Sep 12, 1983 - Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma ...
pp.2355-2361, 1983
The EMBO Journal Vol.2 No.12
Stage-specific embryonic antigens (SSEA-3 and -4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells Reiji Kannagi, Nancy A. Cochran, Fumitsugu Ishigami, Sen-itiroh Hakomori, Peter W. Andrews1, Barbara B. Knowles1 and Davor Solterl* Biochemical Oncology, Fred Hutchinson Cancer Research Center, Seattle, WA 98104, and 'The Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104, USA Communicated by C.F. Graham Received on 12 September 1983
Two monoclonal antibodies (MC631 and MC813-70) raised against 4- to 8-cell stage mouse embryos and a human teratocarcinoma cell line, respectively, detect the stage-specific embryonic antigens, the previously defined SSEA-3 and SSEA4, described herein. These antibodies were both reactive with a unique globo-eries ganglioside with the structure shown below: a
NeuAca2- 3Gal3l- 3GalNAc3l- 3Galal- 4Galo1- 4GIcl1- ICer b
The antibodies were found to recognize sequential regions of this ganglioside, i.e., MC813-70 recognizes the terminal 'a' structure whereas antibody MC631 recognizes the internal 'b' structure. Thus, a set of two antibodies defines this unique embryonic antigen. During differentiation of human teratocarcinoma 2102Ep cells, the globo-series glycolipids defined by these antibodies decrease and the lacto-eries glycolipids, reacting with the SSEA-1 antibody appear. This antigenic conversion suggests that a shift of glycolipid synthesis from globo-series to lacto-series glycolipids occurs during differentiation of human teratocarcinoma and perhaps of pre-mplantation mouse embryos. Key words: embryonic antigens/glycolipids/human terato-
carcinoma/monoclonal antibodies/mouse embryos Introduction Changes in cell surface molecules during the course of murine embyronic development have been analyzed and documented using immunological methods (Jacob, 1977; Solter and Knowles, 1979). Several of the developmentally regulated antigens expressed by pre- and peri-implantation stages of mouse embryos are also found on the surface of murine teratocarcinoma cells. Many of these antigenic determinants are carbohydrate in nature and include epitopes of the Forssman antigen (Stern et al., 1978; Willison et al., 1982), globoside (Willison et al., 1982) and the stage-specific embryonic antigens SSEA-1 and -3 (Solter and Knowles, 1978, 1979; Hakomori et al., 1981; Gooi et al., 1981; Kannagi et al., 1982b, 1983b; Shevinsky et al., 1982). The cell surface phenotype of human teratocarcinoma cell lines undergoing differentiation has also been analyzed under *To whom reprint requests should be sent.
(cl IRL Press Limited, Oxford, England.
the assumption that as changes in the phenotype of differentiating murine teratocarcinoma cell lines reflect those of the developing mouse embryo, so changes in antigenic phenotype of human teratocarcinoma cells might provide clues to those occurring during early human embryonic development. Interestingly, some of the mouse embryonic antigens are detected on human teratocarcinoma cells. For instance, an antigen detected on the embryonal carcinoma (EC) cells of the F9 murine teratocarcinoma cell line, using syngeneic antisera to F9 cells, has also been detected on some human teratocarcinoma cell lines (Holden et al. 1977; Hogan et al., 1977). The monoclonal antibody defined antigen, SSEA-1, which first appears on the 8-cell stage mouse embryo, was also detected on some cells in a variety of human teratocarcinomaderived cell lines (Andrews et al., 1980). However, SSEA-1 is not expressed on the human EC cell surface but rather on that of some differentiated derivatives of EC cells (Andrews et al., 1982; Damjanov et al., 1982). Human EC cells do express SSEA-3, an antigenic determinant detected on zygote and cleavage-stage mouse embryos (Shevinsky et al., 1982). The detection of embryonic antigens common to mouse embyros and human teratocarcinomas may reflect the conservation of expression of such antigenic determinants on functionally related cells from different species. During the course of differentiation of human EC cells, there is a transition from the SSEA-3 + /SSEA-1 - to the SSEA-1 + phenotype, with a diminished expression of SSEA-3 (Andrews et al., 1982). Such a phenotypic transition is also observed during pre-implanation development of the mouse embryo (Shevinsky et al., 1982). As a first step to exploring the functional implications of the change in cell surface carbohydrates, we have characterized these changes on a molecular basis and find that the cell surface phenotype reflects a profound shift of synthesis of glycolipid core structure upon differentiation. Here we present a further characterization of the glycolipidbearing SSEA-3 and describe a new, related stage-specific embryonic antigen, SSEA-4. We find that both SSEA-3 and SSEA-4 are epitopes of a globolipid unique to human teratocarcinoma cells.
