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1.
Int J Mol Sci ; 23(18)2022 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-36142595

RESUMO

The gangliosidoses GM2 are a group of pathologies mainly affecting the central nervous system due to the impaired GM2 ganglioside degradation inside the lysosome. Under physiological conditions, GM2 ganglioside is catabolized by the ß-hexosaminidase A in a GM2 activator protein-dependent mechanism. In contrast, uncharged substrates such as globosides and some glycosaminoglycans can be hydrolyzed by the ß-hexosaminidase B. Monogenic mutations on HEXA, HEXB, or GM2A genes arise in the Tay-Sachs (TSD), Sandhoff (SD), and AB variant diseases, respectively. In this work, we validated a CRISPR/Cas9-based gene editing strategy that relies on a Cas9 nickase (nCas9) as a potential approach for treating GM2 gangliosidoses using in vitro models for TSD and SD. The nCas9 contains a mutation in the catalytic RuvC domain but maintains the active HNH domain, which reduces potential off-target effects. Liposomes (LPs)- and novel magnetoliposomes (MLPs)-based vectors were used to deliver the CRISPR/nCas9 system. When LPs were used as a vector, positive outcomes were observed for the ß-hexosaminidase activity, glycosaminoglycans levels, lysosome mass, and oxidative stress. In the case of MLPs, a high cytocompatibility and transfection ratio was observed, with a slight increase in the ß-hexosaminidase activity and significant oxidative stress recovery in both TSD and SD cells. These results show the remarkable potential of CRISPR/nCas9 as a new alternative for treating GM2 gangliosidoses, as well as the superior performance of non-viral vectors in enhancing the potency of this therapeutic approach.


Assuntos
Gangliosidoses GM2 , Doença de Tay-Sachs , Desoxirribonuclease I/metabolismo , Fibroblastos/metabolismo , Proteína Ativadora de G(M2) , Gangliosídeo G(M2)/genética , Gangliosídeo G(M2)/metabolismo , Gangliosidoses GM2/genética , Gangliosidoses GM2/metabolismo , Gangliosidoses GM2/terapia , Edição de Genes , Globosídeos/metabolismo , Glicosaminoglicanos/metabolismo , Hexosaminidase A/metabolismo , Humanos , Lipopolissacarídeos/metabolismo , Lipossomos/metabolismo , Doença de Tay-Sachs/genética , Doença de Tay-Sachs/metabolismo , Doença de Tay-Sachs/terapia , beta-N-Acetil-Hexosaminidases/metabolismo
2.
Cell Rep ; 40(7): 111181, 2022 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-35977490

RESUMO

The molecular repertoire promoting cancer cell plasticity is not fully elucidated. Here, we propose that glycosphingolipids (GSLs), specifically the globo and ganglio series, correlate and promote the transition between epithelial and mesenchymal cells. The epithelial character of ovarian cancer remains stable throughout disease progression, and spatial glycosphingolipidomics reveals elevated globosides in the tumor compartment compared with the ganglioside-rich stroma. CRISPR-Cas9 knockin mediated truncation of endogenous E-cadherin induces epithelial-to-mesenchymal transition (EMT) and decreases globosides. The transcriptomics analysis identifies the ganglioside-synthesizing enzyme ST8SIA1 to be consistently elevated in mesenchymal-like samples, predicting poor outcome. Subsequent deletion of ST8SIA1 induces epithelial cell features through mTORS2448 phosphorylation, whereas loss of globosides in ΔA4GALT cells, resulting in EMT, is accompanied by increased ERKY202/T204 and AKTS124. The GSL composition dynamics corroborate cancer cell plasticity, and further evidence suggests that mesenchymal cells are maintained through ganglioside-dependent, calcium-mediated mechanisms.


Assuntos
Glicoesfingolipídeos , Neoplasias Ovarianas , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Feminino , Gangliosídeos/metabolismo , Globosídeos/metabolismo , Glicoesfingolipídeos/metabolismo , Humanos , Transdução de Sinais
3.
Front Immunol ; 13: 926220, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35844525

