Qualitative Differences in Position of Sialylation - Cancer Research

(CANCER RESEARCH 46, 1395-1402, March 1986]

Qualitative Differences in Position of Sialylation and Surface Expression of

Glycolipids between Murine Lymphomas with Low Metastatic (Eb) and

High Metastatic (ESb) Potentials and Isolation of a NovelDisialoganglioside (GD1a) from Eb Cells1

Kimie Murayama2, Steven B. Levery, Volker Schirrmacher, and Sen-itiroh Hakomori

Program of Biochemical Oncology/Membrane Research, Fred Hutchinson Cancer Research Center, and the Departments of Pathobiology, Microbiology, and Immunology,University of Washington, Seattle, Washington 98104 [K. M., S. B. L, S-i. H.], and Institut fÃ¼rImmunologie und Genetik, Deutsches Krebsforschungszentrum,Heidelberg, West Germany [V. S.]

ABSTRACT

Glycolipids of murine lymphoma cell lines with low metastatic(Eb) and high metastatic (ESb) potentials have been investigated.The Eb cell line was characterized by a high quantity of gangli-

otriaosylceramide (Gg3), gangliotetraosylceramide (Gg4), GM1b,and a new type of disialoganglioside, termed GD1a. In contrast,the high metastatic ESb cell line was characterized by theabsence of these glycolipids and instead by the presence ofGM3, GM2, GM1a, GD1a, and GDib gangliosides. A clear cellsurface reactivity with monoclonal antibody anti-Gg3 (2D4) was

observed only in Eb cells. Thus, Eb cells are distinct from ESbcells in their ability to add the GalNAc residue to LacCer, supplying Gg3 for synthesis of a series of glycolipids via an asialogan-

gliotetraosyl pathway, while ESb cells are capable of synthesizing GM3, which initiates synthesis of ganglio-series gangliosides

GM2, GM1a, GD1a, and GD1b. While disialogangliosides of ESbcells were identified as GD,,, and GD,,â€ža disialogangliosideisolated from Eb cells was characterized as having a novelstructure (referred to as GD1n)as follows:

Gal/31 ->3GalNAciS1 ->4Gal/81 ->4Glc/31 ->1 Cer

3 6

Ã® ÃŽSAÂ«2 SAa2

Thus, Eb and ESb cells are clearly different in their qualitativesialylation patterns, i.e., the position of sialic acid residues. Cellsurface labeling with galactose-oxidase/NaB[3H]4 revealed a high

exposure of Gg3 and Gg4 at the Eb cell surface, while both labelswere absent in ESb cells. In contrast, ESb cells showed asubstantial label at GM1a, which was greatly enhanced aftersialidase treatment.

INTRODUCTION

Aberrant glycoslylation of both glycolipids and glycoproteinsin tumor cells has been well established (for reviews, see Refs.1 and 2), although the cell biological significance of aberrantglycosylation in tumor cells is not known. Comparisons of the

Received 8/26/85; revised 11/13/85; accepted 11/15/85.The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by a grant from the National Institutes of Health,

CA20026.2 Present address: Central Laboratory of Medical Sciences, Juntendo University

School of Medicine, Tokyo, Japan.

glycosylation patterns in tumor cells having different metastaticpotentials have been made using variant cell lines with differentmetastatic potentials derived from a single cell line. Most of thesestudies have been based on comparisons of radioactivity profilesof metabolic labeling, cell surface labeling, or radioactive lectin-blotting pattern for glycoproteins separated on sodium dodecylsulfate polyacrylamide gel electrophoresis, and/or comparison ofglycolipids separated on thin-layer chromatography (for reviews,

see Refs. 3 and 4). No information on the carbohydrate structures present in cells with different metastatic potentials hasbeen described. This paper describes the structural basis of thequalitative glycosylation differences between two lymphoma celllines with different metastatic potentials established previouslyas Eb and ESb (4-6) and the presence of a novel disialoganglioside, designated GD1n,3in non-metastatic Eb cells.

MATERIALS AND METHODS

Cells. A murine T-lymphoma cell line derived from a methylcholan-threne-induced lymphoma in DBA/2 mice (H-2d), with a defined antigen-

icity and metastatic potential, was established and termed L5178Y/ES(5). From this clone, the high metastatic subclone ESb and the lowmetastatic subclone Eb were isolated (6). Phenotypes of these variantswere stable and were transferred in February 1983 from Heidelberg,West Germany to Seattle, Washington. Eb cells were propagated inRPMI medium supplemented with 10% fetal calf serum, and ESb cellsused in this study were grown under the same conditions as above withan additional supplement of 5 x 10~5 M 2-mercaptoethanol as described

previously. The biological properties of these variants have been detailedby Schirrmacher et al. (7). More recently, however, ESb cells in ourlaboratory have been adapted to grow in the absence of 2-mercaptoeth

anol. Although glycolipid analysis of ESb cells reported in this paper wasmade of the culture described above, the same glycolipid pattern hasbeen observed in those cells grown in the absence of mercaptoethanol.4

Furthermore, those ESb cells grown in the absence of 2-mercaptoethanolshowed the same high host-killing ability (or metastatic potential) as didthe non-adapted ESb cells, and it was confirmed that the host-killingability of adapted ESb cells is higher than that of Eb cells.4 In vitro

doubling times of Eb and ESb cells (either non-adapted or adapted cells)

were approximately the same, although the Eb cells had a slightly shorterdoubling time (16 h) than ESb cells (18 h). Both cells were seeded 2 x

3This assignment is based on Svennerholm's designation (46). GD, denotes

disialogangliosides having a gangliotetraosyl core structure. Since the position ofsialic acid in this new ganglioside is not at the internal Gal (Il-Gal) but on the GalNAc(IM-GalNAc), the suffix following GD, should be different from the a, b series. Thisnew ganglioside is now designated GDiâ€ž.One of the frog brain gangliosides isolatedby Ohashi (47) contains two sialic acids at the GalNAc and can be designatedGDW.

