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1.
Clin Chim Acta ; 505: 1-5, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32070726

RESUMO

BACKGROUND: The secretor type α(1,2)fucosyltransferase gene (FUT2) is known to be rich in population-specific polymorphisms. However, genetic variations of FUT2 have not been well examined in Latin American populations in which nonsecretors are rare. METHODS: Conventional polymerase chain reactions and direct sequencing were performed to detect single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) of FUT2 in Mexicans including Americans of Mexican ancestry, Puerto Ricans, Caribbeans, and Colombians. FUT2 alleles were determined by cloning into plasmids or PHASE software. The impact of uncharacterized missense SNPs on the enzyme activity were examined by transient transfection assays and estimated by several software programs. RESULTS: Three alleles, Se357, Se, and se428, were common, and the frequency of nonsecretors was relatively low in the studied populations. We also encountered several alleles specific to Africans, Europeans, and South and East Asians including a South Asian-specific sedel. In contrast to the in silico prediction, a transient expression study suggested that both of two missense SNPs, 235G > A and 304G > A, did not impair the enzyme activity. CONCLUSIONS: The allelic polymorphism of FUT2 suggests that the modern Latin American populations were formed via genetic admixture among Native Americans and populations whose ancestors migrated from other continents. In this study, we have observed a discrepancy between in silico and functional analyses for FUT2 for the first time. Therefore, experimental functional analysis is required for evaluation of SNPs of FUT2.


Assuntos
Fucosiltransferases/genética , Alelos , Grupo com Ancestrais do Continente Asiático , Simulação por Computador , Variações do Número de Cópias de DNA , Grupo com Ancestrais do Continente Europeu , Fucosiltransferases/análise , Expressão Gênica , Frequência do Gene , Humanos , Índios Sul-Americanos , América Latina/epidemiologia , Mutação de Sentido Incorreto/genética , Reação em Cadeia da Polimerase , Polimorfismo Genético , Polimorfismo de Nucleotídeo Único
2.
Nat Commun ; 11(1): 973, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080177

RESUMO

Core-fucosylation is an essential biological modification by which a fucose is transferred from GDP-ß-L-fucose to the innermost N-acetylglucosamine residue of N-linked glycans. A single human enzyme α1,6-fucosyltransferase (FUT8) is the only enzyme responsible for this modification via the addition of an α-1,6-linked fucose to N-glycans. To date, the details of substrate recognition and catalysis by FUT8 remain unknown. Here, we report the crystal structure of FUT8 complexed with GDP and a biantennary complex N-glycan (G0), which provides insight into both substrate recognition and catalysis. FUT8 follows an SN2 mechanism and deploys a series of loops and an α-helix which all contribute in forming the binding site. An exosite, formed by one of these loops and an SH3 domain, is responsible for the recognition of branched sugars, making contacts specifically to the α1,3 arm GlcNAc, a feature required for catalysis. This information serves as a framework for inhibitor design, and helps to assess its potential as a therapeutic target.


Assuntos
Fucosiltransferases/química , Fucosiltransferases/metabolismo , Biocatálise , Sequência de Carboidratos , Domínio Catalítico , Cristalografia por Raios X , Glicosilação , Guanosina Difosfato/metabolismo , Humanos , Análise em Microsséries , Modelos Moleculares , Polissacarídeos/química , Polissacarídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Domínios de Homologia de src
4.
Cell Prolif ; 53(2): e12745, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31889361

