THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U S A . Val. 258, No. 14, Issue of July 25, pp. 8934-8942, 1983

New Globoseries Glycosphingolipids in Human Teratocarcinoma Reactive with the Monoclonal Antibody Directed to a Developmentally Regulated Antigen, Stage-specific Embryonic Antigen 3*

(Received for publication, February 8, 1983)

Reiji KannagiS, Steven B. LeveryS, Fumitsugu IshigamiS, Sen-itiroh HakomoriSj, Lynne H. ShevinskyB, Barbara B. Knowlesll((, and Davor SolterB** From the $Division of Biochemical Oncology, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104 and the lIWistar Institute for Anatomy and Biology, Philadelphia, Pennsylvania 19104

Glycolipids in a cultured human teratocarcinoma cell line (2102Ep) were investigated. The major glycolipids in these cells are globoseries glycolipids having the following structures:

G.lal~G~181~Gl~Bl*lCer (!, Gb3, Pk-antigen)

C~1NAcPI*3C~lal~C~lPl~Cl~Pl*lCer (b, Gbq. P-antigen)

G.lP1*3C.1NAcBl*3G~lal~4G.lPl~ClcDI*1Cer ( 5 )

Pucal*2C~1PI*3)C~1NAcP1*3G~1a1~G~1~1~G1cBI*lCcr ($1

AcuAcaZ*3G~lBl*3C~lNAcP1~3G~lal~G~lPl~c1~Pl*1Cer (2 )

Synthesis of these structures by serial addition of gal- actose, fucose, and N-acetylneuraminic acid to globo- side (Gb,) in this teratocarcinoma is obvious, although further elongation of Gb4 in human cells and tissues has not been previously found with the exception of the presence of a small quantity of Forssman glycolipid in some tissues in the human population (Fs’ group) and in some human cancers. The latter four glycolipids (b-c), with the common internal structure R-, 3GalNAcB1+3Galal+4R’, were all reactive to a mon- oclonal antibody directed to the 4- to 8-cell stage of murine embryos, known as the stage-specific embry- onic antigen 3 (SSEA-3 (Shevinsky, L. H., Knowles, B. B., Damjanov, I., and Solter, D. (1982) Cell 30, 697-705)); structure (c) showed the strongest reactiv- ity. These findings, together with the demonstration of the glycolipid nature of SSEA-1 antigen (Kannagi, R., Nudelman, E., Levery, S. B., and Hakomori, S . (1982) J. Biol. Chem. 257, 14865-14874), indicate that cell surface glycolipids play significant roles as differentia- tion antigens during the course of embryogenesis.

~

Changes in cell surface molecules have been observed dur- ing the process of embryogenesis and/or differentiation of cultured teratocarcinoma cells (see Refs. 1 and 2 for review). Many of these developmentally regulated antigens are car- bohydrate in nature and include ABH, Forssman, globoside, Ii, and SSEA-1’ (3-9). Such antigenic determinants may be

* This work was supported by National Institutes of Health Grants CA19224, CA10815, and HD12847. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 Recipient of National Institues of Health Grant CA20026 in support of this work.

11 Recipient of National Science Foundation Grant CA18470 in support of this work.

** Recipient of National Science Foundation Grant PCM 81.18801 in support of this work.

The abbreviations used are: SSEA, stage-specific embryonic an-

carried by lipids and/or by protein molecules. Precise under- standing of the glycoconjugates at the surface of embryos and teratocarcinoma cells is of increasing interest as these mole- cules may be important to the developmental potential of the cells. This paper describes characterization of the glycolipids in a cultured human teratocarcinoma, 2102Ep, cell line.

It has been previously reported that human teratocarci- noma cell lines express an embryonic antigen, SSEA-3, de- tected by a monoclonal antibody raised against 4- to 8-cell stage mouse embryos (10). The antigen is expressed in a stage-specific manner during early mouse embryogenesis and a change in the expression of SSEA-3 is also detected during the course of differentiation of human teratocarcinoma cells (11). The presence of an embryonic antigen common to mouse oocytes, mouse embryos, and human teratocarcinoma cell lines is of interest because of the possibility of conserved expression of such an antigenic determinant on functionally related cells from different species. The SSEA-3 antigenic determinant appears to be carbohydrate in nature and carried by both membrane glycolipids and glycoproteins (10). We have now purified glycolipids from human teratocarcinoma cells and studied their structure and reactivity with this monoclonal antibody.

MATERIALS AND METHODS

Cells and Antibodies”B102Ep cells were derived from a surgical specimen of a primary testicular germ-cell tumor containing embry- onic carcinoma and yolk sac elements (11, 12). The cells were main- tained in Dulhecco’s modified minimal essential medium supple- mented with 10% fetal bovine serum. For glycolipid analysis, 100 ml of packed cells were prepared from multiple harvests of cells grown in 15-cm plastic culture dishes. The monoclonal antibody to SSEA- 3, the product of a rat splenocyte fused with mouse myeloma cells (rat IgM) was prepared as described previously (10). A monoclonal antibody to Pk was a gift from J. Wiels, M. Lipinski, and T. Tursz, Institut Gustave-Roussy, Villejuif, France (13).

tigen; TLC, thin layer chromatography; HPTLC, high performance thin layer chromatography; HPLC, high performance liquid chro- matography; GC-MS, gas chromatography-mass spectrometry; PBS, phosphate-buffered saline, pH 7.4; HexCer, hexosylceramide Glcpl+ 1Cer; LacCer, lactosylceramide, Gal@l-AGlc@l+lCer; Gba, globo- triaosylceramide (ceramide trihexoside, CTH), Galal-*4Galpl+ 4Glcpl+lCer; Ghr, globotetraosylceramide (globoside), GalNAcBl- 3Gala1+4Gal@1+4Glc@1-*1Cer; iGha, isoglobotriaosylceramide, Galal+3Gal@l+4Glc@l-+lCer; iGh,, isoglobotetraosylceramide (cytolipin R), GalNAc@l+3Galal-+3Gal@1+4Glc~1+1Cer; L c ~ , lactotriaosylceramide (amino-CTH), GlcNAc~1-+3Gal@l~Glc@l+ 1Cer; nLc4, neolactotetraosylceramide (paragloboside), Ga1@1+ 4GlcNAc@l~3Gal@1-AG1c@l+lCer; Gg3, gangliotriaosylceramide (asialo GM,), GalNAc~l~4Gal@l+4Glc@l+lCer; Gg,, ganglio- tetraosylceramide (asialo GM,), Gal@l-+3GalNAc@l+4Gal@l-+ 4Glc@l+lCer; IV3aNeuAcGg4, GM,!,, NeuAca2+3Galpl”+3Gal- NAc@l-ulGal@14Glcpl+lCer; GL, glycolipid; PNA, peanut agglu- tinin.