' J. Wiels and S. Hakomori, unpublished observation.

CANCER RESEARCH VOL. 46 MARCH 1986

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DIFFERENCES IN GLYCOLIPIDS BETWEEN LYMPHOMAS WITH LOW METASTATIC AND HIGH METASTATIC POTENTIALS

105/ml and were harvested after 2 days at a cell population density of106/ml. This is the later stage of the logarithmic growth phase. (The

stationary phase of cell population growth for both Eb and ESb cells wasapproximately 1.5 x 106/ml.)

Cell Surface Labeling. Approximately 1 ml of lymphoma was harvested by centrifugation and washed twice with PBS5 at pH 7.4, and acell suspension with a density of 1 x 107 cells/0.2 ml PBS, pH 7.0, was

prepared. Aliquots of the cell suspension were treated with sialidase,trypsin, or a combination of sialidase and trypsin according to theconditions described previously (8, 9). Cells without enzyme treatmentor those treated with sialidase and trypsin were oxidized with 20 units/20 n\ of galactose oxidase, followed by washing with Mcllvaine's phos

phate-citrate buffer, pH 7.4, and cells were labeled by addition of 50 ^l(2.5 mCi) of NaB[3H]4 for 30 min at room temperature. The cell suspen

sion was then mixed with 50 n\ of PBS, pH 7.4, containing 100 ITIMcoldNaB[3H]4. After 15 min, cells were washed twice with PBS, and pelletswere stored at -20Â°C until use. Labeled cells were analyzed for glyco-

lipids after extraction with organic solvent as described below. Aliquotsof labeled cells were analyzed by sodium dodecyl sulfate-polyacrylamideslab gel electrophoresis. The surface-labeled glycolipids, separated on

HPTLC, were detected by fluorography as described previously (9).Extraction and Subsequent Fractionation of Glycolipids. Packed

cells (1 ml volume) were extracted with 5 ml isopropanol-hexane-water

(50:25:20) by sonication for 5 min and centrifugea. The pellet was againextracted with 5 ml of the same solvent mixture by sonication for 2 minand centrifuged. Subsequently, the pellet was extracted by sonication in5 ml of chloroform-methanol (2:1). Extracts were combined, evaporatedto dryness, dissolved in 12 ml chloroform-methanol (2:1), and partitioned

according to the modified method of Folch ef a/. (10) to separate theupper and lower phases. The lower phase was re-partitioned four timeswith the theoretical upper phase (chloroform-methanol-0.1% sodium

chloride, 1:10:10, v/v/v), and the combined upper phases (10) weredialyzed through a Spectrapor dialysis tube (Spectrum Medical Industries, Los Angeles, CA) and evaporated to dryness with ethanol in arotary evaporator. The mixture was dissolved in chloroform-methanol-water (8:60:32, v/v/v) and fractionated by DEAE-Sephadex chromatog-

raphy (8 ml column for glycolipid extracted from 1 ml cells) into neutralglycolipids and gangliosides (11). Gangliosides were further separatedinto mono-, di-, and trisialosyl fractions by step-wise elution with 0.015,

0.15, and 0.45 M ammonium acetate as described previously (11). Eachfraction was dialyzed and lyophilized. Glycolipids in the Folch's lower

phase were purified by an acetylation procedure (12). Gangliosides andneutral glycolipids isolated from the upper and the lower phases wereanalyzed by HPTLC using Baker's HPTLC plate (J. T. Baker, Co.,

Phillipsburg, NJ), as described previously (14,18).Antibodies Applied for Glycolipid Characterization and Determi

nation of Cell Surface Reactivity. The following monoclonal antibodieswere used for characterization of glycolipids by immunostaining according to the method described previously by Magnani ef a/. (13) andmodified by Kannagi ef al. (14) on Baker's HPTLC plates: 2D4 antibody

directed to Gg3 (asialo GM2) (15); 1B2 antibody defining A/-acetyllacto-samine (16); the antibody 38-13 defining GalÂ«1-Â»4Gal/31->4Gal structure(Gb3) (17); and the antibody to SSEA-4 directed to NeuAca2->3Gal/31->

3GalNAc01-Â»3GalÂ«1â€”Â»R(18) were established in this laboratory asdescribed previously. These antibodies were also used for cytofluoro-

metric determination of cell surface antigens as described previously

(19).Fractionation and Purification of Glycolipids. Each glycolipidfraction

separated by chromatography on Florisil column as acetates or on DEAE-

Sephadex column as free glycolipids was further separated into components by HPLC on latrobeads 6RS-8010 column in an isopropanol-

hexane-water system as described previously (20). Briefly, the columnwas equilibrated by pumping a solvent mixture, isopropanol-hexane-

5The abbreviations used are: PBS, phosphate-buffered saline; HPTLC, high-performance thin-layer chromatography; HPLC, high-performance liquid chromatography; NK, natural killer.

