RESUMO
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.
Assuntos
Fibroblastos , Fucose , Fucosiltransferases , Receptor Notch1 , Humanos , Fibroblastos/metabolismo , Fucose/metabolismo , Fucosiltransferases/metabolismo , Fucosiltransferases/genética , Receptor Notch1/metabolismo , Receptor Notch1/química , Família de Proteínas EGF/metabolismoRESUMO
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.
Assuntos
Escherichia coli , Fucosiltransferases , Engenharia Metabólica , Trissacarídeos , Escherichia coli/genética , Escherichia coli/metabolismo , Trissacarídeos/metabolismo , Trissacarídeos/biossíntese , Trissacarídeos/química , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Humanos , OligossacarídeosRESUMO
The fucosylation of glycoproteins regulates diverse physiological processes. Inhibitors that can control cellular levels of protein fucosylation have consequently emerged as being of high interest. One area where inhibitors of fucosylation have gained significant attention is in the production of afucosylated antibodies, which exhibit superior antibody-dependent cell cytotoxicity as compared to their fucosylated counterparts. Here, we describe ß-carbafucose, a fucose derivative in which the endocyclic ring oxygen is replaced by a methylene group, and show that it acts as a potent metabolic inhibitor within cells to antagonize protein fucosylation. ß-carbafucose is assimilated by the fucose salvage pathway to form GDP-carbafucose which, due to its being unable to form the oxocarbenium ion-like transition states used by fucosyltransferases, is an incompetent substrate for these enzymes. ß-carbafucose treatment of a CHO cell line used for high-level production of the therapeutic antibody Herceptin leads to dose-dependent reductions in core fucosylation without affecting cell growth or antibody production. Mass spectrometry analyses of the intact antibody and N-glycans show that ß-carbafucose is not incorporated into the antibody N-glycans at detectable levels. We expect that ß-carbafucose will serve as a useful research tool for the community and may find immediate application for the rapid production of afucosylated antibodies for therapeutic purposes.
Assuntos
Cricetulus , Fucose , Fucose/metabolismo , Animais , Células CHO , Glicosilação , Humanos , Trastuzumab/farmacologia , Trastuzumab/metabolismo , Fucosiltransferases/metabolismo , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacosRESUMO
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.
Assuntos
Antígeno B7-H1 , Fucosiltransferases , Imunoterapia , Neoplasias Hepáticas , Fucosiltransferases/metabolismo , Fucosiltransferases/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/tratamento farmacológico , Humanos , Camundongos , Animais , Antígeno B7-H1/metabolismo , Imunoterapia/métodos , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Evasão Tumoral , Microambiente Tumoral , Evasão da Resposta Imune , Linhagem Celular TumoralRESUMO
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.
Assuntos
Dermatite Atópica , Fucosiltransferases , Galactosídeo 2-alfa-L-Fucosiltransferase , Camundongos Knockout , Adulto , Animais , Feminino , Humanos , Masculino , Camundongos , Citocinas/metabolismo , Dermatite Atópica/imunologia , Modelos Animais de Doenças , Epiderme/imunologia , Epiderme/patologia , Epiderme/metabolismo , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Escherichia coli , Fucosiltransferases , Engenharia Metabólica , Trissacarídeos , Escherichia coli/genética , Escherichia coli/metabolismo , Trissacarídeos/metabolismo , Trissacarídeos/biossíntese , Engenharia Metabólica/métodos , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Lactose/metabolismo , Bacteroides/genética , Bacteroides/metabolismo , Fermentação , OligossacarídeosRESUMO
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.
Assuntos
Antibacterianos , Fucosiltransferases , Galactosídeo 2-alfa-L-Fucosiltransferase , Microbioma Gastrointestinal , Probióticos , Animais , Camundongos , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Probióticos/administração & dosagem , Antibacterianos/farmacologia , Bifidobacterium longum subspecies infantis/genética , Bifidobacterium longum subspecies infantis/metabolismo , Polissacarídeos/metabolismo , Interações entre Hospedeiro e Microrganismos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Bifidobacterium/genética , Bifidobacterium/metabolismoRESUMO
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.
