Biochimit~ et Biophysica A cta. 1083 (IqOlI 153-160 ,i~ 1991 EisevierScience PublishersB.V.0005-2760/91/$03.50 ADONIS 0005276091001525
153
Metabolism of cholesterol, phosphatidylethanolamine and stearylamine analogues of G M1 ganglioside by rat glioma C6 cells T a d e u s z P a c u s z k a a n d Peter H. F i s h m a n Membrane Biochemistry Section. Laboratory o] Molecular cmd Cellular Neurobiolo~'. National Institute of Neurological Disorders and Stroke. Tile National Institutes of Health. Bethesda, MD I U.S.A.)
(Received 1 November1990l Key words: G~,llganglioside: Metabolism:Cholera toxin Tritium-labeled neoglycolipids consisting of the oligosaccharide of gang!i~side GMt attached to cholesterol (GMIOSNH-X-CHOL), phosphatidylethanolamine (G~tOS-PE) and stearylamine (G,~IIOSNHCts) were synthesized and their uptake and metabolism by G Mr-deficient rat glioma C6 cells were determined. When the neoglyculipids were added to serum.free culture medium, all three were rapidly taken up by the ceils and initially inserted into the plasma membrane based on their resistance to trypsin and their ability, to bind cholera toxin. With time, the neoglycolipids underwent internalization as the ratio of cell-associated radioactivity, to cell surface toxin binding increased: this process was slow for G M,OSNH-X-CHOL and G MtOS-PE and rapid for G MtOSNHC ts. Analysis of lipids cxtracted from the cells indicated that the neoglyeolipids also underwent metabolism to G nt~OS-based analogues. In addition, G ~uOSNHX-CHOL and GMtOSNHCts were degraded to their GM2OS-based analogues, whereas GMzOS-PE was not detected. In contrast, large amounts of 3H were recovered in the medium from cells treated with GMtOS-PE and the label was associated with material that behaved neither as an oligosaccharide or a neoglyeulipid. In the presence of manensin or chloroquine, metabolism of the three neoglycolipids was inhibited. Thus, G .~ttOS-based neoglycolipids were taken up by the cells, internalized and sorted both to the Golgi apparatus (sialylated to G Dt,OS-based analogues) and to lysosomes (hydrolyzed to GM2OS-based analogues). The rate and extent of these processes, however, were strongly influenced by the nature of lipid moiety.
Introduction
for cellular
glycosyltransferases and
glycosidases
[1,5,9,10]. Apparently, some of these metabolic processes
It is well established that exogenous gangliosides added to the culture medium are taken up by cu!tured cells and initially inserted into the plasma taembrane [1-5]. The newly acquired gangliosides can endow the cells with the ability to bind toxins [1,31 and viruses [61, to organize fibronectin into an extracellular matrix [7], and in the case of neurotumor cells to extend neurite-like processes [8]. In addition, the exogenously incorporated gangliosides can undergo metabolism and are substrates
involve internalization of the plasma membrane and intracellular sorting through the Golgi apparatus and lysosomes [9,10]. Recently, we synthesized several neoglycolipids by coupling the oligosaccharide released from G u t to cholesterol, phosphatidylethanolamine and octadecylamine and demonstrated that these neoglycolipids are taken up by GM:deficient rat glioma C6 cells and function as receptors for cholera toxin [ll]. In the present report, we describe the kinetics of incorporation of these neoglycolipids by rat glioma C6 cells and their metabolism.
Abbreviations: GMI, 113Neu5Ac'GgOse4Cer; GDt~. 1v~Ncu5Ac-, ll3NeuSAc-GgOseaCer; GM2. ll3Neu5Ac-GgOse3Cer; GM|OS, Gol~OS and GM2OS, correspondingoligosaccharides: GMIOSNH2 and GDlaOSNH2,correspondingl-amine-l-deoxyglucitolderivatives; PE0phosphatidylethanolamine;CHOL.cholesterol; abbreviationsand structures of neoglycolipidsare shownin Fig, I.
Material and Methods
Correspondence: P.H. Fishman, Park Bldg: Rm. 411, National Inst. Health, Bethesda, MD 20892, U.S.A.
Materials
Cholera toxin was purchased from List Biological Laboratories (Campbell, CA) and for iodination from ICN Biomedicals (Cleveland, OH). Monensin and Fibrio cholerae neuraminidase were from Calbiochem Corp.
154 (San Diego, CA). Bovine testes /~-galactosidase was from Boehringer Mannheim (Indianapolis, IN); crystallized trypsin from Worthington Biochemical Corp. (Freehold, N J); and chloroquine was from Sigma Chemical Co. (St. Louis, MO). NaB3H4 (15 Ci/mmol) was from New England Nuclear (Boston, MA) and NaB3H3CN (3.8 Ci/mmol) was from Amersham Corp. (Arlington Height, IL).
Preparation of neoglycolipids GM~ and Grit ~ were purified from bovine brain [12]. GMIOS and GMIOSNH 2 were prepared as described previously [13]. The same basic methods were used to prepare GDt~OS, reductively aminate it and to purify the GDI,,OSNH2. The only change was to elute the GDI~OS from the Dowex l-X2 column with 0.4 M pyridinium acetate buffer (pH 5.0). GD~OSNH-XCHOL, GDlaOS-PE and GDtaOSNHCIs for standards were synthesized by the procedures detailed for GM1OS derivatives [11] with the following minor modification. The fil;:l reaction mixtures were taken to dryness and partitioned [14]; the lower layers were extracted once with theoretical upper phase and the combined upper phases were taken to dryness on a rotary evaporator (below 30°C). The reaction products then were further purified on silica gel and Sephadex G-25 superfine columns [11]. Tritium-labelled GM1OSNH-X-CHOL, GM1OS-PE and G M10SN H C is were prepared by the same methods used for the unlabelled derivatives. Briefly, GM1OS was reductively aminated using NaB3H4 and the 3H-labeled G ~ , O S N H 2 was used to make the cholesterol derivative. GM~OS was coupled to PE and octylamine via reductive amination in the presence of NaB3H3CN. 3H-labeled GMEOS-PE, GMEOSNH-X-CHOL and GM2OSNHC~s were prepared from the above compounds by removal of the terminal galactose with flgalactosidase as described by Distler and Jourdian [15]. Briefly, each compound (3 • 10' dpm) was dissolved in 0.1 ml of chloroform/methanol (1 : 1, v/v) with 25 #g of Triton X-100. The sample was taken to dryness and the residue was suspended in 50 /tl of sodium citratephosphate buffer (pH 4.3) containing 10 munits of fl-galactosidase. After an overnight incubation at 37 o C, the samples were desalted on Sephadex G-25 superfine columns. The radioactive reaction products were not purified further. Cells and cell culture Rat glioma C6 cells were cultured as described recently [11,13]. Cells were grown in either cluster dishes (24 × 16-ram wells) or 75 cm 2 flasks until confluent (80-100 /~g protein per well or 3-4 mg protein per flask). The cells were washed with phosphate-buffered saline at room temperature and fresh serum-free medium containing 0.1 /tM 3H-labeled neoglycolipids was ad-
ded. The neoglycolipids were stored at - 8 0 ° C in organic solvents and portions were taken to dryness under sterile conditions and dissolved in the medium with brief immersion in an ultrasonic cleaning bath. In experiments with chloroquine (100 p.M) and monensin (5 /~M), the cells were exposed to the drugs 30 rain before any treatment and kept in the presence of the drugs throughout the experiment. For most of the metabolic experiments, the cells were cultured in the presence of the neoglycolipids for 1 or 24 h after which the medium was changed and the cells were incubated in the absence of neoglycolipids for an additional 18 h or as indicated.
Lipid extraction and analysis At the end of an experiment, the cells were washed with ice-cold phosphate-buffered saline, detached with a plastic scraper or released with trypsin (0.05% for 5 min at 3 7 ° C [3]), and centrifuged at 500 × g at 4 ° C for 5 rain. The cell pellets were extracted with chloroform and methanol as described [16]. The lipid extracts were takes to dryness under nitrogen and further purified on small columns of Sephadex G-25 superfine and Unisil [11]. Typically, 0.2 g of Sephadex and 0.15"g of Unisil packed in disposable glass Pasteur pipets were used for lipids extracted from cells isolated from one flask. The purified lipids were analyzed by thin-layer chromatography on precoated silica gel plates (E. Merck, Cat. No. 5725) in solvent system A, chloroform/methanol/0.25% CaCI 2 ( 6 0 : 3 5 : 8 , v/v). The purified lipid extracts also were treated with neuraminidase [17], desalted on Sephadex G-25 [12] and analyzed by thin-layer chromatography. Radioactive material on the chromatograms was detected with a Bioscan System 200 imaging scanner (Bioscan, Washington, DC). Other methods Sialic acid was determined with resorcinol [18]. Carbohydrate compositional analysis following acid hydrolysis of neoglycolipids was by anion exchange chromatography with pulsed amperometric detection [19] as outlined in the Dionex workbook and manual (Dionex Corp., Sunnyville, CA). Oligosaccharides were analysed by thin-layer chromatography using solvent system B, n-propanol/0.1% CaCI2 (6 : 2, v/v). Labeled degradation products in the culture medium were analyzed by gel filtration column chromatography [9] except Bio-Gel P-4 was used. Protein was measured by the method of Lowry et al. [20]. Results
Characterization of neoglycolipids The specific radioactivity of the 3H-labeled GMtOSNH-X-CHOL was 0.67 Ci/mmol, whereas GMIOS-PE was 3.5 C i / m m o l and GMIOSNHCIs was 2.5 C i / m m o l
155
CH20H Gal/It--3GaINAc/I1--4Ga~/]1"O
?
