H-1-NMR STUDIES OF A BIOSYNTHETIC LACTO-GANGLIO HYBRID GLYCOSPHINGOLIPID - CONFIRMATION OF STRUCTURE, INTERPRETATION OF ANOMALOUS CHEMICAL-SHIFTS, AND EVIDENCE FOR INTERRESIDUE AMIDE AMIDE HYDROGEN-BONDING

Glycosphingolipids bearing GlcNAc-beta-1 --> 3 and GalNAc-beta-1 --> 4 linked to beta-Gal of lactosylceramide (lacto-ganglio hybrids), first isolated from a murine myelogenous leukemia cell line [Kannagi, R., Levery, S. B., & Hakomori, S. (1984) J. Biol. Chem. 259, 8444-8451], have since been found as normal components of mullet roe and English sole liver. In order to clarify the biosynthetic pathways responsible for its occurrence both as a product of normal tissues and as a possible mammalian cancer-associated antigen, the lacto-ganglio hybrid core structure LcGg4Cer was synthesized from Lc3Cer using a GalNAc-beta-1 --> 4 transferase preparation from English sole liver. A preliminary characterization of the enzyme, which may be identical to the GalNAc T-1 responsible for synthesis of GM2 ganglioside, is presented. The enzymatically synthesized product was analyzed by 1- and 2-D H-1 NMR spectroscopy, confirming its primary structure as GalNAc-beta-1 --> 4-(GlcNac-beta-1 --> 3)Gal-beta-1 --> 4Glc-beta-1 --> 1 Cer. In addition to assigning all nonexchangeable glycosyl proton resonances, measurements of several properties of the amide NH protons, including chemical shift, coupling constants, exchange rates, and temperature shift coefficients, were obtained and compared to those in the simpler constituent triglycosylceramides, Lc3- and Gg3Cer. An approximate three-dimensional structure for LcGg4Cer is proposed, consistent with all data obtained, which should be useful in discussing the results of H-1 NMR analysis of compounds containing this core tetrasaccharide. The structure is characterized by an unusual arrangement of terminal N-acetylhexosamine residues, resulting in a pi-H hydrogen-bonding interaction between their acetamido groups.