Generation of murine monoclonal antibodies specific for N-glycolylneuraminic acid-containing gangliosides.

We generated two murine monoclonal antibodies (MAbs) specific for mono- and disialylgangliosides having N-glycolylneuraminic acid (NeuGc) as their sialic acid moiety, respectively, by immunizing C3H/HeN mice with these purified gangliosides adsorbed to Salmonella minnesota followed by fusion with mouse myeloma cells. By use of a wide variety of glycolipids, including NeuGc-containing gangliosides, the precise structures recognized by these two antibodies were elucidated through enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatography. One MAb, GMR8, which was generated by immunizing the mice with purified GM3(NeuGc), reacted specifically with gangliosides having NeuGc alpha 2----3Gal- terminal structures, such as GM3(NeuGc), IV3NeuGc alpha-Gg4Cer, IV3NeuGc alpha-nLc4Cer, V3NeuGc alpha-Gb5Cer, and GD1a(NeuGc, NeuGc). None of the other gangliosides having internal NeuGc alpha2----3Gal- sequences, such as GM2(NeuGc) and GM1(NeuGc), nor corresponding gangliosides having NeuAc alpha 2----3Gal- sequences, nor neutral glycolipids were recognized. Thus, the epitope structures recognized by the MAb were found to be strictly NeuGc alpha 2----3Gal- terminal structures. In contrast, the other MAb, GMR3, which was generated by immunizing the mice with purified GD3(NeuGc-NeuGc-) adsorbed to the bacteria, reacted specifically with gangliosides having NeuGc alpha 2----8NeuGc alpha 2----3Gal- terminal sequences, such as GD3(NeuGc-NeuGc-), IV3NeuGc alpha 2-Gg4Cer, IV3NeuGc alpha 2-nLc4Cer, and V3NeuGc alpha 2-Gb5Cer, but did not react with corresponding gangliosides having NeuAc as their sialic acid moiety or with the neutral glycolipids tested. The epitope structures recognized by the MAb were suggested to be NeuGc alpha 2----8NeuGc alpha 2----3Gal- terminal structures. Using these MAbs, we determined the distribution of such gangliosides in the spleen, kidney, and liver of several mice strains. Novel gangliosides reactive with these MAbs were detected in these tissues.     

Identification of N-glycolylneuraminic acid-containing gangliosides of cat and sheep erythrocytes. 252Cf fission fragment ionization mass spectrometry in the analysis of glycosphingolipids

A number of gangliosides were isolated from cat and sheep erythrocytes for use in analyzing the specificity of a panel of human anti-heterophile monoclonal antibodies. The structures of these compounds were determined by a combination of different procedures, including sugar analysis, glycosidase treatment, periodate oxidation, TLC immunostaining, methylation analysis, and mass spectrometry. These methods identified the cat erythrocytes gangliosides (C1 and C2) as N-glycolylneuraminic acid (NeuGc)-containing hematosides; C1 was shown to be NeuGc alpha 2----8NeuGc alpha 2----3Gal beta I----4Glc-Cer [NeuGc)2GD3) and C2 to be NeuAc alpha 2----8NeuGc alpha 2----3Gal beta 1----4Glc-Cer [NeuAc-NeuGc-)GD3). The two sheep gangliosides (S1 and S2) were found to be novel glycolipids based on the paragloboside sequence; S1 was identified as NeuGc alpha 2----8NeuGc alpha 2----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer [NeuGc)2-disialylparagloboside) and S2 as NeuAc alpha 2----8NeuGc alpha 2----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer [NeuAc-NeuGc-)-disialylparagloboside). Structural analysis of these compounds was aided by the use of 252Cf fission fragment ionization time-of-flight mass spectrometry. This method provided easily interpretable spectra on methylated derivatives which were particularly useful in determining the sialic acid composition of the gangliosides and the sequence of their disialosyl side chains.     

Different fine binding specificities of monoclonal antibodies to disialosylganglioside GD2

