RESUMO
Adult stem cells are critical for the maintenance of tissue homeostasis. However, how the proliferation and differentiation of intestinal stem cells (ISCs) are regulated remains not fully understood. Here, we find a mutant, stum 9-3, affecting the proliferation and differentiation of Drosophila adult ISCs in a forward genetic screen for factors regulating the proliferation and differentiation ISCs. stum 9-3 acts through the conserved Notch signaling pathway, upstream of the S2 cleavage of the Notch receptor. Interestingly, the phenotype of stum 9-3 mutant is not caused by disruption of stumble (stum), where the p-element is inserted. Detailed mapping, rescue experiments and mutant characterization show that stum 9-3 is a new allele of O-fucosyltransferase 1 (O-fut1). Our results indicate that unexpected mutants with interesting phenotype could be recovered in forward genetic screens using known p-element insertion stocks.
Assuntos
Alelos , Proteínas de Drosophila/fisiologia , Drosophila/genética , Fucosiltransferases/fisiologia , Células-Tronco/metabolismo , Animais , Intestinos/citologiaRESUMO
FUT2, a protein that uses l-fucose to mediate fucosylation of intestinal epithelial cells, is one of the detected gene variants in IBD patients. We aimed to investigate whether exogenous l-fucose could be an enteral nutritional supplement to protect intestinal barrier function. The effect of l-fucose on the restoration of epithelial barrier function in both the DSS-induced colitis mouse model and LPS-stimulated Caco-2 cells was investigated, and the impact on fucosylation of epithelial cells was examined. The severity of DSS-induced colitis was significantly reduced by l-fucose. Restoration of epithelial barrier function by l-fucose was detected. Direct l-fucose-mediated protection of tight junctions was observed in Caco-2 cells. Moreover, exogenous l-fucose promoted the exogenous metabolic pathway of l-fucose, and fucosylation of epithelial cells both in vivo and in vitro. Moreover, knockout of the FUT2 gene restrained fucosylation and the protective effect of l-fucose on barrier function. The severity of colitis was not improved by l-fucose in Fut2 knockout mice. Therefore we conclude that exogenous l-fucose protects intestinal barrier function and relieves intestinal inflammation via upregulation of FUT2-mediated fucosylation of intestinal epithelial cells.
Assuntos
Colite/prevenção & controle , Células Epiteliais/efeitos dos fármacos , Fucose/farmacologia , Fucosiltransferases/fisiologia , Inflamação/prevenção & controle , Mucosa Intestinal/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Animais , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Sulfato de Dextrana/toxicidade , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
Glycosylation represents one of the most abundant posttranslational modification of proteins. Glycosylation products are diverse and are regulated by the cooperative action of various glycosyltransferases, glycosidases, substrates thereof: nucleoside sugars and their transporters, and chaperons. In this article, we focus on a glycosyltransferase, α1,6-fucosyltransferase (Fut8) and its product, the core fucose structure on N-glycans, and summarize the potential protective functions of this structure against emphysema and chronic obstructive pulmonary disease (COPD). Studies of FUT8 and its enzymatic product, core fucose, are becoming an emerging area of interest in various fields of research including inflammation, cancer and therapeutics. This article discusses what we can learn from studies of Fut8 and core fucose by using knockout mice or in vitro studies that were conducted by our group as well as other groups. We also include a discussion of the potential protective functions of the keratan sulfate (KS) disaccharide, namely L4, against emphysema and COPD as a glycomimetic. Glycomimetics using glycan analogs is one of the more promising therapeutics that compensate for the usual therapeutic strategy that involves targeting the genome and the proteome. These typical glycans using KS derivatives as glycomimetics, will likely become a clue to the development of novel and effective therapeutic strategies.
