An Activation-associated Ganglioside in 'Rat Thymocytes*

During the activation of rat thymocytes elicited by stimulation with 12-0-tetradecanoylphorbol-13-acetate (TPA) and calcium ionophore A23187, the total amount of gangliosides per cell was demonstrated to increase, reaching a maximum level several times higher than that of resting cells at 48 h after activation when measured at 24-h intervals. The amount of the overwhelm- ingly predominant ganglioside in resting thymocytes, G1,(NeuGc,NeuGc) (Nohara, K., Suzuki, M., Inagaki, F., and Kaya, K. (1991) J. Biochem. (mkyo) 110, 274-2781, was found to increase further as a result of activation. Furthermore, another ganglioside, which was barely recognizable in resting thymocytes, was found to increase in activated and proliferating thymocytes to a level similar to that of Glc. This activation-associated ganglioside was isolated and its structure examined. On the basis of the results of compositional analysis, methylation analysis, sialidase hydrolysis followed by detec- tion with cholera toxin B subunit on TLC, and proton NMR spectroscopy, this ganglioside was clarified to be a rare species of Gib containing two N-glycolylneur- aminic acid residues. On the other hand, when the thymocytes were activated using concanavalin A (Cod) as a stimulant, the amount of gangliosides per

During the activation of rat thymocytes elicited by stimulation with 12-0-tetradecanoylphorbol-13-acetate (TPA) and calcium ionophore A23187, the total amount of gangliosides per cell was demonstrated to increase, reaching a maximum level several times higher than that of resting cells at 48 h after activation when measured at 24-h intervals. The amount of the overwhelmingly predominant ganglioside in resting thymocytes, G1,(NeuGc,NeuGc) (Nohara, K., Suzuki, M., Inagaki, F., and Kaya, K. (1991) J. Biochem. (mkyo) 110, 274-2781, was found to increase further as a result of activation. Furthermore, another ganglioside, which was barely recognizable in resting thymocytes, was found to increase in activated and proliferating thymocytes to a level similar to that of Glc. This activation-associated ganglioside was isolated and its structure examined. On the basis of the results of compositional analysis, methylation analysis, sialidase hydrolysis followed by detection with cholera toxin B subunit on TLC, and proton N M R spectroscopy, this ganglioside was clarified to be a rare species of Gib containing two N-glycolylneuraminic acid residues.
On the other hand, when the thymocytes were activated using concanavalin A ( C o d ) as a stimulant, the amount of gangliosides per cell was increased more strikingly than that in thymocytes activated with TPA and A23187. In the Cod-activated thymocytes, many other gangliosides, in addition to G D l C and Gib- (NeuGcaeuGc), were demonstrated to appear in large amounts. The cause of this difference in gangliosides between thymocytes activated with a combination of TPA and A23187 and those activated with C o d is also discussed.
Resting T lymphocytes or thymocytes become activated when their T cell antigen receptors (TCR)l recognize antigens presented by accessory cells. The interaction between TCR and antigen triggers numerous biological processes including activation of protein kinase C and elevation of the intracellular calcium level. The latter two events induce interleukin-2 (IL-2) synthesis and IL-2 receptor expression, and eventually cell proliferation (1). These activated and proliferating T lymphocytes play an important role in various immune responses as effector cells.
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "udoertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Much evidence has been obtained that gangliosides on the plasma membrane associate with the pathway of T lymphocyte and thymocyte activation, and modulate the activation and proliferation process (2)(3)(4)(5). Other evidence suggests that gangliosides inhibit the expression of CD4 molecules on helper T cells (6). It has also been reported that a ceramide analog, which inhibits glycolipid biosynthesis, suppresses mitogen-induced proliferation of T cells and a T cell clone (7). However, in these previous studies, information was not fully obtained on the compositions of the gangliosides actually present on the lymphocytes or thymocytes and their alterations during cell activation. To establish definitive functions of gangliosides in the cell activation, such information will be prerequisites.
Recently, we clarified that a unique hlb-derived disialoganglioside, G1,(NeuGc,NeuGc), is the overwhelmingly predominant ganglioside of rat thymocytes, most of which are considered to be resting cells (8). In the present study, we found and characterized a ganglioside that appears in activated and proliferating rat thymocytes obtained by stimulation with 12-0tetradecanoylphorbol-13-acetate (TPA) and calcium ionophore A23187. Recent studies have revealed that a combination of TPA and a calcium ionophore mimics antigenic stimulation via TCR by activating protein kinase C and augmenting cytosolic calcium, thus inducing proliferation (1). On the other hand, lectin mitogens such as concanavalin A (ConA) have frequently been used to activate T lymphocytes and thymocytes. These lectins are presumed to interact with TCR to activate the cells (9). In this investigation, we also demonstrated that there is a distinctive difference between the gangliosides appearing in activated thymocytes after stimulation with TPA and A23187 and those in cells stimulated with C o d .
