Acylation of Exogenous Glycosylsphingosines by Intact Neuroblastoma

THE JOURNAL OF BIOLOGICAL CHEMLWRY Vol. 265, No. 36, Issue of December 25, pp. 22217-22222,199O Q 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Prrnted m U.S.A.

Acylation of Exogenous Glycosylsphingosines by Intact Neuroblastoma (NCB-20) Cells*

(Received for publication, April 20, 1990)

Robert G. Farrer and Glyn DawsonS From the Departments of Pediatrics, Biochemistry, and Molecular Biology, Joseph P. Kennedy, Jr. Mental Retardation Research Center, University of Chicago, Chicago, Illinois 60637

Acylation of exogenously added galactosylsphingosine was demonstrated in intact NCB-20 neuroblastoma cells, a cell line that normally does not synthesize galactosylceramide. Labeling of cells with [3H] palmitic acid for 6 h in the presence of 100 pM exogenous galactosylsphingosine (GalSph) resulted in a more than 3-fold increase in the incorporation of label into the ceramide monohexoside fraction relative to controls. This increase, which was almost entirely due to the incorporation of labeled nonhydroxy fatty acid into galactosylceramide, was linear over a concentration range of l-100 MM galactosylsphingosine and for the first 5 h after the addition of galactosylsphingosine. Similarly, the addition of 100 pM glucosylsphingosine resulted in a 3-fold increase of label incorporated into glucosylceramide. Incubation of cells with 100 pM GalSph and labeled fatty acids of various chain lengths revealed that the acylation of GalSph was specific for medium chain (C16-C18) nonhydroxy fatty acids, suggesting that this was an enzyme-mediated reaction. The enzymatic nature of GalSph acylation was further demonstrated when cells were incubated for 72 h with 15 pM [3H]galactosylsphingosine labeled in the galactose moiety. [3H]Galactosylceramide containing only medium chain non-hydroxy fatty acids accumulated linearly with time reaching a maximum at 48 h and was observed to be further metabolized to ceramide dihexoside. This acylation reaction may be potentially important for the removal of glycosylsphingosines in the cell.

Evidence that cells can generate lysosphingolipids is suggested by the presence of endogenous lysosphingolipids in a number of tissues and cell types. For example, free sphingosine has been detected in rat liver (l), porcine epidermis (2), several murine tissues (3), and in human neutrophils (4) and promyelocytic leukemic cells (5). Glycosylated forms of sphingosine (lysoglycosphingolipids) have been reported to be present at low levels in normal mouse spinal cord (6), human brain (7), and in a human epidermoid carcinoma cell line (8). The most dramatic evidence of intracellular lysosphingolipid generation, however, is seen in cases of inherited sphingolipid

* This work was supported in part by United States Public Health Service Grant HD-06426, a grant from the Multiple Sclerosis Society, and Developmental Biology Training Grant HD-07136 postdoctoral fellowship award (to R. G. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed: Dept. of Pediat- rics, Box 82, University of Chicago, 5841 S. Maryland Ave., Chicago, IL 60637. Tel: 312-702-6430.

storage diseases where severalfold accumulations of these compounds have been observed (9). For example, galactosylsphingosine has been found in elevated levels in humans (10, 11) and mice (6, 11) with an inherited deficiency of galactosylceramide$-galactosidase activity. A similar accumulation of glucosylsphingosine in Gaucher tissue (12, 13) and lyso- GM2* in Tay-Sach’s brain (14) has also been reported.

Although the mechanism(s) of formation and functional significance of intracellular lysoglycosphingolipids have yet to be determined, it is possible that glycosylated forms of sphingosine may serve as intermediates in minor biosynthetic pathways. Recently, an alternative explanation for their presence was suggested by studies showing that sphingosine and other long chain bases could act as potent inhibitors of protein kinase C both in vitro and in intact cells (see Refs. 15 and 16 for recent reviews). These studies, together with the findings that lysosphingolipids can also modulate protein kinase C independent events (17-22), suggest that lysosphingolipids may serve a regulatory function in the cell. However, in order for lysosphingolipids to act as intracellular modulators of enzymes located in subcellular compartments such as the plasma membrane, mechanisms for their rapid generation and removal must exist in these compartments. Other than the nonlysosomal formation of sphingosine from ceramide that has been suggested by in vitro studies with various rat tissues (23, 24), there is little evidence for such mechanisms.

