Thiogalactopyranosides are resistant to hydrolysis by α-galactosidases.

Fluorescently tagged glycosides containing terminal α(1→3) and α(1→4)-linked thiogalactopyranosides have been prepared and tested for resistance to hydrolysis by α-galactosidases. Eight fluorescent glycosides containing either galactose or 5-thiogalactose as the terminal sugar were enzymatically synthesized using galactosyltransferases, with lactosyl glycosides as acceptors and UDP-galactose or UDP-5'-thiogalactose, respectively, as donors. The glycosides were incubated with human α-galactosidase A (CAZy family GH27, a retaining glycosidase), Bacteroides fragilis α-1,3-galactosidase (GH110, an inverting glycosidase), or homogenates of MCF-7 human breast cancer cells or NG108-15 rat glioma cells. Substrate hydrolysis was monitored by capillary electrophoresis with fluorescence detection. All compounds containing terminal O-galactose were readily degraded. Their 5-thiogalactose counterparts were resistant to hydrolysis by human α-galactosidase A and the enzymes present in the cell extracts. B. fragilis α-1,3-galactosidase hydrolyzed both thio- and O-galactoside substrates; however, the thiogalactosides were hydrolyzed at only 1-3 % of the rate of O-galactosides. The hydrolytic resistance of 5-thiogalactose was also confirmed by an in vivo study using cells in culture. The results suggest that 5-thiogalactosides may be useful tools for the study of anabolic pathways in cell extracts or in single cells.

Enzymatic synthesis of Gb3 and iGb3 ceramides.

Gb3 and iGb3 are physiologically important trihexosylceramides with a terminal alpha-d-Galp-(1-->4)-beta-d-Galp- and alpha-d-Galp-(1-->3)-beta-d-Galp sequence, respectively. In particular iGb3 is attracting considerable attention as it is believed to serve as a ligand for natural killer T cells. Whether or not iGb3 is present in humans and which enzyme might be responsible for its synthesis is at present a matter of lively debate. In the current investigation we evaluated human blood group B galactosyltransferase (GTB) for its ability to catalyze the formation of iGb3 from lactosylceramide and UDP-Galp. GTB is a retaining glycosyltransferase that in vivo catalyzes the transfer of galactose from UDP-Galp donors to OH-3 of Galp on the H-antigen (alpha-l-Fucp-(1-->2)-beta-d-Galp) acceptor forming the blood group B antigen. GTB tolerates modifications in donor and acceptor substrates and its ability to accept lactosides as acceptors makes it a possible candidate for iGb3 production in humans. For comparison iGb3 and Gb3 were also synthesized from the same acceptor using an alpha-(1-->3)- and alpha-(1-->4)-specific galactosyltransferase, respectively. All the enzymes tested catalyzed the desired reactions. Product characterization by NMR analysis clearly differentiated between the alpha-Galp-(1-->3)-Galp and alpha-Galp-(1-->4)-Galp product, with the GTB product being identical to that of the alpha-(1-->3)-GalT-catalyzed reaction. The rate of transfer by GTB however was very low, only 0.001% of the rate obtained with a good substrate, H antigen disaccharide (octyl alpha-l-Fucp-(1-->2)-beta-d-Galp). This is too low to account for the possible formation of the iGb3 structure in humans in vivo.

Immunologic glycosphingolipidomics and NKT cell development in mouse thymus.

