NeuAc as their sialic acid residue or neutral glycolipids were completely unreactive. Consequently, it was suggested that MAb GMR3 recognizes disialylgangliosides containing NeuGccu2-+8NeuGccu2+3Galterminal structures. of various gangliosides with MAb TLC immunostaining GMR8. We also determined the binding reactivity of the MAb with authentic gangliosides containing NeuGc as their sialic acid moiety by immunostaining on TLC (Fig. 3). GM3(NeuGc) and GDla(NeuGc, NeuGc) showed strong reactivity. Neither GMB(NeuGc) nor GMl(NeuGc) was stained. Gangliosides isolated from bovine brain and Ml4 human melanoma cells were completely unreactive. With a large panel of NeuGc-containing gangliosides, it was found that IV3NeuGccr-Gg&er, IV3NeuGccunLc&er, and V3NeuGca-Gb&!er were stained as well as

12

3

4

5

FIG. 4. TLC immunostaining of gangliosides containing the NeuGcGal-sequence with MAb GMRS. Purified gangliosides (approximately 1 nmol as sialic acid content) were chromatographed with chloroformmethanol-0.22% CaC12 in water (55x45:10, v/v) and immunostaining with MAb GMR.8. (1) GM3(NeuGc) of horse erythrocytes, (2) GMB(NeuGc) of mouse (ICR) liver, (3) IV3NeuGco-Gg&er of mouse spleen, (4) IVsNeuGccu-nLc,Cer of bovine erythrocytes, (5) V’NeuGcaGb&er of mouse kidney.

GM3(NeuGc) with the MAb (Fig. 4). MAb GMR8 reacted specifically with gangliosides having an external disaccharide (NeuGccu2+3Gal-) sequence. None of the other gangliosides having NeuAc as their sialic acid moiety nor neutral glycolipids were recognized (data not shown). TLC immunostaining of various authentic gangliosides with MAb GMR3. MAb GMR3 was generated by immunizing the mice with purified ganglioside GD3(NeuGc-NeuGc-). Among four GD3 isomers, the MAb reacted only with GD3(NeuGc-NeuGc-) (Fig. 5). The other three GD3 isomers containing NeuAc as their sialic acid residue were unreactive. Analysis of a larger panel of gangliosides having the NeuGccu2+8NeuGcar2+3Galsequence, such as IV3NeuGccu2-Gg,Cer, IV3NeuGcaznLc&er, and V3NeuGcaz-Gb&er, revealed that the epitope of MAb GMR3 is an external trisaccharide (NeuGca2+8NeuGccu2+3Gal-) sequence on several core structures such as ganglio series, neolacto series, and glob0 series (Fig. 6). The reactivities of these two MAbs with various authentic gangliosides are summarized in Table II. A GM3 GM2

e

GD3

)

GO2

*

Oriclln

B

b 1234512345

FIG. 5. TLC immunostaining of standard gangliosides with MAb GMR3 to GDB(NeuGc-NeuGc-). (A) Ganglioside fraction (10 nmol as sialic acid content) from human melanoma cell line Ml4 and the same amount of four isomers of ganglioside GD3 were chromatographed with chloroform-methanol-0.22% CaClr in water (55:45:10, v/v) and visualized with resorcinol. (1) gangliosides of M14, (2) GD3(NeuAcNeuAc-), (3) GDB(NeuAc-NeuGc-), (4) GD3(NeuGc-NeuAc-), (5) GD3(NeuGcNeuGc-). (B) The same ganglioside fractions (total 1 nmol/lane) ‘were chromatographed similarly and immunostained with the MAb.

MONOCLONAL

ANTIBODIES

TO

GM3 (G)

FIG. 6. TLC immunostaining of gangliosides containing NeuGcNeuGc-Galsequence with MAb GMR3. Purified gangliosides (approximately 5 nmol as sialic acid content) were chromatographed with chloroform-methanol-0.22% CaClx in water (55:45:10, v/v) and immunostained with MAb GMR3. (1) GD3(NeuGc-NeuGc-) of bear erythrocytes, (2) GD3(NeuGcNeuGc-) of cat erythrocytes, (3) IV3NeuGccu,-Gg,Cer of mouse thymoma, (4) IVsNeuGca,-nLc,Cer of sheep erythrocytes, (5) V3NeuGca,-Gb&er of mouse kidney.

