Monoclonal Antibody AA4, Which Inhibits Binding of IgE to High ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Val. 264, No. 22, Issue of August 5, pp. 13267-13272,1989 Printed in U.S.A.

Monoclonal Antibody AA4, Which Inhibits Binding of IgE to High Affinity Receptors onRat Basophilic LeukemiaCells, Binds to Novel &-Galactosyl Derivativesof Ganglioside GDlb* (Received for publication, January 19, 1989)

Nenghua GuoS, Guor R. Hers, Vernon N. Reinholdj, Michael J. Brennanll, ReubenP. Siraganianll, and Victor GinsburgSII From the $Laboratory of Structural Biology, National Institute of Diabetes and Digestive and Kidney Diseases and Vlaboratory of Immunology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892 and the $Division of Biological Sciences, Harvard School of Public Health, Boston, Massachusetts021 15

Mouse monoclonal antibody AA4 inhibits the bindingthat bind IgE immunoglobulins with high affinity (1). The of IgE to high affinity IgE receptors on the rat baso- cross-linking of these receptors by either anti-receptor antiphilic leukemia cell line RBL-2H3. As shown by im- bodies or by antigen binding to the IgE receptor complexes munostaining of thin layer chromatograms, antibody triggers the cells to degranulate and secrete inflammatory AA4 binds avidly to two disialogangliosides (antigen I mediators. The high affinity receptor consists of three differand antigen 11) that occur in this cell line. The two ent noncovalently linked subunits called a, p, and y. The a antigens were purified by anion exchange chromatogsubunit is heavily glycosylated, exposed on the outer surface raphy followed byshort-bedcontinuousthin-layer of the cell, and is responsible for IgE binding. In contrast, the chromatography. About 230 pg of antigen I and 60 pg p and y subunits span the plasma membrane with portions of antigen 11were obtained from2 0 g (wet weight)of exposed to thecytoplasm and to theoutside of the cell. leukemia cells. The structures of both purified antigens Four monoclonal antibodies were recently isolated that were determined tobe a-galactosyl derivatives of the ganglioside GDlbby fastatom bombardment-massspec- inhibit the binding of IgE to high affinity IgE receptors on trometry, bychemical ionization-mass spectrometryof rat basophilic leukemia cells (2). Three of the four react with permethylated samples, by gas chromatography-mass the receptors at sites close to or identical to the IgE-binding spectrometry of partially methylated alditol acetates, site. There was reciprocal cross-inhibition between these anand by treatment with exoglycosidases and mild acid tibodies and IgE; the number of antibody molecules bound per cell was similar to the number of high affinity receptors, hydrolysis. The structureof antigen I is: and the antibodies degranulated the cells. The fourth antiGalal-3Ga~1-3GalNAc@l-4Gal@l-4Glc@l-lCer body, mAb’AA4, differed from the other three in several 3 ways: IgE and the other mAb did not inhibit the binding of I mAb AA4; the number of molecules of this antibody bound NeuAca2-8NeuAca2 per cell was 14-fold greater than the number of high affinity Antigen I1 has an additional a-galactosyl residue as receptors; and mAbAA4 not only did not degranulate the follows: cells but, on the contrary, inhibited histamine release (2). Moreover, when proteins from radiolabeled cells were precipGalal-3Galal-3Gal@1-3GalNAc~l-4Ga~l-4Glc@l-lCer itated with mAb AA4and analyzed by sodium dodecyl sulfate3 polyacrylamide gel electrophoresis followed by autoradiograI phy, although several peptides were detected, none of them NeuAca2-8NeuAca2 were derived from the receptor. Also, mAb AA4 did not bind Theceramide of antigen I containsapproximately to purified receptor subunits that had been transferred to equal amounts of C24:0, C22:0, C20:0, C18:0, and nitrocellulose. These data indicate that the structuresbound C16:O N-acyl fattyacids. The ceramide base is predom- by mAb AA4are close to but notcovalently linked to the high inantly sphingosine along with a small amountof dih- affinity IgE receptors. ydrosphingosine. In contrast, the ceramideof antigen I1 contains mainly C24:O N-acyl fatty acid with much The abbreviations used are: mAb, monoclonal antibody; FABlower amountsof C22:0, C20:0, and C18:O fatty acids. Moreover, the ceramide base is approximately 55% MS, fast atom bombardment-mass spectrometry; DCI-MS, desorption chemical ionization-mass spectrometry; GM1, Gal81-3GalNAcplsphingosine and 45% dihydrosphingosine. No unsatu- 4[NeuAca2-3]Gal~l-4Glc~l-lceramide; GM2, GalNAcpl-Q[NeuAcaPrated N-acyl fatty acids were detected in either anti-3]Gal~l-4Glc~1-lceramide; GM3, NeuAca2-3Gal~l-4Glc(3l-lceragen. mide; GD~,,NeuAca2-3Gal(31-3GalNAc~l-4[NeuAca2-3]Gal~l-4Glc

Rat basophilic leukemia cells have cell surface receptors

* 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” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 11 To whom correspondence should be addressed NIH, Bldg. 10, Rm. 110, Bethesda, MD 20892.

