Accumulation of Unique Globo-series Glycolipids in PC 12h Pheochromocytoma Cells*

we reported the presence of a unique globo-series glycolipid as one of the major neutral glycolipid: Gala1-3Galal-4Galj31-4Glc~1-1’Cer, in the subcloned PC 12h pheochromocytoma cells Biochemistry 27,5335-5340). Recently we found that the subcloned PC 12h cells accumulated other unusual neutral glycolipids. In order to characterize these gly- colipids, PC 12h cells were subcutaneously trans- planted into rats. The induced tumor tissue accumulated four minor neutral glycolipids, which were pu- rified by droplet counter-current, Iatrobeads column, and preparative thin-layer chromatographies. These glycolipid structures were determined by fast atom bombardment-mass spectrometry, proton nuclear magnetic resonance spectroscopy, permethylation and 1:4, v/v) and methanol. The complex glyco- lipids were recovered in the ch1oroform:methanol (1:4, v/v) fraction. The effluent was evaporated to dryness. The dried material was then applied to the TLC plates and the plates were developed with 1-propanol, 28% ammonium hydroxide, water (701020, v/v) and then the bands were scraped separately from the plates and glycolipids were isolated as described above. Final purification of neutral glyco- lipids A to D was achieved by the Sephadex LH-20 column (0.8 X 48 cm) with methanol as the eluting solvent (18). Analytical Procedures-Compositional analysis was carried out by gas-liquid chromatography (GLC) and nuclear magnetic resonance (NMR) spectroscopy. Neutral sugars, amino sugars, fatty acids, and long-chain bases were analyzed as (7, 18).

It is well known that the glycolipid composition changes dramatically during cell differentiation and oncogenic transformation (2, 3). In recent years, many tumor-associated glycolipids have been characterized in several tumor tissues as well as established tumor cell lines (4). The PC 12 pheochromocytoma cells are a clonal line of rat adrenal medullary tumor cells and display many properties associated with normal adrenal chromaffin cells (5,6). We recently reported that PC 12 cells contained many fucosylated gangliosides (7) and these fucosylated gangliosides were purified and characterized * This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan (to T. A.) and by Grant NS11853 from the National Institutes of Health (to R. K. Y.). 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" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 5 To whom correspondence should be addressed. from an induced tumor, which was produced by subcutaneous transplantation of the subcloned PC 12h cells into rats. These unusual gangliosides were characterized as fucosyl-GM1, fucosyl-GDlb, and the corresponding fucosylated gangliosides with blood group B determinant (8). In addition, we have reported that the PC 12 cells contained predominantly globoside in the neutral glycolipid fraction (7). Schwarting et al. (9) also found globoside as the major neutral glycolipid in the PC 12 cells maintained in culture. In addition, we reported that glycolipid composition changed dramatically during subcloning and that the subcloned PC 12h cells accumulated another unusual neutral glycolipid Galal-3Galal-4Galp1-4Glcpl-1'Cer' (10). We also found that the subcloned PC 12h cells contained many complex neutral glycolipids having unusual chromatographic behavior.
We have isolated four unique glycolipids from an induced tumor and characterized their structures. These unique glycolipids are characteristic of the presence of repetitive Galal-3 residues in the molecule. Breimer et al. (11) first reported complex neutral glycolipids carrying repetitive Galal-3 residues in normal rat small intestine and they suggested that these unique glycolipids accumulated in the subcloned PC 12h cells were also present in normal rat small intestine (11,12).

EXPERIMENTAL PROCEDURES
Cell Culture and Induced Tumor Tissue-PC 12h cells were subcloned by Dr. Hatanaka, Mitsubishi Kasei Institute of Life Science, Machida, Japan, in which the tyrosine hydrolase activity was significantly increased in the presence of the physiological concentration of nerve growth factor (13). In order to obtain sufficient tissues to isolate and characterize the neutral glycolipids, the PC 12h cells were subcutaneously transplanted into New England Deaconess Hospital rats. Conditions of the cell culture and induced tumor tissue were described previously (7,8).

Isolation of Complex Neutral Glycolipids-The isolation procedures
for the neutral glycolipids in the cells were described previously (7,14).