Assuntos
Materiais Biomiméticos/uso terapêutico , Sulfato de Queratano/química , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Antígenos de Superfície/fisiologia , Materiais Biomiméticos/química , Fucose/metabolismo , Fucosiltransferases/fisiologia , Glicosilação , Humanos , Lectinas Tipo C/antagonistas & inibidores , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Lectinas Tipo C/fisiologia , Lectinas de Ligação a Manose/antagonistas & inibidores , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Lectinas de Ligação a Manose/fisiologia , Camundongos , Camundongos Knockout , Terapia de Alvo Molecular/métodos , Polissacarídeos/química , Polissacarídeos/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/metabolismoRESUMO
Helminth parasites secrete a wide variety of immunomodulatory proteins and lipids to dampen host immune responses. Many of these immunomodulatory compounds are modified with complex sugar structures (or glycans), which play an important role at the host-parasite interface. As an example, the human blood fluke Schistosoma mansoni produces highly fucosylated glycan structures on glycoproteins and glycolipids. Up to 20 different S. mansoni fucosyltransferase (SmFucT) genes can be found in genome databases, but thus far only one enzyme has been functionally characterized. To unravel the synthesis of highly fucosylated N-glycans by S. mansoni, we examined the ability of ten selected SmFucTs to modify N-glycans upon transient expression in Nicotiana benthamiana plants. All enzymes were localized in the plant Golgi apparatus, which allowed us to identify the SmFucTs involved in core fucosylation and the synthesis of complex antennary glycan motifs. This knowledge provides a starting point for investigations into the role of specific fucosylated glycan motifs of schistosomes in parasite-host interactions. The functionally characterized SmFucTs can also be applied to synthesize complex N-glycan structures on recombinant proteins to study their contribution to immunomodulation. Furthermore, this plant expression system will fuel the development of helminth glycoproteins for pharmaceutical applications or novel anti-helminth vaccines.
Assuntos
Fucosiltransferases/metabolismo , Nicotiana/metabolismo , Schistosoma mansoni/metabolismo , Animais , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/imunologia , Fucosiltransferases/fisiologia , Glicoproteínas/metabolismo , Glicosilação , Proteínas de Helminto/imunologia , Proteínas de Helminto/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Parasitos/metabolismo , Polissacarídeos/química , Schistosoma mansoni/genética , Schistosoma mansoni/parasitologia , Nicotiana/parasitologiaRESUMO
PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer. It is an aggressive malignancy associated with poor prognosis because of recurrence, metastasis, and treatment resistance. Aberrant glycosylation of cancer cells triggers their migration and invasion and is considered one of the most important prognostic cancer biomarkers. The current study aimed to identify glycan alterations and their relationship with the malignant potential of PDAC. METHODS: Using a lectin microarray, we evaluated glycan expression in 62 PDAC samples. Expression of fucosyltransferase 8 (FUT8), the only enzyme catalyzing core fucosylation, was investigated by immunohistochemistry. The role of FUT8 in PDAC invasion and metastasis was confirmed using an in vitro assay and a xenograft peritoneal metastasis mouse model. RESULTS: The microarray data demonstrated that core fucose-binding lectins were significantly higher in carcinoma than in normal pancreatic duct tissues. Similarly, FUT8 protein expression was significantly higher in carcinoma than in normal pancreatic duct tissues. High FUT8 protein expression was significantly associated with lymph-node metastases and relapse-free survival. FUT8 knockdown significantly reduced the invasion in PDAC cell lines and impaired peritoneal metastasis in the xenograft model. CONCLUSIONS: The findings of this study provide evidence that FUT8 plays a pivotal role in PDAC invasion and metastasis and might be a therapeutic target for this disease.