EXPERIMENTAL PROCEDURES Materials-Six-to 9-week-old male Wistar rats supplied by Clea Japan (Tokyo) were used for all the experiments.
Preparation of Activated Thymocytes-Thymocytes were prepared from rat thymuses by teasing out through a stainless steel mesh into RPMI 1640 medium supplemented with 12 m~ HEPES, 5 x M 2-mercaptoethanol, 100 unitdml penicillin, 100 pg/ml streptomycin, and 10% fetal calf serum (complete medium) and washed once. The cells were cultured at a density of 3 x 106/ml in complete medium containing TPA (10 nglml), A23187 (60 ng/ml), and supplementary rat IL-2 (2 units/ml) or with ConA (8 pg/ml) at 37 "C in humidified 5% CO,. After incubation for the indicated period, the activated and proliferating cells were harvested, washed three times with PBS, and subjected to investigation of their gangliosides. Preparation of Gangliosides--Total glycolipids were extracted from cells and partitioned by the method of Svennerholm and Fredman (12). The crude glycolipids obtained in the upper phase were treated with 0.5 N NaOH in methanol at 40 "C for 1 h, dialyzed, and evaporated. An aliquot was used for two-dimensional TLC. The other portion was fractionated by DEAE-Sephadex column chromatography (13). After neutral lipids had been eluted with 10 volumes of chlorofodmethanoV water (3:7:1), gangliosides were separated into mono-, di-, tri-, and tetrasialogangliosides by stepwise elution with 10 volumes each of chlorofodmethanol (3:7) containing a 0.1 volume of 0.2, 0.5, or 2.0 M aqueous ammonium acetate.
Isolation of the Activation-associated Ganglioside (GXbThe activation-associated ganglioside, tentatively named GX, was isolated from the disialogangliosides of activated thymocytes stimulated with TPA and A23187 by preparative TLC using a solvent system of 1-propanol, 28% NH,OH, water (75525). GX recovered from the plates was punfied with a Sep-Pak CIS cartridge (14).
Compositional Analysis and Methylation Analysis-Gangliosidebound sialic acids were quantified by the thiobarbituric acid method using NeuGc as a standard (15). Carbohydrate and fatty acid compositions were analyzed as described previously (IO, 16). Methylation analyses were performed according to the previous method (8).
Sialidase Hydrolysis Followed by Detection with Cholera Toxin B Subunit on TLC-Gangliosides were hydrolyzed with C. perfringens sialidase as described previously (10). After the products had been chromatographed on a TLC aluminum sheet, the sheet was divided into two pieces, and the products on one of the pieces were detected with 20% sulfuric acid. The other piece of the sheet was immersed in 0.5% poly-(isobutyl methacrylate) in chloroform for 1 min and dried. Then, it was incubated with biotinylated cholera toxin B subunit (1500 dilution in PBS) for 1 h, washed three times with PBS, and incubated with Vectastain ABC solution for 1 h. After washing three times, the peroxidase activity was detected with 4-chloro-l-naphthol/H202 solution (17).
NMR Spectroscopy-Proton NMR spectra were obtained with a JEOL JNM-GX400 NMR spectrometer as described previously (10.16).

Alteration in the Amount of
Gangliosides-When the thymocytes were cultured with TPA and A23187 as stimulants, and with supplementary IL-2 to ensure growth, the cells proliferated efficiently as shown in Fig. 1. Blast transformation was observed in all cultures at 24 h onward after activation when measured at 24-h intervals (data not shown). In the activated thymocytes, the amount of gangliosides per cell was demonstrated to increase to a maximum level several times higher than that of resting cells at 48 h after activation (Fig. 1).
On the other hand, when the thymocytes were stimulated with C o d , the harvested cells slightly decreased at 24 h after activation and then increased up to 96 h. The increase in the amount of gangliosides per cell was found to be much greater in the thymocytes activated with C o d than in those activated with TPA and A23187. Fig. 1 shows a representative result of three experiments, in which all of the fenomena described above were reproducible.
Changes in the Composition of Gangliosides-The compositions of the gangliosides from resting and activated thymocytes were examined by two-dimensional thin-layer chromatography. In resting thymocytes (Fig. 2 A ) , hlC(NeuGc,NeuGc) was the overwhelmingly predominant ganglioside, as reported previously (8). Upon stimulation with TPA and A23187 (Fig. 2 B ) , a ganglioside with a slower TLC mobility than that of G D~~, and hardly recognizable in resting thymocytes, was found to be strikingly increased, becoming a major ganglioside in addition to hlC. This activation-associated ganglioside, tentatively named GX, was isolated and characterized as described below.
When the gangliosides from 1.2 x lo9 resting thymocytes and those from 1.6 x los activated cells were compared on respective chromatograms (Fig. 2, A and B ) , hlc was also found to accumulate upon stimulation with TPA and A23187. No discernible change in ganglioside composition was evident from 24 to 72 h after activation (data not shown).