The tumor tissues, 100 g, were homogenized in 1 liter of chloroform:methanol (2:1, v/v), followed by successive extractions with 1 liter each of ch1oroform:methanol (1:l and 1:2, v/v) and methanol. The combined extracts were evaporated, dissolved in 2 liters of ch1oroform:methanol:water (8:4:3, v/v) and then partitioned. The lower chloroform layer was washed twice with Folch's theoretical upper phase (15). The combined upper phase lipids were evaporated and dialyzed against distilled water for 3 days, followed by lyophilization. The residue was then dissolved in 200 ml of chloroform:methanol:water (30604.5, v/v) and applied to a DEAE-Sepha-The abbreviations used are: Cer, ceramide; Fuc, fucose; FAB, fast atom bombardment; GLC, gas-liquid chromatography; EI, electron impact ionization. The neutral glycolipid nomenclature used follows the system of the IUPAC-IUB recommendations (1). dex A-25 column (acetate form; 100-ml bed volume). Neutral lipids containing the complex neutral glycolipids were eluted with 600 ml of methanol and evaporated to dryness in vacuo, and then subjected to droplet counter-current chromatography (DCC). The DCC was performed with 250 glass columns (2 mm inner diameter X 40 cm) by the ascending method using the upper layer of a solvent mixture of ch1oroform:benzene:methanokwater (50:25:6530, v/v) as the mobile phase and the lower phase of the same solvent mixture as the stationary phase (16,17). The glycolipid sample was dissolved in 10 ml of the lower phase of the mixture and applied to the DCC column. The effluent was collected in 3.5-ml fractions. In a separate experiment, acetone-insoluble lipids obtained from the lower phase on Folch's partitioning were also applied to the DCC column to yield small amounts of the complex neutral glycolipids. The neutral glycolipid fraction containing complex glycolipids were combined, evaporated to dryness, and then applied to an Iatrobeads column (42 g, 1.2 X 120 cm). The column was eluted with an 800-ml linear gradient elution system prepared from chloroform, methanol, 2.5 N ammonium hydroxide (60401 and 30704, v/v). The effluent was collected in fractions of 7 ml. Final purification of the neutral glycolipids was achieved by preparative thin-layer chromatography using two different solvent systems. Briefly, the TLC plates were developed with 1propanokwater (7525, v/v). The bands were scraped separately from the TLC plates. The silica gel containing the complex neutral glycolipids was applied to a mini column of Iatrobeads (400 mg) and then each glycolipid was eluted successively with 6 ml each of chloroform:methanol (1:2 and 1:4, v/v) and methanol. The complex glycolipids were recovered in the ch1oroform:methanol (1:4, v/v) fraction. The effluent was evaporated to dryness. The dried material was then applied to the TLC plates and the plates were developed with 1propanol, 28% ammonium hydroxide, water (701020, v/v) and then the bands were scraped separately from the plates and glycolipids were isolated as described above. Final purification of neutral glycolipids A to D was achieved by the Sephadex LH-20 column (0.8 X 48 cm) with methanol as the eluting solvent (18).
Analytical Procedures-Compositional analysis was carried out by gas-liquid chromatography (GLC) and nuclear magnetic resonance (NMR) spectroscopy. Neutral sugars, amino sugars, fatty acids, and long-chain bases were analyzed as described previously (7, 18).
Exoglycosidase Digestion-Glycolipid A, about 20 pg, was dissolved in 100 pl of 0.05 M citrate-phosphate buffer (pH 5.5) containing 10% sodium taurocholate and 30 pl of a-fucosidase (EC 3.2.1.51,50 units/ ml, Seikagaku-Kogyo, Tokyo, Japan) from C. lampas was added. The mixture was incubated for 48 h at 37 "C, and then the reaction was terminated by the addition of 0.5 ml of methanol. Glycolipid B, about 20 pg, was dissolved in 60 pl of 0.1 M sodium citrate buffer (pH 5.0) containing 10% sodium taurocholate and 30 pl of P-N-acetylhexosaminidase (EC 3.2.1.52, 25 units/0.77 ml of 3.2 M ammonium sulfate, Sigma) from coffee beans was added. The mixture was incubated for 12 h at 37 "C and the reaction was terminated by the addition of 0.5 ml of methanol. Glycolipids C and D, about 40 pg each, were dissolved in 100 pl of sodium-citrate buffer (pH 5.0) containing 10% sodium taurocholate and 10 pl of a-galactosidase (EC 3.2.1.22, 50 units/l.2 ml of 3.2 M ammonium acetate, Sigma) from green coffee beans was added. The mixture was incubated for 30 min at 37 "C. Half of this reaction mixture was treated with 0.5 ml of methanol and the other half was further incubated for 12 h at 37 "C. The reaction was terminated by the addition of 0.5 ml of methanol. All these glycolipid products were further purified by a Sephadex LH-20 column (0.8 X 48 cm) to remove the salts and enzymes, and then examined by TLC.