One mechanism by which intracellular lysosphingolipids may be rapidly removed is by direct acylation to N-acylsphin- gosines. In view of the current interest in the regulatory properties of the lysosphingolipids, we have taken the ap- proach of examining this aspect of the metabolism of simple lysoglycosphingolipids by intact cells. In this paper, we report the enzymatic acylation of exogenously administered glycosylsphingosines by the neuroblastoma cell line NCB-20.

EXPERIMENTAL PROCEDURES

Materials-[9,10-“H]Palmitic acid (60 Ci/mmol) was purchased from Du Pont-New England Nuclear; [S,IO”H]myristic acid (48 Ci/ mmol) and [5,6,8,9,11,12,14,15-3H]arachidonic acid (201.5 Ci/mmol) were obtained from Amersham Corp.; [l-“Clstearic acid (55 mCi/ mmol) was from American Radiolabeled Chemicals, Inc. (St. Louis, MO); [‘%]lignoceric acid (57 mCi/mmol) was a generous gift of Dr. Inderiit Sinah. Universitv of South Carolina Medical School. Charles- ton, SC. Galactosylsphingosine, glucosylsphingosine, and &labeled fatty acids were purchased from Sigma. The two glycosylsphingosines each gave a single orcinol and fluorescamine positive band when chromatographed on borate-impregnated silica gel G TLC plates in a solvent system of chloroform/methanol/water (100:42:6, v/v). Cell

1 The abbreviations used are: GM,, I13NeuAcGgOse&er; CMH, ceramide monohexoside; CDH, ceramide dihexoside; GalSph, galactosylsphingosine; GalCer, galactosylceramide; GlcSph, glucosylsphingosine; GlcCer, glucosylceramide; TLC, thin layer chromatography; EMS, Matalon’s modified Eagle’s medium.

22217

22218 Acylation of Glycosylsphingosines

culture media (EMS) and fetal calf serum (FCS) were from GIBCO. Analytical TLC plates (silica gel 60) were from Whatman.

Cell Culture-Mouse neuroblastoma x 18 day Chinese hamster embryo brain explant hybrid cell line NCB-20, obtained from Dr. John Minna, VA Hospital, Washington, D. C., were grown in EMS supplemented with 5% FCS and 60 /*g/ml gentamycin. Cells (passage number between 5 and 20) were cultured as monolayers on Costar 6 well plastic tissue culture dishes.

Metabolic Labeling and Glycosylsphingosine Treatment of CelLs- Cells were incubated in 1 ml of EMS containing 5% FCS and 2 &i of [“H]palmitic acid. For determining fatty acid specificity, the cells were incubated with 25, 50, and 100 gM concentrations (constant specific activity) of [“Hlmyristic, [“Clstearic, [RH]arachidonic, or [ ‘“Cllignoceric acids. Glycosylsphingosines were added to the cultures in 10 ~1 of 50% ethanol to give the desired final concentration. Control cultures received 10 ~1 of 50% ethanol. After 6 h the incubations were stopped by removing the cells from the culture dish and placing on

GlcCer

GalCer

A B C NCB

FIG. 1. Absence of GalCer in neutral lipid extracts from NCB-20 cells. Neutral lipids were extracted from cells as described under “Experimental Procedures” and separated on borate-impregnated TLC plates with the solvent system chloroform/methanol/ concentrated ammonia (65:25:5, v/v). Glycolipids were visualized with orcinol spray. Standard lanes: A, neutral glycosphingolipids; B, GlcCer; C, GalCer.

CDH _

ice. Cells were collected by centrifugation and washed twice with cold phosphate-buffered saline. The final washed cell pellets were stored at -20 “C until further analysis.

For [“Hlgalactosylsphingosine feeding experiments, cells were incubated with 1 ml of EMS containing 1% FCS to slow cell prolifera- tion during the course of the experiments. [3H]Ga1actosy1sphingosine, diluted with unlabeled galactosylsphingosine to a specific activity of 38,480 dpm/nmol, was added to the culture media to give a final concentration of 15 pM (0.26 &i/ml).