F ~ A(
~OFOH • O CH2" NHV'~O
~
TABLE 11 Rate of uptake of [ ~tl]GmtOSNH-X-CIIOL I~v rat glioma C6 cells and effect on cholera toxin binding Details are the same as those in Table I except the cells were incubated with 0.1 p.M [3HIGMIOSNH-X-CHOL(75000 dpm/well).
o
NeuSAca2 GMIOSNH-X-CHOL
CH2OH Galpl--SGaINAcpl~4Ga1111"0 OH ,
NeuSAco2
CH2--NH--CH2-- CH2--
%o
OH
0~¢x t - v ~ ,
L 0 AA/X,,~VVV~
GMIOS-PE
CH2OH Oalpl--3Ga*NAc/n--4Galpl,
3
t NeuSAca2
J~C_/
Incubation time (mini
C'H2-- NH/V~/~/XAJXAA/X
OH
0 1 5 15 30 60 120 180
[3HIGMtOSNHX-CHOL incorporated (fmol/well)
tzSI-cholera toxin bound (fmol/well)
Ratio =
356+ 13 2025± 57 3797±191 4890±126 6127±237 7719±489 9861 ±430
3.1± 0.9 68.9_+ l.S 397 ± 3.1 873 ± 9.1 1003 _+21 1163 ±21 1 143 ±24 l 122 ± 16
5.4 5.3 4.4 4.9 5.3 6.8 7.6
a Ratio corrected for toxin bound to untreated cells,
GMIOS-NHCI8
Fig. I. Structure and labelling of 13HlGMiOS-based neoglycolipids.
and the location of the 3H is shown in Fig. l. Based on radioscanning after thin-layer c h r o m a t o g r a p h y (Fig. 2, left panels), each a p p e a r e d to be highly homogeneous. Each of the 3H-labeled G u t O S - b a s e d neoglycolipids comigrated with its unlabeled counterpart (Fig. 2, lanes 2). All the [ 3 H ] G u t O S - b a s e d neoglycolipids were susceptible to fl-galactosidase and the corresponding reaction products, p r e s u m a b l y GM2OS-based derivatives, were more mobile on thin-layer c h r o m a t o g r a p h y (Fig. 2, fight panels). T h e difference in mobility was similar to that observed for G m c o m p a r e d to G u t (see standards in lanes 1). In contrast, the unlabeled disialyl derivatives had a slower mobility than the corresponding m o n o -
TABLE I Rate of uptake of [JH].GMtO.~;-PE by rat glioma C6 cells and effect on cholera toxin binding Cells in cluster dishes were incubated in 0.5 ml tff serum-frec medium containing with I p.M [3HIGMtOS-PE(440000 cpm/well} at 37°C for the indicated times. The cells then were washed three times and either assayed for specific t251-eholera toxin binding ll3l or dissolved in 1 M NaOH and analyzed for neoglycolipidincorporation by liquid scintillation spectroscopy. Cell protein was determined and the values were normalized to 100 pg/well. Values are means ± S.D. of triplicate determinations. Incubation time (rain)
I3HI-GM~OS-PE incorporated (fmol/well)
t251-cholera toxin bound (fmol/well)
610± 26 2670± 70 5080+ 105 5265:t: 42 7730±110
2.0± 0.5 168 ± 6.2 489 ± l l 661 +21 822 +10 948 ±23
0 I
5 15 30 60
= Ratio corrected for toxin bound to untreated cells.
sialyl derivatives (Fig. 2, lanes 2) which is consistent with the observed migration of GDt a c o m p a r e d to GMt. C a r b o h y d r a t e composition analyses of Got=OS-based neoglycolipids gave m o l a r ratios of sialic a c i d : g a l a c tose : galactosamine or 2 : 2 : 1 with glucose not exceeding 0.2. U p t a k e o f neoglycolipids by rat glioma C6 cells T h e ['~H]G~tjOS-PF-,, [ ~ H ] G M t O S N H - X - C H O L and [ 3 H ] G M t O S N H C t s were rapidly taken up by rat glioma C6 cells as measured by 3H incorporation and increased t25I-cholera toxin binding (Tables L II and Ill). As each toxin molecule can bind to 5 G M t O S molecules [21], initially most of the incorporated neoglycolipids appeared to he on the cell surface for both derivatives as had been observed earlier for GMt [9]. After 15 rain, however, cells exposed to G MtOSN H C t s began to bind
TABLE
Details are the same as in Table l except the cells were incubated with 0.1 aM [3HIGM~OSNHCla(2780~30dpm/well). Incubation time (rain)
[~HIGMI OSNHCIs incorporated (fmol/well)
Iz~l-cholera toxin bound (fmol/well)
O l 5
3 0 6 + 34 1 5 8 9 ± 259
4.5 ± 0,9 66.6± 2.3 465 ± 19
15 30 60 120 180
3642+ 40 5435± 36 76634- 85 10790-&607 14480± 137
Ratio a
3.7 5.5 7.7 6.4 8.2
111
Rate of uptake of [~H]GMIOSNHCI s by rat glioma C6 cells and effect on cholera toxin binding
557 491 384 382 284
+ 6.4 + 5.6 ± 6.9 ±20 ± 5.9
Ratio corrected for toxin bound to untreated cells.
4.95 3.45
6.5 ll.l 20.0 28.2 51.0
156 less 1251-toxin even though the amount of 3H-label incorporated continued to increase for up to 3 h (Table Ill). As had been found for [3H]GM~ [3], the labeled neoglycolipids appeared to be stably inserted into the cell membrane. When the cells were detached from the culture vessels with 0.05% trypsin, slightly more radioactivity was recovered in the cell pellets than when cells were detached by scraping (unpublished data).
neoglycolipids were purified, resolved by thin-layer chromatography and detected by radioscanning (Fig. 3). In addition to the original neoglycolipids, new radioactive compounds were detected. The slower migrating compounds corresponded to the GDtaOS-based standards and presumably were the result of biosynthetic conversion of the G ~ O S - b a s e d neoglycolipids by the cells. The faster migrating compounds corresponded to the [3H]GM2OS-based standards and presumably resulted from enzymatic hydrolysis of the parental compounds. The quantities of these metabolic products
Analyses of neoglycolipids taken up by rat glioma C6 cells Lipid extracts from cells incubated with 3H-labeled
iA
,I Origin 1
i! i
2 :...,
.......
,, ....
.,.,.;
B
qp
I I
..... 1
l..-.7.,, r~,,,..,
ic
t
l Orig in 2
t,., ,-..~|
l i
I i ::
~
-
A
i
!
J-! .....
~R
lOrigin
Fig. 2. Analysis of unlabeled and 3H-labeled neoglycolipids by thin-layer chromatography and radiochromagraphic scanning• [3HlGMlOS-based neoglycolipids were incubated without (left panels) and with (right panels) .8-galactosidase and (5-10).10 4 dpm of each ware separated by thin-layer chromatography in solvent A along with standard gangliosides (lanes 1 from top to bottom: GM3, GM2, GM1, GOla) and neoglycolipid standards (lanes 2 upper and lower: GMIOS- and GDhOS-based) as described under Materials and Methods. The radiolabelod compounds were detected by radioscanning and the standards detected with orcinol. (A) [3H]GM~OSNH-X-CHOL (left) and its fl-galactosidas¢product (right); lane 2, GMtOSNH-X-CHOL and GD~OSNH-X-CHOL; (B) [3H]GM~OS-PE (left) and its //-galactosidase product (right); lane 2, GM~OS-PE and G.~OS-PE: (C) [3HIGM~OSNHC~s (left) and its .8-galactosidase product (right); lane 2, GMjOSNHC~s. GD~=OSNHCIs and GD~=OS (right above origin).