8934

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Human Teratocarcinuma SSEA-3 Glycolipids 8935

Extraction and Purification of Glycolipids-Packed cells were ho- mogenized and extracted with 20 volumes of chloroform/methanol (2:1, 1:1, and 1:2, v/v). After Folch's partition (14), the lower layer glycolipids were freed from phospholipid contamination by acetyla- tion (15). Upper layer and lower layer glycolipids were pooled and subjected to DEAE-Sephadex column chromatography to separate neutral and acidic glycolipids (16). Further purification of the glyco- lipids was performed by high performance liquid chromatography with a Varian HPLC system (model 5000, Varian Associates Inc., Walnut Creek, CA), using a column (1 X 50 cm) of Iatrobeads (IRS 8010, 10-p diameter, Iatron, Tokyo) and eluted with a gradient of isopropyl alcohol/hexane/water (17). The solvent composition for gradient elution is shown in the legend to Fig. 1.

Carbohydrate Analysis-Purified glycolipid was permethylated (18) and isolated by gel filtration on a column of Sephadex LH-20 in chloroform-methanol (1:l). Permethylated neutral glycolipids were hydrolyzed in 90% acetic acid containing 0.5 N sulfuric acid a t 80 "C for 6-8 h (19). Methanolysis was performed to obtain derivatives of sialic acid of the acidic glycolipids (20). Partially methylated alditol acetates were analyzed by chemical ionization gas chromatography- mass spectroscopy using a Finnigan 3300 mass spectrometer with a 6110 data system. A DB-5 column (J and W Scientific Co., Rancho- Cordova, CA) was used for gas chromatographic analysis. Alterna- tively, an OV-225 column (Supelco Inc., Bellefonte, PA) was used for separation of 2,4,6-0-Me3- from 3,4,6-0-Me3-Gal.

'H-NMR was performed with deuterium-exchanged 100- to 150-pg purified glycolipids dissolved in 0.4 ml of dimethyl sulfoxide-d6 con- taining 2 1 D20 and 1% trimethylsilane (25) and recorded with a 500- MHz NMR spectrometer (model WM-500, Bruker, W. Germany) in the Fourier-transform mode usingquadrature detection, an excitation pulse angle of 90', and collecting 16,000 data points for a 4-KHz spectral width.

Purified fig n-galactosidase was a gift from Drs. Y.-T. Li and S.-C. Li, Tulane University, New Orleans, LA. &Galactosidases from jack bean, Escherichia coli and Aspergillus niger, and jack bean 8-N- acetylhexosaminidase were obtained from Sigma; Charonia lampas 8- galactosidase was purchased from Miles Biochemicals (Elkhart, IN). Defucosylation of glycolipids was done with 0.1 N trichloroacetic acid at 100 "C for 1 h, and desialylation was performed in 1% acetic acid a t 100 "C for 1 h.

Immunological Reactivities of Glycolipids-The reactivity of the glycolipid with antibodies was ascertained by three different methods. TLC immunostaining was performed as described previously (8, 21). Briefly, glycolipid was chromatographed on HPTLC plates (Si-HPF plates, J. T. Baker Chemical Co.) and reacted successively with 1:lOOO diluted monoclonal SSEA-3 antibody or Pk antibody (both rat IgM), 1:lOOO rabbit anti-rat IgM (p-chain specific), and '251-labeled protein A solution. After washing, TLC plates were subjected to autoradiog- raphy. Solid phase radioimmunoassay was performed with vinyl assay strips (Costar, Cambridge, MA). Glycolipids (-50 ng/well) were dissolved in ethanol with phosphatidylcholine and cholesterol (250 ng and 125 ng/well), dried a t 37 'C to achieve adsorption to the bottom of each well. After treatment with PBS containing 5% bovine serum albumin, the well was reacted successively with 1:500 diluted monoclonal SSEA-3 antibody (IgM), 1:lOOO diluted rabbit anti-rat IgM, and "'I-protein A solution. After washing, the radioactivity of each well was measured by a y-scintillation counter. The cell-binding inhibition test was performed as follows: 5 X lo4 2102Ep cells were incubated for 1 h a t 37 "C with a 1:lOOO dilution of SSEA-3 antibody (50 plltube) in the presence or absence of liposome suspensions (50 pl/tube) containing various amounts of glycolipids. Liposomes were made from 50 pg of glycolipid, 200 pg of phosphatidylcholine, 150 pg of cholesterol, and 7.5 pg of dicetylphosphate, suspended by sonica- tion in 0.25 ml of PBS. Serial dilution was made by diluting this liposome suspension with PBS. After incubation a t room temperature for 1 h, liposomes and unreacted antibody were removed by washing the cells three times with PBS. The cells were then incubated with 50 plltube of a 1:lOOO dilution of second antibody (rabbit anti-rat IgM) at room temperature for 1 h, washed, and reacted with 9 - labeled protein A solution. Radioactivity absorbed to the cells was determined with a y-scintillation counter.

RESULTS

Purification of 2102Ep Teratocarcinoma Glycolipids on HPLC-A typical elution pattern, on HPLC, of neutral and acidic glycolipids prepared from the teratocarcinoma cells is shown in Fig. 1. The neutral glycolipid fraction apparently

contained six glycolipids (each appeared as double spots on TLC), tentatively named GL-1 to GL-6 according to their mobility on TLC. Each glycolipid was purified to homogeneity by HPLC using a shallow gradient system (Fig. la) as shown in Fig. 2. GL-1 eluted in fractions 7-9, GL-2 in fractions 12- 14, GL-3 in fractions 21-29, GL-4 in fractions 37-42, GL-5 in fractions 45-48, and GL-6 in fractions 51-54. The acidic glycolipid fraction apparently contained only one glycolipid showing double spots (GL-7), which was eluted in fractions 53-57 with the same gradient system (Fig. lb). Purified gly- colipids GL-1 to GL-4, GL-6, and GL-7 showed a single spot on TLC after acetylation. Acetylated GL-5 was separated into three spots having very similar RF values, 0.67, 0.64, and 0.61 (solvent system, dichloroethane/acetone/water, 60:40:0.1, v/ v) and tentatively termed GL-5a, 5b, and 5c. GL-5b was a

- c +GL-l

=GL-2

Z G L - 3

Z G L - 5 +GL-6

GL-7

CC' -30 40 sv a, KJ C'C

Fraction No.

