Possible role of ceramide in defining structure and function of ...

Proc. Nati Acad. Sci. USA

Vol. 79, pp. 3470-3474, June 1982 Biochemistry

Possible role of ceramide in defining structure and function of membrane glycolipids (neolacto-series glycolipid/developmental antigen/fatty acid/terminal carbohydrate structure/glycosyltransferase)

REIJI KANNAGI, EDWARD NUDELMAN, AND SEN-ITIROH HAKOMORI Biochemical Oncology, Fred Hutchinson Cancer Research Center, and Departments of Pathobiology, Microbiology and Immunology of the University of Washington,

Seattle, Washington 98104 Communicated by Eloise 1R. Giblett, March 8, 1982

ABSTRACT A possible role of ceramide in defining the carbohydrate structure ofglycolipids and the expression of glycolipid function has been proposed, on the basis of our finding that the ceramide composition of "lacto-series" glycosphingolipid isolated from human erythrocytes shows a remarkable correlation with the terminal carbohydrate structure: (i) The ceramides of three glycosphingolipids with LeX (or x) determinant [GalPI1-4(Fucal -*3)GlcNAc] had almost exclusively 16:0 fatty acid; in contrast, the ceramide of its positional isomer H determinant had mainly 20-24:0 fatty acids. (ii) The ceramide of two glycosphingolipids with NeuAca2-*6Gal structure was predominantly of 16:0 fatty acid, in contrast to that of its positional isomer NeuAca2-*3Gal residue, in which the ceramide had 20-24:0 fatty acids. These results, together with our previous observation that ceramide composition of mouse lymphoma and myelocytic leukemia MI cells affects their antigenicity, suggest that ceramide structure may define the organization ofglycosyltransferases for synthesis of the carbohydrate determinants and may affect the organization and orientation of the carbohydrate chain in membranes, eliciting or suppressing the reactivity to its ligand. Because these glycolipids with Lex and NeuAca2--6Gal structures are developmentally regulated and are highly expressed in certain tumors, ceramide composition may affect development, differentiation, and onco-

type 0 human adult peripheral blood as described (6). The extract was evaporated to dryness, dissolved in chloroform/ methanol, 2:1 (vol/vol), and partitioned four times according to a modification ofFolch's procedure (7). The neutral glycolipid and ganglioside fractions were separated from the Folch upper phase by DEAE-Sephadex chromatography (8), and each fraction was purified by low-pressure chromatography in a Varian HPLC apparatus with a column of Iatrobeads (100 X 1 cm; 6RS860, 60-,m-diameter; Iatron Laboratory, Tokyo). The column was eluted with a gradient of isopropyl alcohoVhexane/water, 55:40:5 to 55:30:15, followed by 55:25:20 for 100 min and 55:20:25 for 100 min with a flow rate of 2 ml/min. The separation pattern was similar to that described in ref. 6. The fractions eluted between H2 and H3 glycolipids and eluted after H3 glycolipid were tentatively called "Y fraction" and "Z fraction", respectively. The Y fraction contained four components, tentatively termed Y1-Y4 glycolipids. One of these (Y2) was reactive with antibody to stage-specific embryonic antigen 1 (SSEA-1) (9), which reacts with glycolipids having Lex [Gal31-> 4(Fuca1->3)GlcNAc31-->R] structure (10) and is inhibitable by oligosaccharides with this sequence (11). The Y2 glycolipid was purified by HPLC according to the method of Watanabe and Arao (12), using a modified solvent system, namely, a shallow gradient of isopropyl alcohoVhexane/water from 55:37:8 to 55:35:10 with the column of latrobeads (6RS-810, 10 ,um-diameter, column size 50 x 1 cm) at about 100 pounds/inch2 (690 kPa) to obtain a 2.0 ml/min flow. The Z fraction contained three major components that separated on TLC, designated Z1, Z2, and Z3 glycolipids. The Z1 glycolipid, which was reactive with anti-SSEA-1 antibody, was purified by using a gradient of isopropyl alcohoVhexane/water from 55:33:12 to 55:30:15, followed by elution with 55:25:20. The third glycolipid component reactive with anti-SSEA-1 antibody was also isolated from the fraction eluted with H4 glycolipid. Details of the isolation procedure of these glycolipids will be described elsewhere. The G2, G4, G6, and G7 gangliosides of human erythrocytes (13) were purified from the monosialoganglioside fraction by HPLC under the same conditions as described above. The carbohydrate structure of these glycolipids was determined by a combination of (i) methylation analysis, (ii) direct probe mass spectrometry of methylated compounds, (iii) endo-,B3galactosidase digestion, and (iv) stepwise degradation with exoglycosidases (6, 13, 14). Fatty acid composition of these purified glycolipids was determined by gas chromatography on a 6 foot X 1'8 inch (182

