RESUMEN
Fucosyltransferase 2 (FUT2) gene, which regulates the formation of Histoblood group antigens, could determine the human susceptibility to norovirus. This study aimed to investigate the correlation between FUT2 gene polymorphism and susceptibility to norovirus gastroenteritis in Han Chinese population. A total of 212 children patients with acute gastroenteritis were enrolled. The stool and serum samples were collected respectively. We used the qPCR method to detect the norovirus infection status from the stool samples, and we used serum samples to detect the FUT2 polymorphism. A case-control study was conducted to investigate the three common SNPs polymorphisms (rs281377, rs1047781, and rs601338) of FUT2 gene with sanger sequencing method. The results indicated that the homozygous genotypes and mutant allele of rs1047781 (A385T) would downgrade the risk of norovirus gastroenteritis in Chinese Han population (AA vs. TT, odds ratio [OR] = 0.098, 95% confidence interval [CI] = 0.026-0.370, p = 0.001; AA + AT vs. TT, OR = 0.118. 95% CI = 0.033-0.424, p = 0.001; A vs. T, OR = 0.528, 95% CI = 0.351-0.974, p = 0.002). There were no significant difference of rs281377 (C357T) and rs601338 (G428A) polymorphisms between norovirus positive and norovirus negative groups (p > 0.05). The haplotype T-T-G was less susceptible (OR = 0.49, 95% CI = 0.31-0.79, p = 0.0034) to norovirus infection compared to other haplotypes. Our results investigated the relationship between the FUT2 gene polymorphisms and norovirus susceptibility in Han Chinese population, and firstly revealed that children with homozygous genotypes and mutant alleles of FUT2 rs1047781 (A385T) were less susceptible to norovirus gastroenteritis.
Asunto(s)
Pueblo Asiatico , Infecciones por Caliciviridae , Fucosiltransferasas , Galactósido 2-alfa-L-Fucosiltransferasa , Gastroenteritis , Predisposición Genética a la Enfermedad , Genotipo , Norovirus , Polimorfismo de Nucleótido Simple , Humanos , Fucosiltransferasas/genética , Infecciones por Caliciviridae/genética , Infecciones por Caliciviridae/virología , Infecciones por Caliciviridae/epidemiología , Femenino , Masculino , Gastroenteritis/virología , Gastroenteritis/genética , Estudios de Casos y Controles , Preescolar , Norovirus/genética , Pueblo Asiatico/genética , Lactante , China/epidemiología , Niño , Heces/virología , Alelos , Haplotipos , Pueblos del Este de AsiaRESUMEN
BACKGROUND: Helicobacter pylori infection is a significant pathogen in gastrointestinal diseases. Previous studies have identified single-nucleotide polymorphisms (SNPs) are factors associated with H. pylori infection. Notably, Leb and Sialyl-Lex antigens, regulated by the FUT3 and FUT6 genes, play a crucial role in H. pylori infection. This study aimed to investigate the correlation between FUT3 and FUT6 gene polymorphisms and H. pylori infection in the Han population of northern China. MATERIALS AND METHODS: An immunoturbidimetric assay was employed to detect H. pylori infection, categorizing subjects into infected and noninfected groups. Gene variants were identified through sequencing. Finally, FUT3 and FUT6 gene polymorphisms were analyzed to assess their association with H. pylori infection. RESULTS: The frequency of the T allele (rs778805) and the G allele (rs61147939) in the infection group was significantly higher than that in the noninfection group (63.4% vs. 55.1%, p = 0.045; 55.2% vs. 47.0%, p = 0.042, respectively). In the infection group, the frequency of the AA genotype (rs3745635) in the recessive model, the TT genotype (rs778805) in the recessive model, and the GG genotype (rs61147939) in the recessive model were significantly higher than the noninfection group (5.8% vs. 2.3%, p = 0.042; 41.9% vs. 29.3%, p = 0.022; 34.9% vs. 20.5%, p = 0.0068, respectively). The frequency of the A13 haplotype and the A13/A13 diplotype of the FUT6 gene was significantly higher in the infection group than in the noninfection group (55.56% vs. 46.32%, p = 0.019; 34.94% vs. 20.30%, p = 0.045, respectively). The rs778805-rs17855739-rs28362459-rs3745635 combination was identified as the best interaction model (p < 0.05). CONCLUSIONS: This study suggests that FUT3 and FUT6 gene polymorphisms are significantly associated with H. pylori infection in the Han Chinese from northern China.
