galactosyl residue at the terminal a-GalNAc residue of blood group A determinant and are .... the acetates were separated on Merck HPTLC plates (Silica Gel 60,.
THEJOURNALO F 16,
BIOLOGICAL CHEMISTRY
Vol. 262, No. 29, Issue of October 15, pp. 14228-14234,1987 Printed in U.S.A .
1987 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation and Characterization of Novel Glycolipids with Blood Group A-related Structures: Galactosyl-A and Sialosylgalactosyl-A* (Received for publication, June 15, 1987)
Henrik ClausenSS, Steven B. Leveryn, Edward D. Nudelman, Mark Stroud, Mary Ellen K. Salyan, and Sen-itiroh HakomoriST 1) From the Biomembrane Institute, Departments of $Pathobiologyand llchemistry, University of Washington, Seattle, Washington 98119
Two glycosphingolipids with the following’novel structures have been isolated from human blood cells and characterized by NMR, direct probe mass spectrometry, fast atom bombardmentmass spectrometry, and methylation analysis: Gal@1+3GalNAca1+3G~1+4GlcNAc@1+3Ga~l+4Glc@1+1Cer (i)
2
T
Fucal
NeuAca2~3Ga~l~3GalNAcal-r3Ga~l~4GlcNAc@l+3Gal@1~4Glc@1+1Cer (ii)
2
*
Fuca 1
Both structure i and structure ii are characterized by substitution of &galactosyl or sialosyl-8galactosyl residue at the terminal a-GalNAc residue of blood group A determinant and are therefore specific products associated with the blood group A phenotype.
Blood group A determinant has been well established as a trisaccharide, GalNAca1-+3[Fuccu1+2]Gal~1-*R,which has been considered to be located exclusively at the terminus of type 1 or type 2 chain (1-3), a mucin disaccharide core (type 3 chain) (4,5 ) , or globotetraosecore (type 4 chain) (6, 7). Recently, however, extended A structures, such as repetitive A (GalNAcal+3[Fucal-+2]Gal~1~3GalNAcal+3[Fucal+ 2IGalP1-R) and A-associated H (Fucal-+2Gal@l+3GalNAcoc1”+3[Fuc~1+2]Gal~l-+R), have been isolated and characterized from the glycolipid fraction of human erythrocytes. The repetitiveA represents themajor A,-specific determinant (8, 9), while A-associated H represents an A,-associated antigen (10, 11). In addition, two novel A-substituted structures have now been isolated and characterized as glycolipids. One has been characterized as galactosyl-A, and the other has been identified as sialosylgalactosyl-A. This paper describes the isolation and characterization of these two glycolipids. MATERIALS ANDMETHODS
Antibodies-Two monoclonal antibodies, HH8 and HH9, directed to the galactosyl-A antigen were raised and used to probe for this antigen in blood cell glycolipids during purification (see below). The antibodies were generated by immunization with galactosyl-A” glycolipid, which was obtained by successive exoglycosidase treatment of A” type 3 chain (repetitive A) (9).’ The antibody HH8 is specific for Galpl+3GalNAcal+R residue presentin galactosyl-A glyco-
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 3 Supported by a fellowship from Ingeborg og Leo Dannin Fonden and Vera og Carl Johan Michaelsens Legat, Denmark. (1 Supported by Research Grant OIG CA42505 from the National Cancer Institute.
