New gangliosides from human erythrocytes.

We have identified a number of gangliosides from human erythrocytes that have not previously been detected in these cells, including two new compounds. The gangliosides were separated into monosialo- and disialoganglioside fractions by DEAE-column chromatography. Two monosialogangliosides that have not been previously detected in these cells are GM2 and GM1. Two other monosialogangliosides have the same carbohydrate structure, NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4) Glc-Cer, but they contain different fatty acids. The compound with higher chromatographic mobility (MG-5) contains a predominance of C22 and C24 fatty acids, whereas the principal fatty acid of the slower compound (MG-6) is C16. Both gangliosides are receptors for human anti-p and anti-Gd cold agglutinins. Six disialogangliosides not identifed previously in human red cells include GD3, GD1a, GD1b, DG-3, (formula: see text). The latter two are newly identified compounds and DG-4 contains a sugar sequence that has not been described previously, sialic acid residues linked to different hydroxyl groups of the same galactose.


NeuAc(a2-3)Gal(~1-4)GlcNAc(~l
The latter two are newly identified compounds and DG-4 contains a sugar sequence that has not been described previously, sialic acid residues linked to different hydroxyl groups of the same galactose. Although the major gangliosides in brain and extraneural tissues have been well characterized (2-4), structures of many less abundant gangliosides remain to be elucidated. Many of these minor components possess interesting biological and immunological properties. Of the nine gangliosides that Watanabe et ul. recently isolated from human erythrocytes, gan-* This research was supported by Research Grant AI 17712 from the National Institutes of Health, Grant Q-832 from the Robert A. Welch Foundation, and Grants 3967 and 6521 from the Swedish Medical Research Council. 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.
To whom correspondence should be addressed.
gliosides with lactonorhexaosyl and lactoisooctaosyl structures were shown to be i and I antigens, respectively (5-7).
We reported previously (8) that an anti-p cold agglutinin was inhibited by sialosyllactoneotetraosylceramide, the most abundant ganglioside of human erythrocytes. We found subsequently (9) that two less abundant gangliosides from human erythrocytes, identified as MG-5 and MG-6 in this study, are more potent inhibitors of this antibody and of anti-Gd antibodies than sialosyllactoneotetraosylceramide. In this report we describe the isolation and characterization of six disialogangliosides and two monosialogangliosides that have not been identified previously in human erythrocytes. We have also identified a unique disialoganglioside structure that contains a galactosyl residue with sialic acid substituents on two separate hydroxyl groups.
The purified monosialo-and disialogangliosides isolated from human erythrocytes are shown in Figs. 1 and 2, respectively, and their approximate relative abundance is presented in Table I.   MG-I-This ganglioside, which was not detected in the original mixture (Fig. I), co-migrated with brain GM2 and was similar to the latter in carbohydrate composition (Table 11) and in its resistance to Vibrio cholerae neuraminidase. The structure was confirmed by methylation analysis (Table 111) and partial acid hydrolysis of native ganglioside. The desialylated MG-1 was identified as gangliotriaosylceramide on the basis of chromatographic mobility and complement fixation with purified anti-gangliotriaosylceramide. Thus, the oligosaccharide structure of MG-1 appears identical with that of brain GMZ.
MG-3-This ganglioside co-migrated with brain G M~ and was similar to the latter in carbohydrate composition ( Table  11) and in its resistance to V. cholerae neuraminidase. Partially 0-methylated hexitol and hexosaminitol acetates identified in the hydrolysates of permethylated ganglioside were identical with the products obtained from brain GM1 (Table 111). On treatment with Arthrobacter ureafaciens neuraminidase, MG-3 yielded a desialylated glycolipid which was identified as gangliotetraosylceramide on the basis of chromatographic mobility and complement fixation with purified anti-gangliotetraosylceramide.
MG-5 and MG-6"These two gangliosides occur in approximately equal concentration and together comprise approximately 5% of the monosialoganglioside fraction (Table I).