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Fucosiltransferasas , Predisposición Genética a la Enfermedad , Infecciones por Helicobacter , Helicobacter pylori , Polimorfismo de Nucleótido Simple , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/microbiología , Fucosiltransferasas/genética , Humanos , Masculino , China/epidemiología , Persona de Mediana Edad , Femenino , Helicobacter pylori/genética , Adulto , Anciano , Adulto Joven , Genotipo , Frecuencia de los GenesRESUMEN
NOTCH1 is a transmembrane receptor interacting with membrane-tethered ligands on opposing cells that mediate the direct cell-cell interaction necessary for many cell fate decisions. Protein O-fucosyltransferase 1 (POFUT1) adds O-fucose to Epidermal Growth Factor (EGF)-like repeats in the NOTCH1 extracellular domain, which is required for trafficking and signaling activation. We previously showed that POFUT1 S162L caused a 90% loss of POFUT1 activity and global developmental defects in a patient; however, the mechanism by which POFUT1 contributes to these symptoms is still unclear. Compared to controls, POFUT1 S162L patient fibroblast cells had an equivalent amount of NOTCH1 on the cell surface but showed a 60% reduction of DLL1 ligand binding and a 70% reduction in JAG1 ligand binding. To determine if the reduction of O-fucose on NOTCH1 in POFUT1 S162L patient fibroblasts was the cause of these effects, we immunopurified endogenous NOTCH1 from control and patient fibroblasts and analyzed O-fucosylation using mass spectral glycoproteomics methods. NOTCH1 EGF8 to EGF12 comprise the ligand binding domain, and O-fucose on EGF8 and EGF12 physically interact with ligands to enhance affinity. Glycoproteomics of NOTCH1 from POFUT1 S162L patient fibroblasts showed WT fucosylation levels at all sites analyzed except for a large decrease at EGF9 and the complete absence of O-fucose at EGF12. Since the loss of O-fucose on EGF12 is known to have significant effects on NOTCH1 activity, this may explain the symptoms observed in the POFUT1 S162L patient.
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Fibroblastos , Fucosa , Fucosiltransferasas , Receptor Notch1 , Humanos , Fibroblastos/metabolismo , Fucosa/metabolismo , Fucosiltransferasas/metabolismo , Fucosiltransferasas/genética , Receptor Notch1/metabolismo , Receptor Notch1/química , Familia de Proteínas EGF/metabolismoRESUMEN
The FUT2 gene encodes an enzyme called α-1,2-fucosyltransferase, which is involved in the formation of blood group antigens AB0(H) and is also involved in the processes of vitamin B12 absorption and its transport between cells. FUT2 gene polymorphisms are associated with vitamin B12 levels in the body. Vitamin B12 deficiency associated with hyperhomocysteinemia is a major risk factor for cardiovascular diseases (CVDs), which are one of the main causes of death in patients after kidney transplantation. The aim of our study was to determine the impact of the rs602662 (G>A) polymorphism of the FUT2 gene on the functionality of transplanted kidneys and the risk of CVD in patients after kidney transplantation. The study included 402 patients treated with immunosuppression (183 patients taking cyclosporine (CsA) and 219 patients taking tacrolimus (TAC)). The analysis of the FUT2 rs602662 (G>A) polymorphism was performed using real-time PCR. Patients with CsA were more likely to be underweight (1.64% vs. 0.91%) and obese (27.87% vs. 15.98%), while those taking TAC were more likely to be of normal weight (39.27%) or overweight (43.84%). No statistically significant differences were observed comparing the mean blood pressure, both systolic and diastolic. The renal profile showed a higher median urea nitrogen concentration in patients with CsA (26.45 mg/dL (20.60-35.40) vs. 22.95 mg/dL (17.60-33.30), p = 0.004). The observed frequency of rs602662 alleles of the FUT2 gene was similar in the analyzed groups. The A allele was present in 43.7% of patients with CsA and 41.1% of those taking TAC (OR = 0.898; 95% CI: 0.678-1.189; p = 0.453). In the group with CsA, the GG genotype was present in 32.2% of patients, the GA in 48.1% and the AA in 19.7%. A similar distribution was obtained in the TAC group: GG-33.8%, GA-50.2%, and AA-16.0%. An association of genotypes containing the G allele with a higher incidence of hypertension was observed. The G allele was present in 65% of people with hypertension and in 56% of patients with normal blood pressure (p = 0.036). Moreover, the evaluation of the renal parameters showed no effect of the FUT2 polymorphism on the risk of organ rejection because the levels of creatinine, eGFR, potassium, and urea nitrogen were prognostic of successful transplantation. Our results suggest that the rs6022662 FUT2 polymorphism may influence the risk of cardiovascular diseases.