genesis.

Glycosphingolipids are glycosides of N-fatty acyl-sphingosine,* whose trivial name is ceramide. They are membrane components and the majority are assumed to be present at the outer leaflet ofplasma membranes, although their exact organization in membrane remains unknown (1, 2). Because of their possible role in cellular interaction, differentiation, and oncogenesis (1), as well as their function as antigens through their sugars (3-5), much attention has been paid to their carbohydrate structure. Because the ceramides themselves have been ignored or regarded simply as the anchoring point to membranes, their functional role as part of glycolipids has received little attention. On the basis ofthe present study, in which the ceramide composition of a large variety of erythrocyte glycolipids was analyzed, and on the basis of previous findings on ceramide composition of ganglio-N-triaosylceramide in mouse lymphoma variants and in mouse myelocyte Ml, we suggest that ceramides may play an important role in selection of glycosylation and in defining the carbohydrate reactivity to its ligands. MATERIALS AND METHODS Erythrocyte stroma and the total lipid extract with isopropyl alcohol/hexane/water, 55:20:25 (vol/vol), were prepared from

Abbreviations: SSEA-1, stage-specific embryonic antigen 1; Cer, ceramide; GgOse3Cer, gangliotriaosylceramide; CMH, ceramide monohexoside.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

* D-erythro-1,3-Dihydroxy-2-(alkylacetamido)4,5-trans-octadecene or

its analogs.

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Proc. Natl. Acad. Sci. USA 79 (1982)

Biochemistry: Kannagi et aL x 0.3 cm) (inner diameter) column of SE-30 on 80/100 Supelcoport (Supelco, Bellefonte, PA) after methanolysis as described (14).

RESULTS Glycolipids with Lex, H, NeuAc 2-3, and 2-a6 Gal Residues of Human Erythrocytes. Two glycolipids of human erythrocytes reacting with the monoclonal antibody directed toward the differentiation antigen of teratocarcinoma and early mouse embryo (SSEA-1) were isolated and their structures were elucidated as shown in Table 1 (item 1). The structure was determined as a fucosyl 1--3 substitution at the penultimate GlcNAc residue of lacto-N-norhexaosylceramide (Y2 glycolipid; Table 1, item la) and lacto-N-noroctaosylceramide (Zi glycolipid; Table 1, item lb) by the methods described in Materials and Methods. The third glycolipid component reacting with the monoclonal anti-SSEA-1 antibody was also isolated. Isolation and characterization of these glycolipids will be described in detail elsewhere. Two types of H-active glycolipid, H2 and H3 (Table 1, items 2a and 2b) have been characterized as described (15). H2 and Y2 have identical structures except that the fucose residue of H2 is linked to the terminal Gal through al->2 linkage and that of Y2 is linked to the penultimate GlcNAc residue through al->3 linkage. Two gangliosides having a NeuAca2-46Gal residue have been isolated and characterized, previously referred to as G4 and G7 (13) (Table 1, items 3a and 3b). Another two gangliosides having a NeuAca2-*3Gal residue have been iso-