lipids, and HH9 is specific for the sequence Galpl+3GalNAcal+ 3[Fucal+2]Gal. Details of production and characterization of these antibodies will be described elsewhere.’ Glycolipid Preparation-Pooled, outdated human whole blood was lysed in tap water containing 0.2% acetic acid and ice overnight. Whole blood cell membranes, including those of white cells, were prepared by continuous centrifugation on a Sharplesscentrifuge, and total lipids of whole blood cell membranes were extracted with isopropyl alcohol/hexane/water (55:25:20, v/v/v; lower phase) as described previously (12). The extracts were evaporated in a rotary evaporator to dryness and dissolved in chloroform/methanol (2:1, v/ v), and the “upper phase glycolipid fraction” was prepared from the total lipid extract as described previously (12, 13). The neutral and acidic glycolipid fractions were separated by DEAE-Sephadex (A-25) chromatography (14). Purification of Galactosyl-A”-The upper neutral glycolipid fraction was fractionated by low-pressure high performance liquid chromatography (HPLC)3on a 1X 50-cm column of porous silica gel, Iatrobeads 6RS-8060 (60-pm particles; Iatron Chemical Co., Tokyo, Japan) (15). The elution was programmed from isopropyl alcohol/hexane/water (55:405 to 55:20:25, v/v/v) during 200 min with a flow rate of 3 ml/ min. Each 6-ml fraction was collected on a fraction collector (total of 600mlof eluate collected over 100 fractions). Each fraction was analyzed by high performance thin-layer chromatography (HPTLC)
A” is A determinant carried by nLcl, and Ab is A determinant carried by nLc6. A”, GalNAcal+3[Fucal+2]Gal~l+4GlcNAc~l+ 3Galpl+4Glc+Cer; Ab, GalNAcal+3[Fucal+2]Gal~l+4GlcNAc~l+3Gal~l4GlcNAc~l+3Gal~l+4Glc+Cer; type 3 chain A”, G a l N A c ~ l + 3 [ F u c a l ~ 2 ] G a l ~ l + 3 G a l N A c ~ l ~ 3 [ F u c a l + 2 ] G a l ~l-+4GlcNAcpl+3Gal~l4Glc+Cer; type 3 chain Ab, GalNAca1+3[Fuca1~2]Gal~1+3GalNAcal+3[Fucal+2]Gal~l+4GlcNAc~l+3Gal~l+4GlcNAc~l+3Gal~l+4Glc~Cer (see Refs. 3,19). The abbreviated desienations of glycolipids are according to IUPACIUC nomenclature (G). Clausen, H., Stroud, M., Parker, J., and Hakomori, S. (1987)Mol. Immunol., in press. The abbreviations used are: HPLC, high performance liquid chromatography; TLC, thin-layer chromatography; HPTLC, high performance thin-layer chromatography; FAB, fast atom bombardment.
14228
”
14229
Galactosyl-A and Sialosylgalactosyl-A
B
A
9r
HH9'
.
v GOI -AA?"/ ",
c.L
-GOI-AO-
Ab-
,NeuAc-Gol-A> GOI-~b
0 A I A2 AI A2 A, A2 3 4 5 neutrol
-
mono-siolyl
.."
--.
-
neulrol
-GOI-AO I
*
-
....."
. . "
I
mono-siolyl
u
desiolyloted desiolyloted FIG. 1. HPTLC-immunostaininganalysis of total glycolipidextracts and purified components.Panel A, orcinol/H2S04 reaction. Panel B, TLC immunostaining with HH9 performed as described previously (8,10,20). Lanes 0, A], and A2 are theupper neutral glycolipid or the ganglioside fractions prepared from 0, AI and A2 blood cells, respectively. Lanes for neutral glycolipids, those for gangliosides, and those for sialidase-treated gangliosides are indicated at thebottom. Lanes, 1 and 2, galactosyl-A"and Ab glycolipids obtained by successive exoglycosidase treatment of type 3 chain A", Ab, respectively; lane 3, purified 2,3-sialosylgalactosyl-A";lane 4, neuraminidasetreated purified 2,3-sialosylgalactosyl-A"(upper dark band is sodium taurocholate); lane 5,purified galactosyl-A". Plates developed in chloroform/methanol/water (504010, v/v/v, 0.05% CaClZ,w/v). Mobilities of A", Ab, and A' regions and of galactosyl-A", galactosyl-Ab,and 2,3-sialosylgalactosyl-A' are indicated. in a solvent of chloroform/methanol/water(504010, v/v/v). A crude fraction containing mainly nLcs, galactosyl-A", A",H2 type 2 chain, and otherminor unidentified components was eluted in tubes50-52. The fraction was rechromatographed on high-pressure HPLC through Iatrobeads 6RS-8010 column (10-pm particles; see above) as described of isopropyl alcohol/ previously (8, 10, 16, 17), usingagradient hexane/water from 55:405 to 55:30:15 (v/v/v). A fraction containing mostly nLcs and galactosyl-A" was obtained and