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Glycobiology Pages 885-890  

Variations among cell lines in the synthesis of sphingolipids in de novo and recycling pathways
Introduction
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References

Variations among cell lines in the synthesis of sphingolipids in de novo and recycling pathways

Variations among cell lines in the synthesis of sphingolipids in de novo and recycling pathways

Baiba K.Gillard1,4, Rhonda G.Clement1, Donald M.Marcus1,2,3

Departments of 1Medicine and 2Microbiology and Immunology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, USA

Received on December 16, 1997; revised on January 29, 1998; accepted on February 17, 1998

There are several pathways for the incorporation of sugars into glycosphingolipids (GSL). Sugars can be added to ceramide that contains sphinganine (dihydrosphingosine) synthesized de novo (pathway 1), to ceramide synthesized from sphingoid bases produced by hydrolysis of sphingolipids (pathway 2), and into GSL recycling from the endosomal pathway through the Golgi (pathway 3). We reported previously the surprising observation that SW13 cells, a human adrenal carcinoma cell line, synthesize most of their GSL in pathway 2. We now present data on the synthesis of GSL in four additional cell lines. Approximately 90% of sugar incorporation took place in pathway 2, and 10% or less in pathway 1, in human foreskin fibroblasts and NB41A3 neuroblastoma cells. In contrast, approximately 50-90% of sugar incorporation took place in pathway 1 in C2C12 myoblasts. The C2C12 cells divide more rapidly and synthesize 10-14 times as much GSL as the other three cell lines. In C6 glioma cells, ~30% of sugar incorporation occurred in pathway 1 and 60% in pathway 2. There was no relation between the utilization of pathways for GSL and sphingomyelin synthesis in foreskin fibroblasts and C2C12 cells. In both cells pathways 1 and 2 each accounted for 50% of incorporation of choline into sphingomyelin. In five of the six cell lines that we have studied, most GSL synthesis takes place in pathway 2. We suggest that when the need for synthesis is relatively low, as in slowly dividing cells, GSL are synthesized predominantly from sphingoid bases salvaged from the hydrolytic pathway. When cells are dividing more rapidly, the need for increased synthesis is met by upregulating the de novo pathway.

Key words: glycosphingolipids/sphingomyelin/biosynthesis/ pathways of metabolism

Introduction

Sphingolipids (Table I) are found in all membranes in eukaryotic cells (Hakomori, 1993). In addition to their role in modulating membrane organization and fluidity (Hakomori, 1993), many other functions of these compounds and their metabolites have been described in recent years. These properties include modulation of signaling by cell membrane receptors, regulation of cell growth, regulation of cell-cell and cell-substrate interactions, and the role of sphingolipid metabolites as second messengers, particularly in mediating apoptotic signals. These topics have been the subject of several recent reviews (Hakomori, 1993; Spiegel et al., 1993; Hannun, 1994; Hakomori and Igarashi, 1995; Kolesnick and Fuks, 1995; Spiegel and Merrill, 1996). Although there is a voluminous literature on bioactive metabolites of sphingolipids, much less attention has been paid to new data on the synthesis of these molecules.

Table I. Structures of glycosphingolipids and sphingomyelina
Neutral glycosphingolipids
GlcCer Glc[beta]1-Cer
LacCer Gal[beta]1-4Glc[beta]1-Cer
Gb3Cer Gal[alpha]1-4Gal[beta]1-4Glc[beta]1-Cer
Gb4Cer GalNAc[beta]1-3Gal[alpha]1-4Gal[beta]1-4Glc[beta]1-Cer
Gangliosides
GM3 NeuAc[alpha]2-3Gal[beta]1-4Glc[beta]1-Cer
GM2 GalNAc[beta]1-4[NeuAc[alpha]2-3]Gal[beta]1-4Glc[beta]1-Cer
GM1 Gal[beta]1-3GalNAc[beta]1-4[NeuAc[alpha]2-3]Gal[beta]1-4Glc[beta]1-Cer
GD1a NeuAc[alpha]2-3Gal[beta]1-3GalNAc[beta]1-4[NeuAc[alpha]2-3]Gal[beta]1-4Glc[beta]1-Cer
Sphingomyelin
SM (CH3)3N+CH2CH203P--Cer
aAbbreviations: Gal, d-galactose; Glc, d-glucose; GalNAc, N-acetyl-d-galactosamine; GlcNAc, N-acetyl-d-glucosamine; NeuAc, N-acetyl-neuraminic acid; Cer, ceramide (N-acyl-sphingosine).