The fine structural specificities of six monoclonal antibodies (MAbs) to ganglioside GD2, GalNAc beta 1----4(NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta 1----4Glc-Cer, were studied. The binding specificities of these MAbs were found to differ from each other by virtue of their binding to structurally related authentic standard glycolipids as revealed by three different assay systems, including enzyme immunostaining on thin-layer chromatography, enzyme-linked immunosorbent assay, and immune adherence inhibition assay. The MAbs examined could be divided into three binding types. MAbs A1-201, A1-410, and A1-425 bound specifically to ganglioside GD2 and none of the other gangliosides tested. Two other MAbs (A1-245 and A1-267) reacted not only with GD2, but also with several other gangliosides having the sequence NeuAc alpha 2----8NeuAc alpha 2----3Gal (GD3, GD1b, GT1a, GT1b, and GQ1b). The reactivities with these gangliosides varied to some degree. In addition, these MAbs were found to react with both GD3(NeuAc-NeuAc) and GD3(NeuGc-NeuAc), but not with GD3(NeuAc-NeuGc) or GD3(NeuGc-NeuGc). The last MAb (A1-287) also reacted with several other gangliosides but with lower avidity than A1-245 and A1-267. These findings suggest that each MAb to ganglioside GD2 may have an individual binding specificity and avidity. These MAbs represent potentially useful reagents for analyzing the function of GD2 on cell surface membranes, and provide a system for precisely studying the interactions between an anti-ganglioside antibody and the binding epitope of the antigenic determinant.     

Gangliosides as paramyxovirus receptor. Structural requirement of sialo-oligosaccharides in receptors for hemagglutinating virus of Japan (Sendai virus) and Newcastle disease virus

A sensitive assay system for receptor activity of gangliosides to paramyxovirus was developed. This system involves incorporation of gangliosides into neuraminidase-treated chicken erythrocytes (asialoerythrocytes) followed by estimation of virus-mediated agglutination and hemolysis. The asialoerythrocytes coated with I-active ganglioside (Sia alpha 2-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer) were effectively agglutinated by hemagglutinating virus of Japan (HVJ, Sendai virus). The hemolysis of the asialoerythrocytes mediated by HVJ was restored to the highest level by labeling the cells with gangliosides possessing lacto-series oligosaccharide chains, i.e., I-active ganglioside, N-acetylneuraminosylparagloboside (SiaPG(NeuAc)), and i-active ganglioside (Sia alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-Cer). The specific receptor activity of ganglioside GD1a possessing a gangliotetraose chain was lower than those of the gangliosides described above. Gangliosides GM3, GD3, GM1a, GD1b, SiaPG(NeuGc) showed little effect on the restoration of HVJ-mediated hemolysis. On infection with Newcastle disease virus (NDV), the highest specific restoration of lysis was found in chicken asialoerythrocytes coated with SiaPG(NeuAc or NeuGc) and GM3(NeuAc or NeuGc), whereas those coated with I-active ganglioside, GD3, GM1a, and GD1b showed very low NDV-mediated hemolysis. The above results indicate that the determinants of receptor for HVJ contain sialylated branched and/or linear lacto-series oligosaccharides carried by I,i-active gangliosides and SiaPG(NeuAc) and sialosylgangliotetraose chain carried by GD1a. The determinants for NDV are carried by SiaPG(NeuAc or NeuGc) containing linear lacto-series oligosaccharide and GM3(NeuAc or NeuGc). The absence of detectable binding of free oligosaccharides obtained from I-active ganglioside and sialoglycoprotein GP-2 isolated from bovine erythrocyte membranes as HVJ receptor (Suzuki, Y., et al. J. Biochem. (1983) 93, 1621-1633; (1984) 95, 1193-1200) indicates that HVJ recognizes the sialooligosaccharides oriented out of the lipid bilayer in the cell membranes where the hydrophobic ceramide or peptide backbone of the receptor is integrated.     

Acidic glycolipids from dolphin kidney

The composition and contents of acidic glycolipids in the kidney of a striped dolphin (Stenella coeruleoalba, the order Cetacea, whales) were determined. The following eight acidic glycolipids were isolated and characterized: SM4s (124.2 nmol/g tissue), SM3 (8.7), GM3 (NeuAc) (12.3), GM3 (NeuGc) (31.6), GD3 (NeuAc-NeuAc) (14.7), GD3 (NeuAc-NeuGc) (II3 alpha(NeuAc alpha 2-8NeuGc)-LacCer) (9.8), GD3 (NeuGc-NeuAc) (II3 alpha(NeuGc alpha 2-8NeuAc)-LacCer) (5.3), and GD3 (NeuGc-NeuGc) (15.8). The assignment of the four types of GD3 was further confirmed as described below. Evidence indicating 2-8 linkages of the disialosyl residues of GD3 was obtained on methylation analysis of sialic acid. GD3 (NeuAc-NeuAc) and GD3 (NeuGc-NeuAc) were degraded to GM3 (NeuAc), and GD3 (NeuAc-NeuGc) and GD3 (NeuGc-NeuGc) yielded GM3 (NeuGc) on mild acid hydrolysis. Fragment ions characteristic of the carbohydrate and lipophilic moieties of the permethylated GD3 were observed in direct inlet-electron impact-mass spectra. The presence of these four types of GD3 in a tissue has not been reported previously. GD3 contained non-hydroxy (69-84%) and hydroxy fatty acids (16-31%) with 16-24 carbons. The long chain base of all GD3, except GD3 (NeuGc-NeuAc) (not analyzed), consisted of 4-sphingenine (d18:1) and 4-hydroxysphinganine (t18:0) in almost equal amounts. The total amount of renal lipid-bound acidic groups (sulfate and sialic acid) of the dolphin (190 mumol/animal) is considerably higher than that of a terrestrial mammal (88 mumol/animal) with a body weight comparable to that of the dolphin. This deviation suggests that the amount of renal acidic amphiphiles required to maintain the osmotic balance of body fluids in marine mammals might be higher than that in terrestrial ones.     