Negative Ion Fast Atom Bombardment (FAB)-Mass Spectromety-FAB-mass spectra were obtained by a JEOL-HX110 high resolution mass spectrometer equipped with a FAB ion source and JMA-5000 computer system (JEOL, Tokyo, Japan). Xenon gas was used at a 6 kV neutral beam. The sample, 50 pg, was dissolved in 20 pl of ch1oroform:methanol (l:l, v/v). About 2 pl of the solution was applied to a stainless steel holder (1 X 4 mm). Triethanolamine, about 2 pl, was added and then analyzed (19).
Permethylation Study-Permethylation of the neutral glycolipids was carried out by a modification of the procedure of Ciucanu and Kerek (21) as previously described (8,lO). The permethylated glycolipids were hydrolyzed in 90% acetic acid containing 0.7 N hydrochloric acid at 80 "C for 18 h (22). The methylated sugars were reduced with sodium borohydride and then acetylated (23). The alditol acetate derivatives of sugars were analyzed by GLC-electron impact ionization (EI) mass spectrometry (Shimadzu QP-1000) on a fused silica capillary column (0.32 mm X 25 m) of 5% phenylmethylsilicone (Hewlett Packard Co., Palo Alto, CA) or DB-225 (J & W Scientific, Inc., Cordova, CA), with the temperature programmed at a rate of 5 "C/min from 180 to 220 "C (10).

RESULTS
The neutral glycolipid patterns of the subcloned PC 12h cells and tumor tissues induced by PC 12h cell transplantation are shown in Fig. 1. The glycolipid patterns in the tumor tissue were quite similar with that of the PC 12h cells (Fig. 1,  lanes 2 and 3). As compared with the parent PC 12 cells, the PC 12h cells contained the complex neutral glycolipids, which might be expressed during cellular subcloning (10). In the present study, we have isolated these complex glycolipids from the induced tumor tissues. As shown in Fig. 1, the glycolipids, A to D, were found to be homogeneous on TLC with both neutral and basic solvent systems. The yields of these glycolipids, A to D, starting from 100 g of tumor tissue, were 1.0, 0.5, 0.7, and 0.9 mg, respectively. The amount of these glycolipids was approximately 8% of the total neutral glycolipids. Table I shows compositional analysis of the carbohydrates, fatty acids, and long-chain bases. Glycolipid A was found to contain glucose, galactose, fucose, and long-chain base in the ratio of 1:3:1:1. Glycolipid B contained 1 mol of N-acetylgalactosamine instead of the fucose in glycolipid A. Glycolipids C and D were found to contain glucose, galactose, and long chain base in the molar ratios of 1:4:1 and 1:5:1, respectively, and they did not contain any fucose or hexosamine. These glycolipids contained mainly palmitic, stearic, behenic, lignoceric, and nervonic acid, but not a-hydroxy fatty acids.
They were found to contain c18 sphingenine (over 92%) as the major long-chain base, and they did not contain any phytoshingosine.