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Enfermedades Cardiovasculares , Fucosiltransferasas , Galactósido 2-alfa-L-Fucosiltransferasa , Trasplante de Riñón , Polimorfismo de Nucleótido Simple , Humanos , Fucosiltransferasas/genética , Trasplante de Riñón/efectos adversos , Masculino , Femenino , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/etiología , Persona de Mediana Edad , Adulto , Factores de Riesgo , Predisposición Genética a la Enfermedad , Genotipo , Inmunosupresores/uso terapéutico , Inmunosupresores/efectos adversos , Ciclosporina/uso terapéutico , Ciclosporina/efectos adversos , Tacrolimus/efectos adversos , Tacrolimus/uso terapéuticoRESUMEN
BACKGROUND AND AIMS: The immunosuppressive tumor microenvironment (TME) plays an essential role in cancer progression and immunotherapy response. Despite the considerable advancements in cancer immunotherapy, the limited response to immune checkpoint blockade (ICB) therapies in patients with hepatocellular carcinoma (HCC) remains a major challenge for its clinical implications. Here, we investigated the molecular basis of the protein O-fucosyltransferase 1 (POFUT1) that drives HCC immune evasion and explored a potential therapeutic strategy for enhancing ICB efficacy. METHODS: De novo MYC/Trp53-/- liver tumor and the xenograft tumor models were used to evaluate the function of POFUT1 in immune evasion. Biochemical assays were performed to elucidate the underlying mechanism of POFUT1-mediated immune evasion. RESULTS: We identified POFUT1 as a crucial promoter of immune evasion in liver cancer. Notably, POFUT1 promoted HCC progression and inhibited T-cell infiltration in the xenograft tumor and de novo MYC/Trp53-/- mouse liver tumor models. Mechanistically, we demonstrated that POFUT1 stabilized programmed death ligand 1 (PD-L1) protein by preventing tripartite motif containing 21-mediated PD-L1 ubiquitination and degradation independently of its protein-O-fucosyltransferase activity. In addition, we further demonstrated that PD-L1 was required for the tumor-promoting and immune evasion effects of POFUT1 in HCC. Importantly, inhibition of POFUT1 could synergize with anti-programmed death receptor 1 therapy by remodeling TME in the xenograft tumor mouse model. Clinically, POFUT1 high expression displayed a lower response rate and worse clinical outcome to ICB therapies. CONCLUSIONS: Our findings demonstrate that POFUT1 functions as a novel regulator of tumor immune evasion and inhibition of POFUT1 may be a potential therapeutic strategy to enhance the efficacy of immune therapy in HCC.
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Antígeno B7-H1 , Fucosiltransferasas , Inmunoterapia , Neoplasias Hepáticas , Fucosiltransferasas/metabolismo , Fucosiltransferasas/genética , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/tratamiento farmacológico , Humanos , Ratones , Animales , Antígeno B7-H1/metabolismo , Inmunoterapia/métodos , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Escape del Tumor , Microambiente Tumoral , Evasión Inmune , Línea Celular TumoralAsunto(s)
Sistema del Grupo Sanguíneo ABO , Alelos , Pueblo Asiatico , Fucosiltransferasas , Galactósido 2-alfa-L-Fucosiltransferasa , Fenotipo , Humanos , Fucosiltransferasas/genética , Pueblo Asiatico/genética , Sistema del Grupo Sanguíneo ABO/genética , China , Masculino , Femenino , Pueblos del Este de AsiaRESUMEN
This study aimed to determine the function of LINC00511 in Nod-Like Receptor Pyrin Domain 3 inflammasome-mediated chondrocyte pyroptosis via the regulation of miR-9-5p and FUT 1. Chondrocyte inflammatory injury was induced by treating chondrocytes with LPS. Afterwards, the levels of IL-1ß and IL-18, the expression of NLRP3, ASC, Caspase-1, and GSDMD, cell viability, and LDH activity in chondrocytes were assessed. LINC00511 expression in LPS-treated chondrocytes was detected, and LINC00511 was subsequently silenced to analyse its role in chondrocyte pyroptosis. The subcellular localization of LINC00511 was predicted and verified. Furthermore, the binding relationships between LINC00511 and miR-9-5p and between miR-9-5p and FUT1 were validated. LINC00511 regulated NLRP3 inflammasome-mediated chondrocyte pyroptosis through the miR-9-5p/FUT1 axis. LPS-treated ATDC5 cells exhibited elevated levels of inflammatory injury; increased levels of NLRP3, ASC, Caspase-1, and GSDMD; reduced cell viability; increased LDH activity; and increased LINC00511 expression, while LINC00511 silencing inhibited the NLRP3 inflammasome to restrict LPS-induced chondrocyte pyroptosis. Next, LINC00511 sponged miR-9-5p, which targeted FUT1. Silencing LINC00511 suppressed FUT1 by upregulating miR-9-5p. Additionally, downregulation of miR-9-5p or overexpression of FUT1 neutralized the suppressive effect of LINC00511 knockdown on LPS-induced chondrocyte pyroptosis. Silencing LINC00511 inhibited the NLRP3 inflammasome to quench Caspase-1-dependent chondrocyte pyroptosis in OA by promoting miR-9-5p and downregulating FUT1.