157 varied for the different neoglycolipids. F o r GMIOS-PE, the biosynthetic p r o d u c t was the major o n e as the b r e a k d o w n p r o d u c t , i.e., GM2OS-PE, was barely detected. In contrast, the d e g r a d a t i o n p r o d u c t s of GM1OSNH-X-CHOL and GMIOSNHCls predominated. F o r the latter neoglycolipid, an additional even faster m i g r a t i n g c o m p o u n d was detected. T h e n a t u r e of this new c o m p o u n d is u n k n o w n ; it is unlikely to be G M 3 O S N H C t s as rat g l i o m a C6 are u n a b l e to breakd o w n exogenously i n c o r p o r a t e d [ 3H ] G M2 [9]. T h e slower m i g r a t i n g radioactive c o m p o u n d s were susceptible to Vibrio cholerae n e u r a m i n i d a s e , whereas the GMIOSb a s e d derivatives a n d the faster m i g r a t i n g c o m p o u n d s were resistant. Effect of chloroquine and monensin B o t h c h l o r o q u i n e (100 F M ) a n d m o n e n s i n (5 F M ) inhibited a l m o s t completely the m e t a b o l i s m of the 3Hlabeled neoglycolipids b y cat g l i o m a C6 cells (Fig. 3). T h e effect o f m o n e n s i n w a s even m o r e p r o n o u n c e d t h a n t h a t o f chloroquine. W i t h either drug, the Go~,,OS- or G M2OS-based c o m p o u n d s were barely detectable. Release of radioactivity into the medium Cells were i n c u b a t e d with 3H-labeled neoglycolipids for 1 h, w a s h e d a n d c u l t u r e d in fresh m e d i u m for 18 h at w h i c h time th~ cultures were analyzed for distribution of radioactivity (Table IV). In the case o f
TABLE tV Distribution of radioactivity in cultures of rat glioma cells treated with 3H-labelled neoglvcolipids Cells in 75 cm2 fla~ks were incubated for I h at 37°C in serum-free medium containing 0.t ,aM of the indicated 3H-labelled neoglycolipid, washed and incubated in fresh medium for t8 h. The cultures then •~ere analyzed for distribution of 3H in the medium, the cellular lipid extract, and the delipidated celt residue. Neoglycolipid
Medium (pmol/flask}
Lipid extract
Cell residue
[3HIG M1OSNH-X-CHOL [3HIG M~OSNHCIs [3HIG MtOS-PE
13.8 12.5 27.9
220.4 213.3 39.2
t.l 1.0 4.3
[ 3 H ] G M I O S N H - X - C H O L a n d [ 3 H ] G M I O S N H C t s , less than 10% of the 3H w a s f o u n d in the culture m e d i u m , over 90% was recovered in lipid extract of the cells, a n d only a trace was f o u n d associated with the cell residue. In contrast, 39% of the 3H w a s recovered in the m e d i u m from cells treated with [3H]GMIOS-PE, 55% w a s f o u n d in the lipid extract a n d 6% w a s associated with the cell residue. This radioactivity in the m e d i u m w a s further analyzed by gel filtration c o l u m n c h r o m a t o g r a p h y (Fig. 4). T h e material w a s r e t a i n e d b y the c o l u m n a n d m o s t of it eluted slightly a h e a d of the G M t O S N H 2 s t a n d a r d . T h e p e a k was freeze-dried a n d analyzed b y thin-layer MONENSIN -
.
.
.
-
. -
GM2OSNH
X
GMIOSNH
x
GD,aOSNH-
Chol
ChoI X
Chol
-. O,,g,n
.
.
.
.
.
.
.~ .
~ .c
-
~GIClOS
BE
.iI 't....... ~ GolaOS-PE
I -
---
_
~
-
GM2OSNH-C,s
~ GM1OSNH -- GD,aOSNH
C18
Cta
-. o , , g . .
Fig. 3. Metabolism of 3H-labelled neoglycolipids taken up by rat glioma C6 ceils: effects of chloroquine and monensin. Cells were incubated with 3H-labelled neoglycolipids in the absence (Control) and presence of either 100 ,aM chloroquine or 5 ,aM monensin as indicated under Materials and Methods. The labeled lipids were extracted front the cells, purified, separated by thin-layer chromatography and detected by radioscanning. Between 5 and 10-104 dpm were applied per lane.
158 15000
~ 10000
5ooo
do
6o
oo
Fractions
Fig. 4. Gel filtration columnchromatographyof radioactive degradation products released to the culture mediumby rat glioma C6 cells incubated with [3H]GMIOS-PE.Cells were incubated with [3HIGM: OS-PE for 1 h. washedand incubated in freshserum-freemediumfor 18 h. The latter medium was freeze-dried, dissolved in 50 mM ammoniumbicarbonate(pH 7.8) and a portion(105cpm)applied to a Bio-Gel P-4 column whichwas eluted with the same solution. Fractions were collected and counted for 3H. The column was calibrated with bluedextran(B.D.),G MIOSNH2 and glucose(GIc).
chromatography in both solvents systems A and B. Upon radioscanning, the major peak of radioactivity was detected at the origin in both solvent systems. Thus, it was unlikely to be either a glycolipid or a simple oligosaccharide. One possibility is that it is a phosphoor phosphoglycerol-oligosaccharide metabolic product of [3HIG M~OS-PE. Discussion We found that G MiOS-based neoglycolipids were rapidly taken up from the medium by rat glioma C6 cells and initially inserted into the plasma membrane. These conclusions were supported by the following observations: (1) The 3H-labeled neo~;lycolipids were not released from the cells by trypsin. We had demonstrated previously that [3H]GMt and a [3H]GMt derivative in which the fatty acid had been replaced by acetate also were not released from rat glioma C6 ells by trypsin [3]. Others have reported that gangliosides taken up by cultured cells are in both trypsin-sensitive and -resistant states [2,4,8]. In most of these studies, however, the gangliosides are added in medium containing serum and serum proteins may be binding the gangliosides and preventing their insertion into the membrane [22]. In all of our studies, we exposed the cells to gangliosides (or neoglycolipids) in serum-free medium: and (2) When the cells were exposed to the neoglycolipids, they rapidly acquired new cholera toxin binding sites. Initially, the molar ratio of neoglycolipid incorporated to toxin bound was close to the theoretical ratio of 5:1 [21]. This is consistent with most of the neoglycolipid molecules being on the outer leaflet of the plasma membrane bilayer and mobile in the plane of the membrane in
order to permit multivalent binding of the toxin [23]. We have shown previously that the affinity of cholera toxin for neoglycolipids inserted into C6 membranes is the same as that for inserted GM~ and consistent with a single class of high affinity binding sites [11]. Although the neoglycolipids initially were inserted into the plasma membrane, with time they appeared to undergo internalization. For [~H]GMtOSNH-X-CHOL and [3H]GMIOS-PE, the ratio of neoglycolipid to toxin bound slowly increased with time In contrast, when cells were exposed to [3H]GMtOSNHCts, the ratio increased rapidly and the number of toxin binding sites actually decreased even though the amount of cell-associated [3H]GMtOSNHCIs continued to increase. The basis of this phenomenon is not clear. Large amounts of GMIOSNHCI~ within the plasma membrane may stimulate rapid and possibly selective endocytosis in order to clear from the cell surface this potentially toxic substance [24]. It is unlikely that it was due to the rapid metabolism of [3H]GMtOSNHC1s as no metabofic products were detected after a 2-h incubation. Formation of [ ~H]G M2OSN HCts was detected after 4 h, but it represented less than 20% of the [3H]Gr~tOSNHCms extracted from the cells (unpublished data). It has been reported by this laboratory [1,9] as well as by others [5,10] that exogenous gangliosides are metabolized by cultured cells, being converted to both less complex and more complex gangliosides. Presumably, the former are due to the action of cellular glycohydrolases located in lysosomes and the latter to that of glycosyltransferases located in the Golgi apparatus. We found that neoglycolipids incorporated into rat glioma C6 cells also were converted into both more complex and less complex analogues of the parent compounds. The more complex products had the same mobility on thin-layer chromatograms as the Go~aOS-based standards and were susceptible to Vibrio cholerae neuraminidase. The less complex products derived from [aH]GMIOSNH-X-CHOL and [3H]GMtOSNHCts corresponded to the GMzOS-based standards and were the major metabolic products as had been observed for [3H]GMt taken up by rat glioma C6 cells [9]. In contrast, no [3H]GM2OS-PE was detected in cultures treated with [3H]GMtOS-PE even though the latter compound was hydrolyzed by fl-galactosidase in vitro (see Fig. 2). A substantial amount of 3H was released into the culture medium which indicated that the [3H]GMIOS-PE was metabolized by the cells. One possibility is that the major degradative pathway is via phospholipases and not glycohydrolases. The effects of monensin and chloroquine provide further support that the neoglycolipids are being internalizcd, then undergoing intracellular sorting and finally being metabolized. Monensin has been shown to inhibited the biosynthesis of endogenous gangliosides in