lated and characterized; these were previously referred to as G2 and G6 (13) (Table 1, items 4a and 4b). The carbohydrate structures of these gangliosides were confirmed as described (15). Fatty Acid Composition and TLC Mobility of Each Glycolipid and its Degradation Product. Fatty acid composition of the glycolipids in Table 1 is shown in Table 2, and some of the gas chromatography patterns are shown in Fig. 1. A remarkable association between the fatty acid profile and the terminal carbohydrate structure was found. These glycolipids with Lex terminal structure, Y2 and Z1, contained predominantly 16:0 fatty acid, in striking contrast to those with H terminal structure, which contained predominantly 22:0, 24:1, and 24:0 fatty acids (Fig. 1, Table 2). A similar correlation was found between gangliosides with NeuAc 2-+6 Gal terminal structure (G4 and G7) and those with NeuAc 2-)3 Gal terminal structure (G2 and G6). The former was characterized by the predominance of 16:0 fatty acid (Fig. ic and Table 2) and the latter contained mainly 24:1 and 24:0 fatty acids (Fig. ld, Table 2). The defucosylated core from H2 had a higher TLC mobility than that from Y2 (see Fig. 2a, lanes 2, 3, and 4), consistent with a shorter fatty acid in Y2 than in H2. Similarly, desialylated G2 had a higher mobility than. did desialylated G4 (Fig. 2b, lanes 2 and 3). When degraded by endo-fgalactosidase, the glycolipids listed in Table 1 yielded a glucosylceramide (ceramide monohexoside, CMH),with obviously different TLC mobilities: those glycolipids with LeX structure and those with NeuAca2 --)6Gal structure were degraded into a lower band glucosylceramide, and those with H structure and with NeuAca2-*

Table 1. Structures of glycolipids from erythrocyte membranes used in this study 1. LeX-active series a. Y2 glycolipid GalBl-.4GlcNAcP1-.3Gall1-*4GlcNAcp1--*3Galf1--*4Glcl-*lCer Fucal /" b. Zi glycolipid

3

Galfl1-*4GlcNAc31--+3GalIl1-4GlcNAc31-.3Gal/l--*.4GlcNAc31--*3Galf31--4Glcl1-4lCer 3

FucalA/ 2. H-active series a.

H2 glycolipid

2 Galpl--14GlcNAclB13Gal lB134GlcNAcplB13Gall814Glcl-lCer

Fucal"' 2

b. H3 glycolipid

GalBl-*4GlcNAcfpl E 3

6Gall14GlcNAcl13Gal,14Glcl-lCer

FucalA GalBl1+4GlcNAcB1 /

3. NeuAc 2-*6 series a. NeuAc 2-*6 PG (G4)

b. NeuAc 2--6 nHC (G7)

6Galpl314GlcNAcpl

>3Gall14Glcl ylCer

4GlcNAcl3

3Galdl14GlcllCer

NeuAc2,"

6Galp8 s4GlcNAcl13Galpl3 NeuAc2,"

4. NeuAc 2--3 series

3Galfl3-*4GlcNAcfl1- 3Gall31--4Glcl--lCer

a. NeuAc 2-+3 PG (G2)

b. NeuAc 2--3 nHC (G6)

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NeuAc2/

3GalBl-.4GlcNAcf81-.3Galfl1-4GlcNAcl--*3Galpl--14Glc1--lCer

NeuAc2/

Cer, ceramide; PG, paragloboside; nHC, norhexaosylaceramide; NeuAc, N-acetylneuraminic acid.

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Proc. Natl. Acad. Sci. USA 79 (1982)