Asunto(s)
Condrocitos , Fucosiltransferasas , MicroARNs , Proteína con Dominio Pirina 3 de la Familia NLR , Piroptosis , ARN Largo no Codificante , Condrocitos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , MicroARNs/genética , MicroARNs/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Inflamasomas/metabolismo , Lipopolisacáridos , Humanos , Línea Celular , Animales , Ratones , Supervivencia Celular , Interleucina-1beta/metabolismo , Interleucina-18/metabolismo , Interleucina-18/genética , Caspasa 1/metabolismo , Caspasa 1/genéticaRESUMEN
OBJECTIVE: This study aims to disclose how loss of fucosyltransferase 2 (Fut2) impacts intestinal inflammation through cGAS-STING pathway that is closely associated with gut microbiota, and which microbial metabolite improves colitis in Fut2 deficiency. METHODS: Chronic colitis was induced in intestinal epithelial Fut2 knock out mice (Fut2â³IEC), whose intestinal inflammation and activity of cGAS-STING pathway were evaluated. 16S rRNA sequencing and metabolomics were performed using intestinal samples. 2-oxindole was used to treat RAW264.7 cells and Fut2â³IEC mice with colitis (Fut2â³IEC-DSS) to investigate the effect of 2-oxindole on cGAS-STING response and intestinal inflammation. RESULTS: Fut2 loss exacerbated chronic colitis in mice, manifested by declined body weight, reduced colon length, increased disease activity index (DAI) and more colon injury in Fut2â³IEC-DSS mice compared with WT-DSS (wild type mice with colitis). Lack of Fut2 promoted activation of cGAS-STING pathway. Fut2 deficiency had a primary impact on colonic microbiota, as shown by alteration of microbial diversity and structure, as well as decreased Lactobacillus. Metabolic structure and tryptophan metabolism in colonic luminal microbiota were also influenced by Fut2 loss. Fut2 deficiency also led to decreased levels of aryl hydrocarbon receptor (AHR) and its ligand 2-oxindole derived from tryptophan metabolism. 2-oxindole compromised cGAS-STING response through activating AHR in macrophages, and protected against intestinal inflammation and overactive cGAS-STING pathway in Fut2â³IEC-DSS mice. CONCLUSION: Fut2 deficiency promotes cGAS-STING pathway through suppressing 2-oxindole-AHR axis, ultimately facilitating the susceptibility to chronic colitis.
Asunto(s)
Colitis , Fucosiltransferasas , Microbioma Gastrointestinal , Proteínas de la Membrana , Ratones Noqueados , Nucleotidiltransferasas , Oxindoles , Transducción de Señal , Animales , Humanos , Masculino , Ratones , Enfermedad Crónica , Colitis/inducido químicamente , Colitis/inmunología , Colon/patología , Colon/inmunología , Colon/metabolismo , Sulfato de Dextran , Modelos Animales de Enfermedad , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Fucosiltransferasas/deficiencia , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Células RAW 264.7 , Receptores de Hidrocarburo de Aril/metabolismo , Receptores de Hidrocarburo de Aril/genéticaRESUMEN
Probiotics have gained significant attention as a potential strategy to improve health by modulating host-microbe interactions, particularly in situations where the normal microbiota has been disrupted. However, evidence regarding their efficacy has been inconsistent, with considerable interindividual variability in response. We aimed to explore whether a common genetic variant that affects the production of mucosal α(1,2)-fucosylated glycans, present in around 20% of the population, could explain the observed interpersonal differences in the persistence of commonly used probiotics. Using a mouse model with varying α(1,2)-fucosylated glycans secretion (Fut2WT or Fut2KO), we examined the abundance and persistence of Bifidobacterium strains (infantis, breve, and bifidum). We observed significant differences in baseline gut microbiota characteristics between Fut2WT and Fut2KO littermates, with Fut2WT mice exhibiting enrichment of species able to utilize α(1,2)-fucosylated glycans. Following antibiotic exposure, only Fut2WT animals showed persistent engraftment of Bifidobacterium infantis, a strain able to internalize α(1,2)-fucosylated glycans, whereas B. breve and B. bifidum, which cannot internalize α(1,2)-fucosylated glycans, did not exhibit this difference. In mice with an intact commensal microbiota, the relationship between secretor status and B. infantis persistence was reversed, with Fut2KO animals showing greater persistence compared to Fut2WT. Our findings suggest that the interplay between a common genetic variation and antibiotic exposure plays a crucial role in determining the dynamics of B. infantis in the recipient gut, which could potentially contribute to the observed variation in response to this commonly used probiotic species.
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Antibacterianos , Fucosiltransferasas , Galactósido 2-alfa-L-Fucosiltransferasa , Microbioma Gastrointestinal , Probióticos , Animales , Ratones , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Probióticos/administración & dosificación , Antibacterianos/farmacología , Bifidobacterium longum subspecies infantis/genética , Bifidobacterium longum subspecies infantis/metabolismo , Polisacáridos/metabolismo , Interacciones Microbiota-Huesped , Ratones Endogámicos C57BL , Ratones Noqueados , Bifidobacterium/genética , Bifidobacterium/metabolismoRESUMEN
3-Fucosyllactose (3-FL), an important fucosylated human milk oligosaccharide in breast milk, offers numerous health benefits to infants. Previously, we metabolically engineered Escherichia coli BL21(DE3) for the in vivo biosynthesis of 3-FL. In this study, we initially optimized culture conditions to double 3-FL production. Competing pathway genes involved in in vivo guanosine 5'-diphosphate-fucose biosynthesis were subsequently inactivated to redirect fluxes toward 3-FL biosynthesis. Next, three promising transporters were evaluated using plasmid-based or chromosomally integrated expression to maximize extracellular 3-FL production. Additionally, through analysis of α1,3-fucosyltransferase (FutM2) structure, we identified Q126 residues as a highly mutable residue in the active site. After site-saturation mutation, the best-performing mutant, FutM2-Q126A, was obtained. Structural analysis and molecular dynamics simulations revealed that small residue replacement positively influenced helical structure generation. Finally, the best strain BD3-A produced 6.91 and 52.1 g/L of 3-FL in a shake-flask and fed-batch cultivations, respectively, highlighting its potential for large-scale industrial applications.