RESUMO

Toll-like receptor 4 (TLR4)-mediated changes in macrophages reshape intracellular lipid pools to coordinate an effective innate immune response. Although this has been previously well-studied in different model systems, it remains incompletely understood in primary human macrophages. Here we report time-dependent lipidomic and transcriptomic responses to lipopolysaccharide (LPS) in primary human macrophages from healthy donors. We grouped the variation of ~200 individual lipid species measured by LC-MS/MS into eight temporal clusters. Among all other lipids, glycosphingolipids (glycoSP) and cholesteryl esters (CE) showed a sharp increase during the resolution phase (between 8h or 16h post LPS). GlycoSP, belonging to the globoside family (Gb3 and Gb4), showed the greatest inter-individual variability among all lipids quantified. Integrative network analysis between GlycoSP/CE levels and genome-wide transcripts, identified Gb4 d18:1/16:0 and CE 20:4 association with subnetworks enriched for T cell receptor signaling (PDCD1, CD86, PTPRC, CD247, IFNG) and DC-SIGN signaling (RAF1, CD209), respectively. Our findings reveal Gb3 and Gb4 globosides as sphingolipids associated with the resolution phase of inflammatory response in human macrophages.


Assuntos
Globosídeos , Lipopolissacarídeos , Macrófagos , Cromatografia Líquida , Humanos , Macrófagos/imunologia , Espectrometria de Massas em Tandem
4.
Biochem Biophys Res Commun ; 557: 247-253, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-33894410

RESUMO

Accumulation of amyloid-ß peptide (Aß) in neuronal cells and in the extracellular regions in the brain is a major cause of Alzheimer's disease (AD); therefore, inhibition of Aß accumulation offers a promising approach for therapeutic strategies against AD. Aß is produced by sequential proteolysis of amyloid precursor protein (APP) in late/recycling endosomes after endocytosis of APP located in the plasma membrane. Aß is then released from cells in a free form or in an exosome-bound form. Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli. Recently, we found that one of the Stx subtypes, Stx2a, has a unique intracellular transport route after endocytosis through its receptor-binding B-subunit. A part of Stx2a can be transported to late/recycling endosomes and then degraded in a lysosomal acidic compartment, although in general Stx is transported to the Golgi and then to the endoplasmic reticulum in a retrograde manner. In this study, we found that treatment of APP-expressing cells with a mutant Stx2a (mStx2a), lacking cytotoxic activity because of mutations in the catalytic A-subunit, stimulated the transport of APP to the acidic compartment, which led to degradation of APP and a reduction in the amount of Aß. mStx2a-treatment also inhibited the extracellular release of Aß. Therefore, mStx2a may provide a new strategy to inhibit the production of Aß by modulating the intracellular transport of APP.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Membrana Celular/efeitos dos fármacos , Endossomos/metabolismo , Lisossomos/metabolismo , Transporte Proteico/efeitos dos fármacos , Toxina Shiga II/farmacologia , Animais , Células CHO , Domínio Catalítico/genética , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , Globosídeos/química , Humanos , Mutação , Fosfatidilcolinas/química , Proteínas Recombinantes , Toxina Shiga II/química , Toxina Shiga II/genética , Triexosilceramidas/química
5.
PLoS Pathog ; 17(4): e1009434, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33878123

RESUMO

The glycosphingolipid (GSL) globoside (Gb4) is essential for parvovirus B19 (B19V) infection. Historically considered the cellular receptor of B19V, the role of Gb4 and its interaction with B19V are controversial. In this study, we applied artificial viral particles, genetically modified cells, and specific competitors to address the interplay between the virus and the GSL. Our findings demonstrate that Gb4 is not involved in the binding or internalization process of the virus into permissive erythroid cells, a function that corresponds to the VP1u cognate receptor. However, Gb4 is essential at a post-internalization step before the delivery of the single-stranded viral DNA into the nucleus. In susceptible erythroid Gb4 knockout cells, incoming viruses were arrested in the endosomal compartment, showing no cytoplasmic spreading of capsids as observed in Gb4-expressing cells. Hemagglutination and binding assays revealed that pH acts as a switch to modulate the affinity between the virus and the GSL. Capsids interact with Gb4 exclusively under acidic conditions and dissociate at neutral pH. Inducing a specific Gb4-mediated attachment to permissive erythroid cells by acidification of the extracellular environment led to a non-infectious uptake of the virus, indicating that low pH-mediated binding to the GSL initiates active membrane processes resulting in vesicle formation. In summary, this study provides mechanistic insight into the interaction of B19V with Gb4. The strict pH-dependent binding to the ubiquitously expressed GSL prevents the redirection of the virus to nonpermissive tissues while promoting the interaction in acidic intracellular compartments as an essential step in infectious endocytic trafficking.