FIG. 1. Purification of human teratocarcinoma 2102Ep gly- colipids on HPLC. a, neutral glycolipids; b, acidic glycolipids (gan- gliosides). Gradient system for HPLC was from isopropyl alcohol/ hexane/water, 55:43:2 (v/v/v) to 55:3015 for 200 min a t a flow rate of 3 ml/min. The pressure was maintained between 44 to 85 atm throughout the chromatography. Eluate was collected at every 2 min (6 ml). Glycolipids in each fraction were detected by TLC stained with orcinol/HzS04 reagent. Solvent system for TLC was chloroform/ methanol/water (60:35:8, v/v/v). C, total neutral glycolipids; C', total acidic glycolipids prepared from the teratocarcinoma cells, serving as mobility controls.

FIG. 2. HPTLC pattern of purified glycolipids of human teratocarcinoma 2102Ep cells. Lane I , total neutral glycolipids; lane 2, total acidic glycolipids (gangliosides); lane 3, purified GL-3; lane 4, GL-4; lane 5, GL-5; lanes 6 and 7, GL-5a and 5c after acetylation purification; lane 8, GL-6; lane 9, GL-7. Solvent system, chloroform/methanol/water (6035:8, v/v/v); stained with orcinol/ &SO4 reagent.

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

8936 Human Teratocarcinoma S S E A S Glycolipids

1

\- I

: d-2 Lt s T n

E - ??' Y

FIG. 3. Limited mass chromatogram of partially 0-methyl- ated hexitol and hexosaminitol acetates obtained from hy- drolysis of permethylated human teratocarcinoma 2102Ep

minor component and GL-5a and c were the major glycolipids, comprising about 55 and 30% of the GL-5 glycolipid, respec- tively. Only GL-5a and 5c (Fig. 2, lanes 6 and 7) were further analyzed.

Chemical Characterization of GL-1 to 4-GL-1 and 2 showed the same TLC mobilities as HexCer and LacCer standards prepared from human erythrocytes. Based on this finding and the following results on GL-3 to 7, the structure of these two glycolipids must be Glc@l+lCer and Galfi14Glcfil"rlCer.

GL-3 was the major glycolipid in these cells, comprising about 60% of total glycolipids. GL-3 had the same TLC mobility as that of standard Gbn (CTH) of human erythro- cytes, both in the free form and in an acetylated form. Direct probe mass spectrometry showed characteristic ions for Hex- (m/z 219, 187, 155), Hex-0-Hex- (m/z 423,391), and Hex-0- Hex-0-Hex- (m/z 627, 595, data not shown). Methylation analysis showed the presence of 2,3,4,6-0-Me4-Gal (terminal Gal), 2,3,6-O-Men-Gal ( 4 G a l l + ) , and 2,3,6-0-Men-Glc ( 4 G l c l + ) a s shown in Fig. 3a. GL-3 was cleaved by fig LY-

galactosidase, yielding a glycolipid having the same TLC mobility as GL-2. GL-3 was strongly reactive with a mono- clonal Pk antibody (Fig. 46). From these findings, the struc- ture of GL-3 is identified a s G a l ~ l 4 G a l f i l 4 G l c ~ l + l C e r (Gbn).

GL-4 comigrated with a Gb, standard prepared from human erythrocytes on TLC. Direct probe mass spectrometry showed the characteristic ions for HexNAc- (m/z 260,228), HexNAc- 0-Hex- (m/z 464,432), and HexNAc-0-Hex-0-Hex- (m/z 668, 636, data not shown). Methylation analysis (Fig. 3) showed

U b ~ * - c - GL-7

c ,GL-2

G L-3

,GL-4

ZGL-5 -GL-6

c

c

t o r i g i n

lipids with monoclonal (a) SSEA-3 and ( 6 ) P* antibodies. Total FIG. 4. Immunostaining of human teratocarcinoma glyco-

neutral glycolipids from teratocarcinoma 2102Ep cells were chromat- ographed on a HPTLC plate with solvent system of chloroform/ methanol/water (603523, v/v/v) and stained by TLC-immunostaining technique (see under "Materials and Methods"). For orcinol staining pattern of total neutral glycolipids, see Fig. 2, lune I.

glycolipids. a, GL-3; 6, GL-4; c, GL-5c; d , GL-6 ( d - I , DB-5 column; d-2, OV-225 column); e, GL-7 (e -I , after acetolysis; e-2, after meth- anolysis). A mixture of acetic acid and sulfuric acid was used for the hydrolysis of GL-3 to GL-7 (19). Methanolysis was performed with an aliquot of perrnethylated GL-7 to obtain derivatives of sialic acid residues. Gas chromatography was performed on a DB-5 bonded phase fused silica capillary column with oven temperature pro- grammed from 140-240 "C a t 4 "C/min except in d-2, where the OV- 225 column was used for the separation of 2,4,6-0-Me3-Gal, and in e- 2 where the temperature was programmed a t 230 "C isothermal (DB- 5 column). Abscissa, scan number; ordinate, the sum of the intensities of the ions a t m/z [MH-GO]' and [MH-321' for 0-methylhexitol acetates, and a t m/z [MH-60]+ and MH' for 0-methylhexosaminitol acetates.

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

0

m a

Human Teratocarcinoma SSEA-3 Glycolipids 8937

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

8938 Human Teratocarcinoma SSEA-3 Glycolipids

the presence of 3,4,6-0-Me:)-GalNAc (terminal GalNAc), 2,4,6-0-Me:&al (+3Gall+), 2,3,6-0-Me:&al ( 4 G a l l + ) , and 2,3,6-0-Men-Glc ( 4 G l c l + 1 ) . GL-4 was completely cleaved by @-N-acetylhexosaminidase from jack bean and yielded glycolipid spots having the same mobility as GL-3 doublets on TLC. Thus, the structure of GL-4 is identified as GalNAc@l~3Galcul4Ga1@1-4Glc~l-lCer (Gb,).

Chemical Characterization of GL-5-GL-5 had a TLC mo- bility very similar to the standard asialo GM, prepared from bovine brain and also comigrated with IV:'/3GalNAcnLc4 (X, glycolipid, Ref. 22) prepared from human erythrocytes. Direct probe mass spectrometry of GL-5c (Fig. 5a) showed the char- acteristic ions for the structure Hex- (m/z 219,187,155), Hex- 0-HexNAc- (m/z 464,432), and Hex-0-HexNAc-0-Hex- (m/ z 872, 840). The other ions which can arise from the internal structure, -0-HexNAc-0-Hex- (m/z 450, 418), were also ob- served. The strong m/z 228 ion is the characteristic product from R-SHexNAcl- structure as reported (23, 24). The other ions observed are derived from the ceramide portion (m/z 364 from sphingosine, and others from varous species of fatty acyl chain, m/z 294, 322, 350, 549, 577). GL-5a showed essentially the same results on direct probe mass spectrometry (data not shown) as GL-5c. The only difference observed between GL-5c and GL-5a is that the ions m/z 661, 659,406, and 404 for ceramides containing C24:O and C24:l fatty acid were dominant in GL-5a and the ions m/z 549 and 294, which are characteristic for ceramides having C16:O fatty acid, were dominant in GL-5c.