Assuntos
Carcinoma Ductal Pancreático/patologia , Fucosiltransferases/metabolismo , Fucosiltransferases/fisiologia , Metástase Linfática/genética , Invasividade Neoplásica/genética , Neoplasias Pancreáticas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Fucosiltransferases/genética , Expressão Gênica , Glicosilação , Humanos , Lectinas , Camundongos , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Polissacarídeos/genética , Polissacarídeos/metabolismo , Análise Serial de ProteínasRESUMO
INTRODUCTION: Trophoblast proliferation and invasion are essential for embryo implantation and placentation. Protein glycosylation is one of the most common and vital post-translational modifications, regulates protein physical and biochemical properties. FUT8 is the only known fucosyltransferase responsible for catalyzing α1,6-fucosylation in mammals, and α1,6-fucosylated glycoproteins are found to participate in various physiopathological processes. However, whether FUT8/α1,6-fucosylation modulates the functions of trophoblastic cells remains elusive. METHODS: FUT8 in human placenta villi during 6-8 gestational weeks and trophoblastic cells were detected by Western blot and immunofluorescent staining. α1,6-fucosylation in tissues or cells were measured by Lectin LCA (Lens culinaris) fluorescent staining and Lectin blot. FUT8 expression was down-regulated by siRNA transfection in JAR and JEG-3â¯cells, and cell viability, motility and invasiveness ability were detected by the functional experiments. α1,6-fucosylation of insulin-like growth factor receptor (IGF-1R) was examined by immunoprecipitation, and the amount of phosphorylated IGF-1R was detected in FUT8 down-regulated JAR cells. RESULTS: Human placenta villi and trophoblastic cells expressed FUT8/α1,6-fucosylation. Knockdown FUT8 by siRNA transfection suppressed the proliferation, epithelial-mesenchymal transition, migration and invasion of JAR and JEG-3â¯cells. Furthermore, we found that FUT8 modified the α1,6-fucosylation of IGF-1R, and regulated IGF-1 dependent activation of IGF-1R, MAPK and PI3K/Akt signaling pathways in JAR cells. CONCLUSIONS: Our results implicate a critical role for FUT8 in maintaining the normal functions of trophoblastic cells, suggesting manipulating FUT8 may be an effective approach in pregnancy.
Assuntos
Fucosiltransferases/fisiologia , Trofoblastos/fisiologia , Linhagem Celular , Proliferação de Células , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Receptor IGF Tipo 1/metabolismo , Transdução de SinaisRESUMO
PURPOSE: As an important glycosyltransferase, fucosyltransferase IV (FUT4) is abnormally upregulated in different types of cancers, but its clinical role remains inexplicit. This work aimed to determine the predictive ability of FUT4 in lung adenocarcinoma (LUAD) after curative resection, as well as to explore the role of a possible FUT4 molecular mechanism on LUAD malignant behavior. METHODS: A total of 273 LUAD patients after curative resection with complete clinicopathological and RNAseq data from The Cancer Genome Atlas (TCGA) cohort were collected. Correlation of FUT4 with overall survival (OS) was analyzed based on TCGA and further validated by online "Kaplan-Meier Plotter" database and IHC in 70 LUAD patients recruited in the First Hospital of China Medical University cohort. Multivariate Cox regression analysis and 1000 bootstrapping were performed to verify the predictive value of FUT4. Gene set enrichment assay (GSEA) was performed to investigate the biological characteristics. Correlation between PD-1 and FUT4 was analyzed based on TCGA cohort and validated by IHC on cohort from our hospital. RESULTS: Increased FUT4 expression led to reduced overall survival (OS) of LUAD patients based on TCGA (p = 0.006 and 0.001 for dichotomous and trichotomous modeling, respectively) and externally validated in KMPLOTTER (p = 0.01) and by IHC based on cohort from our hospital (p = 0.005 and p = 0.019 for dichotomous and trichotomous modeling, respectively). FUT4 overexpression was an independent high risk factor for OS along with advanced pT stage and pTNM stage (p = 0.001, p = 0.037, and p < 0.001, respectively). GSEA revealed that FUT4 overexpression might correlate with shortened survival of LUAD patients by promoting cell proliferation via ERBB signaling, and suppressing immune response-related pathways. FUT4 expression positively correlated with PD-1 in TCGA (p = 0.026) and validated by IHC on cohort from our hospital (p = 0.029). CONCLUSIONS: Increased FUT4 expression led to reduced OS in operable LUAD. FUT4 showed significant correlation with immune response and PD-1 expression.