81-lceramide; GDlb; Gal~l-3GalNAc~l-4[NeuAca2-8NeuAca2-3]Gal @1-4Glc@l-lceramide;G D ~GalNAc~l-4[NeuAca2-8NeuAca2-3]Gal , p1-4Glcpl-lceramide; GD3, NeuAca2-8NeuAca2-3Gal~l-4Glcpll c e r a m i d e ;G ~ l b , NeuAca2-3Gal~l-3GalNAc~l-4[NeuAca28NeuAca2-3]Gal~l-4Glc~l-lceramide; GT3, NeuAca2-8NeuAca28NeuAca2-3Gal~l-4G1c~l-lceramide; GBlb, NeuAca2-8NeuAca23Gal~l-3GalNAc~l-4[NeuAca2-8NeuAca2-3]Gal~l-lceramide; FucGM1, Fucal-2Gal~l-3GalNAc~l-4[NeuAca2-3]Gal~l-4Glc~llceramide; FUC-GDlb, Fucal-2Gal~l-3GalNAc~l-4[NeuAca28NeuAca2-3]Gal~l-4G1c~l-lceramide; asialo-GMl,Galpl-

3GalNAc~l-4Gal~1-4G1c~l-lceramide.

13267

13268

a-Galactosyl Derivatives of Ganglioside GDlb

form:methanol (2:l by volume). The lipid solution was dried, the residue dissolved in ch1oroform:methanol:water (3060:8 by volume), and neutral and acidic lipids were separated as follows. The solution was passed through a column of DEAE-Sepharose CL-GB in the bicarbonate form (1.6 X 20 cm) equilibrated with the same solvent. Neutral glycolipidswere eluted with 80 ml of the same solvent EXPERIMENTALPROCEDURES followed by 400 ml of methanol. Gangliosides containing 1, 2, 3, and Materials-Monoclonal antibody AA4 belonging to the IgG sub- more sialyl residues were eluted with 400 ml of 0.01, 0.02, 0.04, 0.1, class is produced by a hybridoma obtained by fusing spleen cells from and 0.5 M ammonium bicarbonate in methanol. Elution of antigens a mouse immunized with the rat basophilic leukemia cell line RBL- was monitored by immunostaining thin-layer chromatograms as de2H3 with a myeloma cell line (2). The antibody is precipitated from scribed above. Antigen I and antigen 11, which were eluted by 0.04 M ascitic fluid with 50% saturated ammonium sulfate and purified by ammonium bicarbonate, were separated from each other and further purified by short-bed, continuous development thin-layer chromatogchromatography on DE52 (Whatman) followed by gel filtration on Bio-Gel A-1.5 m (Bio-Rad). Affinity purified goat antibodies to mouse raphy (10) using solvent B. Aliquots of the purified antigens chroIgG (Jackson Immunoresearch Laboratories, Avondale, PA) were matographed on glass HPTLC plates were visualized with resorcinol labeled with "'I by the Iodogen method (3). Monoclonal antibody reagent and quantitated by densitometric scanning using 0.15-2 pg against asialo-GM1 waskindly supplied by Dr. Donald Marcus, Baylor of ganglioside GD~, aasstandard. Mild Acid Hydrolysis-Glycolipids were desialylated by hydrolysis Collegeof Medicine, Houston, TX. Glycolipid standards were obM formic acid for 1 h a t 100 "C. Formic acid was tained from Supelco (Bellefonte, PA) or from Bachem (Torrence, in 0.2mlof1.0 CA). Galal-4Gal wasfrom Sockerbolaget Fine Chemical Division removed by the addition and evaporation of 1.0 ml of methanol three times. (Arlov, Sweden). Enzymatic Hydrolysis-The gangliosides were hydrolyzed byneurThin-layer Chromatography-Glycolipidswere chromatographed on glass or aluminum-backed high performance thin-layer chroma- aminidase as follows. About 10 pg of purified antigen I or antigen I1 tography plates (Silica Gel 60, E. Merck, Darmstadt, West Germany) was incubated at 37"C for 18 h with 0.05 units of Clostridium either in chloroform:methanol:0.25% KC1 in HzO (5:4:1 by volume) perfringens neuraminidase type X (Sigma) in 100 p1 of 0.05 M sodium (solvent A) or in chloroform:methanol:2.5 N ammonium hydroxide, acetate buffer, pH 5.5, containing 0.15 M NaCl and 0.009 M CaC12. The reaction was stopped by adding 2 ml of ch1oroform:methanol (1:l 0.25% KC1 in Hz0 (5:4:1 by volume) (solvent B). Immunostaining of Glycolipid Antigens-Glycolipid antigens were by volume). The reaction mixtures were evaporated to dryness and detected on thin-layer chromatograms by autoradiography as follows dissolved in 0.5 mlof ch1oroform:methanol:water (3050%by volume) volume) (4). Glycolipids were chromatographed as described above. The dried and loaded on a DEAE-Sepharose column (0.5mlbed chromatogram was soaked for 1 min in a 0.1% solution of polyiso- previously equilibrated with the same solvent. The reaction product butylmethacrylate (Polysciences) in hexane. After drying in air, the was eluted from the column with ammonium bicarbonate in methanol chromatogram was sprayed with buffer A (0.05 M Tris, 0.15 M NaCl, and desalted using Sephadex G-25 as described above. The reaction pH 7.8, with 1%bovine serum albumin and 0.1% sodium azide) and products were quantitated asdescribed above for the purified antigens ~ used as a standard. immediately soaked in the same buffer until all of the silica gel was except that ganglioside G Mwas Glycolipids were hydrolyzed by a-galactosidase as follows. To 10 wet. The plate was then removed and overlayed (60 pl/cm2) with monoclonal antibody