Alexandre Pons

Galectin-4 and sulfatides in apical membrane trafficking in enterocyte-like cells

We have previously reported that 1-benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside (GalNAcα-O-bn), an inhibitor of glycosylation, perturbed apical biosynthetic trafficking in polarized HT-29 cells suggesting an involvement of a lectin-based mechanism. Here, we have identified galectin-4 as one of the major components of detergent-resistant membranes (DRMs) isolated from HT-29 5M12 cells. Galectin-4 was also found in post-Golgi carrier vesicles. The functional role of galectin-4 in polarized trafficking in HT-29 5M12 cells was studied by using a retrovirus-mediated RNA interference. In galectin-4–depleted HT-29 5M12 cells apical membrane markers accumulated intracellularly. In contrast, basolateral membrane markers were not affected. Moreover, galectin-4 depletion altered the DRM association characteristics of apical proteins. Sulfatides with long chain-hydroxylated fatty acids, which were also enriched in DRMs, were identified as high-affinity ligands for galectin-4. Together, our data propose that interaction between galectin-4 and sulfatides plays a functional role in the clustering of lipid rafts for apical delivery.

Occurrence of ceramides and neutral glycolipids with unusual long-chain base composition in purified rat liver mitochondria.

The free ceramide content of rat liver mitochondria was found to be 1.7 nmol/mg protein and outer membranes contained a three‐fold higher concentration than inner membranes. The mitochondrial content in neutral glycolipids was 0.6 nmol/mg protein. The long‐chain bases found in free ceramides were d18:1 sphingosine, d18:0 3‐ketosphinganine and t21:1 phytosphingosine in increasing order. In contrast, 3‐ketosphinganine was the only base of glucosylceramide and lactosylceramide of inner membranes, whereas d18:1 sphingosine was the major long‐chain base of glucosylceramide of outer membranes.

Diversity of the human erythrocyte membrane sialic acids in relation with blood groups

The composition of the human erythrocyte membrane (RBC) glycoprotein‐ and glycolipid‐bound sialic acids of A, B, AB and O type donors was studied using a new method (Zanetta et al., Glycobiology 11 (2001) 663–676). In addition to Neu5Ac as the major compound, Kdn, Neu5,9Ac2, Neu5,7Ac2, Neu (de‐N‐acetylated‐Neu5Ac), Neu5Ac8Me, Neu5Ac9Lt, Neu4,5Ac2, Neu5,8Ac29Lt and Neu5Ac8S were characterised. Among these different compounds, Neu5Ac8Me, Neu5Ac9Lt, Neu4,5Ac2, Neu5,8Ac29Lt and Neu5Ac8S have never been described and quantitatively determined before in human tissues or cells. Neu5Gc and its O‐alkylated or O‐acylated derivatives were not detected.

N-glycosylation potential of maize: The human lactoferrin used as a model

In order to determine the N-glycosylation potential of maize, a monocotyledon expression system for the production of recombinant glycoproteins, human lactoferrin was used as a model. The human lactoferrin coding sequence was inserted into the pUC18 plasmid under control of the wheat glutenin promoter. Maize was stably transformed and recombinant lactoferrin was purified from the fourth generation seeds. Glycosylation was analysed by gas chromatography, lectin detection, glycosidase digestions and mass spectrometry. The results indicated that both N-glycosylation sites of recombinant lactoferrin are mainly substituted by typical plant paucimannose-type glycans, with β1,2-xylose and α1,3-linked fucose at the proximal N-acetylglucosamine, and that complex-type glycans with Lewisa determinants are not present in maize recombinant lactoferrin.

Localisation and distribution of O-acetylated N-acetylneuraminic acids, the endogenous substrates of the hemagglutinin-esterases of murine coronaviruses, in mouse tissue

Infections by mouse hepatitis viruses result in disease of the liver, the gastrointestinal tract, respiratory tract, and the central nervous system. Coronaviruses related to mouse hepatitis virus express a hemagglutinin-esterase surface glycoprotein, which specifically hydrolyses either 5-N-acetyl-4-O-acetyl neuraminic acid (Neu4,5Ac2) or 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac2). Moreover, these sialic acids represent potential cellular receptor determinants for murine coronaviruses. Until now, the distribution of these sialic acids in mouse brain was not thoroughly investigated. Particularly Neu4,5Ac2 was not yet found in mouse brain. Using a sensitive method of gas chromatography coupled to mass spectrometry in the electron impact mode of ionization this manuscript demonstrates the occurrence of 13 different sialic acids varying in their alkyl and acyl substituents in mouse tissues including 5-N-acetyl-4-O-acetyl-9-O-lactyl-neuraminic acid (Neu4,5Ac29Lt), 5-N-acetyl-9-O-lactyl-neuraminic acid (Neu5Ac9Lt), 5-N-acetyl-8-O-methyl-neuraminic acid (Neu5Ac8Me) and the 1,7-lactone (Neu5Ac1,7L) of neuraminic acid. Neu4,5Ac2, relatively abundant in the gut, was present as a minor compound in all tissues, including liver, olfactory lobe, telencephalon, metencephalon and hippocampus. Neu5,9Ac2 was also found in these tissues, except in the liver. It is suggested that these sialic acids represent the endogenous substrate and receptor determinants for murine coronaviruses.