Assuntos
Capsídeo/metabolismo , Endocitose/imunologia , Glicoesfingolipídeos/metabolismo , Parvovirus B19 Humano/genética , Proteínas do Capsídeo/efeitos dos fármacos , Proteínas do Capsídeo/metabolismo , Endocitose/fisiologia , Globosídeos/metabolismo , Humanos , Parvovirus B19 Humano/patogenicidade , Receptores Virais/efeitos dos fármacos , Receptores Virais/metabolismo , Vírion/efeitos dos fármacos , Vírion/metabolismo , Internalização do Vírus/efeitos dos fármacos
6.
Mol Genet Metab ; 132(4): 234-243, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33642210

RESUMO

BACKGROUND: Recent years have witnessed a considerable increase in clinical trials of new investigational agents for Fabry disease (FD). Several trials investigating different agents are currently in progress; however, lack of standardisation results in challenges to interpretation and comparison. To facilitate the standardisation of investigational programs, we have developed a common framework for future clinical trials in FD. METHODS AND FINDINGS: A broad consensus regarding clinical outcomes and ways to measure them was obtained via the Delphi methodology. 35 FD clinical experts from 4 continents, representing 3389 FD patients, participated in 3 rounds of Delphi procedure. The aim was to reach a consensus regarding clinical trial design, best treatment comparator, clinical outcomes, measurement of those clinical outcomes and inclusion and exclusion criteria. Consensus results of this initiative included: the selection of the adaptative clinical trial as the ideal study design and agalsidase beta as ideal comparator treatment due to its longstanding use in FD. Renal and cardiac outcomes, such as glomerular filtration rate, proteinuria and left ventricular mass index, were prioritised, whereas neurological outcomes including cerebrovascular and white matter lesions were dismissed as a primary or secondary outcome measure. Besides, there was a consensus regarding the importance of patient-related outcomes such as general quality of life, pain, and gastrointestinal symptoms. Also, unity about lysoGb3 and Gb3 tissue deposits as useful surrogate markers of the disease was obtained. The group recognised that cardiac T1 mapping still has potential but requires further development before its widespread introduction in clinical trials. Finally, patients with end-stage renal disease or renal transplant should be excluded unless a particular group for them is created inside the clinical trial. CONCLUSION: This consensus will help to shape the future of clinical trials in FD. We note that the FDA has, coincidentally, recently published draft guidelines on clinical trials in FD and welcome this contribution.


Assuntos
Ensaios Clínicos como Assunto , Terapia de Reposição de Enzimas , Doença de Fabry/tratamento farmacológico , Rim/metabolismo , Adulto , Consenso , Técnica Delfos , Doença de Fabry/genética , Doença de Fabry/metabolismo , Doença de Fabry/patologia , Feminino , Globosídeos/uso terapêutico , Glicolipídeos/uso terapêutico , Humanos , Isoenzimas/genética , Rim/efeitos dos fármacos , Rim/patologia , Masculino , Pessoa de Meia-Idade , Qualidade de Vida , Esfingolipídeos/uso terapêutico , Resultado do Tratamento , Triexosilceramidas/uso terapêutico , alfa-Galactosidase/genética
7.
Toxins (Basel) ; 13(2)2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673393

RESUMO

Human kidney epithelial cells are supposed to be directly involved in the pathogenesis of the hemolytic-uremic syndrome (HUS) caused by Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC). The characterization of the major and minor Stx-binding glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), respectively, of primary human renal cortical epithelial cells (pHRCEpiCs) revealed GSLs with Cer (d18:1, C16:0), Cer (d18:1, C22:0), and Cer (d18:1, C24:1/C24:0) as the dominant lipoforms. Using detergent-resistant membranes (DRMs) and non-DRMs, Gb3Cer and Gb4Cer prevailed in the DRM fractions, suggesting their association with microdomains in the liquid-ordered membrane phase. A preference of Gb3Cer and Gb4Cer endowed with C24:0 fatty acid accompanied by minor monounsaturated C24:1-harboring counterparts was observed in DRMs, whereas the C24:1 fatty acid increased in relation to the saturated equivalents in non-DRMs. A shift of the dominant phospholipid phosphatidylcholine with saturated fatty acids in the DRM to unsaturated species in the non-DRM fractions correlated with the GSL distribution. Cytotoxicity assays gave a moderate susceptibility of pHRCEpiCs to the Stx1a and Stx2a subtypes when compared to highly sensitive Vero-B4 cells. The results indicate that presence of Stx-binding GSLs per se and preferred occurrence in microdomains do not necessarily lead to a high cellular susceptibility towards Stx.