solution (5 pg/ml in buffer A) and incubated pg of glycolipid wasadded 0.14 ml ofsodium citrate-phosphate buffer, for 1 h at room temperature. The chromatogram was washed by pH 5.7, containing 0.5 mg/ml of sodium taurocholate. The sample dipping in four successive changes of cold phosphate-buffered saline was sonicated for 20 s, and then0.5 unit of coffee bean a-galactosidase (Boehringer Mannheim) was added. After incubation at 37 "C for a t 1-min intervals and overlayed with buffer A containing 2 x IO6 48 h, the reaction was stopped by adding 2.0ml of chlorocpm/ml 'Z51-labeledgoat antibodies to mouse IgG. After 1 h at room form:methanokHzO (60:30:4.5 by volume), the sample was desalted temperature, the chromatogram was washed as before in cold phosusing Sephadex G-25, and the products were analyzed by chemical phate-buffered saline, dried, and exposed to XAR-5 x-ray film (Eaststaining and immunostaining of chromatograms as described above. man Kodak). Mass Spectral Analysis-Fast atom bombardment-mass spectromSolid Phase Radioimmunoassay-The binding of antibody to gly- etry (FAB-MS)and desorption chemical ionization-mass spectromecolipid was measured by solid phase radioimmunoassay as follows try (DCI-MS) of permethylated samples were carried out on a VG (5). Glycolipid in 50 pl of methanol was added to wells of a round- ZAB-SE double-focusing mass spectrometer (VG Instruments, Bevbottom polyvinylchloride microtiter plate (Dynatech), and the solu- erly, MA) operating at 8 KeV as described previously (11).For FABtions were dried under vacuum. The wells werethen filled with buffer MS, approximately 2 pg of sample was mixedwith 1pl of thioglycerol A. After 30 min, the wells were emptied and toeach was added 50 pl matrix before being loaded on the stainless steel target. The target of monoclonal antibody in buffer A (5 pglml). The wells were covered was bombarded with xenon atoms with a kinetic energy of 8 KeV. with parafilm, incubated 1h at room temperature, washed three times For DCI-MS, approximately 1pg of sample was loaded on to theDCI with buffer A, and then toeach was added about 100,000cpm of lZ5I- wire. A mixture of 88% carbon dioxide and 12% methanol was used labeled goat antibodies to mouse IgG antibody in 50 p1 of buffer A. as the reagent gas. After 1 h, the wellswere washed six times with cold phosphateFor linkage analysis, partially methylated alditol acetates were buffered saline, cut from the plate, and assayed for1''' ina y prepared and analyzed by a Hewlett-Packard5890 gaschromatograph scintillation spectrometer. and a Finnigan MAT-312gas chromatograph-mass spectrometer. Isolation of Antigen I and Antigen ZZ (ti-Rat basophilic leukemia Each peak separated on a30 m DB-5 capillary column was analyzed cells (RBL-2H3) were grown in culture as described previously (7), by electron ionization mass spectrometry. The column temperature and 4 X lo6 cells were injected subcutaneously into 3-5-day-old was maintained a t 75 "C for 1 min and then programmed to raise to Wistar-Furth rats. Tumors were collected after 15-17 days and im- 150 'C at a rate of 20 "C/min and finally to 300 "C at a rate of 2 'C/ mediately frozen. The tumors (20 g, wet weight) were homogenized min. Sugar linkages were identified by comparing relative retention in aDounce homogenizerwith 60 ml of HzO at 4 "C. The homogenate times and electron ionization mass spectra with those from a standard was added to 200 ml of methanol with constant stirring. Chloroform, compound. 100 ml, was added and the mixture was stirred at room temperature for 90 min. The homogenate was centrifuged at 1500 X g for 15 min. RESULTS The pellet was rehomogenized in 80 ml of HzO, added to 300 ml of Isolation of Ganglioside Antigens from Rat Basophilic Leuch1oroform:methanol (1:2 by volume), stirred a t room temperature, and centrifuged as before. The supernatant solutions were combined, kemia Cells-The antigens were extracted from the cells and evaporated to dryness, and submitted to a Folch partition (8). The purified by DEAE-Sepharose chromatography and short-bed, combined upper phases were evaporated to dryness. The residue was continuous development thin-layer chromatography as dedissolved in ch1oroform:methanol:HzO (6030:4.5, by volume), and scribed under "Experimental Procedures." mAb AA4 binds to lipids were separated from salt and other non-lipid contaminants as follows (9): the solution was passed through a column of Sephadex two gangliosides (antigen I and antigen 11) in lipid extracts of G-25 (1 X 12 cm) equilibrated with the same solvent. Lipids were rat leukemia cells as revealed by immunostaining thin-layer eluted with 100 ml of the same solvent followed by 35 ml of chloro- chromatograms (Fig. 1, lane 6). Both antigens are found in

