DOCSLIB.ORG

Galactosyltransferase Gene Led to the Deficiency of Gb3 Biosynthesis

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Galactosyltransferase Gene Led to the Deficiency of Gb3 Biosynthesis A single base insertion of the 4-α- galactosyltransferase gene led to the deficiency of Gb3 biosynthesis M.TANAKA,N.YAMASHITA,J.TAKAHASHI,F.HIRAYAMA,Y.TANI, AND H. SHIBATA cDNAs for α 1,4 galactosyltransferase (A4GALT) have been Lactosylceramide (CDH) is the common precursor isolated. To explore the molecular basis of the p phenotype in for glycosphingolipids (GSLs), which are components Japanese donors, we analyzed the A4GALT gene sequences of normal and p phenotype samples. The coding region in the of the plasma membranes and intracellular membranes A4GALT gene for DNA sequencing was amplified by PCR of all vertebrate cells. Different series of GSLs are amplification. A4GALT expression vectors for individual mutants 3 were constructed by PCR amplification of the coding region using formed depending on which sugars are added to CDH. primers and subsequent subcloning into an expression vector. The Pk (Gb3/CD77) and P (Gb4) are high-incidence expression of Gb3/CD77 antigen on the cell surface was evaluated antigens expressed on RBCs, platelets, lymphocytes, by flow cytometry and by immunochemical techniques. All 4 individuals with the p phenotype were found to have a single base fibroblasts, and uroepithelial cells. Individuals with insertion (A4GALT/insC) at the same nucleotide position. Neither the p phenotype lack Gb3, Gb4, and P1 antigens, the transfectant cells with a mutant gene (A4GALT/insC) of donor presumably as a result of a defective enzyme, 1,4- origin or those with a synthesized mutant gene (A4GALT/insC-Mu) α expressed Gb3 antigen indicating that the presence of galactosyltransferase (A4GALT) that is responsible for A4GALT/insC diminished the A4GALT enzyme activity. In addition, the synthesis of Gb3 from CDH5,6 (Fig. 1). Naturally an allele-specific PCR (ASP) system was developed in which donors occurring antibodies are formed against the missing of the p phenotype with A4GALT/insC can be unambiguously discriminated from normal donors. Based on the finding that a carbohydrate structures and can cause hemolytic single base insertion (A4GALT/insC) diminishes A4GALT activity,an transfusion reactions.4 ASP assay was developed to detect individuals with this particular cDNAs for A4GALT have been isolated.3,7,8 Five p phenotype. Immunohematology 2006;22:23–29. mutations of the A4GALT gene,T>A at nucleotide 548 Key Words: P blood group system,p phenotype,α 1,4- (M183K), G>A at 560 (G187D), C>T at 752 (P251L), galactosyltransferase C>T at 290 (S97L), and G>A at 783 (W261Stop), were reported to destroy enzyme activity, leading to the p The Globoside blood group system comprises the P phenotype. Silent and missense mutations, G>A at antigen and the Globoside blood group collection nucleotide 903 (P301P), G>A at 987 (T329T), and A>G includes the Pk and LKE antigens.1 The P blood group at 109 (M37V), with no effect on A4GALT activity,were system contains the P1 antigen.1 Five phenotypes (P ,P , 1 2 3,9 k k also found. In addition, Koda et al. found a novel one- P2 ,P1 , and p) are possible, depending on the presence or absence of the three antigens (P1, P, and Pk).2 The base insertion at positions 1026 to 1029 (A4GALT/ insC, p[1026insC]) in two Japanese p individuals and a presence of all three antigens results in the P1 CTT deletion at nucleotides 237 to 239 in one Japanese phenotype, but absence of the P1 antigen causes the P2 phenotype. If both P1 and P antigens are absent, the p individual.10 In this study, we independently k phenotype P2 arises. Absence of P antigen but presence identified the same A4GALT/insC genes that Koda et al. k k of P1 and P antigens results in the P1 phenotype. found in four Japanese