further purified through preparative HPTLC as acetate. Acetylation was performed in pyridine/acetic anhydride (2:l) a t room temperature for 18 h, and the acetates were separated on Merck HPTLC plates (Silica Gel 60, Merck, Darmstadt, West Germany) in a solvent of dichloroethane/ acetone/water (50501, v/v/v) (10). The location of glycolipids on HPTLC was detected by Primulin spray (Aldrich) followed byobservation under UV light (18). Glycolipid acetates were extracted from HPTLC silica gel by sonicationin chloroform/methanol/water (2:1:0.15) and deacetylated in chloroform/methanol (2:l) containing 0.2% sodium methoxide in methanol a t room temperature for 15 min (19). The purification was, a t each step, monitored by TLC immunostaining using the monoclonal antibodies HH8 and HH9, using a modified procedure (12) originally described by Magnani et al. (20). Purification of Sialosylgalactosyl-A"-The monosialosyl fraction collected by elution of the DEAE-Sephadex using 0.05 M ammonium acetate in methanol was fractionated by HPLC as described above. The programs of HPLC on Iatrobeads6RS-8060 column, followed by HPLConIatrobeads 6RS-8010 columns, were identical to those described above. Thus, a crude fraction containing 2,3-sialosyl-nLc6 and a minor component, which was identified as sialosylgalactosylA", was obtained. Further purification of sialosylgalactosyl-A" was performed by sequential preparative HPTLC in solvent systems of chloroform/methanol/water (504010, v/v/v, 0.05% CaC12,w/v) and l-propanol/28%ammonia/water (6:1:1.5). Sialidase treatment of monosialosyl gangliosides was performed by incubation of 10-100 pg(crude fractions)or 2 pg (purified components) of glycolipid dissolved in 30 pl of 0.1 M acetate buffer (pH 4.5) containing 30 pg of sodium deoxytaurocholate added with 0.05 unit/ml of neuraminidase from Clostridium perfringem type X (Sigma). Glycolipid Characterization-500 MHz protonnuclear magnetic resonance ('H NMR) spectra were recorded with a Bruker WM-500 spectrometer equipped with an Aspect 2000 computer and pulse programmer, operating in the Fourier transform mode with quadrature detection. Spectra were recorded a t 308 and 328 f 2 K on a deuterium-exchanged sample (200 pg) dissolved in 0.4 ml of dimethyl sulfoxide-d6containing 2% D20 (21) and 1%tetramethylsilane as a chemical shift reference. Other parameters and data treatmentwere as described previously (22).
FIG.2. HPTLC of isolated galactosyl-A" and other glycolipids. Acetylated compounds are separated on HPTLC plate developed in dichloroethane/acetone/water(50501). Lane A h b , difucosyl type 1chain A; lane Hz, type 2 chain HZ;lane n k 6 , two lactonorhexaosylceramide variants; lane Gal-A", purified galactosyl-@. Plate visualized with orcinol/H2S04reaction. Mass spectrometry was performed using a JEOL HX-110 (Jeol Ltd., Tokyo) and DA-5000 data system (Jeol, Ltd., Tokyo). Negative ion fast atom bombardment (FAB)-mass spectra of native sialosylGal-A" wereacquired in the accumulation mode using aXenon beam source and triethanolamine matrix. Sodium iodide in glycerol was used in the negative ion FABmode as the calibration standard. Further conditions are described in the legend to Fig. 5. A portion (100 pg) of glycolipid was methylated (23), and 80% of this material was hydrolyzed in 0.5 N H2S04,90% acetic acid, reduced with NaB2H4, and acetylated according to published procedures (22, 24-26). Partially 0-methylated, N-methylated deoxyhexitol, hexitol, and hex-
Galactosyl-A and Sialosylgalactosyl-A
14230
A FIG. 3. HPTLC of isolated sialosylgalactosyl-A'. Native compounds are separated onHPTLC. Panel A, plate developed in chloroform/methanol/ water (5040:10, v/v/v), 0.05% CaClz (w/ v). Panel B, plate developed in l-propanol/ammonia/water (61:1.5). Lanes At and A*, monosialosyl-glycolipid fraction of A, and AZ erythrocytes, respectively. Lane 1, purified 2,3-sialosylgalactosyl-A", lune 2, 2,3-sialosylnorhexaosylceramide (fast-migrating component); lune 3, 2,3-sialosylnorhexaosylceramide (slow-migrating component). Plates visualized with orcinol/HzSO, reaction.
