FUT2 promotes colorectal cancer metastasis by reprogramming fatty acid metabolism via YAP/TAZ signaling and SREBP-1.

Colorectal cancer (CRC) ranks as the second most lethal cancer worldwide because of its high rate of metastasis, and approximately 20% of CRC patients have metastases at initial diagnosis. Metabolic reprogramming, a hallmark of cancer cells, has been implicated in the process of metastasis. We previously demonstrated that fucosyltransferase 2 (FUT2) promotes the malignancy of CRC cells, however, the underlying mechanisms remain unclear. Here, bioinformatic analysis revealed that FUT2 is associated with the malignant phenotype and fatty acid metabolism in CRC. FUT2 knockdown decreased glucose uptake and de novo fatty acid synthesis, which in turn inhibited the proliferation and metastasis of CRC cells. Mechanistically, FUT2 promotes YAP1 nuclear translocation and stabilizes mSREBP-1 by fucosylation, thus promoting de novo fatty acid synthesis in CRC cells. In summary, this study demonstrates that FUT2 promotes the proliferation and metastasis of CRC cells by reprogramming fatty acid metabolism via YAP/TAZ signaling and SREBP-1, indicating that FUT2 might be a potential target for developing therapeutic strategies against CRC.

FUT6 Suppresses the Proliferation, Migration, Invasion, and Epithelial-Mesenchymal Transition of Esophageal Carcinoma Cells via the Epidermal Growth Factor Receptor/Extracellular Signal-Regulated Kinase Signaling Pathway.

Esophageal cancer (ESCA) is a high-incidence disease worldwide, of which the 5-year survival rate remains dismal since the cellular basis of ESCA remains largely unclear. Herein, we attempted to examine the manifestation of fucosyltransferase-6 (FUT6) in ESCA and the associated mechanisms. The GSE161533 dataset was used to analyze a crucial gene in ESCA. The expression of FUT6 was investigated in normal esophageal epithelial cells and ESCA cell lines. Following FUT6 knockdown or overexpression, cell proliferation, migration, invasion, and levels of epithelial­mesenchymal transition (EMT)-related and epidermal growth factor receptor (EGFR)/extracellular signal-regulated kinase (ERK) signaling pathway-related proteins were evaluated using CCK-8, Transwell, and Western blotting with antibodies against EGFR, p-EGFR, E-cadherin, Vimentin, N-cadherin, ERK1/2, and p-ERK1/2), respectively. EGF was administered to stimulate the EGFR/ERK signaling pathway, followed by the assessment of cellular activity. Database analysis revealed that FUT6 was downregulated in the ESCA cells. Our study indicated that FUT6 is suppressed in various ESCA cell lines. Moreover, cell proliferation, invasion, migration, and EMT-related protein levels were conspicuously enhanced or restrained by FUT6 disruption or overexpression. FUT6 overexpression suppressed the malignant activities of the cells when stimulated by EGF, including inhibition of cell growth, movement, invasion, and EMT advancement, as well the reduction the levels of EGFR/ERK pathway proteins. In conclusion, FUT6 can suppress the EGFR/ERK signaling pathway activated by EGF, leading to the potential attenuation of ESCA cell proliferation, invasion, migration, and EMT.

Terminal α1,2-fucosylation of glycosphingolipids by FUT1 is a key regulator in early cell-fate decisions.

The embryonic cell surface is rich in glycosphingolipids (GSLs), which change during differentiation. The reasons for GSL subgroup variation during early embryogenesis remain elusive. By combining genomic approaches, flow cytometry, confocal imaging, and transcriptomic data analysis, we discovered that α1,2-fucosylated GSLs control the differentiation of human pluripotent cells (hPCs) into germ layer tissues. Overexpression of α1,2-fucosylated GSLs disrupts hPC differentiation into mesodermal lineage and reduces differentiation into cardiomyocytes. Conversely, reducing α1,2-fucosylated groups promotes hPC differentiation and mesoderm commitment in response to external signals. We find that bone morphogenetic protein 4 (BMP4), a mesodermal gene inducer, suppresses α1,2-fucosylated GSL expression. Overexpression of α1,2-fucosylated GSLs impairs SMAD activation despite BMP4 presence, suggesting α-fucosyl end groups as BMP pathway regulators. Additionally, the absence of α1,2-fucosylated GSLs in early/late mesoderm and primitive streak stages in mouse embryos aligns with the hPC results. Thus, α1,2-fucosylated GSLs may regulate early cell-fate decisions and embryo development by modulating cell signaling.