Results Reactivity of monoclonal antibodies with embryos and cells MC813-70, selected for reactivity with the human teratocarcinoma cell surface, was screened on a panel of human and mouse cell lines (Table I). The only reactivity observed was with human erythrocytes and with the K562 erythromyeloid leukemia-derived cell line (Lozzio and Lozzio, 1975); this reactivity, in indirect radioimmunoassay (RIA), was 2- to 3-times above the background counts but < l10o of the counts bound to an equivalent number of cells from the human teratocarcinoma cell lines. MC813-70 reacts homogeneously with mouse oocytes and with all cells of all mouse pre-implantation embryo stages up to and including the early blastocyst; in expanded blastocysts, reactivity is restricted to the inner cell mass; the trophoblast is negative (data not shown). This reactivity pattern on cells and embryos 2355
R. Kannagi el al.
Table 1. Reactivity of NIC813-70 on huntnlL and nIoLse Cells
PositiV c
testted
Ccli lines, H Lnt1n Teratocarciroma
3
Hepatonta
()
Fibroblast (normal Er\ throliekemflia T-cell leukemiiia NMv eloid leuklemiia s
anfd
traInsformed)
()
0
NIouse Teratocarcinooma
Fibroblasts Parietal v olk
() sac
carcillomz
Fig. 1. GIlycolipid pattern ol hmLriiani teratocarcinomna 21()2Ep cells VisLali,cd with orcinol/H2SO4 reagents, (a); t.l.c.-imlLmunostairlingsw ith NIC631, (b); and NIC813-70, (c). Lanes I and 2, total glvcolipids in 2102Ep cells; lane 3, a standard GMib puritied from mouse lymphoma cells (major two
()
Erythroleu kemia
1)
hands). Solvent sNystem, chloroform/methanol/water (60:35:9,
Normal peripheral blood cells' Huiman
Erythrocy tes
61'
6
lymphocytes
0
6
GranuloCVstCs
()
6
0
4
0
4
8
(8
NIouse'1 Ervthrocs tes Lcukocvtcs
Motuse embryos' Oocyte through Inner cell nmass Trophectoderim
early
otf
blastocvst
expanided blastocvst
tCell lines and peripheral blood cells
svere
tested
in
8
8
0
8
incirect RIA.
"Reactivity on comparable number and/or volume of cells was
< 10%O of the reactivity xsith human teratocarcinoma cells. 'Peripheral blood from six different donors of differenit ABO type atndt sex. "Peripheral blood from f'our different mouse strains. 'Embryos derived from eight different inbred mouse strain.s. ApproximatelN 50 embryos f'rom each stage and strain were analvied bh indirect
inmmunofluoresencce.
corresponds to that previously found with MC63 1 which defines SSEA-3 (Shevinsky et al., 1982). Glycolipids in human teratocarcinoina 2102Ep cells Extracts of human teratocarcinoma 2102Ep cells were found to contain six major neutral glycolipids (GL-1 6) and one major ganglioside (GL-7) (Figure la, lane 1) (Kannagi et al., 1983b). The carbohydrate structure of these glycolipids was determined by enzymatic hydrolysis, methylation analysis, mass spectrometry, and proton n.m.r. spectroscopy after extensive purification by h.p.l.c. The details of the structural study are described elsewhere (Kannagi et al., 1983b). All of these glycolipids were identified to be of the globoseries, i.e., containing the common core structure Galoxl4Galo3I - 4Glc or GalNAcflI-3GalcvI--4Gal (see Table II). Reactivity of glycolipids with MC631 and MC813-70 MC63 1 reacted with GL-5 and GL-7 on immunostaining of t.l.c. plates (Figure lb); faint staining of GL-4 was also noted. The results of solid-phase RIA of the purified glycolipids also showed that this antibody is highly reactive with GL-5 and -
2356
v).