RESUMO

OBJECTIVES: The transformation of cytotrophoblasts into mesenchymal-like extravillous trophoblasts is necessary for successful embryo implantation, and the inadequate transformation may cause abortion. Epiregulin, which is a new growth factor, plays important roles in the reproductive processes. The glycosylation of many proteins in reproduction processes is critical. Protein O-fucosyltransferase 1 (poFUT1) is the key enzyme for the biosynthesis of O-fucosylation on the specific glycoproteins. Urokinase-type plasminogen activator (uPA) contains O-fucosylated domain on Thr18 . However, the functions of epiregulin and poFUT1 in the trophoblast epithelial-mesenchymal transition (EMT) process, the regulatory mechanism of epiregulin on poFUT1 and the resulting O-fucosylated uPA remain unclear. MATERIALS AND METHODS: We employed ELISA and Western blot to detect serum levels of epiregulin and poFUT1 from non-pregnancy women, pregnancy women and abortion patients. Using two trophoblast cell lines and a mouse pregnancy model, we investigated the underlying mechanisms of epiregulin and poFUT1 in trophoblast EMT process. RESULTS: Serum levels of epiregulin and poFUT1 were higher in pregnant women compared with non-pregnant women, and their levels were significantly decreased in abortion patients compared with pregnant women. The results showed that epiregulin upregulated poFUT1 expression and increased O-fucosylation on uPA, which further activated the PI3K/Akt signalling pathway, facilitating EMT behaviour of trophoblast cells and embryo implantation in the mouse pregnant model. CONCLUSIONS: Level of epiregulin and poFUT1 is lower in abortion patients than early pregnancy women. Epiregulin promotes trophoblast EMT through O-fucosylation on uPA catalysed by poFUT1. Epiregulin and poFUT1 may be suggested as the potential diagnostic biomarkers and useful treatment targets for abortion.


Assuntos
Epirregulina/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Fucosiltransferases/metabolismo , Trofoblastos/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Adulto , Animais , Linhagem Celular , Feminino , Glicosilação , Humanos , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Gravidez , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia
5.
Int J Cancer ; 146(7): 1979-1992, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31411736

RESUMO

Removal of colorectal adenomas is an effective strategy to reduce colorectal cancer (CRC) mortality rates. However, as only a minority of adenomas progress to cancer, such strategies may lead to overtreatment. The present study aimed to characterize adenomas by in-depth molecular profiling, to obtain insights into altered biology associated with the colorectal adenoma-to-carcinoma progression. We obtained low-coverage whole genome sequencing, RNA sequencing and tandem mass spectrometry data for 30 CRCs, 30 adenomas and 18 normal adjacent colon samples. These data were used for DNA copy number aberrations profiling, differential expression, gene set enrichment and gene-dosage effect analysis. Protein expression was independently validated by immunohistochemistry on tissue microarrays and in patient-derived colorectal adenoma organoids. Stroma percentage was determined by digital image analysis of tissue sections. Twenty-four out of 30 adenomas could be unambiguously classified as high risk (n = 9) or low risk (n = 15) of progressing to cancer, based on DNA copy number profiles. Biological processes more prevalent in high-risk than low-risk adenomas were related to proliferation, tumor microenvironment and Notch, Wnt, PI3K/AKT/mTOR and Hedgehog signaling, while metabolic processes and protein secretion were enriched in low-risk adenomas. DNA copy number driven gene-dosage effect in high-risk adenomas and cancers was observed for POFUT1, RPRD1B and EIF6. Increased POFUT1 expression in high-risk adenomas was validated in tissue samples and organoids. High POFUT1 expression was also associated with Notch signaling enrichment and with decreased goblet cells differentiation. In-depth molecular characterization of colorectal adenomas revealed POFUT1 and Notch signaling as potential drivers of tumor progression.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Fucosiltransferases/genética , Proteínas Oncogênicas/genética , Adenoma/genética , Adenoma/metabolismo , Adenoma/patologia , Biomarcadores Tumorais , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patologia , Neoplasias Colorretais/metabolismo , Progressão da Doença , Fucosiltransferases/metabolismo , Humanos , Proteínas Oncogênicas/metabolismo , Reprodutibilidade dos Testes , Microambiente Tumoral
6.
Methods Mol Biol ; 2043: 25-43, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31463900