159 cultured cells [25-27] a n d is k n o w n to disrupt vesicular t r a n s p o r t through the Golgi a p p a r a t u s [28]. C h l o r o q u i n e causes alkalization of lysosomes a n d thus inhibits lysospinal hydrolases [29]. Both c h l o r o q u i n e a n d m o n e n s i n also inhibit e n d o s o m a l acidification a n d thus m a y interfere with n o r m a l intracellular vesicular trafficking ]30]. In the presence of these drugs, the internalized [3H]Gm~OS-based neoglycolipids m a y be u n a b l e to be sorted either to the Golgi or the lysosomes. U s i n g a fluorescent PE analog, Sleight a n d P a g a n o d e m o n s t r a t e d t h a t the a n a l o g is initially i n c o r p o r a t e d into the o u t e r layer of the p l a s m a m e m b r a n e of cultured cells a n d then u n d e r g o e s internalization at 3 7 ° C to internal m e m b r a n e s by two s e p a r a t e processes [31]. Delivery of the a n a l o g to the G o l g i a p p a r a t u s involves endoeytosis, whereas delivery to the nuclear envelope a n d m i t o c h o n d r i a involves a r a p i d t r a n s b i l a y e r movem e n t of the fluorescent PE across the p l a s m a m e m b r a n e . A r o u n d 40% of the fluorescent PE is internalized b y 30 rain a n d the a n a l o g is rapidly d e g r a d e d by the cells with a half-life of 1 h. In contrast, G M t O S - P E is internalized very slowly with a half-life of a r o u n d 3 h [11] a n d b a s e d o n o u r present results, a p p e a r s to be d e g r a d e d very slowly. W i t h its large, negatively c h a r g e d h y d r o p h i l i c h e a d g r o u p , G M I O S - P E is unlikely to undergo transbilayer movement. T h e u p t a k e of exogenous cholesterol by cultured cells is even m o r e complex. T h e most studied p a t h w a y involves the r e c e p t o r - m e d i a t e d e n d o c y t o s i s of low density lipoproteins [321. L a n g e a n d Matthies [33] reported t h a t w h e n a d d e d to lipoprotein-deficient m e d i u m in tracer a m o u n t s , r a d i o l a b e l e d cholesterol is rapidly t a k e n u p be c u l t u r e d h u m a n fibroblasts. O v e r 95% is associated with the o u t e r layer o f the p l a s m a m e m b r a n e a n d there is n o significant t r a n s f e r into the cells or esterification for up to 16 h at 3 7 ° C (less t h a n 0.5%). Interestingly, w h e n carrier cholesterol (25 p,M) was a d d e d to the m e d i u m , s o m e of the e x o g e n o u s cholesterol a p p e a r e d to u n d e r g o e n d o c y t o s i s a n d esterification. In contrast, we f o u n d t h a t w h e n rat g l i o m a C6 are i n c u b a t e d with 0.1 p.M G M I O S N H - X - C H O L for 1.5 h, w a s h e d a n d i n c u b a t e d in fresh m e d i u m , the derivative is internalized with a half-life of 2 h [11]. This is consistent with the increase in the ratio o f neoglyeolipid i n c o r p o r a t e d to toxin b o u n d t h a t o c c u r r e d with time of i n c u b a t i o n (Table IlL W e also observed that over 50% of the [ 3 H I G M t O S N H - X C H O L taken u p b y the cells u n d e r w e n t m e t a b o l i s m to m o r e a n d less c o m p l e x derivatives b y 18 h. W e c a n d r a w the following c o n c l u s i o n s that f r o m o u r studies: (i) Despite the differences in lipid moieties, the G m ~ O S - b a s e d neoglycolipids initially are inserted a n d o r g a n i z e d in the outlet leaflet o f the p l a s m a m e m b r a n e in a m a n n e r that s u p p o r t s multivalent b i n d i n g of cholera toxin a n a l o g o u s to GM~; (2) Despite the differences in lipids, they gain access to a n d are substra,,:s /or C M P sialic acid : G ml sialyltransferase, a Golgi e n z y m e [12,341
present in rat glioma C6 cells [9], a n d for two of the three neoglycolipids, lysosomal fl-galactosidase [29]; (3) The lipid moieties strongly influence the rate of internalization a n d possibly their s u b s e q u e n t sorting. T h i s was p a r t i c u l a r l y evident for [ 3 H I G M t O S N H - X - C H O L a n d [3H]GM~OSNHC~s w h i c h were taken u p b y the cells at similar rates, were metabolized to b o t h m o r e a n d less complex analogues, a n d h a d almost identical distributions of radioactivity i n t o the m a j o r pools of medium, lipid extract a n d delipidated cell residue (see T a b l e IV). Yet, [ 3 H ] G M t O S N H C l a w a s m u c h m o r e rapidly internalized t h a n [ 3 H | G M t O S N H - X - C H O L based o n cholera toxin binding. In contrast, 13H]GMt OS-PE, which w a s t a k e n u p a n d internalized at a rate similar to [ 3 H ] G M I O S N H - X - C H O L , w a s not d e g r a d e d to GM2OS-PE a n d its d i s t r i b u t i o n of radioactivity was distinct f r o m the o t h e r two neoglycolipids, with a large fraction a p p e a r i n g in the m e d i u m . P r e s u m a b l y , this reflects a different p a t h w a y of intraeellular sorting. References I Fishman. P.H., Moss, J. and Vaughan, M. (19761 J. Biol. Chem. 251, 4490-4494. 2 Callies, R., Schwarzmann, G,. Rad~k. K., Siegen, R. and Wiegandt, H. (1977) Eur. J. Biochem. 80, 425-432, 3 Fishman, P.H., Pacu~ka, T., Horn, B. and Moss. J. (19801J. Biol. ('hem. 255, 7657-7664. 4 Schwarzmann. G., Hoffman-Bleihauer, P., Schubert, J., Sandhoff, K. and Mar~.h, D. (19831 Biochemistry 22, 5041-5048. 5 Masserini. M., Guiliani, A., Palestini, P., Acquoni, D., Pillo, M., Chigorno, V, and Tettamanti, G. (1990) Biochemistry 29, 69"/-701. 6 Markwdl, M.A.K.. Moss, J., Horn, B.E., Fishman, P.H. and Svennerholm, L. (1986) Virology 155, 356-364. 7 Spiegel, S., Yamada. K.M, Horn, B.E., Moss, J. and Fishman, P.H. (19851J. Cell. Biol. 100. 721-726. 8 Fact:i, L., Leon, A., Toffano, G. Sonnino, S., Ghidoni, R. and Tettamant. G. (19841 J. Neurochem. 42, 299-305. 9 Fishman, P.H., Bradley, R.M., Horn, B.E. and Moss, J. (1983) J. Lipid Res. 24, lOOT-lOll. 10 Sonderfeld. S., Conzelmann, E.. Sch,.varzmann, G., Burg, J.. Hinrichs, U. and Sandhoff. K. 11985) Eur. J. Biochem. 149, 247-255. II Pacuszka, T., Bradley, R.M. and Fishman, P.H. (1991) Biochemistry 30, 2563-2570. 12 Pacuszka, T.. Dullard, R.O., Nishimura, R.N.. Brady, R.O. and Fishman, P,H. (19781J. Biol. Chem. 253, 5839-5846. 13 Pacuszka, T. and Fishman. P.H. (19901 J. Biol. Chem. 265, 76737678. 14 Folch-Pi, J,, Lee:,. M. and SIoan Stanley. G.G, (19571 J. Biol. (-'hem. 226, 497 509. 15 Distler, JJ. and Jourdian, G.W. (19731 J. Biol. Chem. 248, 67726780. 16 Miller-Podraza, H.. Bradley, R.M. and Fishman, P.H. (19821 Biochemistry 21. 3260-3265. 17 Mullin, B.R.. Pacuszka, T., Lee, G., Kohn. UD., Brady, R.O. and Fishman, P.H. (1978) Science 199, 77-79. 18 Svennerholm. L. and Fredman. P. (1990) Biochim. Biophys. Acta 671.97-109. 19 ['lardy, M.R. (19891 Methods Enzymol. 179, 76-82. 211 Lowry, O.H., Rosebough, N.J., Farr. A.L. and Randall, R.J, (1951) J. Biol. Chem. 193, 265-275.