GL-7 and to a lesser degree with GL-4 (see Figure 2a). No reaction was detected with lacto- or ganglio-series glycolipids, including Gg3, Gg4, GM,a, GMlb, GDia, -GalnLc4 (IV3,B-GalnLc4), 3-GalNAcnLc4 (X2 glycolipid; Kannagi et al., 1982a) 3-GalNAc-globoside (IV3 3-GalNAc Gb4) (Ando et al., 1982) and sialosyl paragloboside (Table II, and Figure 2a legend). Inhibition of binding of MC631 to 2102Ep cells by glycolipid liposomes also indicated that both GL-5 and GL-7 are equally reactive with this antibody (see Figure 3a); the reactivity of GL-4 with this antibody was less prominent. A very weak reaction was observed with GL-6 and Forssman antigen. These results indicate that MC63 1 recognizes the internal core structure of GL-7 as well as other globo-series glycolipids, i.e., R-Galf3l - 3GalNAco3I - 3GaloI - 4Galo l - R. MC813-70 reacts with GL-7 (Figure ic), the major ganglioside of the human teratocarcinoma cells, identified structurally in Table II. This finding was confirmed by solid-phase RIA and inhibition of antibody binding to the cells by glycolipid liposomes (Figures 2b and 3b). Since the terminal sugar sequence of GL-7 is identical to some ganglio-series glycolipids, such as GMlb and GDi,, the reactivity of MC813-70 with these gangliosides was determined. As expected, a significant cross-reaction was observed with GMlb on t.l.c.-immunostaining (Figure lc, lane 3). However, this antibody showed only a faint reaction with GMlb and GD1, on solid-phase RIA and almost no reaction in the inhibition of antibody binding to cells. These results indicate that antibody MC813-70 mainly recognizes the terminal structure of GL-7, i.e., NeuAcce2-3GalIl-3GalNAc3l-R, which is shared with a few ganglio-series glycolipids including GMlb and GDla. The reactivity of the antibody, however, is also influenced by the internal structure to some degree. In contrast, MC631 does not react with GMit and GDla, consistent with the observation that this antibody reacts exclusively with the core oligosaccharide of the globo-series glycolipids. Thus, MC613 and MC813-70 recognize different antigenic determinants present on the same molecule. Despite the coincident reactivity of these antibodies with the cells and embryos tested, MC813-70 defines a novel stage-specific embryonic antigen, SSEA-4. Only GL-7, the major ganglioside of human teratocarcinomas, reacts with both antibodies, i.e.,
Glycolipids of human teratocarcinoma cells
Table 11. Structures of glxcolipids of human teratocarcinoma 2102Ep cells and other reference glycolipids used in this study
Glycolipid
Reactivity with: MC631 MC813-70
Structure
Glycolipids in human teratocarcinoma 2102Ep cells GL-3 (Gb3) Galcal-4GalfIl-4Glc,31 -lCer GL-4 (Gb4) GalNAco l - 3Galcsl -4Gal3 I -4GlcflI -I Cer GL-5 Gal,BI- 3GalNAco3l- 3Galal-4Galj3l - 4GlcflI - ICer GL-6 Fuces I-2GalolI -3GalNAco3l -3Galal-4Gal3l -4Glco3l - ICer GL-7 NeuAcce2- 3Gal301- 3GalNAcflI - 3Gala I -4Gal,l -4Glc l - Ie La
-
-
+ + +
± ++
+
Reference glycolipids Forssman Cytolipin R Asialo GM2 (Gg3) Asialo GM1 (Gg4)
GM1a GMlb
GDla X2 glycolipid
GalNAcal -3GalNAc3l -3Galal -4Gal3l -4Glcol- ICer
+
GalNAc,Bl-3Galcel-3Gal,3l-4Glcjl-ICer
4
GalNacaIl-4GalfIl-4Glc Il--ICer Gal,BI -3GalNAcaI - 4GalB I -4GlcIB - I Cer Gal,Bl-3GalNAco3l -4(NeuAcc22-3)Galo3I-4Glcf3l - lCer NeuAcc22-3Gal,31-3GalNAc I -4Galo3I - 4Glcf3l- lCer NeuAca2-3Galo3l - 3GaJNAco3l-4(NeuAca2- 3)Galo3l- 4GlcflI - lCer GalNAc3l -3Gal3l -4GlcNAc3l- 3Gal3l -4Glcfl - lCer