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Escherichia coli , Fucosiltransferasas , Ingeniería Metabólica , Trisacáridos , Escherichia coli/genética , Escherichia coli/metabolismo , Trisacáridos/metabolismo , Trisacáridos/biosíntesis , Trisacáridos/química , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Humanos , OligosacáridosRESUMEN
Genetic engineering plays an essential role in the development of cell lines for biopharmaceutical manufacturing. Advanced gene editing tools can improve both the productivity of recombinant cell lines as well as the quality of therapeutic antibodies. Antibody glycosylation is a critical quality attribute for therapeutic biologics because the glycan patterns on the antibody fragment crystallizable (Fc) region can alter its clinical efficacy and safety as a therapeutic drug. As an example, recombinant antibodies derived from Chinese hamster ovary (CHO) cells are generally highly fucosylated; the absence of α1,6-fucose significantly enhances antibody-dependent cell-mediated cytotoxicity (ADCC) against cancer cells. This chapter describes a protocol applying clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) approach with different formats to disrupt the α-1,6-fucosyltransferase (FUT8) gene and subsequently inhibit α-1,6 fucosylation on antibodies expressed in CHO cells.
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Sistemas CRISPR-Cas , Cricetulus , Fucosa , Fucosiltransferasas , Edición Génica , Células CHO , Animales , Edición Génica/métodos , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Glicosilación , Fucosa/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Cricetinae , HumanosRESUMEN
3-Fucosyllactose (3-FL) is an important fucosylated human milk oligosaccharide (HMO) with biological functions such as promoting immunity and brain development. Therefore, the construction of microbial cell factories is a promising approach to synthesizing 3-FL from renewable feedstocks. In this study, a combinatorial engineering strategy was used to achieve efficient de novo 3-FL production in Escherichia coli. α-1,3-Fucosyltransferase (futM2) from Bacteroides gallinaceum was introduced into E. coli and optimized to create a 3-FL-producing chassis strain. Subsequently, the 3-FL titer increased to 5.2 g/L by improving the utilization of the precursor lactose and down-regulating the endogenous competitive pathways. Furthermore, a synthetic membraneless organelle system based on intrinsically disordered proteins was designed to spatially regulate the pathway enzymes, producing 7.3 g/L 3-FL. The supply of the cofactors NADPH and GTP was also enhanced, after which the 3-FL titer of engineered strain E26 was improved to 8.2 g/L in a shake flask and 10.8 g/L in a 3 L fermenter. In this study, we developed a valuable approach for constructing an efficient 3-FL-producing cell factory and provided a versatile workflow for other chassis cells and HMOs.
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Escherichia coli , Fucosiltransferasas , Ingeniería Metabólica , Trisacáridos , Escherichia coli/genética , Escherichia coli/metabolismo , Trisacáridos/metabolismo , Trisacáridos/biosíntesis , Ingeniería Metabólica/métodos , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Lactosa/metabolismo , Bacteroides/genética , Bacteroides/metabolismo , Fermentación , OligosacáridosRESUMEN
The presence of the blood group H2 antigen on the membrane of red blood cells determines blood type O in individuals and this H2 antigen serves as a precursor to the A and B antigens expressed in blood types A and B, respectively. However, the specific involvement of ABH antigens in skin diseases is unknown. Therefore, we aim to investigate the expression of ABH antigens in skin tissue of patients with atopic dermatitis (AD) and MC903-induced AD-like mice. We demonstrated that the expression of ABH antigen is primarily located in the granular and horny layers of the skin in healthy control individuals. However, in patients with AD, the expression of the ABH antigen was absent or diminished in these layers, while the H2 antigen expression increased in the spinous layers of the affected skin lesions. Then, we investigated the biological function of blood group H antigen mediated by fucosyltransferase 1 (Fut1) in the skin, utilizing an AD mouse model induced by MC903 in wild-type (WT) and Fut1-knockout mice. After the application of MC903, Fut1-deficient mice, with no H2 antigen expression on their skin, exhibited more severe clinical signs, increased ear swelling, and elevated serum IgE levels compared with those of WT mice. Additionally, the MC903-induced thickening of both the epidermis and dermis was more pronounced in Fut1-deficient mice than that in WT mice. Furthermore, Fut1-deficient mice showed a significantly higher production of interleukin-4 (IL-4) and IL-6 in skin lesions compared with that of their WT counterparts. The expression of chemokines, particularly Ccl2 and Ccl8, was notably higher in Fut1-deficient mice compared with those of WT mice. The infiltration of CD4+ T cells, eosinophils, and mast cells into the lesional skin was significantly elevated in Fut1-deficient mice compared with that in WT mice. These findings demonstrate the protective role of H2 antigen expression against AD-like inflammation and highlight its potential therapeutic impact on AD through the regulation of blood group antigens.