Northern hybridization analysis of VH gene expression in murine monoclonal antibodies directed to cancer-associated ganglioside antigens having various sialic acid linkages

The expression of the VH genes in 46 murine hybridoma cells that secrete mAb directed to the cancer-associated carbohydrate Ag, especially acidic glycolipids such as gangliosides and sulfated glycoplipids, was analyzed by Northern hybridization of poly(A)+ RNA of hybridoma with cDNA probes for nine VH gene families. Different hybridomas tended to express VH genes of the same family when the cognate Ag had the same or similar carbohydrate structures; i.e., the VH genes of the J558 family (group 1) were preferentially expressed in the mAb directed to various gangliosides that have NeuAc alpha (or NeuGc alpha) 2-3 and/or 2-8 linkage (71%), the most common linkage of sialic acid residues in the gangliosides of higher animals, and the hybridomas directed to sulfated glycolipids also expressed mainly the VH genes of the J558 family (80%). In contrast, the five mAb directed to various gangliosides with NeuAc alpha 2-6 linkage were exclusively encoded by the VH genes of Q52 family (group 2, 100%), and three antibodies directed to gangliosides with a NeuAc alpha 2-9 linkage all expressed genes of J606 family (group 6, 100%). The VH family usage was largely correlated with the linkage of sialic acid residues in the cognate carbohydrate Ag, but was not correlated at all with the difference in the fine specificities toward the core neutral carbohydrate chain, to which the sialic acid residues were attached. These findings suggest that the VH gene family in these anticarbohydrate antibodies is selected, depending primarily on the linkage of the sialic acid residues in carbohydrate Ag; these residues form the immunodominant sugar residue in the respective antigenic determinant.     

Gangliosides modulate sodium transport in cultured toad kidney epithelia

Cultured A6 epithelial cells from toad kidney form confluent monolayers with tight junctions separating the apical and basolateral membranes. These two membrane domains have distinct compositions and functions. Thus, sodium is actively transported across the epithelia from the apical to basolateral surface via amiloride-inhibitable sodium channels located in the apical membrane. Sodium transport is stimulated by vasopressin, cholera toxin, and 8-bromo-cAMP applied to the basolateral surface where the receptors, adenylate cyclase, and Na+/K+-ATPase are located. In a previous study (Spiegel, S., Blumenthal, R., Fishman, P.H., and Handler, J.S. (1985) Biochim. Biophys. Acta 821, 310-318), we demonstrated that exogenous gangliosides inserted into the apical membrane of A6 epithelia do not redistribute to the basolateral membrane. With the ability to vary selectively the ganglioside composition of the apical membrane, we examined the effects of gangliosides on sodium transport in A6 epithelia. When the apical surface of A6 epithelia were exposed to exogenous gangliosides, sodium transport in response to vasopressin, cholera toxin, and 8-bromo-cAMP was enhanced compared to epithelia not exposed to gangliosides. The effect was observed with bovine brain gangliosides, NeuAc alpha 2----3Gal beta 1----3GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4Glc beta 1----Cer (GD1a) and Gal beta-1----3GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4Glc beta 1----Cer (GM1), but not with the less complex ganglioside, Neu-Ac alpha 2----3Gal beta 1----4Glc beta 1----Cer (GM3). We examined A6 cells for endogenous gangliosides and found that, whereas GM3 was a major ganglioside, only trace amounts of GM1 and GD1a were present. Based on cell surface and metabolic labeling studies, these gangliosides were synthesized by the cells and were present on the apical as well as the basolateral surface. Bacterial sialidase, which hydrolyzes more complex gangliosides to GM1, was used to modify the endogenous gangliosides on the apical surface; after sialidase treatment, the epithelia were more responsive to vasopressin, cholera toxin, and 8-bromo-cAMP. Thus, gangliosides may be modulators of sodium channels present in the apical membrane of epithelial cells.     