Table 2. Fatty acid composition (wt %) of neolacto-series glycolipids from type 0 human erythrocytes NeuAc 2--3 NeuAc 2--6 species species LAX species H species 2-6 2-3 2-+6 23 nHC H2 H3 nHC Fatty acid Y2 Zi PG PG 0.4 0.1 0.0 0.3 0.2 0.0 0.1 14:0 2.1 0.4 0.0 0.0 0.6 0.2 0.0 14:1 0.0 0.0 2.1 14.0 9.8 11.5 55.6 39.7 16:0 69.9 88.3 0.4 0.8 1.3 1.6 0.4 1.4 0.9 16:1 4.3 4.7 6.5 3.2 11.7 16.6 10.4 16.6 18:0 5.9 1.3 2.8 1.8 2.2 6.2 4.2 9.4 18:1 4.3 0.9 2.0 1.8 4.1 1.1 1.4 1.6 20:0 1.6 0.4 0.1 0.6 0.6 2.8 12 20:1 3.2 0.4 16.6 1.1 24.0 17.7 5.6 3.6 22:0 1.1 -14.1 2.1 6.4 0.0 4.7 2.2 4.2 12.4 22:1 4.3 48.4 32.6 0.6 19.6 26.1 4.9 5.0 24:0 1.4 21.4 14.7 24:1 0.6 14.5 12.8 6.3 4.8 1.4 2.8 3.4 Others 0.4 6.3 4.4 3.5 4.7 0.5 20 + 22 + 24 (a) 87.3 72.4 4.7 67.5 60.3 25.2 28.6 13.0 9.3 24.2 26.2 35.1 71.6 66.7 14 + 16 + 18 (b) 86.5 94.6 9.39 2.99 0.05 2.58 1.72 0.35 0.43 a/b 0.15 PG, paragloboside; nHC, norhexaosylaceramide.

3Gal structure were degraded into an upper band glucosylceramide (Fig. 3). The third component, reacting with anti-SSEA1 antibody and identified as having Lex structure, was found to have a ceramide with short fatty acids, because it gave a lower band glucosylceramide on endo-f3-galactosidase treatment (data not shown).

DISCUSSION The results clearly indicate that in these glycolipids there is a close correlation between the terminal carbohydrate structure and fatty acid composition of the ceramide. The glycolipids with the terminal structure of either Lex [GalP1--4(Fuca1-l 3)GlcNAc] or NeuAca2-->6Gal were characterized by the presence of ceramide having short fatty acids. In striking contrast, those with H structure (Fucal-+2Gal) or NeuAca2-,3Gal ter-

minal structure had ceramide with longer fatty acid. In addition, the ceramide of G3 ganglioside (NeuAca2--+3GalNAcP1

-3GallB4GlcNAcl13Gall14GlcpllCer) from human erythrocytes had a predominance of 14:0 fatty acid (16), whereas the glycolipid with the same structure as asialo core of G3 ("X2" glycolipid) had 20-24:0 fatty acids as the major component (6). A selective sialylation of X2 glycolipid with a shorter fatty acid was suggested. These clear-cut correlations between ceramide composition and carbohydrate structure suggest a few possibilities: (i) The glycosyltransferases that synthesize the terminal structure may have an additional substrate specificity toward the fatty acid moiety of ceramide. For this hypothesis, one has to assume an unusual enzyme specificity capable of recognizing both fatty acid chain length and the terminal carbohydrate structure without recognizing the length of the lactosamine core structure. (ii) The glycosyltransferases that synthesize the terminal structure of the glycolipid on the shorter fatty acid b

a

TW

0

Ar---", Glob --PG

HI

Glob H2 -

-

-Y2

-

LI1

do

1

0

10

20

30

0

10

20

30

Time, min

FIG. 1. Gas/liquid chromatography of fatty acid methyl esters from Y2 glycolipid (a), H2 glycolipid (b), G4 ganglioside (c), and G2 ganglioside (d).

H3

2

3

4

5

1 23 4

FIG. 2. Thin-layer chromatogram of defucosylated H2'and Y2 glycolipids or desialylated G2 and G4 gangliosides. (a) Lanes 1 and 5, total upper neutral glycolipid fraction from type O human erythrocytes; lane 2, defucosylated H2; lane 3, defucosylated Y2; lane 4, defucosylated H2 + Y2. (b) Lanes 1 and 4, total upper neutral glycolipids from type 0 human erythrocytes; lane 2, desialylated G2; lane 3, desialylated G4. Solvent system was chloroform/methanol/water, 60:35:8 (vol/vol). Glob., globoside; PG, paragloboside.

