THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Soeiety for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 26, Issue of September 15, pp. 18810-18813,1992 Printed in U.S.A.

A Novel Ganglioside Expressed by Mouse Hematopoietic Cell Lines* (Received for publication, April 21, 1992)

Michio ShimamuraSO, Manami Okug, and Tatsuya YamagataP From the Laboratories of fMoleculnr ImmunoloPv and IiGlycoconjugate Research, Mitsubishi Kasei Institute of Life Sciences, I 1 Minamiooya, Machida, T&yo 194, Japan

I"

Mouse progenitor T cell-derived cell lines were es- tablished by fusion of cells of hematopoietic organs such as bone marrow and fetal liver with T lymphoma (BW6147) to determine their characteristic cell-sur- face components. The hybridomas with the phenotype of Thy-l*,CD3-,CD4-,CDS- and expression of T cell receptor gene mRNA (BM216 and FL339) were se- lected for progenitor T cell-derived cell lines, and their ganglioside compositions were studied. A ganglioside component with a mobility slightly faster than that of bovine brain GD1. on high-performance thin-layer chromatography was found in the cell extracts of these cell lines as one of the most abundant components and was absent in the extract of the parental cell line (BW6147). The structure of the ganglioside was deter- mined to be: NeuAca-Gal&Gal&Gala-Gal&Glc@- ceramide. Gangliosides with such a sequence have never been found before, suggesting the possibility that the ganglioside is expressed as a surface marker of the cells in hematopoietic organs committed to a specific cell lineage, presumably to T cell lineage.

All blood cells are derived from the multipotent stem cells present in hematopoietic organs such as bone marrow in adults and liver in fetuses (1, 2). Hematopoietic stem cells become committed to a specific lineage under the influence of the hematopoietic microenvironment (3, 4) and thus differ- entiate into mature hematopoietic cells. During this process, cell-to-cell interactions between stem cells and stromal cells of the hematopoietic organs constituting the hematopoietic microenvironment are implicated to be important.

One of the cell-surface components participating in such cell-to-cell interactions may be the glycosphingolipids (5). So far, the glycosphingolipid compositions of rat bone marrow cells (6,7) and human leukemic cell lines (8,9), each of which represent hematopoietic cells at different stages of develop- ment (lo), have been reported.

In this study, an attempt was made to determine the gly- cosphingolipids characteristic of hematopoietic cells. Mouse hematopoietic cell lines were established; their glycosphingo- lipid compositions were compared with those of the cells from lymphoid organs; and a possible candidate for a hematopoietic cell-specific ganglioside was found.

EXPERIMENTAL PROCEDURES

Mice-BALB/c mice (nu/nu and +/+) were purchased from Sankyo Lab0 Service Co.

* This work was supported in part by Grant-in-Aid 02259103 for Scientific Research on Priority Areas from the Ministry of Education, Science, and Culture 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 "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Establishment and Characterization of Cell Lines Derived from Hematopoietic Cells-Cells from BALB/c mouse (nulnu) bone mar- row (8-week old) or BALB/c (+/+) fetal liver (gestational day 14) were fused with an AKR lymphoma line, BW5147 ( l l ) , in the pres- ence of polyethylene glycol (12).' Hybridomas resistant to hypoxan- thine, aminopterin, and thymidine were cloned by the limiting dilu- tion method. The cell lines thus established were maintained at 37 "C in 10% COZ in RPMI 1640 medium supplemented with fetal calf serum (12%), 2-mercaptoethanol (5 X lo-' M), penicillin (50 units/ ml), and streptomycin (50 pglml). The surface phenotypes of the cultured cell lines were determined by immunofluorescence flow cytometry using a FACScan (Becton Dickinson). T cell receptor (TCR)' gene transcription was examined by Northern blot analysis (14). The TCR p-gene probe (86T5 cDNA) (15) and the TCR y-gene probe (8/10-271.1 cDNA) (16) were kindly presented by Dr. H. Yagita (Juntendo University). The TCR &gene probe prepared from KN12 genomic DNA (17) was obtained from Dr. Y. Takagaki (Mitsubishi Kasei Institute of Life Sciences).