Tissue-specific expression of GM3(NeuGc) and GD3(NeuGc) in epithelial cells of the small intestine of strains of inbred rats. Absence of NeuGc in intestine and presence in kidney gangliosides of brown Norway and spontaneously hypertensive rats

The ganglioside composition of the epithelial cells of the small intestine was investigated in 15 strains of inbred rats. Most of these strains had GM3 as the only detectable ganglioside. In addition to GM3, small amounts of GD3 were found in four strains, AVN, BN, DA, and LE. The fatty acid content of the ceramide portion was composed of a large, although variable, percentage of 2-hydroxy fatty acids. The sphingoid base was always C18-4D-hydroxysphinganine. The highly prominent sialic acid was N-glycolylneuraminic acid (NeuGc) in most strains. However in two strains, Brown Norway (BN) and spontaneously hypertensive rats (SHR), NeuAc was the only sialic acid of the gangliosides of the intestinal epithelium. The analysis of the ganglioside composition of the epithelium of the small intestine of the first generation hybrids of SHR with DA and BN, respectively, demonstrated that the expressions of GM3 (NeuGc) and GD3 were genetically transmitted as dominant traits and that BN and SHR were likely to carry the same deficient gene that led to the expression of GM3(NeuAc) instead of GM3(NeuGc) in the small intestine. For comparison, the sialic acid composition of kidney gangliosides was analyzed in some strains. 21-23% of the kidney gangliosides was GM3(NeuGc) in all tested strains, including BN and SHR. Therefore, the ganglioside composition of the intestinal epithelium could vary in the rat species, and the defect of N-glycolylneuraminic acid was not only strain-specific but also occurred in a tissue-specific way among strains of inbred rats.     

Glycosphingolipids of normal bovine and enzootic bovine leukosis lymph node cells

We analyzed glycosphingolipids from normal lymph node cells of seven cattle and lymph node cells of eight cattle with enzootic bovine leukosis. The neutral glycosphingolipids and gangliosides were analyzed by thin-layer chromatography. Both normal and tumorous lymph node cells had GlcCer, LacCer, and GbOse3Cer as major neutral glycosphingolipids. In the ganglioside fraction, GM3 was the predominant component in both normal and tumorous lymph node cells, and another component, ganglioside Gx fraction, was also prominent in tumorous lymph node cells. The structure of this ganglioside Gx fraction was elucidated by thin-layer chromatography, sugar analysis, neuraminidase digestion, and permethylation studies. This ganglioside Gx fraction was found to be a mixture of four ganglioside species. The structures of individual gangliosides Gx (1 to 4) were characterized as follows. 1: GD3, NeuAc alpha 2-8NeuAc alpha 2-3Gal1-4Glc-Cer. 2: GD3, NeuAc alpha 2-8NeuGc alpha 2-3Gal1-4Glc-Cer. 3: GD3, NeuGc alpha 2-8NeuAc alpha 2-3Gal1-Glc-Cer. 4: GD3, NeuGc alpha 2-8NeuGc alpha 2-3Gal1-4Glc-Cer. These GD3 species may be formed as a result of the induced synthesis inassociation with malignant transformation.     

Enhanced binding of the neural siglecs, myelin-associated glycoprotein and Schwann cell myelin protein, to Chol-1 (alpha-series) gangliosides and novel sulfated Chol-1 analogs

Extended glycoconjugate binding specificities of three sialic acid-dependent immunoglobulin-like family member lectins (siglecs), myelin-associated glycoprotein (MAG), Schwann cell myelin protein (SMP), and sialoadhesin, were compared by measuring siglec-mediated cell adhesion to immobilized gangliosides. Synthetic gangliosides bearing the alpha-series determinant (NeuAc alpha2,6-linked to GalNAc on a gangliotetraose core) were tested, including GD1alpha (IV(3)NeuAc, III(6)NeuAc-Gg(4)OseCer), GD1alpha with modified sialic acid residues at the III(6)-position, and the "Chol-1" gangliosides GT1aalpha (IV(3)NeuAc, III(6)NeuAc, II(3)NeuAc-Gg(4)OseCer) and GQ1balpha (IV(3)NeuAc, III(6)NeuAc, II(3)(NeuAc)(2)-Gg(4)OseCer). The alpha-series gangliosides displayed enhanced potency for MAG- and SMP-mediated cell adhesion (GQ1balpha > GT1aalpha, GD1alpha > GT1b, GD1a > GM1 (nonbinding)), whereas sialoadhesin-mediated adhesion was comparable with alpha-series and non-alpha-series gangliosides. GD1alpha derivatives with modified sialic acids (7-, 8-, or 9-deoxy) or sulfate (instead of sialic acid) at the III(6)-position supported adhesion comparable with that of GD1alpha. Notably, a novel GT1aalpha analog with sulfates at two internal sites of sialylation (NeuAcalpha2,3Galbeta1,4GalNAc-6-sulfatebeta1, 4Gal3-sulfatebeta1,4Glcbeta1,1'ceramide) was the most potent siglec-binding structure tested to date (10-fold more potent than GT1aalpha in supporting MAG and SMP binding). Together with prior studies, these data indicate that MAG and SMP display an extended structural specificity with a requirement for a terminal alpha2, 3-linked NeuAc and great enhancement by nearby precisely spaced anionic charges.     