Negative ion FAB mass spectra of glycolipids A to D showed prominent dehydrogenated molecular ion (M-H)which corresponded to glycolipid molecular species containing fatty acids with chain lengths ranging from C160 to C240 and C18 sphingenine (Fig. 2). All glycolipids showed the same fragment ions corresponding to ceramide (  hexose should be attached to glycolipid C. FAB mass data suggested that glycolipids A to C were ceramide pentasaccharide having different terminal sugar residues, and glycolipid D was ceramide hexasaccharide having a terminal hexose, as: A, ceramide-Hex-Hex-Hex-Hex-deoxyHex; B, ceramide-Hex-Hex-Hex-Hex-HexNAc; C, ceramide-Hex-Hex-Hex-Hex-Hex; and D, ceramide-Hex-Hex-Hex-Hex-Hex-Hex. Fig. 3 shows the TLC of glycolipid products from these isolated unknown glycolipids, A to D, after digestion with various exoglycosidases in the presence of sodium taurocholate. As shown in Fig. 3A, glycolipid A was digested by the treatment of a-fucosidase to yield Galal-3Galal-4Gal/3l-4Glc@l-l'Cer (lane 4 ) , which was already characterized in the PC 12h cells as previously described (10). Glycolipid B was also found to produce the same glycolipid product following the treatment of 8-N-acetylhexosaminidase (Fig. 3B, lane 6). Glycolipids C and D were found to produce lactosyl ceramide, globotriaosyl ceramide, and Galal-3Galal-4Gal/31-4Glc/31-1'Cer following the treatment of a-galactosidase for 30 min (Fig. 3C, lanes 8 and 11). These glycolipids, C and D, produced lactosyl ceramide after the incubation for 12 h with a-galactosidase (Fig. 3C, lanes 9 and 12).
Analysis by capillary GLC-E1 mass spectrometry of these glycolipids, A to D, revealed the presence of 1,3,5-tri-O-acetyl-   1 7 s 9 1 0 1 1 1 2 4   FIG. 3. Thin-layer chromatograms of glycolipid products from isolated glycolipids A to D following the treatment of various exoglycosidases. Lane 1, neutral glycolipid mixtures from pig erythrocyte membranes; lane 2, isolated glycolipid A; lane 3, glycolipid products from glycolipid A after a-fucosidase (C. lampus) treatment; lane 4, Gal~1-3Galal-4Gal~l-4Glc~1-l'Cer from the PC 12h cells (10); lane 5, isolated glycolipid B lane 6, glycolipid products from glycolipid B after P-N-acetylhexosaminidase (green coffee beans) treatment; lane 7, isolated glycolipid C in this study; lanes 8 and 9, glycolipid products from glycolipid C after a-galactosidase (green coffee beans) digestion for 30 min and 12 h, respectively; lane 10, isolated glycolipid D; lanes 11 and 12, glycolipid products from glycolipid D after a-galactosidase (green coffee beans) digestion for 30 min and 12 h, respectively. The fast-running band in lanes 3, 6, 8, 9, 11, and 12 was sodium taurocholate. The plates were developed with 1-propanol, 28% ammonium hydroxide, water (701020, v/v). The glycolipid bands were stained with orcinol-sulfuric acid reagent.  (Table  11). One-dimensional NMR spectrum of glycolipids A to C showed the presence of five protons in the anomeric region (4-5 ppm) (Fig. 4, A-C). In glycolipid D, six protons were found in this region (Fig. 40). From the chemical shifts and coupling constants, the glycosyl H-1 signals of glycolipids A, B, and C were tentatively assigned as shown in Table 111. The glycosyl H-1 signals of glycolipids A and C were confirmed by two-dimensional NMR spectroscopy (data not shown). In the case of glycolipid D, the assignment of the glycosyl H-1 signals corresponding to the repetitive Galal-3 residue was quite difficult.

DISCUSSION
In a previous paper we have described that a new globoseries glycolipid, Galal-3Gala1-4Gal/3l-4Glc~l-l'Cer, is accumulated in the subcloned PC 12h cells. PC 12h cells also accumulated other complex neutral glycolipids. In order to obtain these glycolipids from the tumor tissues induced on the PC 12h cells, we used the combination of Folch's partitioning method and an anion exchange column chromatography. Most of these complex neutral glycolipids were recovered with the upper phase upon Folch's partitioning. In addition, the DCC method should be valuable, because the contamination of phospholipids and the concentration of simple glycolipids, such as ceramide mono-, di-, and trisaccharides are quite low in the complex glycolipid fraction (16,17). Attempts to isolate the complex neutral glycolipids A to D from the induced tumor tissues by Iatrobeads column chromatography or by TLC with the solvent systems using ch1oroform:methanol or by TLC with the one-dimensional solvent system were not successful. Final separation of glycolipids A to D was achieved by preparative high performance TLC with two developments using both neutral and ammoniacontaining solvent systems. For the analyses of glycolipids by FAB mass spectrometry and NMR spectroscopy, the Sephadex LH-20 column proves to be useful to remove contaminants, such as lower molecular substances and silica gels, which are derived from TLC or Iatrobeads column chromatography (18).