-
(IV3 /3-GalNAcnLc4) X3b glycolipid (IV3 O-GalNAcGb4)
nt
-
-
+
-
4
-
nt
GalNAcflI - 3GalNAcolI - 3Galal-4Galo3l - 4Glc, I - lCer
-
nt
Galf3l - 3Galj3l -4GlcNAcf3l- 3Galo3l-4Glco l - ICer
-
nt
NeuAca2- 3Galo3I -4GlcNAc,BI - 3GalflI - 4Glco I -1lCer
-
3-galactosyl paragloboside (IV3 3-GalnLc4) Sialosyl paragloboside (IV3 NeuAcnLc4) nt: not tested 20
b 10
50
0l
0.5
5
50 0
Glycolipid (ng/well)
0.01
0.1
1
1.0
0 0.01
Inhibitor Glycolipid Fig. 2. Reactivity of glycolipids present in human teratocarcinoma 2102Ep cells and other reference glycolipids with monoclonal antibodies as ascertained by solid-phase RIA with MC631 (a) and MC813-70 (b). 0, GL-7; ,GL-5; x, GL4 (Gb4); ^, Forssman glycolipid (lV3cGalNAc Gb4); GL-6; A, GD1a, *, GMlb. GL-3 (Gb3), GM,b, GD1a, X2 glycolipid, X3b glvcolipid (para-Forssman), 3-galactosyl paragloboside, and sialosyl paragloboside showed no reaction with MC631 (not shown in a); and Forssman glycolipid and GL-6 showed no reactivity with MC813-70 (not shown in b). L,
0.1
(jgg/tube)
Fig. 3. Reactivity of glycolipids present in human teratocarcinoma 2102Ep cells and other reference glycolipids with monoclonal antibodies as ascertained by cell binding inhibition test with MC631 (a) and MC813-70 (b). GL-6; 0, GL-7; 0, GL-5; x, GL4 (Gb4); 1, Forssman glycolipid; A, GD1a; *, GMlb. GL-3, GMlb, and GDja showed no inhibition with MC631 (not shown in a); and Forssman glycolipid and GL-6 showed no inhibition with MC813-70 (not shown in b). Ii,
embryos following
also obtained with fertilized eggs and cleavage-stage embryos (data not shown). Glycolipid changes associated with differentiation of 2102Ep
MC631 and MC813-70 are both equivalently reactive with unfertilized eggs isolated from random-bred ICR mice (Figure 4). As predicted from the epitope structure, pretreatment of unfertilized eggs with neuraminidase does not affect their reactivity with MC63 1, whereas reactivity with MC813-70 is significantly reduced (Figure 4). Identical results
cells Human EC cells like oocyte and early cleavage-stage mouse embryos, express globo-series glycolipid antigens, such as SSEA-3 and SSEA-4, and do not express the lacto-series glycolipids SSEA-1. Upon differentiation of these stem cells, and at late 8-cell stage of the developing mouse embryos,
contains both epitopes. Detection of SSEA -3 and -4 neuraminidase
were on mouse
treatment
2357
R. Kannagi et al.
SSEA-1 positive cells appear (Andrews et al., 1982; Solter and Knowles, 1978). Since these changes have only been examined at the cell surface, where the carbohydrate moiety is associated with both glycoprotein and glycolipid, it was imnportant to determine whether binding of anti-SSEA-1 is paralleled by a qualitative change in total specific glycolipids. We have previously shown that human EC cells can be induced to differentiate and express SSEA-1 by plating at low cell density (Andrews et al., 1982). Samples of viable cloned 2102Ep cells grown at high and at low density were incubated with monoclonal antibodies to SSEA-1 and SSEA-3 and
I I Fig. 4. Unfertilized mouse eggs were pretreated ( + ) or not ( ) with neuraminidase, incubated with MC631 and MC813-70 and examined using indirect immunofluorescence. There is no change in reactivity with MC631 following neuraminidase pretreatment, while the sanie treatment abolishes reactivity with MC813-70.