water (55:40:5, v/v/v), for 150 min (1.0 ml/min); then the glycolipidsample, dissolved in 220 Â¡Aof chloroform-methanol-water (60:35:8), was

injected onto the column. The flow rate was set as 0.5 ml/min, and thegradient elution program was set from Â¡sopropanol-hexane-water(55:40:5) to isopropanol-hexane-water (55:25:20) during 150 min and

was subsequently eluted with the last solvent mixture until 200 min.One-mi fractions were collected every 2 min in a fraction collector, and

2 n\ of each fraction were used for detection of glycolipids on a HPTLCplate. Glycolipids were detected by 0.2% orcinol-10% sulfuric acid. The

fractions thus separated were further purified on HPTLC, and the bandsseparated were revealed by Primulin spray (21), followed by extractionwith isopropanol-hexane-water (50:25:20, v/v/v) on sonication.

Characterization of Glycolipid Components. Glycolipids were characterized by methylation analysis (22) with identification of partiallymethylated aminosugars by gas chromatography and mass spectrometry(23, 24). The procedure has been greatly improved by the use of acapillary column with selected ion chemical ionization mass chromatography as described previously (25). Enzymatic degradation with sialidasewith or without ionic detergent (e.g., sodium taurocholate) was used todistinguish the sialosyl residue at C-3 of Il-Gal from that linked to C-3 ofIV-Gal or C-6 of NI-GalNAc. Approximately 10 /Â¿gof glycolipid with or

without addition of 20 Â¿igof sodium deoxytaurocholate were dissolvedin chloroform-methanol (2:1, v/v) and evaporated under nitrogen stream.

The residue was dissolved in 10 ^l of 100 mM acetate buffer, pH 4.5,and 10 M! of Clostridium perfringens sialidase (5 units/ml) were added.The mixture was incubated for 18 h at 37Â°C, followed by purificationthrough a "Bond-Elut C18" column (Analytichem International, Harbor

City, CA).

RESULTS

Glycolipid Profiles of Eb and ESb Lymphoma

The total glycolipids present in the upper phase and thosepresent in the lower phase of Folch's partition are shown in Fig.

1. The major component present in the lower phase of Eb cellswas identified as gangliotriaosylceramide (Gb3) (Fig. 1, lane 6),which was strongly stained by monoclonal antibody 2D4 (seesubsequent section on "Immunostaining of Glycolipids"). In con

trast, ESb cells did not contain Gg3 but contained GM3 as amajor component (Fig. 1, lane 7). The lower phase from Eb cellextract contained several weaker bands, which will be described

. â€” ~ 4Â» }LacCer

Gg3-<

GM3-

GM1b{z:

GDlaâ€”

=}Gg3â€”Glob-GM3

â€”GM2--GMla

--GDlb--GT

1 234567Fig. 1. Thin-layer chromatography pattern of murine lymphoma Eb and ESb.

Lane 7, reference Gg3from guinea pig erythrocytes. Lane 2, reference GM3fromdog erythrocytes. Lane 3, glycolipids in Folch's upper phase fraction of Eb. Lane4, the same fraction of ESb. Lane 5, lower neutral glycolipids of 0-erythrocytes.Lane 6, glycolipids in Folch's lower phase fraction of Eb. Lane 7, the same fraction

of ESb.


1396




subsequently. The upper fraction from ESb cell extract containeda series of gangliosides that had the same HPLTC mobility asGM3, GM2, GMia, GDia, and GD,b (Fig. 1, lane 4). The neutralglycolipid from the Eb upper phase was identified as gangliotet-

raosylceramide (Gg<), but no glycolipids were detectable in theupper neutral fraction of ESb cell extract.

Two major gangliosides were detected in the upper phasefrom Eb cell extract, a doublet with the same HPTLC mobility asthat of GMi and a slower-migrating band with a HPTLC mobilityslower than that of GD1a(Fig. 2, lane 3). These ganglioside bandswere susceptible to sialidase and were degraded into Gg4 (Fig.2, lane 4). Of various gangliosides detected in the extract of ESbcells, the band corresponding to GM3 was degraded into lacto-

sylceramide by sialidase, while the bands with slower HPTLCmobility were degraded into GM1a (Fig. 2, lane 5).

Gangliosides of Eb and ESb were further fractionated on HPLCin an isopropanol-hexane-water system into six fractions. The

pattern of gangliosides separated on HPLC is shown in Fig. 3.The major ganglioside component from Eb cells, representingfractions 3, 4, and 5, and a slow-migrating major component infraction 6 were isolated in a pure state. This slow-migrating

component, although partially eluted in the monosialogangliosidefraction, was found abundantly in the disialoganglioside fractionon DEAE-Sepharose chromatography. The proportion eluted inthe mono- and disialoganglioside fractions varied, and the gan

gliosides eluted in these two fractions showed identical chemicalproperties. In contrast, the monosialoganglioside fraction of ESbcells separated on HPLC is shown in the right panel of Fig. 3.These components were identified as GM3, GM2, GMia, GDia,and GD1b, as described subsequently, and were purified ashomogeneous bands, as shown in Fig. 4.

