Biosynthesis and shedding of murine lymphoma gangliosides

Biochimica et Biophysics Acta, 1170 (1993) 283-290 0 1993 Elsevier Science Publishers B.V. All rights

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283 resewed 00052760/93/$06.00

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Biosynthesis and shedding of murine lymphoma gangliosides Ruixiang Li a, Douglas Gage b and Stephan Ladisch a,* a Center for Cancer and Transplantation Biology, Children S National Medical Center, and Departments of Pediatrics and Biochemistry /Molecular Biology, George Washington University School of Medicine, 111 Michigan Avenue, NW., Washington DC 20010 (USA) and b Department of Biochemistry and the NIH-MSU Mass Spectrometry Facility, Michigan State University, East Lansing, MI (USA) (Received

Key words:

Ganglioside;

Ganglioside

biosynthesis;

20 May 1993)

Ganglioside

shedding;

Ceramide;

Murine

lymphoma

cell

Ganglioside biosynthesis and subsequent shedding are a potential mechanism contributing to tumor cell escape from the host immune response. As a first step in identifying active molecular species, structural characterization and quantification of the purified individual cellular and shed gangliosides of YAC-1 murine lymphoma cells were undertaken. These studies uncovered three striking changes in ganglioside metabolism in cells passaged in vivo, compared with cells cultured in vitro. (i) Marked in vitro, inhibition of GalNAcG,,, synthesis: GILln, was present in an equal proportion to its biosynthetic product GalNAcG,,, but was present in a 6-fold higher concentration in vivo. (ii) Marked inhibition of NeuGc synthesis: NeuGc, present in vitro in an up to 7-fold higher concentration than its biosynthetic precursor NeuAc, was decreased in relative concentration in vivo (1:l). (iii) Selectivity of shedding: ganglioside shedding in vitro was generalized with respect to both carbohydrate structure and ceramide structure (mainly d18:1-C24:l and d18:1-C16:0), while in vivo, there was selective shedding of gangliosides containing in vivo, and the selective shedding NeuGc and the shorter chain fatty acid. The reduced synthesis of NeuGc and of GalNAcG,,, of more polar ganglioside species, also in vivo, show that the extracellular environment can markedly affect cellular ganglioside metabolism.

Introduction sialic acid-containing glycosphinGangliosides, golipids, are synthesized in the Golgi complex, and are transported to, and incorporated into, the cell plasma membrane. These molecules are then shed [l-3] into the extracellular milieu, particularly by some proliferating cells such as tumor cells [4-61. Shedding of tumor cell gangliosides may have important biological consequences, since a number of these molecules have potent immunosuppressive activity [4,7,8]. A striking example of tumor ganglioside shedding is found in the YAC-1 murine lymphoma. The gangliosides shed by this lymphoma were the first tumor gangliosides shown

* Corresponding author. Fax: + 1 (202) 884 5685. Abbreviations: CAD-MS/MS, collisionally activated dissociation tandem-mass spectrometry; FAB-MS, fast atom bombardment-mass spectrometry; FCS, fetal calf serum; HPLC, high-performance liquid chromatography; HPTLC, high-performance thin-layer chromatography; LBSA, Lipid-bound sialic acid; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid; Gangliosides are abbreviated according to the nomenclature of Svennerholm (Adv. Exp. Med. Biol. (1980) 125, 11).

to be immunosuppressive, inhibiting mitogen- and antigen-induced lymphoproliferation at micromolar concentrations [4]. Curiously, by high-performance thinlayer chromatography (HPTLC), the patterns of gangliosides of YAC-1 cells propagated in vitro were not identical to those of gangliosides of the same cells proliferating in vivo [41. Therefore, we felt it was important to determine the complete structure of individual molecules homogeneous not only in carbohydrate structure [93 but also in ceramide structure, and to study the metabolism of these immunologically active molecules. This has recently become feasible with the development of both reversed-phase HPLC technology which we have adapted for the isolation of small amounts of tumor gangliosides [lo], and of negative ion fast atom bombardment collisionally activated dissociation tandem mass spectrometry (FAB CAD-MS/MS) which is capable of determining ceramide structure on very small quantities of sample [ll]. We have now completed a comprehensive study of structure, synthesis, and shedding of YAC-1 murine lymphoma cellular gangliosides. This study reveals that a change of the extracellular microenvironment, i.e., from in vitro to in vivo, can result in striking changes in cellular ganglioside metabolism.