The existence of several potential pathways for the synthesis of sphingolipids (Figure 1) is well known, reviewed by (Schwarzmann and Sandhoff, 1990; Hoekstra and Kok, 1992; Sandhoff and van Echten, 1994; Tettamanti and Riboni, 1994). However, information about the relative contributions of each pathway to the synthesis of these compounds is limited. The initial steps of sphingolipid synthesis occur on the cytoplasmic face of the endoplasmic reticulum (ER) (Merrill, Jr. and Wang, 1992; Mandon et al., 1992; Futerman et al., 1990). In the de novo pathway (pathway 1), 3-ketosphinganine is synthesized by the condensation of serine with palmitoyl-CoA, and it is rapidly reduced to sphinganine (dihydrosphingosine) which is acylated to form dihydroceramide. Although sphingosine was thought previously to be a biosynthetic intermediate in sphingolipid biosynthesis, it is currently regarded solely as a product of sphingolipid catabolism (Merrill, Jr. and Wang, 1986; Rother et al., 1992; Smith and Merrill, Jr., 1995). Dihydroceramide may be converted to ceramide by a desaturase, which is also located on the cytoplasmic face of the ER, that inserts a double bond into sphinganine (Michel et al., 1997). In some cells most of the newly synthesized sphingolipids contain dihydroceramide, rather than ceramide, for several hours (Merrill, Jr. and Wang, 1986; Smith and Merrill, Jr., 1995), but in other cells most of the dihydroceramide is immediately converted to ceramide (Michel et al., 1997). The next step in GSL synthesis, addition of glucose to dihydroceramide or ceramide, takes place on the cytoplasmic face of the cis-Golgi (Trinchera et al., 1991; Futerman and Pagano, 1991; Jeckel et al., 1992). The Glc(dihydro)Cer is then transported either directly to the plasma membrane (Warnock et al., 1994), or into the Golgi lumen where the sugar chains are extended as the GSL are transported from the cis- to trans-Golgi (van Echten et al., 1990; Young, Jr. et al., 1990; Hoekstra and Kok, 1992; Young, Jr., 1993; van Echten and Sandhoff, 1993).


Figure 1. Proposed pathways for the biosynthesis and recycling of sphingolipids. De novo biosynthesis of dihydroceramide and ceramide take place in pathway 1 (A), and this pathway is inhibited by [beta]-chloroalanine. In pathway 2, sphingosine and sphinganine produced by hydrolysis of sphingolipids are reutilized for synthesis of sphingolipids (B). Fumonisin B1 inhibits pathways 1 and 2. In pathway 3, native or partially hydrolyzed GSL recycle through the Golgi apparatus.

In pathway 2 (Figure 1), sphingosine and sphinganine, arising in lysosomes from hydrolysis of sphingolipids, can be reacylated to form (dihydro)ceramide and then transported to the Golgi and further metabolized as discussed above. A third pathway involves the transport of native or partially hydrolyzed GSL from endosomes to the Golgi, where the sugar chains may be elongated (Schwarzmann and Sandhoff, 1990; Tettamanti and Riboni, 1994). An estimate of the quantity of sugar incorporated into GSL in the three pathways can be obtained by the use of two inhibitors of sphingolipid synthesis, [beta]-chloroalanine, which inhibits the synthesis of sphinganine (pathway 1) (Medlock and Merrill, Jr., 1988; Merrill, Jr. and Wang, 1992), and fumonisin B1, which inhibits the acylation of sphinganine and sphingosine (pathways 1 and 2) (Merrill, Jr. and Wang, 1992; Merrill, Jr. et al., 1993a,b).

The SW13 cell line was established from a human adrenal carcinoma (Leibovitz et al., 1973), and it consists of a mixture of cells with and without vimentin (vim+ and vim-) intermediate filaments (IF) (Hedberg and Chen, 1986). Cloned SW13 vim- cells synthesize less GSL than vim+ cells (Gillard et al., 1994). The defect in the vim- cells is principally in the recycling pathways (2 and 3) (Gillard et al., 1996). We were also surprised to find that in both vim- and vim+ cells, most of the sugar was incorporated into GSL in the recycling pathways rather than in the de novo pathway.

Since our observations were the first analyses of the relative contribution of the three pathways to GSL biosynthesis, we extended our studies to four additional cell lines. We now report that most sugar is incorporated into GSL in the recycling pathways in three additional cell lines, human foreskin fibroblasts, C6 glioma cells and NB41A3 neuroblastoma cells, and in the de novo pathway in C2C12 myoblast cells. We also present data on the pathways of choline incorporation into sphingomyelin in four cell lines.