Assuntos
Células Epiteliais/metabolismo , Globosídeos/metabolismo , Córtex Renal/metabolismo , Toxina Shiga I/toxicidade , Toxina Shiga II/toxicidade , Triexosilceramidas/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Células Epiteliais/patologia , Infecções por Escherichia coli/microbiologia , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Córtex Renal/patologia , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/patologia , Cultura Primária de Células , Ligação Proteica , Toxina Shiga I/metabolismo , Toxina Shiga II/metabolismo , Escherichia coli Shiga Toxigênica/metabolismo , Escherichia coli Shiga Toxigênica/patogenicidade , Células Vero
8.
J Biol Chem ; 296: 100299, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33460651

RESUMO

The human Gb3/CD77 synthase, encoded by the A4GALT gene, is an unusually promiscuous glycosyltransferase. It synthesizes the Galα1→4Gal linkage on two different glycosphingolipids (GSLs), producing globotriaosylceramide (Gb3, CD77, Pk) and the P1 antigen. Gb3 is the major receptor for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli. A single amino acid substitution (p.Q211E) ramps up the enzyme's promiscuity, rendering it able to attach Gal both to another Gal residue and to GalNAc, giving rise to NOR1 and NOR2 GSLs. Human Gb3/CD77 synthase was long believed to transfer Gal only to GSL acceptors, therefore its GSL products were, by default, considered the only human Stx receptors. Here, using soluble, recombinant human Gb3/CD77 synthase and p.Q211E mutein, we demonstrate that both enzymes can synthesize the P1 glycotope (terminal Galα1→4Galß1→4GlcNAc-R) on a complex type N-glycan and a synthetic N-glycoprotein (saposin D). Moreover, by transfection of CHO-Lec2 cells with vectors encoding human Gb3/CD77 synthase and its p.Q211E mutein, we demonstrate that both enzymes produce P1 glycotopes on N-glycoproteins, with the mutein exhibiting elevated activity. These P1-terminated N-glycoproteins are recognized by Stx1 but not Stx2 B subunits. Finally, cytotoxicity assays show that Stx1 can use P1 N-glycoproteins produced in CHO-Lec2 cells as functional receptors. We conclude that Stx1 can recognize and use P1 N-glycoproteins in addition to its canonical GSL receptors to enter and kill the cells, while Stx2 can use GSLs only. Collectively, these results may have important implications for our understanding of the Shiga toxin pathology.


Assuntos
Galactosiltransferases/química , Globosídeos/química , Toxina Shiga I/química , Triexosilceramidas/química , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Animais , Sítios de Ligação , Células CHO , Sequência de Carboidratos , Cricetulus , Escherichia coli Êntero-Hemorrágica/química , Escherichia coli Êntero-Hemorrágica/patogenicidade , Galactose/química , Galactose/metabolismo , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Expressão Gênica , Globosídeos/biossíntese , Globosídeos/metabolismo , Glucose/química , Glucose/metabolismo , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Toxina Shiga I/metabolismo , Toxina Shiga II/química , Toxina Shiga II/metabolismo , Triexosilceramidas/biossíntese
11.
Int J Mol Sci ; 22(18)2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34576167

RESUMO

Shiga toxin (Stx) is released by enterohemorrhagic Escherichia coli (EHEC) into the human intestinal lumen and transferred across the colon epithelium to the circulation. Stx-mediated damage of human kidney and brain endothelial cells and renal epithelial cells is a renowned feature, while the sensitivity of the human colon epithelium towards Stx and the decoration with the Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα1-4Galß1-4Glcß1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcß1-3Galα1-4Galß1-4Glcß1-1Cer) is a matter of debate. Structural analysis of the globo-series GSLs of serum-free cultivated primary human colon epithelial cells (pHCoEpiCs) revealed Gb4Cer as the major neutral GSL with Cer (d18:1, C16:0), Cer (d18:1, C22:1/C22:0) and Cer (d18:1, C24:2/C24:1) accompanied by minor Gb3Cer with Cer (d18:1, C16:0) and Cer (d18:1, C24:1) as the dominant lipoforms. Gb3Cer and Gb4Cer co-distributed with cholesterol and sphingomyelin to detergent-resistant membranes (DRMs) used as microdomain analogs. Exposure to increasing Stx concentrations indicated only a slight cell-damaging effect at the highest toxin concentration of 1 µg/mL for Stx1a and Stx2a, whereas a significant effect was detected for Stx2e. Considerable Stx refractiveness of pHCoEpiCs that correlated with the rather low cellular content of the high-affinity Stx-receptor Gb3Cer renders the human colon epithelium questionable as a major target of Stx1a and Stx2a.