Sialylation of glycoprotein oligosaccharides with N-acetyl-, N-glycolyl-, and N-O-diacetylneuraminic acids

Four common sialic acids (Sia), NeuAc, N-glycolyl-neuraminic acid (NeuGc), 4-O-acetyl-N-acetylneuraminic acid (4-O-Ac-NeuAc), and 9-O-Ac-NeuAc were examined for activation to their corresponding CMP-sialic acid conjugates and subsequently for their transfer to glycoprotein oligosaccharides by purified mammalian sialyltransferases. CMP-sialic acid synthetases from calf brain and from bovine and equine submaxillary glands were found to convert NeuAc, NeuGc, and 9-O-Ac-NeuAc to their corresponding CMP-sailic acids. In contrast, no conversion of 4-O-Ac-NeuAc to CMP-4-O-Ac-NeuAc was observed for any of the three synthetases examined. A new procedure for the preparation of CMP-9-O-Ac-NeuAc, CMP-NeuGc, and CMP-NeuAc in high yield and purity was developed, using the calf brain CMP-sialic acid synthetase. Each of these derivatives was tested as donor substrates for six mammalian sialyltransferases purified from porcine, rat, and bovine tissues, including a bovine GalNAc alpha 2,6 sialyltransferase whose purification is described in this report. The sialyltransferases examined represent those which form the Sia alpha 2,6Gal beta 1,4-GlcNAc-, Sia alpha 2,3Gal beta 1,3(4)GlcNAc-, Sia alpha 2,3Gal beta 1,3-GalNAc- and Sia alpha 2,6GalNAc- sequences found on N-linked and O-linked oligosaccharides of glycoproteins. CMP-NeuAc and CMP-NeuGc were equally good donor substrates for all six sialyltransferases. However, transfer of 9-O-Ac-NeuAc from CMP-9-O-Ac-NeuAc varied from only 10% to nearly 70% that of the transfer of NeuAc from CMP-NeuAc. Results are viewed to define the relative roles of direct transfer of these sialic acids and modification of glycosidically bound NeuAc in glycoproteins.     

Monoclonal antibody detects monosialogangliosides having a sialic acid alpha 2----3-galactosyl residue

A murine monoclonal antibody (mAb), designated mAb 202, was generated using a human melanoma cell line, UCLASO-M14 as the immunogen. mAb 202 reacted with two (GM2 and GM3) of the four (GM2, GM3, GD2, and GD3) gangliosides expressed by M14. Several authentic monosialogangliosides, including GM4, GM3, GM2, GM1, GM1b, and sialylparagloboside were then tested for their binding to 202 mAb by the immune adherence inhibition assay, TLC-enzyme immunostaining, and enzyme-linked immunosorbent assay. All showed positive binding but in varying degrees. GM4 showed the strongest affinity. No significant differences of reactivity were observed between the sialic acid derivatives, N-acetyl and N-glycolyl, in these gangliosides. Disialogangliosides such as GD3, GD2, GD1a, and GD1b, trisialoganglioside GT1b, and neutral glycolipids including GlcCer, GalCer, LacCer, GbOs3Cer, GbOs4Cer, GgOs3Cer, GgOs4Cer, and nLcOs4Cer were all negative. These results indicate that the 202 mAb detects sialyl alpha 2----3Gal residue in the monosialoganglioside, irrespective of the internal structure. Since GM4 is not expressed by M14 cells, the terminal disaccharide (sialyl alpha 2----3Gal) in GM3 and/or GM2 must have been the epitope responsible for the generation of the antibody.     