The present paper shows that mAbAA4 binds avidly to two disialogangliosides found in the rat basophilic leukemia cells. The two disialogangliosides were isolated and identified to be novel a-galactosyl derivatives of the ganglioside GDlb.

a-Galactosyl Derivatives of Ganglioside G D , ~ the disialoganglioside fraction eluted from the DEAE-Sepharose column (Fig. 1, lane 10). The gangliosides in this fraction, revealed by orcinol, are shown in Fig. 1, lane 3. The two antigens isolated by thin-layer chromatography and revealed by orcinol are shown in Fig. 1, lanes 4 and 5. The single orcinol bands were coincident with the antigen bandsdetected by immunostaining chromatograms developed with solvent A (Fig. 1, lanes 13-20) or with solvent B (data not shown). About 230 pg ofantigen I and60 pg of antigen I1 were obtained from 20 g wet weightof cells. As measured by immunostaining, mAb AA4 binds to antigens I and I1 with approximately equal avidity (Fig. 1, lanes 13-20). Mass Spectral Analysis of Antigens I and 11-Positive FABMS analysis of permethylated antigen I provided the spectra in Fig. 2, A and B, which corresponds to the carbohydrate composition He%-HexNAcl-NeuAc2.The several ions in the molecular ion region, MH+ (m/z 2475, 2447, 2419, 2391), are distributed onthe basis of different N-acyl fatty acid residues. A better assessment of this fatty acid distribution can be observed at greater abundance with a series of ceramide fragments starting at m/z 660 (C24:O) and decreasing by 28 Da, (m/z 632, 604, 576). These fragments correspond to the

Sulfatide

-

GM3

-

GM2 GM1 GO3 GDla GDlb

GTlb Origin

-

. .

#

1t

-

1

2 3

4

5

6

7

8 10 9

0.”. 11 12 13 14 15 16 17 18 19 20

FIG.1. Purification of antigen I and antigen I1 by anion exchange and thin layer chromatography. Glycolipids from rat basophilic leukemia cells were fractionated by DEAE-Sepharose chromatography followed by short-bed,continuous development thinlayer chromatography as described under “ExperimentalProcedures.” A, glycolipids visualized with orcinol; B, glycolipid antigens visualized by immunostaining with antibody AA4. Lane 1, standard glycolipids, 0.5 pg each, as indicated at the left margin; lane 2, unfractionated glycolipids from 10 mg (wet weight) of cells; lane 3, disialoganglioside fraction from 10 mg (wet weight) of cells; lane 4, 0.2 pg of purified antigen I; lane 5, 0.2 pg of purified antigen 11; lane 6, unfractionated glycolipids from 2 mg (wet weight) of cells; lanes 7-12, aliquots of anion-exchange column fractions representing 2 mg (wet weight) of cells as follows: lane 7, neutral glycolipids, and lanes 8-12, 0.01, 0.02, 0.04, 0.1, and 0.5 M ammonium bicarbonate eluates, respectively; lanes 13-26, 10,2,0.2, and 0.02 ng of purified antigen I, respectively; and lanes 17-20, 10, 2, 0.2, and 0.02 ng of purified antigen 11, respectively. lenm

FIG.2. Mass smctra of antigen I. A, positive fast atom bombardment mass spectrum of antigen I scanned from 350 to 3050 atomic mass units. Abundance amplified 2X from m/z 2300 to m/z 3000. All masses have been corrected to monoisotopic values; and B, positive fast atom bombardment mass spectrum of antigen I replotted from 350 to 1000 atomic mass units to observe greater spectral detail.