.
..
_,..*.-
B
1-
NeuAc-Gal-AaL rc 2,3 NeuAc nLc6
,2,3NeuAcnLc6 -NeuAc-Gal-Aa
A1 A2 1 2 3 osaminitol acetates were analyzed by gas chromatography-electron impact mass spectrometry using a Hewlett-Packard 5890A gas chromatograph interfaced to a 5970B mass selective detector under conditions described in the legend to Fig. 6. Derivatives were identified by characteristic fragmentationpatterns (24-27) and retention times, verified by coinjection with standards when necessary. The remainder of the permethylated glycolipid was analyzed by electron impact and positive ion FAB-mass spectrometry using the JEOL HX-llO/DA-5000 spectrometer. In both cases, CsI/NaI was used in the positive FAB mode as the calibration standard. Further conditions are described in the legends to Figs. 7 and 8.
A1 A2 1 2 3 YI
NeuNAca2+3GolB1+3GolNAcal+3 Golpl-4GlcNAcpl~3Gol~l~4Glc~l~lCer A Fucal+Z
JJI
Y
II
I
R
N VSb
RESULTS
Presence ofGalactosyl-A" and Sialosylgalactosyl-A" Glycolipids in A Blood Cells TLC immunostaining of neutral glycolipids with monoclonal antibodies HH8 and HH9, directed to the galactosyl-A antigen, showed two major bands co-migrating with galactosyl-A" and galactosyl-Ab and very weak staining of slower migrating bands. These bands were present in glycolipids from A, and A, blood cells, but were absent in glycolipids from B and 0 blood cells. In addition, desialylation of monosialosyl glycolipid fractions from AI and A, blood cells but not 0 or B blood cells resulted in HH8- and HH9-reactive bands, indicating that thegalactosyl-A glycolipids were present in erythrocytes as sialosyl derivatives (Fig. 1). Purification of Galactosyl-A" An HPLC fraction migratingas one major band on HPTLC in the solvent system chloroform/methanol/water (50:40:10, v/v/v) and reactive with HH8 and HH9antibodies was purified by preparative HPTLC as acetate (see "Materials and Methods"). The fraction contained nLc6 as the major component, which was separated from two minor components on HPTLC as acetate. TLC immunostaining after deacetylation revealed the presence of a slow-migrating band reactive with the antibodies (Fig. 1). The purified galactosyl-A" as acetate showed a single band on HPTLC and hada slower mobility than acetatesof nLc6, HP,and ALeb (Fig. 2). Purification of Sialosylgalactosyl-A" The presence of sialosylgalactosyl-A"was monitored by the reactivity with HH8 and HH9 antibodies after treatmentwith sialidase. TheTLC mobility of sialosylgalactosyl-A" was found to be similar to that of 2,3-sialosyl-nLc6. By repeated HPLC, a fraction migrating as one major band in the solvent system chloroform/methanol/water (504010, v/v/v) was obtained (Fig. 1).This component co-migrated on HPTLC with 2,3-sialosyl-nLc6 in chloroform/methanol/water system
4:O
5.0 blppml
FIG. 4. Downfield region of resolution-enhanced 'H NMR spectrum of sialosyl-galactosyl-ABglycosphingolipid from A erythrocytes in dimethyl sulfoxide-&, 2%DzO at 328 f 2 K. Concentration approximately 0.3 mM; 12,000 scans accumulated. Arabic numbers refer t o protons of residues indicated by Roman numerals in the structure drawn at thetop of the panel. Resonances marked by R- are from the sphingosine backbone, while the triplet marked Cis is from the cis-vinyl protons of unsaturated fatty acids. Footnotes: a, a-GalNAc H-1, H-2, H-3 connectivities established by decoupling experiment (H-2 irradiated during acquisition); b, a-Fuc H-5 established by decoupling from methyl doublet a t 1.096 ppm (reproduced in inset); c, tentative assignment for a-GalNAc H-5.