Syngeneic monoclonal antibody against melanoma antigen with interspecies cross-reactivity recognizes GM3, a prominent ganglioside of B16 melanoma

It has previously been reported that a mouse (C57BL/6) monoclonal antibody, M2590, was established against syngeneic melanoma B16 cells, which was shown to react only with melanoma cells from various species but not with other tumor cells or normal tissues (Taniguchi, M., and Wakabayashi, S. (1984) Gann 75, 418-426). In the present study, the specificity of M2590 antibody was shown to be directed to a saccharide arrangement (NeuAc alpha 2-3Gal beta 1-4Glc (or -GlcNAc)) of gangliosides by three different assay systems including enzyme immunostaining on thin layer plates, sandwich radioimmunoassay, and enzyme-linked immunoadsorbent assays using a variety of glycolipids with known structures. Neither gangliosides having NeuGc terminus, including NeuGc alpha 2-3Gal beta 1-4Glc-ceramide and NeuGc alpha 2-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-ceramide, nor ganglio series gangliosides carrying NeuAc reacted with the antibody. An M2590 antibody-reactive antigen was isolated from B16 melanoma cells, and its structure was determined to be NeuAc alpha 2-3Gal beta 1-4Glc-ceramide by fast atom bombardment mass spectrometry, methylation analysis, and exoglycosidase treatment. The ceramide was composed of d18:1 as its long-chain base and C16:0, C24:1, and C24:0 as major fatty acids. The same ganglioside was also detected in the culture supernatant of the melanoma cells as shedding antigen.     

A new monoclonal antibody directed to sialyl alpha 2-3lactoneotetraosylceramide and its application for detection of human gastrointestinal neoplasms

A new monoclonal antibody (NS24) directed to the N-acetylneuraminyl alpha 2-3Gal beta 1-4GlcNAc residue in type II sugar chain of N-acetylneuraminyllactoneotetraosylceramide [sialylparagloboside, IV3(NeuAc)nLc4Cer] was prepared by hybridoma technique. Liposomes composed of dipalmitoylphosphatidylcholine, cholesterol, IV3(NeuAc)nLc4Cer, and lipopolysaccharides from Salmonella minnesota R595 were used for immunization with IV3(NeuAc)nLc4Cer isolated from human erythrocytes. This method allowed the fusion of spleen cells of immunized mouse with myeloma cells only three days after immunization. NS24 reacted specifically to both naturally occurring and chemically synthesized IV3-(NeuAc)nLc4Cer, whereas it has no reactivity to structurally related gangliosides, such as IV6(NeuAc)nLc4Cer, N-glycolylneuraminyl alpha 2-3lactoneotetraosylceramide [IV3(NeuGc)-nLc4Cer], i-active ganglioside [VI3(NeuAc)nLc6Cer], I-active ganglioside [VIII3(NeuAc)-VI3(NeuAc)IV6kladoLc8Cer], GM4(NeuAc), GM3(NeuAc), GM3(NeuGc), GM1b(NeuAc), GD3-(NeuAc), other ganglio-series gangliosides, sulfatide, and paragloboside (nLc4Cer). Synthetic N-acetylneuraminyl alpha 2-3lactotetraosylceramide [IV3(NeuAc)Lc4Cer] and its asialo-derivative (Lc4Cer) carrying type I sugar chain also showed no reaction with NS24. One to 100 pmol of IV3(NeuAc)nLc4Cer was detected dose-dependently by a thin-layer chromatography/enzyme immunostaining procedure. Human gastric carcinomas showed positive reactions with NS24 immunochemically and histochemically. NS24 reacted preferentially with poorly differentiated adenocarcinomas rather than well differentiated ones.     

Structures of glycosphingolipids isolated from human granulocytes. The presence of a series of linear poly-N-acetyllactosaminylceramide and its significance in glycolipids of whole blood cells

Structures of glycolipids isolated from human granulocytes were elucidated by fast atom bombardment-mass spectrometry, methylation analysis, and exo- and endoglycosidase treatment. All neutral glycolipids, with saccharide residues ranging from 2 to 10, were found to have linear N-acetyllactosaminyl backbones. The majority of neutral glycolipids contain one or two fucosyl residues attached to N-acetylglucosamine residues through the Fuc alpha 1----3 linkage and were reactive with the monoclonal antibody specific to Gal beta 1----4(Fuc alpha 1----3)GlcNAc, the Lex structure. Their general structure can be expressed as follows: (formula; see text) where n = 0-3. Glycolipids containing sialic acid (gangliosides) were also found to have linear N-acetyllactosaminyl backbones with sialic acid joined to this backbone by either alpha 2----3 or alpha 2----6 linkage. The gangliosides have the following general structure: (formula; see text) where n = 0-3. The ceramide was composed of sphingosine with d18:1 as the long-chain base and C16:0 (as a major component) or C24:1 (as a minor component) fatty acid. Analysis of glycolipids isolated from granulocytes, erythrocytes, and whole blood cells revealed that, among the glycolipids prepared from the whole blood cells, dihexaosylceramide, lactoneotetraosylceramide, and the above described linear lactoneo series neutral glycolipids are present in granulocytes but barely present in erythrocytes.     