Characterization of Gangliosides from Eb Cells

GM1b. The major ganglioside present in extracts of Eb cellswas characterized as GMib based on the following results: (a) itmigrated on HPTLC in three different solvents like GM1bgangliosides extracted from L5178 cells as previously isolated andcharacterized (26); (b) it was hydrolyzed by C. perfringens sialidase in the presence and absence of Triton X-100 into a com-

Eb Esb

GMlb=:::GDia- â€”GDlbâ€”

lLacCer

GM2â€” -GMia

1 23456Fig. 2. Thin-layer chromatography pattern of gangliosides of Eb and ESb, before

and after sialidase treatment. Lanes 1 and 2 are reference compounds, as identifiedin the left margin. Lane 3, gangliosides of Eb cells before sialidase treatment. Lane4, the same as in lane 3 after sialidase treatment. Lane 5, gangliosides of ESb cellsbefore sialidase treatment. Lane 6, the same as in lane 5 after sialidase treatment.

Cl234567RC1234567 CFig. 3. Separation of gangliosides from Eb and ESb cells on HPLC in an

isopropanol-hexane-water system. Lane C, control glycolipids from top to bottomare GM3, GM2, GM,, GD,., GD,b, and GT from bovine brain. Lane R, glycolipids ofhuman teratocarcinoma 2102. Lanes 1-7 of the left are gangliosides from Eb cellseluted on latrobeads 6RS-8010 column. Lanes 1-7 on the right are gangliosidesfrom ESb cells eluted on latrobeads 6RS-8010 column. A gradient elution fromisopropanol-hexane-water (55:40:5) to isopropanol-hexane-water (55:25:20) wasperformed as described in the text. Fractions were collected in a fraction collectorat a flow rate of 0.5 ml/min/tube. From ESb cells: tubes 13-17, fraction 1 (fast-migrating component of GM3); 18-22, fraction 2 (fast- and slow-migrating components of GM3); 23-27, fraction 3 (GM2); 28-34, fraction 4 (fast- and slow-migratingcomponents of GM2); 35-45, fraction 5 (GM,.); 46-52, fraction 6 (fast- and slow-migrating components of GMU, and a part of GD,.); and 53-68, fraction 7 (GD,.,GD,b, and GT). From Eb cells: fraction 3 (fast-migrating GM,b), fraction 4 (middle-migrating GM,D), and fraction 5 (a new ganglioside, GD, J.

4 R3 R4 6 7 R< 8 9 R2 10 11 12

Fig. 4. Purified gangliosides from Eb and ESb cells and their degradationpatterns on sialidase treatment. Lane fÃ1, upper neutral glycolipids of humanerythrocytes. Lane R2, Gg4 (asialo GM,; upper band] and GM, (lower band). LaneR3, GM3 (upper band), GM2 (middle band), and GM, (lower band) isolated frombovine brain. Lane R4, GD,. (upper band), GD,b (middle band), and GT,b (lowerband) isolated from bovine brain. Lane 7, fraction 4 ganglioside of Eb cells. Lane 2,fraction 5 ganglioside of Eb cells. These bands were identified as GM,b withdifferent ceramide compositions. Lane 3, fraction 4 ganglioside of ESb cells. Lane4, fraction 5 ganglioside of ESb cells. These bands were identified as GM2 andGM,, respectively, on methylation analysis (see text). Lane 6, fraction 6 gangliosideof Eb cells, which has mobility between GD,. and GD,b and was identified as anew type of disialoganglioside (termed GD,â€ž)as described in the text. Lane 7,fraction 6 ganglioside of ESb cells (identified as GD,B). Lane 8, fraction 4 gangliosideof Eb cells (GM,b) hydrolyzed with C. perfringens sialidase in the absence ofdetergent. Lane 9, fraction 6 ganglioside of Eb cells (new disialoganglioside, GD,â€ž)cleaved with C. perfringens sialidase in the presence of detergent. Lane 10, GD,bganglioside of ESb cells. Lane 11, GD,b ganglioside of ESb cells cleaved withsialidase in the presence of detergent. Lane 72, a subfraction of fraction 7 of ESbcells prepared by HPTLC and treated with C. perfringens sialidase and detergent.Note that it completely withstands sialidase.

ponent with the same HPTLC mobility as gangliotetraosylcer-amide (Gg4 or asialo-GM,) (see Fig. 4, lane 8); and (c) methylationanalysis gave 2,4,6-tri-O-methyl-Gal, 2,3,6-tri-O-methyl-Gal,2,3,6-tri-O-methyl-Glc, and 4,6-di-O-methyl-GalNAcMe (Fig. 5A).


1397




Am/z292264262234B

292264262234C

Â¡292iÂ«Â»

Â¡264262234m/z

34829.Â«[|4

/0.0Â«3203

305ÃŽ

-ÃŽ348KM

r 320u.

re

3 3051292\

348979?

320j

305292m/z

421T*"

393361Â¡

333421393361333421Ã¯'5"

393361I

333

Fig. 5. Selected ion chromatograms of partially O-methylated hexitol and hex-osaminitol acetates from the hydrolysates of permethylated glycolipids, separatedon a DB-5 column (temperature program 140Â°C-250Â°Cat 4Â°C/min) and detected

by methane chemical ionization mass spectrometry (mass spectrometry cycle time,1 s). A, GM,0 (lower band) from Eb cells (upper band gave a virtually identicalchromatogram); B, GM2; C, CM,, from ESb cells. Peate identified were: peak 1,2,3,4,6-tetra-O-Me-Gal;pea/(2,2,3,6-tri-O-Me-Gal;peai(3,2,4,6-tri-O-Me-Glc;pea/(4, 2,4,6-tri-O-Me-Gal; pea* 5, 2,6-di-O-Me-Gal; peak 6, 3,4,6-tri-O-Me-GalNAcMe;and peak 7, 4,6-di-O-Me-GalNAcMe. Italicized numbers are retention times inminutes. Identifications were made on the basis of appropriate MH+, (MH-32)*, and(MH-60)* ions, and retention indices were compared with authentic standards andconfirmed, if necessary, by co-injection.