analyzed by flow cytofluorimetry. Less than 5% of 2102Ep cells in the high cell density cultures are SSEA-1 positive, whereas - 50W of the cells in the low cell density culture are SSEA-1 positive. More than 90% of the cells in both high and low density cultures are SSEA-3 positive. The cultures of high and low cell density were analyzed with repect to their glycolipid pattern, and to the presence or absence of SSEA-1 and SSEA-3 in the glycolipid extract (Figure 5). The presence of GL-1 through GL-7 was observed in both types of cultures and no qualitative differences were detectable by orcinolsulfuric acid reaction of the t.l.c. plates. When glycolipids separated on t.l.c. plates were incubated with MC631, the radioactive spots corresponding to GL-5 and GL-7 were reduced in the 2102Ep cl.2A6 low density differentiated cultures (Figure 5b). In contrast, when the glycolipids, separated on t.l.c. plates, were incubated with SSEA-1 antibody, a clear qualitative change was detected, i.e., glycolipids from differentiated cultures showed a strongly positive band reacting to SSEA-1 (Figure 5c). The t.l.c. mobility of the band in differentiated cultures was identical to the Zl-glycolipid of human erythrocytes (see Figure 5c, lane c). The amount of this SSEA-l-active glycolipid in differentiated cells is small; only a faint orcinol reaction was found corresponding to this band. These observations indicate that the amount of SSEA-3 -active glycolipids is reduced and new synthesis of SSEA-l-active glycolipids is induced during the course of differentiation of human EC cells. Discussion Using monoclonal antibodies, we have identified three stage-specific embryonic antigens (Solter and Knowles, 1978; Shevinsky et al., 1982; this report). SSEA-l is the fucosylated type 2 chain previously known as the X or Lex hapten (Gooi et al., 1981; Hakomori et al. 1981; Kannagi et al., 1982b). SSEA-3 and SSEA-4 are also carbohydrate moieties of glyco-
Fig. 5. Decrease of SSEA-3 glycolipids and appearaincc of SSEA-1 glYcolipids associated with the differentiation of humani teratocarcinoma 2102Ep cell clones. (a) Orcinol/H2SO4 staining pattern; (b) t.l.c.-immunostaining with SSEA-3 (MC631) antibody; (c) with SSEA-1 antibodY. Lane 1, 2102Ep cl. 2A6 cells plated at high cell density (undifferentiated type); lane 2, cl. 2A6 cells plated at low cell density (differentiated type); cl. 4D3 plated at high cell density (undifferentiated type); lane c, human type 0 erythrocyte neutral glycolipid mixtures in the aqueous layer of Folch's partition serving as control. Solvent system, chloroforrm/methanol/w!ater (60:35:9, v v v). For abbreviations and structure of human teratocarcinoma glycolipids, see Table I. Y2, Zl and Z2a glycolipids are SSEA-1 active glycolipids found in human erythrocytes having the following structures: Y2 glycolipid, Gal31-4(Fuc"l -3)GIcNAc31 -3Gaj3l 4GlcNAc3l - 3Gald3- 4Glc31l ICer; Zl glycolipid, Gaj3l -4(Fuca I -3)GlcNAcj31 - 3GaIj3l - 4GlcNacI 3Galj3l- 4GlcNAcj3l -3Gal31- 4Glcj3I- Cer; Z2a glycolipid, Gal(31 - 4(Fuc( I - 3)GlcNAc311- 3Gal3l -4(FucY I- 3)GlcNAc3 1- 3Gall 1- 4GIcNAcj31- 3Gaij3 1- 4Glcq3I - I Cer. For details see Kannagi et -
al. (1982b).
2358
Glycolipids of human teratocarcinoma cells
lipids. The antibody directed to SSEA-1 was produced by immunization with mouse EC cells and is reactive with mouse embryos; the antibody to SSEA-3 was prepared against 4- to 8-cell stage mouse embryos and reacts with human EC cells; the antibody to SSEA-4 was prepared to human EC cells and reacts with mouse embryos. Thus, a combination of the monoclonal antibody approach and the use of human and mouse EC cells has proven to be a powerful tool for detecting the cell surface molecules present on the limited amounts of cells available from the embryo. The fact that these antigenic determinants were identified on glycolipid molecules implies that carbohydrates may play a major, as yet unidentified, role during early embryogenesis. The globo-structure has been found only in glycolipids, and not in glycoproteins of F9-teratocarcinoma (Willison et al., 1982). We, however, find that the SSEA-3 and SSEA-4 determinants are associated with a variety of cell surface glycopeptides (Shevinsky et al., 1982; and unpublished results). The specificity of monoclonal antibodies for defined carbohydrate sequences offers an advantage over lectins for their use in carbohydrate determination; in addition, they are not spontaneously toxic. Since the range of sugar sequences recognized by a single monoclonal antibody is usually within three to four monosaccharide units, a complete recognition of carbohydrate sequences longer than three to four monosaccharide units may require more than one monoclonal antibody with the ability to recognize sequential regions. Our monoclonal antibodies, MC813-70 and MC631, represent a good example of a set with such a sequential recognition. A combination of these two antibodies defines a new type of