RESUMO

Metalloproteinases of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin type 1 repeats) superfamily are extensively modified with glycan moieties. Glycosylation occurs as these enzymes are trafficked through the endoplasmic reticulum (ER) and Golgi apparatus on their way to the extracellular space and includes N-linked glycosylation, O-linked fucosylation and C-linked mannosylation. This chapter focuses on O-linked fucose, which is added to properly folded thrombospondin type 1 repeats (TSRs) in the ER by protein O-fucosyltransferase 2 (POFUT2) and elongated to a Glucoseß1-3Fucose disaccharide by ß3-glucosyltransferase (B3GLCT). Knockout of POFUT2 results in embryonic lethality in mice, and inactivating mutations in B3GLCT cause Peters plus syndrome, a congenital disorder of glycosylation in humans. Addition of the disaccharide by POFUT2 and B3GLCT stabilizes folded TSRs, enhancing folding in the ER and secretion efficiency of several ADAMTS proteins from cells. Thus, POFUT2 and B3GLCT both function as an ER quality control pathway for folding of TSRs in ADAMTS proteins. In this chapter we describe in detail the methods developed to analyze secretion defects of ADAMTS proteins upon loss of either POFUT2 or B3GLCT. The methods described include creation of CRISPR/Cas9-mediated knockout cell lines of POFUT2 and B3GLCT and use of these cell lines to analyze and quantify secretion defects of ADAMTS proteins.


Assuntos
Proteínas ADAMTS/metabolismo , Fucose/metabolismo , Fucosiltransferases/genética , Galactosiltransferases/genética , Glucosiltransferases/genética , Proteínas ADAMTS/química , Animais , Retículo Endoplasmático/metabolismo , Fucosiltransferases/metabolismo , Galactosiltransferases/metabolismo , Técnicas de Inativação de Genes , Glucosiltransferases/metabolismo , Glicosilação , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Dobramento de Proteína
7.
Biomed Pharmacother ; 121: 109605, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31706102

RESUMO

Bladder cancer (BC) brings a heavy burden to afflicted patients worldwide. In order to find new diagnostic markers and therapeutic targets for this disease, we investigated the role of a novel lncRNA, AC114812.8, in bladder cancer progression. Clone formation and CCK-8 assays were used to detect the proliferative capacity of the cells, and the transwell assay was used to explore their invasion and migration abilities. Wound healing experiments were also used to detect cell migration. Luciferase reporter assays were used to investigate the interactions between lncRNA, target gene and miRNA. The expression of FUT4 and marker genes related to epithelial-mesenchymal transition was explored through western blot analysis. Our findings revealed that AC114812.8 was significantly upregulated in BC and could markedly facilitate the proliferation, migration, and invasion of bladder cancer cells both in vitro and in vivo. Furthermore, duel-luciferase reporter assay revealed that AC114812.8 could regulate the FUT4 expression level by sponging miR-371b-5p to facilitate BC progression. We detected the levels of EMT-related biomarkers in AC114812.8-overexpressing BC cells by western blot analysis and found that AC114812.8 could promote EMT process. Rescue experiments showed that miR-371b-5p could rescue the effect of AC114812.8 on proliferation and metastasis of BC. Our results suggest that AC114812.8 could be a novel prognostic biomarker and therapeutic target for bladder cancer.


Assuntos
Progressão da Doença , Fucosiltransferases/biossíntese , MicroRNAs/biossíntese , RNA Longo não Codificante/biossíntese , Neoplasias da Bexiga Urinária/metabolismo , Animais , Linhagem Celular Transformada , Linhagem Celular Tumoral , Fucosiltransferases/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , RNA Longo não Codificante/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
8.
Int J Oncol ; 55(5): 1033-1048, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31793656