Lipid Extraction and Sphingolipid Isolation-Cell pellets were sus- pended by sonication (Heat Systems-Ultrasonics, mode1 W185) in 200 ~1 of water, and the protein content of each suspension was determined by the method of Lowry et al. (25) with bovine serum albumin as standard. The remaining cell sonicate was extracted with 2 ml of chloroform/methanol (1:1, v/v), and the resulting pellet was washed with 1 ml chloroform/methanol (l:l, v/v). Neutral glycosphingolipids were isolated by phase partitioning (26) combined with alkaline methanolysis to destroy alkali-labile glycerophospholipids. For [3H]GalSph feeding experiments, the pH of the upper phase was adjusted to 9.0 with concentrated NH,OH to allow complete recovery of GalSph in the lower phase.

Neutral glycosphingolipids were separated by TLC on lo-cm silica gel G plates developed in the indicated solvent systems. Borate- impregnated TLC plates were prepared as described by Igisu et al. (27). Fluorographic analysis of labeled sphingolipids separated on TLC plates was performed by spraying the plates with ENRHANCE (Du Pont-New England Nuclear) and exposing to X-Omat AR 2 film (Kodak). Radioactivity was quantitated by scintillation counting of the individual bands scraped from the TLC plate.

Preparation of fH]Galactosylsphingosine-[3H]Galactosylceram- ide, labeled by the galactose oxidase-sodium [3H]borohydride proce- dure, was generously supplied by Dr. A. Horwitz (University of Chicago). Removal of the amide-linked fatty acids from the [3H] galactosylceramide was accomplished by alkaline degradation essen- tially by the method of Taketomi and Yamakawa (28). Briefly, the [“Hlgalactosylceramide was hydrolyzed in 1 N KOH in 90% l-butanol for 2 h at 117 “C, and the resulting hydrolysate was partitioned with an equal volume of water. The butanol phase was evaporated and subjected to Unisil column chromatography to separate the unreacted [“Hlgalactosylceramide from the [3H]galactosylsphingosine. [3H]Ga- lactosylsphingosine was quantitated by the fluorescamine method of Higgins (29). The final specific activity of the purified [3H]ga1acto- sylsphingosine was 61,198 dpm/nmol.

RESULTS

Endogenous Neutral Glycosphingolipids of NCB-20 Cells- To show that NCB-20 cells do not normally synthesize galactosylceramide (GalCer), we extracted the neutral glyco-

;GlcCer \

GalCer /

A 0 1 2

STDS

FIG. 2. Increased [3H]palmitic acid incorporation into CMH by intact NCB-20 cells during treatment with GalSph. Cells were incubated for 6 h in medium containing 2 rCi/ml [3H]palmitic acid in the presence or absence of 100 pM GalSph. Neutral glycosphingolipids were isolated and separated by TLC with the solvent system chloroform/methanol/water (100:42:6, v/v). Visualization of labeled sphingolipids by fluorography from GalSph treated (lane 1) and control (lane 2) cells shows increased labeling of CMH (arrow). Standard lanes: Aneutral glycosphingolipids; B, GalCer. Standards were visualized with orcinol reagent.

I : + ,

1 2 3 A B C

STDS

FIG. 3. Qualitative fluorographic determination of labeled ceramide monohexoside species. CMH bands from TLC separa- tions of neutral glycosphingolipid were scraped, eluted, and reapplied to a borate-impregnated TLC plate which was developed three times with chloroform/methanol/water (144:25:2.8, v.v). Lane I, CMH from [“Hlpalmitic acid-labeled control cells; lane 2, CMH from cells labeled with [:‘H]palmitic acid in the presence of 100 pM GalSph; lane 3, CMH from cells incubated with [“H]GalSph. Standard (STDS) lanes: A, GalCer; B, hydroxy fatty acid GalCer (phrenasin); C, GlcCer. Standards were visualized with orcinol reagent.