160 21 Fishman, P.H.. Moss, J. and Osborne, J.C., Jr. (1978) Biochemistry 17, 711-716. 22 Fishman. P.H., Bradley. R.M.. Moss, J. and Manganiello, V.C. (1978) J. Lipid Res. 19, 77-81. 23 Fishman. P.H. (1990) in (Moss, J. and Vaughan, M,, eds.). ADPRibosylating Toxins and G Proteins: Insights into Signal Transduction. American Society of Microbiology, pp. 127-140. Washington. 24 llannun. Y.A. and Bell. R.M. (1987) Science 235, 670-674. 25 Miller-Podraza, H. and Fishman. P.H. (1984) Biochim. Biophys. Acta 804. 44-51. 26 Saito. M., Saito, M. and Rosenbcrg. A. (1985) Biochemistry 24. 3054-3059. 27 Van Echten. G. and Sandhoff. K. (1989) J. Neurochem. 52, 207-214,
28 Tartakoff, A.M. (1983) Cell 32, 1026-1028. 29 DeDuve, C. (1983) Eur. J. Biochem. 137, 391-397. 30 Mellman, I., Fuchs. R. and Helenias, A. (1986) Annu. Rev. Biochem. 55, 663-7O0. 31 Sleight. R.G. and Pagano, R.E. (19851 J. Biol. Chem. 260. 11461154. 32 Goldstein, J.L., Anderson, R.G.W.. and Brown, M.S. 09791 Nature 279. 679-684. 33 Lange, Y.. and Matthies. H.J.G. {1985) J. Biol. Chem. 260,1462414630. 34 Pohlentz. G., Klein, D.. Schwarzmann, G.. Sehmitz, D. and Sandhoff. K. (1988) Proc. Natl. Acad. Sci. USA 85. 7044-7048.
153
Metabolism of cholesterol, phosphatidylethanolamine and stearylamine analogues of G M1 ganglioside by rat glioma C6 cells T a d e u s z P a c u s z k a a n d Peter H. F i s h m a n Membrane Biochemistry Section. Laboratory o] Molecular cmd Cellular Neurobiolo~'. National Institute of Neurological Disorders and Stroke. Tile National Institutes of Health. Bethesda, MD I U.S.A.)
(Received 1 November1990l Key words: G~,llganglioside: Metabolism:Cholera toxin Tritium-labeled neoglycolipids consisting of the oligosaccharide of gang!i~side GMt attached to cholesterol (GMIOSNH-X-CHOL), phosphatidylethanolamine (G~tOS-PE) and stearylamine (G,~IIOSNHCts) were synthesized and their uptake and metabolism by G Mr-deficient rat glioma C6 cells were determined. When the neoglyculipids were added to serum.free culture medium, all three were rapidly taken up by the ceils and initially inserted into the plasma membrane based on their resistance to trypsin and their ability, to bind cholera toxin. With time, the neoglycolipids underwent internalization as the ratio of cell-associated radioactivity, to cell surface toxin binding increased: this process was slow for G M,OSNH-X-CHOL and G MtOS-PE and rapid for G MtOSNHC ts. Analysis of lipids cxtracted from the cells indicated that the neoglyeolipids also underwent metabolism to G nt~OS-based analogues. In addition, G ~uOSNHX-CHOL and GMtOSNHCts were degraded to their GM2OS-based analogues, whereas GMzOS-PE was not detected. In contrast, large amounts of 3H were recovered in the medium from cells treated with GMtOS-PE and the label was associated with material that behaved neither as an oligosaccharide or a neoglyeulipid. In the presence of manensin or chloroquine, metabolism of the three neoglycolipids was inhibited. Thus, G .~ttOS-based neoglycolipids were taken up by the cells, internalized and sorted both to the Golgi apparatus (sialylated to G Dt,OS-based analogues) and to lysosomes (hydrolyzed to GM2OS-based analogues). The rate and extent of these processes, however, were strongly influenced by the nature of lipid moiety.
Introduction
for cellular
glycosyltransferases and
glycosidases
[1,5,9,10]. Apparently, some of these metabolic processes
It is well established that exogenous gangliosides added to the culture medium are taken up by cu!tured cells and initially inserted into the plasma taembrane [1-5]. The newly acquired gangliosides can endow the cells with the ability to bind toxins [1,31 and viruses [61, to organize fibronectin into an extracellular matrix [7], and in the case of neurotumor cells to extend neurite-like processes [8]. In addition, the exogenously incorporated gangliosides can undergo metabolism and are substrates
involve internalization of the plasma membrane and intracellular sorting through the Golgi apparatus and lysosomes [9,10]. Recently, we synthesized several neoglycolipids by coupling the oligosaccharide released from G u t to cholesterol, phosphatidylethanolamine and octadecylamine and demonstrated that these neoglycolipids are taken up by GM:deficient rat glioma C6 cells and function as receptors for cholera toxin [ll]. In the present report, we describe the kinetics of incorporation of these neoglycolipids by rat glioma C6 cells and their metabolism.
Abbreviations: GMI, 113Neu5Ac'GgOse4Cer; GDt~. 1v~Ncu5Ac-, ll3NeuSAc-GgOseaCer; GM2. ll3Neu5Ac-GgOse3Cer; GM|OS, Gol~OS and GM2OS, correspondingoligosaccharides: GMIOSNH2 and GDlaOSNH2,correspondingl-amine-l-deoxyglucitolderivatives; PE0phosphatidylethanolamine;CHOL.cholesterol; abbreviationsand structures of neoglycolipidsare shownin Fig, I.
Material and Methods
Correspondence: P.H. Fishman, Park Bldg: Rm. 411, National Inst. Health, Bethesda, MD 20892, U.S.A.
Materials
Cholera toxin was purchased from List Biological Laboratories (Campbell, CA) and for iodination from ICN Biomedicals (Cleveland, OH). Monensin and Fibrio cholerae neuraminidase were from Calbiochem Corp.
154 (San Diego, CA). Bovine testes /~-galactosidase was from Boehringer Mannheim (Indianapolis, IN); crystallized trypsin from Worthington Biochemical Corp. (Freehold, N J); and chloroquine was from Sigma Chemical Co. (St. Louis, MO). NaB3H4 (15 Ci/mmol) was from New England Nuclear (Boston, MA) and NaB3H3CN (3.8 Ci/mmol) was from Amersham Corp. (Arlington Height, IL).
Preparation of neoglycolipids GM~ and Grit ~ were purified from bovine brain [12]. GMIOS and GMIOSNH 2 were prepared as described previously [13]. The same basic methods were used to prepare GDt~OS, reductively aminate it and to purify the GDI,,OSNH2. The only change was to elute the GDI~OS from the Dowex l-X2 column with 0.4 M pyridinium acetate buffer (pH 5.0). GD~OSNH-XCHOL, GDlaOS-PE and GDtaOSNHCIs for standards were synthesized by the procedures detailed for GM1OS derivatives [11] with the following minor modification. The fil;:l reaction mixtures were taken to dryness and partitioned [14]; the lower layers were extracted once with theoretical upper phase and the combined upper phases were taken to dryness on a rotary evaporator (below 30°C). The reaction products then were further purified on silica gel and Sephadex G-25 superfine columns [11]. Tritium-labelled GM1OSNH-X-CHOL, GM1OS-PE and G M10SN H C is were prepared by the same methods used for the unlabelled derivatives. Briefly, GM1OS was reductively aminated using NaB3H4 and the 3H-labeled G ~ , O S N H 2 was used to make the cholesterol derivative. GM~OS was coupled to PE and octylamine via reductive amination in the presence of NaB3H3CN. 3H-labeled GMEOS-PE, GMEOSNH-X-CHOL and GM2OSNHC~s were prepared from the above compounds by removal of the terminal galactose with flgalactosidase as described by Distler and Jourdian [15]. Briefly, each compound (3 • 10' dpm) was dissolved in 0.1 ml of chloroform/methanol (1 : 1, v/v) with 25 #g of Triton X-100. The sample was taken to dryness and the residue was suspended in 50 /tl of sodium citratephosphate buffer (pH 4.3) containing 10 munits of fl-galactosidase. After an overnight incubation at 37 o C, the samples were desalted on Sephadex G-25 superfine columns. The radioactive reaction products were not purified further. Cells and cell culture Rat glioma C6 cells were cultured as described recently [11,13]. Cells were grown in either cluster dishes (24 × 16-ram wells) or 75 cm 2 flasks until confluent (80-100 /~g protein per well or 3-4 mg protein per flask). The cells were washed with phosphate-buffered saline at room temperature and fresh serum-free medium containing 0.1 /tM 3H-labeled neoglycolipids was ad-
ded. The neoglycolipids were stored at - 8 0 ° C in organic solvents and portions were taken to dryness under sterile conditions and dissolved in the medium with brief immersion in an ultrasonic cleaning bath. In experiments with chloroquine (100 p.M) and monensin (5 /~M), the cells were exposed to the drugs 30 rain before any treatment and kept in the presence of the drugs throughout the experiment. For most of the metabolic experiments, the cells were cultured in the presence of the neoglycolipids for 1 or 24 h after which the medium was changed and the cells were incubated in the absence of neoglycolipids for an additional 18 h or as indicated.