284 Materials

and Methods

YAC-I murine lymphoma cell culture and tumor growth YAC-1 murine lymphoma cells were propagated in suspension culture in RPM1 medium supplemented with 2 mM L-glutamine (GIBCO Laboratories, USA) and 10% heat-inactivated fetal calf serum (FCS) (Hyclone Laboratories, USA), or in HB104 serum-free medium (Hana Biologics, Alameda, CA). The cells were seeded in 20 ml medium in 75 cm’ flasks, and cultured in a humidified 5% CO,:95% air atmosphere. Cell viability was > 98% as assessed by trypan blue dye exclusion throughout these experiments. To harvest cells, the cell suspension was first removed from culture flasks and the flasks were rinsed with phosphatebuffered saline. The rinses were added to the cell suspension and centrifuged at 300 X g for 10 min. The cell pellet was then washed with phosphate-buffered saline, while the supernatant was centrifuged at higher speed (1000 X g) for 10 min to obtain the cell-free culture supernatant to quantify the shed gangliosides

[41. YAC-1 lymphoma cells propagated in vivo were obtained by passaging these cells in the peritoneal cavity (5 . lo6 cells/mouse) of syngeneic mice [4] or by injecting YAC-1 cells subcutaneously (2.5 . lo6 cells/ mouse) in the dorsum of mice to produce solid tumors. Isolation and quantification of gangliosides Total lipid extracts of cell pellets and culture supernatants were obtained by chloroform/ methanol extraction. Gangliosides were purified by di-isopropyl ether/l-butanol partition [12] followed by Sephadex G-50 gel filtration. The purified gangliosides were quantified by the modified resorcinol method [13] as nmol lipid-bound sialic acid (LBSA). HPTLC analysis of gangliosides was performed, using 10 X 20 cm precoated silica-gel 60 HPTLC plates (Merck, Darmstadt, Germany). The plates were developed in chloroform/ methanol/0.25% aqueous CaCl, . 2H,O (60:40:9) and were stained with resorcinol 1141. High-performance liquid chromatography (HPLC) purification of gangliosides Total YAC-1 lymphoma cell gangliosides were first separated by normal phase HPLC to yield molecular species homogeneous in carbohydrate structure [I5]. This was accomplished by dissolving 50-70 nmol of total gangliosides in 100 ~1 water, and by chromatographing the solution on a LiChrosorb-NH, column (250 mm length, 4 mm i.d., Merck, Darmstadt, Germany) using the Perkin-Elmer Isopure HPLC system. The eluting solvent system was composed of acetonitrile/5 mM Sorensen’s phosphate buffer (83:17), pH 5.6 (solvent A), and acetonitrile/20 mM Sorensen’s phosphate buffer (l:l), pH 5.6 (solvent B). A linear