p individuals and Absence of all three antigens results in the p phenotype. unambiguously showed that A4GALT/insC is Individuals with RBCs of the p phenotype are very rare.2 responsible for diminishing A4GALT activity. IMMUNOHEMATOLOGY, VOLUME 22, NUMBER 1, 2006 23 M.TANAKA ET AL. PCR amplification and DNA sequencing The coding region in the A4GALT gene was amplified for DNA sequencing using original oligonucleotide primers (GALT01, GW-GALT11, and GW-GALT12), as shown in Figure 2. For direct sequencing, PCR amplification was performed with initial denaturing at 95°C for 9 minutes followed by 30 cycles at 94°C for 1 minute, 58°C or 62°C (for primer combinations GALT01/ GW-GALT11 and GALT01/ GW- GALT12, respectively) for 30 seconds, and 72°C for 1 minute. The PCR products were excised from 1.1% agarose gels (Cambrex, East Rutherford, NJ) stained with ethidium bromide, and purified using Spin Columns (Bio-Rad, Hercules, CA). A cycle sequencing kit (BigDye Terminator v3.1 Cycle Sequencing Kit, Applied Biosystems, Foster City, CA) and a genetic analyzer (ABI PRISM 3100 Genetic Analyzer, Applied Biosystems) were used for direct DNA sequencing with capillary electrophoresis. Fig. 1. Pathways for the biosynthesis of Pk (Gb3) and P (Gb4) antigens. CDH is the common precursor consisting of lipid and carbohydrate. Pk (Gb3) and P (Gb4) belong to the Globoside blood group collection and the Globoside blood group system, respectively. GSLs are components of the plasma membrane and intracellular membranes of all vertebrate cells. GSLs are synthesized by sequential actions of glycosyltransferases. Gb3 is synthesized by A4GALT from LacCer. Gb3 has been characterized on RBCs as the Pk antigen of the Globoside blood group collection. Gb4 is synthesized by 1,3-N-acetylglucosaminyl- transferase from Gb3 and has been characterized on RBCs as the P antigen. The p phenotype lacks A4GALT, which causes the Fig. 2. Genomic structure of exon 3 of the A4GALT gene and four sets deficiency of Gb3 biosynthesis. of primers used for PCR. Primers (GALT01/GW-GALT11 and GALT01/GW-GALT12) were used for PCR amplification for sequencing and primers (GW-GALT01/GW-GALT11 and GW- Materials and Methods GALT01/GW-GALT12) were used for PCR amplification for expression. Primers (Ins01, Ins02, and Ins11) were used for the Blood samples and DNA preparation amplification of ASP. Four p phenotype and 50 normal blood samples were obtained from Japanese volunteer donors. DNAs Construction of expression vectors were prepared from the samples by QIAamp DNA A4GALT expression vectors for the A4GALT gene Blood Kit (Qiagen, Chatsworth, CA) and dissolved in derived from normal (A4GALT/WT) or p phenotype H2O to a concentration of 100 ng/L. (A4GALT/insC) individuals were constructed by PCR amplification of the coding region using the primers Blood group serology (GW-GALT01 and GW-GALT11) for pcDNA Gateway The RBC phenotypes were determined by standard directional TOPO expression kits (Invitrogen, Carlsbad, serologic techniques with our inhouse collections of CA), as shown in Figure 2, and subsequent insertion of antisera and test RBCs. the PCR products into the pcDNA3.2/V5/GW/D-TOPO 24 IMMUNOHEMATOLOGY, VOLUME 22, NUMBER 1, 2006 A4GALT/insC led to deficiency of G3b biosynthesis vector (Invitrogen). The A4GALT/insC-Mu vector was Japanese donors and from four Japanese p phenotype constructed as follows: First, the coding region in a donors determined using serologic methods. Primers normal A4GALT gene was amplified using the primers (Ins01, Ins02, and Ins11) for A4GALT genes and (GW-GALT01 and GW-GALT12), as shown in Figure 2. primers for β-actin (internal control) were used for ASP The products were then subcloned into the pKF18K amplification. PCR amplification was performed with vector and a mutation with one nucleotide insertion C initial denaturing at 95°C for 9 minutes followed by 30 at positions 1026 to 1029 in A4GALT/WT was induced cycles at 94°C for 1 minute, 62°C for 30 seconds, and by Site-Direct Mutagenesis (TaKaRa, Shiga, Japan). 