91 88 78

13269

C22:0, C200, and C180 residues, and the structureof this ion is:

I

R - 0

The ion at m/z 550 is unique in this series because its molecular weight suggests the fragment to be a dihydrosphingosine analog with C160 as theN-acyl moiety with the same complement of sugar residues. Further examination of this spectra (Fig. 2B) andthe fragments, m/z 578, 606, 634, indicates that this analog also has a conjugating series of higher N-acyl fatty acid residues corresponding to C180, C20:0, and C22:O. In addition to the heterogeneity contributed by dihydrosphingosine and N-acyl fatty acids, alkane heterogeneity is commonly observed in the sphingosine base, for example eicosasphingosine (C20) in place of C18 sphingosine. The combined heterogeneity of base and N-acyl residue produces isomeric molecular ions that would not be differentiated by mass spectrometry and difficult to unravel structurally (for example, compare C18 sphingosine and C22 N-acyl residue versus C20 eicosphingosine and C20 N-acyl residue). For antigens I and11, this problem was resolved byDCI-MS using CO, and methanol as the reagent gas. These more energetic ionization methods (charge exchange) induce cleavage between the sphingosine C2 and C3 carbon skeleton with elimination of the terminal part of the alkane chain, (acleavage to theamide nitrogen) along with any heterogeneity originating in this residue. A comparison of the molecular ion distribution with the ct cleavage fragments following DCI-MS allows differentiation between the two contributing sources of heterogeneity. When antigens I and I1 were studied by this method, it was found that the molecular ion heterogeneity was identical to the ct cleavage fragments which indicated that sphingosine, C18, was the major base along with small amounts of dihydrosphingosine. The oligosaccharide sequence for both samples can be determined by examination of the low mass end of the FAB spectrum, (Figs. 2B and 3B). The ions m/z 376 and 737 are terminal neuraminyl fragments representing the monomer (NeuAc-) and dimer (NeuAc-NeuAc-), respectively. An additional terminating residue (branching) is indicated for each sample with the ions m/z 668 (Fig. 2B) and m/z 872 (Fig. 3 B ) . These fragments can be assigned the composition (HexzHexNAc) and (Hex3-HexNAc), andtheir high abundance suggests glycosidic cleavage proximal to anamino sugar residue. Lower fragment ions, indicative of oligomer sequence,

lye,I I=’

; ;68 ;58

5

a I I@

1588

’,!

I

m

m

3888

358

1Ba

458

5%

5%

688

,.*,

6%

m

78

888

a-Galactosyl Derivatives

13270

FIG. 3. Mass spectra of antigen 11. A, positive fast atom bombardment mass spectrum of antigen I1 scanned from 350 to 3050 atomic mass units. All masses have been corrected to monoisotopic values. B, positive fastatom bombardment mass spectrum of antigen I1 replotted from 350 to 1000 atomic units to observe greater spectral detail.

of Ganglioside GDlb

88-

g n.

H

6858

S "

%

7 8 8 8 8 8 M, 2

MI2

TABLE I Gas-liquid chromatography retention time of alditol acetates

GD1b

Is

Alditol acetate derivative

"7

GDlb

min

Terminal Gal 13.49 13.49 13.52 13.50 13.51 4-Linked 14.61 14.62 14.63 Glc 15.43 15.43 3,4-Linked 15.44 15.45 Gal 3-Linked GalN 15.73 15.72 4-Linked Gal 14.58 14.52 3-Linked Gal 14.74 14.73 14.74 .. ..

czD'?

I

II

Antigen I

.'. z..

Antigen I Antigen I1

G M ~ GL4

D.2

f

E.

Parent compound

: .

Antigen II

FIG. 4. Gas liquid chromatography of alditol acetates prepared fromGDII,,antigen I, and antigen 11. Labeled peaks are as follows: C16alkane internal standard(S);alditol acetates of terminal galactose ( A ) , 4-linked galactose ( B ) ,4-linked glucose ( C ) , 3-linked galactose (D), and 3-linked N-acetylgalactosamine ( E ) .Sample concentration data were obtained from a printout of integrated peak areas. The peaks S, C, and D are signalsaturated in the two antigen samples and do not reflect relative sample concentration.

were not detected for either the tri- or tetrasaccharide (Figs. 2B and 3B, respectively), and this would be anticipated (12). To study these structures in greater detail, linkage analysis was performed on GDlb,antigen 1 and antigen 11 (Fig. 4). Alditol acetates were prepared from GMl,GL4, and the disaccharide Galal-4Gal toprovide retention timesfor comparison (Table I). For GDlb,the peaks A, B, D, and E were identical in retention time with terminal galactose, 4-linked glucose,

14.61

14.61

15.71 15.71

3,4-linked galactose, and a3-linked N-acetylgalactosamine. A comparison of these results with those obtained with antigen I and antigen I1 indicated an identical distribution of alditol acetates with the exception of a new residue detected in both antigens, 3-linked galactose (peak C). Peak area integration and a comparison of 3-linked galactose to terminal galactose for the appropriatecompounds indicated that relative amount of 3-linked galactose was higher in antigen I1 than in antigen I (1.9 compared to 1.3, respectively). From a consideration of the M, and thelinkage analysis data, the structureof antigen I and I1 can be accounted for by the attachment to GDlbof single 3-linked galactose in antigen I and two 3-linked galactose residues in antigen 11. Positive FAB-MS of permethylated antigen I1 (Fig. 3A) showed a major protonated molecular ion (MH+) with a smaller distribution of satellite peaks indicating N-acyl fatty acid heterogeneity. The most abundant ion, m/z 2679, is 204 Da higher than the antigen I (C14:O N-acyl group) and corAs discussed responds to Hexs-HexNAcl-NeuNAcz-Cer. above, the threemajor glycosidic cleavagefragments, m/z 376, 737, and 872 (Fig. 3B), correspond to NeuAc, NeuAc-NeuAc, and Hex3-HexNAc,respectively. The comparison of data from antigens I and I1 and the known structure ofGDlb suggests that antigen I is Gal(l-3)Gal(1-3)GalNAc(l-4)[NeuNAc(28)NeuNAc(2-3)]Gal(l-4)Glc(l-1)Cer and that antigen I1 is:

Gal(l-3)Gal(l-3)Gal(1-3)GalNAc(1-4)[NeuNAc(2-8)NeuNAc(2-3)]Gal(1-4)Glc(l-l)Cer. Examination of the low mass end of the positive FAB mass spectrum indicates the N-acyl fatty acid distribution of mainly C24:0, with smaller amounts of C22:O ( m / z 6321, C20:O (m/z 604), C180 ( m / z 576). Interestingly, there is more dihydrosphingosine in antigen I1 than in antigen I. In aspecific case, when the C24:O N-acyl analogs are compared, the ratio of sphingosine to dihydrosphingosine is approximately 55:45. The ion series at m/z 662, 634, 606, 578, and 550, represents the N-acyl residues of C24:0, C22:0, C20:0, C18:0, and C16:0, respectively, for the dihydrosphingosinehomolog.No unsaturated fatty acids were detected conjugated to either antigen. Degradation of Antigens Z and ZI by Enzymatic and Acid

a-Galactosyl Derivatives of Ganglioside G D , ~ Hydrolysis-Both antigens I and I1 yield gangliosides with the chromatographic mobility of GDlb (Fig. 5, lanes 3-6) when treated with a-galactosidase. These resultsshow that 1 galactosyl residue in antigen I and 2 galactosyl residues in antigen I1 are a-linked. The binding of mAb AA4 is almostcompletely abolished by this treatment (Fig. 5, lanes 7-10). Treatment of both antigen I and antigen I1 with a-galactosidase followed by desialylation by mild acid hydrolysis (Fig. 5, lanes 12 and 13), but not by treatment with a-galactosidase alone (Fig. 5, lanes 14 and 15))results in the formation of glycolipids that bind a specific anti-asialo-GM1 antibody and have the same chromatographic mobility as authenticasialo-GM1(Fig. 5,lane 11). Desialylation by mild acid hydrolysis rather than by treatment with neuraminidase is necessary in order to remove both sialyl residues. One sialyl residue in both antigens, and in the gangliosides derived from both antigens by treatment with a-galactosidase, is resistant to neuraminidase as shown by the formation of faster-migrating, resorcinol-positive glycolipids upon their treatment with neuraminidase (Fig. 6 A ) . The monosialoganglioside products do not bind antibody (Fig. 6B). Structure of Antigens Z and ZZ-The mass spectral analysis of antigens I and I1 indicate that they are both galactosyl derivatives of the ganglioside GDIb. These data,along with the results of enzymatic and acid hydrolysis, indicate that the structure of antigen I is: Galal-3Gal@1-3GalNAc@1-4Gal@l-4Glc~1-1Cer,

13271

B sulfatida-

GM3GM2GM1GD3GDla-

.

GO1 bGTlb-

.

Origin-

1

2

3

4

5

6

7 8 9

FIG. 6. Effect of neuraminidase on purified antigen I and antigen 11. The antigens were treated with neuraminidase as described under “Experimental Procedures.” A, glycolipids visualized with resorcinol; B , glycolipid antigens visualized by immunostaining with antibody AA4. Chromatogram was developed with solvent A. Lane 1, standard gangliosides, 0.5 pg each, as indicated in the left margin; lanes 2 and 6, 0.5 pg and 10 ng of purified antigen I, respectively; lanes 3 and 7,0.5 pg and 10 ng of antigen I treated with neuraminidase, respectively; lanes 4 and 8,0.5 pg and 10 ng of purified antigen 11, respectively; lanes 5 and 9,0.5 pg and 10 ng of antigen I1 treated with neuraminidase, respectively.

’I-

O

n

3

I

NeuAca2-8NeuAca2

and that thestructure of antigen I1 is Galal-3Gala1-3Gal@1-3GalNAc@1-4Gal@1-4Glc@l-1Cer 3

I

NeuAca2-8NeuAca2

GANGLIOSIDE h g )

GM3GM2GMlGD1aGDlbGT1bGQ1b“r Origin‘

FIG. 7. Binding of antibody AA4 to purified glycolipids. Binding was measured by solid phase radioimmunoassay as described under “Experimental Procedures.” Binding to purified antigen I before (0)and after (B)treatment with neuraminidase, to antigen I1 (O), to ganglioside GoIb (El), and to gangliosides GM3, G Y ~ , G M ~ , G D ~ , G D ~GDI,, , G n , Gmb, GQlb, FUC-GMI, or FUC-GDlb (A).