Upon stimulation with ConA ( Fig. 2 0 , many species of gangliosides, in addition to hlc and GX, were demonstrated to appear in a large amount. As GX was subsequently demonstrated to be a disialoganglioside, as mentioned below, the presence of GX in the ConA-activated thymocytes was verified by TLC of the disialogangliosides obtained by DEAE-Sephadex column chromatography (data not shown).
Characterization of the Activation-associated Ganglioside GX " O n DEAE-Sephadex column chromatography, ganglioside GX, which increased markedly in the thymocytes activated with TPA and A23187, was obtained in the disialoganglioside fraction. GX was isolated from the other disialogangliosides by preparative TLC. The TLC profile of the isolated GX is shown in Fig. 3. GX showed a similar mobility to that of GDlb(NeuAc,NeuAc) in a neutral solvent system (Fig. 3 A ) and migrated more slowly than hlC(NeuGc,NeuGc) in a solvent system containing ammonium hydroxide (Fig. 3B). Compositional analyses showed that GX contained Gal, Glc, GalNAc, and N-glycolylneuraminic acid in a molar ratio of  From the results of compositional analysis, sialidase hydrolysis, and methylation analysis, GX was suggested to be G l b containing two N-glycolylneuraminic acid residues. The proton NMR spectrum also supported this proposed structure of GX On the basis of the above results, GX was concluded to be GDlb containing two N-glycolylneuraminic acid residues.

DISCUSSION
The composition and/or amount of glycosphingolipids in lymphocytes have long been suggested to alter during the cell activation based on measurements of incorporation of radioactive carbohydrate precursors (18)(19)(20). In another study using antiasialo-G1 antibody, expression of asialo-h1 was found to be induced on mouse splenic T cells by stimulation with mitogens (21). However, the quantitative changes in the composition of gangliosides on the lymphocytes before and after cell activation have not been fully clarified. In the present study, we clearly demonstrated changes in both the amount and composition of gangliosides in rat thymocytes accompanying cell activation. As summarized in Fig. 6, it was shown that the amount of Glc, the predominant ganglioside of resting thymocytes, increased further and that, moreover, the activation-associated ganglioside confirmed to be Glb(Neu&,Neu&) appeared as the major ganglioside following activation of thymocytes with TPA and A23187. Although culture medium containing fetal calf serum was used in this study, the accumulation of hlc and Glb(Neu&,Neu&) was ascertained not to be due to incorporation of gangliosides in fetal calf serum by confirming the absence of the two gangliosides in the serum by TLC (data not shown). On the issue of involvement of gangliosides in thymocyte activation, the role of the accumulated gangliosides Glc and hlb(Neu&,Neu&) should be important.
Moreover, we demonstrated that there is a distinctive difference between the ganglioside compositions of thymocytes activated with a combination of TPA and A23187, and those activated with ConA. That is, when the thymocytes were activated using C o d , many other gangliosides, in addition to hlc and Glb(Neu&,Neu&), were found to appear in large amounts. Possible reasons for this difference are as follows: 1) different populations of thymocytes expressing different sets of gangliosides proliferated upon stimulation with a combination of TPA and A23187, and with C o d , respectively; 2) C o d , a lectin, elicited additional events not essential for cell activation by interacting with certain sugar structures besides TCR on the cell surface, inducing additional glycolipid synthesis; 3) whereas C o d triggered all the reactions originally associated with cell activation by interacting with TCR in the activated populations and induced many species of gangliosides, TPA and A23187 stimulated only part of the signal pathway, triggering the synthesis of only Glc and Glb(Neu&,Neu&). Further studies will be needed in order to examine these possibilities. Such studies should help to clarify the significance of accumulation of specific gangliosides during thymocyte activation.
Recently, characterization of the gangliosides present on murine lymphoid cells has been reported. Miithing et d . (22,23) isolated radioactive gangliosides from splenic T cells of CBNJ mice, which were activated with ConA and metabolically labeled with radioactive carbohydrates, and their findings indicated the presence of h l b , GalNAc-hlb, and GI,. All these gangliosides have the unique feature of synthesis through  A23187 (solid arrow). The boldface arrow indicates that the resulting gangliosides were present in large amounts. asialo-G, and then Gib, which is a common pathway for the synthesis of hlc. More recently, Nakamura et al. (24) reported the occurrence of hlc as the major disialoganglioside in WHT/Ht mouse thymocytes. Moreover, they tentatively identified the other major disialoganglioside in the thymocytes as the rare species of hlb, hlb(NeuGc,NeuGc), by sialidase hydrolysis followed by TLC immunostaining (24). These reports, taken together with our present study and a previous report on hlc in rat thymocytes (€41, suggest that rats and mice have similar pathways of ganglioside synthesis in T lineage lymphoid cells, at least partly. Data on the structures of gangliosides in T lineage lymphocytes obtained from rats and mice may be mutually suggestive, although still fragmentary with regard to the changes occurring during cell maturation and cell activation with various mitogens.