Assuntos
Condrócitos , Fucosiltransferases , MicroRNAs , Proteína 3 que Contém Domínio de Pirina da Família NLR , Piroptose , RNA Longo não Codificante , Condrócitos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Inflamassomos/metabolismo , Lipopolissacarídeos , Humanos , Linhagem Celular , Animais , Camundongos , Sobrevivência Celular , Interleucina-1beta/metabolismo , Interleucina-18/metabolismo , Interleucina-18/genética , Caspase 1/metabolismo , Caspase 1/genéticaRESUMO
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.
Assuntos
Colite , Fucosiltransferases , Microbioma Gastrointestinal , Proteínas de Membrana , Camundongos Knockout , Nucleotidiltransferases , Oxindóis , Transdução de Sinais , Animais , Humanos , Masculino , Camundongos , Doença Crônica , Colite/induzido quimicamente , Colite/imunologia , Colo/patologia , Colo/imunologia , Colo/metabolismo , Sulfato de Dextrana , Modelos Animais de Doenças , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Fucosiltransferases/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Células RAW 264.7 , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Hidrocarboneto Arílico/genéticaRESUMO
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.
Assuntos
Sistemas CRISPR-Cas , Cricetulus , Fucose , Fucosiltransferases , Edição de Genes , Células CHO , Animais , Edição de Genes/métodos , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Glicosilação , Fucose/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Cricetinae , HumanosRESUMO
As a member of glycosyltransferases, fucosyltransferase 8 (FUT8) is essential to core fucosylation and has been considered as a potential therapeutic target for malignant tumors, including colorectal cancer (CRC). Based on the identification of key binding residues and probable conformation of FUT8, an integrated strategy that combines virtual screening and chemical optimization was carried out and compound 15 was identified as a potent FUT8 inhibitor with novel chemical structure and in vitro antitumor activity. Moreover, chemical pulldown experiments and binding assays confirmed that compound 15 selectively bound to FUT8. In vivo, compound 15 showed promising anti-CRC effects in SW480 xenografts. These data support that compound 15 is a potential FUT8 inhibitor for CRC treatment and deserve further optimization studies.
Assuntos
Antineoplásicos , Neoplasias Colorretais , Descoberta de Drogas , Inibidores Enzimáticos , Fucosiltransferases , Fucosiltransferases/antagonistas & inibidores , Fucosiltransferases/metabolismo , Humanos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Animais , Relação Estrutura-Atividade , Camundongos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Estrutura Molecular , Ensaios de Seleção de Medicamentos Antitumorais , Relação Dose-Resposta a Droga , Proliferação de Células/efeitos dos fármacos , Camundongos Nus , Linhagem Celular Tumoral , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Neoplasias Experimentais/metabolismo , Simulação de Acoplamento MolecularRESUMO
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.
Assuntos
Diferenciação Celular , Neoplasias Colorretais , Glicoesfingolipídeos , Polissacarídeos , Humanos , Glicoesfingolipídeos/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Polissacarídeos/metabolismo , Linhagem Celular Tumoral , Colo/metabolismo , Glicosilação , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Fucosiltransferases/metabolismo , Fucosiltransferases/genética , Glicômica/métodos , Regulação Neoplásica da Expressão GênicaRESUMO
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.
Assuntos
Sistemas CRISPR-Cas , Cricetulus , Edição de Genes , Células CHO , Animais , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Anticorpos Monoclonais/genética , Proteínas Recombinantes/genética , Técnicas de Inativação de Genes/métodos , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Cricetinae , Engenharia Genética/métodosRESUMO
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.
