Two Paralogous Gb3/CD77 Synthases in Birds Show Different Preferences for Their Glycoprotein and Glycosphingolipid Substrates

Most glycosyltransferases show remarkable gross and fine substrate specificity, which is reflected in the old one enzyme-one linkage paradigm. While human Gb3/CD77 synthase is a glycosyltransferase that synthesizes the Galα1→4Gal moiety mainly on glycosphingolipids, its pigeon homolog prefers glycoproteins as acceptors. In this study, we characterized two Gb3/CD77 synthase paralogs found in pigeons (Columba livia). We evaluated their specificities in transfected human teratocarcinoma 2102Ep cells by flow cytofluorometry, Western blotting, high-performance thin-layer chromatography, mass spectrometry and metabolic labelling with 14C-galactose. We found that the previously described pigeon Gb3/CD77 synthase (called P) can use predominately glycoproteins as acceptors, while its paralog (called M), which we serendipitously discovered while conducting this study, efficiently synthesizes Galα1→4Gal caps on both glycoproteins and glycosphingolipids. These two paralogs may underlie the difference in expression profiles of Galα1→4Gal-terminated glycoconjugates between neoavians and mammals.

Metabolic labelling of cancer cells with glycodendrimers stimulate immune-mediated cytotoxicity.

The recruitment of antibody naturally present in human blood stream at the surface of cancer cells have been proved a promising immunotherapeutic strategy to fight cancer. Antibody recruiting molecules (ARMs)...

Understanding selectivity of metabolic labelling and click-targeting in multicellular environments as a route to tissue selective drug delivery

The efficiency of metabolic labelling and click-chemistry to attach nanoparticles to cell surfaces is different between different cell populations in co-culture formats.

Vulpeculin: a novel and abundant lipocalin in the urine of the common brushtail possum, Trichosurus vulpecula

Lipocalins are a family of secreted proteins. They are capable of binding small lipophilic compounds and have been extensively studied for their role in chemosignalling in rodent urine. Urine of the common brushtail possum ( Trichosurus vulpecula ) contains a prominent glycoprotein of 20 kDa, expressed in both sexes. We have isolated this protein and determined its primary sequence by mass spectrometry, including the use of metabolic labelling to resolve the leucine/isoleucine isobaric ambiguity. The protein sequence was identified as a lipocalin, and phylogenetic analysis grouped the protein with other marsupial lipocalin sequences in a phylogenetic clade distinct from established cross-species lipocalin sub-families. The pattern of expression in possum urine and the similarity in sequence and structure to other lipocalins suggests this protein may have a role in brushtail possum chemosignalling.

APOL1 polymorphism modulates sphingolipid profile of human podocytes

Apolipoprotein L1 (APOL1) wild type (G0) plays a role in the metabolism of sphingolipids, glycosphingolipids, sphingomyelin and ceramide, which constitute bioactive components of the lipid rafts (DRM). We asked whether APOL1 variants (APOL1-Vs) G1 and G2 carry the potential to alter the metabolism of sphingolipids in human podocytes. The sphingolipid pattern in HPs overexpressing either APOL1G0 or APOL1-Vs was analysed by using a thin mono- and bi-dimensional layer chromatography, mass-spectrometry and metabolic labelling with [1-3H]sphingosine. HP G0 and G1/G2-Vs exhibit a comparable decrease in lactosylceramide and an increase in the globotriaosylceramide content. An analysis of the main glycohydrolases activity involved in glycosphingolipid catabolism showed an overall decrease in the activeness of the tested enzymes, irrespective of the type of APOL1-Vs expression. Similarly, the high throughput cell live-based assay showed a comparable increased action of the plasma membrane glycosphingolipid-glycohydrolases in living cells independent of the genetic APOL1 expression profile. Importantly, the most significative modification of the sphingolipid pattern induced by APOL1-Vs occurred in DRM resulted with a drastic reduction of radioactivity associated with sphingolipids. G1/G2-Vs present a decrease amount of globotriaosylceramide and globopentaosylceramide compared to G0. Additionally, ceramide at the DRM site and lactosylceramide in general, showed a greatest fall in G1/G2 in comparison with G0. Additionally, the levels of glucosylceramide decreased only in the DRM of human podocytes overexpressing G1/G2-Vs. These findings suggest that altered sphingolipidsprofiles may contribute to the deranged functionality of the plasma membrane in APOL1 risk milieu.