Assuntos
Colo/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Globosídeos/metabolismo , Toxina Shiga/metabolismo , Triexosilceramidas/metabolismo , Linhagem Celular , Células Cultivadas , Cromatografia em Camada Delgada , Glicoesfingolipídeos/metabolismo , Humanos , Espectrometria de Massas , Sintaxina 1/metabolismo
12.
Int J Mol Sci ; 22(18)2021 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-34576250

RESUMO

Anderson-Fabry disease (AFD) is a rare disease with an incidenceof approximately 1:117,000 male births. Lysosomal accumulation of globotriaosylceramide (Gb3) is the element characterizing Fabry disease due to a hereditary deficiency α-galactosidase A (GLA) enzyme. The accumulation of Gb3 causes lysosomal dysfunction that compromises cell signaling pathways. Deposition of sphingolipids occurs in the autonomic nervous system, dorsal root ganglia, kidney epithelial cells, vascular system cells, and myocardial cells, resulting in organ failure. This manuscript will review the molecular pathogenetic pathways involved in Anderson-Fabry disease and in its organ damage. Some studies reported that inhibition of mitochondrial function and energy metabolism plays a significant role in AFD cardiomyopathy and in kidney disease of AFD patients. Furthermore, mitochondrial dysfunction has been reported as linked to the dysregulation of the autophagy-lysosomal pathway which inhibits the mechanistic target of rapamycin kinase (mTOR) mediated control of mitochondrial metabolism in AFD cells. Cerebrovascular complications due to AFD are caused by cerebral micro vessel stenosis. These are caused by wall thickening resulting from the intramural accumulation of glycolipids, luminal occlusion or thrombosis. Other pathogenetic mechanisms involved in organ damage linked to Gb3 accumulation are endocytosis and lysosomal degradation of endothelial calcium-activated intermediate-conductance potassium ion channel 3.1 (KCa3.1) via a clathrin-dependent process. This process represents a crucial event in endothelial dysfunction. Several studies have identified the deacylated form of Gb3, globotriaosylsphingosine (Lyso-Gb3), as the main catabolite that increases in plasma and urine in patients with AFD. The mean concentrations of Gb3 in all organs and plasma of Galactosidase A knockout mice were significantly higher than those of wild-type mice. The distributions of Gb3 isoforms vary from organ to organ. Various Gb3 isoforms were observed mainly in the kidneys, and kidney-specific Gb3 isoforms were hydroxylated. Furthermore, the action of Gb3 on the KCa3.1 channel suggests a possible contribution of this interaction to the Fabry disease process, as this channel is expressed in various cells, including endothelial cells, fibroblasts, smooth muscle cells in proliferation, microglia, and lymphocytes. These molecular pathways could be considered a potential therapeutic target to correct the enzyme in addition to the traditional enzyme replacement therapies (ERT) or drug chaperone therapy.


Assuntos
Células Endoteliais/metabolismo , Doença de Fabry/tratamento farmacológico , Doença de Fabry/metabolismo , MicroRNAs/metabolismo , Animais , Autofagia , Circulação Cerebrovascular , Constrição Patológica , Terapia de Reposição de Enzimas , Doença de Fabry/fisiopatologia , Globosídeos/química , Glicolipídeos/metabolismo , Humanos , Lisossomos/química , Camundongos , Microcirculação , Mitocôndrias/metabolismo , Isoformas de Proteínas , Transdução de Sinais , Esfingolipídeos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Triexosilceramidas/química , Triexosilceramidas/metabolismo , alfa-Galactosidase/metabolismo
13.
Transfus Med ; 31(1): 48-54, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33314439

