Glycolipid extracts from various human cancer tissues and cell lines showed the presence of a slowmigrating glycolipid component which was strongly reactive with monoclonal antibody (mAb) NCC-ST42 1 (raised against human gastric adenocarcinoma) and weakly cross-reactive with anti-Le“ mAbs. The slow-migrating glycolipid was isolated from human colonic adenocarcinoma cell line colo205 grown in nude mice, and was purified by high-performance liquid chromatography followed by preparative thinlayer chromatography. Its structure was elucidated by sequential enzymatic degradation and thin-layer chromatography immunostaining of the degradation products with various mAbs, ‘H NMR spectroscopy, positive-ion fast atom bombardment mass spectrometry, and methylation analysis. The major slow-migrating component reacting with mAb ST-421 was identified as dimeric Le“, with the structure as follows.

A Lewis-b-active glycosphingolipid containing a repetitive type-1 chain carbohydrate core was isolated from human colonic adenocarcinoma cell line Colo205. This glycosphingolipid was purified by HPLC and preparative high-performance thin-layer chromatography and its structure elucidated by positive-ion fast-atom-bombardment mass spectrometry with collision-induced disassociation, 1H-NMR spectroscopy and methylation analysis. The glycosphingolipid was found to be a trifucosylated derivative of this novel carbohydrate core, having the following structure: [formula; see text].

Lipid extracts of eggs, worms, and cercariae of the parasitic trematode Schistosoma mansoni have been shown to contain a large number of highly immunogenic glycolipids (Weiss, J. B., Magnani, J. L., and Strand, M. (1986) J. Immunol. 136, 4275-4282). Three fractions of schistosome egg glycolipids were selected on the basis of their reactivity with an anti-schistosome monoclonal antibody (128C3/3), which recognizes a developmentally regulated carbohydrate epitope present on both glycolipid and glycoprotein antigens from S. mansoni. These fractions were purified by silica gel chromatography and preparative high performance thin layer chromatography and characterized by monosaccharide, fatty acid, and linkage analysis with gas chromatography-mass spectrometry, as well as by positive and negative ion fast atom bombardment-mass spectrometry. The immunogens were shown to be glycosphingolipids having homologous structures based on a highly novel extension of glucosylceramide. Monosaccharide inhibition studies indicated that the epitope recognized by 128C3/3 resides in an outer region of the immunogens consisting of Fuc2GlcNAc (where Fuc is fucose) repeating units. The largest antigen characterized may have the following structure, based on the evidence presented in this paper.[GRAPHICS]The evidence indicated the existence of a series of glycan structures created by deletions of one or more Fuc1 --> 3 side chains from the above structure.

Glycolipid extracts from various human cancer tissues and cell lines showed the presence of a slow-migrating glycolipid component which was strongly reactive with monoclonal antibody (mAb) NCC-ST-421 (raised against human gastric adenocarcinoma) and weakly cross-reactive with anti-Lea mAbs. The slow-migrating glycolipid was isolated from human colonic adenocarcinoma cell line Colo205 grown in nude mice, and was purified by high-performance liquid chromatography followed by preparative thin-layer chromatography. Its structure was elucidated by sequential enzymatic degradation and thin-layer chromatography immunostaining of the degradation products with various mAbs, 1H NMR spectroscopy, positive-ion fast atom bombardment mass spectrometry, and methylation analysis. The major slow-migrating component reacting with mAb ST-421 was identified as dimeric Lea, with the structure as follows. [formula: see text] Antigens containing this structure and various analogous structures (including enzymatically synthesized Lea/Lex hybrid antigen) were tested with ST-421. While the mAb was equally reactive with dimeric Lea and Lea/Lex, only the former was chemically detectable as the slow-migrating glycolipid from the tumor extract. ST-421 showed less reactivity with simple Lea (III4FucLc4) or extended Lea (V4FucLc6, and/or IV3Gal beta 1----3[Fuc alpha 1----4]GlcNAcnLc4), and was not reactive with Lex/Lex (dimeric Lex). It was concluded, therefore, that the major tumor-associated slow-migrating glycolipid reacting with ST-421 has the dimeric Lea structure shown above. Since extension of lacto-series structure has been shown to be limited to type 2 chain in normal cells and tissues, extended elongation of type 1 chain as shown in this structure represents a novel tumor-associated epitope.

Two novel gas chromatography/mass spectrometry (GC/MS) strategies have been developed to assist in characterization of ganglioside inner esters. In the first, lactonized gangliosides which have been ammonolyzed and permethylated (see previous paper) are subjected to methanolysis and acetylation. This produces partially methylated NeuAc derivatives in which previous involvement of the carboxyl group in an ester linkage is indicated by conversion to an N1, N1-dimethylamide. These can be analyzed by GC/MS using conditions analogous to those used normally in NeuAc methylation analysis, providing glycosidic and ester linkage information in the same procedure. In the second procedure, internally esterified NeuAc hydroxyl groups are located by protection, under neutral conditions, of all free hydroxyl groups of the ganglioside lactone with methoxyethoxymethyl groups, followed by permethylation, methanolysis, and GC/MS analysis of the resulting partially methylated NeuAc methyl ester methyl glycosides as their per-O-trimethylsilylates or per-O-acetates. This procedure has been used to show unambiguously that in GD3 lactones it is the 9-hydroxyl group of the internal NeuAc residue which is esterified by the terminal NeuAc carboxyl. Both of these strategies should have general application in characterizing lactones occurring in gangliosides, NeuAc homo- and heteropolymers, and oligosaccharides or glycopeptides terminated by polysialosyl chains.

A comparison of old and new fast atom bombardment (FAB) mass spectrometric strategies for characterization of ganglioside inner esters (lactones) is presented. Data obtained for lactones of GD3 (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) using negative ion FAB mass spectrometry of underivatized materials, negative ion FAB mass spectrometry following ammonolysis, and positive ion FAB mass spectrometry following ammonolysis and permethylation are presented and discussed. The latter method uses well-known reactions to produce a novel type of ganglioside derivative, highly amenable to analysis by positive ion FAB mass spectrometry, which is introduced to simplify unambiguous location of NeuAc residues involved in ester linkages to other sugars.

A method is described which is suitable for protection of all free hydroxyl groups of a glycosphingolipid under conditions which will not cleave ester linkages, including inner ester linkages characteristic of ganglioside lactones. The protecting methoxyethoxymethyl group is stable in alkaline media, surviving permethylation procedures which introduce a methyl ether at all sites previously acylated. Hydrolysis, reduction, and acetylation then yield alditol acetate derivatives which can be analyzed by conventional GC-MS to locate the methyl ether groups. The method is used to locate the inner esterification site of GM3 lactone.