Analysis of glycosphingolipid-derived oligosaccharides by high pH anion exchange chromatography

As an adjunct to existing thin layer and column chromatographic methods for the identification of glycolipids a method that utilizes the high pH anion chromatographic (HPAEC) analysis of the oligosaccharides released from the glycolipids by endoglycoceramidase has been developed. Using a Dionex Carbo Pak PA1 column and elution with a linear gradient of sodium acetate in 0.2M NaOH, the elution times of eight neutral and fourteen acidic oligosaccharides derived from glycolipids were determined. Under these conditions the neutral oligosaccharides were well separated from each other but some of the acidic oligosaccharides had overlapping elution times. The ganglioside-derived oligosaccharides could be further identified by treating them with sialidase or by mild acid hydrolysis and reanalysing the products by HPAEC. The method was applied to the analysis of mixed bovine brain gangliosides. The procedure provides an additional approach for the initial identification of glycolipids by analysing the component oligosaccharides rather than the intact glycolipids.     

Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system

Endoglycoceramidase (EGCase) is a glycosidase capable of hydrolyzing the β -glycosidic linkage between the oligosaccharides and ceramides of glycosphingolipids (GSLs). Three molecular species of EGCase differing in specificity were found in the culture fluid of Rhodococcus equi (formerly Rhodococcus sp. M-750) and designated EGCase I, II, and III. This study describes the molecular cloning of EGCase I and characterization of the recombinant enzyme, which was highly expressed in a rhodococcal expression system using Rhodococcus erythropolis. Kinetic analysis revealed the turnover number (k(cat)) (k(cat)) of the recombinant EGCase I to be 22- and 1,200-fold higher than that of EGCase II toward GM1a and Gb3Cer, respectively, although the K(m) of both enzymes was almost the same for these substrates. Comparison of the three-dimensional structure of EGCase I (model) and EGCase II (crystal) indicated that a flexible loop hangs over the catalytic cleft of EGCase II but not EGCase I. Deletion of the loop from EGCase II increased the k(cat) of the mutant enzyme, suggesting that the loop is a critical factor affecting the turnover of substrates and products in the catalytic region. Recombinant EGCase I exhibited broad specificity and good reaction efficiency compared with EGCase II, making EGCase I well-suited to a comprehensive analysis of GSLs.     

Gangliosides and β1‐Integrin Are Required for Caveolae and Membrane Domains

Caveolae are plasma membrane domains involved in the uptake of certain pathogens and toxins. Internalization of some cell surface integrins occurs via caveolae suggesting caveolae may play a crucial role in modulating integrin‐mediated adhesion and cell migration. Here we demonstrate a critical role for gangliosides (sialo‐glycosphingolipids) in regulating caveolar endocytosis in human skin fibroblasts. Pretreatment of cells with endoglycoceramidase (cleaves glycosphingolipids) or sialidase (modifies cell surface gangliosides and glycoproteins) selectively inhibited caveolar endocytosis by >70%, inhibited the formation of plasma membrane domains enriched in sphingolipids and cholesterol (‘lipid rafts'), reduced caveolae and caveolin‐1 at the plasma membrane by approximately 80%, and blunted activation of β1‐integrin, a protein required for caveolar endocytosis in these cells. These effects could be reversed by a brief incubation with gangliosides (but not with asialo‐gangliosides or other sphingolipids) at 10°C, suggesting that sialo‐lipids are critical in supporting caveolar endocytosis. Endoglycoceramidase treatment also caused a redistribution of focal adhesion kinase, paxillin, talin, and PIP Kinase Iγ away from focal adhesions. The effects of sialidase or endoglycoceramidase on membrane domains and the distribution of caveolin‐1 could be recapitulated by β1‐integrin knockdown. These results suggest that both gangliosides and β1‐integrin are required for maintenance of caveolae and plasma membrane domains.     

Quantitative GSL-glycome analysis of human whole serum based on an EGCase digestion and glycoblotting method

Glycosphingolipids (GSLs) are lipid molecules linked to carbohydrate units that form the plasma membrane lipid raft, which is clustered with sphingolipids, sterols, and specific proteins, and thereby contributes to membrane physical properties and specific recognition sites for various biological events. These bioactive GSL molecules consequently affect the pathophysiology and pathogenesis of various diseases. Thus, altered expression of GSLs in various diseases may be of importance for disease-related biomarker discovery. However, analysis of GSLs in blood is particularly challenging because GSLs are present at extremely low concentrations in serum/plasma. In this study, we established absolute GSL-glycan analysis of human serum based on endoglycoceramidase digestion and glycoblotting purification. We established two sample preparation protocols, one with and the other without GSL extraction using chloroform/methanol. Similar amounts of GSL-glycans were recovered with the two protocols. Both protocols permitted absolute quantitation of GSL-glycans using as little as 20 μl of serum. Using 10 healthy human serum samples, up to 42 signals corresponding to GSL-glycan compositions could be quantitatively detected, and the total serum GSL-glycan concentration was calculated to be 12.1–21.4 μM. We further applied this method to TLC-prefractionated serum samples. These findings will assist the discovery of disease-related biomarkers by serum GSL-glycomics.     

Konjac ceramide (kCer) regulates keratinocyte migration by Sema3A-like repulsion mechanism

Previously, we proposed the following mechanism for konjac ceramide (kCer)-mediated neurite outgrowth inhibition: kCer binds to Nrp as a Sema3A agonist, resulting in Nrp1/PlexA complex formation and activation of the Sema3A signaling pathway to induce phosphorylation of CRMP2 and microtubule depolymerization. The Sema3A/Nrp1 signaling pathway is known to be also expressed in normal human keratinocytes. To determine whether kCer can function in human keratinocytes as it does in neurites, that is, if it can bind to Nrp1 in place of Sema3A, we studied the effect of kCer on HaCaT cell migration activity. Using a trans-well chamber assay, we compared the effects of Sema3A and kCer on serum-derived cell migration activity. kCer showed Sema3A-like suppression of cell migration activity and induction of cellular Cofilin phosphorylation. In addition, kCer and Sema3A inhibited histamine (His)-enhanced migration of immature HaCaT cells. We have demonstrated that kCer does not interact with histaime receptors H1R or H4R directly, but we speculate that kCer may transduce a signal downstream of the His signaling pathway.