gradient from 100% solvent A to soivent A/solvent B (66:34) over 58 min was used, at a flow rate of 1 ml/ min. The individual gangliosides which were now homogeneous in carbohydrate structure were further separated by reversed-phase HPLC to yield ganglioside species also homogeneous in ceramide structure [16]. Gangliosides (10 nmol) in 25 ~1 water were chromatographed on a reversed-phase LiChrosorb RP-8 column (250 mm length, 4 mm i.d., Merck, Darmstadt, Germany). The solvent system consisted of acetonitrile and 5 mM sodium phosphate buffer (pH 7.0) in a ratio of 55:45 for 10 min and increased linearly to 65:35 over 40 min at a flow rate of 0.52 ml/min. The elution profile was monitored by flow-through detection at 215 nm (for normal phase HPLC) and at 195 nm (for reversed-phase HPLC). Structural characterization of gangliosides by mass spectrometry The carbohydrate structure of gangliosides was characterized by negative-ion fast atom bombardmentmass spectrometry (FAB-MS). Ceramide structure was elucidated by negative ion FAB CAD-MS/ MS without prior derivatization using linked scans at constant B/E [lo]. Approx. 1 ~1 of ganglioside methanol solution (0.1-2.0 nmol/kl) was mixed with 2 ~1 of triethanolamine (matrix) on the FAB probe tip. Ions were formed by bombardment with a 6-keV beam of xenon atoms in a JEOL HX-110 double focusing mass spectrometer. The accelerating voltage was 10 kV and the resolution was 3000. For FAB CAD-MS/MS, helium was used as the collision gas in a cell located in the first field-free region. The [CerO]fragment ion was selected as the precursor ion for FAB CAD-MS/ MS. The helium pressure in the collision cell in both cases was adjusted to reduce the abundance of the precursor by 75%. A JEOL DA-5000 data system generated the linked scans at constant B/E. Metabolic radiolabeling study of YAC-1 ganglioside shedding YAC-1 cells seeded at a density of 2.3 . 10” cells/ml in 20 ml medium were metabolically radiolabeled for 40 h with D-[l-‘4C]galactose (specific activity, 56.5 mCi/ mmol) and D-[ l- “C]glucosamine-HC1 (specific activity, 54.2 mCi/ mmol; New England Nuclear, Boston, MA, USA) [17]. This method results in proportional radiolabeling of all YAC-1 cellular gangliosides [4]. The cells were harvested, washed 3 times with 10 ml medium to remove unincorporated radiolabeled sugars, and cultured in fresh medium. Aliquots of cell suspension were taken to measure shed gangliosides every 12 to 24 h. The radiolabeled gangliosides from the cells and culture supernatants were purified, quantified by p-scintillation counting, and analyzed by

285 HPTLC [18,19].

autoradiography

as

described

previously

Results Purification and carbohydrate structure of YAC-1 gangliosides

The major ganglioside components of YAC-1 murine lymphoma cells, proliferating under different in vitro and in vivo conditions, were separated by normal phase HPLC. The carbohydrate structures of these molecules were confirmed by negative-ion FAB-MS, For example, the total gangIiosides of YAC-1 cells cultured in RPMI-1640 with 10% FCS, the condition which was commonly used [4,9], were separated by normal phase HPLC into two fractions, with retention times of 33.29 and 35.38 min (Fig. 1, top panel). When analyzed by FAB-MS, fraction I yielded two major pseudomolecuIar ions, [M-HI-, at m/z 1642 and 1532 (Fig. 21, with two sets of characteristic fragment ions due to successive elimination of carbohydrate residues: m/z 1335, 1173, 970, 808, 646; m/z 1225, 1063, 860, 698, 536.

Fig. 1. Normal phase and reversed-phase HPLC separation of gangliosides isolated from YAC-1 murine lymphoma cells cultured in vitro (10% FCS-containing medium). Top panel: normal phase HPLC profile of the total cellular gangliosides. Bottom panels: reversedphase HPLC separation of the two ganglioside fractions (1 and II) isolated by normal phase HPLC in the top panel.

0

*

‘

.i

I

500

1000

1500

2000

500

1000

1500

2rJoo

100

E

80

5 9’ P

60

4 .I -6 %

40

20

0

wz Fig. 2. Negative-ion FAEl-MS of YAC-1 murine lymphoma gangiiosides (fractions I and II isolated by normal phase HPLC). The peaks at m/z 1532, 1642, 1735 and 1845 represent the [M-HI- ions of the predominant molecular species, G,,,(d18:1-C16:0), Ghillb(d18:lC24:1), GalNAcGM,,(d18:1-Cl6:0), and GalNAcGr,.&d18:l-C24:1), respectively. All contain ~-giycolylneuraminic acid. Ions at m/z 148, 296, and 591 are matrix (tricthanolamine)-related peaks.