Results

Incorporation of sugars into glycolipids

Data on the incorporation of radiolabeled sugars and serine into GSL during a two-hour period are presented in Figure 2, and calculations of the pathways of biosynthesis are summarized in Table II. The de novo synthesis of (dihydro)ceramide and sphingolipids, as measured by the incorporation of 14C-serine (Figure 2), was inhibited almost completely by [beta]-chloroalanine and fumonisin B1. In contrast, the effect of [beta]-chloroalanine on incorporation of sugars into GSL varies considerably among the cell lines. There was virtually no inhibition of sugar incorporation in the fibroblasts and neuroblastoma cells, ~33% inhibition in the glioma cells and extensive inhibition in C2C12 cells. Fumonisin B1 abolished almost completely sugar incorporation into small GSL, and partially inhibited the synthesis of GD1a and other more complex GSL in C2C12 cells.


Figure 2. Incorporation of 14C-serine, 14C-galactose, and 14C-glucosamine during a 2 h period into GSL of four cell lines, and inhibition by [beta]-chloroalanine and fumonisin B1. Incorporation in the absence of inhibitors (control) is depicted in solid bars; in the presence of [beta]-chloroalanine ([beta]-chloro), hatched bars; and in the presence of fumonisin B1 (fum), open bars. (A) Human foreskin fibroblasts; (B) human NB41A3 neuroblastoma cells; (C) rat C6 glioma cells; (D) mouse C2C12 myoblasts. For inhibition with [beta]-chloroalanine, cells were preincubated for 60 min with 25 mM [beta]-chloro-l-alanine in serine-free Hanks' balanced salt solution (HBSS), washed, and then radiolabeled in complete medium. For inhibition with fumonisin B1, cells were preincubated in 50 µM fumonisin B1 in complete medium for 18-24 h, and then radiolabeled in complete medium in the continued presence of fumonisin. When both inhibitors were used in the same experiment, the last hour of preincubation with fumonisin was also done in serine-free HBSS.

These data were used to calculate pathways of biosynthesis (Table II). There is a striking predominance of pathway 2 in human foreskin fibroblasts and neuroblastoma cells, and, with one exception, very little synthesis in pathways 1 and 3. In NB41A3 cells, a ganglioside with the mobility of GD1a incorporated 41% of its sugars in pathway 3. The small quantity of sugar incorporation in pathway 1 was surprising. With the possible exception of Gb4Cer in NB41A3 cells, no detectable quantities of GSL more complex than GlcCer were synthesized de novo in these two cell lines. In contrast, pathway 1 was predominant in C2C12 myoblasts. These cells synthesized ten times as much GSL as the other cell lines. The pattern of biosynthesis in C6 glioma cells was intermediate between these extremes, approximately one-third of sugar incorporation took place in pathway 1 and two thirds in the recycling pathways. Very little synthesis of GSL took place in pathway 3 in fibroblasts and C6 glioma cells, but appreciable synthesis of GSL with large carbohydrate chains occurred in this pathway in the neuroblastoma and C2C12 cells.

Synthesis of sphingomyelin

There is little information about the relative importance of the de novo and recycling pathways in the synthesis of sphingomyelin. We also wished to compare the pathways used for GSL and sphingomyelin synthesis in the same cell line. For this purpose, we studied two cells that differed markedly in GSL synthesis, fibroblasts and C2C12 cells, and we also analyzed SW13 cells, whose synthesis of GSL has been extensively studied (Gillard et al., 1994; Gillard et al., 1996). We used [beta]-chloroalanine and fumonisin B1 to analyze incorporation of choline into sphingomyelin in pathways 1 and 2. In foreskin fibroblasts and C2C12 myoblasts there was no correlation between pathways used to synthesize sphingomyelin and GSL (Table III). Approximately 50% of sphingomyelin was synthesized in pathway 1 in fibroblasts, compared to 0-10% of sugar incorporation. In C2C12 cells ~50% of choline was incorporated in pathway 1, compared to 48-87% for incorporation of sugars.

In SW13 cells, the pattern of choline incorporation was similar to that of sugar incorporation(Gillard et al., 1996), ~20% in pathway 1 and 80% in pathway 2. Pathways 1 and 2 accounted for essentially all of the incorporation of choline into sphingomyelin. Other potential pathways for synthesis of sphingomyelin, recycling of ceramide from lysosomes to Golgi and resynthesis from ceramide at the plasma membrane (Futerman et al., 1990; van Helvoort et al., 1994), did not make an appreciable contribution in these three cell lines (Table III, column 4).