These glycolipids A to D had a same core structure, Galal-3Gala1-4Gal/31-4Gal~l-l'Cer, which was confirmed by digestion with various exoglycosidases (Fig. 3). The terminal sugars of glycolipids A to C were found to be a-fucose, /3-N-acetylhexosamine, and a-galactose, respectively. Glycolipid D had 2 mol of a-galactose in the nonreducing terminus. Exhaustive digestion of glycolipids C and D by a-galactosidase suggests the presence of lactosyl ceramide core structure. Negative ion FAB mass spectrometry of the glycolipids provides information directly on their molecular weights and sugar sequence. In addition, their fatty acid and long-chain base compositions can be obtained (19,24). NMR spectroscopy can provide information on the assignments of anomeric proton in each sugar residue on the basis of their chemical shifts and coupling constant. We have assigned all ring protons in a globo-series glycolipid; Galal-3Galal-4Gal/3l-4Glcl-1'Cer, which is ac-  ' Glycosyl H-1 signals corresponding to repetitive Galal-3 residues were not identified. cumulated in the subcloned PC 12h cells, by two-dimensional NMR techniques (10). Inagaki et al. (25,26) have recently reported the NMR spectra of globoside and Forssman glycolipid by the two-dimensional homonuclear Hartmann-Hahn technique. These data provide information on the assignment of anomeric protons in the unusual glycolipids A, B, and C (Table 111). The glycosyl H-1 protons and all ring protons in glycolipids A and C were assigned by two-dimensional NMR and homonuclear Hartmann-Hahn techniques.' The GLC-E1 mass spectrometry of the methylated alditol acetates is useful to decide the sugar linkages in the glycolipids using a fused silica capillary column coated with 5% phenylmethylsilicone (8,lO). However, peaks corresponding to -2Gall-and -3Gallin glycolipid A failed to be separated from each other by this column. Finally, we succeeded in identifying the peak of -2Gall-by the use of a fused silica capillary column coated with DB-225.
Breimer et al. (11) and Angstrom et al. (12) suggested the presence of these unusual glycolipids A, C, and D in epithelial cells and glycolipid B in non-epithelial cells in normal rat intestine. They further found that these intestinal glycolipids contained a-hydroxy fatty acids and phytosphingosine. However, they failed to isolate glycolipids A to D in pure form and to characterize them fully. In this study, we have purified these glycolipids and provided evidence on the proposed structure. These glycolipids were also found to contain C18-sphingenine and normal fatty acids ranging from c16:O to C24:O (see Table I), but not phytosphingosines and a-hydroxy fatty acids. We (7) and Schwarting et al. (9) have reported that globoside is a major glycolipid in the PC 12 cells. We also reported that the subcloned PC 12h cells accumulated an unusual globo-series glycolipid Galal-3Galal-4Gal~1-4Glc~l-l'Cer in addition to globoside (10). We assumed that this unusual glycolipid should be present in much low concentrations in the parent PC 12 cells. In addition, we reported that the concentration of globotriaosylceramide was increased in the PC 12h cells (10). The accumulation of the unusual globoseries glycolipids might be due to the induction of a-galacto-I. Shimada, T. Ariga, and F. Inagaki, unpublished observations. syltransferase in the subcloned PC 12h cells and solid tumor cells (10). Schwarting et al. also reported that the metabolic labeling of the original PC 12 cells with [3H]fucose and/or [3H]galactose revealed the incorporation of unknown complex neutral glycolipids (N1 and N2) and that these glycolipids were increased following the treatment of nerve growth factor (9). They were tentatively identified as fucosyl and galactosyl derivatives of globoside for N1 and N2, respectively. In this study, we have proved that these unique glycolipids have a same core structure, Galal-3Galal-4Galal-4Glc~1-1'Cer, but not globoside. It is possible that the accumulation of these unique glycolipids, A to D, in the subcloned PC 12h cells or solid tumor cells may be the consequence of the activation of specific glycosyltransferases during cellular differentiation and/or the neurite outgrowth.