The fragment ions detected at m/z 849, 687 and 484 indicate the presence of a terminal sialic acid containing an additional oxygen atom ([NeuGc-Gal-GalNAcGaIO]-, [NeuGc-Gal-GalNAcOland [NeuGcGaIO]-1. Correspondingly, the fragment ions at m/z 646 and 536 (apparent in the spectrum with expansion) suggest that two major ceramide species exist. These results show that the two most abundant molecular species in fraction I have the same carbohydrate structure, G,,,(NeuGc). Similarly, fraction II yielded two major pseudomolecular ions, EM-HI-, at m/z 1845 and 1735, with two major sets of fragment ions: m/r 1538, 1335, 1173, 970, 808, 646; m/z 1428, 1225, 1063, 860, 698, 536. The fragment ions detected at m /z 1052,890 and 687 were assigned as [GalNAc-(NeuGc-)Gal-GalNAcGalOl-, EGalNAc-(NeuGc-IGal-GalNAcOland [GalNAc-(NeuGc-)GalOl-, respectively. Thus, the two prominent ganglioside components of fraction II have the same carbohydrate structure, that of GalNAcG~~~ (NeuGc). In addition to NeuGc-containing gangliosides, NeuAc-containing GMlb and GalNAcG,,, are also present at very low concentrations (Fig. 2). Gangliosides from cells propagated under the other three growth conditions, serum-free medium in vitro, and ascitic and solid tumor forms in vivo, were also characterized using’ identical methodolo~. In each case, the same ganglioside species found in cells cultured in 10% FCS-containing medium in vitro [9], G,,,

286 and G~INAcG,,~, were detected (mass spectra not shown). Also of note is that every ganglioside purified by normaf phase HPLC to homogeneity of carbohydrate structure had an additional level of structural complexity, in that the mass spectrum of the molecular ion region in each case was heterogeneous. Ceramide structure of YAC-I gangliosides

The multiple peaks in the molecular ion regions of the negative-ion FAB-MS of G,,, and GalNA~G~,~ suggest heterogeneity of the ceramide moiety. Each of the two fractions (I and II) separated by normai phase HPLC yielded two major peaks and a series of minor peaks (Fig. 1, bottom panels) by reversed-phase HPLC, each representing different subspecies of the gangliosides GMu, (fraction I) or GalNAcG,,, (fraction II). These ganglioside molecular species were characterized by negative-ion FAB-MS and FAB CAD-MS/ MS, as shown in Fig. 3 for one major molecular species of GalNAcG,,, isolated by reversed-phase HPLC from fraction II (retention time = 31.92 min, Fig. 1). The ceramide parent ion [CerOl- at m/t 646 was used to produce the daughter S and T ions [ll]. The presence of S ions at m /.z 390 and T ions at m/z 406 indicate that the long chain base is d18:1, the fatty acid is C24:1, and the ceramide structure is d18:1-C24:l. Using this same approach, the major gangliosides isoiated by reversed-phase HPLC from fraction I (with retention time of 9.90 and 34.61 min) and II (with retention time of 9.25 min) (Fig. 1) were analyzed. The results show that the major ceramide structures of each of these two gangliosides, G,,, and GaINAcG,,,, are dlS:I-C16:O and dl&l-C24:l. Some minor ceramide species, including d18:1-C24:0, are also present. The major ceramide structures of gangIiosides from ceIIs propagated under several different in vitro and in vivo conditions were similarly characterized. In each case

.g 5

40

1173

z

600

000

1000

1200

1400

1600

1800

2000

M/Z

Fig. 3. Negative-ion FAB-MS of GalNAcG,,, isolated by reversedphase HPLC. The FAB CAD-MS/MS of the precursor ion [CerO]at m /z 646 is shown in the inset. The product ions, .S (m/z 390) and T (m/z 406) (111, indicate the ceramide structure is dIS:l-C24:l. Ion at m/z 591 is a matrix-related peak.