Purification and characterization of an O-acetylsialic acid-specific lectin from a marine crab Cancer antennarius

A sialic acid-binding lectin with high specificity for 9-O-acetyl- and 4-O-acetylsialic acids was purified from the hemolymph of the California coastal crab, Cancer antennarius, by affinity chromatography using bovine submaxillary mucin coupled to agarose. The binding specificity of the crab lectin distinguishes it from other known sialic acid-specific lectins from Limulus polyphemus and Limax flavus which show a broader range of specificity for sialic acids. The purified lectin is homogenous on sodium dodecyl sulfate-polyacrylamide electropherograms with a subunit molecular weight of about 36 kDa. The specificity of the lectin for O-acetylsialic acids appears to account for the fact that it agglutinates mouse, rat, rabbit, and horse erythrocytes, which contain O-acetylsialic acids on cell surface glycoconjugates, but not human monkey, sheep, goat, and chicken erythrocytes which contain only NeuAc or N-glycolylneuraminic acid (NeuGc). This conclusion was supported by the potent inhibition of hemagglutination by bovine and equine submaxillary mucins which contain 9(7,8)-O-acetyl- and 4-O-acetylsialic acids, respectively, and also by free 9-O-acetyl-N-acetylneuraminic acid (9-O-Ac-NeuAc) and 4-O-Ac-NeuAc relative to NeuAc and NeuGc. Further support for the role of O-Ac-sialic acids in hemagglutination of erythrocytes was obtained by enzymatic modification of human erythrocytes. Sialidase-treated erythrocytes were resialylated with purified sialyltransferases and various CMP-sialic acid donor substrates to contain NeuAc or NeuGc or 9-O-Ac-NeuAc in the Sia alpha 2,3Gal or Sia alpha 2,6Gal linkages. Cells resialylated to contain NeuAc or NeuGc were not agglutinated, but cells resialylated to contain 9-O-Ac-NeuAc were agglutinated with high titer, comparable to that of mice or horse erythrocytes.     

Mouse monoclonal antibodies with specificity for the melanoma-associated ganglioside disialyllactosylceramide (GD3) also react with the structural analogue disialylparagloboside

A mouse monoclonal IgM antibody, 4.2, has previously been shown to bind preferentially to the surface of human malignant melanoma cells and to have specificity for the GD3 ganglioside (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----4GlcCer). Using overlay of antibodies on thin-layer chromatograms with glycolipids of various sources, it was shown that antibody 4.2, a further IgM and two IgG3 mouse monoclonal antibodies, selected on the basis of reactivity with GD3, also bound with similar strength to the structural analogue NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----4GlcNac beta 1----3Gal beta 1----4GlcCer or disialylparagloboside. The SK-MEL 28 melanoma cell line used for immunization was shown to contain a large amount of GD3 but to lack disialylparagloboside. The demonstrated cross-reactivity may be of importance when considering the use of these antibodies for biochemical and medical purposes.     

Purification and enzymatic characterization of CMP-sialic acid: beta-galactosyl1----3-N-acetylgalactosaminide alpha 2----3-sialyltransferase from human placenta

A CMP-NeuAc:Gal beta 1----3GalNAc-R alpha 2----3-sialyltransferase has been purified over 20,000-fold from a Triton X-100 extract of human placenta by affinity chromatography on concanavalin A-Sepharose and CDP-hexanolamine-Sepharose in a yield of 10%. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions revealed that the enzyme consists of a major polypeptide species with a molecular weight of 41,000 and some minor forms with molecular weights of 40,000, 43,000, and 65,000, respectively, which can be resolved partially by gel filtration on Sephadex G-100. Isoelectric focusing revealed that the enzyme occurs in a major and a minor charged form with pI values of 5.0-5.5 and 6.0, respectively. Acceptor specificity studies indicated that the enzyme catalyzes the incorporation of sialic acid from CMP-NeuAc into glycoproteins, glycolipids, and oligosaccharides which possess a terminal Gal beta----3GalNAc unit. Analysis of the structure of the product chain by high-pressure liquid chromatography and thin layer chromatography as well as methylation analysis revealed that a NeuAc alpha 2----3Gal beta 1----3GalNAc sequence is elaborated. The best glycoprotein acceptors are antifreeze glycoprotein and porcine submaxillary asialo/afucomucin. The disaccharide Gal beta 1----3GalNAc-Thr shows values for Km and V which are close to those of the latter glycoprotein. Lactose as well as oligosaccharides in which galactose is linked beta 1----3 or beta 1----4 to N-acetylglucosamine are less efficient acceptors. Of the glycolipids tested only gangliosides GM1 and GD1b served as an acceptor. The enzyme does not show an absolute aglycon specificity, and attaches sialic acid regardless the anomeric configuration of the N-acetylgalactosaminyl residue in the accepting Gal beta 1----3GalNAc unit. By use of specific acceptor substrates it could be demonstrated that the purified enzyme is free from other known sialyltransferase activities. Studies with rabbit antibodies raised against a partially purified sialyltransferase preparation indicated that the enzyme is immunologically unrelated to a Gal beta 1----4GlcNAc-R alpha 2----3-sialyltransferase, which previously had been identified in human placenta (Van den Eijnden, D.H., and Schiphorst, W. E. C. M. (1981) J. Biol. Chem. 256, 3159-3162). Initial-rate kinetic studies suggest that the sialyltransferase operates through a mechanism involving a ternary complex of enzyme, sugar donor, and acceptor. This is the first report on the extensive purification and characterization of a sialyltransferase from a human tissue.     