ganglioside described as GL-7 (IV3 NeuAc 2-3 GalGb4) which has a terminal structure similar to GMlb and GD, and an internal structure identical to globoside. Recognition of internal structures of an oligosaccharide by anti-li antibodies (Watanabe et al., 1979; Feizi et al., 1979) as well as by antiForssman/globoside antibodies (Naiki and Marcus, 1977; Marcus et al., 1981) has been described. Since MC631 and MC813-70 detect antigens on human teratocarcinoma cells and on the same stages of the mouse embryos, it is possible that the major antigen recognized by these antibodies is the GL-7 ganglioside. GL-7 carries the longest carbohydrate chain among the glycolipids detected in the human EC cells; glycolipid antigens with shorter carbohydrate chains, such as Gb3, are frequently not detectable unless the cell membrane is disrupted (Hakomori and Kannagi, 1983). The classical carbohydrate antigens carried by glycolipids, such as Ii and H antigens have also been shown to be expressed in mouse embryos in a stage-specific manner (Kapadia et al., 1981; Knowles et al., 1982). Ii, ABH, and SSEA-1 antigens are carried by a common internal carbohydrate structure consisting of repeating N-acetyllactosamine units, the lactoseries glycolipids. The transition among these antigens observed during the course of embryogenesis has been assumed to occur through an external modification by the addition of a few sugars to the common internal core structure (Gooi et al., 1981). Sequential, stage-dependent expression of globo-series glycolipids such as globotriaosylceramide Gb3, globoside and Forssman antigen has also been demonstrated in the early pre-implantation mouse embryo (Willison et al., 1982). In parallel to the antigenic transition from SSEA-
I
UDP-0( qtoc~
Gb3
Gb4
\UDP-0
\ UDP--
I
Gb3
IO Gb4 2 0,
.-
t
(i)
C@QSCOZ~ (I)
Sequence a
Gb5
0>2c0
O-O .C0-00000~~
4,,
NeuAcGb5
I
(GL7))
O~c 4 04-0-0c~
--
Gb5l NeuAcGb5
QC
Sequence b
(GL7)
Sequence a
Sequence c
Fig. 6. Conversion of glycolipid synthesis from globo-series to lacto-series which occurs associated with differentiation of human teratocarcinoma cells and in the early mouse embryo. Symbols for sugars are as follows: 0 (Glc, 0 Gal, 0 GlcNAc, Q GaINAc, A Fuc, * NeuAc. Symbols linked horizontally represent fi linkage and those linked perpendicularly represent a linkage. The scheme represents a drastic change in the flow of glycolipid synthesis from globoseries to lacto-series during differentiation. The synthesis of globo-series is initiated by addition of a-Gal to lactosylceramide followed by addition of GaINAc, Gal, and Fuc or NeuAc which leads to the formation of the extended globo-series glycolipids (sequence a). SSEA-3 antibody is directed to the extended globo-series glycolipids, and SSEA4 antibody recognizes, cooperatively with anti-SSEA-3, the ganglioside GL-7 (NeuAc GbM). The sites of recognition of SSEA-3 antibody and SSEA4 antibody are shown by solid and dotted lines, respectively. The synthesis of lacto-series glycolipids is initiated by addition of GlcNAc to lactosylceramide followed by chain elongation through alternating addition of Gal and GlcNAc, thus forming a branched (I) or unbranched (i) polylactosamine structure (sequence b). Some of the unbranched polylactosamines, but not the branched polylactosamine (Kannagi et al., 1982b), are converted to the X-hapten structure forming SSEA-1 (sequence c). The major flow in sequence a occurs at the earliest stages of mouse embryogenesis and in undifferentiated human teratocarcinoma cells. Sequence a is reduced by inhibition of Gb3 synthesis, and the major flow is shifted to sequence b by activation of Lc3 synthesis, which eventually leads to sequence c in late cleavage-stage embryos and upon differentiation of human EC cells.
2359
R. Kannagi et a/.
3 + /SSEA-1 to SSEA-3 - /SSEA-l + that occurs in developing mouse embryos, we now report a decrease in SSEA-3 + globo-series glycolipids and the appearance of SSEA-1 + lacto-series glycolipids in differentiating human teratocarcinomas. Thus, not only does a terminal modification of carbohydrate chain occur, as postulated previously (Gooi et al., 1981), but also a dramatic shift from globo-series synthesis to lacto-series synthesis during differentiation (see Figure 6). A crucial change could be the inhibition of UDP-Gal:lactosylceramidef3-galactosyltransferase and activation of UDP-GlcNAc :lactosylceramide3-N-acetylglucosaminyltransferase, thus directing glycolipid synthesis from the globo-series to the lacto-series. A similar change of glycolipid synthesis from ganglio-series through lacto-series to globoseries during the differentiation of the myeloid leukemia cell line M l to macrophages was observed (Kannagi et al., 1 983a). Therefore, a combination of terminal modification and shifting of core structure synthesis from one series to another may result in dramatic modifications of cell surface carbohydrates.