Discussion

Although the existence of the salvage and recycling pathways are well known (Schwarzmann and Sandhoff, 1990; Hoekstra and Kok, 1992; Sandhoff and van Echten, 1994; Tettamanti and Riboni, 1994), textbooks of biochemistry (Stryer, 1995) present only the de novo biosynthetic scheme, because there has been no information about the relative contributions of the various pathways to sphingolipid synthesis. The data in Figure 1 and Table II demonstrate that GSL are synthesized predominantly in pathway 2 in three of the four cell lines examined, in addition to SW13 cells (Gillard et al., 1996). Murine embryonic fibroblasts also synthesize most of their GSL in pathway 2 (B.K.Gillard, R.Clement, E.Colucci-Guyon, C.Babinet, G.Schwarzmann, T.Taki, and D.M.Marcus, unpublished observations). The only cell line in which pathway 1 predominated was C2C12 myoblasts, which divide more rapidly than the other cell lines, and incorporate 10-15 times as much sugar into GSL (Table II). Most of the increase in C2C12 cells is in pathway 1, 38-190 times higher than the other cells, compared to a 2- to 3-fold increase in pathway 2.

Table II. Pathways of sugar incorporation into glycosphingolipids
Cell type GSLa Counts/µg proteinb Percentc
Pathway 1 Pathway 2 Pathway 3
Human foreskin GlcCer 404 ± 32 10 ± 7 90 ± 7 0 ± 0
fibroblasts Gb3Cer 363 ± 32 0 ± 0 99 ± 1 1 ± 1
  GM3 248 ± 17 0 ± 0 95 ± 3 5 ± 3
  Gb4Cer 302 ± 21 0 ± 0 94 ± 3 6 ± 3
  Total 1204  
Mouse NB41A3 GlcCer 210 ± 12 6 ± 6 94 ± 6 0 ± 0
neuroblastoma Gb4Cer 168 ± 11 11 ± 1 89 ± 1 0 ± 0
  GM2-GM1 445 ± 27 0 ± 0 95 ± 0 5 ± 0
  GD1a 56 ± 4 0 ± 0 60 ± 8 41 ± 8
  Total 1052  
Rat C6 glioma GlcCer 141 ± 7 31 ± 2 62 ± 1 7 ± 2
  GM3 1160 ± 58 34 ± 3 59 ± 1 7 ± 2
  Total 1301  
Mouse C2C12 GlcCer 7026 ± 1007 87 ± 1 13 ± 1 1 ± 1
myoblasts GM3 2316 ± 212 81 ± 7 12 ± 6 8 ± 2
  Gb4Cer 1836 ± 248 69 ± 3 17 ± 0 15 ± 3
  GM2-GM1 1850 ± 325 49 ± 5 34 ± 5 18 ± 1
  GD1a 2829 ± 568 48 ± 2 19 ± 2 34 ± 1
  Total 15,857  
aGSL are tentatively identified by their TLC mobility. Only the major GSL of each cell type are included in this table. Number of bands seen on TLC.
bTotal sugar incorporated per microgram protein in control cells. Values are mean ± SEM for two experiments, each done in duplicate or quadruplicate.
c Percent incorporated per pathway, calculated from a combination of [beta]-chloroalanine and fumonisin B1 inhibition data, as described in Materials and methods. Values are mean ± SEM.

Table III. Pathways of choline incorporation into sphingomyelina
Cell type Percent
Pathway 1 Pathway 2 Pathway 3
Human foreskin fibroblasts 40 ± 5 58 ± 8 1 ± 2
Mouse C2C12 myoblasts 49 ± 1 43 ± 1 9 ± 1
SW13 clone HV2 20 ± 6 80 ± 6 1 ± 1
SW13 clone 1HF5 7 ± 2 93 ± 2 1 ± 1
aCell cultures were preincubated for 1 h with 25 mM [beta]-chloroalanine or 24 h with 50 µM fumonisin B1 and labeled with 14C-serine or 14C-choline for 3 or 6 h. Results are representative of two to three experiments, each done in duplicate. Values: Pathway 1, mean ± SEM; Pathways 2 and 3, mean ± SD

The very small amount of GSL synthesis in pathway 1 by the foreskin fibroblasts and NB41A3 neuroblastoma cells is striking. There are several possible explanations for this observation. The amount of synthesis in pathway 1 could be underestimated if newly synthesized GSL were hydrolyzed within the 2 h incubation period. Studies performed previously with SW13 cells indicated a substantial decrease in radioactivity in GlcCer during a 2 h chase period (unpublished observations). Another possibility is that in these cells most of the GlcCer, which is synthesized on the cytoplasmic face of the cis-Golgi, is transported directly to the plasma membrane (Warnock et al., 1994), and very little is transported into the Golgi lumen.