Biochemistry: Kannagi et aL a

sf

sf CMHCDH CTH Gb

Proc. Natl. Acad. Sci. USA 79 (1982)

b a

CMH CDH

CTH Gb 0

-

i-

-

0

1 2 34

12 3 4

FIG. 3. Thin-layer chromatogram of CMHI obtained by hydrolysis with endo-/-galactosidase from Escherichia freundii. Reaction mixture was partitioned according to the method of Folch et al. (7) and lower-phase lipids were chromatographed. (a) Lanes 1 and 2, CMH from Y2 glycolipid; lanes 3 and 4, CMH from H2 glycolipid. (b) Lane 1, control CMH purified from bovine brain; lane 2, control ceramide dihexoside (CDH), ceramide trihexoside (CTH), and globoside (Gb) purified from human erythrocytes; lane 3, CMH from G2 ganglioside; lane 4, CMH from G4 ganglioside. Solvent system was chloroform/ methanol/water, 100:40:6 (vol/vol). o, Origin; -sf, solvent front.

(i.e., LeX or NeuAca2--6Gal) may be preferentially associated with a lipid bilayer region rich in precursor glycolipids with a short fatty acid chain-i.e., nonrigid or fluid lipid bilayer. In contrast, the glycosyltransferases that synthesize the terminal structure of the glycolipid with the longer fatty acid chain (i.e., H or NeuAca2--3Gal) may react preferentially in a membrane domain with more rigid properties. Besides the fluidity ofmembrane, the fatty acid chain length of ceramide affects membrane thickness, which may also affect the association of the enzymes with precursor glycolipids. Because glycolipids and glycosyltransferases are membrane bound and are essentially lipophilic, it is reasonable to assume that each transferase may have different and specific lipophilic properties (lipophilism). (iii) It is possible that a given molecular species of ceramide is synthesized coincidentally at the same stage as the synthesis of a specific carbohydrate chain during the process of differentiation. Because it has been shown that glycolipids with LeX or those with NeuAca2--6Gal structure represent embryonic or fetal antigens (9) and could be the major component of fetal tissue (17), the synthesis of their determinants could be greatest in the embryonic stage and decline during the course of development with incidental decline in the synthesis of a ceramide having 16:0 fatty acid. It is known that the average chain length of ceramide fatty acid increases during the course of development or differentiation of brain (18, 19) or intestinal epithelia (20). Similar mechanisms can be applied to differentiation of human erythrocytes. However, the possibility of such a coincidental but independent change of ceramide and carbohydrate synthesis seems to be remote. (iv) Conceivably, the fatty acid composition of ceramide could be developmentally regulated, such that the change of ceramide composition might alter the organization and association of glycosyltransferases, resulting in the selective synthesis of the terminal carbohydrate structure. This fourth possibility is a combination of the second and third possibilties and could explain the change of glycolipid structure associated with differentiation and development. The correlation between ceramide and carbohydrate structure as described in this paper is based on human erythrocyte glycolipid within the same lacto-series structure. A correlation