RESUMO

OBJECTIVES: This study aimed to investigate single-nucleotide variants (SNVs) associated with P1 expression among Thai blood donors and develop a genotyping method using multiplex polymerase chain reaction (PCR) to predict P1 blood group status. BACKGROUND: The α1,4-galactosyltransferase (A4GALT), also called Gb3/CD77 synthase or P1/Pk synthase enzyme, is encoded by the A4GALT gene and catalyses the transfer of galactose from uridine diphosphate-galactose to lactosylceramide, creating the Pk antigen (Gb3). The same enzyme synthesises the P1 antigen by adding terminal galactose to paragloboside. The A4GALT transcripts are elevated in P1 , and different SNVs in transcription factor-binding regions of A4GALT correlate with P1 and P2 phenotypes. MATERIAL AND METHODS: A total of 218 blood samples from Thai blood donors at the Thammasat University Hospital were tested for the P1 antigen using the conventional tube technique. Genomic DNA was extracted, and non-coding regions of A4GALT were sequenced and analysed. A multiplex PCR assay was developed and validated to identify P1-associated SNVs and was subsequently tested on 1022 Thai DNA samples of unknown P1 antigen status. RESULTS: In the tested cohort (n = 218), P1 and P2 phenotypes were found in 24.77% and 75.23% of donors, respectively. Moreover, three SNVs-rs8138197 (C/T), rs2143918 (T/G) and rs5751348 (G/T)-correlated 100% with both phenotypes. Finally, findings agreed with serological phenotyping and DNA sequencing results, confirming their validity for predicting P1 antigen positivity. CONCLUSIONS: This study confirmed that three SNVs also correlated with P1 /P2 phenotypes among Thais, as expected. A multiplex PCR found that SNVs rs2143918 (T) and rs5751348 (G) predicted blood group P1 and is an accurate, reproducible, cost-effective and less time-consuming alternative to traditional methods.


Assuntos
Doadores de Sangue , Galactosiltransferases/genética , Genótipo , Técnicas de Genotipagem , Globosídeos/genética , Reação em Cadeia da Polimerase Multiplex , Polimorfismo de Nucleotídeo Único , Feminino , Humanos , Masculino , Tailândia
14.
Tohoku J Exp Med ; 252(1): 1-8, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32814720

RESUMO

Carbohydrate antigens are associated with carcinogenesis, cancer invasion, and metastasis and their expression reflect biological activities of various cancers. We previously reported that expression of disialosyl globopentaosyl ceramide (DSGb5), one of carbohydrate antigens, in radical prostatectomy specimens independently predicted biochemical recurrence (i.e., elevating serum prostate specific antigen without recurrent lesions in the image) after radical prostatectomy. However, it is important to evaluate the prognosis at the diagnosis. In this study we investigated DSGb5 expression in prostate biopsy specimens to develop a novel biomarker for providing appropriate management. Between 2005 and 2011, patients who underwent both prostate biopsy and radical prostatectomy in our institution were included. The median follow-up period was 88 months. DSGb5 expression was assessed by immunohistochemical staining and defined 116 patients as high DSGb5 expression (42 patients) or low DSGb5 expression (74 patients). High DSGb5 expression was significantly associated with lymphovascular invasion in radical prostatectomy specimens on both univariate and multivariable analyses (p = 0.028, 0.027). On multivariable analysis, Gleason Score in prostatectomy specimen, positive resection margin, and DSGb5 expression in the biopsy specimen were independently associated with biochemical recurrence-free survival following radical prostatectomy (p = 0.004, 0.008, 0.024). When targeting only patients with negative resection margin, DSGb5 expression was significantly associated with biochemical recurrence-free survival on both univariate and multivariable analyses (p = 0.006, 0.007). DSGb5 expression in prostate biopsy specimens is predictive of lymphovascular invasion and biochemical recurrence-free survival following radical prostatectomy. DSGb5 is a potential biomarker for preoperatively predicting oncological outcomes of prostate cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Globosídeos/metabolismo , Recidiva Local de Neoplasia/patologia , Próstata/metabolismo , Próstata/patologia , Prostatectomia , Idoso , Biópsia , Intervalo Livre de Doença , Humanos , Masculino , Margens de Excisão , Pessoa de Meia-Idade , Próstata/cirurgia
15.
J Biol Chem ; 295(42): 14305-14324, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-32796033

RESUMO

Streptococcus suis is part of the pig commensal microbiome but strains can also be pathogenic, causing pneumonia and meningitis in pigs as well as zoonotic meningitis. According to genomic analysis, S. suis is divided into asymptomatic carriage, respiratory and systemic strains with distinct genomic signatures. Because the strategies to target pathogenic S. suis are limited, new therapeutic approaches are needed. The virulence factor S. suis adhesin P (SadP) recognizes the galabiose Galα1-4Gal-oligosaccharide. Based on its oligosaccharide fine specificity, SadP can be divided into subtypes PN and PO We show here that subtype PN is distributed in the systemic strains causing meningitis, whereas type PO is found in asymptomatic carriage and respiratory strains. Both types of SadP are shown to predominantly bind to pig lung globotriaosylceramide (Gb3). However, SadP adhesin from systemic subtype PN strains also binds to globotetraosylceramide (Gb4). Mutagenesis studies of the galabiose-binding domain of type PN SadP adhesin showed that the amino acid asparagine 285, which is replaced by an aspartate residue in type PO SadP, was required for binding to Gb4 and, strikingly, was also required for interaction with the glycomimetic inhibitor phenylurea-galabiose. Molecular dynamics simulations provided insight into the role of Asn-285 for Gb4 and phenylurea-galabiose binding, suggesting additional hydrogen bonding to terminal GalNAc of Gb4 and the urea group. Thus, the Asn-285-mediated molecular mechanism of type PN SadP binding to Gb4 could be used to selectively target S. suis in systemic disease without interfering with commensal strains, opening up new avenues for interventional strategies against this pathogen.