Lipid extraction and analysis At the end of an experiment, the cells were washed with ice-cold phosphate-buffered saline, detached with a plastic scraper or released with trypsin (0.05% for 5 min at 3 7 ° C [3]), and centrifuged at 500 × g at 4 ° C for 5 rain. The cell pellets were extracted with chloroform and methanol as described [16]. The lipid extracts were takes to dryness under nitrogen and further purified on small columns of Sephadex G-25 superfine and Unisil [11]. Typically, 0.2 g of Sephadex and 0.15"g of Unisil packed in disposable glass Pasteur pipets were used for lipids extracted from cells isolated from one flask. The purified lipids were analyzed by thin-layer chromatography on precoated silica gel plates (E. Merck, Cat. No. 5725) in solvent system A, chloroform/methanol/0.25% CaCI 2 ( 6 0 : 3 5 : 8 , v/v). The purified lipid extracts also were treated with neuraminidase [17], desalted on Sephadex G-25 [12] and analyzed by thin-layer chromatography. Radioactive material on the chromatograms was detected with a Bioscan System 200 imaging scanner (Bioscan, Washington, DC). Other methods Sialic acid was determined with resorcinol [18]. Carbohydrate compositional analysis following acid hydrolysis of neoglycolipids was by anion exchange chromatography with pulsed amperometric detection [19] as outlined in the Dionex workbook and manual (Dionex Corp., Sunnyville, CA). Oligosaccharides were analysed by thin-layer chromatography using solvent system B, n-propanol/0.1% CaCI2 (6 : 2, v/v). Labeled degradation products in the culture medium were analyzed by gel filtration column chromatography [9] except Bio-Gel P-4 was used. Protein was measured by the method of Lowry et al. [20]. Results
Characterization of neoglycolipids The specific radioactivity of the 3H-labeled GMtOSNH-X-CHOL was 0.67 Ci/mmol, whereas GMIOS-PE was 3.5 C i / m m o l and GMIOSNHCIs was 2.5 C i / m m o l
155
CH20H Gal/It--3GaINAc/I1--4Ga~/]1"O
?
F ~ A(
~OFOH • O CH2" NHV'~O
~
TABLE 11 Rate of uptake of [ ~tl]GmtOSNH-X-CIIOL I~v rat glioma C6 cells and effect on cholera toxin binding Details are the same as those in Table I except the cells were incubated with 0.1 p.M [3HIGMIOSNH-X-CHOL(75000 dpm/well).
o
NeuSAca2 GMIOSNH-X-CHOL
CH2OH Galpl--SGaINAcpl~4Ga1111"0 OH ,
NeuSAco2
CH2--NH--CH2-- CH2--
%o
OH
0~¢x t - v ~ ,
L 0 AA/X,,~VVV~
GMIOS-PE
CH2OH Oalpl--3Ga*NAc/n--4Galpl,
3
t NeuSAca2
J~C_/
Incubation time (mini
C'H2-- NH/V~/~/XAJXAA/X
OH
0 1 5 15 30 60 120 180
[3HIGMtOSNHX-CHOL incorporated (fmol/well)
tzSI-cholera toxin bound (fmol/well)
Ratio =
356+ 13 2025± 57 3797±191 4890±126 6127±237 7719±489 9861 ±430
3.1± 0.9 68.9_+ l.S 397 ± 3.1 873 ± 9.1 1003 _+21 1163 ±21 1 143 ±24 l 122 ± 16
5.4 5.3 4.4 4.9 5.3 6.8 7.6
a Ratio corrected for toxin bound to untreated cells,
GMIOS-NHCI8
Fig. I. Structure and labelling of 13HlGMiOS-based neoglycolipids.
and the location of the 3H is shown in Fig. l. Based on radioscanning after thin-layer c h r o m a t o g r a p h y (Fig. 2, left panels), each a p p e a r e d to be highly homogeneous. Each of the 3H-labeled G u t O S - b a s e d neoglycolipids comigrated with its unlabeled counterpart (Fig. 2, lanes 2). All the [ 3 H ] G u t O S - b a s e d neoglycolipids were susceptible to fl-galactosidase and the corresponding reaction products, p r e s u m a b l y GM2OS-based derivatives, were more mobile on thin-layer c h r o m a t o g r a p h y (Fig. 2, fight panels). T h e difference in mobility was similar to that observed for G m c o m p a r e d to G u t (see standards in lanes 1). In contrast, the unlabeled disialyl derivatives had a slower mobility than the corresponding m o n o -
TABLE I Rate of uptake of [JH].GMtO.~;-PE by rat glioma C6 cells and effect on cholera toxin binding Cells in cluster dishes were incubated in 0.5 ml tff serum-frec medium containing with I p.M [3HIGMtOS-PE(440000 cpm/well} at 37°C for the indicated times. The cells then were washed three times and either assayed for specific t251-eholera toxin binding ll3l or dissolved in 1 M NaOH and analyzed for neoglycolipidincorporation by liquid scintillation spectroscopy. Cell protein was determined and the values were normalized to 100 pg/well. Values are means ± S.D. of triplicate determinations. Incubation time (rain)
I3HI-GM~OS-PE incorporated (fmol/well)
t251-cholera toxin bound (fmol/well)
610± 26 2670± 70 5080+ 105 5265:t: 42 7730±110
2.0± 0.5 168 ± 6.2 489 ± l l 661 +21 822 +10 948 ±23
0 I
5 15 30 60
= Ratio corrected for toxin bound to untreated cells.
sialyl derivatives (Fig. 2, lanes 2) which is consistent with the observed migration of GDt a c o m p a r e d to GMt. C a r b o h y d r a t e composition analyses of Got=OS-based neoglycolipids gave m o l a r ratios of sialic a c i d : g a l a c tose : galactosamine or 2 : 2 : 1 with glucose not exceeding 0.2. U p t a k e o f neoglycolipids by rat glioma C6 cells T h e ['~H]G~tjOS-PF-,, [ ~ H ] G M t O S N H - X - C H O L and [ 3 H ] G M t O S N H C t s were rapidly taken up by rat glioma C6 cells as measured by 3H incorporation and increased t25I-cholera toxin binding (Tables L II and Ill). As each toxin molecule can bind to 5 G M t O S molecules [21], initially most of the incorporated neoglycolipids appeared to he on the cell surface for both derivatives as had been observed earlier for GMt [9]. After 15 rain, however, cells exposed to G MtOSN H C t s began to bind
TABLE
Details are the same as in Table l except the cells were incubated with 0.1 aM [3HIGM~OSNHCla(2780~30dpm/well). Incubation time (rain)
[~HIGMI OSNHCIs incorporated (fmol/well)
Iz~l-cholera toxin bound (fmol/well)
O l 5
3 0 6 + 34 1 5 8 9 ± 259
4.5 ± 0,9 66.6± 2.3 465 ± 19
15 30 60 120 180
3642+ 40 5435± 36 76634- 85 10790-&607 14480± 137
Ratio a
3.7 5.5 7.7 6.4 8.2
111
Rate of uptake of [~H]GMIOSNHCI s by rat glioma C6 cells and effect on cholera toxin binding
557 491 384 382 284
+ 6.4 + 5.6 ± 6.9 ±20 ± 5.9
Ratio corrected for toxin bound to untreated cells.
4.95 3.45
6.5 ll.l 20.0 28.2 51.0
156 less 1251-toxin even though the amount of 3H-label incorporated continued to increase for up to 3 h (Table Ill). As had been found for [3H]GM~ [3], the labeled neoglycolipids appeared to be stably inserted into the cell membrane. When the cells were detached from the culture vessels with 0.05% trypsin, slightly more radioactivity was recovered in the cell pellets than when cells were detached by scraping (unpublished data).
neoglycolipids were purified, resolved by thin-layer chromatography and detected by radioscanning (Fig. 3). In addition to the original neoglycolipids, new radioactive compounds were detected. The slower migrating compounds corresponded to the GDtaOS-based standards and presumably were the result of biosynthetic conversion of the G ~ O S - b a s e d neoglycolipids by the cells. The faster migrating compounds corresponded to the [3H]GM2OS-based standards and presumably resulted from enzymatic hydrolysis of the parental compounds. The quantities of these metabolic products
Analyses of neoglycolipids taken up by rat glioma C6 cells Lipid extracts from cells incubated with 3H-labeled
iA
,I Origin 1
i! i
2 :...,
.......
,, ....
.,.,.;
B
qp
I I
..... 1
l..-.7.,, r~,,,..,
ic
t
l Orig in 2
t,., ,-..~|
l i
I i ::
~
-
A
i
!
J-! .....
~R
lOrigin
Fig. 2. Analysis of unlabeled and 3H-labeled neoglycolipids by thin-layer chromatography and radiochromagraphic scanning• [3HlGMlOS-based neoglycolipids were incubated without (left panels) and with (right panels) .8-galactosidase and (5-10).10 4 dpm of each ware separated by thin-layer chromatography in solvent A along with standard gangliosides (lanes 1 from top to bottom: GM3, GM2, GM1, GOla) and neoglycolipid standards (lanes 2 upper and lower: GMIOS- and GDhOS-based) as described under Materials and Methods. The radiolabelod compounds were detected by radioscanning and the standards detected with orcinol. (A) [3H]GM~OSNH-X-CHOL (left) and its fl-galactosidas¢product (right); lane 2, GMtOSNH-X-CHOL and GD~OSNH-X-CHOL; (B) [3H]GM~OS-PE (left) and its //-galactosidase product (right); lane 2, GM~OS-PE and G.~OS-PE: (C) [3HIGM~OSNHC~s (left) and its .8-galactosidase product (right); lane 2, GMjOSNHC~s. GD~=OSNHCIs and GD~=OS (right above origin).