RESUMO

The formation of distant metastasis resulting from vascular dissemination is one of the leading causes of mortality in non­small cell lung cancer (NSCLC). This metastatic dissemination initiates with the adhesion of circulating cancer cells to the endothelium. The minimal requirement for the binding of leukocytes to endothelial E­selectins and subsequent transmigration is the epitope of the fucosylated glycan, sialyl Lewis x (sLex), attached to specific cell surface glycoproteins. sLex and its isomer sialyl Lewis a (sLea) have been described in NSCLC, but their functional role in cancer cell adhesion to endothelium is still poorly understood. In this study, it was hypothesised that, similarly to leukocytes, sLe glycans play a role in NSCLC cell adhesion to E­selectins. To assess this, paired tumour and normal lung tissue samples from 18 NSCLC patients were analyzed. Immunoblotting and immunohistochemistry assays demonstrated that tumour tissues exhibited significantly stronger reactivity with anti­sLex/sLea antibody and E­selectin chimera than normal tissues (2.2­ and 1.8­fold higher, respectively), as well as a higher immunoreactive score. High sLex/sLea expression was associated with bone metastasis. The overall α1,3­fucosyltransferase (FUT) activity was increased in tumour tissues, along with the mRNA levels of FUT3, FUT6 and FUT7, whereas FUT4 mRNA expression was decreased. The expression of E­selectin ligands exhibited a weak but significant correlation with the FUT3/FUT4 and FUT7/FUT4 ratios. Additionally, carcinoembryonic antigen (CEA) was identified in only 8 of the 18 tumour tissues; CEA­positive tissues exhibited significantly increased sLex/sLea expression. Tumour tissue areas expressing CEA also expressed sLex/sLea and showed reactivity to E­selectin. Blot rolling assays further demonstrated that CEA immunoprecipitates exhibited sustained adhesive interactions with E­selectin­expressing cells, suggesting CEA acts as a functional protein scaffold for E­selectin ligands in NSCLC. In conclusion, this work provides the first demonstration that sLex/sLea are increased in primary NSCLC due to increased α1,3­FUT activity. sLex/sLea is carried by CEA and confers the ability for NSCLC cells to bind E­selectins, and is potentially associated with bone metastasis. This study contributes to identifying potential future diagnostic/prognostic biomarkers and therapeutic targets for lung cancer.


Assuntos
Antígeno Carcinoembrionário/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Selectina E/metabolismo , Fucosiltransferases/metabolismo , Neoplasias Pulmonares/metabolismo , Antígeno Sialil Lewis X/metabolismo , Idoso , Carcinoma Pulmonar de Células não Pequenas/patologia , Adesão Celular/fisiologia , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Polissacarídeos/metabolismo
9.
Exp Oncol ; 41(4): 318-322, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31868335

RESUMO

This study aimed to investigate the effects of hypoxia and serum deprivation on regulation of fucosyltransferase-3 (FUT3) expression in breast cancer cells. MATERIALS AND METHODS: FUT3 expression was evaluated in T47D and MCF7 cells. Transcriptional and protein analysis was performed under hypoxia and serum deprivation conditions after 6 and 24 hours; and after 24 and 48 hours, respectively. RESULTS: In T47D cells, experimental conditions induced a significant decrease in FUT3 expression at both, transcriptional and protein levels, while in MCF7 cells the same conditions induced a significant increase of FUT3 expression. CONCLUSIONS: Regulation of FUT3 expression under hypoxic and serum deprivation conditions may be involved in the acquisition of advantages related to apoptosis resistance and metastasis promotion, according to the intrinsic differences presented by T47D and MCF7 cells.


Assuntos
Neoplasias da Mama/genética , Fucosiltransferases/genética , Regulação Neoplásica da Expressão Gênica , Apoptose , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Células MCF-7 , Soro/metabolismo , Hipóxia Tumoral
10.
Clin Lab ; 65(12)2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31850709

RESUMO

BACKGROUND: A case of a para-Bombay phenotype caused by a compound heterozygous mutation in the FUT1 gene was identified in this study. METHODS: We performed an agglutination examination of anti-H serum and secretor status to assess the presence of soluble blood group substances. Genotyping of ABO and FUT1 genes was also performed. RESULTS: Our results showed the presence of A and H antigens in saliva. Based on these results, the patient in the present case was diagnosed with the para-Bombay A phenotype. Direct DNA sequencing of the ABO gene indicated A1v/O1vgenotype. FUT1 gene sequence analysis revealed that the patient harbored the compound heterozygous mutation, c.881_882delTT (p.Phe294Cysfs*40) and c.658C>T (p.Arg220Cys). CONCLUSIONS: Improper identification of this phenotype may cause inappropriate transfusions because this particular blood group may be mislabeled as group O. Therefore, blood bank staff should be well trained to solve the discrepancy between cell and serum grouping in the para-Bombay phenotype.


