Human blood group B-active ganglio-glycosphingolipid in rat glioma

Biochimica et Biophysics Acta, 1170 (1993) 25-31 0 1993 Elsevier Science Publishers B.V. All rights reserved

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Human blood group B-active ganglio-glycosphingolipid in rat glioma Keiji Suetake a, Shinsei Gasa b, Takao Taki ‘, Masahiko Chiba a, Toshiaki Yamaki a, Yukihiro Ibayashi a and Kazuo Hashi a aDepartment of Neurosurgery and b Department of Chemistry, School of Medicine, Sapporo Medical University, Sapporo (Japan) and ’ Department of Biochemistry, Tokyo Medical and Dental University, Tokyo (Japan) (Received

Key words:

Glycosphingolipid;

8 June 1993)

Ganglioglycolipid;

B-active

lipid; Glioma;

(Rat)

A human blood group B-active glycosphingolipid, belonging to the ganglio-series, was isolated from rat glioma cell line RG2 subcutaneous isografts. The oligosaccharide structure of the glycosphingolipid was completely characterized as Galal-3(Fuccul2)Gal~l-3GalNAc~l-4Gal~l-4Glc~l-l’ceramide by NMR spectrometry, negative fast atom bombardment-mass spectrometry, sequential degradation by glycosidases and methylation analysis. Human blood group B antigenicity and the activity of this glycosphingolipid were confirmed by immunostaining on thin-layer chromatography and the inhibition of hemagglutination, respectively. Although the lipid has been detected in rat granuloma, bone marrow cells, spleen, thymus, ascites hepatoma cells and gastric mucosa, this is the first report of the occurrence of the B-active lipid in glioma.

Introduction The human blood group-active glycosphingolipids (GSLs), part of the ABH blood group system, are found in copious quantities in human erythrocytes and the gastrointestinal epithelium of mammals [l]. The structures of the GSLs in the rat gastrointestinal tract have been characterized [2-61. A human blood B-active neutral GSL with a ganglio-skeleton has been isolated from normal rat tissues (bone marrow cells, spleen, thymus and gastric mucosa) and tumor cells (granuloma and ascites hepatoma cells) [5,7,8]. This characteristic B-active GSL for rat organs is not detected in the normal rat brain. However, a neutral ganglio-GSL (gangliotetraosylceramide (Gg,Cer)) was

Correspondence to: K. Suetake, Department of Chemistry, School of Medicine, Sapporo Medical University, Chuo-ku, Sl W17, Sapporo 060, Japan. Fax: +81 116125861. Abbreviations: 2D-NMR, 2-Ndimensional-NMR; 2D-COSY, 2-dimensional-correlated spectroscopy; FAB-MS, fast atom bombardmentmass spectrometry; TLC, thin-layer chromatography; HPTLC, highperformance TLC; GC-MS, gas chromatography-mass spectrometry; GSL, glycosphingolipid; Me,SO-d6, ‘H,-dimethyl sulfoxide; CMW, chloroform/methanol/water; STDC, sodium taurodeoxycholate; Glc, glucose; Gal, galactose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; Fuc, fucose. Nomenclature and abbreviations of glycosphingolipids follow .those recommended by the IUPAC-IUB Nomenclature Commission (Lipids (1977) 12, 455-468).

recently detected in the normal brain with an immunological technique [9]. Several ordinary gangliosides in glioma tissues and cells have been isolated and identified [lO,lll. Human glioma tissue was found, in general, to contain a low concentration of ganglioside, increased proportions of GM,, GM, and GD, and decreased proportions of the major normal brain gangliosides [lo]. However, examinations of several glioma cell lines grown in culture revealed large variations in the ganglioside composition [ 111. New gangliosides, mono- and di-sialolactotetraosylceramide, were isolated and identified in murine xenografts of a human glioma cell line [12] and human glioma tissues [13]. However, neutral GSLs in glioma have not been examined in detail, though several studies have identified glucosylceramide (GlcCer), lactosylceramide (LacCer), globotetraosylceramide (Gb,Cer), neolactotetraosylceramide (nLc,Cer), Gg,Cer and their biosynthetic precursors [14-201. We have examined a neutral GSL with a longer sugar chain in rat glioma subcutaneous isografts than the above tetraosylceramides, and shown, for the first time, a B-active GSL in the ganglio-series. Materials

and Methods

Materials

DEAE-Sephadex A-25 was purchased from Pharmacia (Uppsala, Sweden), latrobeads from latron Labora-