Methylation analysis of GL-5c showed the presence of 2,3,4,6-0-Me4-Gal (terminal Gal), 4,6-0-Me2-GalNAc (-3GalNAc1+), 2,4,6-0-Me:&al (+3Gall+), 2,3,6-0-Mecl- Gal (4Gal1+) , and 2,3,6-0-Me:l-Glc (-4Glcl+) as shown in Fig. 3c. The same neutral sugar derivatives were detected by GC-MS using an OV-225 capillary column (amino sugar derivatives do not elute from OV-225 capillary). Permethyl- ated GL-5a yielded exactly the same partially methylated alditol acetates as GL-5c on GC-MS analysis (data not shown). These results indicate that the subtle difference in TLC mobilities between GL-5a and 5c is due to the difference in the fatty acid composition of ceramide moieties. From these findings, the structure of GL-5a and 5c must be identical with galactosyl 1-3 Gb,.

The anomeric structure of the terminal galactose residue in GL-5a and c was difficult to determine. Neither glycolipid was cleaved with jack bean @-galactosidase, although a nLc4 standard bearing the Gal@l4GlcNAc-terminus and a Gg, standard having a Gal@1-3GalNAc-terminus were com- pletely cleaved under the same conditions. Purified n-galac- tosidase from fig and @-galactosidase from E. coli or from A. niger also failed to cleave GL-5a and c. The only enzyme that cleaved GL-5a and c was &galactosidase from C. lampas, as shown in Fig. 6. The complete hydrolysis of 20 pg of GL-5a or c was obtained with 20 pl of enzyme (1 unit/ml) after overnight incubation, yielding glycolipids having the same TLC mobility as the upper and lower spots of GL-4. Since this enzyme has so far not been utilized to determine the anomeric structure of glycolipid carbohydrates, a strict control study was performed. Under the same conditions, the enzyme preparation degraded nLc4 into LC?, Gal@l-3nLc4 into Lcn, and Gg, to Ggn. Gbn prepared from human erythrocytes having a G a l n l 4 G a l terminus, and iCb:, having a Galnl"r3Gal terminus, prepared by enzymatic degradation of iGb, (cytoli- pin-R) obtained from rat kidney, were not cleaved by the enzyme preparation. These findings confirm the specificity of the enzyme toward the @-galactose terminus. Based on these findings, we conclude that the terminal galactose in GL-5a and c is the @-anomer. The finding that GL-Sa and c react

with PNA lectin can be taken as additional evidence for the @-Gal terminus (data not shown). Thus, the entire structure of GL-5a and c must be Gal@1+3GalNAc@1+3Galcul+ 4Gal@1+iGlcBl+1Cer.

This structure was further confirmed by the results of 'H- NMR study for the anomeric reasonances of underivatized GL-5a (Fig. 7a and Table I ) . Three resonances corresponded to the first three saccharide units of Gb, given by Dabrowski et al. (25): 4.80 ppm, Galnl-4; 4.26 ppm, Galfil-4; 4.17 ppm, Glc@1+1Cer. In their work and in ours, these three reso- nances were demonstrated to be shifted very little by further elongation of the saccharide chain, for the compounds tested. In addition, the B-Glc doublet can be seen to be broadened and distorted, most probably by the presence of the cu-galac-

/ 2 3 4 FIG. 6. HPTLC pattern of GL-5 after hydrolysis with C.

lampus &galactosidase. Lane 1, control GL-5a: lane 2, G I A a cleaved with the enzyme: lane 3 , control GL-5c: lane 4, GL-5c cleaved with the enzyme. Each glycolipid (5 pg) was incubated with 0.02 unit of enzyme in 20 pl of 0.1 M sodium citrate huffer, pH 4.0, with 1 mg/ ml of sodium taurodesoxycholate at 37 "C for overnight. Solvent system, chloroform/methanol/water (60:35:8. v/v/v); stained with orcinol/H2S04 reagent. Tc', sodium taurodesoxycholate; Gh,, globo- side.

--Y;oFlAc~~I-3) G & " S

a -3Gol(al-4). - ~ F Q I ( ~ I - ~ ) . b . -4uC(@l

"IKW

I1 I

,I ' \ L I"

" ~- - ~ _ - - -~-- 50 0 5

SIPPMI

FIG. 7. Anomeric region proton NMR spectra of human ter- atocarcinoma glycolipids. a, GL-5a; b, CL-6; c, GL-7. Each gly- colipid in 0.4 ml of dimethyl sulfoxide-& (25); 1000 pulse (CL-5a and 7) or 500 pulse (GL-6) at 29 "C. A line-broadening program (LB = -1.0 Hz) was applied before transformation of free induction decays.

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Human Teratocarcinoma SSEA-3 Glycolipids 8939

TABLE I Glycosyl H-I chemical shifts (ppm from trimethylsilnne) and 3J1,2 coupling constants (Hz) of glycolipids from human

terutocurcinoma cells Fucal-+P Gal@l+3 GalNAc@1+3 G a l a 1 4 G a l B 1 4 Glc@l-lCer

Glycolipid 6 ( J ) 6 ( J ) 8 ( J ) 6 ( J ) 6 ( J ) 6 ( J )

Gb4" 4.52 (8.1) 4.81 (3.6) 4.26 (7.7) 4.16 (7.7) GL-5 4.20 (7.3) 4.61 (8.3) 4.80 (3.9) 4.26 (7.3) 4.17 (7.3) GL-6 4.95 (2.4) 4.46 (7.3) 4.46 (7.3) 4.80 (3.5) 4.26 (6.3) 4.17 (7.8) GL-7 (NeuAcu2-+3) 4.24 (7.8) 4.57 (8.3) 4.81 (2.9) 4.26 (7.8) 4.19 (8.3)

a Data on Gb4 (globoside) was taken from Dabrowski et al. (25).

tose H-5 resonance, which is poorly resolved, but apparently very close to its position in Gb4 (-4.16 uersus 4.14 ppm) (25). The resonance at 4.61 ppm of GL-5a was assigned to the penultimate GalNAcPl-3 H-1, shifted downfield (A6 = 0.09 ppm) from its position in Gb4, as would be expected for glycosylation of this residue (25). One additional signal in the region for H-1 of &galactose or glucose units, at 4.20 ppm, is assigned to the terminal GalPl-3 of GL-5. Its coupling constant (J1,* = 7.3 Hz) confirms its assignment to a P anomer. In view of the fact that this resonance appears to be shifted upon further elongations of the saccharide chain (see later discussion), we consider this assignment to be of fairly high reliability.

