Identification of Novel Gangliosides Containing Lactosaminyl-GM1 Structure from Rat Spleen*

Two novel monosialogangliosides were isolated from rat spleen. The structures of the gangliosides (shown below, where NeuNGc is N-glycolylneuraminic acid) were characterized by compositional analysis, methylation analysis, hydrolyses with exoglycosidases, direct probe fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectrometry. A novel structure common to both gangliosides was N-acetyllactosaminyl-GM1 LacN-GM1; where GM1 is II3NeuAc-GgOse4Cer), and this is the first paper to report the occurrence of a new group of gangliosides. [formula: see text] Furthermore, in a monosialoganglioside fraction of rat spleen, the occurrence of a ganglioside having two lactosamine units (Gal alpha 1-3(Gal beta 1-4GlcNAc beta 1-3)2-GM1(NeuAc) (alpha Gal-(LacN)2-GM1] was suggested. These gangliosides have a unique structure, which includes the ganglio series ganglioside core and the extended modification characteristic of the lacto series.

Two novel monosialogangliosides were isolated from rat spleen. The structures of the gangliosides (shown below, where NeuNGc is N-glycolylneuraminic acid) were characterized by compositional analysis, methylation analysis, hydrolyses with exoglycosidases, direct probe fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectrometry. A novel structure common to both gangliosides was N-acetyllactosaminyl-GM1 LacN-GM1; where GW is I13NeuAc-GgOse&er), and this is the first paper to report the occurrence of a new group of gangliosides.
Gangliosides are characteristic cell membrane components and are involved in a variety of cellular events, including differentiation, maturation, and transformation (1,2).
Important functions of these molecules as antigens, and immunomodulators have also been recognized (3). Recently, gangliosides of murine spleen have been the subject of several investigations (4-6). In rat spleen, two major gangliosides were shown to correspond to GM3 ' and GD1, on TLC. The occurrence of minor gangliosides, which increased with the progress of development, was also demonstrated (4). However, further characterization of these gangliosides has not been carried out. We have been trying to establish the chemical structures of gangliosides present in rat spleen.
During this investigation, we found a series of novel gangiio- HCl, and fatty acid methyl esters were extracted with hexane (10). Methyl glycosides in the methanolic phase were subjected to N-acetylation according to Kozulic et al. (11) and were then trimethylsilylated (10). The carbohydrate derivatives were analyzed on a Hewlett-Packard HP-1 fused silica capillary column (0.32 mm x 25 m) using a splitless injection mode. The column temperature was kept at 120 "C for 1.5 min and then raised to 300 "C at 6 "C/min. The fatty acid methyl esters were analyzed by GLC under the same conditions as above and were then further examined by gas chromatography-mass spectrometry with a JEOL JMS-DX 300. Sialic acid species were determined by GLC (12). Methyl&ion Analysis-Gangliosides (5 Hg as sialic acid) were methylated according to Ciucanu and Kerek (13). Partially methylated alditol acetates from the permethylated gangliosides were prepared as described by Yang and Hakomori (14) except that acetolysis was performed with 0.7 N HCI in 80% acetic acid at 75 "C for 18 h." GLC analysis of the derivatives was carried out on the HP-1 capillary column mentioned above, with a temperature program beginning at 120 "C for 1.5 min followed by raising the temperature by 10 "C/min to 160 "C and then by 4 "C/min to 280 "C. The methylated alditol acetates were also identified by gas chromatography-mass spectrometry (15,16).
Prior to permethylation of degradation products of gangliosides with exoglycosidases, the gangliosides were subjected to preparative TLC using chloroform/methanol/water (60:35:8) as the developing solvent. The gangliosides were detected with iodine vapor, recovered from the plate with chloroform/methanol/water (3:7:1), and purified with a Sep-Pat Cl8 cartridge (17). Enzymatic Hydrolysis-Stepwise hydrolyses of Ml4 with exoglycosidases were carried out as follows. 15

RESULTS
Three novel monosialogangliosides were isolated from rat. spleen and tentatively named M13, M14, and Ml5 in order of decreasing mobility on TLC. TLC profiles obtained with two different solvent systems are shown in Fig. 1. Ml3 migrated slightly faster than GoI,, Ml4 between GD1, and GDlb, and Ml5 near GTlb with the neutral solvent system A (Fig.  la)  Ocontaining solvent system B than with solvent system A, in contrast to the reference di-and trisialogangliosides (Fig. lb). Ml3 and Ml4 showed a single band on TLC obtained with solvent systems A-C. Although Ml5 showed a single band on TLC with the three solvent systems, the results of exoglycosidase treatment revealed that the fraction contained two components as mentioned below. Yields of M13-Ml5 were 120, 810, and 920 rg from 2.2 kg of wet tissue, respectively.