72°C for 1 minute. PCR products were excised from 1.1% agarose gels (Cambrex, East Rutherford, NJ) Transfection and expression stained with ethidium bromide. Namalwa cells (Pk– cells) were purchased from a cell bank (The Japan Health Sciences Foundation, Results Tokyo, Japan). Cells were harvested after 7 days’ Mutation in the A4GALT gene of Japanese p culture with 10% FCS in RPMI-1640 medium. The phenotype donors A4GALT expression vectors were transfected into the The results of direct sequencing revealed that all harvested Namalwa cells by electroporation using four Japanese donors with p phenotype in our center GenePulser (Bio-Rad). After 48 hours in culture, the showed a single C insertion at positions 1026 to 1029 transfectants were harvested and cultured with 10% in the A4GALT gene (A4GALT/insC, Fig. 3 right). No FCS in RPMI-1640 medium containing neomycin (750 other reported mutations, deletions, and insertions, or µg/mL) for use in further analysis. missense mutations presented in Table 1 were found. The A4GALT/insC alleles were therefore speculated to Flowcytometric analysis have caused a reading frameshift and loss of the stop The Gb3 antigen expression on the surface of the codon, resulting in a loss of enzyme activity. transfected cells was evaluated by FACS Calibur (Becton Dickinson, Franklin Lakes, NJ) using monoclonal anti-CD77/Gb3 antibody (MCA579, Serotec, Oxford, UK) and FITC-labeled anti-rat IgM. Thin-layer chromatography and immunostaining assay Glycolipids extracted from the transfectant cells were used for thin-layer chromatography (TLC) after lysis and chloroform-methanol (2:1) treatment.6 Twenty µg of glycolipids was applied to a TLC plate (Merck,Whitehouse Station, NJ) which was developed for 1 hour using a solvent system of chloroform, methanol, and water (65:35:8). The TLC plate was stained with 0.5% orcin (SIGMA,St.Louis,MO) in H2SO4 and baked at 120°C in an oven. TLC immunostaining was performed using anti-CD77/Gb3.
Load more

Recommended publications
  • Supplemental Figure 1. Vimentin
    Double mutant specific genes Transcript gene_assignment Gene Symbol RefSeq FDR Fold- FDR Fold- FDR Fold- ID (single vs. Change (double Change (double Change wt) (single vs. wt) (double vs. single) (double vs. wt) vs. wt) vs. single) 10485013 BC085239 // 1110051M20Rik // RIKEN cDNA 1110051M20 gene // 2 E1 // 228356 /// NM 1110051M20Ri BC085239 0.164013 -1.38517 0.0345128 -2.24228 0.154535 -1.61877 k 10358717 NM_197990 // 1700025G04Rik // RIKEN cDNA 1700025G04 gene // 1 G2 // 69399 /// BC 1700025G04Rik NM_197990 0.142593 -1.37878 0.0212926 -3.13385 0.093068 -2.27291 10358713 NM_197990 // 1700025G04Rik // RIKEN cDNA 1700025G04 gene // 1 G2 // 69399 1700025G04Rik NM_197990 0.0655213 -1.71563 0.0222468 -2.32498 0.166843 -1.35517 10481312 NM_027283 // 1700026L06Rik // RIKEN cDNA 1700026L06 gene // 2 A3 // 69987 /// EN 1700026L06Rik NM_027283 0.0503754 -1.46385 0.0140999 -2.19537 0.0825609 -1.49972 10351465 BC150846 // 1700084C01Rik // RIKEN cDNA 1700084C01 gene // 1 H3 // 78465 /// NM_ 1700084C01Rik BC150846 0.107391 -1.5916 0.0385418 -2.05801 0.295457 -1.29305 10569654 AK007416 // 1810010D01Rik // RIKEN cDNA 1810010D01 gene // 7 F5 // 381935 /// XR 1810010D01Rik AK007416 0.145576 1.69432 0.0476957 2.51662 0.288571 1.48533 10508883 NM_001083916 // 1810019J16Rik // RIKEN cDNA 1810019J16 gene // 4 D2.3 // 69073 / 1810019J16Rik NM_001083916 0.0533206 1.57139 0.0145433 2.56417 0.0836674 1.63179 10585282 ENSMUST00000050829 // 2010007H06Rik // RIKEN cDNA 2010007H06 gene // --- // 6984 2010007H06Rik ENSMUST00000050829 0.129914 -1.71998 0.0434862 -2.51672
    [Show full text]
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD
    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
    [Show full text]
  • Datasheet: VPA00585 Product Details
    Datasheet: VPA00585 Description: RABBIT ANTI A4GALT Specificity: A4GALT Format: Purified Product Type: PrecisionAb™ Polyclonal Isotype: Polyclonal IgG Quantity: 100 µl Product Details Applications This product has been reported to work in the following applications. This information is derived from testing within our laboratories, peer-reviewed publications or personal communications from the originators. Please refer to references indicated for further information. For general protocol recommendations, please visit www.bio-rad-antibodies.com/protocols. Yes No Not Determined Suggested Dilution Western Blotting 1/1000 PrecisionAb antibodies have been extensively validated for the western blot application. The antibody has been validated at the suggested dilution. Where this product has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Further optimization may be required dependant on sample type. Target Species Human Species Cross Reacts with: Mouse Reactivity N.B. Antibody reactivity and working conditions may vary between species. Product Form Purified IgG - liquid Preparation Rabbit polyclonal antibody purified by affinity chromatography Buffer Solution Phosphate buffered saline Preservative 0.09% Sodium Azide (NaN3) Stabilisers 2% Sucrose Immunogen Synthetic peptide directed towards the middle region of human A4GALT External Database Links UniProt: Q9NPC4 Related reagents Entrez Gene: 53947 A4GALT Related reagents Synonyms A14GALT, A4GALT1 Page 1 of 2 Specificity Rabbit anti Human A4GALT antibody recognizes A4GALT, also known as CD77 synthase, GB3 synthase, P blood group (P one antigen), P(k) antigen synthase, P1/Pk synthase, UDP-galactose:beta-D-galactosyl-beta1-R 4-alpha-D-galactosyltransferase, alpha-1,4-N- acetylglucosaminyltransferase, alpha4Gal-T1 or globotriaosylceramide synthase.
    [Show full text]
  • Zebrafish As Research Model to Study Gaucher Disease
    Cover Page The handle http://hdl.handle.net/1887/137851 holds various files of this Leiden University dissertation. Author: Lelieveld, L.T. Title: Zebrafish as research model to study Gaucher disease: Insights into molecular mechanisms Issue date: 2020-10-20 Summary ● Nederlandse samenvatting List of publications ● Curriculum Vitae Dankwoord + 250 251 Summary he primary goal of this thesis has been to evaluate zebrafish as vertebrate animal model for the investigation of lysosomal storage disorders, in particular Gaucher Tdisease (GD). Deficiency of glucocerebrosidase (GCase) degrading glucosylceramide (GlcCer) in lysosomes constitutes the molecular basis of GD. Zebrafish are an appealing model organism to study genetic disorders due to their facile maintenance, their ability to generate hundreds of off-spring which rapidly develop, are transparent and fit in a 96-wells plate. Zebrafish appear to be particularly interesting to study GD since they produce the lipid GlcCer, their genome contains an orthologue of the human GBA gene and an active acid β-glucosidase is expressed. Zebrafish also express many of the candidate modifier proteins, such as non-lysosomal Gba2 and lysosomal acid ceramidase (ACase). Research described in this thesis primarily focused on optimizing and using several biochemical and genetic techniques to study the catalytic features of zebrafish GCase and the consequences of its defect in zebrafish larvae and adults. In addition, the impact of two other enzymes, non-lysosomal GBA2 and lysosomal acid ceramidase, on GCase-deficient zebrafish received attention. Chapter 1 provides an overview of the biological function, synthesis and catabolism of (glyco)sphingolipids (GSLs). Defects in one of the enzymes responsible for degradation of these lipids lead to a group of orphan diseases called lysosomal storage disorders (LSDs).