Invariant NKT cells are a hybrid cell type of Natural Killer cells and T cells, whose development is dependent on thymic positive selection mediated by double positive thymocytes through their recognition of natural ligands presented by CD1d, a nonpolymorphic, non-MHC, MHC-like antigen presenting molecule. Genetic evidence suggested that beta-glucosylceramide derived glycosphingolipids (GSLs) are natural ligands for NKT cells. N-butyldeoxygalactonojirimycin (NB-DGJ), a drug that specifically inhibits the glucosylceramide synthase, inhibits the endogenous ligands for NKT cells. Furthermore, we and others have found a beta-linked glycosphingolipid, isoglobotriaosylceramide (iGb3), is a stimulatory NKT ligand. The iGb3 synthase knockout mice have a normal NKT development and function, indicating that other ligands exist and remain to be identified. In this study, we have performed a glycosphingolipidomics study of mouse thymus, and studied mice mutants which are deficient in beta-hexosaminidase b or alpha-galactosidase A, two glycosidases that are up- and downstream agents of iGb3 turnover, respectively. Our mass spectrometry methods generated a first database for glycosphingolipids expressed in mouse thymus, which are specifically regulated by rate-limiting glycosidases. Among the identified thymic glycosphingolipids, only iGb3 is a stimulatory ligand for NKT cells, suggesting that large-scale fractionation, enrichment and characterization of minor species of glycosphingolipids are necessary for identifying additional ligands for NKT cells. Our results also provide early insights into cellular lipidomics studies, with a specific focus on the important immunological functions of glycosphingolipids.

Solvent-free enzymatic synthesis of fatty alkanolamides.

An environmentally benign and volume efficient process for enzymatic production of alkanolamides is described. Immobilized Candida antarctica lipase B, Novozym435, was used to catalyze the condensation of lauric acid with monoethanolamine. The reaction temperature of 90 degrees C was required to keep the reactants in a liquid state. Stepwise addition of the amine minimized problems caused by the formation of a highly viscous amine/fatty acid ion-pair. The enzyme was both very active and stable under the reaction conditions, with about half of the activity remaining after 2 weeks. The maximum amide yield obtained when using equimolar amounts of the reactants was 75%, which could be increased to 95% upon water removal. Special precautions to avoid co-distillation of the amine were required. Two different strategies to avoid the amine loss are presented.

Analysis of fatty acid epoxidation by high performance liquid chromatography coupled with evaporative light scattering detection and mass spectrometry.

Conventionally, epoxidation of unsaturated fatty acids has been studied either with titrimetric methods or in a lengthy procedure involving derivatization followed by gas chromatography (GC). We have developed a more rapid and descriptive analysis procedure for the substances using high performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD). Chemo-enzymatic epoxidation of unsaturated fatty acids (oleic, linoleic and linolenic acid, respectively) has been performed using hydrogen peroxide and immobilized lipase from Candida antarctica (Novozym 435). The fatty acids and their epoxidation products were separated by HPLC on a C-18 reversed-phase column using methanol-water containing 0.05% acetic acid as mobile phase. The method facilitated the simultaneous determination of fatty acids and epoxides differing from each other in the number of epoxide rings, the degree of unsaturation and the position of the epoxide rings and double bonds. An important aspect of the method development was the use of electrospray ionization and tandem mass spectrometry to confirm the structure of the epoxide products. It is suggested that the HPLC method, providing more information about the kind and concentration of fatty acids and their epoxides, represents a powerful complement to the existing methods for monitoring epoxidation processes on fatty acids.

Synthesis of phosphatidylcholine with defined fatty acid in the sn-1 position by lipase-catalyzed esterification and transesterification reaction.

The incorporation of caproic acid in the sn-1 position of phosphatidylcholine (PC) catalyzed by lipase from Rhizopus oryzae was investigated in a water activity-controlled organic medium. The reaction was carried out either as esterification or transesterification. A comparison between these two reaction modes was made with regard to product yield, product purity, reaction time, and byproduct formation as a consequence of acyl migration. The yield in the esterification and transesterification reaction was the same under identical conditions. The highest yield (78%) was obtained at a water activity (a(w)) of 0.11 and a caproic acid concentration of 0.8 M. The reaction time was shorter in the esterification reaction than in the transesterification reaction. The difference in reaction time was especially pronounced at low water activities and high fatty acid concentrations. The loss in yield due to acyl migration and consequent enzymatic side reactions was around 16% under a wide range of conditions. The incorporation of a fatty acid in the sn-1 position of PC proved to be thermodynamically much more favorable than the incorporation of a fatty acid in the sn-2 position.