RIP3 regulates doxorubicin-induced intestinal mucositis via FUT2-mediated α-1,2-fucosylation.

OBJECTIVE: Intestinal mucositis is one of the common side effects of anti-cancer chemotherapy. However, the molecular mechanisms involved in mucositis development remain incompletely understood. In this study, we investigated the function of receptor-interacting protein kinase 3 (RIP3/RIPK3) in regulating doxorubicin-induced intestinal mucositis and its potential mechanisms. METHODS: Intestinal mucositis animal models were induced in mice for in vivo studies. Rat intestinal cell line IEC-6 was used for in vitro studies. RNA­seq was used to explore the transcriptomic changes in doxorubicin-induced intestinal mucositis. Intact glycopeptide characterization using mass spectrometry was applied to identify α-1,2-fucosylated proteins associated with mucositis. RESULTS: Doxorubicin treatment increased RIP3 expression in the intestine and caused severe intestinal mucositis in the mice, depletion of RIP3 abolished doxorubicin-induced intestinal mucositis. RIP3-mediated doxorubicin-induced mucositis did not depend on mixed lineage kinase domain-like (MLKL) but on α-1,2-fucosyltransferase 2 (FUT2)-catalyzed α-1,2-fucosylation on inflammation-related proteins. Deficiency of MLKL did not affect intestinal mucositis, whereas inhibition of α-1,2-fucosylation by 2-deoxy-D-galactose (2dGal) profoundly attenuated doxorubicin-induced inflammation and mucositis. CONCLUSIONS: RIP3-FUT2 pathway is a central node in doxorubicin-induced intestinal mucositis. Targeting intestinal RIP3 and/or FUT2-mediated α-1,2-fucosylation may provide potential targets for preventing chemotherapy-induced intestinal mucositis.

Core fucosylation within the Fc-FcγR degradation pathway promotes enhanced IgG levels via exogenous L-fucose.

α1,6-Fucosyltransferase (Fut8) is the enzyme responsible for catalyzing core fucosylation. Exogenous L-fucose upregulates fucosylation levels through the GDP-fucose salvage pathway. This study investigated the relationship between core fucosylation and immunoglobulin G (IgG) amounts in serum utilizing WT (Fut8+/+), Fut8 heterozygous knockout (Fut8+/-), and Fut8 knockout (Fut8-/-) mice. The IgG levels in serum were lower in Fut8+/- and Fut8-/- mice compared with Fut8+/+ mice. Exogenous L-fucose increased IgG levels in Fut8+/- mice, while the ratios of core fucosylated IgG versus total IgG showed no significant difference among Fut8+/+, Fut8+/-, and Fut8+/- mice treated with L-fucose. These ratios were determined by Western blot, lectin blot, and mass spectrometry analysis. Real-time PCR results demonstrated that mRNA levels of IgG Fc and neonatal Fc receptor, responsible for protecting IgG turnover, were similar among Fut8+/+, Fut8+/-, and Fut8+/- mice treated with L-fucose. In contrast, the expression levels of Fc-gamma receptor Ⅳ (FcγRⅣ), mainly expressed on macrophages and neutrophils, were increased in Fut8+/- mice compared to Fut8+/+ mice. The effect was reversed by administrating L-fucose, suggesting that core fucosylation primarily regulates the IgG levels through the Fc-FcγRⅣ degradation pathway. Consistently, IgG internalization and transcytosis were suppressed in FcγRⅣ-knockout cells while enhanced in Fut8-knockout cells. Furthermore, we assessed the expression levels of specific antibodies against ovalbumin and found they were downregulated in Fut8+/- mice, with potential recovery observed with L-fucose administration. These findings confirm that core fucosylation plays a vital role in regulating IgG levels in serum, which may provide insights into a novel mechanism in adaptive immune regulation.