Reactivities of MAbs with gangliosides from spleen, kidney, and liver of several mice strains. Gangliosides isolated from the spleen, kidney, and liver of four mice strains-DBA/2, CBA/N, BALB/c, and C57BL/6-were developed on TLC and stained with these two MAbs. Several gangliosides reactive with MAb GMR8 were detected in the spleen of these mice; three major bands correspond to GM3(NeuGc), IV3NeuGca-Gg&er, and GDla(NeuGc, NeuGc) (Fig. 7A). Also, one major ganglioside reactive with MAb GMR3 was found in all the species tested (Fig. 7B). The ganglioside was characterized as IV3NeuGcaz-Gg&er. This ganglioside had the same

TABLE Reactivities

of Two

MAbs

with

II Various

Authentic

Glycolipids MAb

Ganglioside” GM3(NeuGc) GMB(NeuGc) GMl(NeuGc) IV3NeuGca-Gg,Cer IVsNeuGca-nLc,Cer V3NeuGca-Gb,Cer GDla(NeuGc, NeuGc) GDB(NeuGc-NeuGc-) GD3(NeuAc-NeuGc-) GDB(NeuGc-Net&-) IV3NeuGccr2-Gg,Cer IV3NeuGccu,-nLc&er V3NeuGcaz-Gb&er

GMR3

+++* + ++ ++ ++ ++ + + -

GMR3

+++ + + ++ ++ +

’ All of the gangliosides have NeuGc as their sialic acid moiety except the two GD3 isomers which contain both NeuGc and NeuAc. No other glycolipids, such as gangliosides having NeuAc as their sialic acid moiety (GM4, GM3, GM2, GMl, IVsNeuAco-Gg&er, IV3NeuAca-nLc&er, GD3, 0-AC-GD3, GD2, GDlb, GTlb, GQlb, and WNeuAcar-nLc&er) or the neutral glycolipids (LacCer, GbsCer, Gb&er, IV3GalNAccu-Gb,Cer, Gg,Cer, and Gg&er) tested, were detected. * +++, strong; ++, moderate; +, weak or trace; -, none.

431

GANGLIOSIDES

1

2

3

4

m1a (~33

cm3 G-G-)

1

2

3

4

FIG. 7. TLC immunostaining of gangliosides from spleen of several mice strains. Ganglioside fractions equivalent to 50 mg of tissue (wet weight) from the spleens of several mice strains were chromatographed with chloroform-methanol-0.22% CaCl, in water (55:45:10, v/v) and immunostained with MAbs GMR8 and GMRB. (A) MAb GMR8, (B) MAb GMRB. GM3(G), GM3(NeuGc); GDla(G,G), GDla(NeuGc, NeuGc); GD3(G-G-), GDB(NeuGc-NeuGc-). (1) DBA/Z, (2) CBA/N, (3) BALB/c, (4) C57BL/6. Hybridoma supernatants (1:lO diluted) were used as MAbs.

migration rate as standard IV3NeuGccrz-Gg&er of mouse thymoma. After sialidase treatment, the ganglioside was converted to GAl, which was determined by a specific MAb GMR24 for GA1 (data not shown). There was no significant heterogeneity in the expression of spleen gangliosides reactive with these MAbs among these mice strains. Three gangliosides reactive with MAb GMR8 were detected in the kidney of these mice strains (Fig. 8A). Two major gangliosides were GM3(NeuGc) and V3NeuGccuGb&er, whereas one minor ganglioside is unknown. The reactive patterns of these gangliosides were different among these strains of mice. Two strains of mice, DBA/ 2, and CBA/N, contained these three gangliosides, whereas the other two strains showed only one ganglioside, GM3(NeuGc). Two major gangliosides reactive with MAb GMR3 were found in these kidneys (Fig. 8B). Also, heterogeneity in the expression of these gangliosides was shown among these strains. Two strains of mice, DBA/2 and CBA/N, showed one reactive ganglioside (upper band) that migrated between GDla and GTlb on TLC, whereas the other two strains, BALB/c and C57BL/6 showed one ganglioside (lower band) that comigrated

A

B

-ca* Origin

*

. GM3 (G)