in vitro

Total content a Ganglioside distribution (%I ’ G Mlb NeuAc NeuGc GalNAcGM,, NeuAc NeuGc

in viva

10% FCS medium

serum-free medium

ascites form

solid tumor

18.6

25.8

14.6

28.4

46 7 39 54 12 42

51 8 43 49 4 45

87 44 43 13 9 4

78 48 30 14 7 I

Mean nmol LBSA per IO” cells (n = 2-4) or per 0.1 gram solid tumor. The relative concentrations of each species were quantified by integration of the HPLC elution profile. Each value represents the mean of five determinations, the SE. was uniformly < 12% of the mean. Other minor species were also present in solid tumor; altogether they comprised < 8% of the total gangliosides.

the ceramide C24:l.

structures

were d18:1-C16:O and d18:1-

Ir$?uence of the cellular environment composition

on gangiioside

The first evidence suggesting that cell growth environment influences ganglioside composition is found in the original HPTLC pattern of unseparated total gangliosides of YAC-1 Iymphoma cells; cells cultured in vitro had a pattern that was quite different from the pattern of gangliosides of cells propagated in vivo [4]. Subsequently, ganglioside differences by HPTLC-immunostaining have also been found in cells cultured in serum-containing and serum-free medium [20]. By direct HPLC isolation and separation, and negative-ion FAB-MS and FAB-MS/MS, we have identified the molecular differences not only in ganglioside synthesis but also in ganglioside shedding, under several in vitro and in vivo growth conditions. For these comprehensive comparative studies, cells were propagated in vitro in FCS-containing or serumfree medium, and in vivo for 1 week either in ascites form or as a solid tumor. The total content andrelative distribution of individual gangliosides in cells under these four conditions are given in Table I. Several striking changes in cellular ganglioside expression depended on the conditions of cell propagation. YAC-1 cells cultured in vitro in serum-free medium and in vivo as a solid tumor had a slightly higher total ganglioside content than did the cells propagated under the other conditions. However, the major difference was found in qualitative characteristics; variations in the

287 relative concentrations of individual gangliosides were quite significant. First, the two major gangliosides, were present in a different G Mlb and GalNAcG,,,, ratio in vitro than in vivo. As shown in Table I, GMlb was present in equal proportion to GalNAcG,,, (1:l) under both in vitro conditions. In contrast, in both in vivo conditions G,,, was present in an approx. 6-fold higher concentration than was GalNAcG,,,. Since GalNAcG,,, is the biosynthetic product of GM,,,, these findings suggest that YAC-1 cells propagated in vivo have markedly inhibited addition of N-acetylgalactosamine to G,,,. The second major difference was found in sialic acid structure. Quantification of the NeuAc- and NeuGccontaining carbohydrate subspecies of the two major gangliosides, GMlb and GalNAcG,,,, gave unexpected results. In vitro, gangliosides containing NeuGc were 4-7-fold more prominent than those containing NeuAc (Table I). In vivo, NeuGc containing gangliosides were present in a much lower concentration relative to NeuAc-containing gangliosides (1:l). To exemplify this point, NeuGc contributes more than 80% of total sialic acid to gangliosides of cells cultured in vitro in medium containing 10% FCS, while NeuGc comprises only half of total sialic acid of gangliosides of YAC-1 cells propagated in ascitic form in vivo. Since NeuGc is formed by the hydroxylation of NeuAc [21-231, these findings suggest a second major alteration in ganglioside metabolism of YAC-1 lymphoma cells, i.e., a marked reduction in the proportion of NeuGc, suggesting decreased activity of the hydroxylation system in cells propagated in vivo. It is unlikely that such changes are due to the selection of a particular cell subpopulation. This is supported by the fact that the ganglioside pattern of cells cultured in vitro was restored when cells from a solid tumor or from ascites fluid (i.e., in vivo) were recultured in vitro. Furthermore, these changes in ganglioside metabolism are unlikely due to normal lymphocytes infiltrating the tumor or present in the ascites fluid of a tumor bearing mouse because normal lymphocytes contain G,,, and GalNAcG,,, in an approx. equal ratio [24]. Thus these cells could not cause a shift in the ratio of YAC-1 gangliosides G,,, and GalNAcG,,, from 1:l in vitro to 6:l in vivo. The third structural aspect of interest is ceramide. Two main ceramide species, d18:1-C16:O and d18:1C24:1, were identified in each ganglioside. The relative ratio of these two ceramide species was constant and equal (1:l) in each of the four major gangliosides of YAC-1 lymphoma cells, whatever the different in vitro or in vivo growth conditions. The only additional prominent ceramide species was d18:1-C24:O which was detectable in G,,, (NeuGc) of cells cultured in vitro. Thus, in contrast to large differences in relative shedding of various ceramide species (see below), qual-