Both gangliosides contained galactose, glucose, glucosamine, and N-acetylneuraminic acid in approximate molar ratios of 2.8:1.0:1.8:0.8 (Table 11). On treatment with V. cholerae neuraminidase, MG-5 and MG-6 yielded the asialoglycolipids. The TLC migration rates relative to lactoneotetraosylceramide (RLNnT) were 0.58 and 0.49, respectively. Treatment of the asialoglycolipids with jack bean p-galactosidase gave products with RLN,,T 0.80 and 0.70, respectively, and hydrolysis of these products by jack bean P-N-acetylhexosaminidase yielded compounds with R L N n T 1.0 and 0.85, respectively. Both compounds were identified as lactoneotetraosylceramide by complement fixation with purified anti-lactoneotetraosylceramide antibody (33).
These two gangliosides were shown by direct inlet mass spectrometry to have identical carbohydrate structures and to differ in their ceramide moieties. Both fractions were analyzed as permethylated, permethylated-reduced, and permethylated-reduced-trimethylsilylated derivatives. The mass spectrum of the latter derivative of MG-6 is shown in Fig. 3. Molecular ions are obtained for the major molecular species of MG-6 containing normal C16:O fatty acids and C18:l sphingosine base at m/z 2233. Ions containing the fatty acid (C16) plus the complete carbohydrate chain are seen at m/z 1981. A number of terminal saccharide fragments as well as rearrangement fragments containing the fatty acid plus part of the carbohydrate chain are present in the spectra of all three derivatives, confirming the structure given in Fig. 3.
Although no molecular ions were recorded for the corresponding derivatives of MG-5, the mass spectra (not shown) nevertheless give conclusive evidence for a ganglioside with a carbohydrate part identical with that of MG-6 but with a different ceramide composition. The major fatty acids of MG-5 are C22:0, C24:1, and C24:O normal fatty acids (Table IV).
DG-I-This ganglioside displayed two bands that co-migrated with the two bands of human spleen GD3 and that were similar to the latter in carbohydrate composition. Removal of both sialic acid residues by V. cholerae neuraminidase or mild acid hydrolysis yielded lactosylceramide. Periodate borohydride treatment followed by mild methanolysis (24) yielded equimolar amounts of intact NeuAc and NeuAc-7 indicating a NeuAc(2-8)NeuAc sequence.
The mass spectra (not shown) of permethylated, permethylated-reduced, and permethylated-reduced-trimethylsilylated DG-1 were similar to those published previously for GD3 of bovine retina (37). The complete sugar sequence of DG-1 was thus established to be identical with that of G D~. The mass spectra further show that DG-1 predominantly contains normal C16:O fatty acid but also minor amounts of C16:O and C18:O hydroxy fatty acids (Table IV).
DG-2"This ganglioside co-migrated with brain Go,, and was similar to the latter in carbohydrate composition (Table  111). On treatment with V. cholerae neuraminidase, DG-2 yielded a ganglioside that co-migrated on TLC with brain GMl. Periodate borohydride treatment of DG-2 converted both sialic acid residues to the lower homologue NeuAc-7 which

TABLE IV
Approximate fatty acid composition of gangliosides in human erythrocytes Determined by mass spectrometry by measuring the relative intensity of fatty acid peaks preferably in spectra of methylated-reduced derivatives. indicated that the sialic acids were on two different sugar residues. On methylation analysis (Table   111), DG-2 gave products identical with the products obtained from brain GD~,.
The sugar sequence was confirmed by mass spectra of permethylated, permethylated-reduced, and permethylatedreduced-trimethylsilylated DG-2 which were found to be similar to those published (38) for Gol, from brain. The approximate fatty acid composition is shown in Table IV. DG-3"This ganglioside co-migrated with DG-4 on TLC with ch1oroform:methanol:water solvent systems and preparative TLC with a chloroform:methanol:2.5 N NH40H, 60409 (v/v) solvent was necessary to purify it. Carbohydrate analysis revealed that DG-3 contained galactose, glucose, glucosamine, and sialic acid in an approximate molar ratio of 2:1:1:2 ( Table   11). On treatment with V. cholerae neuraminidase, the ganglioside lost both sialic acids and the asialoglycolipid comigrated with lactoneotetraosylceramide. Periodate borohydride treatment followed by mild methanolysis yielded equimolar amounts of intact NeuAc and the NeuAc-7 which indicated that the two sialic acid residues are linked by a 2-8 bond. The products of methylation analysis of DG-3 were identical with those (Table   111) obtained from 2,3sialosyllactoneotetraosylceramide.