1

,. 2

,,^.” 3

.I_ 4

. _-603 (G-G-)

1

_2

mm ..-.. . 3

-” 4

FIG. 8. TLC immunostaining of gangliosides from kidney of several mice strains. Ganglioside fractions equivalent to 50 mg of tissue (wet weight) from kidneys of several mice strains were chromatographed with chloroform-methanol-0.22% CaCl, in water (55:45:10, v/v) and immunostained with MAbs GMR8 and GMRB. (A) GMR8, (B) GMRB. GM3(G), GM3(NeuGc); GD3(G-G-), GD3(NeuGc-NeuGc-). (1) DBA/ 2, (2) CBA/N, (3) BALB/c, (4) C57BL/6. Hybridoma supernatants (1:lO diluted) were used as MAbs.

432

OZAWA,

KAWASHIMA,

with GQlb. The former ganglioside was characterized as V3NeuGcazGb5Cer; the latter one is entirely unknown. These results clearly indicate that kidney ganglioside profiles have heterogeneous expression in these mice strains. On the other hand, no liver gangliosides reactive with either MAb GMR8 or MAb GMR3 were detected in these mice strains (data not shown). DISCUSSION

In this paper, we describe the generation of mouse MAbs specific for gangliosides containing NeuGc as their sialic acid moiety. We have recently developed an improved method for generating MAbs to gangliosidess by immunizing C3H/HeN mice with purified gangliosides. In fact, we have already generated two sets of hybridomas producing MAbs specific for a-pathway (M. Kotani et al., manuscript in preparation) and b-pathway ganglio-series gangliosides (12), respectively, by immunizing the mice with these purified gangliosides. It is noteworthy that mouse MAbs specific for NeuGc-containing gangliosides have been generated by the same procedure even though the antibody responses to NeuGc-containing gangliosides develop minimum titers in C3H/HeN mice (9). This is the first description of murine MAbs reacting specifically with NeuGc-containing gangliosides GM3 and GD3, respectively (Table II). The binding specificity of the MAb GMR8 was highly restricted for several gangliosides having NeuGccu2+3Galterminal structures. The antibody was completely unreactive with corresponding gangliosides having NeuAc-type sialic acid and neutral glycolipids. On the other hand, MAb GMR3 reacted exclusively with disialylgangliosides having NeuGccu2+8NeuGca2+3Galterminal structures. Further characterization with gangliosides having the trisaccharide structures in internal position will elucidate the precise binding specificity of the MAb, although these studies are not available at present. The binding specificities of these MAbs were elucidated by ELISA and TLC immunostaining. In general, TLC immunostaining results agreed well with those obtained by ELISA; however, a number of discrepancies were found. For example, although neither GM2(NeuGc) nor GD3(NeuGc-NeuGc-) reacted with MAb GMRB in TLC immunostaining, both were weakly detected by ELISA. Also, GD3(NeuAcNeuGc-) and GD3(NeuGc-NeuAc-) were barely positive with the MAb GMRS. These findings confirmed our previous observation that ELISA is more sensitive than TLC immunostaining (15). Our results suggest that it may be difficult to generate MAbs specific for GM3(NeuGc) and GD3(NeuGc-NeuGc-), since a number of gangliosides share the terminal structure of gangliosides GM3(NeuGc), i.e., NeuGccu2+3Gal-, and GD3(NeuGc-NeuGc-), i.e., NeuGcLu2+8NeuGccu2+3Gal-. In fact, the binding specificities of MAbs to GM3 and GD3, both of which are