Acylution of Glycosykphingosines 22219

i

01 0 1 2 3 4 5 6

time (h)

1 3

Oi 0 10 20 30 40 50 60 70 80 90100

GalSph (/A)

FIG. 4. Time course and concentration curve of label accumulation in CMH of cells treated with GalSph. Cells were incubated in medium containing 2 pCi of t3H]palmitic acid either in the presence (filled circles) or absence (filled square) of 100 PM GalSph for increasing times up to 6 h (A) or with increasing concentrations of GalSph for 6 h (B). Neutral glycosphingolipids were isolated and separated by TLC as described in Fig. 2. CMH bands were scraped from the TLC plates and radioactivity quantitated by scintillation counting. Each point represents the mean + S.D. from triplicate determinations. (Error bars are too small to be seen at low concentrations).

TABLE I

Effect of 100 ELM GalSph treatment on PH]palmitic acid incorporation into neutral glycosphingolipids

Cells were incubated for 6 h in medium containing 2 &i/ml [3H] palmitic acid either in the absence (control) or presence (treated) of 100 fiM GalSph. Neutral sphingolipids were isolated as described under “Experimental Procedures” and separated by TLC in the solvent system chloroform/methanol/water (100:42:6, v/v). Radioac- tivity was quantitated by scintillation counting of the individual bands scraped from the TLC plate. Values are means f S.D. from triplicate determinations. CTH, ceramide trihexoside.

Glycosphingolipid Control Treated Percent

CMH CDH CTH

dpm/mg protein 10,148 f 1,535 33,936 + 2,375 334

4,998+424 7,705 t- 1,554 154 170,307 + 6.610 179,339 f 30.264 105

Globoside 45,202 f 6;216 52,860 + 4,412 117

sphingolipids from these cells and chromatographed this lipid fraction on a borate-impregnated TLC plate. Orcinol staining of the TLC plate shows no positive bands in the GalCer region (Fig. 1). Similar extraction and separation of neutral glycosphingolipids from cells labeled for 24 h with [3H]galactose showed no labeled GalCer bands upon fluorographic analysis (not shown). We therefore could use this cell line to measure acylation of exogenously added GalSph without de nouo synthesis contributing to the labeling of GalCer.

Effect of Exogenous Glycosylsphingosines on PHJPalmitic Acid Zncorporation into Neutral Glycosphingolipids-When

0 0 Cl 3 0 2 4T

J - T g m’ a

FIG. 5. Effect of exogenously added GlcSph on [3H]palmitic acid incorporation into GlcCer by intact NCB-20 cells. Cells were incubated for 6 h in medium containing 2 &X/ml [3H]palmitic acid either in the absence or presence of 100 PM GlcSph. Neutral glycosphingolipids were separated on borate-impregnated TLC plates with the solvent system chloroform/methanol/water (100:42:6, v/v). GlcCer bands were scraped and radioactivity quantitated by scintillation counting. Bars represent mean it S.D. from triplicate determinations.

incubated for 6 h in media containing [3H]palmitic acid and 100 WM GalSph, NCB-20 cells remained intact to trypan blue and incorporated 334% more radioactivity into the ceramide monohexoside (CMH) fraction (GalCer + glucosylceramide (GlcCer)) relative to controls (Fig. 2). To show that this increase was due to the acylation of GalSph rather than increased GlcCer synthesis, the CMH band was eluted and rechromatographed on borate-impregnated TLC plates with chloroform/methanol/water (144:25:2.8, v/v) to separate the various species of CMH as described by Morel1 and Radin (30). The resulting fluorogram (Fig. 3, lanes 1 and 2) demonstrates the presence of labeled GalCer containing nonhydroxy medium chain (Cl6-C18) fatty acids after incubation of the cells with exogenous GalSph (Fig. 3, lane 2). There was no detectable label in a-hydroxy GalCer or long chain (X22) GalCer. Control NCB-20 cultures showed detectable labeling only in GlcCer (Fig. 3, lane I), confirming that these cells do not synthesize GalCer.

We examined the behavior of this reaction in intact cells with time and increasing concentrations of exogenous GalSph. The increased incorporation of radioactivity from [3H]palmitic acid into CMH was linear for at least the first 5 h after the addition of 100 PM GalSph (Fig. 4A) indicating that the exogenously added GalSph was readily and continually made available to the acylating system within the cell, possibly reaching a plateau after 5 h. Formation of CMH (GalCer) was also linear upon exposure of the cells to increasing concentrations of GalSph (Fig. 4B).