~ig~-~er~Q~~~~~e thin-layer chromatogCl plates iSilica-gel $O), HPTLC alurninum sheets (Silica-gel 60) and

galaclssidase

Cjack

nal antibody derived fro agnostic System otiaer reagents were of analytical grade.

analysis of the purified GSE was of SkBlnes et al. 01 acetates were

an biue dye exclu-

saline were subcutaneQus &grafts were harvested abQut 1 mont later and the tumors were stsred at - 80°C until use. Extraction and separation of G&L,6 The ratio of solvent ixtures is expressed by v e. The tUHlQI-S (1235 et weight) were treated WI

acetone to yield an acetone pQwder wi

eraeetylated and e~~Q~a~ogra~he~ on an lacolurm by stepwise elution wit cetone with mising the polarity. ~a~Qgra~by was repeated to obtain ~Q~Qge~Q~~ aeety deacetylized by aP treatment an to structural analysis. Qgraphed on an HP piate, developed with C (5.5 : 45 : IO>, and visualize

Fig. 1. Thin-layer c~romat~g~a~~y of nestral GSL and Gk-l from rat glioma subcutaneous isografts. The neutral GSL fradion aad the purified GL-1 from rat glioma were chromatographed on TLC, developed with CMW (55: 45 : IO) aid stained 5y orcinoi-sulfuric acid. Std, standard GSLs; lane 1, neutral GSL mixt?lre from rat glioma; lane 2, purified GL-1; lane 3, crude B-active GSL is&%ed previously from rat boce marrow cells. In Std, C contains GaKei and GlcCe:.

27

analyzed by gas chromatography-mass (GC-MS) as reported previously [25].

spectrometry

Immunostaining on TLC

Immunostaining of GSLs on an HPTLC aluminum plate was performed as prevously described [25] by a modification of the procedure of Magnani et al. [26].

Analysis of lipid moiety

The fatty acid component and the long-chain base were separately analyzed from the methanolyzates of the purified GSL by GC-MS as methyl ester and O-trimethylsilyl derivatives, respectively, as reported previously [251.

Enzymatic degradation

The enzymatic reaction of the purified GSL as well as chromatography of the product was performed in the same manner as in a previous report [S].

FueH5

T

A

NAc

Fuc-C6 T

(I -Fuc F.A. 1

Gal(V)-GalWO-GalNAc~llI~-Gal(JI1-Glc(I >-Cer FucO’I)

Fig. 2. lD- and 2D-NMR spectra Methods. Panel A, lD-spectrum;

of GL-1 lD- and 2D-NMR spectra of GG1 (approx. 1 mg) were measured as described F.A., fatty acyl olefinic protons, Panel B, 2D-COSY spectrum. Some contours assigned e.g., 11z2 show the connectivity between H-l and H-2 on Glc.

under Materials and to the 2D-spectrum,

Reference glycosphingolipids P-GalNAc in GbsCerC31) Gg,Cer(32) F-GlcNAc in HP FucaLc,Cer(33) IV FucmLc,Cer(33)

4.56 4.566

4.02 3.768

3.53 3.647

3.76 3.792

4.60 4.64

3.49 3.45

3.81 3.53

3.21 3.49

a Value in parenthesis show coupling constant (J in Hz). nd: not determined.

the -active GSL isolated p marrow cells ES].At least 5 G Gb,Cer and GE-1 were not y

esldts NeutraE GSLs in rat gliorna

Whole neutral GSLs in rat glioma s~b~~ta~eo~s isografts are shown in Fig. I, together wit GSL (abbreviated as GL-I> from rat glioma and crude B-active GSL from rat bone marrow cehs. The ordinary GSLs, GalCer, GlcCer, LacCer, Gb,Cer, Lc,Cer, Gb,Cer and nLc,Cer, were obtained aft silica-gel column chromatography, followe mation of those structures by NMR spect shown). From these determinations of r-rents and data in other reports [Is-201, it was apparent that the glioma had three biosynthetic series called the globe-, neolacto-, and gangho-GSLs. GL-1 was separated from other GSLs to give apm-ox. 5 mg of the gravity per 1 kg of wet ghoma tissue. The mobility of the purified GL-I was similar to that of

presence

of anomeri

a-Gal and a-fucose

Fig. 3. Negative fast atom bombardment-mass

spectpilm of GL-i.

of GE-l rotons due to

showed t:x?