Assuntos
Proteínas de Bactérias , Escherichia coli , Fucosiltransferases , Guanosina Difosfato Fucose , Engenharia Metabólica , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Escherichia coli/genética , Escherichia coli/metabolismo , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Guanosina Difosfato Fucose/metabolismo , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Helicobacter pylori/enzimologia , Oligossacarídeos/metabolismo , Oligossacarídeos/biossínteseRESUMO
SEMA7A belongs to the Semaphorin family and is involved in the oncogenesis and tumor progression. Aberrant glycosylation has been intricately linked with immune escape and tumor growth. SEMA7A is a highly glycosylated protein with five glycosylated sites. The underlying mechanisms of SEMA7A glycosylation and its contribution to immunosuppression and tumorigenesis are unclear. Here, we identify overexpression and aberrant N-glycosylation of SEMA7A in head and neck squamous cell carcinoma, and elucidate fucosyltransferase FUT8 catalyzes aberrant core fucosylation in SEMA7A at N-linked oligosaccharides (Asn 105, 157, 258, 330, and 602) via a direct proteinâprotein interaction. A glycosylated statue of SEMA7A is necessary for its intra-cellular trafficking from the cytoplasm to the cytomembrane. Cytokine EGF triggers SEMA7A N-glycosylation through increasing the binding affinity of SEMA7A toward FUT8, whereas TGF-ß1 promotes abnormal glycosylation of SEMA7A via induction of epithelial-mesenchymal transition. Aberrant N-glycosylation of SEMA7A leads to the differentiation of CD8+ T cells along a trajectory toward an exhausted state, thus shaping an immunosuppressive microenvironment and being resistant immunogenic cell death. Deglycosylation of SEMA7A significantly improves the clinical outcome of EGFR-targeted and anti-PD-L1-based immunotherapy. Finally, we also define RBM4, a splice regulator, as a downstream effector of glycosylated SEMA7A and a pivotal mediator of PD-L1 alternative splicing. These findings suggest that targeting FUT8-SEMA7A axis might be a promising strategy for improving antitumor responses in head and neck squamous cell carcinoma patients.
Assuntos
Neoplasias de Cabeça e Pescoço , Semaforinas , Humanos , Glicosilação , Carcinoma de Células Escamosas de Cabeça e Pescoço , Linfócitos T CD8-Positivos/metabolismo , Fucosiltransferases/metabolismo , Microambiente Tumoral , Proteínas de Ligação a RNA/metabolismo , Antígenos CD/metabolismo , Semaforinas/metabolismo , Proteínas Ligadas por GPI/metabolismoRESUMO
Fucosyltransferase 8 (Fut8) and core fucosylation play critical roles in regulating various biological processes, including immune response, signal transduction, proteasomal degradation, and energy metabolism. However, the function and underlying mechanism of Fut8 and core fucosylation in regulating adult neurogenesis remains unknown. We have shown that Fut8 and core fucosylation display dynamic features during the differentiation of adult neural stem/progenitor cells (aNSPCs) and postnatal brain development. Fut8 depletion reduces the proliferation of aNSPCs and inhibits neuronal differentiation of aNSPCs in vitro and in vivo, respectively. Additionally, Fut8 deficiency impairs learning and memory in mice. Mechanistically, Fut8 directly interacts with integrin α6 (Itga6), an upstream regulator of the PI3k-Akt signaling pathway, and catalyzes core fucosylation of Itga6. Deletion of Fut8 enhances the ubiquitination of Itga6 by promoting the binding of ubiquitin ligase Trim21 to Itga6. Low levels of Itga6 inhibit the activity of the PI3K/Akt signaling pathway. Moreover, the Akt agonist SC79 can rescue neurogenic and behavioral deficits caused by Fut8 deficiency. In summary, our study uncovers an essential function of Fut8 and core fucosylation in regulating adult neurogenesis and sheds light on the underlying mechanisms.