itative aspects of ceramide synthesis seem not affected by changes in culture conditions. Influence shedding

of the cellular environment

on ganglioside

Shedding of gangliosides is another dynamic aspect of ganglioside metabolism in tumor cells. To determine the influence of cell growth conditions on shedding, an experimental model is required in which gangliosides, once shed, can be recovered and analyzed. In vitro, this is most feasible using serum-free medium, since the only gangliosides in the culture medium will be those synthesized by the cells. In vivo, the analysis of mouse ascites fluid in which the lymphoma cells are proliferating also provides information about ganglioside shedding, particularly its qualitative aspects. Quantification of ganglioside shedding in vitro in serum-free medium showed a rate of 240 pmol/lO* cells per h, or approx. 1% of total cellular gangliosides per h. A similar rate (300 pmol/108 cells per h) was estimated for cells cultured in serum-containing medium (10% FCS), suggesting that the presence of serum does not markedly alter the shedding rate. Kinetic measurements, using metabolic radiolabeling methods applied to cells cultured in vitro, show that ganglioside shedding is linear with time, indicating a continuous release of ganglioside molecules from the cell surface (Fig. 4). In vivo, the total ganglioside concentration in an ascites fluid sample was 14.3 nmol/ml. A minor fraction, 1.5 nmol/ml, consisted of host-derived gangliosides, mainly G,,. The recovery of a resultant net total of 12.8 nmol shed gangliosides/ml ascites fluid, which contained approx. 10’ YAC-1

l

OV

0

I

12

I

24 Culture

I

I

36 Duration

48

I

60

(hour)

Fig. 4. Kinetics of ganglioside shedding of YAC-1 murine lymphoma cells. Cells were radiolabeled for 40 h, harvested, washed and cultured in fresh medium. The radiolabeled gangliosides were purified from the cells and conditioned medium of aliquots taken every 12 h. Cellular and supernatant ganglioside-associated radioactivity was quantified by p-scintillation counting. Comparison of these paired values allowed calculation of% of shed gangliosides. Experiment 1 (01, Experiment 2 (A), each point represents the mean of Tao determinations (range 5 10% of the mean).

288 TABLE

II

Comparison YAC-I cells

of shedding

of the major gangliaside ceramide species by

cells

G M,h

in viva h

in vitro a

Gangliosides

conditioned medium

cells

ascites fluid

23,

7, 14’ 21

0JeuAc)

d18: 1X24: 1 d18: 1X16:0

4’ 4

3 3

21

,44

G,,, (NeuGc) d18: lC24:

1

d18: 1X16:0 dl8: I-C24:O GalNAcG,,, (NeuAc) d18: l-C24: 1 dlS:l-Clh:O G~INAcG~,~ (NeuGc) d18:1-C24: 1 d18: X16:0

16

16

18,

23,62

14)43

l3)4O

25 ,43

39’