Materials and methods Cells, eIn)rvos and a17tibodies The clonally derived humnan EC cell lines, 2102Ep cl. 2A6 and 4D3, have been described (Andrews et a/. 1980, 1982; Wang et al., 1980). When maintained at a high cell density (seeded at > 5 x 106 cells/75 cm2 flask), cultures contained mostly EC cells, which are SSEA-3 ', and only few SSEA-1 ' diff'erentiated cells; when plated at a low cell density (105 cells/75 cm2 tlask), many non-EC, SSEA-l + diff'erentiated cells appear although EC cells persist. Maximum prodLtCtion of' SSEA-1 cells is achieved 5 - 7 days after seeding, after which EC cells begin to overgrow. Mouse embryos were isolated and reactivity with monoclonal antibodics examined as described (Shevinsky et al., 1982). When indicated, zonia pellUcidiat'ree embryos were exposed to neuraminidase (from Vihrio cholerae, Calbiochemn, La Jolla, CA), 1t) U/ml of' Whitten miiediuLm, f'or h at 37°C before reacting with antibodies. Monoclonial antibody to SSEA-3 (MNC631) was derived by t'flsion of SP2/0 mouse mveloma cells sith splenocytes f'rom a Fisher rat immunliZed sith 4- to 8-cell stage, zona pellucida-free mouse embryos (Shevinskv et al.. 1982). Monoclonal antibody to SSEA-4 (MC813-70) was derived f'rom the fusion of splenocytes of a BALB/c mouse, immuIlized with 2102Ep cells, with SP2/0 myeloma Cells. Supernatants f'rom wells containinag growing colonies, sere tested tor reactivits sith 2102Ep cells by indirect RIA aild sith prcimplantation mou.se ernbrvos bs indirect immunofluorescenrce (Shesinskv et a!., 1982). Clone MC813-70, which secreted IgG3 antibody reactive with both 2102Ep atid mouse embryos, sas selected t'or further study. Hybrid cells producing M,C631 andi NIC813-70 sere injected into pristane-primed nudc mice or BALB/c mnice, respectivelv. Ascites fluid svas removed, clarified bv cecntrifugation and stored at - 70°C. Cell lines and humain peripheral bloocd cells swere tested by ani inidirect R1A. Llvtraciiot1 and pafri/catian1 o/)gf' col0ipis' Packed 2102Ep cells (100 nl), wvere homiiogenizeed and extraIcted sith 20 v'olumeCs ot chlorof'orm/methaniol (2:1, 1:1, and 1:2, v/ v). At'ter Folch's palrtition (Folch-Pi el aI!., 1951), the loscr laver glvcolipids s\ere freed f'rom phospholipid conitaiinationi by acetylation (Saito 'and Hakomori, 1971). Upper and lower laver glycolipids were pooled and subjected to DEAE-Sephadex column chromatography to separate neuLtral and aLcidic glyeolipids (YuL aInd Ledeen, 1972). FuLrther puiritfication ot' the glycolipids svas perf'ormled by h.p.l.c. with a Vanrin hf.p.l.c. s,stem (Model 5000, Varnin Associates IInc, Walnut Creek, CA), Llsing a Colrnin (I cm x 50cm) of Iatrobeads (6RS 8010, It) pum diatmeter, latron L iboratories, Inc., Kandai-kU Tokyo) and oLuted sith a gradient ot' isopropsl ilcohol/hexane/wsater (Kannagi et a!., 1982b; Watanabe and Arao, 1981). CGlcolipids were preparec fromrl 2102Ep el. 2A6 and el. 4D3 cells harvested f'rom high cell population densits ctiltures (I ml packed Volume) aind t'rom losV cell populationi density cuLltures (2 ml packed Volumrle) ot' 2102Ep cl. 2A6. Thc percentage of vilblc cells rcaictiig svith antibody to SSEA-1 land SSEA-3 wsas determined bv indircct immunulofltuorescenlce aind analysis swith the Ortho C sVtofluorograf 50H. Ilelefren ttl (li}i(.s' aused in thiis studi VariOLIs glycolipicds, sith thte knosn stRICturcs liNtcci I 'ablec1, hav\ hbeen
