Profiling of glycosphingolipids with SCDase digestion and HPLC-FLD-MS.

Lipid components of cells and tissues feature a large diversity of structures that present a challenging problem for molecular analysis. Glycolipids from mammalian cells contain glycosphingolipids (GSLs) as their major glycolipid component, and these structures vary in the identity of the glycan headgroup as well as the structure of the fatty acid and sphingosine (Sph) tails. Analysis of intact GSLs is challenging due to the low abundance of these species. Here, we develop a new strategy for the analysis of lyso-GSL (l-GSL), GSL that retain linkage of the glycan headgroup with the Sph base. The analysis begins with digestion of a GSL sample with sphingolipid ceramide N-deacylase (SCDase), followed by labelling with an amine-reactive fluorophore. The sample was then analyzed by HPLC-FLD-MS and quantitated by addition of an external standard. This method was compared to analysis of GSL glycans after cleavage by an Endoglycoceramidase (EGCase) enzyme and labeling with a fluorophore (2-anthranilic acid, 2AA). The two methods are complementary, with EGCase providing improved signal (due to fewer species) and SCDase providing analysis of lyso-GSL. Importantly the SCDase method provides Sph composition of GSL species. We demonstrate the method on cultured human cells (Jurkat T cells) and tissue homogenate (porcine brain).

Persistent reduction in sialylation of cerebral glycoproteins following postnatal inflammatory exposure.

BACKGROUND: The extension of sepsis encompassing the preterm newborn's brain is often overlooked due to technical challenges in this highly vulnerable population, yet it leads to substantial long-term neurodevelopmental disabilities. In this study, we demonstrate how neonatal neuroinflammation following postnatal E. coli lipopolysaccharide (LPS) exposure in rat pups results in persistent reduction in sialylation of cerebral glycoproteins. METHODS: Male Sprague-Dawley rat pups at postnatal day 3 (P3) were injected in the corpus callosum with saline or LPS. Twenty-four hours (P4) or 21 days (P24) following injection, brains were extracted and analyzed for neuraminidase activity and expression as well as for sialylation of cerebral glycoproteins and glycolipids. RESULTS: At both P4 and P24, we detected a significant increase of the acidic neuraminidase activity in LPS-exposed rats. It correlated with significantly increased neuraminidase 1 (Neu1) mRNA in LPS-treated brains at P4 and with neuraminidases 1 and 4 at P24 suggesting that these enzymes were responsible for the rise of neuraminidase activity. At both P4 and P24, sialylation of N-glycans on brain glycoproteins decreased according to both mass-spectrometry analysis and lectin blotting, but the ganglioside composition remained intact. Finally, at P24, analysis of brain tissues by immunohistochemistry showed that neurons in the upper layers (II-III) of somatosensory cortex had a reduced surface content of polysialic acid. CONCLUSIONS: Together, our data demonstrate that neonatal LPS exposure results in specific and sustained induction of Neu1 and Neu4, causing long-lasting negative changes in sialylation of glycoproteins on brain cells. Considering the important roles played by sialoglycoproteins in CNS function, we speculate that observed re-programming of the brain sialome constitutes an important part of pathophysiological consequences in perinatal infectious exposure.

NEU1 and NEU3 enzymes alter CD22 organization on B cells.

The B cell membrane expresses sialic-acid-binding immunoglobulin-like lectins, also called Siglecs, that are important for modulating immune response. Siglecs have interactions with sialoglycoproteins found on the same membrane (cis-ligands) that result in homotypic and heterotypic receptor clusters. The regulation and organization of these clusters, and their effect on cell activation, is not clearly understood. We investigated the role of human neuraminidase enzymes NEU1 and NEU3 on the clustering of CD22 on B cells using confocal microscopy. We observed that native NEU1 and NEU3 activity influence the cluster size of CD22. Using single-particle tracking, we observed that NEU3 activity increased the lateral mobility of CD22, which was in contrast to the effect of exogenous bacterial NEU enzymes. Moreover, we show that native NEU1 and NEU3 activity influenced cellular Ca2+ levels, supporting a role for these enzymes in regulating B cell activation. Our results establish a role for native NEU activity in modulating CD22 organization and function on B cells.