In addition to overall differences among cell lines in the utilization of the three pathways, there are some differences among GSL in the same cell line. Within experimental error, no GlcCer is synthesized in pathway 3 in the cells studied in this report and in SW13 cells (Gillard et al., 1996). This indicates that either ceramide is not recycled from endosomes to the Golgi, or that all GlcCer synthesized from recycling ceramide is used to synthesize larger GSL. The reutilization of ceramide would not be inhibited by fumonisin B1, and since this compound usually inhibits GSL synthesis by 90-95% (Figure 2), it is likely that recycling of ceramide is very limited. Another point of interest is that more GSL with long than short carbohydrate chains are synthesized in pathway 3 in neuroblastoma and C2C12 cells. This observation suggests that addition of terminal galactose and sialic acid residues is facilitated by the recycling process.

Sphingomyelin is synthesized by the transfer of phosphorylcholine from phosphatidylcholine to ceramide or dihydroceramide (reviewed by Merrill, Jr. and Jones, 1990). The principal site of sphingomyelin synthesis is in the lumen of the cis- and medial Golgi (Merrill, Jr. and Wang, 1992; Mandon et al., 1992; Futerman et al., 1990; Koval and Pagano, 1991), where phosphorylcholine is added to (dihydro)ceramide. Sphingomyelin can also be synthesized at the plasma membrane (Futerman et al., 1990; van Helvoort et al., 1994), but the magnitude of this pathway is probably small in comparison to synthesis in the Golgi. The data in Table III document the extensive use of pathway 2 in sphingomyelin synthesis in all four cell lines studied. There is no correlation between the use of pathways 1 and 2 for GSL and sphingomyelin synthesis in foreskin fibroblasts and C2C12 cells. Approximately equal quantities of sphingomyelin are made in pathways 1 and 2 in both cells, in contrast to the predominant use of pathway 2 for GSL synthesis in fibroblasts, and the predominance of pathway 1 in C2C12 cells. Independent regulation of GSL and sphingomyelin synthesis (Merrill, Jr. et al., 1993b; Yokoyama et al., 1995) is not surprising in view of the more rapid turnover of phospholipids and their diverse roles in metabolism.

In view of the differences among cells in their utilization of pathways 1 and 2 (Table II), it is interesting to consider why these differences exist, and how the activities of the pathways might be regulated. The generation of sphinganine and sphingosine from hydrolysis of sphingolipids is a concomitant of the constitutive flow of a large quantity of plasma membrane components through the endocytic pathway. It has been estimated that an entire plasma membrane equivalent may recycle in 1 h in a phagocytic cell, 2 h in a fibroblast-like cell, and in ten h in a lymphocyte (reviewed by (Thilo, 1994). Most plasma membrane constituents are recycled back to the plasma membrane unchanged from early endosomes and sorting vesicles, and some of the membrane components recycle through the Golgi. Although only a very small fraction, 5% or less, is hydrolyzed in late endosomes and lysosomes (Thilo, 1994), the absolute amount of material hydrolyzed is appreciable because of the large volume of endocytic flow. In view of the constitutive nature of the endocytic process, the cell can conserve energy by reutilizing the products of sphingolipid hydrolysis, and minimizing the de novo synthesis of sphinganine.

The rate limiting step in de novo biosynthesis of sphinganine is the synthesis of 3-ketosphinganine (Figure 1), which is catalyzed by palmitoylserine transferase (reviewed by) (Merrill, Jr. and Jones, 1990). Regulation of the concentration or activity of this enzyme could provide a means of modulating the de novo pathway. When the need for synthesis of new membranes is limited, e.g., in nonphagocytic cells that are not dividing rapidly, most of the need for sphingoid bases for synthesis of GSL can be met by salvage in pathway 2. When the requirement for synthesis of cell membranes is greater, as in rapidly dividing cells, the rate of de novo synthesis (pathway 1) can be increased. This hypothesis is consistent with our observation that of the cells we studied, only the rapidly dividing cell line C2C12 synthesized most of its GSL de novo. The hypothesis could be tested by comparing the pathways used for GSL synthesis in slowly and rapidly dividing cells.