The carbohydrate constituents of Ml3 and Ml4 were: Gal, Glc, GalNAc, GlcNAc, and NeuNGc at molar ratios of 2.9:1:1.1:0.9:1.1 and 4.1:1:1.2:1.0:1.1, respectively. These gangliosides were characterized by the presence of both N-acetylgalactosamine and N-acetylglucosamine. Ml5 also contained two types of hexosamine, and the carbohydrate constituents were Gal, Glc, GalNAc, GlcNAc, and NeuAc at a molar ratio of 5.3:1:1.0:2.1:1.0. The sialic acid in Ml3 and Ml4 was identified as N-glycolylneuraminic acid, and that of gangliosides in Ml5 was found only in the N-acetyl form.
Churacterization of Ml3 and Ml4-Methylation analysis of Ml3 (Table I) showed the presence of terminal galactose with the detection of 2,3,4,6-tri-O-Me-Gal.
The sequential treatment of Ml4 with (Y-and &galactosidases (M14b) produced 3,4,6-tri-O-Me-GlcNAc instead of 3,6-di-0-Me-GlcNAc, indicating the presence of N-acetylglucosamine residues at the nonreducing terminus. The disappearance of 2,3,4,6-tri-O-Me-Gal and the reduction of 1 mol of 2,4,6-tri-O-Me-Gal were also shown. When Ml4 was subjected to exoglycosidase treatment (Fig.  2), the terminal galactose was liberated by a-galactosidase. The product showed exactly the same RF value as that of Ml3 and was further sequentially hydrolyzed with @galactosidase and P-N-acetylhexosaminidase, being converted to a ganglioside with the same TLC mobility as Ghll(NeuNGc galactose of Ml3 was shown to possess the /3-anomerit configuration by b-galactosidase treatment. The product also gave the same TLC mobility as the product after sequential treatment of Ml4 with (Y-and /?-galactosidases. From the results of methylation analysis and exoglycosidase treatment, Ml3 and Ml4 were suggested to contain the terminal sequences Gal@l-4GlcNAc@l-and GalLul-3GalBl-4GlcNAc/31-attaching to the external galactose of the GILI1(NeuNGc) core through l-3 linkages, respectively.
To confirm the proposed structures of Ml3 and M14, the gangliosides were examined by negative-ion fast atom bombardment mass spectrometry and lH NMR spectrometry. As shown in Fig. 3B), the main pseudo molecular ion ((M -H)-) of Ml4 was demonstrated at m/z 2170. The mass number is 1 unit higher than that calculated for the proposed structure with C24:l fatty acid and Cl8 sphingenine, which is due to the isotope effect. This assignment was supported by the fatty  Dabrowski et al. (18). (tuGal-nLc,Cer, cu-galactosyllacto-N-neotetraosylceramide). acid composition (see Table III), which indicated that C24:l is the major fatty acid in M14. Fragment ions due to the successive elimination of carbohydrates were also detected at m/z 2007,1845,1642,1480,1277,970,808, and 646 and were assigned as noted in Fig. 3B. The presence of the fragment ion at m/z 1277 clearly demonstrated that the sialic acid is linked to the innermost galactose and that the core structure of the ganglioside is Ghll, not Grullb. In the mass spectrum of Ml3 (Fig. 3 A), the main pseudo molecular ion was detected at m/z 2007, which is consistent with the value calculated for the proposed structure of Ml3 with C24:l fatty acid and Cl8 sphingenine. Characteristic fragment ions were also detected and were assigned as shown (Fig. 3).
The anomeric proton regions of the 'H NMR spectra of Ml3 and Ml4 are shown in Fig. 4, and the assignment of each signal is given in Fig. 4 and Table II. Ml3 showed six distinctive signals of anomeric protons. The signals at 4. 16, 4.29, and 4.85 ppm were in positions almost identical to those of GM,(NeuNGc) and were assigned to p-GlcI, P-GalII, and /3-GalNAcIII, respectively. The signal at 4.23 ppm showed a chemical shift virtually identical to the terminal galactose of GM1(NeuNGc), assigned to the terminal P-GalVI; and the signal at 4.30 ppm was assumed to be P-GalIV with a downfield shift due to glycosylation. The signal at 4.66 ppm, which is a region for P-N-acetylglucosamine (18), must belong to p-GlcNAcV. In the spectrum of M14, four signals showed exactly the same chemical shifts as those of M13: 4. 16, 4.29, 4.66, and 4.85 ppm, assigned to ,&GlcI, P-GalII, P-GlcNAcV, and P-GalNAcIII, respectively. The overlapping signals around 4.30 ppm were assumed to be P-GalIV and P-GalVI. The remaining signal at 4.86 ppm showed good agreement in chemical shift and coupling constant to that of the anomeric proton of terminal a-galactose in a-galactosyllacto-N-neotetraosylceramide reported by Dabrowski et al. (18) and was assigned to a-GalVII in M14.