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Dermatitis Atópica , Fucosiltransferasas , Galactósido 2-alfa-L-Fucosiltransferasa , Ratones Noqueados , Adulto , Animales , Femenino , Humanos , Masculino , Ratones , Citocinas/metabolismo , Dermatitis Atópica/inmunología , Modelos Animales de Enfermedad , Epidermis/inmunología , Epidermis/patología , Epidermis/metabolismo , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Ratones Endogámicos C57BLRESUMEN
The aim of this study was to identify effective genetic markers for the Antigen Processing Associated Transporter 1 (TAP1), α (1,2) Fucosyltransferase 1 (FUT1), Natural Resistance Associated Macrophage Protein 1 (NRAMP1), Mucin 4 (MUC4) and Mucin 13 (MUC13) diarrhea-resistance genes in the local pig breeds, namely Shanghai white pigs, Fengjing pigs, Shawutou pigs, Meishan pigs and Pudong white pigs, to provide a reference for the characterization of local pig breed resources in Shanghai. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLR) and sequence sequencing were applied to analyze the polymorphisms of the above genes and to explore the effects on the immunity of Shanghai local pig breeds in conjunction with some immunity factors. The results showed that both TAP1 and MUC4 genes had antidiarrheal genotype GG in the five pig breeds, AG and GG genotypes of the FUT1 gene were detected in Pudong white pigs, AA antidiarrheal genes of the NRAMP1 gene were detected in Meishan pigs, the AB type of the NRAMP1 gene was detected in Pudong white pigs, and antidiarrheal genotype GG of the MUC13 gene was only detected in Shanghai white pigs. The MUC13 antidiarrhea genotype GG was only detected in Shanghai white pigs. The TAP1 gene was moderately polymorphic in Shanghai white pigs, Fengjing pigs, Shawutou pigs, Meishan pigs and Pudong white pigs, among which TAP1 in Shanghai white pigs and Shawutou pigs did not satisfy the Hardy-Weinberg equilibrium. The FUT1 gene of Pudong white pigs was in a state of low polymorphism. NRAMP1 of Meishan pigs and Pudong white pigs was in a state of moderate polymorphism, which did not satisfy the Hardy-Weinberg equilibrium. The MUC4 genes of Shanghai white pigs and Pudong white pigs were in a state of low polymorphism, and the MUC4 genes of Fengjing pigs and Shawutou pigs were in a state of moderate polymorphism, and the MUC4 genes of Fengjing pigs and Pudong white pigs did not satisfy the Hardy-Weinberg equilibrium. The MUC13 gene of Shanghai white pigs and Pudong white pigs was in a state of moderate polymorphism. Meishan pigs had higher levels of IL-2, IL-10, IgG and TNF-α, and Pudong white pigs had higher levels of IL-12 than the other pigs. The level of interleukin 12 (IL-12) was significantly higher in the AA genotype of the MUC13 gene of Shanghai white pigs than in the AG genotype. The indicator of tumor necrosis factor alpha (TNF-α) in the AA genotype of the TAP1 gene of Fengjing pigs was significantly higher than that of the GG and AG genotypes. The indicator of IL-12 in the AG genotype of the Shawutou pig TAP1 gene was significantly higher than that of the GG genotype. The level of TNF-α in the AA genotype of the NRAMP1 gene of Meishan pigs was markedly higher than that of the AB genotype. The IL-2 level of the AG type of the FUT1 gene was obviously higher than that of the GG type of Pudong white pigs, the IL-2 level of the AA type of the MUC4 gene was dramatically higher than that of the AG type, and the IgG level of the GG type of the MUC13 gene was apparently higher than that of the AG type. The results of this study are of great significance in guiding the antidiarrhea breeding and molecular selection of Shanghai white pigs, Fengjing pigs, Shawutou pigs, Meishan pigs and Pudong white pigs and laying the foundation for future antidiarrhea breeding of various local pig breeds in Shanghai.