Assuntos
Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/cirurgia , Fucosiltransferases/fisiologia , Antígenos CD15/fisiologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/cirurgia , Receptor de Morte Celular Programada 1/fisiologia , Adenocarcinoma de Pulmão/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/imunologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas Proto-Oncogênicas/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais , Análise de SobrevidaRESUMO
The fucose salvage pathway is a two-step process in which mammalian cells transform L-fucose into GDP-L-fucose, a universal fucose donor used by fucosyltransferases to modify glycans. Emerging evidence indicates the fucose salvage pathway and the fucosylation of proteins are altered during melanoma progression and metastasis. However the underlying mechanisms are not completely understood. Here, we report that the fucose salvage pathway inhibits invadopodia formation and extracellular matrix degradation by promoting α-1,2 fucosylation. Chemically or genetically increasing the fucose salvage pathway decreases invadopodium numbers and inhibits the proteolytic activity of invadopodia in WM793 melanoma cells. Inhibiting fucosylation by depleting fucokinase abrogates L-fucose-mediated inhibition of invadopodia, suggesting dependence on the fucose salvage pathway. The inhibition of invadopodium formation by L-fucose or ectopically expressed FUK could be rescued by treatment with α-1,2, but not α-1,3/α-1,4 fucosidase, implicating an α-1,2 fucose linkage-dependent anti-metastatic effect. The expression of FUT1, an α-1,2 fucosyltransferase, is remarkably down-regulated during melanoma progression, and the ectopic expression of FUT1 is sufficient to inhibit invadopodium formation and ECM degradation. Our findings indicate that the fucose salvage pathway can inhibit invadopodium formation, and consequently, invasiveness in melanoma via α-1,2 fucosylation. Re-activation of this pathway in melanoma could be useful for preventing melanoma invasion and metastasis.
Assuntos
Matriz Extracelular/metabolismo , Fucose/metabolismo , Fucosiltransferases/fisiologia , Melanoma/metabolismo , Proteínas de Neoplasias/fisiologia , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Podossomos/fisiologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Fucose/farmacologia , Fucosiltransferases/deficiência , Fucosiltransferases/genética , Vetores Genéticos/farmacologia , Glicosilação , Humanos , Melanoma/fisiopatologia , Redes e Vias Metabólicas , Invasividade Neoplásica , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Podossomos/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/farmacologia , Regulação para Cima , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
Formation of appropriate gut microbiota is essential for human health. The first two years of life is the critical period for this process. Selection of mutualistic microorganisms of the intestinal microbiota is controlled by the FUT2 and FUT3 genes that encode fucosyltransferases, enzymes responsible for the synthesis of fucosylated glycan structures of mucins and milk oligosaccharides. In this review, the mechanisms of the selection and maintenance of intestinal microorganisms that involve fucosylated oligosaccharides of breast milk and mucins of the newborn's intestine are described. Possible reasons for the use of fucose, and not sialic acid, as the major biological signal for the selection are also discussed.
Assuntos
Antígenos de Grupos Sanguíneos , Fucose , Microbioma Gastrointestinal , Leite Humano/química , Feminino , Fucosiltransferases/fisiologia , Humanos , Lactente , Recém-Nascido , Leite Humano/microbiologia , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
Association of aberrant glycosylation with melanoma progression is based mainly on analyses of cell lines. Here we present a systems-based study of glycomic changes and corresponding enzymes associated with melanoma metastasis in patient samples. Upregulation of core fucosylation (FUT8) and downregulation of α-1,2 fucosylation (FUT1, FUT2) were identified as features of metastatic melanoma. Using both in vitro and in vivo studies, we demonstrate FUT8 is a driver of melanoma metastasis which, when silenced, suppresses invasion and tumor dissemination. Glycoprotein targets of FUT8 were enriched in cell migration proteins including the adhesion molecule L1CAM. Core fucosylation impacted L1CAM cleavage and the ability of L1CAM to support melanoma invasion. FUT8 and its targets represent therapeutic targets in melanoma metastasis.