On HPTLC in chloroform-methanol-calcium chloride, this com

ponent gave a duplet representing a component with two different classes of ceramide.

A Novel Disialoganglioside (GD1a). The second slow-migrat

ing component was identified as having a new structure, asshown in Table 1 (boldface print) based on the following findings:(a) it migrated on HPTLC between GD1a and GD1b (Fig. 4, lane6); (b) it was hydrolyzed with C. perfringens sialidase and byweak acid (1% acetic acid) to a component with the same HPTLCmobility as that of gangliotetraosylceramide (Gg4 or asialo-GM,)

(see Fig. 4, lane 9); (c) the two sialic acid residues were notlinked together through Â«2â€”Â»8linkage; rather, each sialic acidwas linked to a different position of the core structure, since only4,7,8,9-tetra-O-methyl-A/-acetylmethyl neuraminic acid but not4,7,9-tri-O-methyl-A/-acetylmethyl neuraminic acid were yieldedon methylation analysis (see Fig. 6). No A/-glycolylneuraminic acid

derivatives were detected; (d) on sugar analysis after methanol-ysis followed by trimethylsilylation, the ratio of Gal/GalNAc/GIcwas 2:1:1; no fucose or GlcNAc was detected, (e) 2,3,6-tri-O-methyl-Gal, 2,4,6-tri-O-methyl-Gal, 2,3,6-tri-O-methyl-Glc, and 4-mono-O-methyl-GalNAcMe, but no trace amounts of 4,6-di-O-methyl-GalNAcMe and other aminosugar derivatives, were

yielded on hydrolysis of the permethylated ganglioside (see Fig.7). The chemical ionization mass spectrum of 4-0-methyl-Gal-

NAcMe is shown in Fig. 8; and (f) the desialylated core structureshowed identical HPTLC mobility as gangliotetraosylceramide(Gg4; asialo-GMi) but did not give any other component reactivewith 1B2, 1B9, and SSEA-3 antibodies.

Characterization of Gangliosides from ESb Cells

The major gangliosides present in the extract of ESb cellswere characterized as GM3, GM2, GM1a, and GD1a,based on thefollowing results: (a) The component having a HPTLC mobilityidentical to that of GM3 was characterized as GM3, since sialidasetreatment and weak acid hydrolysis converted it to lactosylcer-

amide (Fig. 2, lane 6) with liberation of sialic acid; (b) the secondcomponent was identified as GM2, since it had the same HPTLCmobility as that of GM2 and was not hydrolyzed by sialidase (Fig.2, lane 6). Furthermore, the structure was confirmed by methylation analysis. 3,4,6-tri-O-methyl-GalNAcMe, 2,6-di-O-methyl-Gal, and 2,3,6-tri-O-methyl-Glc were identified on gas chroma-tography-mass spectrometry (see Fig. 5B). No di-O-methyl-Galnor 2,3,4-tetra-O-methyl-Gal was detected. On acid hydrolysis,

it gave a component with the same HPTLC mobility and reactivityas Gg3; (c) the third component was identified as GM1a by itsmethylation pattern and its insusceptibility to sialidase in theabsence of detergent (Fig. 2, lane 6). 2,3,4,6-tetra-O-methyl-Gal,4,6-di-O-methyl-GalNAcMe, 2,6-di-O-methyl-Gal, and 2,4,6-tri-O-methyl-GIc were identified on gas chromatography-mass spec

trometry in the hydrolysate of permethylated compound (see Fig.5C); and (d) two slowest-migrating components were identified

as GD1a and GDib, since they were converted to GM1a aftersialidase treatment in the absence of ionic detergent and gaveon methylation analysis 2,4,6-tri-O-methyl-Gal, 4,6-di-O-methyl-GalNAcMe, 2,6-di-O-methyl-Gal, and 2,3,6-tri-O-methyl-Glc (data

not shown). No gangliosides with the same methylation patternas that of GMu, or GO,,,, as detected in Eb cells, were found inESb cells. The fraction 6 ganglioside from ESb cells had a HPTLCmobility identical to that of GD1b, but it was not hydrolyzed bysialidase even in the presence of ionic detergent (Fig. 4, lane 12).The compound remains to be characterized in a future study.