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Diarrea , Animales , Porcinos/genética , China , Diarrea/genética , Diarrea/veterinaria , Fucosiltransferasas/genética , Proteínas de Transporte de Catión/genética , Cruzamiento , Galactósido 2-alfa-L-Fucosiltransferasa , Mucina 4/genética , GenotipoRESUMEN
Chinese hamster ovary (CHO) cells are the commonly used mammalian host system to manufacture recombinant proteins including monoclonal antibodies. However unfavorable non-human glycoprofile displayed on CHO-produced monoclonal antibodies have negative impacts on product quality, pharmacokinetics, and therapeutic efficiency. Glycoengineering such as genetic elimination of genes involved in glycosylation pathway in CHO cells is a viable solution but constrained due to longer timeline and laborious workflow. Here, in this proof-of-concept (PoC) study, we present a novel approach coined CellEDIT to engineer CHO cells by intranuclear delivery of the CRISPR components to single cells using the FluidFM technology. Co-injection of CRISPR system targeting BAX, DHFR, and FUT8 directly into the nucleus of single cells, enabled us to generate triple knockout CHO-K1 cell lines within a short time frame. The proposed technique assures the origin of monoclonality without the requirement of limiting dilution, cell sorting or positive selection. Furthermore, the approach is compatible to develop both single and multiple knockout clones (FUT8, BAX, and DHFR) in CHO cells. Further analyses on single and multiple knockout clones confirmed the targeted genetic disruption and altered protein expression. The knockout CHO-K1 clones showed the persistence of gene editing during the subsequent passages, compatible with serum free chemically defined media and showed equivalent transgene expression like parental clone.
Asunto(s)
Sistemas CRISPR-Cas , Cricetulus , Edición Génica , Células CHO , Animales , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Anticuerpos Monoclonales/genética , Proteínas Recombinantes/genética , Técnicas de Inactivación de Genes/métodos , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Cricetinae , Ingeniería Genética/métodosRESUMEN
Alterations in the glycomic profile are a hallmark of cancer, including colorectal cancer (CRC). While, the glycosylation of glycoproteins and glycolipids has been widely studied for CRC cell lines and tissues, a comprehensive overview of CRC glycomics is still lacking due to the usage of different samples and analytical methods. In this study, we compared glycosylation features of N-, O-glycans, and glycosphingolipid glycans for a set of 22 CRC cell lines, all measured by porous graphitized carbon nano-liquid chromatography-tandem mass spectrometry. An overall, high abundance of (sialyl)Lewis antigens for colon-like cell lines was found, while undifferentiated cell lines showed high expression of H blood group antigens and α2-3/6 sialylation. Moreover, significant associations of glycosylation features were found between the three classes of glycans, such as (sialyl)Lewis and H blood group antigens. Integration of the datasets with transcriptomics data revealed positive correlations between (sialyl)Lewis antigens, the corresponding glycosyltransferase FUT3 and transcription factors CDX1, ETS, HNF1/4A, MECOM, and MYB. This indicates a possible role of these transcription factors in the upregulation of (sialyl)Lewis antigens, particularly on glycosphingolipid glycans, via FUT3/4 expression in colon-like cell lines. In conclusion, our study provides insights into the possible regulation of glycans in CRC and can serve as a guide for the development of diagnostic and therapeutic biomarkers.
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Diferenciación Celular , Neoplasias Colorrectales , Glicoesfingolípidos , Polisacáridos , Humanos , Glicoesfingolípidos/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Polisacáridos/metabolismo , Línea Celular Tumoral , Colon/metabolismo , Glicosilación , Antígenos del Grupo Sanguíneo de Lewis/metabolismo , Fucosiltransferasas/metabolismo , Fucosiltransferasas/genética , Glicómica/métodos , Regulación Neoplásica de la Expresión GénicaRESUMEN
Lacto-N-difucohexaose II (LNDFH II) is a typical fucosylated human milk oligosaccharide and can be enzymatically produced from lacto-N-tetraose (LNT) by a specific α1,3/4-fucosyltransferase from Helicobacter pylori DMS 6709, referred to as FucT14. Previously, we constructed an engineered Escherichia coli BL21(DE3) with a single plasmid for highly efficient biosynthesis of LNT. In this study, two additional plasmids harboring the de novo GDP-L-fucose pathway module and FucT14, respectively, were further introduced to construct the strain for successful biosynthesis of LNDFH II. FucT14 was actively expressed, and the engineered strain produced LNDFH II as the major product, lacto-N-fucopentaose (LNFP) V as the minor product, and a trace amount of LNFP II and 3-fucosyllactose as very minor products. Additional expression of the α1,3-fucosyltransferase FutM1 from a Bacteroidaceae bacterium from the gut metagenome could obviously enhance the LNDFH II biosynthesis. After optimization of induction conditions, the maximum titer reached 3.011 g/L by shake-flask cultivation. During the fed-batch cultivation, LNDFH II was highly efficiently produced with the highest titer of 18.062 g/L and the productivity yield of 0.301 g/L·h.