Analysis of endogenous NOTCH1 from POFUT1 S162L patient fibroblasts reveals the importance of the O-fucose modification on EGF12 in human development.

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.

BMSCs promote alveolar epithelial cell autophagy to reduce pulmonary fibrosis by inhibiting core fucosylation modifications.

BACKGROUND: Idiopathic pulmonary fibrosis (PF) is a chronic progressive interstitial lung disease characterized by alveolar epithelial cell (AEC) injury and fibroblast activation. Inadequate autophagy in AECs may result from the activation of several signaling pathways following AEC injury, with glycoproteins serving as key receptor proteins. The core fucosylation (CF) modification in glycoproteins is crucial. Mesenchymal stem cells derived from bone marrow (BMSCs) have the ability to regenerate damaged tissue and treat PF. This study aimed to elucidate the relationship and mechanism of interaction between BMSCs, CF modification, and autophagy in PF. METHODS: C57BL/6 male mice, AEC-specific FUT8 conditional knockout (CKO) mice, and MLE12 cells were administered bleomycin (BLM), FUT8 siRNA, and mouse BMSCs, respectively. Experimental techniques including tissue staining, Western blotting, immunofluorescence, autophagic flux detection, and flow cytometry were used in this study. RESULTS: First, we found that autophagy was inhibited while FUT8 expression was elevated in PF mice and BLM-induced AEC injury models. Subsequently, CKO mice and MLE12 cells transfected with FUT8 siRNA were used to demonstrate that inhibition of CF modification induces autophagy in AECs and mitigates PF. Finally, mouse BMSCs were used to demonstrate that they alleviate the detrimental autophagy of AECs by inhibiting CF modification and decreasing PF. CONCLUSIONS: Suppression of CF modification enhanced the suppression of AEC autophagy and reduced PF in mice. Additionally, through the prevention of CF modification, BMSCs can assist AECs deficient in autophagy and partially alleviate PF.

Impact of fucosyltransferase 1-mediated epidermal blood group antigen H on anti-inflammatory response in atopic dermatitis.

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.

A metabolic inhibitor blocks cellular fucosylation and enables production of afucosylated antibodies.

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.

Intestinal persistence of Bifidobacterium infantis is determined by interaction of host genetics and antibiotic exposure.

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.

Combinatorial Optimization Strategies for 3-Fucosyllactose Hyperproduction in Escherichia coli.

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.

Combinatorial Engineering of Escherichia coli for Enhancing 3-Fucosyllactose Production.

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.

Role of Long Intergenic Nonprotein-Coding RNA 00511 in Nod-Like Receptor Protein Pyrin Domain 3-Induced Chondrocyte Pyroptosis via the MicroRNA-9-5p/FUT1 Axis.

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.

Protein O-fucosyltransferase 1 promotes PD-L1 stability to drive immune evasion and directs liver cancer to immunotherapy.

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.

Fut2 deficiency aggravates chronic colitis through 2-oxindole-AHR mediated cGAS-STING pathway.

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 (Fut2△IEC), 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 Fut2△IEC mice with colitis (Fut2△IEC-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 Fut2△IEC-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 Fut2△IEC-DSS mice. CONCLUSION: Fut2 deficiency promotes cGAS-STING pathway through suppressing 2-oxindole-AHR axis, ultimately facilitating the susceptibility to chronic colitis.

Application of the CRISPR/Cas9 Gene Editing Method for Modulating Antibody Fucosylation in CHO Cells.

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.

Discovery of novel FUT8 inhibitors with promising affinity and in vivo efficacy for colorectal cancer therapy.

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.

Accelerated generation of gene-engineered monoclonal CHO cell lines using FluidFM nanoinjection and CRISPR/Cas9.

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.

(Sialyl)Lewis Antigen Expression on Glycosphingolipids, N-, and O-Glycans in Colorectal Cancer Cell Lines is Linked to a Colon-Like Differentiation Program.

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.

Engineering Escherichia coli for Highly Efficient Biosynthesis of Lacto-N-difucohexaose II through De Novo GDP-l-fucose Pathway.

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.