From the results mentioned above, the structures of Ml3 and Ml4 were concluded to be as shown below.  3 Ml3 contains the GM, core structure and is extended by a neolacto-type disaccharide (N-acetyllactosamine). Ml4 is a derivative of Ml3 with an a-galactosyl substitution.
The fatty acid compositions of Ml3 and Ml4 are summarized in Table III. They are characterized by the predominance of saturated and unsaturated C24 fatty acids including C24:2. No hydroxy fatty acid was detected in these gangliosides.
Characterization of Ml5 by Sequential Exoglycosidase Treatment and Methylation Analysis-Ganglioside Ml5 was converted into two distinctive components on TLC by a-galactosidase treatment (Fig. 5). Both were hydrolyzed with /3galactosidase, and two products were obtained. The predom-inant product, the lower band in Fig. 5 (lane b), was further and successively hydrolyzed with P-N-acetylhexosaminidase and P-galactosidase and was converted to a ganglioside corresponding to Ghll(NeuAc) by /3-N-acetylhexosaminidase. The other product, the upper band in Fig. 5 (lane 5) was stable to treatment with P-galactosidase and ,&N-acetylhexosaminidase. The results of the enzymatic hydrolyses together with the result of the compositional analysis suggested that the major ganglioside in Ml5 contains the terminal sequences Gala-(Galp-GlcNAcp-)z substituted to GMl(NeuAc). Further analysis will be required to show the structure of another ganglioside in M15.
The methylation analysis of Ml5 gave identical derivatives to those of Ml4 (Table I). The higher ratios of 2,4,6-tri-O-Me-Gal and 3,6-di-0-Me-GlcNAc in Ml5 than in Ml4 were consistent with a structure containing a repeating 3GalPl-4GlcNAcPl-unit in the substitution extended on GM1. From the results mentioned above, the structure of one of the two gangliosides in Ml5 was deduced to be as follows.

DISCUSSION
Generally, N-acetylgalactosamine is the exclusive hexosamine constituent of ganglio-and globo series glycolipids, and N-acetylglucosamine is that of lacto series glycolipids. In this study, two novel monosialogangliosides containing both Nacetylgalactosamine and N-acetylglucosamine were isolated from rat spleen. These gangliosides (LacN-GM1 and aGal-LacN-GILI1) were clearly demonstrated to be novel ones, whose structures consist of a GM1 core extended by Galbl-4GlcNAc@l-3 and Galal-3Gal@l-4GlcNAc@l-3 sequences, respectively. Moreover, the third novel ganglioside, which was suggested to contain an a-galactosylated repeating N-acetyllactosamine unit, was found in the same tissue (aGal-(LacN)z-GMl). Recently, hybrid-type neutral glycolipids (19) and gangliosides (20) containing both neolacto-and ganglio-type structures have been isolated. The glycolipids, named "lactoganglio series" (19) or "neolacto-ganglia series" (20), contain the branching structure -GlcNAc@l-3(GalNAc@l-4)Galpl-4Glcpl-lCer, and the neolacto-type sugar chain is further extended. On the other hand, the novel group (lactosaminyl-GM~ group) gangliosides characterized in this study have a unique structure with a ganglio series ganglioside core and a neolacto-type sequence, which are combined without branching.
The ganglioside aGal-(LacN)z-GM1 was suggested to occur as one of two gangliosides, which co-migrated on TLC with all the solvent systems examined. We noticed the occurrence of these two gangliosides from the results of sequential exoglycosidase treatment.
One of them was proposed to be a member of the lactosaminyl-G1lll group, containing the Galal-3(Gal/31-4GlcNAcpl-3)z sequence extended on G,,. The results of compositional analysis and methylation analysis of the fraction were almost consistent with those from the proposed structure mentioned above. Therefore, the unknown ganglioside co-migrating with aGal-(LacN)B-GIVI1 is suggested to contain a structure with carbohydrate constituents and linkages similar to those of aGal-(LacNh-GM1. It is notable that LacN-GM1 and aGal-LacN-GM1 contain N-glycolylneuraminic acid, whereas aGal-(LacN)z-GM1 contains N-acetylneuraminic acid. For some reason, the sialic acid in aGal-(LacN)Z-G,, may be hardly modified by the monooxygenase which converts N-acetylneuraminic acid to the N-glycolyl form (21). Whether glycosyltransferases involved in the formation of the lacto series can synthesize lactosaminyl-GM, group gangliosides from GM1 remains unanswered. It has been reported (22) that mouse myelogenous leukemia cells contain lacto series glycolipids, the syntheses of which were enhanced upon differentiation of the cells into macrophage-like cells. If the enzymes involved in lacto series synthesis could synthesize lactosaminyl-GM1 group gangliosides, the expression may be associated with cell differentiation such as that of monocytes to macrophages in the spleen. Further studies are required to clarify the biosynthetic pathways including hydroxylation of sialic acid in lactosaminyl-GM1 group gangliosides.