between ceramide composition and carbohydrate structure

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among different molecular species has been well known in brain glycosphingolipids-i.e., cerebrosides have longer-chain fatty acids and their a-hydroxylated derivatives (21), whereas gangliosides have shorter-chain fatty acids (22). These two are entirely different molecular species, and this comparison is in a different category as described in this paper. In human erythrocytes globoside has mainly long-chain fatty acids, and the blocking of globoside synthesis such as in blood group p individuals leads to an accumulation of lactosylceramide having long-chain fatty acids (23). It is of interest to study whether or not these correlations between ceramide composition and carbohydrate structure are preserved in transformed malignant tissues. An unusual ceramide has been found in a glycolipid accumulated in human cancer. A ceramide pentasaccharide with LeX determinant (lacto-N-fucopentaosyl III ceramide) that accumulates in some human adenocarcinomas has a unique ceramide with 16:0 or 16:0-a-OH fatty acid and trihydroxysphinganine (phytosphingosine) (24). In addition, a fucosylceramide, a unique glycolipid present in certain human colonic cancers, also has a unique ceramide with 14 fatty acid and icosasphingenine (25). These unique ceramides may selectively associate with specific fucosyltransferases. Those glycolipids described in Table 1 are characterized by the predominance of either a short (14-16 carbons) or a long (20-24 carbons) fatty acid. In contrast, many simpler glycolipids such as mono- to tetrahexaosylceramides contain ceramides with both short and long fatty acids in about equal quantity, showing double or triple bands on TLC. In general, the upper band represents glycolipids having the longer fatty acids and the lower band represents those with the shorter fatty acids or a-hydroxyl derivatives. Glycosyltransferases involved in synthesis of these simpler glycolipids may have no preferential association with special-ceramide species. The triple bands of gangliotriaosylceramide of mouse lymphoma L5178Y (26) have been identified as those having three classes of fatty acids-i. e., 22-24:0, 16:0, and 16:0-a-OH (27). It is assumed, therefore, that glycosyltransferases involved in the synthesis of this glycolipid have no apparent ceramide specificity. An apparent specificity of glycosyltransferases toward the ceramide structure may be more obvious for those involved in synthesis of the terminal structure of a relatively long carbohydrate chain. It is also possible that ceramides may have an effect on the reactivity of the carbohydrate chain of a glycolipid. As previously described (26), the mouse lymphoma L5178Y was found to have three bands of gangliotriaosylceramide (GgOse3Cer). The high expressor clone consistently had three bands of GgOse3Cer with different fatty acids (20-24:0, 16:0, and 16:0a-OH), whereas the low expressor clone always lacked the third band-i.e., 16:0-a-OH fatty acid. Thus the cellular reactivity to anti-GgOse3Cer was closely related to the ceramide species rather than to the chemical quantity of the glycolipid (26). The presence of a similar multiple molecular species of GgOse3Cer was noticed in fully differentiated macrophages, which showed strong reactivity with anti-GgOse3Cer antibody. Conversely, their precursor Ml cell,t at a less differentiated stage, had a single molecular species of GgOse3Cer but lacked the second or third molecular species and had a much lower reactivity with anti-GgOse3Cer antibody (unpublished data). These results indicate that ceramide composition may well define the orientation and organization of glycolipid carbohydrate chains assembled in the lipid bilayer. The reactivity of glycolipids at the cell surface with their ligands is defined by multiple factors. The reactivity of N1L cell t Mouse

myelocytic leukemia cell line established by Ichikawa (28) which differentiates into macrophages under certain conditions.

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Biochemistry: Kannagi et al.

globoside with galactose oxidase was greatly enhanced when cells were pretreated with a lectin such as concanavalin A (29). The reactivity of ganglio-N-triaosylceramide on L5178Y cells with galactose oxidase and with specific antibodies was greatly enhanced when cells were treated with a sialidase (unpublished data). These results suggest that the reactivity of membrane glycolipid is greatly influenced by the organization and dynamic state of coexisting glycoproteins on the surface membranes. Now we have proposed another factor that affects the reactivity of glycolipids, namely the ceramide structure. We propose as a working hypothesis that ceramide composition and structure may define the association with a multiglycosyltransferase system (30) in membranes and may affect the cooperativity of the enzymes (31). The ceramide portion may also influence the orientation of the determinant structure, which greatly affects the reactivity of glycolipids with various ligands. A well-concerted relationship between the terminal sugar structure and ceramide composition as found in normal cells and tissues could be disarranged in certain pathological processes such as cancer. A breakdown in the normal relationship between ceramide and sugar synthesis may lead to the altered expression of glycolipid antigenicity in cancer cells. This investigation has been supported by Research Grants CA-20026 and CA-19224 from the National Institutes of Health. 1. Yamakawa, T. & Nagai, Y. (1978) Trends Biochem. Sci. 3, 128-131; 2. Hakomori, S. (1981) Annu. Rev. Biochem. 50, 733-764. 3. Hakomori, S. (1981) Sem. HematoL 18, 39-62. 4. Marcus, D. M., Kundu, S. K. & Suzuki, A. (1981) Sem. Hematol 18, 63-71. 5. Marcus, D. M. & Schwarting, G. A. (1976) Adv. ImmunoL 23, 203-240. 6. Kannagi, R., Fukuda, M. N. & Hakomori, S. (1982) J. BioL Chem. 257, in press. 7. Folch-Pi, J., Arsov, S. & Meath, J. A. (1951)J. BOL Chem. 191, 819-831.