Assuntos
Sistema ABO de Grupos Sanguíneos/genética , Fucosiltransferases/genética , Mutação , Idoso , Feminino , Genótipo , Heterozigoto , Humanos , Fenótipo
11.
PLoS Genet ; 15(11): e1008497, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31747390

RESUMO

The lipopolysaccharide O-antigen structure expressed by the European Helicobacter pylori model strain G27 encompasses a trisaccharide, an intervening glucan-heptan and distal Lewis antigens that promote immune escape. However, several gaps still remain in the corresponding biosynthetic pathway. Here, systematic mutagenesis of glycosyltransferase genes in G27 combined with lipopolysaccharide structural analysis, uncovered HP0102 as the trisaccharide fucosyltransferase, HP1283 as the heptan transferase, and HP1578 as the GlcNAc transferase that initiates the synthesis of Lewis antigens onto the heptan motif. Comparative genomic analysis of G27 lipopolysaccharide biosynthetic genes in strains of different ethnic origin revealed that East-Asian strains lack the HP1283/HP1578 genes but contain an additional copy of HP1105 and JHP0562. Further correlation of different lipopolysaccharide structures with corresponding gene contents led us to propose that the second copy of HP1105 and the JHP0562 may function as the GlcNAc and Gal transferase, respectively, to initiate synthesis of the Lewis antigen onto the Glc-Trio-Core in East-Asian strains lacking the HP1283/HP1578 genes. In view of the high gastric cancer rate in East Asia, the absence of the HP1283/HP1578 genes in East-Asian H. pylori strains warrants future studies addressing the role of the lipopolysaccharide heptan in pathogenesis.


Assuntos
Infecções por Helicobacter/genética , Lipopolissacarídeos/genética , Antígenos O/genética , Neoplasias Gástricas/genética , Grupo com Ancestrais do Continente Asiático , Fucosiltransferases/genética , Fucosiltransferases/imunologia , Glucanos/genética , Glicosiltransferases/genética , Glicosiltransferases/imunologia , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/genética , Helicobacter pylori/imunologia , Helicobacter pylori/patogenicidade , Humanos , Antígenos do Grupo Sanguíneo de Lewis/genética , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Mutagênese , Antígenos O/imunologia , Neoplasias Gástricas/epidemiologia , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/patologia
12.
J Korean Med Sci ; 34(39): e258, 2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31602828

RESUMO

Para-Bombay phenotypes are rare blood groups that have inherent defects in producing H antigens associated with FUT1 and/or FUT2. We report the first case of para-Bombay blood type in a Southeast Asian patient admitted at a tertiary hospital in Korea. A 23-year-old Indonesian man presented to the hospital with fever and was diagnosed with a disseminated nontuberculous mycobacterium infection and anemia. During blood group typing for blood transfusion, cell typing showed no agglutination with both anti-A and anti-B reagents. Serum typing showed strong reactivity against B cells and trace agglutination pattern with A1 cells. His red blood cells failed to react with anti-H reagents. Direct sequencing of FUT1 and FUT2 revealed a missense variation, c.328G>A (p.Ala110Thr, rs56342683, FUT1*01W.02), and a synonymous variant, c.390C>T (p.Asn130=, rs281377, Se357), respectively. This highlights the need for both forward and reverse grouping.