Assuntos
Cognição , Fucosiltransferases , Integrina alfa6 , Neurogênese , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Animais , Fucosiltransferases/metabolismo , Fucosiltransferases/genética , Neurogênese/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Integrina alfa6/metabolismo , Integrina alfa6/genética , Cognição/fisiologia , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Diferenciação Celular , Proliferação de Células , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
2'-Fucosyllactose (2'-FL), one of the major human milk oligosaccharides, was produced in several engineered microorganisms. However, the low solubility of α-1,2-fucosyltransferase (α1,2-FucT) often becomes a bottleneck to produce maximum amount of 2'-FL in the microorganisms. To overcome this solubility issue, the following studies were conducted to improve the soluble expression of α1,2-FucT. Initially, hydrophobic amino acids in the hydrophilic region of the 6 α-helices were mutated, adhering to the α-helix rule. Subsequently, gfp11 was fused to the C-terminal of futC gene encoding α1,2-FucT (FutC), enabling selection of high-fluorescence mutants through split-GFP. Each mutant library was screened via fluorescence activated cell sorting (FACS) to separate soluble mutants for high-throughput screening. As a result, L80C single mutant and A121D/P124A/L125R triple mutant were found, and a combined quadruple mutant was created. Furthermore, we combined mutations of conserved sequences (Q150H/C151R/Q239S) of FutC, which showed positive effects in the previous studies from our lab, with the above quadruple mutants (L80C/A121D/P124A/L125R). The resulting strain produced approximately 3.4-fold higher 2'-FL titer than that of the wild-type, suggesting that the conserved sequence mutations are an independent subset of the mutations that further improve the solubility of the target protein acquired by random mutagenesis using split-GFP.
Assuntos
Escherichia coli , Citometria de Fluxo , Fucosiltransferases , Proteínas de Fluorescência Verde , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Solubilidade , Trissacarídeos/metabolismo , Galactosídeo 2-alfa-L-Fucosiltransferase , Mutação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
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.
Assuntos
Microbioma Gastrointestinal , Tolerância Imunológica , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares , Polissacarídeos , Linfócitos T Reguladores , Animais , Linfócitos T Reguladores/imunologia , Camundongos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Feminino , Polissacarídeos/metabolismo , Lactobacillus , Fucosiltransferases/metabolismo , Fucosiltransferases/genética , Leite Humano/imunologia , Humanos , Fucose/metabolismo , Animais Recém-Nascidos , Camundongos Endogâmicos C57BL , LeiteRESUMO
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].
Assuntos
Neoplasias dos Ductos Biliares , Movimento Celular , Proliferação de Células , Colangiocarcinoma , Progressão da Doença , Transição Epitelial-Mesenquimal , Fucosiltransferases , Regulação Neoplásica da Expressão Gênica , Transição Epitelial-Mesenquimal/genética , Colangiocarcinoma/genética , Colangiocarcinoma/patologia , Colangiocarcinoma/metabolismo , Humanos , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Neoplasias dos Ductos Biliares/metabolismo , Linhagem Celular Tumoral , Animais , Proliferação de Células/genética , Movimento Celular/genética , Camundongos Nus , Camundongos , Camundongos Endogâmicos BALB C , Regulação para Cima/genética , MasculinoRESUMO
Lacto-N-fucopentaose V (LNFP V) is a typical human milk pentasaccharide. Multi-enzymatic in vitro synthesis of LNFP V from lactose was reported, however, microbial cell factory approach to LNFP V production has not been reported yet. In this study, the biosynthetic pathway of LNFP V was examined in Escherichia coli. The previously constructed E. coli efficiently producing lacto-N-tetraose was used as the starting strain. GDP-fucose pathway module and a regio-specific glycosyltransferase with α1,3-fucosylation activity were introduced to realize the efficient synthesis of LNFP V. The α1,3/4-fucosyltransferase from Bacteroides fragilis was selected as the best enzyme for in vivo biosynthesis of LNFP V from nine candidates, with the highest titer and the lowest by-product accumulation. A beneficial variant K128D was obtained to further enhance LNFP V titer using computer-assisted site-directed mutagenesis. The final strain EW10 could produce 25.68 g/L LNFP V by fed-batch cultivation, with the productivity of 0.56 g/L·h.