Materials and methods

Cells

Early passage primary human foreskin fibroblasts were obtained from Drs. Olivia M. Pereira-Smith and Susan F. Venable (Department of Molecular Virology, Baylor College of Medicine). C2C12 mouse muscle myoblast cells (ATCC No. CRL 1772) were obtained from Dr. Y. Capetanaki (Department of Cell Biology, Baylor College of Medicine). C6 rat glioma cells (ATCC No. CCL107), and NB41A3 mouse neuroblastoma cells (ATCC No. CCL 147) were purchased from the American Type Culture Collection (Rockville, MD). SW13 cells were described previously (Gillard et al., 1994).

Radiolabeling and analysis of sphingolipids

Our procedures for metabolic radiolabeling of cells, and purification and analysis of GSL, ceramide, and sphingomyelin, were described previously (Gillard et al., 1994; Gillard et al., 1996). Metabolic radiolabeling of subconfluent cells was performed for 2 h. It was initiated by addition of radioactive precursors to the culture medium (containing 10% fetal calf serum) to give final concentrations of 14C-galactose and 14C-glucosamine, 2 µCi/ml each; 14C-serine, 5-10 µCi/ml; and 14C-choline, 2 µCi/ml. For inhibition with [beta]-chloroalanine, cells were preincubated for 60 min with 0-25 mM [beta]-chloro-l-alanine (Sigma) in serine free Hanks' balanced salt solution (HBSS), washed, and then radiolabeled in complete medium (Medlock and Merrill, Jr., 1988; Gillard et al., 1996). For inhibition with fumonisin B1, cells were preincubated with 0-50 µM fumonisin B1 (Sigma) in complete medium for 18-24 hr, for 60 min in HBSS (concurrent with the [beta]-chloro-l-alanine preincubation), and then radiolabeled in complete medium in the continued presence of fumonisin (Merrill, Jr. et al., 1993a,b; Gillard et al., 1996).

Glycolipid composition of samples was analyzed by TLC, on high performance Silica Gel 60. Plates were developed in chloroform/methanol/2.5 M NH4OH (aq) 90:10:2 or 80:20:2 (v:v:v) for analysis of ceramide, in chloroform/methanol/0.25% CaCl2 (aq) 60:35:8 for analysis of GSL and sphingomyelin. Identification of GSL was based on chromatographic mobility in comparison to the mobility of standard sphingolipids, phospholipids and fatty acids present on the same plate. The amount of radioactivity in each band was determined by direct imaging of the TLC plates onto Phosphor screens, and quantitation on the Molecular Dynamics Phosphorimager system (Baylor College of Medicine Phosphorimager Core Facility).

Estimation of sugar incorporation in the three pathways

As described previously (Gillard et al., 1996),the quantity of sugar incorporated into GSL, or cholineinto sphingomyelin in the three pathways was estimated as follows: Pathway 1 (de novo) = amount inhibited by [beta]-chloroalanine. Pathway 2 (recycling of sphingosine and sphinganine) = amount inhibited by fumonisin B1 minus amount inhibited by [beta]-chloroalanine. Pathway 3 (recycling of GSL through the Golgi) = amount not inhibited by fumonisin B1. A correction for incomplete inhibition of pathways 1 and 2 was based on the amount of residual serine incorporation into ceramide in the presence of the inhibitors. In general, the inhibition was over 95% (Figure 2).

Acknowledgments

We thank Dr. Alfred H. Merrill, Jr., for helpful discussions, Dr. Takao Taki for assistance in identification of radioactive ganglioside standards, and Mrs. Brenda Galena for her expert assistance with the manuscript. The work was supported by Research Grants AI 17712 from the National Institutes of Health, and BE-88B from the American Cancer Society.

Abbreviations

Glycosphingolipid structures are abbreviated according to the IUPAC-IUB Commission on Biochemical Nomenclature, 1977, except that ganglio series gangliosides are abbreviated according to Svennerholm (Svennerholm, 1964), and the suffix Cer is used in place of OseCer. Cer, ceramide; Gal, d-galactose; Glc, d-glucose; GalNAc, N-acetyl-d-galactosamine; NeuAc, N-acetyl-neuraminic acid; GSL, glycosphingolipid(s); IF, intermediate filaments; and ER, endoplasmic reticulum.

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3To whom correspondence should be addressed
4Present address: Department of Pediatrics, Division of Infectious Diseases, University of Texas-Houston Medical School, JFB 1.739, 6431 Fannin, Houston, TX 77030

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