Assuntos
Sistema ABO de Grupos Sanguíneos/genética , Fucosiltransferases/genética , Grupo com Ancestrais do Continente Asiático/genética , Transfusão de Sangue , Humanos , Indonésia , Masculino , Mutação de Sentido Incorreto , Infecções por Mycobacterium não Tuberculosas/diagnóstico , República da Coreia , Análise de Sequência de DNA , Centros de Atenção Terciária , Adulto Jovem
13.
Oncol Rep ; 42(6): 2473-2485, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31638246

RESUMO

In the current era of precision medicine, there is a general consensus that the anatomical site is an important factor in the management of colorectal cancer (CRC). To investigate the underlying molecular mechanisms between proximal and distal CRC and to identify the responsible genes, we analyzed the gene expression patterns of colorectal tumors from two microarray datasets, GSE39582 and GSE14333, on the NCBI Gene Expression Omnibus and the RNA­seq data from TCGA. Weighted coexpression network analysis (WGCNA) was applied to construct a gene coexpression network. The red module in GSE39582 and the dark­gray module from the TCGA dataset were found to be highly correlated with the anatomical site of CRC. A total of 12 hub genes were found in two datasets, 2 of which PLAG1 like zinc finger 2 (PLAGL2) and protein O­fucosyltransferase 1 (POFUT1) were common and upregulated in tumor samples in CRC. The module with the highest correlation provided references that will help to characterize the difference between left­sided and right­sided CRC. The survival analysis of PLAGL2 and POFUT1 expression revealed differences between proximal and distal CRC. Gene set enrichment analysis based on those two genes provided similar results: GPI anchor biosynthesis and peroxisome and selenoamino acid metabolism. PLAGL2 and POFUT1, which have the highest correlation with tumor location, may serve as biomarkers and therapeutic targets for the precise diagnosis and treatment of CRC in the future.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Proteínas de Ligação a DNA/genética , Fucosiltransferases/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Colo/patologia , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/mortalidade , Biologia Computacional , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Conjuntos de Dados como Assunto , Feminino , Fucosiltransferases/antagonistas & inibidores , Fucosiltransferases/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/genética , Humanos , Masculino , Medicina de Precisão/métodos , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Reto/patologia , Análise de Sobrevida , Análise Serial de Tecidos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Regulação para Cima
14.
Int J Mol Sci ; 20(18)2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31500188

RESUMO

Past work has shown that the protein O-fucosyltransferase 1 (POFUT1) is involved in mammal myogenic differentiation program. Pofut1 knockdown (Po -) in murine C2C12 cells leads to numerous elongated and thin myotubes, suggesting significant defects in secondary fusion. Among the few pathways involved in this process, NFATc2/IL-4 is described as the major one. To unravel the impact of POFUT1 on secondary fusion, we used wild-type (WT) C2C12 and Po - cell lines to follow Myf6, Nfatc2, Il-4 and Il-4rα expressions during a 120 h myogenic differentiation time course. Secreted IL-4 was quantified by ELISA. IL-4Rα expression and its labeling on myogenic cell types were investigated by Western blot and immunofluorescence, respectively. Phenotypic observations of cells treated with IL-4Rα blocking antibody were performed. In Po -, we found a decrease in nuclei number per myotube and a downexpression of Myf6. The observed downregulation of Nfatc2 is correlated to a diminution of secreted IL-4 and to the low level of IL-4Rα for reserve cells. Neutralization of IL-4Rα on WT C2C12 promotes myonuclear accretion defects, similarly to those identified in Po -. Thus, POFUT1 could be a new controller of myotube growth during myogenesis, especially through NFATc2/IL-4 signaling pathway.


Assuntos
Fucosiltransferases/genética , Regulação da Expressão Gênica , Interleucina-4/metabolismo , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Fatores de Transcrição NFATC/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular/genética , Linhagem Celular , Técnicas de Silenciamento de Genes , Camundongos , Fatores de Transcrição NFATC/genética , Receptores Notch/metabolismo
15.
Anal Sci ; 35(12): 1333-1340, 2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31423004