Assuntos
Adesinas Bacterianas/metabolismo , Globosídeos/metabolismo , Fatores de Virulência/metabolismo , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência de Carboidratos , Portador Sadio , Globosídeos/química , Glicoesfingolipídeos/análise , Glicoesfingolipídeos/química , Glicoesfingolipídeos/metabolismo , Pulmão/metabolismo , Meningite/microbiologia , Meningite/patologia , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Fenótipo , Compostos de Fenilureia/química , Compostos de Fenilureia/metabolismo , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Streptococcus suis/metabolismo , Suínos , Doenças dos Suínos/microbiologia , Doenças dos Suínos/patologia , Fatores de Virulência/química , Fatores de Virulência/genética
16.
J Org Chem ; 85(24): 15920-15935, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-32567311

RESUMO

The total synthesis of the oligosaccharide moiety of disialosyl globopentaosylceramide (DSGb5 Cer), a dominant ganglioside isolated from malignant renal cell carcinoma tissues, is reported. The synthetic strategy relies on a chemical α(2,6)-sialylation at the internal GalNAc unit of a Gb5 pentasaccharide backbone that furnishes a Neu5Acα(2,6)GalNAc-linked hexasaccharide, suitable for an enzymatic α(2,3)-sialylation of the terminal Gal residue to construct a heptasaccharide glycan. Convergent access to this key α(2,6)-sialylated hexasaccharide was also achieved through a [3+3] glycosylation building upon a Galß(1,3)[Neu5Acα(2,6)]GalNAc-based trisaccharide donor and a Gb3 acceptor. The synthetic DSGb5 glycan bearing a 6-azidohexyl aglycon at the reducing end could undergo further regioselective functionalization. This approach represents a viable chemoenzymatic method for accessing complex ganglioside glycans and should be useful for the synthesis and biological investigation of DSGb5 derivatives.


Assuntos
Globosídeos , Polissacarídeos , Glicosilação , Oligossacarídeos
17.
Int J Mol Sci ; 21(10)2020 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-32455599

RESUMO

Glycosphingolipids containing very-long-chain fatty acids (VLCFAs) regulate several immune responses, such as cytokine production, immune signaling, and antibody induction. We previously reported that stimulation with an inflammatory mediator, TNF-α, promotes the expression of glycosphingolipids in vascular endothelial cells. The major component is globotetraosylceramide containing VLCFAs (Gb4Cer-VLCFAs), but its role in inflammatory responses has not been fully investigated. In this study, the antibody-inducing properties of Gb4Cer-VLCFAs were analyzed using serum and hybridoma cells generated from Gb4Cer-VLCFA-immunized mice. The reactivity of serum antibodies against Gb4Cer indicated that immunization with Gb4Cer-VLCFAs immediately induced the production of anti-Gb4Cer antibodies. Over 81% of hybridomas generated from the splenocytes of an immunized mouse produced anti-Gb4Cer antibodies, a subset of which recognized an epitope shared by Gb4Cer and its precursor globotriaosylceramide (Gb3Cer). Further biochemical analyses of established monoclonal antibodies revealed that these antibodies included IgM and IgG3, which specifically react with Gb4Cer and Gb3Cer. These results indicate that immunization with Gb4Cer-VLCFAs can efficiently induce the production of anti-Gb4Cer and -Gb3Cer antibodies by B cells.