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prepared in this laboratory. Forssmani glycolipid (IV3teGalNAcGb4) wias prepared trom sheep erythrocvtes. Asialo GMN2 (Gg3) was prepared trom guinea pig crythrocytes. Asialo CGN1 (Gg4), GN11z (113 NeuAcC,g4), atid GD,, (1131V3 NeuAcGg4) were prepared trom bovine brain. GMib1 (IV3 NeuAcG,g4) was prepared from mouse lymphoma l.5178. Sialosyl paragloboside (IV3 NeuAcrllc4), X2 glycolipid (IV3/3GalNAcniLc4) (Kannagi et a!., 1982a), s3galactosyl paragloboside (IV3i3GalnLc4) (Stellner anid Hakonmori, 1973) and X3b glycolipid (para-Forssnmani glycolipid; IV313GIaNAcGb4) (Ando et a!., 1982) wNrere isolated ftrom human erythroc te membranes. Cytolipin R (globoisotetraose; i'Gb4) was prepared from rat kidney (Siddiqui et a!., 1972) and svas a gitt of the late Dr. J. Kawanami. General methods for- the preparation of' various glycolipids were described previously (1iakoniori and Siddiqui, 1974). All of these glycolipids svere ptirit'ied on high-presstire hl.p.l.c. as described above. nultnnuno!ogwCal reactivi'ties of0/ glvcolipids T.l.c.-imrimunostaining of glycolipids swithnmonocloinal antibodies was perf'ormed as described (Magnaani et al., 1980) modit'ied on high-performailice t..c. (HPT'LC) plates (Kaninagi et a/., 1982b). Briefly, glycolipids were chromatographed on HPTLC plates (SiHPF plates, J.T. Baker Chemical Co., Phillipsburg, NJ), blocked by bovine serum albtimii (BSA) and inctibated successively with 1:10() diltited monocloinal antibody, 1:1000 rabbit anti-rat IgM (t-chain-specit'ic), and/or 1251-labeled protein A solution. At'ter swashing, t.l.c. plates sere autoradiographed. Solid-phase RIA was perf'ormed with vinyl assay strips (Costar, Cambridge, M\A). Purit'ied glycolipids (50 ng/well) were dissolved in ethanol with phosphatidyrlcholine acnd cholesterol (250 ng and 125 ng/ well, respectively), and absorbed to the bottom ot' each swell bv drying at 37°C. At'ter treatment wsith phosphate-btiffered saline (PBS) containing 5"'% BSA, the wvell was inciubated successiyelv swith 1:500 diluted moiioclonal antibody, 1:1000 diluitecd rabbit anti-rat IgM, and/or 121 -labeled protein A solution. Af'ter wsashing, thc radioactivity of' each well wvas measured in a gamma counter. The cell-binding inhibition test was pert'ormed as follows: 5 x 104 2102Ep cells svere incubated t'or 1 hiat 37°C with a 1:1000 dilution ot' monioclonial antibody (50 p1l/tube) in the presence or absence ot' liposome suspensions (50 pAl/tube) containing yariouLs amounts ot' glycolipids. Liposomes were niade t'rom 50 pg of purified glycolipid, 200 ptg of phosphatidylcholine, 150 ptg of cholesterol, and 7.5 pg of dicetylphosphate, su.spended by sonication in t).25 ml ot' PBS. Thi.s fiposomeCSIspension sswas serially diluted in PBS. Af'ter incubation at room tempetature for h, thc cells were sashed three times wyith PBS and incubated with 50 ptl/ttibe ot' ai 1:1()00 dilution of' a rabbit aiiti-rat IgN1 at roomii temperature for 1 h, sashed, and finally incuibated vith i251 labeled protein A soltition. Radioactivity adsorbed to tle cells swas determiined in a
gamma
counter.
Acknowledgements TFhis studyi has been suIpported by research grants f'rom the National InstitutCs of Health CA-20026, GNM-23100 (to S.H.), CA-29894 (to P.W.A.), CA-18470 (to B.B.K.), CA-10815 (B.B.K. and D.S.), HD-12487 awil the National Science l~ouindationi Grant PCMI 81-18801 (to D.S.).
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