Chemical Characterization of GL-6-GL-6 had almost the same TLC mobility as neolactonorhexaosylceramide, suggest- ing it to be a ceramide hexasaccharide. Direct probe mass spectrometry of GL-6 showed the characteristic ions for deoxyHex- (m/z 189, 157), deoxyHex-0-Hex- (m/z 393, 3611, deoxyHex-0-Hex-0-HexNAc- (m/z 639, 607) and deoxyHex- 0-Hex-0-HexNAc-0-Hex- (m/z 843,811) as shown in Fig. 5b. The strong m/z 228 ion was also observed. The other ions which can arise from the internal structures, -0-HexNAc-O- Hex- and/or -0-Hex-0-HexNAc- (m/z 450, 418) and -0- HexNAc-0-Hex-0-Hex- and/or -0-Hex-0-HexNAc-0-Hex- (m/z 654) were also observed.

Methylation analysis showed the presence of 2,3,4-0-Me3- Fuc (terminal Fuc), 4,6-O-Me2-GalNAc (-3GalNAcl4) and 2,3,6-0-Mes-Glc (-4Glcl+) (Fig. 3d). As to the derivatives of the galactose residues, GL-6 contained 2,3,6-0-Me3-Gal and twice the amount of 2,4,6-0-Me3-Gal and/or 3,4,6-0-Me3- Gal as shown in Fig. 3d-1. Since the peaks of 2,4,6-0-Me3- Gal and 3,4,6-0-Me3-Gal overlapped on the DB-5 column, an OV-225 column was utilized for the separation of those com- pounds. With this column, 2,4,6-0-Me3-Gal elutes signifi- cantly earlier than with DB-5, and 3,4,6-0-Me3-Gal overlaps with 2,3,6-0-Me3-Gal, confirming the presence of 2,4,6-0- Me:3-Gal, as shown in Fig. 3d-2. From these findings, it is obvious that GL-6 contains equimolar amounts of 2,4,6-0- Me:]-Gal, 2,3,6-0-Me3-Gal, and 3,4,6-0-Me3-Gal (-+3Gall+, +4Gall+, and +2Gall+), respectively. Thus, GL-6 yielded the same partially methylated alditol acetates as those from GL-5a or 5c; the only differences are the presence of terminal fucose and 2-substituted galactose residues, with the concom- itant disappearance of the terminal galactose residue which was present in GL-5a and 5c. After defucosylation, GL-6 yielded glycolipids having the same mobility as GL-Sa and 5c. The conversion of GL-6 to GL-5 by defucosylation in 0.1 N trichloroacetic acid was quantitative. Based on these findings, the structure of GL-6 is proposed to be a fucosylated GL-5, Fucal~2Gal~l-+3GalNAc~1+3Galal+4Gal~1+ 4Glc/31+lCer.

This structure was further confirmed with the 'H-NMR study. As shown in Fig. 7b, the spectrum of GL-6 contained six anomeric resonances, two of which coincide at 4.46 ppm. Three resonances from internal aGal, @Gal, and PGlc were unchanged from their positions in the spectrum of GL-5 and

were assigned similarly. The additional signal at 4.95 pprn had the extreme downfield position and vicinal coupling con- stant (J1,* = 2.4 Hz), which is compatible with a terminal Fucal-t2 residue. The proton signals at 4.46 ppm, therefore, correspond to the GalNAcP1+3 H-1 which has shifted upfield (A6 = -0.15 ppm) and the Galpl-3 H-1 which has shifted downfield (A6 = 0.26 ppm) from their positions in the GL-5a spectrum (Table I). The exact reason for the large changes in chemical shifts for these residues is not clear at present, but an analogous effect of terminal fucosylation on the anomeric resonances of internal sugar residues is reported with a type 1 chain H-active glycolipid and has been ascribed to the effect of steric crowding upon fucosylation at the terminus (26). The type 1 chain H-terminal trisaccharide differs from that of GL-6 only at the C-4 configuration of the internal HexNAc. This difference should not alter the gross relative stereochem- istry of the substitutents. The analogy is supported by the presence of a quartet at 4.07 ppm, the position assigned for H-5 of Fucal-2 of the type 1 chain H-terminal. For the type 2 chain H-terminal, this resonance was found at 4.00 ppm (26). The other signal in this region (Fig. 7b), a triplet centered at 4.10 ppm, is most probably the H-5 of a-galactose reso- nance, shifted upfield (A6 = -0.06) from its position in GL- 5. This shift can be taken as evidence for a very long range effect of fucosylation on the steric alignment of the glycosyl chain.

Chemical Characterization of Ganglioside GL-7"The gan- glioside GL-7 showed a similar mobility to GMlb standard on TLC. GMIb was prepared from mouse lymphoma L5178Y. Direct probe mass spectrometry showed characteristic ions for the structure NeuAc- (m/z 376, 344, 312), NeuAc-O-Hex- (m/z 580, 548, 516) and NeuAc-0-Hex-0-HexNac- (m/z 825, 793, 765) (Fig. 5c). Other ions arising from the internal structures -0-Hex-0-HexNAc- and/or -0-HexNAc-0-Hex- (m/z 450, 418) and -0-HexNAc-0-Hex-0-Hex- and/or -0- Hex-0-HexNAc-0-Hex- (m/z 654) were observed. Small m/z 737 and 705 fragments were also observed, compatible with a NeuAc-0-NeuAc-terminus.

GC-MS analysis of the products of methanolysis showed t h e p r e s e n c e of 4 , 7 , 8 , 9 - 0 - M e 4 - N e u A c M e ( t e r m i n a l NeuAc2-+) and a very small amount of 4,7,9-0-Me3-NeuAcMe (4NeuAcZ-t) as shown in Fig. 3e-2. Methylation analysis after hydrolysis in acetic acid-sulfuric acid showed the pres- ence of 4,6-O-Me2-GalNAc (+3GalNAcl+), 2,3,6-0-Me3-Gal (+4Gall+), 2,3,6-0-Me3-Glc (4Glc l+) , and twice the amount of 2,4,6-0-Me3-Gal (-3Gall+) (Fig. 3e-I). The peak of 2,4,6-0-Me3-Gal in this case contained no 3,4,6-0-Me3-Gal as ascertained by chromatography on an OV-225 column. GL- 7 yielded a glycolipid showing double spots on TLC, having the same mobility as GL-5a and 5c after desialylation with 1% acetic acid (see Fig. 2). Thus, the structure of GL-7 is elucidated to be NeuAca2+3Gal@l+3GalNAc~l+3Gal~l+ 4 G a l ~ l " + 4 G l c ~ l ~ 1 C e r accompanied by a small amount of the disialylated derivative.

