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

Nontypeable Haemophilus influenzae (NTHI) are a 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 α2,3- sialylosylparagloboside (IV³NeuAc-nLcOse₄Cer, 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 α2,3 (human macrophages) or α2,6 (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 NeuAcα2-6Galβ1-4GlcNAcβ1-3Galβ1- 4Glcβ1-1Cer (α2,6-sialosylparagloboside, nLM1). Collectively, our studies denote NTHI-binding gangliosides as lacto/neolacto series structures.