Extraction and Fractionation of Glycosphingolipids-Bone marrow, thymus, and spleen cells from 4-week-old BALB/c mice or cultured cell lines (hematopoietic cell-derived hybridomas and the parental line, BW5147) were extracted sequentially with chloroform/metha- nol/water (2:l:O and 3:6:1). The extracts were combined and subjected to chromatography on a DEAE-Sephadex A-25 column equilibrated in chloroform/methanol/water (30:60:8) (18). The acid fraction eluted from the column with chloroform, methanol, 0.8 M CH3COONa (3060:8) was further fractionated by high-performance liquid chro- matography (HPLC) using a chromatograph (Jasco Trirotar VI) with a TSK-gel Amide 80 column (Tosoh). The gangliosides eluted with a linear gradient of acetonitrile, 5 mM KH,PO, (pH 5.5) from 9010 to 6040 were detected by monitoring the UV absorption at 210 nm. The flow rate was 0.5 ml/min.

Ganglioside Composition Analysis by High-performance Thin-layer Chromatography (HPTLC)-The acidic fractions of the extracts de- scribed above were spotted on HPTLC plates (Merck); developed in chloroform, methanol, 0.02% CaC1, (6:41), and then visualized with the resorcinol HCl reagent.

Saccharide Composition Analysis-The saccharide composition of glycosphingolipids were determined by gas-liquid chromatography (19) after methanolysis in 0.5 N methanolic HCl at 65 "C for 16 h (20), re-N-acetylation, and trimethylsilylation. Gas-liquid chromatog- raphy was carried out using a gas chromatograph (Shimadzu GC-SA) equipped with a fused silica capillary column (DB-5, J&W Scientific CO.). 'H NMR Spectroscopy-"H NMR spectra of glycosphingolipids

were obtained with a Bruker AM-500 spectrometer. Determination of Saccharide Sequence of Glycosphingolipids-Sac-

charide sequences of glycolipids were determined as previously de- scribed (21). Briefly, glycosphingolipid samples were digested with endoglycoceramidase (presented by Dr. M. Ito, Mitsubishi Kasei Institute of Life Sciences) (22), and the resulting oligosaccharides were 2-aminopyridylated according to the method of Hase et al. (23).

' Shimamura, M., Oku, M., and Yamagata, T., Abstracts of the 7th

August 5, 1989, p. 209 (abstr.). International Congress of Immunology, Berlin, Germany, July 30-

The abbreviations used an: TCR, T cell receptor; HPLC, high- performance liquid chromatography; HPTLC, high-performance thin-layer chromatography; Sia(Hex)&er, mouse hematopoietic cell line-derived monosialopentahexosylceramide; GD1., IV3-NeuAca, 113- NeuAca-gangliotetraosylceramide (Gg4Cer); Ghllt,, IV3-NeuAca-gan- gliotetraosylceramide.

18810

A Novel Ganglioside in Hematopoietic Cell Lines 18811 h

- r s

E, + 1 8 s

FIG. 1. Northern blot analysis of TCR gene transcripts of hematopoietic cell-derived hybridomas. One microgram of mRNA from each cell line was analyzed with a TCR &gene constant region probe, TCR Cb (17). NB21 is a y8-type T cell-derived hybrid- oma (13) expressing the full-size mRNA of the TCR &gene, and it is shown as a positive control. BM216 and FL339 expressed TCR 6- gene immature mRNA with a somewhat shorter length than that of NB21. The parental cell line (BM5147) possesses rearranged TCR a- genes, and its TCR &genes has been looped out (24). Thus, the expression of TCR 6-gene mRNA observed in the hybridomas was due to transcription from the TCR &genes of the hematopoietic cells immortalized as BM216 and FL339. These hybridomas expressed TCR &gene mRNA of BW5147 origin, but no TCR y-gene mRNA (data not shown).

A

0

FIG. 2. Ganglioside composition of mouse lymphoid organs ( A ) and hematopoietic cell-derived hybridomas ( B ) as ana- lyzed by HPTLC. *, Sia(Hex)&er; BBG, bovine brain ganglioside mixture; 216, BM216; BW, BW5147; 339, FL339; BM, bone marrow; Thy, thymus; Spl, spleen; 113-NeuAca-gangliotetraosylceramide; GDI), I13-NeuAca~-gangliotetraosylceramide; Gnb, IV3-NeuAca, 113- NeuAca2-gangliotetraosylceramide.

The fluorescence-labeled oligosaccharides were digested sequentially with exoglycosidases and fractionated by HPLC using a Trirotar VI chromatograph, a TSK-gel Amide 80 column, and an FP-210 fluores- cence spectrometer (Jasco). The analytical conditions were the same as those conducted for the preparation of the gangliosides.