13

11

2 2

2 2

25, 20’ 45

26, 24’ 50

were shed in proportion to its expression in the cell membrane. Similarly, with respect to ceramide structure, although the ceramides of dlS:l-C24:l and d18:1C16:O in G,,, (NeuGcf and G,,, (NeuAc) were present in roughly equal concentrations in the cells, in the surrounding ascites fluid the concentration of gangliosides with dl&l-Cl60 ceramide was about twice as high as that of gangliosides with dl&l-C24:l ceramide. Together, these results show the selective shedding in vivo of the relatively more polar ganglioside species, those which contain NeuGc (vs. NeuAc) and shorter fvs. longer) chain fatty acids. Discussion

4 5

1 1

2 3

2 4

” Cells cultured for 72 h in serum-free medium (final total ganglioside concentration = 141 pmol/ml medium). ’ Cells were injected into the peritoneal cavity of syngeneic mice and the ascites fluid was recovered 7 days later. The ganglioside concentration in the fluid was 14.3 nmol/ml, including 1.5 nmol/ml host-derived GM,. The net shed gangliosides were 12.8 nmol/ml. The relative concentrations of individual ceramide species were quantified as % of total gangliosides by integration of HPLC elution profile and by relative abundance in the mass spectra. The data represent the mean of three measurements, of which the SE. was uniformly < 10% of the mean.

cells/ml, documents that significant shedding also occurs in vivo. Quantitatively significant shedding both in vitro and in vivo allows definitive experimental determination of the qualitative aspects of shedding, i.e., setectivity with respect to ganglioside structure. Nine gangliosides of different combined carbohydrate and ceramide structure were isolated from YAC-1 cells, culture supernatant, and ascites fluid, and structurally characterized (Table II>. Comparison of the structures and quantities of the individual cellular and shed gangliosides of YAC-1 cells cultured in serum-free medium shows that each ganglioside identified in the cell was shed in vitro in proportion to its concentration in the cell membrane. This was true with respect to both carbohydrate and ceramide structure. Therefore, shedding is generalized in vitro. In contrast, substantial selectivity of shedding was observed in vivo. This selectivity is linked to both carbohydrate and ceramide structure. Regarding the carbohydrate structure, while the two major species, G M,h (NeuGc) and G,,, (NeuAc), were present in cells in a ratio of 1:1, they were present in a ratio of 3:l (62 to 21% of total gangliosides) in the ascites fluid surrounding these cells (Table II). In other words, shedding of NeuGc-containing ganglioside species was three times what would be expected if each molecule

Gangliosides shed by YAC-1 lymphoma cells have potent inhibitory effects on the in vitro cellular immune response at micromolar concentrations [4]. Therefore, elucidation of the structures and metabolism of these molecules is important. it was recently reported that the carbohydrate structures of gangliosides isolated from YAC-1 cells cultured in vitro are mainly G M,b and GalNAcG,,, 191. Here, we have fully characterized the ceramide structure as well as the carbohydrate structure of these gangliosides. Moreover, we have assessed the metabolism of gangliosides from YAC-1 cells grown under several different conditions, both in vitro and in vivo. The quantification and structural characterization of individual gangliosides by reversed-phase HPLC and negative-ion FAB CAD-MS/ MS [lo] reveal dramatic changes in ganglioside metabolism of YAC-1 cells under these different conditions. In vitro, the major gangliosides are G,,, and GaINAcG Mlbt present in an equal ratio; however, in vivo, only G,,, is the major ganglioside component. In vitro NeuGc-containing gangliosides are predominant, whereas NeuAc-containing gangliosides account for more than half of the total gangliosides in vivo. These results suggest a marked inhibition of GalNAcG,,, synthesis and of hydroxylation of NeuAc, and show that intracellular ganglioside metabolism can be modified by the extracellular microenvironment. Gangliosides G,,, and GalNAcG,,, have been detected in murine T l~phocytes [24], murine lymphoma cells [25], murine-derived macrophage-Iike WEHIcells [26], mouse spleen 1271, normal human brain [28] and Tay-Sachs brain [29]. Several facts are known about the metabolic interrelationships between these molecules. First, GalNAcG,,, is synthesized from and G Mlb by the addition of N-acetylgalactosamine, this process was observed in association with the maturation or stimulation of T lymphocytes [30]. Secondly, NeuGc is synthesized by hydro~lation of CMP-NeuAc [21-231. Our findings and previous results lead us to propose an overall biosynthetic pathway for the YAC-1 murine lymphoma gangliosides (Fig. 5): three enzymes are involved in the synthesis of the ganglioside

