Sexual differences in galactose metabolism: galactosyl ceramide galactosidase and other galactosidases in mouse kidney.
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ABSTRACT:
SUBMITTER: Lin YN
PROVIDER: S-EPMC1166066 | biostudies-other | 1973 Dec
REPOSITORIES: biostudies-other
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Project description:The β-galactosidases from Lactobacillus reuteri L103 (Lreuβgal), Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 (Lbulβgal), and Bifidobacterium breve DSM 20281 (Bbreβgal-I and Bbreβgal-II) were investigated in detail with respect to their propensity to transfer galactosyl moieties onto lactose, its hydrolysis products D-glucose and D-galactose, and certain sugar acceptors such as N-acetyl-D-glucosamine (GlcNAc), N-acetyl-D-galactosamine (GalNAc), and L-fucose (Fuc) under defined, initial velocity conditions. The rate constants or partitioning ratios (kNu/kwater) determined for these different acceptors (termed nucleophiles, Nu) were used as a measure for the ability of a certain substance to act as a galactosyl acceptor of these β-galactosidases. When using Lbulβgal or Bbreβgal-II, the galactosyl transfer to GlcNAc was 6 and 10 times higher than that to lactose, respectively. With lactose and GlcNAc used in equimolar substrate concentrations, Lbulβgal and Bbreβgal-II catalyzed the formation of N-acetyl-allolactosamine with the highest yields of 41 and 24%, respectively, as calculated from the initial GlcNAc concentration.
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Project description:A library of dimeric CD1d ligands, containing two ?-galactosyl ceramide (?-GalCer) units linked by spacers of varying lengths has been synthesised. The key dimerisation reactions were carried out via copper-catalysed click reactions between a 6"-azido-6"-deoxy-?-galactosyl ceramide derivative and various diynes. Each ?-GalCer dimer was tested for its ability to stimulate iNKT cells.
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Project description:1. Analysis of transport of d-galactose was complicated by metabolism of the compound but appeared to have two components: a substrate-saturable component and a diffusion component. At low substrate concentration (<1mm) active transport was observed. Accumulation of galactose was largely independent of Na(+) concentration. The apparent K(m) for this component was 0.2mm. At substrate concentrations above 1mm the active transport system appeared saturated and further increases in substrate concentration resulted in a linear increase in the rate of galactose accumulation, but no concentration gradient was formed. 2. d-[1-(14)C]Galactose (2mm) was metabolized to (14)CO(2) by rat kidney-cortex slices incubated at 37 degrees C, at the rate of 68nmol/h per 100mg of tissue. 3. Intracellular components from such incubations were separated into a neutral fraction, the only major labelled component being galactose, and a phosphorylated fraction. 4. Phosphorylated metabolites found in galactose-incubated slices increased with increasing substrate concentration and achieved a limiting value of 0.42mm after 60min of incubation. 5. Galactose uptake was inhibited by anaerobiosis, dinitrophenol and phlorrhizin. 6. Methyl alpha-d-glucoside and d-glucose partially inhibited galactose uptake only at ratios of 100:1. 7. The presence of pyruvate did not decrease galactose metabolism although it did decrease production of (14)CO(2) from [1-(14)C]galactose. Gluconeogenesis occurred in the presence of pyruvate and (14)C from galactose was found in glucose. 8. Rat kidney-cortex slices metabolized 2mm-[1-(14)C]galactonate to (14)CO(2) at a rate of 20nmol/h per 100mg of tissue.
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Project description:1-Aminophytosphingosine and 6-aminogalactosyl phytosphingosine were prepared in 61% and 40% yield libraries with 44 carboxylic acids showed that a 4-butylbenzoic acid-derived product exe, respectively. Glycosylation using benzoyl-protected lipid resulted in better a-selectivity for ceramide analogs, but the yield was less than that obtained with benzyl moieties. Screening the amide rted less cytotoxicity. These analogs were purified for validation of immunological potencies and the a-GalCer analog but not the sphingosine analog stimulated human iNKT cell population.
