Nontypeable Haemophilus influenzae-binding gangliosides of human respiratory (HEp-2) cells have a requisite lacto/neolacto core structure

Nontypeable Haemophilus influenzae (NTHI) area major cause of human infections. We previously demonstrated high affinity and high specificity binding of NTHI to minor gangliosides of human respiratory (HEp-2) cells and macrophages, but not to brain gangliosides. We further identified the NTHI-binding ganglioside of human macrophages as alpha 2,3-sialylosylparagloboside (IV(3)NeuAc-nLcOse(4)Cer, nLM1), which possesses a neolacto core structure that is absent in brain gangliosides. This supported a hypothesis that lacto/neolacto core carbohydrates are critical for NTHI-ganglioside binding. To investigate, we determined the core carbohydrate structure of NTHI-binding gangliosides of HEp-2 cells, through multiple approaches, including specific enzymatic degradation, mass spectral analysis and gas-liquid chromatography. Our analyses denote the following critical structural attributes of NTHI-binding gangliosides: (1) a conserved lacto/neolacto core structure; (2) requisite sialylation, which may be either internal or external, with alpha 2,3 (human macrophages) or alpha 2,6 (HEp-2 cells) anomeric linkages; (3) internalized galactose residues. Mass spectral and gas chromatographic analyses confirm that NTHI-binding gangliosides of HEp-2 cells possess lacto/neolacto carbohydrate cores and identify the structure of the major peak as NeuAc alpha 2-6Ga1 beta 1-4G1cNAc beta 1-3Ga1 beta 1-4Glc beta 1-1Cer (alpha 2,6-sialosylparagloboside, nLM1). Collectively, our studies denote NTHI-binding gangliosides as lacto/neolacto series structures. 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.