Assuntos
Fucosiltransferases/genética , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Animais , Fucosiltransferases/metabolismo , Fucosiltransferases/fisiologia , Inativação Gênica , Glicoproteínas/metabolismo , Glicosilação , Humanos , Melanoma/patologia , Camundongos , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Biologia de Sistemas/métodosRESUMO
Generation of functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) has been a long-sought-after goal for use in hematopoietic cell production, disease modeling, and eventually transplantation medicine. Homing of HSPCs from bloodstream to bone marrow (BM) is an important aspect of HSPC biology that has remained unaddressed in efforts to derive functional HSPCs from human PSCs. We have therefore examined the BM homing properties of human induced pluripotent stem cell-derived HSPCs (hiPS-HSPCs). We found that they express molecular effectors of BM extravasation, such as the chemokine receptor CXCR4 and the integrin dimer VLA-4, but lack expression of E-selectin ligands that program HSPC trafficking to BM. To overcome this deficiency, we expressed human fucosyltransferase 6 using modified mRNA. Expression of fucosyltransferase 6 resulted in marked increases in levels of cell surface E-selectin ligands. The glycoengineered cells exhibited enhanced tethering and rolling interactions on E-selectin-bearing endothelium under flow conditions in vitro as well as increased BM trafficking and extravasation when transplanted into mice. However, glycoengineered hiPS-HSPCs did not engraft long-term, indicating that additional functional deficiencies exist in these cells. Our results suggest that strategies toward increasing E-selectin ligand expression could be applicable as part of a multifaceted approach to optimize the production of HSPCs from human PSCs.
Assuntos
Movimento Celular , Células-Tronco Hematopoéticas/fisiologia , RNA Mensageiro/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Selectina E , Fucosiltransferases/fisiologia , Glicosilação , Transplante de Células-Tronco Hematopoéticas , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos Endogâmicos NOD , Camundongos SCID , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genéticaRESUMO
Sarcopenia, the loss of muscle mass and strength during normal aging, involves coordinate changes in skeletal myofibers and the cells that contact them, including satellite cells and motor neurons. Here we show that the protein O-fucosyltransferase 1 gene (Pofut1), which encodes a glycosyltransferase required for NotchR-mediated cell-cell signaling, has reduced expression in aging skeletal muscle. Moreover, premature postnatal deletion of Pofut1 in skeletal myofibers can induce aging-related phenotypes in cis within skeletal myofibers and in trans within satellite cells and within motor neurons via the neuromuscular junction. Changed phenotypes include reduced skeletal muscle size and strength, decreased myofiber size, increased slow fiber (type 1) density, increased muscle degeneration and regeneration in aged muscles, decreased satellite cell self-renewal and regenerative potential, and increased neuromuscular fragmentation and occasional denervation. Pofut1 deletion in skeletal myofibers reduced NotchR signaling in young adult muscles, but this effect was lost with age. Increasing muscle NotchR signaling also reduced muscle size. Gene expression studies point to regulation of cell cycle genes, muscle myosins, NotchR and Wnt pathway genes, and connective tissue growth factor by Pofut1 in skeletal muscle, with additional effects on α dystroglycan glycosylation.
Assuntos
Envelhecimento/fisiologia , Fucosiltransferases/fisiologia , Neurônios Motores/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Junção Neuromuscular/fisiologia , Células Satélites de Músculo Esquelético/fisiologia , Animais , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/citologia , Fibras Musculares Esqueléticas/citologia , Junção Neuromuscular/patologia , Fenótipo , Receptores Notch/metabolismo , Sarcopenia/etiologia , Sarcopenia/metabolismo , Sarcopenia/patologia , Células Satélites de Músculo Esquelético/citologia , Transdução de SinaisRESUMO
The fucosyltransferases are key enzymes in cell surface antigen synthesis during multidrug resistance (MDR) development. The aim of the present study was to analyze the alteration of α(1,2)-fucosyltransferase involved in MDR development in human chronic myeloid leukemia (CML). FUT1 was overexpressed in three CML/MDR cell lines and peripheral blood mononuclear cells (PBMC) of CML patients. However, no significant changes of FUT2 were observed. The altered levels of FUT1 had a significant impact on the phenotypic variation of MDR of K562 and K562/ADR cells, the activity of EGFR/MAPK pathway and P-glycoprotein (P-gp) expression. Blocking the EGFR/MAPK pathway by its specific inhibitor PD153035 or EGFR small interfering RNA (siRNA) resulted in the reduced MDR of K562/ADR cells. This study indicated that α(1,2)-fucosyltransferase involved in the development of MDR of CML cells probably through FUT1 regulated the activity of EGFR/MAPK signaling pathway and the expression of P-gp.