The sugar sequence was confirmed by direct probe mass spectrometry of permethylated (not shown), permethylatedreduced (not shown), and permethylated-reduced-trimethylsilylated derivatives ( are rearrangement sequence ions (for explanation see formula above the spectra in Fig. 4) which, together with characteristic fragments in the spectra of the other two derivatives indicated that the oligosaccharide structure of DG-3 is identical with that of disialosylparagloboside isolated previously from hu-  (39). An estimate of the fatty acid pattern of DG-3 is given in Table IV. DG-4"This ganglioside was purified by preparative TLC in an ammonia solvent system (Fig. 2) from a column fraction that also contained DG-3. Carbohydrate analysis (Table 11) indicated that DG-4 contained galactose, glucose, galactosamine, and sialic acids in an approximate molar ratio of 3:1:1:2. On treatment with V. cholerae neuraminidase, DG-4 lost both sialic acids to give a neutral glycolipid which co-migrated on TLC with gangliotetraosylceramide in ch1oroform:methanokwater solvent system (60305 or 6035:8, v/v). The asialoglycolipid obtained from DG-4 by neuraminidase treatment fixed complement with purified anti-gangliotetraosylceramide antibody as well as gangliotetraosylceramide (0.5 pg/ml of each glycolipid was needed for fixation of complement). This suggests that the terminal disaccharide of desialylated DG-4 has the same terminal Gal(fil-3)GalNAc moiety as ganglio-N-tetraosylceramide, but the terminal galactose residue of desialylated DG-4 could not be hydrolyzed by jack bean &galactosidase (6). However, desialylated DG-4 could be completely hydrolyzed by the use of Charonia lampas P-galactosidase (50 milliunits of enzyme/50 pg of glycolipid) (40) to a product that co-migrated on TLC with human erythrocyte globotetraosylceramide. The hydrolyzed product was identified as globotetraosylceramide by TLC autoradiographic procedure with purified anti-globotetraosylceramide (41).
Methylation analysis (Table 111) revealed the presence of a 3,6-disubstituted galactose in the native compound, and the disappearance of this compound and the appearance of an unsubstituted terminal galactose (2,3,4,6-tetra-O-methylgalactitol) in the neuraminidase-treated glycolipid ( Table 111).
The presence of sialic acid residues linked to the 3-and 6hydroxyl groups of the terminal galactose is also supported by absence of native NeuAc following periodate borohydride treatment.
The DG-4 fraction was analyzed as permethylated, meth- The peak at m/z 1920 is a secondary ion originating from m/z 2253 by the loss of one NeuAc and the rearrangement of one TMS group. The ions at m/z 1587 and 1515 are similarly produced by the loss of two NeuAc and rearrangement of two TMS groups or one TMS group and one hydrogen atom, respectively; m/z 818 is a rearrangement ion containing the C24 fatty acid plus the proximal two hexoses. The rearrangement of one hydrogen (+1) indicates a non-branched chain; m/z 1022 is the analogous ion with three hexoses in a straight chain. The small peak at m/z 1253 is indicative for a further extension of the straight carbohydrate chain with one hexosamine. Terminal NeuAc is seen at m/z 406 and m/z 374. The mass spectral data discussed so far are consistent with a structure (presented above the spectra) with two NeuAc residues attached to the same penultimate hexose. The position of the branching point is further strengthened by information m/e from the spectrum of the methylated-reduced derivatives in Fig. 6. The very intense peaks at m/z 1443 and 1415 are ions containing the fatty acid (C24, C22) plus the carbohydrate chain from which two terminal NeuAc are lost and replaced by two hydrogen atoms. The somewhat less intense peaks a t m/z 1253 and 1225 are similar fragments, but with an additional loss of one hexose and the uptake of only one hydrogen atom. This strongly indicates that the branching point is located on this last hexose. A number of additional diagnostic fragments indicated in the spectra and fragmentation formulas of all three derivatives (Figs. 5-7) provide further support for the presented structure.