(panel A , Fig. 3), butwas clearly separated from 2,3-sialosylnLc6 in 1-propanol, 28% ammonia/water (6k1.5) (panel B, Fig. 3). Thus, sialosylgalactosyl-A" was separated from 2,3sialosyl-nLc6 by preparative HPTLC in the latter solvent system. The slowest migrating minor component was thus isolated. On desialylation by sialidase, the component was converted to a glycolipid having the same HPTLC mobility as galactosyl-& (lane 4, panel A , Fig. l),and the band was strongly stained by antibody HH9 (lane 4, panel B , Fig. 1). The component was subjected to structuralcharacterization. Structural Characterization of Galactosyl-A" Since the chemical quantity of galactosyl-A" was extremely limited, its structure hasbeen identified based on the following findings. (i) The purified component showed the same mobility as galactosyl-A" prepared from type 3 chain A" by sequential treatment with a-N-acetylgalactosaminidase and a-L-fucosidase. The structure of the standard galactosyl-A" was characterized previously by 'H NMR spectrometry (10). (ii) The purified component was strongly and specifically reactive with monoclonal antibodies HH8 and HH9, which
Sialosylgalactosyl-A and
Galactosyl-A
14231
TABLEI ' H NMR anomeric chemical shifts (ppm) for A erythrocyte glycosphingolipids in dimethyl sulforide-d6, 2% DzO [NeuAca2-31 f Gal(31-3GalNAcal
___t
3Gal~1~4GlcNAc~1+3Ga1(31~4Glc~l--rlCer
Fucal-2
308 f 2K Gal-A" NeuAc-Gal-A""
VI1
VI
V
IV
111
11
I
4.304 4.337
5.029 4.990
5.218 5.237
4.378 4.374
4.628 4.600
4.260 4.277
4.170 4.166
328 ? 2K 4.183 5.062 5.203 4.398 4.662 4.280 4.306 Gal-A" 5.042 5.209 4.393 4.625 4.338 4.295 4.172 NeuAc-Gal-Mb ' Additional resonances: Fuc V-5, 4.132; Fuc V-6, 1.086; GalNAc VI-2, 4.228 GalNAc VI-NAc, 1.802; GlcNAc 111-NAc, 1.818; NeuAc H-3,, 2.757; NeuAc NAc, 1.887. 'Additional resonances: Fuc V-5, 4.138; Fuc V-6, 1.096; GalNAc VI-2, 4.225; GalNAc VI-NAc, 1.815; GlcNAc 111-NAc, 1.825; NeuAc H-3.,, 2.750; NeuAc H-3,,, 1.405; NeuAc NAc, 1.886.
FIG. 5. Molecular ion region of negative ion FAB-mass spectrum of I native sialosyl-Gal-A' (5-10 pg). In- a set, theoretical ion distribution calcu- t lated for an (M - H)- consisting of one " c NeuAc, one deoxy-Hex,fourHex,two HexNAc, d18:l sphingosine, and 24:O R fatty acid (C, HI,, 0 4 5 N4; nominal mass, Ub 2139 atomic mass units); calculated n monoisotopic peak: 2140.153 atomic da mass units; found 2140.147; error, 2.8 n ppm. Note that the peak height ratios C C differ from the theoretical, probably due to noise. Acceleration voltage, 10 kV; resolution, 3000; scan range 100-3000 atomic mass units in 37.5 s, 50-s total cycle time; 3 scans accumulated.
d18-1,24.0 (M-H)2140.1 d18:1,24l(M-H)-2138l35
a . . . .
