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.

Regulation of intracellular activity of N-glycan branching enzymes in mammals.

Most proteins in the secretory pathway are glycosylated, and N-glycans are estimated to be attached to over 7000 proteins in humans. As structural variation of N-glycans critically regulates the functions of a particular glycoprotein, it is pivotal to understand how structural diversity of N-glycans is generated in cells. One of the major factors conferring structural variation of N-glycans is the variable number of N-acetylglucosamine branches. These branch structures are biosynthesized by dedicated glycosyltransferases, including GnT-III (MGAT3), GnT-IVa (MGAT4A), GnT-IVb (MGAT4B), GnT-V (MGAT5), and GnT-IX (GnT-Vb, MGAT5B). In addition, the presence or absence of core modification of N-glycans, namely, core fucose (included as an N-glycan branch in this manuscript), synthesized by FUT8, also confers large structural variation on N-glycans, thereby crucially regulating many protein-protein interactions. Numerous biochemical and medical studies have revealed that these branch structures are involved in a wide range of physiological and pathological processes. However, the mechanisms regulating the activity of the biosynthetic glycosyltransferases are yet to be fully elucidated. In this review, we summarize the previous findings and recent updates regarding regulation of the activity of these N-glycan branching enzymes. We hope that such information will help readers to develop a comprehensive overview of the complex system regulating mammalian N-glycan maturation.

Intestinal commensal microbiota and cytokines regulate Fut2+ Paneth cells for gut defense.

Paneth cells are intestinal epithelial cells that release antimicrobial peptides, such as α-defensin as part of host defense. Together with mesenchymal cells, Paneth cells provide niche factors for epithelial stem cell homeostasis. Here, we report two subtypes of murine Paneth cells, differentiated by their production and utilization of fucosyltransferase 2 (Fut2), which regulates α(1,2)fucosylation to create cohabitation niches for commensal bacteria and prevent invasion of the intestine by pathogenic bacteria. The majority of Fut2- Paneth cells were localized in the duodenum, whereas the majority of Fut2+ Paneth cells were in the ileum. Fut2+ Paneth cells showed higher granularity and structural complexity than did Fut2- Paneth cells, suggesting that Fut2+ Paneth cells are involved in host defense. Signaling by the commensal bacteria, together with interleukin 22 (IL-22), induced the development of Fut2+ Paneth cells. IL-22 was found to affect the α-defensin secretion system via modulation of Fut2 expression, and IL-17a was found to increase the production of α-defensin in the intestinal tract. Thus, these intestinal cytokines regulate the development and function of Fut2+ Paneth cells as part of gut defense.

Crystal structure of mango α1,3/α1,4-fucosyltransferase elucidates unique elements that regulate Lewis A-dominant oligosaccharide assembly.

In various organisms, α1,3/α1,4-fucosyltransferases (CAZy GT10 family enzymes) mediate the assembly of type I (Galß1,3GlcNAc) and/or type II (Galß1,4GlcNAc)-based Lewis structures that are widely distributed in glycoconjugates. Unlike enzymes of other species, plant orthologues show little fucosyltransferase activity for type II-based glycans and predominantly catalyze the assembly of the Lewis A structure [Galß1,3(Fucα1,4)GlcNAc] on the type I disaccharide unit of their substrates. However, the structural basis underlying this unique substrate selectivity remains elusive. In this study, we investigated the structure-function relationship of MiFUT13A, a mango α1,3/α1,4-fucosyltransferase. The prepared MiFUT13A displayed distinct α1,4-fucosyltransferase activity. Consistent with the enzymatic properties of this molecule, X-ray crystallography revealed that this enzyme has a typical GT-B fold-type structure containing a set of residues that are responsible for its SN2-like catalysis. Site-directed mutagenesis and molecular docking analyses proposed a rational binding mechanism for type I oligosaccharides. Within the catalytic cleft, the pocket surrounding Trp121 serves as a binding site, anchoring the non-reducing terminal ß1,3-galactose that belongs to the type I disaccharide unit. Furthermore, Glu177 was postulated to function as a general base catalyst through its interaction with the 4-hydroxy group of the acceptor N-acetylglucosamine residue. Adjacent residues, specifically Thr120, Thr157 and Asp175 were speculated to assist in binding of the reducing terminal residues. Intriguingly, these structural elements were not fully conserved in mammalian orthologue which also shows predominant α1,4-fucosyltransferase activity. In conclusion, we have proposed that MiFUT13A generates the Lewis A structure on type I glycans through a distinct mechanism, divergent from that of mammalian enzymes.

Splenocytes with fucosylation deficiency promote T cell proliferation and differentiation through thrombospondin-1 downregulation.