Assuntos
Fucosiltransferases/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Regulação para Baixo , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/metabolismo , Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
Oxidative damage by reactive oxygen species (ROS) is a major cause of sperm dysfunction. Excessive ROS generation reduces fertilization and enhances DNA damage of spermatozoa. Interaction between spermatozoa and oviductal epithelial cells improves the fertilizing ability of and reduces chromatin damage in spermatozoa. Our previous data showed that oviductal epithelial cell membrane proteins interact with the human spermatozoa and protect them from ROS-induced reduction in sperm motility, membrane integrity and DNA integrity. Sperm fucosyltransferase-5 (sFUT5) is a membrane carbohydrate-binding protein on human spermatozoa. In this study, we demonstrate for the first time that sFUT5 is involved in human spermatozoa-oviduct interaction and the beneficial effects of such interaction on the fertilizing ability of human spermatozoa. Anti-sFUT5 antibody-treated spermatozoa had reduced binding to oviductal membrane proteins. It is consistent with the result that affinity-purified sFUT5 is bound to the epithelial lining of human oviduct and to the immortalized human oviductal epithelial cell line, OE-E6/E7. Pretreatment of spermatozoa with anti-sFUT5 antibody and oviductal membrane proteins with sFUT5 suppressed the protective action of oviductal membrane proteins against ROS/cryopreservation-induced oxidative damage in spermatozoa. Asialofetuin, a reported sFUT5 substrate, can partly mimic the protective effect of oviductal epithelial cell membrane proteins on sperm motility, membrane and DNA integrity. The results enhance our understanding on the protective mechanism of oviduct on sperm functions.
Assuntos
Tubas Uterinas/enzimologia , Fucosiltransferases/fisiologia , Estresse Oxidativo , Comunicação Celular , Criopreservação , Fragmentação do DNA , Células Epiteliais/enzimologia , Feminino , Humanos , Masculino , Espécies Reativas de Oxigênio , Preservação do Sêmen , Motilidade dos Espermatozoides , Espermatozoides/citologia , Espermatozoides/enzimologia , Espermatozoides/fisiologiaRESUMO
OBJECTIVES: Angiogenesis contributes to the pathogenesis of rheumatoid arthritis. Fucosyltransferases (Futs) are involved in angiogenesis and tumour growth. Here, we examined the role of Fut1 in angiogenesis and K/BxN serum transfer arthritis. METHODS: We examined Fut1 expression in human dermal microvascular endothelial cells (HMVECs) by quantitative PCR. We performed a number of angiogenesis assays to determine the role of Fut1 using HMVECs, Fut1 null (Fut1(-/-)), and wild type (wt) endothelial cells (ECs) and mice. K/BxN serum transfer arthritis was performed to determine the contribution of Fut1-mediated angiogenesis in Fut1(-/-) and wt mice. A static adhesion assay was implemented with RAW264.7 (mouse macrophage cell line) and mouse ECs. Quantitative PCR, immunofluorescence and flow cytometry were performed with Fut1(-/-) and wt ECs for adhesion molecule expression. RESULTS: Tumour necrosis factor-α induced Fut1 mRNA and protein expression in HMVECs. HMVECs transfected with Fut1 antisense oligodeoxynucleotide and Fut1(-/-) ECs formed significantly fewer tubes on Matrigel. Fut1(-/-) mice had reduced angiogenesis in Matrigel plug and sponge granuloma angiogenesis assays compared with wt mice. Fut1(-/-) mice were resistant to K/BxN serum transfer arthritis and had decreased angiogenesis and leucocyte ingress into inflamed joints. Adhesion of RAW264.7 cells to wt mouse ECs was significantly reduced when Fut1 was lacking. Fut1(-/-) ECs had decreased intercellular adhesion molecule-1 (ICAM-1) expression at mRNA and protein levels compared with wt ECs. ICAM-1 was also decreased in Fut1(-/-) arthritic ankle cryosections compared with wt ankles. CONCLUSIONS: Fut1 plays an important role in regulating angiogenesis and ICAM-1 expression in inflammatory arthritis.