289

GAI

GA1

(NeuGc)

Ghjb

I Ill

tm 4

GalNAcGMl b

GalNAc GM1 b in vitro

I

in vivo

Fig. 5. Proposed pathway for YAC -1 murine lymphoma ganglioside biosynthesis. Differences in the relative expression of these pathways predominant; in vitro and in vivo are indicated: expressed; - - - not expressed. See text for detailed discussion.

GalNAcG,,, from the precursor neutral glycosphingolipid, G,,. The first enzyme (I) converts NeuAc to NeuGc by hydroxylation of CMP-NeuAc [23]. The second enzyme (II) is sialyltransferase, catalyzing the addition of sialic acid (NeuAc or NeuGc) to GA,, forming G,,, [31]. The third enzyme (III), GalNAc transferase, adds GalNAc to G,,, to yield the final biosynthetic product, GalNAcG,,, [30,32]. In vitro, enzymes I and III appear to be very active; almost all NeuAc is converted to NeuGc which is subsequently incorporated into G,,, and GalNAcG,,, is synthesized in substantial amounts. In contrast, in vivo, formation of the products of these two enzymes (I and III) in YAC-1 lymphoma cells is reduced. Thus, the hydroxylation of NeuNAc and the synthesis of GalNAcG,,, appear to be inhibited in vivo as evidenced in vivo by a higher concentration of NeuAc and almost absence of GalNAcG,,,. Activity of the sialyltransferase, as indicated by Gr,,,b formation, does not appear to be affected by the change in conditions, strongly arguing against a non-specific effect underlying these observed changes in ganglioside metabolism. An unexpected finding of this study was that the cell microenvironment also affects ganglioside shedding. Previously, we had outlined four different possible mechanisms of shedding of gangliosides [18], but thought they would be invariable within a given cell. YAC-1 cells, which shed significant amounts of gangliosides, shed them in a generalized manner in vitro, with respect to both carbohydrate and ceramide structure. In vivo, however, shedding was selectively greater in the case of NeuGc-containing and shorter chain fatty acid (C16:0)-containing gangliosides. A common chemical feature of these two preferentially shed species is that they are more polar than their counterparts (NeuAc and C24:1), suggesting that the polarity

of the ganglioside molecules may influence the relative rate of shedding, a finding which is consistent with several previous observations of selective glycolipid shedding by tumor cells [33,341. This is of particular importance because our recent study shows that ganglioside species containing shorter chain fatty acyl groups have higher immunosuppressive activity than those containing longer chian fatty acyl groups [35]. Our findings support the previously articulated concept that the external microenvironment of the tumor cell may significantly affect ganglioside synthesis. Comparison of ganglioside composition in human gliomas, medulloblastoma and melanoma biopsies, and cells cultured in vitro and passaged in nude mice [36-381 suggested that the environment of the cell affects ganglioside expression. The present findings extend this concept to the biologically important process of ganglioside shedding. In vitro, ganglioside shedding was generalized with respect to both carbohydrate and ceramide structure, but in vivo it was selective for more polar ganglioside species (again with respect to either carbohydrate or ceramide structure). The detection of both qualitative and quantitative changes in ganglioside synthesis of murine lymphoma cells propagated under in vitro and in vivo conditions is of particular interest because these striking changes in ganglioside metabolism were induced by merely altering the cellular microenvironment. Modification of ganglioside expression and shedding by tumor cells, which could have therapeutic implications, may therefore not require genomic manipulation to be accomplished. Acknowledgements

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