The structure was further confirmed by the results of 'H- NMR study, which showed the presence of similar anomeric

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

8940 Human Teratocarcinoma SSEA-3 Glycolipids

TABLE I1 Binding of the monoclonal SSEAJ antibody to various glycolipids Glycolipids were immobilized at the bottom of vinyl strip and

sequentially reacted with monoclonal SSEA-3 antibody, rabbit anti- rat IgM (p-chain specific), and ‘T-protein A. For details see under “Materials and Methods.”

Glycolipid

GL-3 (Gb3) GL-4 (Gb,) GL-5cb GL-6 GL-7 Forssman Gg4 (asialo GM,) Gg, (asialo GMz) IV3i3GalnLc4 IV3i3GalNAcnLc4

Binding of ’l-Protein A

1.6 n g 6.3 ng 50 ng

0 0 0 922 6041 7609

4778 6658 7773 0 412 6699

2061 5487 0

5331 674 3351

0 0 0 0 0 0 0 0 0 0 0 200

ng glycolipid/well. *GL-5a showed essentially the same reactivity in other experi-

ments.

Inhlbltor Glycollpld (pq/tube)

FIG. 8. Inhibition of binding of the monoclonal SSEA-3 an- tibody to human teratocarcinoma 2102Ep cells by liposomes containing various glycolipids. 0, GL-5a; 0, GL-4; 0, GL-3; A, iGb4 (cytolipin R); W, Gg3 (asialo GMI).

proton resonances as found in GL-5 (Fig. 7c and Table I). The Gal@1+3 H-1 moved downfield by 0.04 ppm, as would be expected upon sialylation of this residue; the GalNAcB1- 3 H-1 moved upfield -0.04 ppm. The characteristic doublet of doublets for the H-3, of sialic acid was found at 2.77 ppm (not shown), which is within the region between 2.77-2.75 ppm. This signal is found for all terminal a2-3 sialylated glycolipids we have tested.’ The position of this resonance also has been used extensively in studies of oligosaccharides and glycopeptides for determination of sialic acid linkage with a high degree of reliability (see for example, Vliegenthart et al. (41) and references cited therein). The triplet a t 4.13 ppm is again assigned to the H-5 of a-galactose which is shifted upfield (A6 = -0.03) less than for GL-6.

Immunological Reactivity of Teratocarcinoma Glycolipids to SSEA-3Antibody-As shown in Table 11, GL-4 to 7 all reacted with the antibody to SSEA-3 in the solid state radioimmu- noassay; the Forssman antigen was also weakly reactive. GL- 5 showed the highest reactivity. Significant reactivity of GL- 4 and GL-5 with the antibody was detected by TLC immu- nostaining using a 1:250 dilution of the antibody (Fig. 4 4 . However, when a 1:lOOO dilution was used, only GL-5 was significantly reactive (data not shown). Results of binding inhibition test with 2102Ep cells also showed that GL-5 had a higher affinity for the antibody than GL-4 (Fig. 8). A weak cross-reaction was observed with iGb4 (cytolipin-R) purified from rat kidney, the isomer of Gb,. A 50% inhibition of binding was obtained with 180 ng of G L - ~ c , 375 ng of GL-4, and 8.6 pg of cytolipin R per tube, respectively.

S. B. Levery, R. Kannagi, and S. Hakomori, unpublished data.

The antibody does not seem to react with the terminal structure of GL-5, which is Galpl+3GalNAc@l+R, since Gg4 having the same terminus did not cross-react with the anti- body. In addition, GL-4, GL-6, GL-7, and Forssman antigen, which have entirely different terminal structures than that of GL-5, clearly cross-reacted with the antibody. The antibody seems to recognize the internal structures of these glycolipids, most probably the R+3GalNAcpl+3Gala14Gal~l+R’, the common internal sequence of these glycolipids. That GL- 5 exhibited higher reactivity than the other glycolipids indi- cates that a favorable comformation of the internal determi- nant, R+3GalNAc~l+3Galal+4Gal/3l+R’, may be ob- tained by the Galpl-3 substitution at the GalNAc residue in the determinant. The finding that the antibody did not react with Gg3 which has the GalNAcpl4Galp-R terminal or IV3BGalNAcnLc4 (X, glycolipid, Ref. 22) which has a GalNAcp1+3Galp+R terminal indicates the importance of the 1-3 linkage between IV-pGalNAc and 111-aGal and the a-anomeric structure of the 111-Gal. The 1 4 linkage between 111-aGal and 11-@Gal also seems important, since the reactiv- ity of iGb, was significantly weaker than that of Gb,.

DISCUSSION

This study was initiated to elucidate the structure of the glycolipid antigen reactive with the monoclonal antibody di- rected to SSEA-3 in human teratocarcinoma. Glycolipids in the teratocarcinoma 2102Ep cells were thoroughly analyzed by direct probe mass spectrometry, methylation analysis, enzymatic digestion, and nuclear magnetic resonance spec- trometry after extensive purification by HPLC. Almost all glycolipids which were visible on TLC by orcinol reaction were characterized, with the exception of one minor glycolipid comigrating with GL-5a and 5c. The proposed carbohydrate structures of these glycolipids are summarized in Table 111. The human teratocarcinoma cell showed a characteristic gly- colipid composition. All of the glycolipids characterized be- longed exclusively to the globoseries glycolipids; no apprecia- ble amounts of ganglio- or lactoseries glycolipids were de- tected. The synthetic pathway of these glycolipids in this cell line appears obvious from the carbohydrate structure of these glycolipids, the sequential conversion of each precursor gly- colipid to a higher glycolipid by the stepwise addition of one terminal sugar residue.

The major terminal product of the synthetic pathway of globoseries glycolipids in human tissue was thought to be Gb, (globoside). The presence of these new structures revises the concept of the globoseries glycolipids in humans and raises the possibility that “extended globoseries” glycolipids, such as GL-5, 6, and 7, could be expressed in undifferentiated human tissues or embryos.