To determine if exposure of the cells to 100 pM GalSph had an effect on the overall synthesis of endogenous glycosphingolipids, the incorporation of [3H]palmitic acid into other neutral glycosphingolipids of the lower phase extract was examined. Of the four most prominent identifiable neutral glycosphingolipids labeled with [3H]palmitate, only CMH and ceramide dihexoside (CDH) showed increased incorporation of radioactivity (330 and 150%, respectively) (Table I). We

22220 Acylation of Glycosylsphingosines

FE. 6. Fluorographic analysis of il

the acylation of exogenously added .A”6”.

GalSph by intact cells with [3H]my- 94m ristic, [“‘Clstearic, [3H]arachidonic, and [‘%]lignoceric acids. Cells were incubated for 6 h in the absence (lanes m’ C = control cells) or presence (lanes T = treated cells) of 100 jtM GalSph and d r -G- T‘ T T ( , , . SC T C T C T three concentrations (2550, or 100 pM) 25 50 100 PM A B C 25 50 100 PM of each of the following labeled fatty acids: [“H]myristic acid (71,040 dpm/

(3HIMyristiC acid Stds [lH]Arachldonic acid

nmol), [Wlstearic acid (28,809 dpm/ nmol), [“Hlarachidonic acid (111,000 dpm/nmol), or [Wllignoceric acid (24,864 dpm/nmol). Constant specific radioactivity was maintained when pre- paring the three concentrations of each fatty acid. Standard lanes are: A, GalCer; R, GlcCer; C, neutral glycosphingolipids. Standard were visualized with orcinol reagent. The arrow designates the location of medium chain fatty acid GalCer

-------

in the [“Clstearic acid panel. _I

r?umm -~cyu!3~~~

25 50 loo PM A s C 25 50 100 PM [wC]Stearic acid Stds ktCILignoceric acid

have shown above that the increase in CMH labeling was due NCB-20 cells, then the acylation reaction should not exhibit to GalCer formation rather than GlcCer synthesis, and we have evidence suggesting that the increase in CDH radioactivity is due to the subsequent conversion by the cell of the newly formed GalCer to a more complex glycolipid co-migrat- ing with lactosylceramide (see [“H]GalSph feeding experiments below). Labeling of the more complex glycolipids, ceramide trihexoside and globoside, showed no differences when compared with control values. These results indicate that the presence of GalSph and its acylation to GalCer does not interfere with fatty acid incorporation into other sphingolipids.

Similar to the acylation of exogenously added GalSph, the addition of 100 pM glucosylsphingosine (GlcSph) to NCB-20 cells incubated with [“Hlpalmitic acid resulted in a 3-fold increase (309% of control) of label incorporated into GlcCer (Fig. 5). Therefore, the lysoglycosphingolipid acylation mechanism is functional in intact neurotumor cells with at least two, albeit closely related, glycosphingolipids.

Zncorporation of Labeled Fatty Acids of Various Chain Lengths into Exogenous GalSph by NCB-20 CelLs-Hammar- Strom (31) claimed that under in vitro conditions, acylation of GalSph would occur through a nonenzymatic transfer of the fatty acid from acyl-CoA to the primary amine of the lysosphingolipid. If this type of mechanism was responsible for the acylation of the exogenously added GalSph by intact

any sort of fatty acid specificity. To determine this, we incubated cells in media containing 100 pM GalSph and either [3H]myristic acid (14:0), [“‘Clstearic acid (l&O), [3H]arachidonic acid (20:4), or [‘4C]lignoceric acid (24:0) and examined label incorporation into GalCer. Incorporation of label into phospholipids was also determined as a measure of the uptake and utilization of the four fatty acids by the cells. For each fatty acid, substantial amounts of label were incorporated into phospholipids (not shown) especially phosphatidylcholine, indicating that the exogenously supplied fatty acids had been taken up by the cells and become available intracellularly for utilization in normal biosynthetic processes.