RESUMO

Human antithrombin (AT) has two isoforms of which the predominant α-form is glycosylated on all four possible glycosylation sites and the lower abundant ß-isoform lacks the oligosaccharide on Asn135. The main oligosaccharide structure of human AT consists of biantennary complex-type oligosaccharides lacking a core fucose. Generally, Chinese hamster ovary (CHO) cells produce recombinant human AT (rhAT) with core-fucosylated oligosaccharides. However, rhAT lacking core-fucose oligosaccharides can be produced by POTELLIGENT® technology, which uses FUT8 knockout CHO cells in production. The rhAT has more variable glycan structures, such as tetra-antennary complex type, high-mannose type, and mannose 6-phosphate species as minor components compared to plasma-derived human AT (phAT). In addition, the site-specific glycan profile was different between two ATs. We evaluated the effect of these properties on efficacy and safety based on a comparison of rhAT made by that technology with phAT in terms of their respective oligosaccharide structures, site-specific oligosaccharide profiles, and the ratio of α- and ß-forms. Although some structural differences were found between the rhAT and phAT, we concluded that these differences have no significant effect on the efficacy and safety of rhAT.


Assuntos
Antitrombinas/química , Antitrombinas/metabolismo , Engenharia Genética/métodos , Oligossacarídeos/química , Plasma/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fucosiltransferases/deficiência , Fucosiltransferases/genética , Técnicas de Inativação de Genes , Glicosilação , Humanos , Proteínas Recombinantes/genética
16.
Genetics ; 213(2): 491-500, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31371405

RESUMO

Axon regeneration following neuronal injury is an important repair mechanism that is not well understood at present. In Caenorhabditis elegans, axon regeneration is regulated by DDR-2, a receptor tyrosine kinase (RTK) that contains a discoidin domain and modulates the Met-like SVH-2 RTK-JNK MAP kinase signaling pathway. Here, we describe the svh-10/sqv-3 and svh-11 genes, which encode components of a conserved glycosylation pathway, and show that they modulate axon regeneration in C . elegans Overexpression of svh-2, but not of ddr-2, can suppress the axon regeneration defect observed in svh-11 mutants, suggesting that SVH-11 functions between DDR-2 and SVH-2 in this glycosylation pathway. Furthermore, we found that DDR-2 is N-glycosylated at the Asn-141 residue located in its discoidin domain, and mutation of this residue caused an axon regeneration defect. These findings indicate that N-linked glycosylation plays an important role in axon regeneration in C. elegans.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Receptor com Domínio Discoidina 2/genética , Fucosiltransferases/genética , Regeneração Nervosa/genética , Receptores Proteína Tirosina Quinases/genética , Animais , Axônios/metabolismo , Axônios/fisiologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Receptores com Domínio Discoidina/genética , Glicosilação , Sistema de Sinalização das MAP Quinases/genética , Mutação , Neurônios/metabolismo
18.
Braz J Med Biol Res ; 52(8): e8522, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31365696

RESUMO

Pancreaticobiliary maljunction (PBM) is associated with high risk of epithelial atypical growth and malignant transformation of the bile duct or gallbladder. However, overall changes in genetic expression have not been examined in children with PBM. Genome-wide expression was analyzed using peripheral blood samples from 10 children with PBM and 15 pediatric controls. Differentially expressed genes (DEGs) were identified using microarray. Bioinformatics analysis was conducted using Gene Ontology and KEGG analyses. The top 5 in the up-regulated genes in PBM were verified with qRT-PCR. Receiver operator characteristic curve analysis was conducted to evaluate the predictive accuracy of selected genes for PBM. The microarray experiments identified a total of 876 DEGs in PBM, among which 530 were up-regulated and the remaining 346 were down-regulated. Verification of the top 5 up-regulated genes (TYMS, MYBPC1, FUT1, XAGE2, and GREB1L) by qRT-PCR confirmed the up-regulation of MYBPC1 and FUT1. Receiver operating characteristic curve analysis suggested that FUT1 and MYBPC1 up-regulation could be used to predict PBM, with the area under the curve of 0.873 (95%CI=0.735-1.000) and 0.960 (95%CI=0.891-1.000), respectively. FUT1 and MYBPC1 were up-regulated in children with PBM, and could be used as potential biomarkers for PBM.