    [Show full text]
  • Lessons Learned from Gaucher and Fabry Diseases
    Journal of Clinical Medicine Communication Altered Sphingolipids Metabolism Damaged Mitochondrial Functions: Lessons Learned from Gaucher and Fabry Diseases Margarita Ivanova Lysosomal and Rare Disorders Research and Treatment Center, Faitfax, VA 22030, USA; [email protected] Received: 9 February 2020; Accepted: 10 April 2020; Published: 14 April 2020 Abstract: Sphingolipids represent a class of bioactive lipids that modulate the biophysical properties of biological membranes and play a critical role in cell signal transduction. Multiple studies have demonstrated that sphingolipids control crucial cellular functions such as the cell cycle, senescence, autophagy, apoptosis, cell migration, and inflammation. Sphingolipid metabolism is highly compartmentalized within the subcellular locations. However, the majority of steps of sphingolipids metabolism occur in lysosomes. Altered sphingolipid metabolism with an accumulation of undigested substrates in lysosomes due to lysosomal enzyme deficiency is linked to lysosomal storage disorders (LSD). Trapping of sphingolipids and their metabolites in the lysosomes inhibits lipid recycling, which has a direct effect on the lipid composition of cellular membranes, including the inner mitochondrial membrane. Additionally, lysosomes are not only the house of digestive enzymes, but are also responsible for trafficking organelles, sensing nutrients, and repairing mitochondria. However, lysosomal abnormalities lead to alteration of autophagy and disturb the energy balance and mitochondrial function. In this
    [Show full text]
  • Cross-Talks of Glycosylphosphatidylinositol Biosynthesis with Glycosphingolipid Biosynthesis and ER-Associated Degradation
    ARTICLE https://doi.org/10.1038/s41467-020-14678-2 OPEN Cross-talks of glycosylphosphatidylinositol biosynthesis with glycosphingolipid biosynthesis and ER-associated degradation Yicheng Wang 1,2, Yusuke Maeda 1, Yi-Shi Liu3, Yoko Takada 2, Akinori Ninomiya 1, Tetsuya Hirata1,2,4, ✉ Morihisa Fujita3, Yoshiko Murakami 1,2 & Taroh Kinoshita 1,2 1234567890():,; Glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipids interact with each other in the mammalian plasma membranes, forming dynamic microdomains. How their interaction starts in the cells has been unclear. Here, based on a genome-wide CRISPR-Cas9 genetic screen for genes required for GPI side-chain modification by galactose in the Golgi apparatus, we report that β1,3-galactosyltransferase 4 (B3GALT4), the previously char- acterized GM1 ganglioside synthase, additionally functions in transferring galactose to the N- acetylgalactosamine side-chain of GPI. Furthermore, B3GALT4 requires lactosylceramide for the efficient GPI side-chain galactosylation. Thus, our work demonstrates previously unex- pected functional relationships between GPI-anchored proteins and glycosphingolipids in the Golgi. Through the same screening, we also show that GPI biosynthesis in the endoplasmic reticulum (ER) is severely suppressed by ER-associated degradation to prevent GPI accu- mulation when the transfer of synthesized GPI to proteins is defective. Our data demon- strates cross-talks of GPI biosynthesis with glycosphingolipid biosynthesis and the ER quality control system. 1 Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan. 2 WPI Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan. 3 Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China.
    [Show full text]
  • 1 SUPPLEMENTAL DATA Figure S1. Poly I:C Induces IFN-Β Expression
    SUPPLEMENTAL DATA Figure S1. Poly I:C induces IFN-β expression and signaling. Fibroblasts were incubated in media with or without Poly I:C for 24 h. RNA was isolated and processed for microarray analysis. Genes showing >2-fold up- or down-regulation compared to control fibroblasts were analyzed using Ingenuity Pathway Analysis Software (Red color, up-regulation; Green color, down-regulation). The transcripts with known gene identifiers (HUGO gene symbols) were entered into the Ingenuity Pathways Knowledge Base IPA 4.0. Each gene identifier mapped in the Ingenuity Pathways Knowledge Base was termed as a focus gene, which was overlaid into a global molecular network established from the information in the Ingenuity Pathways Knowledge Base. Each network contained a maximum of 35 focus genes. 1 Figure S2. The overlap of genes regulated by Poly I:C and by IFN. Bioinformatics analysis was conducted to generate a list of 2003 genes showing >2 fold up or down- regulation in fibroblasts treated with Poly I:C for 24 h. The overlap of this gene set with the 117 skin gene IFN Core Signature comprised of datasets of skin cells stimulated by IFN (Wong et al, 2012) was generated using Microsoft Excel. 2 Symbol Description polyIC 24h IFN 24h CXCL10 chemokine (C-X-C motif) ligand 10 129 7.14 CCL5 chemokine (C-C motif) ligand 5 118 1.12 CCL5 chemokine (C-C motif) ligand 5 115 1.01 OASL 2'-5'-oligoadenylate synthetase-like 83.3 9.52 CCL8 chemokine (C-C motif) ligand 8 78.5 3.25 IDO1 indoleamine 2,3-dioxygenase 1 76.3 3.5 IFI27 interferon, alpha-inducible