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Project description:Ceramides (Cers) and complex sphingolipids with defined acyl chain lengths play important roles in numerous cell processes. Six Cer synthase (CerS) isoenzymes (CerS1-6) are the key enzymes responsible for the production of the diversity of molecular species. In this study, we investigated the changes in sphingolipid metabolism during the differentiation of Madin-Darby canine kidney (MDCK) cells. By MALDI TOF TOF MS, we analyzed the molecular species of Cer, glucosylceramide (GlcCer), lactosylceramide (LacCer), and SM in nondifferentiated and differentiated cells (cultured under hypertonicity). The molecular species detected were the same, but cells subjected to hypertonicity presented higher levels of C24:1 Cer, C24:1 GlcCer, C24:1 SM, and C16:0 LacCer. Consistently with the molecular species, MDCK cells expressed CerS2, CerS4, and CerS6, but with no differences during cell differentiation. We next evaluated the different synthesis pathways with sphingolipid inhibitors and found that cells subjected to hypertonicity in the presence of amitriptyline, an inhibitor of acid sphingomyelinase, showed decreased radiolabeled incorporation in LacCer and cells did not develop a mature apical membrane. These results suggest that hypertonicity induces the endolysosomal degradation of SM, generating the Cer used as substrate for the synthesis of specific molecular species of glycosphingolipids that are essential for MDCK cell differentiation.
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Project description:BACKGROUND:Glycoproteins and glycolipids of some mammalian species contain the disaccharide galactosyl-?-(1,3)-galactose (?-Gal). It is known that ?-Gal is immunogenic in humans and causes glycan-specific IgG and also IgE responses with clinical relevance. ?-Gal is part of the IgE-reactive monoclonal therapeutic antibody cetuximab (CTX) and is associated with delayed anaphylaxis to red meat. In this study, different ?-Gal-containing analytes are examined in singleplex and multiplex assays to resolve individual sensitization patterns with IgE against ?-Gal. METHODS:Three serum groups, ?-Gal-associated meat allergy (MA) patients, idiopathic anaphylaxis (IA) patients with suspected MA, and non-meat-allergic healthy control individuals (HC), were analyzed via singleplex allergy diagnostics and a newly established immunoblot diagnostic system. The new dot blot detection system resolved individual IgE sensitization profiles for ?-Gal-containing analytes CTX, bovine thyroglobulin (Bos d TG), and human serum albumin (HSA)-conjugated ?-Gal. RESULTS:Singleplex allergy diagnostics using the ?-Gal analytes CTX and Bos d TG confirms the history of MA patients in 91% and 88% of the cases, respectively. A novel dot blot-based assay system for the detection of IgE against ?-Gal reveals individual IgE sensitization profiles for ?-Gal-containing analytes. An ?-Gal-associated IgE cross-reactivity profile (IgE against CTX, Bos d TG, and HSA-?-Gal) was identified, which is associated with MA. CONCLUSIONS:Detection of individual sensitization patterns with different ?-Gal-containing analytes provides the basis for an individual allergy diagnosis for ?-Gal-sensitized patients. Higher amounts of ?-Gal in pork and beef innards compared to muscle meat as indicated by a higher staining intensity are a plausible explanation for the difference in allergic symptom severity.
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Project description:Coordinated lipid metabolism contributes to maintaining skin homeostasis by regulating skin barrier formation, immune reactions, thermogenesis, and perception. Several reports have documented the changes in lipid composition in dermatitis, including in atopic dermatitis (AD); however, the specific mechanism by which these lipid profiles are altered during AD pathogenesis remains unknown. Here, we performed untargeted and targeted lipidomic analyses of an AD-like dermatitis model resulting from constitutive activation of Janus kinase 1 (Spade mice) to capture the comprehensive lipidome profile during dermatitis onset and progression. We successfully annotated over 700 skin lipids, including glycerophospholipids, ceramides, neutral lipids, and fatty acids, many of which were found to be present at significantly changed levels after dermatitis onset, as determined by the pruritus and erythema. Among them, we found the levels of ceramides composed of nonhydroxy fatty acid and dihydrosphingosine containing very long-chain (C22 or more) fatty acids were significantly downregulated before AD onset. Furthermore, in vitro enzyme assays using the skin of Spade mice demonstrated the enhancement of ceramide desaturation. Finally, we revealed topical application of ceramides composed of nonhydroxy fatty acid and dihydrosphingosine before AD onset effectively ameliorated the progression of AD symptoms in Spade mice. Our results suggest that the disruption in epidermal ceramide composition is caused by boosting ceramide desaturation in the initiation phase of AD, which regulates AD pathogenesis.