On the basis of the above evidence, the structure of DG-4 must be as follows:
The sugar sequence was further confirmed by direct probe mass spectrometry of permethylated (not shown) and permethylated-reduced derivatives (Fig. 8). Molecular ions (M -1)+ at m/z 2074 are seen in Fig. 8 for the major molecular species containing C180 normal fatty acid and C181 long chain base. The fragment at m/z 1821 contains the complete carbohydrate chain (two NeuAc, three hexoses, and one hexosamine) combined with C180 normal fatty acid; m/z 1488, 1169, 951, 720, and 530 are secondary ions confirming the sequence given. The combined data from spectra of both derivatives are fully consistent with the structure proposed for DG-5 as G D l b . The fragment pattern around the peaks at mjz 1488 and 1169 in Fig. 8 shows the approximate fatty acid composition (Table IV). DG-6"This ganglioside contained galactose, glucose, glu-1 2600 cosamine, and sialic acid in an approximate molar ratio of 4:1:3:2. On treatment with V. cholerae neuraminidase the ganglioside lost both sialic acids, and a neutral glycolipid which co-migrated on TLC with lactoisooctanosylceramide was produced. Periodate borohydride reduction-mild methanolysis treatment of DG-6 yielded only NeuAc-7, which indicated that the two sialic acids are not linked to each other. On methylation analysis (Table III), DG-6 revealed the presence of 3-substituted galactose, 3,6-disubstituted galactose, and 4-substituted glucose in an approximate molar ratio of 3:l:l. The 4-substituted hexosamine was confirmed by the presence of 3,6-di-O-methyl-2-deoxy-(N-methylacetamido)glucitol. The asialoglycolipid obtained from DG-6 reacted with jack bean P-galactosidase to give a product with R L N n~ 0.47. This product, on further reaction with jack bean P-N-acetylhexosaminidase, yielded lactoneotetraosylceramide (RF 1.00) (Fig. 9). The identification of this glycolipid was based on the complement fixation results with purified anti-lactoneotetraosylceramide antibody (33).
The DG-6 fraction was further analyzed by direct inlet mass spectrometry as permethylated (not shown) and permethylated-reduced derivatives (Fig. 10). No useful spectrum was obtained after silylation. The peak at m/z 2109 in Fig. 10 is most probably due to a rearrangement ion containing the major fatty acid (C24:O) combined with a carbohydrate chain composed of five hexoses and three hexosamines. The addition of two hydrogens indicates a loss of additional carbohydrate units in two positions. This means that the original structure is branched. The series of peaks a t m/z 1905, 1701, 1674, and 1470 indicates that the two branches contain together at least two hexoses and one hexosamine. The peaks at mlz 1049 and 818 indicate that the proximal carbohydrate chain is unbranched and contains at least two hexoses and one hexosamine with the sequence given in the formula. The small primary carbohydrate fragments at m/z 1088,900,884, 873, and 857 are also consistent with a branched chain terminal with branches containing at least two hexoses and one hexosamine. The peaks at mlz 436 and 452 indicate in fact that the two branches contain together two hexoses and two hexosamines. 10. Mass spectrum of permethylated-reduced ganglioside DG-6 from human erythrocytes and a simplified formula for interpretation. The conditions of analysis were: electron energy, 34 eV; trap current, 500 pHz; acceleration voltage, 4 kV; ion source temperature, 275 "C at evaporation. Peaks below m/z 80 were not reproduced.

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The spectrum of a permethylated derivative (not shown) showed peaks at m/z 376 and 344 (376-32) for terminal NeuAc but no indication of a NeuAc-NeuAc peak. Ions originating from the terminal trisaccharides(s) were seen a t m/z 825, and 765 (825-60). A number of additional secondary carbohydrate fragments were also present. They originated from the terminal branched structure and are all consistent with the structure given.