Assuntos
Artrite Experimental/metabolismo , Artrite Experimental/fisiopatologia , Fucosiltransferases/fisiologia , Molécula 1 de Adesão Intercelular/metabolismo , Neovascularização Patológica/fisiopatologia , Animais , Artrite Experimental/patologia , Adesão Celular/fisiologia , Linhagem Celular , Células Cultivadas , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Fucosiltransferases/deficiência , Fucosiltransferases/genética , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Knockout , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
OBJECTIVE: Serum lipase activities above the threefold upper reference limit indicate acute pancreatitis. We investigated whether high lipase activity-within the reference range and in the absence of pancreatitis-are associated with genetic single nucleotide polymorphisms (SNP), and whether these identified SNPs are also associated with clinical pancreatitis. METHODS: Genome-wide association studies (GWAS) on phenotypes 'serum lipase activity' and 'high serum lipase activity' were conducted including 3966 German volunteers from the population-based Study-of-Health-in-Pomerania (SHIP). Lead SNPs associated on a genome-wide significance level were replicated in two cohorts, 1444 blood donors and 1042 pancreatitis patients. RESULTS: Initial discovery GWAS detected SNPs within or near genes encoding the ABO blood group specifying transferases A/B (ABO), Fucosyltransferase-2 (FUT2), and Chymotrypsinogen-B2 (CTRB2), to be significantly associated with lipase activity levels in asymptomatic subjects. Replication analyses in blood donors confirmed the association of FUT-2 non-secretor status (OR=1.49; p=0.012) and ABO blood-type-B (OR=2.48; p=7.29×10(-8)) with high lipase activity levels. In pancreatitis patients, significant associations were found for FUT-2 non-secretor status (OR=1.53; p=8.56×10(-4)) and ABO-B (OR=1.69, p=1.0×10(-4)) with chronic pancreatitis, but not with acute pancreatitis. Conversely, carriers of blood group O were less frequently affected by chronic pancreatitis (OR=0.62; p=1.22×10(-05)) and less likely to have high lipase activity levels (OR=0.59; p=8.14×10(-05)). CONCLUSIONS: These are the first results indicating that ABO blood type-B as well as FUT2 non-secretor status are common population-wide risk factors for developing chronic pancreatitis. They also imply that, even within the reference range, elevated lipase activities may indicate subclinical pancreatic injury in asymptomatic subjects.
Assuntos
Sistema ABO de Grupos Sanguíneos/fisiologia , Fucosiltransferases/fisiologia , Estudos de Associação Genética , Estudo de Associação Genômica Ampla , Lipase/sangue , Pancreatite Crônica/epidemiologia , Pancreatite Crônica/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Medição de Risco , Adulto Jovem , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
Carbohydrate structures, including Lewis X (Le(x)), which is not synthesized in mutant mice that lack α1,3-fucosyltransferase 9 (Fut9(-/-)), are involved in cell-cell recognition and inflammation. However, immunological alteration in Fut9(-/-) mice has not been studied. Thus, the inflammatory response of Fut9(-/-) mice was examined using the highly neurovirulent mouse hepatitis virus (MHV) JHMV srr7 strain. Pathological study revealed that inflammation induced in the brains of Fut9(-/-) mice after infection was more extensive compared with that of wild-type mice, although viral titers obtained from the brains of mutant mice were lower than those of wild-type mice. Furthermore, the reduction in cell numbers in the spleens of wild-type mice after infection was not observed in the infected Fut9(-/-) mice. Although there were no clear differences in the levels of cytokines examined in the brains between Fut9(-/-) and wild-type mice except for interferon-ß expression, some of those in the spleens, including interferon-γ, interleukin-6, and monocyte chemoattractant protein 1, showed higher levels in Fut9(-/-) than in wild-type mice. Furthermore, Fut9(-/-) mice were refractory to the in vivo inoculation of endotoxin (LPS) compared with wild-type mice. These results indicate that Le(x) structures are involved in host responses against viral or bacterial challenges.