Assuntos
Anticorpos Monoclonais/imunologia , Globosídeos/imunologia , Animais , Células Cultivadas , Humanos , Hibridomas/imunologia , Imunização/métodos , Camundongos , Baço/imunologia , Fator de Necrose Tumoral alfa/metabolismo
18.
Org Biomol Chem ; 18(16): 3142-3148, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32255449

RESUMO

The human cell surface trisaccharide motifs globotriose and P1 antigen play key roles in infections by pathogenic bacteria, which makes them important synthetic targets as antibacterial agents. Enzymatic strategies to install the terminal α1,4-galactosidic linkage are very attractive but have only been demonstrated for a limited set of analogues. Herein, a new bacterial α1,4 galactosyltransferase from N. weaveri was cloned and produced recombinantly in E. coli BL21 (DE3) cells, followed by investigation of its substrate specificity. We demonstrate that the enzyme can tolerate galactosamine (GalN) and also 6-deoxygalactose and 6-deoxy-6-fluorogalactose as donors, and lactose and N-acetyllactosamine as acceptors, leading directly to analogues of Gb3 and P1 that are valuable chemical probes and showcase how biocatalysis can provide fast access to a number of unnatural carbohydrate analogues.


Assuntos
Galactosídeos/síntese química , Galactosiltransferases/metabolismo , Neisseria/enzimologia , Amino Açúcares/metabolismo , Proteínas de Bactérias , Biocatálise , Clonagem Molecular , Escherichia coli/genética , Galactosamina/metabolismo , Galactosídeos/biossíntese , Galactosiltransferases/isolamento & purificação , Globosídeos/química , Humanos , Lactose/metabolismo , Especificidade por Substrato , Trissacarídeos/química
19.
Methods Mol Biol ; 2132: 257-266, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32306333

RESUMO

LecA/PA-IL (Pfam PF07828) is a soluble galactose-binding lectin from bacterium Pseudomonas aeruginosa. The lectin is specific for α-galactose present on glycosphingolipids of the globoside family and has therefore been proposed to play a role in cell adhesion and in internalization of bacteria in epithelial cells. The lectin has also direct toxic activity. Search for high-affinity inhibitors can be performed on the recombinant lectin, with use of surface plasmon resonance assays and structural studies.


Assuntos
Adesinas Bacterianas/química , Adesinas Bacterianas/metabolismo , Melibiose/metabolismo , Pseudomonas aeruginosa/metabolismo , Aderência Bacteriana , Sítios de Ligação , Cristalografia por Raios X , Galectinas/química , Galectinas/metabolismo , Globosídeos/metabolismo , Modelos Moleculares , Conformação Proteica , Pseudomonas aeruginosa/química , Ressonância de Plasmônio de Superfície
20.
J Biol Chem ; 295(13): 4341-4349, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32029474

RESUMO

Sphingolipid biosynthesis generates lipids for membranes and signaling that are crucial for many developmental and physiological processes. In some cases, large amounts of specific sphingolipids must be synthesized for specialized physiological functions, such as during axon myelination. How sphingolipid synthesis is regulated to fulfill these physiological requirements is not known. To identify genes that positively regulate membrane sphingolipid levels, here we employed a genome-wide CRISPR/Cas9 loss-of-function screen in HeLa cells using selection for resistance to Shiga toxin, which uses a plasma membrane-associated glycosphingolipid, globotriaosylceramide (Gb3), for its uptake. The screen identified several genes in the sphingolipid biosynthetic pathway that are required for Gb3 synthesis, and it also identified the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor widely involved in development and physiology, as being required for Gb3 biosynthesis. AHR bound and activated the gene promoter of serine palmitoyltransferase small subunit A (SPTSSA), which encodes a subunit of the serine palmitoyltransferase that catalyzes the first and rate-limiting step in de novo sphingolipid biosynthesis. AHR knockout HeLa cells exhibited significantly reduced levels of cell-surface Gb3, and both AHR knockout HeLa cells and tissues from Ahr knockout mice displayed decreased sphingolipid content as well as significantly reduced expression of several key genes in the sphingolipid biosynthetic pathway. The sciatic nerve of Ahr knockout mice exhibited both reduced ceramide content and reduced myelin thickness. These results indicate that AHR up-regulates sphingolipid levels and is important for full axon myelination, which requires elevated levels of membrane sphingolipids.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Resistência à Doença/genética , Globosídeos/genética , Receptores de Hidrocarboneto Arílico/genética , Serina C-Palmitoiltransferase/genética , Esfingolipídeos/biossíntese , Triexosilceramidas/genética , Animais , Sistemas CRISPR-Cas/genética , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Genoma Humano/genética , Células HeLa , Humanos , Metabolismo dos Lipídeos/genética , Lipídeos/biossíntese , Lipídeos/genética , Camundongos , Camundongos Knockout , Toxina Shiga/farmacologia , Transdução de Sinais/genética , Esfingolipídeos/genética
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