The presence of a large quantity of “extended globoseries” glycolipids detected in this cell line, including the novel struc- tures GL-5, 6, and 7, may be unique for human teratocarci- noma and embryo; their chemical concentration in adult human cells and tissues must be very low or undetectableP

Previously we described a ganglioside (G5) (39) which has very similar properties to teratocarcinoma GL-7 presented in this study. The TLC mobility of erythrocyte G5 was the same as a standard IV3aNeuAcGg4 and desialylated G5 had a TLC mobility identical with Gg,, similar to the teratocarcinoma GL-7 and GL-5 described in this study. A t that time, erythrocyte G5 was tentatively identified as IV3aNeuAcGg4, since the desialylated G5 reacted with a conventional anti-Gg4 (asialo GM1) antibody. The only difference between eryth- rocyte G5 and IV3aNeuAcGg4 (GMlb) was that the desialylated G5 (supposed to be Gg, at that time) was not cleaved with any exogly- cosidases tested, including the p-galactosidase from jack bean, which readily degraded a standard Gg4 prepared from bovine brain under the same condition (see footnote of Ref. 39). The behavior of desialy-

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Human Teratocarcinoma SSEA-3 Glycolipids 8941

TABLE 111 Proposed carbohydrate structures of glycolipids of human teratocarcinoma 2102Ep cells

GlvcoliDid Structure SSEA-3 activity

GL-3 GL-4 GL-5 GL-6 GL-I

Previously, Kundu et al. (40) described a disialosyl derivative of a glycolipid with a similar sugar sequence to GL-5 as a very minor component of human erythrocyte membranes. The position of carbohydrate linkages and anomeric structure remain to be elucidated. Other examples of the presence of "extended globoseries" glycolipids in human tissue are Forss- man antigen in tissues of a small Forssman positive popula- tion (27) and "para-Forssman antigen" as a very minor com- ponent of human erythrocytes (28).

GL-5 carries the terminal sugar sequence, GalP1- 3GalNAcP-, which is identical with the terminal sequence of Gg, (asialo GM,). Some of the asialo GM1-reactive antibodies may cross-react with GL-5. Because of this terminal sugar sequence, Gg, can react with PNA lectin and has been re- garded as the glycolipid receptor for PNA lectin. GL-5 can be another PNA receptor glycolipid, which is carried by the globoseries core structure. GL-6 carries an H-active terminus. It is known that the H-active terminus in erythrocytes and/ or intestinal tissue is carried by lactoseries and/or neolacto- series core structures (29); the H-terminus carried by ganglio- series glycolipids has also been reported (30). GL-6 is the first example of a globoseries glycolipid which carries the H- terminal structure. The terminal structure of GL-7 is identical with that of IV3aNeuAcGg, (GMlb). It would be of interest to test if the glycosyltransferases involved in the synthesis of the terminal structures carried by the globoseries glycolipids are identical with those active in the synthesis of the same terminal structures which are ordinarily found in ganglio- or lactoseries glycolipids in other human cells and tissues. A glycolipid having the same sugar sequence as GL-5 has been suggested to be present in cultured green monkey cells (31). However, the anomeric structure and/or linkage of sugar residues have not been fully elucidated. The anomeric struc- ture of the terminal Gal in pentaglycosylceramide isolated from green monkey kidney cells was tentatively assigned as P because the glycolipid did not have any blood group B or PI activity. The assignment of the anomeric structure by NMR was difficult because of a shortage of material (31). The chemical basis of the structure of a similar glycolipid to GL- 7 detected in chick muscle has not been described so far (32, 33).

Since the antibody defining SSEA-3 seems to react with the sequence R+3GalNAc/31+3Galal+R', the terminal structure of GL-4 (globoside) and the internal structure of GL-5,6, and 7, it is a useful reagent to detect the globoseries glycolipids. Most carbohydrate-reactive antibodies are di- rected to the terminal sugar structures; however, antibodies reacting to an internal sequence are known; a monoclonal IgM antibody reactive with both globoside and Forssman (34) and various types of Ii-reactive antibodies (29) are good examples. Even though the antibody is directed to the internal structure, its reactivity is indirectly affected by the terminal

lated erythrocyte G5 toward various exoglycosidases is very similar to that of the teratocarcinoma GL-5 in this study. Based on these findings, we predict that the structure of erythrocyte G5 is most probably identical with that of teratocarcinoma GL-7 described in this study.

structure, probably due to changes in the tertiary structure of the internal sugar chain, as suggested by the NMR study.

The presence of SSEA-3 antigens in human teratocarci- noma cells raises the possibility that the antigen is also present in human embryos and plays a role as a stage-specific antigen. The presence of P and Pk antigens on the mouse embryo has been detected using polyclonal antisera (9). It is noteworthy that the structure of SSEA-3 active human gly- colipids described in this paper includes the P-blood group antigen and its further metabolites. Since all the globoseries glycolipids so far characterized play a role as alloantigens in the P-blood group system (35), it could be predicted that some of the new structures found in the human teratocarcinoma cells may display previously uncharacterized antigens in P- blood group system. It is well known that individuals of rare pp-phenotype have a high incidence of abortion, and it is suggested to be due to the reaction of anti-PPIPk antibody in the maternal serum with corresponding antigens in the fetus (36,37). Frequency of the abortions is particularly high at the early stages of pregnancy. I t is possible that P-antigen and/ or other antigens in P-blood group system play a role as stage- specific developmental antigens not only in mouse but also in human embryogenesis, and the frequency of abortion depends upon the variable degree of surface expression of these anti- gens on the fetal cells and tissues during the course of embry- ogenesis.

Recently we have also elucidated the complete structures of the SSEA-1-containing glycolipids (8). This antigenic de- terminant, like SSEA-3, is found on the surface of murine embryo, but it is expressed at a later stage of preimplantation development (7, 10). The finding that both of these antigenic determinants are carbohydrates which can be borne by gly- colipid molecules indicates the importance of changes in the cell surface glycolipids in the developing embryo.

The antigenic transformation from SSEA-3+/SSEA-1- to SSEA-3-/SSEA-l+ status has been also detected during the course of in vitro differentiation system of human teratocar- cinoma cells (11). SSEA-1 antigens are carried by a set of lactoseries glycolipids (S), and SSEA-3 antigens are carried

HexCer-LocCer

b 1 L"-""\.~~~6\~n~~.\~......~ : Neo'ocfo-rer'es, SSEA-I ache,

j m3aFuc-nLc4 VbFuc-nLc6 Vl13aFuc-nLcg ~ mox'mol'Y ~ 01 mordo sloge

..........................................

FIG. 9. Synthetic pathways of SSEA-1 and SSEA-3 active glycosphingolipids. a, the synthetic pathway of globoseries glyco- lipids which leads to the synthesis of a set of SSEA-3 active glyco- lipids; b, the synthetic pathway of neolactoseries glycolipids, fucosy- lation at internal GalNAc residues which leads to the synthesis of a set of SSEA-1 active glycolipids (8). A switching or shift of glycolipid synthesis from pathway a to pathway b is suggested to occur in mouse early embryogenesis.