Immunostaining of Glycolipids Separated on Thin-layer Chro-matography and Those at the Cell Surface

The major difference in composition between glycolipids fromEb cells and ESb cells was the presence of Gg3 in Eb cellsstained by 2D4 antibody, which defines Gg3 (Fig. 9X\, lanes 2and 3). Both Eb and ESb cells were negative with antibody 38-13, which defines Gb3. A positive spot at the position of Gg3 inEb cell glycolipids and other minor spots stained by this antibodyremained unclear (Fig. 96). Eb cells showed a positive band with1B2 antibody, which defines the A/-acetyllactosamine terminus

(Fig. 9C). The glycolipid fractions derived from both Eb and ESbshowed a positive staining with the antibody 813, which definessialosylgalactosyl globoside (Fig. 90). However, the chemical


1398




Table 1Location of sialic acids in mono- and disialoganglioside that are characteristic for high and low metastatic variants of mouse lymphoma cells

Eb cells (low metastatic) ESb cells (high metastatic)

Monos lalo GalÃŸ 1â€”Â»3GaINAcfMâ€”Â»4Ga 1Â£)1â€”>4G1C01â€”Â»1Cer

3

1NeuAcÂ«2 (GMib)

Disialo Gal/31->3GalNAcj31-Â»itGal/S1-Â»itGlc/31-Â»1Cer3 6

Ã® ÃŽNeuAca2 NeuAca2 (GD,â€ž)

G a101 â€”Â»3GalNAc01 â€”Â»4Gal01 â€”Â»4Glc/Ã®lâ€”Â»1Cer

3

1NeuAco2 (CM,,)

Gal/31â€”Â»3GalNAc01 â€”Â»4Gal01 â€”Â»4Glc/31 â€”>1Cer

3 3

1 1NeuAca2 NeuAcÂ«2 (GD,â€ž)

Gal|8l-Â»3GalNAc01â€”Â»4Gal|S1â€”Â»4 Glc/31â€”>1 Cer

3

lNeuAca2â€”Â»8NeuAca2 (GD,b)

Aâ€žI4,7.8,9-OMem/z404376372344Bm/z,

4043764,7.9-0-Me

8-0-AcXl

^4,7,8.9-0-Me344

Fig. 6. Selected ion chromatograms of partially 0-methylated W-methylaceta-mido-neuraminic acid methyl ester methyl glycosides from methanolysis of per-methylated GD,â€ž(A) and GD3 (ÃŸ)for comparison. Separation was performed on aDB-5 column (230Â°Cisothermal), with detection as for Fig. 5 (mass spectrometrycycle time, 1 s). Ions detected were (MH-MeOH)+ and (MH-2MeOH)- for each

derivative (for other details of method, see Refs. 44 and 45, from which this methodis adapted). In a parallel experiment, monitoring of ions appropriate for W-glycolyl

derivatives yielded no detectable peaks.

HH-32.MH-60.NEUTRRU HEK1TOL5100

MH,nH-6n,HÂ£X05i5MJN]TOLS

J,3,6-Me3-0-Glc

LSÃŽsoi" " " ssÃ"̈" '"Â¿Â¿Ã¨

Fig. 7. Full scan gas chromatography/mass spectrometry of partially O-methyl-ated hexitol and hexosaminitol acetates from the hydrolysate of permethylatedGD,,, from Eb cells. Conditions were essentially as described for Fig. 5, but themass range from 50-500 amu was scanned every 2 s. The plot Â¡sa summation ofall appropriate MH+, (MH-32)"1",and (MH-60)* ions. The discontinuity at scan 400

shows an increase in detector voltage prior to elution of hexosaminitol derivatives.The arrow shows the elution position of 4-O-Me-GlcNAcMe under identical conditions.

quantity of the ganglioside stained by this antibody was too smallto be detected by orcinol or resorcinol reaction, and its mobilitywas slower than that of GM1band should be due to the presenceof a trace quantity of sialosylgalactosyl globoside (SSEA-4).

Cell Surface Labeling Pattern of Glycolipids in Eb and ESbCells

Cell surface expression of glycolipid antigens was investigatedby cytofluorometry with various antibodies as described above.

1x10â€”jH2C-OAc

HC-NCi** ;i--.Ac.-7./ AcO-CH

MeO-CHHC-OAc j

B1116,8161

H2C-OAc

HC~N'AC' ,15

-CH-I

se ' 'iob ' 'isi Ã®oÃ

HD 332

420 432

AcOMeO-CH

HC-OAcH2C-OMe

MM* Â» 392IMH-321* = 360IMH-601* â€¢332IM'C2H51+ â€¢420M-CjMeJ* = 432

Fig. 8. Chemical ionization (methane, 300 nM) mass spectra of partially 0-methylated 2-deoxy-2-W-methylacetamido hexitol acetate standards. A, 4-O-Me-GalNAcMe; B, 4,6-di-O-Me-GalNAcMe. The monodeuterio derivative from GD,â€ž

had all major fragments (and ratios) appearing in spectrum A, but with ions (except261,201 ) shifted 1 amu to higher mass, and an identical retention time (determinedby co-injection).

As shown in Fig. 10, 2D4 antibody stained Eb cells intensely butdid not stain ESb cells. All other antibodies were negative.

Cell surface labeling with galactose oxidase-NaB[3H]4 of Eb

cells showed an intense triplet label at Gg3 and a doublet labelat Gg4 (Fig. 11, lane 1). These labels on the Eb cell surface weregreatly intensified on sialidase treatment (Fig. 11, lane 2) butwere not affected by trypsin treatment (Fig. 11, lane 3). A tripletlabeling of Gg3 and its intensification by sialidase treatment werethe same as described previously (27). In contrast, ESb cellsgave only a very low level of activity in neutral glycolipids, whichwas not enhanced after sialidase treatment (Fig. 11, lane 6),whereas a clearly positive label was observed in GM1a ganglioside from ESb cells. The label in GMia was enhanced strongly