29 the anomeric

proton of P-Gal to 4.430 ppm supported a partial structure of substituted galactose at C-2-0 with CY-Fuc, which is a core structure of the human ABH-type blood group glycoconjugate. Further assignment of the protons on GL-1 was carried out by 2D-COSY. The cross contours between geminal or vicinal protons are partially shown in Fig. 2B, where the assignments for connectivity between protons are introduced. These NMR data and the assignments of the protons on GL-1 are summarized in Table I. The chemical shift of H-5 on Fuc was characteristically shifted to a lower field at 4.172 ppm, confirming the Fuccul-2GalPl-structure as compared to that of the corresponding proton on A-type GSLs [25]. Furthermore, the chemical shift of H-4 on N-acetylhexosamine at 3.974 ppm was a clear sign that this sugar was GalNAc, since the proton on N-acetylglucosamine (G~cNAc) had a chemical shift of 3.2-3.5 ppm.

the sugar species. GL-1 was found to have unsubstitued Fuc and Gal, substituted Gal at C-2,3-0, GalNAc at C-3-0, Gal at C-4-O and Glc at C-4-O (data not shown). The presence of GalNAc in GL-1 was further confirmed by amino acid analysis after acid hydrolysis (data not shown). These sugar linkages and sugar species suggested ganglio-GSL as the most probable structure of the sugar chain. Lipid moiety of GL-1

The fatty acid component and the sphingosine base of the GLl were separately analyzed by GC-MS, and the data were summarized in Table II. Judging from the major component of the fatty acid and the sphingosine base, the ion at m/z 664 due to ceramide moiety described in the FAB-MS spectrum was composed of C,,-fatty acyl with one unsaturated bond and phytosphingosine. Enzymatic degradation of GL-1

FAB-MS spectrometry of GL-1 The negative FAB-MS spectrum of GL-1 clearly demonstrated the presence of ions at m/z 1661 (MHI-, 1515 (Gal-Gg,Cer-HI-, 1499 (Fuc-Gg,Cer-HI-, 1353 (Gg,Cer-HI-, 1191 (Gg,Cer-HI-, 988 (LacCerH)-, 826 (GlcCer-H)-, and 664 (Cer-HI-, as shown in

Fig. 3. These fragments revealed the sequence of a sugar moiety with hexose-(6-deoxyhexose)-hexose-Nacetylhexosamine-hexose-hexose(each ion was already characterized in Fig. 3). Methylation analysis of GL-1

Purified GG1 was subjected to methylation analysis to identify the linkages between components as well as ,j!

GlcCer

GL-1 was subjected to exoglycosidase treatment in order to confirm the monosaccharide species at the nonreducing termini of the sugar moiety and the anomeric configurations of the component sugars. After treatment with cr-galactosidase, the position of the enzymatic product of GL-1 had migrated to a position quite similar to that of Fuc-Gg,Cer, which has been purified from rat bone marrow cells [8]. Subsequent hydrolysis of the product with cr-fucosidase, P-galactosidase and p-N-acetylhexosaminidase resulted in the sequential formation of tetra-, tri- and di-saccharyl ceramides, as shown in Fig. 4. The final product of monosaccharyl ceramide was formed from disaccharyl ceramide by the action of the P-galactosidase remain-

::

-

LacCer --)

Fuc-Gg&er GL-1

---) -+

0

M-L--

M

1

2

3

4

5

6

M

S

Fig. 4. Exoglycosidase treatment of GL-1 GL-1 was sequentially stripped of the sugar moieties by exoglycosidase and subjected to TLC. Lane 1, purified GL-1; lane 2, CL-1 with a-galactosidase; lane 3, product of lane 2 with a-fucosidase; lane 4, GL-1 with P-galactosidase, cu-galactosidase and cu-fucosidase; lane 5, product of lane 4 with p-N-acetylhexosaminidase; lane 6, product of lane 5 with P-galactosidase; lanes M and S, standard GSLs containing GL-1 and Fuc-Gb,Cer, and sodium taurodeoxycholate, respectively. The products in lane 2 and 3 were chromatographed on a gel-filtration column prior to TLC. Chromatography was developed with CMW (60: 35 : 81,and respective bands were stained with orcinol-sulfuric acid reagent.