Assuntos
Ductos Biliares/anormalidades , Proteínas de Transporte/genética , Fucosiltransferases/genética , Perfilação da Expressão Gênica , Ductos Pancreáticos/anormalidades , Regulação para Cima/genética , Neoplasias dos Ductos Biliares/etiologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Dilatação Patológica/complicações , Dilatação Patológica/congênito , Feminino , Neoplasias da Vesícula Biliar/etiologia , Humanos , Lactente , Masculino , Análise em Microsséries
19.
Artigo em Inglês | MEDLINE | ID: mdl-31334132

RESUMO

Thrombospondin type I repeat (TSR) domains are commonly O-fucosylated by protein O-fucosyltransferase 2 (PoFUT2), and this modification is required for optimal folding and secretion of TSR-containing proteins. The human malaria parasite Plasmodium falciparum expresses proteins containing TSR domains, such as the thrombospondin-related anonymous protein (TRAP) and circumsporozoite surface protein (CSP), which are O-fucosylated. TRAP and CSP are present on the surface of sporozoites and play essential roles in mosquito and human host invasion processes during the transmission stages. Here, we have generated PoFUT2 null-mutant P. falciparum and Plasmodium berghei (rodent) malaria parasites and, by phenotyping them throughout their complete life cycle, we show that PoFUT2 disruption does not affect the growth through the mosquito stages for both species. However, contrary to what has been described previously by others, P. berghei PoFUT2 null mutant sporozoites showed no deleterious motility phenotypes and successfully established blood stage infection in mice. This unexpected result indicates that the importance of O-fucosylation of TSR domains may differ between human and RODENT malaria parasites; complicating our understanding of glycosylation modifications in malaria biology.


Assuntos
Fucosiltransferases/metabolismo , Plasmodium berghei/enzimologia , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/metabolismo , Animais , Linhagem Celular , Culicidae/parasitologia , Modelos Animais de Doenças , Fucosiltransferases/genética , Glicosilação , Humanos , Estágios do Ciclo de Vida , Malária/parasitologia , Malária/transmissão , Malária Falciparum/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Oocistos/metabolismo , Plasmodium berghei/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Esporozoítos/enzimologia , Esporozoítos/genética , Esporozoítos/crescimento & desenvolvimento , Esporozoítos/metabolismo
20.
Cancer Biomark ; 25(4): 303-311, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31306109

RESUMO

BACKGROUND AND OBJECTIVE: Fucosyltranferase 8 (FUT8), which catalyzes core fucosylation of glycopeptides, plays important roles in cancer development. In this study, we aimed to explore the influence of FUT8 expression on migration ability of human breast cancer cells and its potential mechanisms. METHODS: The core fucosylation levels in normal and FUT8 deficient MCF-7 cells were analyzed by lectin LCA blots. Then, the cell adhesion assay, transwell and wound healing experiments were conducted. The phosphorylation of FAK and core fucosylation of E-cadherin and its downstream integrins in the FAK/integrin pathway were measured. Moreover, the expression levels of nuclear ß-catenin, MMP-2, and MMP-9 were also measured. RESULTS: The core fucosylation levels were significantly reduced by inhibited FUT8. FUT8 deficiency suppressed the adhesion, migration and invasion of MCF-7 cells; the potential mechanisms might involve three aspects. FUT8 deficiency inhibited FAK/integrin pathway by suppressing core fucosylation of E-cadherin. In addition, FUT8 deficiency reduced nuclear ß-catenin accumulation. The suppression of MMP-2 and MMP-9 expression also accounted for FUT8 deficiency inhibiting breast cancer cells migration. CONCLUSIONS: FUT8 deficiency suppressed migration of MCF-7 cells by impacting core fucosylation of E-cadherin and the downstream FAK/integrin pathway. Therefore, FUT8 is a potential biomarker for breast cancer detection and treatment.


Assuntos
Neoplasias da Mama/genética , Fucosiltransferases/deficiência , Integrinas/genética , Neoplasias da Mama/patologia , Movimento Celular , Feminino , Humanos
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