    [Show full text]
  • Novel Intrinsic and Extrinsic Approaches to Selectively Regulate
    Novel Intrinsic and Extrinsic Approaches to Selectively Regulate Glycosphingolipid Metabolism by Mustafa Ali Kamani A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Biochemistry University of Toronto © Copyright by Mustafa Ali Kamani 2013. Novel Intrinsic and Extrinsic Approaches to Selectively Regulate Glycosphingolipid Metabolism. Doctor of Philosophy, 2013. Mustafa Ali Kamani Department of Biochemistry University of Toronto Abstract Glycosphingolipid (GSL) metabolism is a complex process involving proteins and enzymes at distinct locations within the cell. Mammalian GSLs are typically based on glucose or galactose, forming glucosylceramide (GlcCer) and galactosylceramide (GalCer). Most GSLs are derived from GlcCer, which is synthesized on the cytosolic leaflet of the Golgi, while all subsequent GSLs are synthesized on the lumenal side. We have utilized both pharamacological and genetic manipulation approaches to selectively regulate GSL metabolism and better understand its mechanistic details. We have developed analogues of GlcCer and GalCer by substituting the fatty acid moiety with an adamanatane frame. The resulting adamantylGSLs are more water- soluble than their natural counterparts. These analogues selectively interfere with GSL metabolism at particular points within the metabolic pathway. At 40 µM, adaGlcCer prevents synthesis of all GSLs downstream of GlcCer, while also elevating GlcCer levels, by inhibiting lactosylceramide (LacCer) synthase and glucocerebrosidase, respectively. AdaGalCer specifically reduces synthesis of globotriaosylceramide (Gb3) and downstream globo-series GSLs. AdaGalCer also increases Gaucher disease N370S glucocerebrosidase expression, lysosomal localization and activity. AdaGSLs, therefore, have potential as novel therapeutic ii agents in diseases characterized by GSL anomalies and as tools to study the effects of GSL modulation.
    [Show full text]
  • Altered Mrna Expression Levels of the Major Components of Sphingolipid Metabolism, Ceramide Synthases and Their Clinical Implication in Colorectal Cancer
    ONCOLOGY REPORTS 40: 3489-3500, 2018 Altered mRNA expression levels of the major components of sphingolipid metabolism, ceramide synthases and their clinical implication in colorectal cancer SUNG WON JANG1*, WOO-JAE PARK2*, HYEONJI MIN3, TAEG KYU KWON3,4, SEONG KYU BAEK5, ILSEON HWANG6, SHIN KIM3,4 and JONG-WOOK PARK3,4 1Department of Emergency Medicine, Dongsan Medical Center, Keimyung University, Daegu 41931; 2Department of Biochemistry, College of Medicine, Gachon University, Incheon 21999; 3Department of Immunology, School of Medicine and 4Institute of Medical Science, Keimyung University, Daegu 42601; 5Department of Surgery, Dongsan Medical Center, Keimyung University, Daegu 41931; 6Department of Pathology, School of Medicine, Keimyung University, Daegu 42601, Republic of Korea Received January 11, 2018; Accepted September 3, 2018 DOI: 10.3892/or.2018.6712 Abstract. Ceramide synthases (CerSs) synthesize various CERS5 mRNA levels in The Cancer Genome Atlas Colon ceramides of different acyl chain lengths and serve important and Rectal Cancer dataset. Notably, CERS2 and CERS4, as roles in the proliferation and death of cancer cells by regu- well as CERS5 and CERS6 levels, were positively correlated lating sphingolipid metabolism-related signaling pathways. with each other in Korean patients with CRC. However, the The present study investigated the mRNA expression levels mRNA expression levels of these four CerS genes were not of various CerS genes using mRNA expression data from six associated with any clinicopathological characteristics in independent colorectal cancer (CRC) cohorts and a Korean Korean patients with CRC. Finally, overexpressing CERS2 or CRC dataset. Expression levels of CERS2, CERS5 and CERS6 inhibited the in vitro viability of various CRC cells.