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Proteínas Bacterianas , Escherichia coli , Fucosiltransferasas , Guanosina Difosfato Fucosa , Ingeniería Metabólica , Humanos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Vías Biosintéticas , Escherichia coli/genética , Escherichia coli/metabolismo , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Guanosina Difosfato Fucosa/metabolismo , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Helicobacter pylori/enzimología , Oligosacáridos/metabolismo , Oligosacáridos/biosíntesisRESUMEN
Milk glycans play key roles in shaping and maintaining a healthy infant gut microbiota. Core fucosylation catalyzed by fucosyltransferase (Fut8) is the major glycosylation pattern on human milk N-glycan, which was crucial for promoting the colonization and dominant growth of Bifidobacterium and Lactobacillus spp. in neonates. However, the influence of core-fucose in breast milk on the establishment of early-life immune tolerance remains poorly characterized. In this study, we found that the deficiency of core-fucose in the milk of maternal mice caused by Fut8 gene heterozygosity (Fut8+/-) resulted in poor immune tolerance towards the ovalbumin (OVA) challenge, accompanied by a reduced proportion of intestinal RORγt+ Treg cells and the abundance of Lactobacillus spp., especially L. reuteri and L. johnsonii, in their breast-fed neonates. The administration of the L. reuteri and L. johnsonii mixture to neonatal mice compromised the OVA-induced allergy and up-regulated the intestinal RORγt+ Treg cell proportions. However, Lactobacillus mixture supplementation did not alleviate allergic responses in RORγt+ Treg cell-deficient mice caused by Rorc gene heterozygosity (Rorc+/-) post OVA challenge, indicating that the intervention effects depend on the RORγt+ Treg cells. Interestingly, instead of L. reuteri and L. johnsonii, we found that the relative abundance of another Lactobacillus spp., L. murinus, in the gut of the offspring mice was significantly promoted by intervention, which showed enhancing effects on the proliferation of splenic and intestinal RORγt+ Treg cells in in vitro studies. The above results indicate that core fucosylation of breast milk N-glycans is beneficial for the establishment of RORγt+ Treg cell mediated early-life immune tolerance through the manipulation of symbiotic bacteria in mice.
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Microbioma Gastrointestinal , Tolerancia Inmunológica , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Polisacáridos , Linfocitos T Reguladores , Animales , Linfocitos T Reguladores/inmunología , Ratones , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Femenino , Polisacáridos/metabolismo , Lactobacillus , Fucosiltransferasas/metabolismo , Fucosiltransferasas/genética , Leche Humana/inmunología , Humanos , Fucosa/metabolismo , Animales Recién Nacidos , Ratones Endogámicos C57BL , LecheRESUMEN
Cholangiocarcinoma (CCA) is a common gastrointestinal malignancy characterized by a poor prognosis. Considering its prevalence, exploring its underlying molecular biological mechanisms is of paramount clinical importance. In this study, bioinformatics techniques were utilized to analyze CCA sample data obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. The analysis revealed a notable upregulation in FUT4 expression in CCA samples. To further investigate the functional implications of FUT4, in vivo and in vitro experiments were conducted, which demonstrated that FUT4 overexpression significantly enhances the proliferative and migratory capabilities of tumor cells. Subsequent sequencing analysis unveiled a correlation between FUT4 and epithelial-mesenchymal transition (EMT). Indeed, the pioneering discovery of elevated FUT4 expression in CCA was highlighted in this study. Further investigations into the function of FUT4 in CCA provided initial insights into its role in driving cancer progression via EMT. These findings present promising avenues for the diagnosis and treatment of CCA.[Figure: see text].
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Neoplasias de los Conductos Biliares , Movimiento Celular , Proliferación Celular , Colangiocarcinoma , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal , Fucosiltransferasas , Regulación Neoplásica de la Expresión Génica , Transición Epitelial-Mesenquimal/genética , Colangiocarcinoma/genética , Colangiocarcinoma/patología , Colangiocarcinoma/metabolismo , Humanos , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/patología , Neoplasias de los Conductos Biliares/metabolismo , Línea Celular Tumoral , Animales , Proliferación Celular/genética , Movimiento Celular/genética , Ratones Desnudos , Ratones , Ratones Endogámicos BALB C , Regulación hacia Arriba/genética , MasculinoRESUMEN
Disruption of the glycosylation machinery is a common feature in many types of cancer, and colorectal cancer (CRC) is no exception. Core fucosylation is mediated by the enzyme fucosyltransferase 8 (FucT-8), which catalyzes the addition of α1,6-l-fucose to the innermost GlcNAc residue of N-glycans. We and others have documented the involvement of FucT-8 and core-fucosylated proteins in CRC progression, in which we addressed core fucosylation in the syngeneic CRC model formed by SW480 and SW620 tumor cell lines from the perspective of alterations in their N-glycosylation profile and protein expression as an effect of the knockdown of the FUT8 gene that encodes FucT-8. Using label-free, semiquantitative mass spectrometry (MS) analysis, we found noticeable differences in N-glycosylation patterns in FUT8-knockdown cells, affecting core fucosylation and sialylation, the Hex/HexNAc ratio, and antennarity. Furthermore, stable isotopic labeling of amino acids in cell culture (SILAC)-based proteomic screening detected the alteration of species involved in protein folding, endoplasmic reticulum (ER) and Golgi post-translational stabilization, epithelial polarity, and cellular response to damage and therapy. This data is available via ProteomeXchange with identifier PXD050012. Overall, the results obtained merit further investigation to validate their feasibility as biomarkers of progression and malignization in CRC, as well as their potential usefulness in clinical practice.