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

8942 Human Teratocarcinoma SSEA-3 Glycolipids

by a set of globoseries glycolipids. Thus, in terms of glycolipid antigens, SSEA-1 and SSEA-3 antigens belong to entirely different species of glycolipids, and the synthetic pathways for the two antigens are also entirely different (Fig. 9). There- fore, the transition in expression of these antigens observed in mouse embryogenesis and differentiation of human tera- tocarcinoma cells is not due to the simple addition of one or a few sugar residues to pre-existing carbohydrate chains, but involves dynamic changes covering multiple synthetic path- ways of cellular glycolipids, i.e. synthesis of lactoseries and globoseries glycolipids. Thus an extensive change in the syn- thesis of cell surface carbohydrates might occur between the 4-8 cell and morula stages. This type of drastic change in glycolipid synthesis involving more than one synthetic path- way is not necessarily unusual; a similar type of alteration of cellular antigenicity carried by lacto- and globoseries glyco- lipids was reported to occur during the course of differentia- tion of a mouse leukemia cell line (38).

Acknowledgments-We thank Drs. Gary Drobny, Dennis Hare, and Eric Shankland of the University of Washington for assistance in NMR spectrometry.

1. 2.

3. 4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

REFERENCES Jacob, F. (1977) Zmmunol. Reu. 3 3 , 3-32 Solter, D., and Knowles, B. B. (1979) Curr. Top. Deu. Biol. 13 ,

Szulman, A. E. (1980) Curr. Top. Deu. Biol. 14, 127-145 Stern, P. L., Willison, K. R., Lennox, E., GalfrC, G., Milstein, C.,

Secher, D., Ziegler, A,, and Springer, T. (1978) Cell 14, 775- 783

Kapadia, A., Feizi, T., and Evans, M. J. (1981) Exp. Cell. Res.

Solter, D., and Knowles, B. B. (1978) Proc. Natl. Acad. Sci. U. S.

Gooi, H. C., Feizi, T., Kapadia, A., Knowles, B. B., Solter, D., and Evans, J. M. (1981) Nature (Lord.) 292 , 156-158

Kannagi, R., Nudelman, E., Levery, S. B., and Hakomori, S. (1982) J . Bid. Chem. 257,14865-14874

Willison, K. R., Karol, R. A., Suzuki, A., Kundu, S. K., and Marcus, D. M. (1982) J. Zmmunol. 129,603-609

Shevinsky, L. H., Knowles, B. B., Damjanov, I., and Solter, D. (1982) Cell 30,697-705

Andrews, P. W., Goodfellow, P. N., Shevinsky, L. H., Bronson, D. L., and Knowles, B. B. (1982) Znt. J . Cancer 2 9 , 523-531

Wang, N., Trend, B., Bronson, D. L., and Fraley, E. E. (1980) Cancer Res. 4 0 , 796-802

Nudelman, E., Kannagi, R., Hakomori, S., Parsons, M., Lipinski, M., Wiels, J., Fellows, M., and Tursz, T. (1983) Science 2 2 0 ,

139-165

131 , 185-195

A. 7 5 , 5565-5569

509-511 14. Folch, J., Arsov, S., and Meath, J . A. (1951) J. Biol. Chem. 191 ,

15. Saito, T., and Hakomori, S. (1971) J. Lipid Res. 12, 257-259 16. Yu, R. K., and Ledeen, R. W. (1972) J . Lipid Res. 13,680-686 17. Watanabe, K., and Arao, Y. (1981) J. Lipid Res. 2 2 , 1020-1024 18. Hakomori, S. (1964) J. Biochem. (Tokyo) 55 , 205-208 19. Stellner, K., Saito, H., and Hakomori, S. (1973) Arch. Biochem.

20. Rauvala, H., and Karkkainen, J. (1977) Carbohydr. Res. 5 6 , l - 9 21. Magnani, J. L., Smith, D. F., and Ginsburg, V. (1980) Anal.

Biochem. 109, 399-402 22. Kannagi, R., Fukuda, M. N., and Hakornori, S. (1982) J . Biol.

Chem. 257,4438-4442 23. Egge, H., and Hanfland, P. (1981) Arch. Biochem. Biophys. 2 1 0 ,

396-404 24. Hanfland, P., and Egge, H. (1975) Chem. Phys. Lipids 15, 243-

247 25. Dabrowski, J., Hanfland, P., and Egge, H. (1980) Biochemistry

19,5652-5658 26. Dabrowski, J., Hanfland, P., Egge, H., and Dabrowski, U. (1981)

Arch. Biochem. Biophys. 210,405-411 27. Hakomori, S., Wang, S-M., and Young, W. W., Jr. (1977) Proc.

Natl. Acad. Sci. U. S. A. 74 , 3023-3027 28. Ando, S., Kon, K., Nagai, Y., and Yamakawa, T. (1982) in New

Vistas in Glycolipid Research (Makita, A,, Handa, S., Taketomi, T., and Nagai, Y., eds) pp. 71-81, Plenum Publishing Corp., New York

819-831

Biophys. 155,464-472

29. Hakomori, S. (1981) Semin. Hematol. 18,39-62 30. Wiegandt, H. (1973) 2. Physiol. Chem. 354 , 1049-1056 31. Blomberg, J., Breimer, M. E., and Karlsson, K-A. (1982) Biochim.

Biophys. Acta 711,466-477 32. Chien, J-L., and Hogan, E. L. (1980) Fed. Proc. 39 , 2183 (Abstr.

3040) 33. Hogan, E., Happel, R. D., and Chien, J-L. (1982) in New Vistas

i n Glycolipid Research (Makita, A., Handa, S., Taketomi, T., and Nagai, Y., eds) pp. 273-278, Plenum Publishing Corp., New York

34. Naiki, M., and Marcus, D. M. (1977) J. Immunol. 119, 537-539 35. Marcus, D. M., Naiki, M., and Kundu, S. K. (1976) Proc. Natl.

36. Iseki, S., Masaki, S., and Levine, P. (1954) Nature (Lond.) 173 ,

37. Levine, P., and Koch-Elizabeth, A. (1954) Science 120 , 239-241 38. Kannagi, R., Levery, S. B., and Hakomori, S. (1983) Proc. Natl.

39. Watanabe, K., Powell, M. E., and Hakomori, S. (1979) J . B i d .

40. Kundu, S. K., Marcus, D. M., Pascher, I., and Samuelsson, B. E.

41. Vliegenthart, J. F. G., Van Halbeek, H., and Dorland, L. (1981)

Acad. Sci. U. S. A. 73 , 3263-3267

1193-1194

Acad. Sci. U. S. A,, in press

Chem. 254,8223-8229

(1981) Fed. Proc. 4 0 , 1545 (Abstr. 37)

Pure Appl. Chem. 53,45-77

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

SolterR Kannagi, S B Levery, F Ishigami, S Hakomori, L H Shevinsky, B B Knowles and D

stage-specific embryonic antigen 3.monoclonal antibody directed to a developmentally regulated antigen,

New globoseries glycosphingolipids in human teratocarcinoma reactive with the

1983, 258:8934-8942.J. Biol. Chem. 

  http://www.jbc.org/content/258/14/8934Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/258/14/8934.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on October 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from