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Eb Esb

12312312345 1234Fig. 9. Immunostaining of glycolipids from Eb and ESb cells separated on thin-

layer chromatography. A, stained with anti-Gg3 antibody 2D4. Lane 1, referenceGg3 isolated from guinea pig erythrocytes. Weak lower two bands were unidentified.Lane 2, Eb cells. Weak, fast-migrating bands were non-specific staining. Lane 3,ESb cells. Note that major bands corresponding to Gg3 were absent in ESb. B,stained with anti-Gb3 antibody 38-13. Lane 1, reference Gb3. Lane 2, Eb cells. Lane3, ESb cells. C, stained with anti-W-acetyllactosamine antibody 1B2. Lane 1,reference nLcÂ».Lanes 2 and 3, erythrocyte glycolipids of Folch's upper and lower

phases, respectively. Lane 4, Eb cells. Lane 5, ESb cells. D, ganglioside fractionstained with anti-sialosylgalactosylgloboside (anti-GL7 or anti-SSEA-4) (18). LaneÃ•,Eb cell ganglioside. Lane 2, the same as lane 1, treated with sialidase. Lane 3,ESb cell ganglioside. Lane 4, the same as lane 3, treated with sialidase.

O)O

10 100 KX>0

Fluorescence Intensity ( arbitrary unit)Fig. 10. Cytofluorometric pattern of Eb and ESb cells with antibody 2D4 directed

to Gg3. Upper pattern, ESb cells; lower pattern, Eb cells. , cells treated withfirst and second antibody. , cells only. , cells treated with secondantibody only. Ordinate, frequency of cells; abscissa, fluorescence intensity (arbitrary units).

when ESb cells were labeled after sialidase treatment (data notshown).

DISCUSSION

The metastatic potential of tumor cells has been functionallycorrelated with properties of cell surface membranes based ona few lines of experimental evidence using tumor cell systemswith different metastatic potentials. The first line of evidence isthat enzymatic modification of cell surfaces can change themetastatic potentials of cell lines (for a review, see Ref. 3).Secondly, a blocked glycosylation by tunicamycin results inmodified metastatic potentials (28). The fact that mutant cell lineswith different metastatic potentials display clear differences inglycosylation has provided the third line of evidence that cellsurface glycosylation may define metastatic potentials of cancer

I 23456789Fig. 11. Fluorography of cell surface-labeled glycolipids separated on thin-layer

chromatography. Lanes 1-4, Eb cells; lanes 5-8, ESb cells. Cells were labeled withgalactose oxidase and NaB[3H]4 with or without sialidase or trypsin treatments.

Lane 1, intact Eb cells directly labeled. Lane 2, Eb cells treated with sialidase, thenlabeled. Lane 3, Eb cells treated with trypsin, then labeled. Lane 4, Eb cells treatedwith trypsin and sialidase, then labeled. Lane 5, intact ESb cells directly labeled.Lane 6, ESb cells treated with sialidase, then labeled. Lane 7, ESb cells treatedwith trypsin, then labeled. Lane 8, ESb cells treated with trypsin and sialidase, thenlabeled. Lane 9, 3T3 cells transformed with murine sarcoma virus, Kirsten's strain,

directly labeled.

cells (references in Ref. 3). The degree of sialylation in glycopro-teins and glycolipids has been correlated with tumorigenic potentials by Schirrmacher ef al. (4), Yogeeswaran ef a/. (29), andYogeeswaran and Salk (30). A comparison of 31 murine cell lineswith different metastatic potentials showed that higher degreesof sialylation could be functional to metastatic potential, althoughthis simple correlation was not found in other studies (reviewedin Ref. 3). Among various BALB/c 3T3 cell lines transformedwith murine sarcoma virus, the quantity of Gg3 was correlatedwith higher metastatic potentials (31), which is different from theresults in our present study on Eb cells. Of melanoma cell linesselected by their lectin sensitivity, a clear cut distinction ofmetastatic potentials and carbohydrate structures has been described by Finne ef a/. (32). Wheat germ lectin-resistant cell lines,

which have low metastatic potential, were characterized ashaving Lex (Gal,31-Â»4[Fuca1â€”>3]GlcNAc),and wheat germ lec-

tin-sensitive cell lines, which have higher metastatic potential,

were characterized by a predominance of a sialylated structure(SAa2â€”>3Gal/31â€”>4GlcNAc)(32). A similar approach has beenapplied to a DBA-2 tumor called MDAY-D2. The wheat germlectin-resistant variant showed a remarkably reduced metastatic

potential and lack of fucosylation (33).Lectin-binding properties have been utilized extensively to

distinguish the cell surface properties of mouse lymphoma celllines Eb and ESb and the MDAY-D2 series (34). The Eb series

consists of low metastatic Eb, high metastatic ESb, low metastatic adhesion variant ESb-M, and high metastatic revenantESb-M. The low metastatic cell lines (Eb and ESb-M) reactstrongly with soybean lectin, which recognizes GalNAc-Gal, andthe high metastatic cell lines (ESb and ESb-MR) react with peanutlectin, which recognizes Gal-GalNAc. Since soybean lectin reactivity is greatly enhanced after sialidase treatment, high metastatic cell lines should have a cryptic soybean lectin receptor.


1400




The same situation has been found for high metastatic MDAY-

D2, low metastatic MDW40, and highly metastatic revertantMDW40M1 (34). None of these studies, however, chemicallyelucidated the carbohydrate structures isolated from these celllines.