2020 2060
2040
2140 2000 2120
2100
2160
I
M/Z
Structural Characterization of Sialosylgalactosyl-A" 'H NMR-The downfield region of the 500-MHz 'H NMR spectrum of sialosyl-Gal-A" (Fig. 4) is characterized by the =J2-4 ~ ,Hz) ~ presence of seven anomeric resonances,two L U - ( ~ and five P-(3J1.2 = 7-9 Hz); in addition, thepresence of an aNeuAc residue is indicated by a characteristic doublet of doublets for H-3,, (not shown, see Table I). A doublet of 12 14 16 I6 28 22 24 26 Tlme fmln. I doublets at a position characteristic for an a-GalNAc H-2 FIG. 6. Electron impact limited masschromatogram of monresonance (8-10, 21, 28) can be decoupled from the more odeuterated partially 0-methylateddeoxyhexitol,hexitol, and hexosaminitol acetates obtained from the hydrolysis of upfield of the a-anomeric resonances. The identity of the be inferred from the presence permethylated Sialosyl-Gal-A'. Separation was on a 30 M DB-5 other a-anomeric resonance can (J & W Scientific, Rancho Cordova, CA) bonded phase fused silica of a broadened quartet which can be decoupled from a 3capillary column (temperature program, 140 to 250 "C at 4 "C/min) proton doublet far upfield (not shown, see Table I), characusing splitless injection. Electron impact-mass spectrawere acquired teristic of a-Fuc H-5 and CH3, respectively. Three N-acetylfrom 50 to 500 atomic mass units at 0.95 s/scan. The resulting gas methyl resonanceswere found, one in a positioncharacteristic chromatograph-mass spectrometry is plotted as a composite of relefor a-NeuAc (29) and the other two occurring in the region vant structural ions: ordinate, summation of selected ion intensities; residues (Table I) (21, abscissa, retention time in minutes. Peaks identified were: 1, 2,3,4- for a- and P-2-N-acetyl-2-deoxyhexose tri-0-Me-Fuc; 2, 2,3,6-tri-O-Me-Glc; 3, 2,4,6-tri-O-Me-Gal; 4, 4,6-di- 28). The spectrum closely resembles that of Gal-A" obtained 0-Me-Gal; 5 , 3,6-di-O-Me-GlcNAcMe; 6, 4,6-di-O-Me-GalNAcMe. previously from enzymatic defucosylation of type 3 chain H Arrows indicateelution positions of 2,3,4,6-tetra-O-Me-Glc and glycolipid (lo), and the anomeric resonances canbe assigned 2,3,4,6-tetra-O-Me-GaI, respectively. as shown in Fig. 4 by analogy to that spectrum. The chemical shift differences can be attributed to the presence of the for residues NeuAc residue, although the changes in H-1 shifts are directed to the standard galactosyl-A" preparationdeas far apart as P-Gal VI1 and P-GlcNAc I11 are difficult to scribed above.HH8 defines the Gal~l--*3GalNAcal+R struc-rationalize in theabsence of a clear three-dimensionalmodel. ture and HH9 defines theGal~l+3GalNAcal+3[Fuca1-+2] Although the shifts for H-3,, (2.757 ppm at 308 K) and N Gal sequence.' (iii) The same component was yielded after acetyl (1.887 ppm at 308 K) are similar to those found for sialidase treatment of sialosylgalactosyl-A", whose structure terminal a2-3-linked NeuAc in ganglio- and neolacto-series has been fully characterized as described in the subsequent glycolipids (29, 30), assignment of linkage position for the asection. NeuAc residue in this structure based on the 'H NMR spec13
Galactosyl-A and Sialosylgalactosyl-A
14232 R c
I
FIG. 7. Positive ion FAB-mass spectrum of permethylated sialosyl-Gal-A" (-10 pg in glycerol matrix), with proposed fragmentation kV; scheme. Acceleratingvoltage,10 scan range 100-2500 atomic mass units (scan time 32 8, total cycle time 38 s); resolution 3000. Onlythe low mass range shown exhibited peaks above background. The ion at m/z 660 is a fragment consisting of ceramide.