Fatty acid (%b)

Long chain

Base (%I

Nonhydroxy

Hydroxy

As trimethylsilyl derivative

16:O 16:l 18:O l&:1 2O:O

9 I> 1 5 4

2O:l 2210 22:l 24:0 24:l 26:0

I> & I 32 36 I>

a

18:O

tP8:O b d18:lc 1

2O:O

1

24:0

I>

a Determined as acetate ester. b 2-Amino ?,3,4-trihydroxy octadecane ’ 2-Amino 1,3-dihydroxy 4-octadecene

97 3

(phytosphingosine). (sphingenine).

ing in the reaction mixture. These results confirmed the structure predicted from the data of N and FB-MS analyses.

of GE-1 The antigenicity of the purified GIL-1 was estimated in an immunological study on TLC using a monoclonal antibody raised agaimst -active sugar. As demonstrated in Fig. 5, the GL-1 was strongly stained by anti-B antibody together with B-active GSL from rat bone marrow cells, whereas A-active GSL from human

Immunological study

Fig. 6. Immunos~aining elf B-active GSL in rat bra.in a,nd giionia. T:e -active GSL in nentral GSL fractions of rat gliana was examined by imm~~osta~~~~g on TLC using a method similar 90 :hat described in the legend of Fig. 5. Pane A, starned by orcmol-suifuric acid; pane! B, immunostaining. Lanes 1, 2 and 3 in paneis A and B are purified GL-l&e neutral fraction from normai ra! brain and Ihe fraction from glioma, respective iy.

(data mot s&ownb.

A

ibed above, we cmfromrat glioma was

identical to that cells: GalaI4GBc$l-i’ceramide.

-active GSL from rat bone marrow h-rl-2)GaiB1-3GalNAcBd-4e;;alB:-

nostained patters, neutral GSE E&&Ke of &ma was Some

iscussism

the present study9 GSL with human blood group antigenicity -was isolate and its structure, ~a~~~~ at of the sugar moiety, rence of human bhd system has been detected very seldom in tissues or cells. Up to this time, it has identified in human erythrocytes [27j and the gastroium in mammals [I]. These GSLs were long to the la&o- or neolactr-series. In

Fig. 5. Immunostaining of purified CL-1 on TLC Panel A, stained by orcinol-sulfuric acid; Panel B, immunostaining. Lanes 1, 2 and 3 in panels A and J3 are A-active GSL from human erythrocytes, B-active GSL from rat bone marrow cells and CL-1 from rat glioma, respectively.

31 the core of which contains GlcNAc. In a singular study of human blood group B-active GSLs in diseased mammalian tissue, Wherrett et al. isolated neolacto-B GSL as well as lacto-B GSL in the pancreas of a patient with Fabry’s disease [28]. On the other hand, B-active GSL with a ganglio-skeleton had already been found in rat granuloma by Hanada et al. [7], in rat bone marrow cells, spleen, thymus, and ascites hepatoma cells by Taki et al. [8], and in rat gastric mucosa by Hansson et al. [5]. Moreover, B-active ganglioside was detected in precancerous liver and hepatoma in rats fed chemical carcinogens by Holmes and Hakomori [29]. The expression of the B-GSL in glioma might, therefore, be an ectopic phenomenon, since the normal rat brain has no B-GSL. The alteration of synthesis of the GSLs-associated with transformation of rat brain suggests a specific role for membranous GSLs in the regulation of cell growth, differentiation, and cell-cell interaction [30]. In particular, further inquiry about the expression of the cu-galactosyltransferase synthesizing B-GSL as well as enzymes for synthetsis of the precursor is required in the study of rat brain tumorigenicity. Although the ganglioside pattern was simplified in the glioma tissue as compared to that of normal brain, unknown highly fucosylated gangliosides were obtained in addition to ordinary gangliosides. The structural determination of these GSLs is in process. Acknowledgements We are grateful to Dr. Akira Makita, Dr. Koichi Honke, and all the other doctors and laboratory assistants in the Biochemistry Laboratory, Cancer Institute, Hokkaido University School of Medicine for their invaluable guidance and advice concerning our experiments. We also thank Mr. Kim Barrymore for his help in the preparation of this article and Dr. Byung Ook Choi, Mr. Tadashi Okada, Ms. Tomoe Kawakatsu and Ms. Ikumi Kimura in our laboratory for their invaluable laboratory assistance. References McKibbin, J.M. (1978) J. Lipid Res. 19, 131-147. Hansson, G.C., Karlsson, K.A. and Thurin, J. (1980) Biochim. Biophys. Acta 620, 270-280. Breimer, M.E., Hansson, G.C., Karlsson, K.A. and Leffler, H. (1982) J. Biol. Chem. 257, 557-568. Hansson, G.C., Karlsson, K.A. and Thurin, J. (1984) Biochim. Biophys. Acta 792, 281-292.

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