    [Show full text]
  • Fabry Disease: Molecular Basis, Pathophysiology, Diagnostics and Potential Therapeutic Directions
    biomolecules Review Fabry Disease: Molecular Basis, Pathophysiology, Diagnostics and Potential Therapeutic Directions Ken Kok 1 , Kimberley C. Zwiers 1 , Rolf G. Boot 1 , Hermen S. Overkleeft 2, Johannes M. F. G. Aerts 1,* and Marta Artola 1,* 1 Department of Medical Biochemistry, Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands; [email protected] (K.K.); [email protected] (K.C.Z.); [email protected] (R.G.B.) 2 Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands; [email protected] * Correspondence: [email protected] (J.M.F.G.A.); [email protected] (M.A.) Abstract: Fabry disease (FD) is a lysosomal storage disorder (LSD) characterized by the deficiency of α-galactosidase A (α-GalA) and the consequent accumulation of toxic metabolites such as globotriao- sylceramide (Gb3) and globotriaosylsphingosine (lysoGb3). Early diagnosis and appropriate timely treatment of FD patients are crucial to prevent tissue damage and organ failure which no treatment can reverse. LSDs might profit from four main therapeutic strategies, but hitherto there is no cure. Among the therapeutic possibilities are intravenous administered enzyme replacement therapy (ERT), oral pharmacological chaperone therapy (PCT) or enzyme stabilizers, substrate reduction therapy (SRT) and the more recent gene/RNA therapy. Unfortunately, FD patients can only benefit from ERT and, since 2016, PCT, both always combined with supportive adjunctive and preventive therapies to clinically manage FD-related chronic renal, cardiac and neurological complications.
    [Show full text]
  • Supplementary Figure 1. Dystrophic Mice Show Unbalanced Stem Cell Niche
    Supplementary Figure 1. Dystrophic mice show unbalanced stem cell niche. (A) Single channel images for the merged panels shown in Figure 1A, for of PAX7, MYOD and Laminin immunohistochemical staining in Lmna Δ8-11 mice of PAX7 and MYOD markers at the indicated days of post-natal growth. Basement membrane of muscle fibers was stained with Laminin. Scale bars, 50 µm. (B) Quantification of the % of PAX7+ MuSCs per 100 fibers at the indicated days of post-natal growth in (A). n =3-6 animals per genotype. (C) Immunohistochemical staining in Lmna Δ8-11 mice of activated, ASCs (PAX7+/KI67+) and quiescent QSCs (PAX7+/Ki67-) MuSCs at d19 and relative quantification (below). n= 4-6 animals per genotype. Scale bars, 50 µm. (D) Quantification of the number of cells per cluster in single myofibers extracted from d19 Lmna Δ8-11 mice and cultured 96h. n= 4-5 animals per group. Data are box with median and whiskers min to max. B, C, Data are mean ± s.e.m. Statistics by one-way (B) or two-way (C, D) analysis of variance (ANOVA) with multiple comparisons. * * P < 0.01, * * * P < 0.001. wt= Lmna Δ8-11 +/+; het= Lmna Δ8-11 +/; hom= Lmna Δ8-11 -/-. Supplementary Figure 2. Heterozygous mice show intermediate Lamin A levels. (A) RNA-seq signal tracks as the effective genome size normalized coverage of each biological replicate of Lmna Δ8-11 mice on Lmna locus. Neomycine cassette is indicated as a dark blue rectangle. (B) Western blot of total protein extracted from the whole Lmna Δ8-11 muscles at d19 hybridized with indicated antibodies.
    [Show full text]
  • Lineage-Specific Effector Signatures of Invariant NKT Cells Are Shared Amongst Δγ T, Innate Lymphoid, and Th Cells
    Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021 δγ is online at: average * The Journal of Immunology , 10 of which you can access for free at: 2016; 197:1460-1470; Prepublished online 6 July from submission to initial decision 4 weeks from acceptance to publication 2016; doi: 10.4049/jimmunol.1600643 http://www.jimmunol.org/content/197/4/1460 Lineage-Specific Effector Signatures of Invariant NKT Cells Are Shared amongst T, Innate Lymphoid, and Th Cells You Jeong Lee, Gabriel J. Starrett, Seungeun Thera Lee, Rendong Yang, Christine M. Henzler, Stephen C. Jameson and Kristin A. Hogquist J Immunol cites 41 articles Submit online. Every submission reviewed by practicing scientists ? is published twice each month by Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts http://jimmunol.org/subscription http://www.jimmunol.org/content/suppl/2016/07/06/jimmunol.160064 3.DCSupplemental This article http://www.jimmunol.org/content/197/4/1460.full#ref-list-1 Information about subscribing to The JI No Triage! Fast Publication! Rapid Reviews! 30 days* Why • • • Material References Permissions Email Alerts Subscription Supplementary The Journal of Immunology The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. This information is current as of September 26, 2021. The Journal of Immunology Lineage-Specific Effector Signatures of Invariant NKT Cells Are Shared amongst gd T, Innate Lymphoid, and Th Cells You Jeong Lee,* Gabriel J.
    [Show full text]