RESULTS

Mouse progenitor T cells rarely present in hematopoietic organs such as adult bone marrow and fetal liver were im- mortalized by cell fusion with an AKR T lymphoma (BW5147). BM216, a hybridoma derived from a BALB/c (nu/ nu) bone marrow cell, and FL339, a hybridoma derived from a fetal liver cell, possessed the phenotype of Thy-1.2+ (Thy-1 allele of BALB/c), CD3-,CD4-,CD8- and expressed mRNA of the TCR &gene from BALB/c cells (since TCR &genes of the fusion partner (BW5147) had been deleted (24)) (Fig. 1). These observations suggested the possibility that these cell

lines were derived from progenitor T cells (25): The ganglioside compositions of BM216 and FL339 were

examined by HPTLC and compared with those of other lymphoid organs to detect hematopoietic organ-specific gan- gliosides (Fig. 2). The ganglioside shown by an asterisk in Fig. 2, which migrates slightly faster than bovine brain GD~,, was one of the most abundant gangliosides in these cell lines. This component was absent in BW5147, thymus, and spleen cells and was also undetectable in the bulk of the bone marrow cells. This ganglioside thus may possibly be a specific com- ponent of a certain hematopoietic cell lineage, especially a progenitor T cell.

BM216 cells were used as the ganglioside source for further studies. The crude ganglioside fraction prepared from BM216 cells was fractionated by HPLC, and the hematopoietic cell line-specific ganglioside was purified. From 25 liters of BM216 culture fluid, 0.1 mg of the ganglioside was obtained. Saccha- ride analysis by gas-liquid chromatography revealed the pu- rified ganglioside fraction to be composed of sialic acid, galac- tose, and glucose at the ratio of 0.9:4.1:1. Judging from the saccharide analysis combined with its mobility on HPTLC, it was determined that this ganglioside is monosialopentahexo- sylceramide, composed of 1 sialic acid, 4 galactoses, and 1 glucose. Thus, it was designated as Sia(Hex)&er.

The 'H NMR spectrum of Sia(Hex)&er showed the pres- ence of anomeric protons for 1 a-hexose and 4 @-hexoses (Fig. 3). Signals at 4.18 ppm (J = 7.4 Hz) and 4.25 ppm (J = 7.4 Hz) were empirically assigned to the anomeric protons of the reducing-end lactose residue (21, 26-30). Signals at 4.34 ppm (J = 7.9 Hz) and 4.93 ppm (J = 4.4 Hz) were assigned to the anomeric protons of the additional 2 @-galactoses and 1 a- galactose, respectively. The sialic acid species was determined to be N-acetylneuraminic acid due to the presence of an N- acetyl signal at 1.85 ppm. The presence of only two amide protons, each representing those of sialic acid and ceramide residues, respectively, is evidence for the ganglioside having no N-acetylhexosamines.

Finally, the saccharide sequence of Sia(Hex)&er was de- termined as described previously (21). Sia(Hex)&er was di- gested with endoglycoceramidase (22), and the resulting oligo- saccharide was 2-aminopyridylated. The fluorescence-labeled oligosaccharide was then digested with a series of exoglycosi- dases, and the digests were analyzed by HPLC (Fig. 4). The intact oligosaccharide was only susceptible to sialidase, thus showing that the saccharide chain had no branches and that its nonreducing end was an N-acetylneuraminic acid residue (Fig. 4, A and B ) . The asialo-oligosaccharide thus generated was digested with @-galactosidase to give a trisaccharide that was eluted at the same retention time as the standard globo- triaosylceramide (CTH) oligosaccharide (Fig. 4C). The slow rate of the release of the @-galactose residues might be an implication that they were linked to the core oligosaccharide chain by Dl-3 linkage since @-galactosidase from jack bean hydrolyzes Gal@l-4 more preferentially than GalS1-3 residues (31). The asialo-oligosaccharide of Sia(Hex)&er was resistant to digestion with @-galactosidase from Diplococcus pneum- niae, which is capable of cleaving Galpl-4 (but not Gal@l-3) residues (32). Thus, the penultimate @-galactose residue was strongly suggested to possess Dl-3 linkage with another 8- galactose residue. The trisaccharide was digested with a- galactosidase to yield lactose (Fig. 40). A series of these experiments together with the data obtained by 'H NMR spectroscopy indicated that the hematopoietic cell line-de- rived ganglioside (Sia(Hex)&er) is a novel ganglioside (33)

Shimamura, M., Oku, M., Ohta, S., and Yamagata, T. (1992) Immunology, in press.