AND

TAI

NeuAc-type gangliosides, showed similar cross-reactivities with other NeuAc-type gangliosides (16-18). A few human MAbs reacting with NeuGc-containing gangliosides have been reported previously (19). Furukawa et al. reported human MAbs specific for NeuGc-containing gangliosides GM3 and GD3, respectively (19). The fine binding specificity of their human MAb to GMS(NeuGc) was similar to that of murine MAb GMR8. First, both antibodies react only with NeuGc-containing monosialyl derivatives. Second, they show very similar reaction patterns with different NeuGc-type gangliosides. On the other hand, the reactivity of human MAb to GDB(NeuGcNeuGc-) was rather different from that of the murine MAb GMR3 as described in the paper. MAb GMR3 reacted predominantly with only GD3(NeuGc-NeuGc-) among four GD3 isomers, whereas human MAb was reactive with both GD3(NeuGc-NeuGc-) and GD3(NeuAcNeuGc-). Neither human nor murine MAb specific for GD3(NeuGc-NeuGc-) has previously been reported. Having established the specificity of these two MAbs (GMR8 and GMR3) for NeuGc-containing gangliosides, we used them to determine the distribution of such gangliosides in three tissues: spleen, kidney, and liver of several strains of mice. The spleens and kidneys contained gangliosides reactive with these two MAbs, whereas the livers did not contain any gangliosides reactive with them. Extensive studies on the structures of NeuGc-containing gangliosides from tissues of various mice strains have been reported by Suzuki’s group (20-22). They found a number of novel gangliosides containing NeuGc as their sialic acid moiety. The structures of monosialylgangliosides reactive with MAb GMR8 in these spleens and kidneys (Figs. 7A and 8A) have been elucidated except for a kidney ganglioside whose structure is unknown, whereas the structures of disialylgangliosides reactive with MAb GMR3 in these tissues (Figs. 7B and 8B) have not precisely been studied (20, 22). A ganglioside reactive with the MAb GMR3 was detected in these spleens (Fig. 7B). The ganglioside was tentatively characterized as IV3NeuGcar,-GgJer, which has been found in mouse thymoma by Nakamura et al. (23). Recently, this ganglioside has also been detected in the spleen of ICR mice (Suzuki et al., personal communication). Two different gangliosides having NeuGca2+8NeuGca2+3Gal sequences reactive with the MAb GMR3 were detected in these kidneys (Fig. 8B). One has already been characterized as V3NeuGca,-Gb&er by Sekine et al. (22); the other ganglioside has not been reported yet. The structure of the ganglioside is entirely unknown. A further study is needed to elucidate the structure of the ganglioside. On the other hand, no gangliosides reactive with either MAb GMR8 or MAb GMR3 were detected in these livers. These results are consistent with those reported by Hashimoto et al. (24), since the livers determined in this study have been shown to express only one ganglioside,

MONOCLONAL

ANTIBODIES

GMB(NeuGc), which is not reactive with either MAb GMR8 or MAb GMR3. Since the ganglioside content of these tissues is generally extremely low in comparison with that of central nervous system tissues, it is hard to obtain a sufficient amount of purified ganglioside for complete chemical analysis. Thus, these MAbs were proved useful for the structural study of such gangliosides having NeuGc as their sialic acid moiety. It is generally agreed that normal human tissues express only NeuAc, but not NeuGc. In contrast, there have been several reports that some human tumors, including melanoma and colon cancer, express NeuGc-containing gangliosides, using chicken polyclonal antibody specific for them (25-27). Recently, a couple of groups described that human tumors do not express NeuGc-containing gangliosides using murine and human MAbs specific for these gangliosides (7, 19). Miyake et al. reported that no significant amount of GM2(NeuGc) was detected in any of 39 lung carcinoma tumors using murine MAbs specific for GMP(NeuGc) (7). Furukawa et al. have also shown that human melanoma and colon cancer do not express NeuGc-containing gangliosides using two human MAbs specific for these gangliosides, GM3 and GD3 (19). Since it was reported that the content of NeuGc-containing gangliosides is extremely low in human tumors, it is still controversial whether human tumors express NeuGccontaining gangliosides or not. In fact, high antibody titer against NeuGc-containing gangliosides has been found in the sera of cancer patients (28). The murine MAbs described in this paper may be useful for analyzing NeuGccontaining gangliosides in human melanomas and other tumors, since murine MAbs are more suitable than human MAbs for the detection of antigens in human tissues by immunohistochemical examination. These studies are now in progress in our laboratory. ACKNOWLEDGMENTS The authors thank Professor T. Yamakawa (Tokyo College of Pharmacy) and Professor Nagai (Tokyo Metropolitan Institute of Medical Science) for their valuable suggestions and support. We also thank Dr. A. Suzuki and his group (Department of Membrane Biochemistry, Tokyo Metropolitan Institute of Medical Science) for providing NeuGc-containing gangliosides and valuable comments.

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