We have extensively characterized the glycolipids present inEb and ESb cells grown in large quantity. Eb cells contain a highquantity of Gg3 and have a high reactivity at the cell surface withanti-Gg3 monoclonal antibody 2D4. Eb cells lack GM3 but containGg4, GM,b, and a new ganglioside GD1o. Therefore, this cell lineis characterized by its ability to synthesize a series of glycolipidswith a gangliotetrasoyl core pathway (Fig. 12). In contrast, highmetastatic ESb cells are characterized by the absence of Gg3and Gg4 and the presence of a large quantity of GM3, GM2, GM1a,and GD1a. Therefore, this cell line is capable of synthesizingganglio-series gangliosides (Fig. 12). Each component was char

acterized by methylation analysis.Both Eb and ESb cells contain disialogangliosides with similar

HPTLC mobilities but different structures. The disialogangliosideof Eb cells has now been characterized as having a new structurewith a peculiar sialosyl 2â€”Â»SGalNACat the penultimate GalNAc,whereas the disialogangliosides of ESb cells have been characterized as GD1a and GD1b (Fig. 12, Table 1). Similarly, themonosialoganglioside of Eb has been characterized as GM1b,whereas the monosialoganglioside of ESb has been identified asGMia (Fig. 12, Table 1). These mono- and disialoganglioside

structures in Eb and ESb cells are positional isomers as to thesialic acid linkage on the common gangliotetraosyl structure.These results clearly indicate that qualitative differences in theposition of the sialylation linkage to the common core structureis important in defining cell surface properties related to metastatic potential, although the degree of sialylation has also beencorrelated with metastatic potentials of various cells (30, 34).Interestingly, another variant cell line of Eb showing highly malignant properties, termed "Eb-J," showed a similar ganglioside

EB ESB

*OOOÂ©<=- GDla

\mO@O&=^lÂ£'

\ . /O@OÂ©cr=i_Go,,

Â©OÂ©<=^ Gc3 *OÂ©C==-GM5/

Fig. 12. Qualitative differences in the position of sialylation and resulting cellsurface glycosylation pattern between Eb and ESb cells. Eb and ESb cells aredistinct in their ability to sialylate LacCer or add GalNAc to LacCer. Thus, twopathways diverge. In Eb cells, a synthesis of GM,h and GD,â€žthrough Gg3 and Gg4predominates. Therefore, the Eb cell surface profile is characterized by the presenceof positional isomers of GM,., GD,., and GD,0, which are present in ESb cells, andthe presence of neutral glycolipids Gg3 and Gg4, which are absent in ESb cells.

profile as ESb and lacked GM1b and GDi,,.6 Further studies with

ESb-revertants are needed to prove or disprove this possibility.

The terminal structure of GD1nganglioside is common and sharedwith mucin-type glycoproteins, and desialylation of this structure

by sialidase induces the reactivity with peanut lectin. The presence of this structure in Eb is compatible with previous resultson peanut lectin sensitivity (34).

The structure of GO,,, now identified through this study isidentical to a disialoganglioside previously isolated and characterized from frog brain by Ohashi (35), although it was describedin a preliminary form. The ganglioside described by Ohashishowed a similar mobility as GDiâ€ž,which migrates on HPTLCbetween GDia and GD1b.

Glycolipid profiles of rat ascites hepatoma with low and highmalignancy have been studied previously (36, 37). The highlymalignant cell lines contained Gg3, Gg4, and GM1b, which isopposite to the results of the present study. Since the presenceof Gg3 in various mouse cell lines, including YAC and L5178Yvariants, has been correlated with NK cell susceptibility (38, 39)and since NK susceptibility has been related to metastatic potential (40, 41 ), it is reasonable to assume that Eb cells, whichcontain Gg3 and are NK susceptible, show less metastatic potential than ESb cells, which do not have Gg3 and are NKresistant. In rats, however, the presence of Gg3 may not becorrelated with NK susceptibility. Therefore, the finding by Takief al. (37) may not seriously conflict with out present observation.Recently, the transferrin receptor has been regarded as an NKtarget (42), and the transferrin receptor and expression of Gg3have been correlated (43). A cell growth inhibition induced bycross-linking of Gg3 at the cell surface effects dysfunction of

transferrin internalization. A possible relationship betweenexpression of Gg3, the transferrin receptor, and metastatic potentials is open for future study.

Note Added in Proof

After we completed this study, a disialoganglioside having the same structureas GD,â€žas described in this paper was isolated and characterized from rathepatoma AH 7974F [Taki, T., Hirabayashi, Y., Ishikawa, H., Ando, S., Kon, K.,Tanaka, Y., and Matsumoto, M. A novel type of disialoganglioside (GD,.) containingW-acetylneuraminyl(Â«2â€”>6)-W-acetylgalactosaminestructure. Presented at the Satellite Meeting on Neuronal Plasticity and Gangliosides of the International Societyfor Neurochemistry, May 29-31,1985, Abstract Book No. 8, Fidia Research Series,Frontiers in Neuroscience]. Details of this work are in press in J. Biol. Chem., 1986(personal communication from Dr. T. Taki).

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1986;46:1395-1402. Cancer Res Kimie Murayama, Steven B. Levery, Volker Schirrmacher, et al.

) from Eb Cellsα1Isolation of a Novel Disialoganglioside (GDLow Metastatic (Eb) and High Metastatic (ESb) Potentials andExpression of Glycolipids between Murine Lymphomas with Qualitative Differences in Position of Sialylation and Surface

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