18812 A Novel Ganglioside in Hematopoietic Cell Lines

FIG. 3. 'H NMR spectrum of Sia(Hex)&er. The spectrum was measured in a mixture of dimethyl sulf- oxide-& (Daiichi Pure Chemicals) and deuterium oxide (Merck) (99:l) a t 110 "C. Chemical shifts and coupling constants are given in parts/million rel- ative to the residual dimethyl sulfoxide methyl signal (2.50 ppm).

I 0-"

D

.. A & I 1 &-PA 0 10 20 30 (M)

FIG. 4. Determination of saccharide sequence of Sia(Hex)5 Cer by HPLC analysis of 2-aminopyridyl oligosaccharides coupled with enzymatic digestion. A , 2-aminopyridyl- Sia(Hex),Cer oligosaccharide; E, a-neuraminidase (Arthrobacter) di- gest of A (interestingly, the retention time of the resulting asialo- oligosaccharide was later than that of the intact oligosaccharide); C, &galactosidase (jack bean) digest of E: D, a-galactosidase (green coffee) digest of C. Glc, 2-aminopyridylglucose; Lac, 2-aminopyridyl- lactose; CTH, 2-aminopyridylceramide trihexoside oligosaccharide; G,,, 2-aminopyridylglobotetraosylceramide oligosaccharide; PA, 2- aminopyridyl.

with the following structure: NeuAca-GalB-Galp-Gala- GalB-Glcp-Cer.

DISCUSSION

A ganglioside predominantly expressed by a mouse bone marrow cell line (BM216) was found to have the structure of NeuAca-Gal~-Gal~-Gala-Galp"lc~-Cer, which has never been reported before (33). This ganglioside (Sia(Hex)&er) was also found to be present in the cells of mouse fetal liver- derived FL339 and several other cell lines (data not shown), but absent in the fusion partner (BW5147) (Fig. 2B), thus indicating Sia(Hex)&er to be a kind of mouse hematopoietic organ-specific ganglioside.

Here, the possibility that the expression of Sia(Hex)&er was due to activation of the fusion partner BW5147 genes should be taken into account. This is an inevitable problem of the cell fusion method. We assume, however, the possibility to be somewhat small in this case. For the synthesis of glycosphingolipids, the simultaneous activation and coopera- tion of several selective glycosyltransferases (each of which possesses an appropriate specificity and location) is necessary. In the well-characterized AKR thymoma (the origin of BW5147), no products of putative key enzymes for the syn- thesis of Sia(Hex)&er, ceramide-trihexoside P-galactosyl- transferase, and/or &galactoside P-galactosyltransferase have been found (39). Thus, it is more likely that the synthesis of the ganglioside in some hybrid cells depends on the intrinsic nature of certain hematopoietic cells in fetal liver and bone marrow fused with BW5147 cells. A final conclusion may be drawn by the analysis of hematopoietic cell clones or hybri- domas with other tumor lines, if available. We believe that the strategy taken in this study is effective at present, and

A Novel Ganglioside in Hematopoietic Cell Lines 18813

the results may contribute to determining the unknown na- ture of hematopoietic cells.

The cell lines expressing this ganglioside (BM216 and FL339) possess the surface phenotype of Thy-1.2+,CD3-, CD4-,CD8" and also express TCR &gene mRNA (Fig. 1). These features strongly suggest that both lines were derived from progenitor T cells (2~5) .~ It should be stressed that Sia(Hex)&er was not detected by HPTLC in thymocytes and splenocytes, where GMlb and its derivatives are abundant as ganglioside components (34-39). It seems possible from its tissue distribution that Sia(Hex)&er is expressed as a specific marker of progenitor T cells in the lymphoid organs, especially in the hematopoietic organs, and functions in events such as cell differentiation or migration to the thymus (40) or bone marrow (41). This hypothesis may be verified by examining its distribution more sensitively by immunological methods using anti-Sia(Hex)&er antibodies or by analyzing the clonal cell lines derived from thymocytes or splenocytes.

Acknowledgments-We thank Drs. H. Yagita, Y. Takagaki, and M. It0 for providing the antibodies and enzymes. We thank Drs. T. Hayase and H. Aoyama for measuring NMR spectra. We are indebted to S. Ohta, M. Arita, and K. Murakami for technical assistance. We also thank Y. Murakami for secretarial assistance.

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