-

Specificity of Antibody AA4-The binding of mAb AA4 to various purified gangliosides as measured by solid phase immunoassay is shown in Fig. 7. Antigen I and antigen I1 bind 7 8 9 10 11 1213 14 15 the antibody with approximately equal avidity in agreement FIG.5. Effect of a-galactosidase and mild acid hydrolysis with the results of immunostaining thin-layer chromatograms on purified antigenI and antigen II.The antigens were treated (Fig. 1, lanes 13-20). Most of their binding activity is lost with a-galactosidase and with mild acid as described under “Experimental Procedures.” A, glycolipids visualized with orcinol; B , glyco- upon removal of 1 sialyl residue from antigen I by treatment lipid antigens visualized by immunostaining with antibody AA4; C, with neuraminidase in agreement with the results in Fig. 6B. glycolipid antigen visualized with an anti-asialo-GMlantibody. Chro- GDlb binds antibody weakly. Other gangliosides, including matograms A and C were developed with solvent A, and chromato- G M ~ , GGMI, M ~ ,G D ~ , GGD~,, D ~ , Gn, GTlb,Gqlb, Fuc-GM~, and gram B was developed with solvent B using short-bed, continuous FUC-GDlb, do not bind antibody at thelevels tested. chromatography as described under “ExperimentalProcedures.” Lane Distribution of Antigens Z and IZ-Antigens with the chro1, standard gangliosides, 0.5 pg each, as indicated in the left margin; lane 2, unfractionated glycolipids from 10 mg (wet weight) of cells; matographic mobility of antigens I and I1 occur in rat tissue lanes 3 and 4,0.2pg of purified antigen I and antigen I1 treated with as detected by immunostaining (Fig. 8). Substantial amounts a-galactosidase, respectively; lanes 5 and 6,0.2 pg of purified antigens are found in lipid extracts of kidney, liver, and epididymis I and 11, respectively; lanes 7 and 9,20 ng of purified antigens I and (Fig. 8, lanes 3-5) and traces in brain(Fig. 8, lane 2). None is 11, respectively; lanes 8 and 10, 20 ng of purified antigens I and I1 found in extracts of rat erythrocytes (Fig. 8, lane 6). The fasttreated with a-galactosidase, respectively; lane 11,0.2 pg of standard migrating antigen detected in extracts of brain and liver (Fig. asialo-GMl; lanes 12 and 13, 0.2pg of purified antigens I and 11, 8, lanes 2 and 4 ) is presumably GDlb, which binds the antibody respectively, treated with a-galactosidase followed by galactosidase weakly (Fig. 7). Antigens with the chromatographic mobility followed by mild acid hydrolysis; and lanes 14 and 15, 0.2pgof of antigens I and I1 were not detected in corresponding purified antigens I and 11, respectively, treated with a-galactosidase.

.

- 0

.. . 1 2 3 4 5 6

-

m

a-Galactosyl Derivatives of Ganglioside GD16

13272

m” AI

.

Why the anti-ganglioside antibody AA4 inhibits bindingof IgE to high affinity receptors is not clear. Interestingly, the disialoganglioside G Dis~ specifically associated with the ArgGly-Asp receptor on human melanoma cells and is required for the optimal binding of polypeptides containing this sequence to thereceptor (18).Possibly related findings are that gangliosides also modulate the activity of many membrane proteins (19) including receptor-associated tyrosine kinases (20)) protein kinase C (21, 22), and other kinases (23, 24)) thus providing possible mechanisms whereby antiganglioside antibodies modulate cell growth (25, 26).

.