157 varied for the different neoglycolipids. F o r GMIOS-PE, the biosynthetic p r o d u c t was the major o n e as the b r e a k d o w n p r o d u c t , i.e., GM2OS-PE, was barely detected. In contrast, the d e g r a d a t i o n p r o d u c t s of GM1OSNH-X-CHOL and GMIOSNHCls predominated. F o r the latter neoglycolipid, an additional even faster m i g r a t i n g c o m p o u n d was detected. T h e n a t u r e of this new c o m p o u n d is u n k n o w n ; it is unlikely to be G M 3 O S N H C t s as rat g l i o m a C6 are u n a b l e to breakd o w n exogenously i n c o r p o r a t e d [ 3H ] G M2 [9]. T h e slower m i g r a t i n g radioactive c o m p o u n d s were susceptible to Vibrio cholerae n e u r a m i n i d a s e , whereas the GMIOSb a s e d derivatives a n d the faster m i g r a t i n g c o m p o u n d s were resistant. Effect of chloroquine and monensin B o t h c h l o r o q u i n e (100 F M ) a n d m o n e n s i n (5 F M ) inhibited a l m o s t completely the m e t a b o l i s m of the 3Hlabeled neoglycolipids b y cat g l i o m a C6 cells (Fig. 3). T h e effect o f m o n e n s i n w a s even m o r e p r o n o u n c e d t h a n t h a t o f chloroquine. W i t h either drug, the Go~,,OS- or G M2OS-based c o m p o u n d s were barely detectable. Release of radioactivity into the medium Cells were i n c u b a t e d with 3H-labeled neoglycolipids for 1 h, w a s h e d a n d c u l t u r e d in fresh m e d i u m for 18 h at w h i c h time th~ cultures were analyzed for distribution of radioactivity (Table IV). In the case o f
TABLE tV Distribution of radioactivity in cultures of rat glioma cells treated with 3H-labelled neoglvcolipids Cells in 75 cm2 fla~ks were incubated for I h at 37°C in serum-free medium containing 0.t ,aM of the indicated 3H-labelled neoglycolipid, washed and incubated in fresh medium for t8 h. The cultures then •~ere analyzed for distribution of 3H in the medium, the cellular lipid extract, and the delipidated celt residue. Neoglycolipid
Medium (pmol/flask}
Lipid extract
Cell residue
[3HIG M1OSNH-X-CHOL [3HIG M~OSNHCIs [3HIG MtOS-PE
13.8 12.5 27.9
220.4 213.3 39.2
t.l 1.0 4.3
[ 3 H ] G M I O S N H - X - C H O L a n d [ 3 H ] G M I O S N H C t s , less than 10% of the 3H w a s f o u n d in the culture m e d i u m , over 90% was recovered in lipid extract of the cells, a n d only a trace was f o u n d associated with the cell residue. In contrast, 39% of the 3H w a s recovered in the m e d i u m from cells treated with [3H]GMIOS-PE, 55% w a s f o u n d in the lipid extract a n d 6% w a s associated with the cell residue. This radioactivity in the m e d i u m w a s further analyzed by gel filtration c o l u m n c h r o m a t o g r a p h y (Fig. 4). T h e material w a s r e t a i n e d b y the c o l u m n a n d m o s t of it eluted slightly a h e a d of the G M t O S N H 2 s t a n d a r d . T h e p e a k was freeze-dried a n d analyzed b y thin-layer MONENSIN -
.
.
.
-
. -
GM2OSNH
X
GMIOSNH
x
GD,aOSNH-
Chol
ChoI X
Chol
-. O,,g,n
.
.
.
.
.
.
.~ .
~ .c
-
~GIClOS
BE
.iI 't....... ~ GolaOS-PE
I -
---
_
~
-
GM2OSNH-C,s
~ GM1OSNH -- GD,aOSNH
C18
Cta
-. o , , g . .
Fig. 3. Metabolism of 3H-labelled neoglycolipids taken up by rat glioma C6 ceils: effects of chloroquine and monensin. Cells were incubated with 3H-labelled neoglycolipids in the absence (Control) and presence of either 100 ,aM chloroquine or 5 ,aM monensin as indicated under Materials and Methods. The labeled lipids were extracted front the cells, purified, separated by thin-layer chromatography and detected by radioscanning. Between 5 and 10-104 dpm were applied per lane.
158 15000
~ 10000
5ooo
do
6o
oo
Fractions
Fig. 4. Gel filtration columnchromatographyof radioactive degradation products released to the culture mediumby rat glioma C6 cells incubated with [3H]GMIOS-PE.Cells were incubated with [3HIGM: OS-PE for 1 h. washedand incubated in freshserum-freemediumfor 18 h. The latter medium was freeze-dried, dissolved in 50 mM ammoniumbicarbonate(pH 7.8) and a portion(105cpm)applied to a Bio-Gel P-4 column whichwas eluted with the same solution. Fractions were collected and counted for 3H. The column was calibrated with bluedextran(B.D.),G MIOSNH2 and glucose(GIc).
chromatography in both solvents systems A and B. Upon radioscanning, the major peak of radioactivity was detected at the origin in both solvent systems. Thus, it was unlikely to be either a glycolipid or a simple oligosaccharide. One possibility is that it is a phosphoor phosphoglycerol-oligosaccharide metabolic product of [3HIG M~OS-PE. Discussion We found that G MiOS-based neoglycolipids were rapidly taken up from the medium by rat glioma C6 cells and initially inserted into the plasma membrane. These conclusions were supported by the following observations: (1) The 3H-labeled neo~;lycolipids were not released from the cells by trypsin. We had demonstrated previously that [3H]GMt and a [3H]GMt derivative in which the fatty acid had been replaced by acetate also were not released from rat glioma C6 ells by trypsin [3]. Others have reported that gangliosides taken up by cultured cells are in both trypsin-sensitive and -resistant states [2,4,8]. In most of these studies, however, the gangliosides are added in medium containing serum and serum proteins may be binding the gangliosides and preventing their insertion into the membrane [22]. In all of our studies, we exposed the cells to gangliosides (or neoglycolipids) in serum-free medium: and (2) When the cells were exposed to the neoglycolipids, they rapidly acquired new cholera toxin binding sites. Initially, the molar ratio of neoglycolipid incorporated to toxin bound was close to the theoretical ratio of 5:1 [21]. This is consistent with most of the neoglycolipid molecules being on the outer leaflet of the plasma membrane bilayer and mobile in the plane of the membrane in
order to permit multivalent binding of the toxin [23]. We have shown previously that the affinity of cholera toxin for neoglycolipids inserted into C6 membranes is the same as that for inserted GM~ and consistent with a single class of high affinity binding sites [11]. Although the neoglycolipids initially were inserted into the plasma membrane, with time they appeared to undergo internalization. For [~H]GMtOSNH-X-CHOL and [3H]GMIOS-PE, the ratio of neoglycolipid to toxin bound slowly increased with time In contrast, when cells were exposed to [3H]GMtOSNHCts, the ratio increased rapidly and the number of toxin binding sites actually decreased even though the amount of cell-associated [3H]GMtOSNHCIs continued to increase. The basis of this phenomenon is not clear. Large amounts of GMIOSNHCI~ within the plasma membrane may stimulate rapid and possibly selective endocytosis in order to clear from the cell surface this potentially toxic substance [24]. It is unlikely that it was due to the rapid metabolism of [3H]GMtOSNHC1s as no metabofic products were detected after a 2-h incubation. Formation of [ ~H]G M2OSN HCts was detected after 4 h, but it represented less than 20% of the [3H]Gr~tOSNHCms extracted from the cells (unpublished data). It has been reported by this laboratory [1,9] as well as by others [5,10] that exogenous gangliosides are metabolized by cultured cells, being converted to both less complex and more complex gangliosides. Presumably, the former are due to the action of cellular glycohydrolases located in lysosomes and the latter to that of glycosyltransferases located in the Golgi apparatus. We found that neoglycolipids incorporated into rat glioma C6 cells also were converted into both more complex and less complex analogues of the parent compounds. The more complex products had the same mobility on thin-layer chromatograms as the Go~aOS-based standards and were susceptible to Vibrio cholerae neuraminidase. The less complex products derived from [aH]GMIOSNH-X-CHOL and [3H]GMtOSNHCts corresponded to the GMzOS-based standards and were the major metabolic products as had been observed for [3H]GMt taken up by rat glioma C6 cells [9]. In contrast, no [3H]GM2OS-PE was detected in cultures treated with [3H]GMtOS-PE even though the latter compound was hydrolyzed by fl-galactosidase in vitro (see Fig. 2). A substantial amount of 3H was released into the culture medium which indicated that the [3H]GMIOS-PE was metabolized by the cells. One possibility is that the major degradative pathway is via phospholipases and not glycohydrolases. The effects of monensin and chloroquine provide further support that the neoglycolipids are being internalizcd, then undergoing intracellular sorting and finally being metabolized. Monensin has been shown to inhibited the biosynthesis of endogenous gangliosides in
