Two novel isoneolacto-undecaglycosylceramides carrying Galalpha1-->3Lewis(x) on the 6-linked antenna and N-acetylneuraminic acidalpha2-->3 or Galactose alpha1-->3 on the 3-linked antenna, expressed in porcine kidney.
Three sialosylated and three neutral glycosphingolipids sharing a common iso-neolacto core were isolated from porcine kidney cortex. They were purified by preparative HPTLC, and were characterized by partial exoglycosidase hydrolysis followed by thin layer chromatography and immunostaining with anti-Galalpha1-->3Gal, anti-type 2 lactosamine and anti-Lewis(x) antibodies, methylation analysis, MALDI-TOF mass spectrometry and 1H-NMR spectroscopy. Among neutral glycolipids, one was a known structure, VI3VI'3(alphaGal)2-iso-nLc8Cer, and two were novel structures differing by the number of Galalpha3Lewis(x) determinants: VI3VI'3(alphaGal)2V'3alphaFuc-iso-nLc8, and VI3VI'3(alphaGal)2 V3V'3(alphaFuc)2-iso-nLc8. The single Galalpha3Lewis(x) determinant was found on the 6-linked antenna. Among sialosylated glycolipids, two had been previously found in other species and tissues, VI3VI'3(NeuAc)2-iso-nLc8, and VI3NeuAcVI'3alphaGal-iso-nLc8. A novel structure was discovered presenting a Galalpha3Lewis(x) determinant on the 6-linked antenna and a N-acetylneuraminic acid on the 3-linked antenna, VI3NeuAcVI'3alphaGalV'3alphaFuc-iso-nLc8. These results indicate that, in vivo, the porcine kidney alpha3fucosyltransferase synthesizes the Gala3Lewis(x) determinant, acting on the 6-linked before the 3-linked Galalpha3neolactosamine, and appears unable to synthesize the sialosylated Lewis(x) determinant on neolactoseries glycolipids.