,
;8 0 I
"
376 t 344
t 793
FIG. 8. Electron-impact directprobe mass spectrum of permethylated sialosyl-Gal-A' (-10 pg), with proposed fragmentation scheme. Acceleratingvoltage, 10 kV; scanrange 100-3600 atomic mass units (scan time 43.4 s, total cycle time 50 s); resolution 3000, ionization potential37 eV, ionizing current 100 PA. deoxyHexfC 628
I89
I
t
660
157
eM$&
Cer
I
CH-CH=CH-(CHZ)~Z-CH)
=
d l 8 I sphlnqoslne 24 0 fotly acid
?Me 1
c =0 1
SH2 p 2
/
364(365-1)
C21H43
trum alone would not be advisable. Negative Ion FAB-MassSpectrum-In support of the overall composition, the negative ion FAB spectrum of the native glycolipid (Fig. 5 ) gave a pseudomolecular ion (M - H)- at a mass calculated for a structure made up of one NeuAc, one deoxy-Hex, fourHex, andtwo HexNAc alongwith a ceramide composed primarily of d18:l sphingosine and 24:O fatty acid (or d20:l sphingosine and 2 2 0 fatty acid)4 with some 24:l fatty acid-containingspecies also present. Methylation Anulysis-The gas chromatograph-mass spectrometry of partiallymethylatedalditolacetatesobtained from hydrolysis of permethylated sialosyl-Gal-A" (Fig. 6) is consistent with the proposed structure. Qualitatively, it differs Alternative structures of the same molecular weight with dihydrosphingosines or phytosphingosines are ruled out by the presence of sphingosine trans-vinyl signals inthe 'H NMR (Fig. 4).
from that of A" type 2 chain in the occurrence of 4,6-di-0indiMe-GalNAcMe instead of 3,4,6-tri-O-Me-GalNAcMe, cating a sugar linked to the 3-position of that residue. Compared with the pattern expected from Gal-A", there is virtually no 2,3,4,6-tetra-o-Me-Gal, but instead there is an elevated quantity of 2,4,6-tri-O-Me-Gal, suggesting that the NeuAc residue is linked to the terminalp-Gal of that structure. Further evidence for the terminal structureis found in the positive ion FAB-mass spectrum of the intact permethylated glycolipid (Fig. 7). Fragments are found at m/z 376 and 344 (terminal NeuAc), weakly a t m/z 580 (NeuAc-0-Hex-), and abundantly at m/z 825 (-793, NeuAc-0-Hex-0-HexNAc-). Unfortunately, it was not possible to obtain significant ions at this time. However, at higher mass with this technique direct probe electron impact-mass spectrometry gave, in addition to these low mass fragments, ions at m/z 1449, 1417, and 1389 representing the fragment:
Sialosylgalactosyl-A and
Galactosyl-A
14233
NeuAc-O-Hex-O-HexNAc-O-Hex-O-HexNAc-
I
0
I
deoxy-Hex
(see Fig. 8). DISCUSSION
Blood group A determinant was characterized by classic studies by Morgan and Watkins(1)and Kabat andassociates (2) during the1950s. Subsequent studiesfocused on glycolipid antigens of erythrocytesindicatedthatthe complexity of blood group ABH antigens is due to varying carrier chains, and the polymorphism generated by differing branching and elongation status iswell established (3). It has been generally believed that the determinant GalNAcal+3[Fucal+2] GalP1-R is the final productspecified by the A gene-defined a-GalNAc transferase and that no further extension or substitution could occur.With the introduction of the monoclonal antibody approach, the presence of new structures, such as repetitive A and A-associated H describedin the Introduction, has been discovered (8-11). This clearly indicates that further extension and substitution of the classically known A determinantcanindeedtake place. The results of thisstudy indicate the presence of the precursor to the type3 chain H structure, galactosyl-A and itssialosyl derivative, which have beenisolated andcharacterizedashavingthestructures shown below:
FIG. 9. Possible synthetic scheme for type 2 and type 3 chain glycolipids in erythrocytes. Designations 1 , 2 , and 3 indicate limiting steps in the biosynthesis of blood group ABH antigens, which are independent of the ABH glycosyltransferases themselves. Whereas ABH antigen biosynthesis is dependent on precursor synthesis 0 and substrateavailability 0,the biosynthesis of blood group A antigens is more complicated, having an additional site for competition with sialosyltransferases 0.Symbols: 0,GlcNAc; 0,Gal, a, GalNAc; 8,Gal (a1-3); 0,Fuc; 0, NeuAc.