159 cultured cells [25-27] a n d is k n o w n to disrupt vesicular t r a n s p o r t through the Golgi a p p a r a t u s [28]. C h l o r o q u i n e causes alkalization of lysosomes a n d thus inhibits lysospinal hydrolases [29]. Both c h l o r o q u i n e a n d m o n e n s i n also inhibit e n d o s o m a l acidification a n d thus m a y interfere with n o r m a l intracellular vesicular trafficking ]30]. In the presence of these drugs, the internalized [3H]Gm~OS-based neoglycolipids m a y be u n a b l e to be sorted either to the Golgi or the lysosomes. U s i n g a fluorescent PE analog, Sleight a n d P a g a n o d e m o n s t r a t e d t h a t the a n a l o g is initially i n c o r p o r a t e d into the o u t e r layer of the p l a s m a m e m b r a n e of cultured cells a n d then u n d e r g o e s internalization at 3 7 ° C to internal m e m b r a n e s by two s e p a r a t e processes [31]. Delivery of the a n a l o g to the G o l g i a p p a r a t u s involves endoeytosis, whereas delivery to the nuclear envelope a n d m i t o c h o n d r i a involves a r a p i d t r a n s b i l a y e r movem e n t of the fluorescent PE across the p l a s m a m e m b r a n e . A r o u n d 40% of the fluorescent PE is internalized b y 30 rain a n d the a n a l o g is rapidly d e g r a d e d by the cells with a half-life of 1 h. In contrast, G M t O S - P E is internalized very slowly with a half-life of a r o u n d 3 h [11] a n d b a s e d o n o u r present results, a p p e a r s to be d e g r a d e d very slowly. W i t h its large, negatively c h a r g e d h y d r o p h i l i c h e a d g r o u p , G M I O S - P E is unlikely to undergo transbilayer movement. T h e u p t a k e of exogenous cholesterol by cultured cells is even m o r e complex. T h e most studied p a t h w a y involves the r e c e p t o r - m e d i a t e d e n d o c y t o s i s of low density lipoproteins [321. L a n g e a n d Matthies [33] reported t h a t w h e n a d d e d to lipoprotein-deficient m e d i u m in tracer a m o u n t s , r a d i o l a b e l e d cholesterol is rapidly t a k e n u p be c u l t u r e d h u m a n fibroblasts. O v e r 95% is associated with the o u t e r layer o f the p l a s m a m e m b r a n e a n d there is n o significant t r a n s f e r into the cells or esterification for up to 16 h at 3 7 ° C (less t h a n 0.5%). Interestingly, w h e n carrier cholesterol (25 p,M) was a d d e d to the m e d i u m , s o m e of the e x o g e n o u s cholesterol a p p e a r e d to u n d e r g o e n d o c y t o s i s a n d esterification. In contrast, we f o u n d t h a t w h e n rat g l i o m a C6 are i n c u b a t e d with 0.1 p.M G M I O S N H - X - C H O L for 1.5 h, w a s h e d a n d i n c u b a t e d in fresh m e d i u m , the derivative is internalized with a half-life of 2 h [11]. This is consistent with the increase in the ratio o f neoglyeolipid i n c o r p o r a t e d to toxin b o u n d t h a t o c c u r r e d with time of i n c u b a t i o n (Table IlL W e also observed that over 50% of the [ 3 H I G M t O S N H - X C H O L taken u p b y the cells u n d e r w e n t m e t a b o l i s m to m o r e a n d less c o m p l e x derivatives b y 18 h. W e c a n d r a w the following c o n c l u s i o n s that f r o m o u r studies: (i) Despite the differences in lipid moieties, the G m ~ O S - b a s e d neoglycolipids initially are inserted a n d o r g a n i z e d in the outlet leaflet o f the p l a s m a m e m b r a n e in a m a n n e r that s u p p o r t s multivalent b i n d i n g of cholera toxin a n a l o g o u s to GM~; (2) Despite the differences in lipids, they gain access to a n d are substra,,:s /or C M P sialic acid : G ml sialyltransferase, a Golgi e n z y m e [12,341
present in rat glioma C6 cells [9], a n d for two of the three neoglycolipids, lysosomal fl-galactosidase [29]; (3) The lipid moieties strongly influence the rate of internalization a n d possibly their s u b s e q u e n t sorting. T h i s was p a r t i c u l a r l y evident for [ 3 H I G M t O S N H - X - C H O L a n d [3H]GM~OSNHC~s w h i c h were taken u p b y the cells at similar rates, were metabolized to b o t h m o r e a n d less complex analogues, a n d h a d almost identical distributions of radioactivity i n t o the m a j o r pools of medium, lipid extract a n d delipidated cell residue (see T a b l e IV). Yet, [ 3 H ] G M t O S N H C l a w a s m u c h m o r e rapidly internalized t h a n [ 3 H | G M t O S N H - X - C H O L based o n cholera toxin binding. In contrast, 13H]GMt OS-PE, which w a s t a k e n u p a n d internalized at a rate similar to [ 3 H ] G M I O S N H - X - C H O L , w a s not d e g r a d e d to GM2OS-PE a n d its d i s t r i b u t i o n of radioactivity was distinct f r o m the o t h e r two neoglycolipids, with a large fraction a p p e a r i n g in the m e d i u m . P r e s u m a b l y , this reflects a different p a t h w a y of intraeellular sorting. References I Fishman. P.H., Moss, J. and Vaughan, M. (19761 J. Biol. Chem. 251, 4490-4494. 2 Callies, R., Schwarzmann, G,. Rad~k. K., Siegen, R. and Wiegandt, H. (1977) Eur. J. Biochem. 80, 425-432, 3 Fishman, P.H., Pacu~ka, T., Horn, B. and Moss. J. (19801J. Biol. ('hem. 255, 7657-7664. 4 Schwarzmann. G., Hoffman-Bleihauer, P., Schubert, J., Sandhoff, K. and Mar~.h, D. (19831 Biochemistry 22, 5041-5048. 5 Masserini. M., Guiliani, A., Palestini, P., Acquoni, D., Pillo, M., Chigorno, V, and Tettamanti, G. (1990) Biochemistry 29, 69"/-701. 6 Markwdl, M.A.K.. Moss, J., Horn, B.E., Fishman, P.H. and Svennerholm, L. (1986) Virology 155, 356-364. 7 Spiegel, S., Yamada. K.M, Horn, B.E., Moss, J. and Fishman, P.H. (19851J. Cell. Biol. 100. 721-726. 8 Fact:i, L., Leon, A., Toffano, G. Sonnino, S., Ghidoni, R. and Tettamant. G. (19841 J. Neurochem. 42, 299-305. 9 Fishman, P.H., Bradley, R.M., Horn, B.E. and Moss, J. (1983) J. Lipid Res. 24, lOOT-lOll. 10 Sonderfeld. S., Conzelmann, E.. Sch,.varzmann, G., Burg, J.. Hinrichs, U. and Sandhoff. K. 11985) Eur. J. Biochem. 149, 247-255. II Pacuszka, T., Bradley, R.M. and Fishman, P.H. (1991) Biochemistry 30, 2563-2570. 12 Pacuszka, T.. Dullard, R.O., Nishimura, R.N.. Brady, R.O. and Fishman, P,H. (19781J. Biol. Chem. 253, 5839-5846. 13 Pacuszka, T. and Fishman. P.H. (19901 J. Biol. Chem. 265, 76737678. 14 Folch-Pi, J,, Lee:,. M. and SIoan Stanley. G.G, (19571 J. Biol. (-'hem. 226, 497 509. 15 Distler, JJ. and Jourdian, G.W. (19731 J. Biol. Chem. 248, 67726780. 16 Miller-Podraza, H.. Bradley, R.M. and Fishman, P.H. (19821 Biochemistry 21. 3260-3265. 17 Mullin, B.R.. Pacuszka, T., Lee, G., Kohn. UD., Brady, R.O. and Fishman, P.H. (1978) Science 199, 77-79. 18 Svennerholm. L. and Fredman. P. (1990) Biochim. Biophys. Acta 671.97-109. 19 ['lardy, M.R. (19891 Methods Enzymol. 179, 76-82. 211 Lowry, O.H., Rosebough, N.J., Farr. A.L. and Randall, R.J, (1951) J. Biol. Chem. 193, 265-275.
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