Although they are strongly suggestive, the mass spectra alone give no absolute proof for the branching point or the total number of constituent sugars. They do, however, provide evidence of a branched 8-sugar basic structure with two free hydroxyls. The terminal trisaccharide fragments in the spectrum of the permethylated derivatives (NeuAc-hexose-hexosamine) appears to overlap with the basic sequence, which makes the proposed structure highly probable.
The major sphingosine base was C18:1, as indicated by an intense peak a t m/z 364 in the spectrum of permethylated derivative. The major fatty acids were C22:0, C24:1, and C24:O (Table IV)  On the basis of all the evidence described above, the structure of DG-6 can be proposed as shown in Scheme A. 3)

DISCUSSION
Approximately 8045% of the gangliosides of human erythrocytes contain a single sialic acid and 15-20% are disialogangliosides. The monosialogangliosides were studied by Watanabe et al. (5-7), who identified nine compounds. We have identified two additional monosialogangliosides that were not detected previously in erythrocytes. MG-1 and MG-3, which have the same oligosaccharide structures as brain gangliosides GM2 and GM1, respectively. MG-5 and MG-6 have the same oligosaccharide structure as ganglioside Gs identified by Watanabe et ul. (6). MG-5, like GG, contains mostly long chain fatty acids, C22-C24 (Table IV), whereas MG-6 contains mostly C16 fatty acids. Kannagi et al. (42) recently presented data on the relationship between the ceramide and carbohydrate structures of a number of glycolipids. They reported that human erythrocyte monosialogangliosides that contain a terminal NeuAc(a2-6)Gal structure contain mostly short chain fatty acids, whereas compounds with a terminal NeuAc(a2-3) structure had a predominance of long chain fatty acids. The predominance of short chain fatty acids in MG-6, the structure of which has been confirmed by Hako-m~r i ,~ suggests that this relationship may not be as complete as suggested by this group, but it does not detract from the conceptual importance of their proposal. We previously made a similar suggestion about the role of fatty acids in determining the carbohydrate structure of glycolipids (11) based on our analysis of the erythrocytes of the rare p phenotype. These cells cannot synthesize globotriaosylceramide and they contain an excess of lactosylceramide that has long chain fatty acids, and a normal amount of lactosylceramide with short chain fatty acids (11).
The disialogangliosides of human erythrocytes have not been studied previously. Four of the six compounds that we characterized have been isolated previously from other tissues. DG-2 and DG-5 are identical with brain gangliosides GD1, and GDlh, respectively, DG-1 is GD3, and DG-3 is disialosyllactoneotetraosylceramide. It is interesting that both brain gangliosides have a predominance of C16 and C18 fatty acids, and that all of the gangliosides of the lacto and globo series, except MG-6, contain mostly C22 and C24 fatty acids. DG-1 (GDJ is the only ganglioside that contains an appreciable quantity of hydroxy fatty acids. Ganglioside DG-6, which is a new compound, has a branched lactoisooctaosylceramide structure with a terminal sialic acid residue on each branch. Monosialogangliosides with the same basic lactoisoctaosyl structure and with a terminal fucose or Gal(a1-3) substituent on one branch, have been isolated from erythrocytes by Watanabe et ul. ( 5 ) .
Ganglioside DG-4 has a novel structural feature, the presence of sialic acid residues attached to different hydroxyl groups of the same galactose. To the best of our knowledge, this is the first example of this structure in glycoconjugates. This compound is only the second ganglioside described that belongs to the globo series of glycolipids. A ganglioside isolated from chicken muscle (43) has the structure NeuAc(a2-S. I. Hakomori, personal communication.
Including the data in this report, 11 monosialogangliosides and 6 disialogangliosides have been identified in human erythrocytes, and additional compounds have been detected but not purified a t present. Although definitive information about the functions of these compounds is not available, there is an increasing body of data about their potential functions as cell membrane receptors and antigens (2,45). A number of monoclonal antibodies against human tumors are directed against gangliosides, and these compounds isolated from erythrocytes provide a valuable library of defined oligosaccharide structures for analysis of the specificity of antibodies and lectins.