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Project description:Pyruvyl modification of oligosaccharides is widely seen in both prokaryotes and eukaryotes. Although the biosynthetic mechanisms of pyruvylation have been investigated, enzymes that metabolize and degrade pyruvylated oligosaccharides are not well known. Here, we searched for a pyruvylated galactose (PvGal)-releasing enzyme by screening soil samples. We identified a Bacillus strain, as confirmed by the 16S ribosomal RNA gene analysis, that exhibited PvGal-ase activity toward p-nitrophenyl-β-D-pyruvylated galactopyranose (pNP-β-D-PvGal). Draft genome sequencing of this strain, named HMA207, identified three candidate genes encoding potential PvGal-ases, among which only the recombinant protein encoded by ORF1119 exhibited PvGal-ase activity. Although ORF1119 protein displayed broad substrate specificity for pNP sugars, pNP-β-D-PvGal was the most favorable substrate. The optimum pH for the ORF1119 PvGal-ase was determined as 7.5. A BLAST search suggested that ORF1119 homologs exist widely in bacteria. Among two homologs tested, BglC from Clostridium but not BglH from Bacillus showed PvGal-ase activity. Crystal structural analysis together with point mutation analysis revealed crucial amino acids for PvGal-ase activity. Moreover, ORF1119 protein catalyzed the hydrolysis of PvGal from galactomannan of Schizosaccharomyces pombe, suggesting that natural polysaccharides might be substrates of the PvGal-ase. This novel PvGal-catalyzing enzyme might be useful for glycoengineering projects to produce new oligosaccharide structures.
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Project description:Ataxin-2 (human gene symbol ATXN2) acts during stress responses, modulating mRNA translation and nutrient metabolism. Ataxin-2 knockout mice exhibit progressive obesity, dyslipidemia, and insulin resistance. Conversely, the progressive ATXN2 gain of function due to the fact of polyglutamine (polyQ) expansions leads to a dominantly inherited neurodegenerative process named spinocerebellar ataxia type 2 (SCA2) with early adipose tissue loss and late muscle atrophy. We tried to understand lipid dysregulation in a SCA2 patient brain and in an authentic mouse model. Thin layer chromatography of a patient cerebellum was compared to the lipid metabolome of Atxn2-CAG100-Knockin (KIN) mouse spinocerebellar tissue. The human pathology caused deficits of sulfatide, galactosylceramide, cholesterol, C22/24-sphingomyelin, and gangliosides GM1a/GD1b despite quite normal levels of C18-sphingomyelin. Cerebellum and spinal cord from the KIN mouse showed a consistent decrease of various ceramides with a significant elevation of sphingosine in the more severely affected spinal cord. Deficiency of C24/26-sphingomyelins contrasted with excess C18/20-sphingomyelin. Spinocerebellar expression profiling revealed consistent reductions of CERS protein isoforms, Sptlc2 and Smpd3, but upregulation of Cers2 mRNA, as prominent anomalies in the ceramide-sphingosine metabolism. Reduction of Asah2 mRNA correlated to deficient S1P levels. In addition, downregulations for the elongase Elovl1,Elovl4,Elovl5 mRNAs and ELOVL4 protein explain the deficit of very long-chain sphingomyelin. Reduced ASMase protein levels correlated to the accumulation of long-chain sphingomyelin. Overall, a deficit of myelin lipids was prominent in SCA2 nervous tissue at prefinal stage and not compensated by transcriptional adaptation of several metabolic enzymes. Myelination is controlled by mTORC1 signals; thus, our human and murine observations are in agreement with the known role of ATXN2 yeast, nematode, and mouse orthologs as mTORC1 inhibitors and autophagy promoters.
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