Fluorographic analysis of neutral glycosphingolipids separated by TLC on borate-impregnated plates revealed that only [14C]stearic acid and [Yllignoceric acid were incorporated into glycosphingolipids (Fig. 6). However, [i4C]stearic acid was the only fatty acid incorporated into GalCer when GalSph was present (see arrow in Fig. 6). Increasing the concentrations of the labeled fatty acids in the medium from 25 to 100 pM (a concentration range similar to what would be found for palmitic acid in medium containing 5% FCS) yielded greater label incorporation into phospholipids for all four labeled fatty acids and into glycosphingolipids for [‘4C]stearic acid and [“Cllignoceric acid, yet, even at the highest fatty acid concentration, radioactivity was only detected in GalCer with

Acylation of Glycosylsphingosines 22221

Q*IC*r

CDH

04 : : : I : : ! 0 1’0 20 30 40 50 60 70 80

time (h)

FIG. 7. Acylation of exogenously added [3H]GalSph by intact NCB-20 cells. Cells were incubated in medium containing 15 pM [“H]GalSph (38,480 dpm/nmol) for the indicated times. Neutral glycosphingolipids were extracted and separated by TLC with the solvent system chloroform/methanol/water (100:42:6, v/v). A, fluorogram of the TLC plate showing the formation of [3H]GalCer and [:‘H]CDH with time. Standard lanes: A, [3H]GalSph; B, [3H]GalCer. B, quantitation of [3H]GalCer formation determined by scraping the bands from the TLC plate and scintillation counting. All points represent the mean + SD. from triplicate determinations.

[ Vjstearic acid. Thus the acylation of GalSph by intact cells appears to be specific for medium chain (C16-C18) fatty acids, suggesting that the reaction is enzyme mediated.

fH]GalSph Feeding Experiments-To further demonstrate that GalCer formation was the result of direct acylation of the exogenous GalSph, we synthesized [3H]GalSph labeled in the galactose moiety, fed it to cells at a concentration of 15 pM, and followed the formation of [3H]GalCer over a 72-h incubation period. Quantitation of the labeled CMH fraction showed that the formation of [3H]GalCer was linear for the first 48 h (Fig. 7) with an average rate of formation of approximately 10 pmol/mg/h. Between 48 and 72 h, an ap- parent steady state had been reached, presumably between the formation and degradation of [3H]GalCer by the cells. Elution of the [3H]GalCer band from the TLC plate and rechromatographing on a borate-impregnated TLC plate in the solvent system chloroform/methanol/water (144:25:2.8, v/v) showed that the [“H]GalCer formed by the cells was almost exclusively the nonhydroxy medium chain fatty acid form of GalCer (Fig. 3, lane 3), similar to what was found with [“Hlpalmitic acid labeling of the cells treated with unlabeled GalSph.

In addition to the formation of [3H]GalCer from [“HI GalSph, we also noticed the appearance with time of a labeled band that we have tentatively identified as CDH (Fig. 7A) due to its comigration with authentic lactosylceramide in two different solvent systems (not shown). This observation sug- gests that some of the newly formed [“H]GalCer was further metabolized to a more complex glycolipid.

DISCUSSION

The development of sensitive methods for the measurement of lysosphingolipids has provided evidence for the existence

of low concentrations of lysoglycosphingolipids in tissues and cells (6-8). If these compounds serve functional roles, either as biosynthetic intermediates or as transiently generated modulators of enzymes such as protein kinases, then enzymatic mechanisms for their generation and removal must be present in the cell. In this paper, we have presented evidence for the cellular metabolism of glycosylsphingosines to glycosylceramides by intact NCB-20 cells. This reaction may be relevant to the physiologic removal of glycosylsphingosines within the cell.

Although cells may generate low levels of lysosphingolipids, it has been technically difficult to demonstrate any metabolic conversion (acylation) of these endogenous compounds by the cell because of interference from de nouo biosynthetic pathways. For this reason, we chose to study the acylation of exogenous GalSph by the NCB-20 cell line which does not synthesize GalCer, the end product of GalSph acylation. NCB-20 cells showed no signs of toxicity upon treatment with GalSph with respect to either morphological changes or their ability to exclude trypan blue. In contrast, toxic effects have been observed with other cell types upon treatment with GalSph at concentrations below 100 pM (Ref. 32).’