Proton NMR and fast-atom bombardment mass spectrometry analysis of the melanoma-associated ganglioside 9-O-acetyl-GD3

A glycolipid antigen, detected by a monoclonal antibody (ME 311) obtained by immunizing mice with a human metastatic melanoma cell line (WM 46), was isolated and structurally characterized. Using immunostaining on thin-layer chromatograms for monitoring, 1.0 mg of a pure alkali-labile disialoganglioside was obtained from 23 g of packed melanoma cells (WM 164). Fractionation of the lipid extract was done on DEAE-Sepharose columns into total disialogangliosides which were repeatedly separated by high-pressure liquid chromatography. On mild alkaline treatment, the ganglioside was converted to a slower migrating species identical with a ganglioside GD3 isolated from the same source (Neu5Ac alpha 2----8Neu5Ac alpha 2----3Gal beta 1----4Glc beta 1----1-cer-amide) and specifically detected by monoclonal antibody R24. Comparison of the two gangliosides by fast-atom bombardment mass spectrometry (revealing an acetyl group on the terminal sialic acid on the alkali-labile species) and by 1H NMR (indicating the position of the acetyl group) suggested the following structure: Neu5,9Ac2 alpha 2----8Neu5Ac alpha 2----3Gal beta 1----4Glc beta 1----1-ceramide. This is identical with a ganglioside proposed earlier to exist in melanoma cells (Cheresh, D. A., Varki, A. P., Varki, N. M., Stallcup, W. B., Levine, J., and Reisfeld, R. A. (1984) J. Biol. Chem. 259, 7453-7459). Immunostaining with ME 311 antibody of cell extracts on thin-layer chromatography chromatograms revealed only this ganglioside in the melanoma cells, while normal human brain was negative. However, in one of the total ganglioside extracts tested for presence of binding with antibody ME 311, three gangliosides were found to bind. No evidence was obtained for the presence of the antigenic epitope in mucins or glycoproteins of the melanoma cells.     

Production of monoclonal antibodies specific for ganglioside GD3.

Four kinds of anti-GD3 monoclonal antibodies, DSG-1, -2, -3, and -4, of the IgM class were obtained by the immunization of BALB/c mice with enzootic bovine leukosis tumor tissue-derived ganglioside GD3 inserted into liposomes with Salmonella minnesota R595 lipopolysaccharides. The specificities of the monoclonal antibodies obtained were defined by complement-dependent liposome immune lysis assay and by enzyme immunostaining on thin-layer chromatography. The reactivities of the monoclonal antibodies obtained to four ganglioside GD3 variants [GD3(NeuAc-NeuAc), GD3(NeuAc-NeuGc), GD3(NeuGc-NeuAc), and GD3(NeuGc-NeuGc)] were tested. All of the monoclonal antibodies were found to react with GD3(NeuAc-NeuAc) and GD3(NeuAc-NeuGc) but not with GD3(NeuGc-NeuAc) or GD3(NeuGc-NeuGc). Furthermore, various purified glycosphingolipids were used to determine the specificity of these monoclonal antibodies. All 4 antibodies reacted only with ganglioside GD3 [GD3(NeuAc-NeuAc) and GD3(NeuAc-NeuGc)], but not with several gangliosides linking the GalNAc, Gal beta 1-3GalNAc, NeuAc alpha 2-3Gal beta 1-3GalNAc, or NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-3GalNAc residue to the Gal moiety of ganglioside GD3 (GD2, GD1b, GT1b, or GQ1b, respectively), ganglioside GT1a having the same terminal NeuAc alpha 2-8NeuAc alpha 2-3Gal residue as ganglioside GD3, other gangliosides, and neutral glycosphingolipids. These findings suggest that the 4 monoclonal antibodies obtained may be specific for the epitope of NeuAc-alpha 2-8Sia alpha 2-3Gal beta 1-4Glc residue of ganglioside GD3.