GD3GM1-

GDlaGD1b GTlb=

w -

1

REFERENCES

1. Metzger, H., Alcaraz, G., Hohman, R., Kinet, J., Pribluda, V., 0rig.- c-Jand Quarto, R. (1986) Annu. Reu. Immunol. 4,419-470 1 2 3 4 5 6 2. Basciano, L. K., Berenstein, E. H., Kmak, L., and Siraganian, R. P. (1986) J. Biol. Chem. 261, 11823-11831 FIG.8. Occurrence of antigens I and I1 in rat tissues. Chro3. Fraker, P. J., and Speck, J. C., Jr. (1978) Biochem. Biophys. Res. matography with solvent A and immunostaining with mAb AA4 was Commun. 80,849-857 carried out as described under “Experimental Procedures.” A, glyco4. Magnani, J. L., Nilsson, B., Brockhaus, M., Zopf, D., Steplewski, lipids visualized with orcinol; B, glycolipids visualized by immunoZ., Kourowski.. H.,. and Ginsburn, J. Biol. Chem. 257. -. V. (1982) . staining. Lune I , standard glycolipids, 0.5 Kg each, as indicated at the 14365114369 left margin; lanes 2-6, total lipid extracts from 2 mg (wet weight) of 5. Young, W. M., MacDonald, J. R., Nowinski, R. C., and Hakomori, rat brain, kidney, liver, epididymis, and erythrocytes, respectively. S. (1979) J . EXD. Med.150.1008-1009 The position of antigen I (Ag I) and antigen I1 (Ag Z I ) is indicated a t 6. Magnani, L., Spitalnik, S. L., and Ginsburg, V. (1987) Methods the right margin. Orig., origin. Enzymol. 138,195-207 7. Barsumian, E.L., Isersky, C.,Petrino, M. G., and Siraganian, R. extracts from human or mouse tissue. The fast-migrating P. (1981) Eur. J. Zmmunol. 11, 317-323 antigen, however, wasdetected in tissues rich in GDIb, such as 8. Folch, J., Lees, M., and Sloane Stanley, G. H. (1957) J. Biol. Chem. 226,497-509 human brain (data not shown). 9. Wells, M.A., and Dittmer, J. C. (1963) Biochemistry 2, 12591263 DISCUSSION 10. Young, W. W., Jr., and Borgman, C. A. (1987) Methods Enzymol. The mass spectraldatain Figs. 2-4 together with the 138,125-132 enzymatic, chemical, and immunologic data in Figs. 5-7 es- 11. Kuei, J., Her, G. R., Sheely, D. M., and Reinhold, V. N. (1988) Anal. Bwchem. 172,228-234 tablish the structure of antigens I and I1 to be a-galactosyl derivatives of the ganglioside GDlb. Although these structures 12. Egge, H., and Peter-Katalinic, J. (1987) Mass Spectrom. Rev. 6, 331-393 have not been reported before, an a-fucosyl derivative of 13. Ariga, T., Kobayashi, K., Kuroda, Y., Yu, R.K., Suzuki, M., antigen I was recently isolated from rat pheochromocytoma Kitagawa, H., Inagaki, F., and Miyatake, T. (1987) J. Biol. cells (13) and a-galactosyl derivatives of other ganglio-series Chem. 262(29), 14146-14153 glycosphingolipids have beenreported, all from rat tissue (1414. Holmes, E. H., and Hakomori, S. (1982) J. Biol. Chem. 257, 16). The antigens occur in various rat organs (Fig. 8) but not 7698-7703 in corresponding tissues from humans or mice. Thus, these 15. Taki, T., Kimura, H., Gasa, S., Nakamura, M., and Matsumoto, M. (1985) J. Biol. Chem. 260,6219-6225 glycolipids appear to be characteristic of rats. The occurrence 16. Bouhours, J., Bouhours, D., and Hansson, G . C. (1987) J. Biol. of many glycolipids is species-specific (17). Chem. 262,16370-16375 The sugar sequence in antigens I and I1 that is recognized 17. Stults, C. L. M., Sweeley, C. C., and Macher, B. (1989) Methods

J.

by mAb AA4 can be deduced from the data in Figs. 1, 5, 6, and 7. The proximal a-galactosyl residue and the terminal sialyl residue are both required for tight binding of the antibody as removal of the terminal sialyl residue from either antigen results in loss of its binding activity (Figs. 6 and 7)) and GDlb binds the antibody only weakly (Figs. 5B and 7). The terminal a-galactosyl residue of antigen I1 contributes little to binding as antigen I and antigen I1 have about the same avidity for the antibody (Figs. 1, 6, and 7). Thus, the epitope recognized by mAb AA4 in antigens I and I1 can be diagrammatically represented by a dashed line on the antigen I1 sugar sequence as follows: Galal-3Galal-3GalB1-3GalNAcB1-4GalB1-4Glc

\

I

\ NeuAca2-8NeuAca2 ”_

Enzymol., in press Pytela, R., Pierschbacher, M. D., Klier, F. G., Ruoslahti, E., and Reisfeld, R. A. (1987) J. Cell Biol. 105,

18. Cheresh, D.A.,

1163-1173 19. Igarashi, Y., Nojiri, H., Hanai, N., and Hakomori, S. (1989)

Methods Enzymol., in press 20. Hanai, N., Nores, G. A., MacLeod, C., Torres-Mendez, C.-R., and Hakomori, S. J. Bwl. Chem. 263,10915-10921 21. Kreutter, D., Kim, J. Y. H., Goldenring, J. R., Rasmussen, H., Ukomadu, C., DeLorenzo, R. J., and Yu, R. K. (1987) J. Biol. Chem. 262,1633-1637 22. Hannun, Y. A., and Bell, R. M. (1987) Science 235,670-674 23. Goldenring, J. R., Otis, L. C., Yu, R. K., and Dehrenzo, R. J. (1985) J . Neurochem. 44, 1229-1234 24. Chan, K.-F. J. (1988) J. Biol. Chem. 263,568-574 25. Lingwood, C., and Hakomori, S. (1977) Exp. Cell Res. 108,385391 26. Dippold, W. G., Knuth, A., and zum Buschenfelde, K.-H. M. (1984) Cancer Res. 44,806-809