Proc. Nad Acad. Sci. USA 79 (1982) 8. Yu, R. & Ledeen, R. W. (1972)J. Lipid Res. 13, 680-686. 9. Solter, D. & Knowles, B. B. (1978) Proc. NatL Acad. Sci. USA 75, 5565-5569. 10. Hakomori, S., Nudelman, E., Levery, S., Solter, D. & Knowles, B. B. (1981) Biochem. Biophys. Res. Commun. 100, 1578-1586. 11. Gooi, H. C., Feizi, T., Kapadia, A., Knowles, B. B., Solter, D. & Evans, J. M. (1981) Nature (London) 292, 156-158. 12. Watanabe, K. & Arao, Y. (1981) J. Lipid Res. 22, 1020-1024. 13. Watanabe, K., Powell, M. D. & Hakomori, S. (1979) J. Biol Chem. 254, 8223-8229. 14. Laine, R., Stellner, K. & Hakomori, S. (1974) in Methods in Membrane Biology, ed. Korn, E. (Plenum, New York), Vol. 2, pp. 205-247. 15. Watanabe, K., Laine, R. A. & Hakomori, S. (1975) Biochenistry 14, 2725-2733. 16. Watanabe, K. & Hakomori, S. (1979) Biochemistry 24, 5502-5504. 17. Nilsson, O., Mansson, J. E., Tibbin, E. & Svennerholm, L. (1981) FEBS Lett. 133, 197-200. 18. Kishimoto, Y. & Radin, N. W. (1959)J. Lipid Res. 1, 79-82. 19. Rosenberg, A. & Stem, N. (1966) J. Lipid Res. 7, 122-131. 20. Breimer, M. E., Hansson, G. C., Karlsson, K. A. & Leffler, H. (1980) in Cell Surface Glycolipids, ed. Sweeley, C. C. (Am. Chem. Soc., Washington, D.C.), pp. 79-104. 21. Thierfelder, H. & Klenk, E. (1930) Die Chemie der Cerebroside und Phosphatide (Springer, Berlin), pp. 1-60. 22. Klenk, E. (1942) Ber. Dtsch. Chem. Ges. 12, 1632-1636. 23. Marcus, D. M., Naiki, M. & Kundu, S. (1976) Proc. Nati Acad. Sci. USA 73, 3263-3267. 24. Yang, H. J. & Hakomori, S. (1971)J. BioL Chem. 246, 1192-1200. 25. Watanabe, K., Matsubara, T. & Hakomori, S. (1976) J. BioL Chem. 251, 2385-2387. 26. Young, W. W., Jr., Durdik, J. M., Urdal, D., Hakomori, S. & Henney, C. S. (1981)J. Immunol 126, 1-6. 27. Hakomori, S., Urdal, D., Yokota, M. & Young, W. W., Jr. (1980) Proc. Jpn. Soc. Med. Mass Spectrom. 5, 2-23. 28. Ichikawa, S. (1969) J. CelL Physiol 74, 223-234. 29. Gahmberg, C. G. & Hakomori, S. (1975) J. Biol, Chem. 250, 2447-2451. 30. Roseman, S. (1970) Chem. Phys. Lipids 5, 270-297. 31. Kijimoto-Ochiai, S., Yokosawa, N. & Makita, A. (1980) J. BioL Chem. 255, 9037-9046.