Neuraminidases 1 and 3 Trigger Atherosclerosis by Desialylating Low-Density Lipoproteins and Increasing Their Uptake by Macrophages.

Background Chronic vascular disease atherosclerosis starts with an uptake of atherogenic modified low-density lipoproteins (LDLs) by resident macrophages, resulting in formation of arterial fatty streaks and eventually atheromatous plaques. Increased plasma sialic acid levels, increased neuraminidase activity, and reduced sialic acid LDL content have been previously associated with atherosclerosis and coronary artery disease in human patients, but the mechanism underlying this association has not been explored. Methods and Results We tested the hypothesis that neuraminidases contribute to development of atherosclerosis by removing sialic acid residues from glycan chains of the LDL glycoprotein and glycolipids. Atherosclerosis progression was investigated in apolipoprotein E and LDL receptor knockout mice with genetic deficiency of neuraminidases 1, 3, and 4 or those treated with specific neuraminidase inhibitors. We show that desialylation of the LDL glycoprotein, apolipoprotein B 100, by human neuraminidases 1 and 3 increases the uptake of human LDL by human cultured macrophages and by macrophages in aortic root lesions in Apoe-/- mice via asialoglycoprotein receptor 1. Genetic inactivation or pharmacological inhibition of neuraminidases 1 and 3 significantly delays formation of fatty streaks in the aortic root without affecting the plasma cholesterol and LDL levels in Apoe-/- and Ldlr-/- mouse models of atherosclerosis. Conclusions Together, our results suggest that neuraminidases 1 and 3 trigger the initial phase of atherosclerosis and formation of aortic fatty streaks by desialylating LDL and increasing their uptake by resident macrophages.

Isoenzyme-Selective Inhibitors of Human Neuraminidases Reveal Distinct Effects on Cell Migration.

The human neuraminidase enzymes (NEU1, NEU2, NEU3, and NEU4) are a class of enzymes implicated in pathologies including cancer and diabetes. Several reports have linked neuraminidase activity to the regulation of cell migration in cancer cells. Using an in vitro cell migration assay on fibronectin (FN) coated surfaces, we have investigated the role of these enzymes in integrin-mediated cell migration. We observed that neuraminidase inhibition caused significant retardation of cell migration in breast cancer (MDA-MB-231) and prostate cancer (PC-3) cell lines when using inhibitors of NEU3 and NEU4. In contrast, inhibition of NEU1 caused a significant increase in cell migration for the same cell lines. We concluded that the blockade of human neuraminidase enzymes with isoenzyme-selective inhibitors can lead to disparate results and has significant potential in the development of anticancer or wound healing therapeutics.

Neuraminidase-3 Is a Negative Regulator of LFA-1 Adhesion.

Within the plasma membrane environment, glycoconjugate-receptor interactions play an important role in the regulation of cell-cell interactions. We have investigated the mechanism and activity of the human neuraminidase (NEU) isoenzyme, NEU3, on T cell adhesion receptors. The enzyme is known to prefer glycolipid substrates, and we confirmed that exogenous enzyme altered the glycolipid composition of cells. NEU3 was able to modify the sialic acid content of purified LFA-1 in vitro. Enzymatic activity of NEU3 resulted in re-organization of LFA-1 into large clusters on the membrane. This change was facilitated by an increase in the lateral mobility of LFA-1 upon NEU3 treatment. Changes to the lateral mobility of LFA-1 were specific for NEU3 activity, and we observed no significant change in diffusion when cells were treated with a bacterial NEU (NanI). Furthermore, we found that NEU3 treatment of cells increased surface expression levels of LFA-1. We observed that NEU3-treated cells had suppressed LFA-1 adhesion to an ICAM-1 coated surface using an in vitro static adhesion assay. These results establish that NEU3 can modulate glycoconjugate composition and contribute to the regulation of integrin activity. We propose that NEU3 should be investigated to determine its role on LFA-1 within the inflammatory cascade.