Assuntos
Fucosiltransferases/deficiência , Imunidade Inata/imunologia , Lipopolissacarídeos/imunologia , Vírus da Hepatite Murina/imunologia , Animais , Encéfalo/patologia , Encéfalo/virologia , Contagem de Células , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/prevenção & controle , Citocinas/metabolismo , Modelos Animais de Doenças , Fucosiltransferases/genética , Fucosiltransferases/fisiologia , Imunidade Inata/genética , Imunidade Inata/fisiologia , Camundongos , Camundongos Knockout , Camundongos Mutantes , Vírus da Hepatite Murina/isolamento & purificação , Baço/patologia , Baço/virologiaRESUMO
OBJECTIVE: To determine the role of α(1,2)-linked fucosylation of proteins by fucosyltransferase 1 (FUT1) in rheumatoid arthritis (RA) angiogenesis. METHODS: Analysis of α(1,2)-linked fucosylated proteins in synovial tissue (ST) samples was performed by immunohistologic staining. Expression of α(1,2)-linked fucosylated angiogenic chemokine in synovial fluid (SF) was determined by immunoprecipitation and lectin blotting. To determine the angiogenic role of α(1,2)-linked fucosylated proteins in RA, we performed human dermal microvascular endothelial cell (HMVEC) chemotaxis and Matrigel assays using sham-depleted and α(1,2)-linked fucosylated protein-depleted RA SF samples. To examine the production of proangiogenic chemokines by FUT1 in HMVECs, cells were transfected with FUT1 sense or antisense oligonucleotides, and enzyme-linked immunosorbent assay was performed. We then studied mouse lung endothelial cell (EC) chemotaxis using wild-type and FUT1 gene-deficient mouse lung ECs. RESULTS: RA ST endothelial cells showed high expression of α(1,2)-linked fucosylated proteins compared to normal ST. The expression of α(1,2)-linked fucosylated monocyte chemoattractant protein 1 (MCP-1)/CCL2 was significantly elevated in RA SF compared with osteoarthritis SF. Depletion of α(1,2)-linked fucosylated proteins in RA SF induced less HMVEC migration and tube formation than occurred in sham-depleted RA SF. We found that blocking FUT1 expression in ECs resulted in decreased MCP-1/CCL2 and RANTES/CCL5 production. Finally, we showed that FUT1 regulates EC migration in response to vascular endothelial cell growth factor. CONCLUSION: Our findings indicate that α(1,2)-linked fucosylation by FUT1 may be an important new target for angiogenic diseases such as RA.
Assuntos
Artrite Reumatoide/complicações , Fucosiltransferases/fisiologia , Neovascularização Patológica/etiologia , Animais , Movimento Celular , Células Cultivadas , Células Endoteliais/fisiologia , Humanos , Camundongos , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
Fucosylation regulates various pathological events in cells. We reported that different levels of CRT (calreticulin) affect the cell adhesion and metastasis of bladder cancer. However, the precise mechanism of tumour metastasis regulated by CRT remains unclear. Using a DNA array, we identified FUT1 (fucosyltransferase 1) as a gene regulated by CRT expression levels. CRT regulated cell adhesion through α1,2-linked fucosylation of ß1 integrin and this modification was catalysed by FUT1. To clarify the roles for FUT1 in bladder cancer, we transfected the human FUT1 gene into CRT-RNAi stable cell lines. FUT1 overexpression in CRT-RNAi cells resulted in increased levels of ß1 integrin fucosylation and rescued cell adhesion to type-I collagen. Treatment with UEA-1 (Ulex europaeus agglutinin-1), a lectin that recognizes FUT1-modified glycosylation structures, did not affect cell adhesion. In contrast, a FUT1-specific fucosidase diminished the activation of ß1 integrin. These results indicated that α1,2-fucosylation of ß1 integrin was not involved in integrin-collagen interaction, but promoted ß1 integrin activation. Moreover, we demonstrated that CRT regulated FUT1 mRNA degradation at the 3'-UTR. In conclusion, the results of the present study suggest that CRT stabilized FUT1 mRNA, thereby leading to an increase in fucosylation of ß1 integrin. Furthermore, increased fucosylation levels activate ß1 integrin, rather than directly modifying the integrin-binding sites.