One of the problems of studying the metabolism of an exogenously added substrate by intact cells is the inability to control the translocation and distribution of the compound within the cell. In this regard, our results on the time and concentration dependence of the acylation of GalSph in intact NCB-20 cells (Fig. 4) reflect the overall process of uptake, distribution to subcellular compartments, and acylation of GalSph by the cell. We were unable, therefore, to make any estimations of the enzymatic parameters of this reaction.

An early study on the biosynthesis of GalCer by brain microsomes showed that the in vitro acylation of GalSph, originally described by Brady (33), could occur in the absence of microsomes (31). A nonenzymatic acyl transfer from Co-A esters to the free amine of the lysosphingolipid was proposed as an explanation of these results (31). Due to the labile nature of the acyl-CoA thioester linkage, the potential for nonenzymatic acylation has also been encountered in studies of protein acylation, providing there is appropriate access to an acceptor such as a free thiol group. The palmitoylation of rhodopsin by rod outer segments (34) is an example of how nonenzymatic acylation of a protein can occur both in vitro and in viva. In the present study, we provide evidence that the acylation of exogenously added GalSph by intact NCB-20 cells is not the result of this type of nonenzymatic mechanism. Acylation of GalSph by the intact cells exhibited a high degree of specificity for the medium chain nonhydroxy fatty acids palmitic acid (16:0) and stearic acid (l&O). Labeled myristic (14:0), arachidonic (20:4), or lignoceric (24:0) acids, at extra- cellular concentrations equivalent to that of palmitate, were not utilized as substrates for this reaction, despite extensive labeling of phospholipids by the different fatty acids (which demonstrates the uptake and intracellular availability of the fatty acids). This substrate specificity is characteristic of an enzyme-mediated reaction and provides evidence against a nonenzymatic mechanism for the acylation of GalSph by intact NCB-20 cells.

Additional evidence indicating the specificity of fatty acid incorporation into GalCer by NCB-20 cells was provided by experiments in which cells were incubated in medium containing [“H]GalSph. The cells took up the [3H]GalSph and converted it to [“H]GalCer (Fig. 7). Only the lower nonhydroxy fatty acid GalCer band (medium chain, C16-Cl8 fatty acid species of GalCer) was formed, despite the presence of

” G. A. Ristic and G. Dawson, unpublished observations.

22222 Acylation of Glyc

various fatty acids and fatty acid derivatives (acyl-CoAs) within the cell that might serve as donors for this reaction. This [3H]GalCer was identical to the GalCer species formed when cells were incubated with unlabeled GalSph and [3H] palmitic acid (Fig. 3). It seems unlikely that a nonenzymatic mechanism would exhibit this sort of specificity.

It is of interest to note that the addition of [3H]GalSph to intact cells eventually resulted in the formation of labeled CDH (Fig. 7A), suggesting that some of the newly formed GalCer had been further glycosylated to a more complex form, possibly in endoplasmic reticulum-Golgi compartments. This is consistent with reports on the uptake and distribution of N-acylsphingolipids which indicate that internalized glycosphingolipids are not solely directed to lysosomal compartments for degradation, but may also enter anabolic compartments to be converted to more complex sphingolipids (24,35, 36).

Apart from demonstrating that intact NCB-20 cells are able to enzymatically convert exogenously administered glycosylsphingosines to glycosylceramides, we found no clear evidence suggesting the metabolic function of this reaction in the cell. However, the existence of this acyltransferase may explain why cellular levels of lysosphingolipids are so low. The lack of a competitive inhibitory effect on the acylation of other glycosphingolipids in the presence of GalSph (Table I) sug- gests that this reaction does not contribute to the biosynthesis of glycosphingolipids, although we cannot exclude the possi- bility of a minor biosynthetic pathway. The functional significance of this reaction may only be clarified once the enzyme’s subcellular location and the endogenous glycosylsphingosine substrate(s) are determined.

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Acylation of Exogenous Glycosylsphingosines by Intact Neuroblastoma

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