thea-mannosylstructurein glycoproteins. In 1971, a-Gal residue was discovered as an internalresidue in globoside (35) and Forssman antigens (36). Thus, globo-series glycolipids havea uniquestructuralandconformational feature: the presence of a-Gal at an internal position. Discovery of extended A, as described previously,represents anotherexample of a-glycoside at an internalresidue. The conformational and three-dimensional structureof extended A is of great interest,
Gal~l~3GalNAca1~3Gal~1~GlcNAc~1~3Gal~1~Glc~l~lCer 2
f
Fucal
NeuAca2~3Gal~l~3GalNAcal-r3Gal~l~GlcNAc~l~33Gal~l~Glc~l~lCer 2
f
Fucal
The terminal disaccharide Gal(?l+3GalNAcal-+R of the galactosyl-A structure is similar to the classically known 0linkeddisaccharide core structure originallydescribed by ThomasandWinzler (31)isolated from orosomucoid and subsequentlyidentifiedasThomsen-Friedenreichdeterminant (called T antigen), which is exposed in human cancer (32, 33). In fact, immunization of mice with this glycolipid resulted in hybridomas secreting antibodies with properties similar to anti-T specificities. Furthermore, human anti-T antibodies and peanut lectin were found to react with the galactosyl-A glycolipids. Results of these experiments will be described elsewhere.’ In the present study, the monoclonal antibodies were used to probe for galactosyl-A structures in their naturally occurringform. The immunological and possibly structural similarities of the type 3 chain A and H glycolipid series with the A and H determinants carriedby 0-glycosidically linked carbohydrate structures also termed type3 chain (5) are becoming increasingly apparent. Several monoclonal antibodies have been isolated and shown to react with similar structures on both carriers (34). However, it is clear that antibodies specific for the A variants and A-associated glycolipids can be generated (8).*,‘ Among glycoconjugate structures in the animal kingdom, the presence of an a-glycosidic linkage at the internal carbohydrate chain has been extremely rare, with the exception of Clausen, H., Stroud, M., Nudelman, E., Baldwin, M., and Hakomori, S. (1987) Mol. Zmmunol., submitted for publication.
since the presence of a-GalNAc at the internal residue may create a very novel structural feature of the carbohydrate chain. Studies along this line are in progress. The present conceptof the structural relationshipof types 2 and 3 chain ABHIi-active glycolipids inerythrocytesis illustrated in Fig. 9. The novel finding that sialosylation can occur as an intermediate step in the biosynthesis of blood group A antigens provides new insight into the interpretation of deletion or re-expression of blood group A antigens associated with transformation (37). An intriguing possibility exists, at least as far as types 2 and 3 chain A antigens are concerned, that the synthesis of type 3 chain A can be blocked by aberrant sialosylation.
1. 2. 3. 4.
5. 6. 7. 8.
9.
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14234
Sialosylgalactosyl-A and
Galactosyl-A
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