Fucosylation plays a critical role in cell-to-cell interactions and disease progression. However, the effects of fucosylation on splenocytes and their interactions with T cells remain unclear. In this study, we aimed to explore the transcriptome profiles of splenocytes deficient in fucosyltransferase (FUT) 1, an enzyme that mediates fucosylation, and investigate their impact on the proliferation and differentiation of T cells. We analysed and compared the transcriptomes of splenocytes isolated from Fut1 knockout (KO) mice and those from wild-type (WT) mice using RNA-seq. Additionally, we examined the effects of Fut1 KO splenocytes on CD4 T cell proliferation and differentiation, in comparison to WT splenocytes, and elucidated the mechanisms involved. The comparative analysis of transcriptomes between Fut1 KO and WT splenocytes revealed that thrombospondin-1, among the genes related to immune response and inflammation, was the most highly downregulated gene in Fut1 KO splenocytes. The reduced expression of thrombospondin-1 was further confirmed using qRT-PCR and flow cytometry. In coculture experiments, Fut1 KO splenocytes promoted the proliferation of CD4 T cells and drove their differentiation toward Th1 and Th17 cells, compared with WT splenocytes. Moreover, the levels of IL-2, IFN-γ and IL-17 were increased, while IL-10 was decreased, in T cells cocultured with Fut1 KO splenocytes compared with those with WT splenocytes. These effects of Fut1 KO splenocytes on T cells were reversed when thrombospondin-1 was replenished. Taken together, our results demonstrate that splenocytes with Fut1 deficiency promote CD4 T cell proliferation and Th1/Th17 differentiation at least in part through thrombospondin-1 downregulation.

Selective microbial production of lacto-N-fucopentaose I in Escherichia coli using engineered α-1,2-fucosyltransferases.

Lacto-N-fucopentaose I (LNFP I) is the second most abundant fucosylated human milk oligosaccharide (HMO) in breast milk after 2'-fucosyllactose (2'-FL). Studies have reported that LNFP I exhibits antimicrobial activity against group B Streptococcus and antiviral effects against Enterovirus and Norovirus. Microbial production of HMOs by engineered Escherichia coli is an attractive, low-cost process, but few studies have investigated production of long-chain HMOs, including the pentasaccharide LNFP I. LNFP I is synthesized by α1,2-fucosyltransfer reaction to the N-acetylglucosamine moiety of the lacto-N-tetraose skeleton, which is catalyzed by α1,2-fucosyltransferase (α1,2-FucT). However, α1,2-FucTs competitively transfer fucose to lactose, resulting in formation of the byproduct 2'-FL. In this study, we constructed LNFP I-producing strains of E. coli with various α1,2-fucTs, and observed undesired 2'-FL accumulation during fed-batch fermentation, although, in test tube assays, some strains produced LNFP I without 2'-FL. We hypothesized that promiscuous substrate selectivity of α1,2-FucT was responsible for 2'-FL production. Therefore, to decrease the formation of byproduct 2'-FL, we designed 15 variants of FsFucT from Francisella sp. FSC1006 by rational and semi-rational design approaches. Five of these variants of FsFucT surpassed a twofold reduction in 2'-FL production compared with wild-type FsFucT while maintaining comparable levels of LNFP I production. These designs encompassed substitutions in either a loop region of the enzyme (residues 154-171), or in specific residues (Q7, H162, and L164) that influence substrate binding either directly or indirectly. In particular, the E. coli strain that expressed FsFucT_S3 variants, with a substituted loop region (residues 154-171) forming an α-helix structure, achieved an accumulation of 19.6 g/L of LNFP I and 0.04 g/L of 2'-FL, while the E. coli strain expressing the wild-type FsFucT accumulated 12.2 g/L of LNFP I and 5.85 g/L of 2'-FL during Fed-bach fermentation. Therefore, we have successfully demonstrated the selective and efficient production of the pentasaccharide LNFP I without the byproduct 2'-FL by combining protein engineering of α1,2-FucT designed through in silico structural modeling of an α1,2-FucT and docking simulation with various ligands, with metabolic engineering of the host cell.

Association of fucosyltransferase 2 gene polymorphism with the susceptibility to norovirus infection in Han Chinese population.

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.

Cloning, expression and characterisation of a novel mollusc α-1,2-Fucosyltransferase from Crassostrea gigas (CgFUT2).

Glycans containing fucose play crucial roles in cell biology, particularly in recognition processes. In humans, fucose found in H-blood group antigens is recognized by various pathogens, thereby influencing host-pathogen interactions. However, in invertebrate biology the specific functions of these modifications and the corresponding glycosyltransferases are not fully elucidated. Therefore, cloning these glycosyltransferases from different model systems will provide valuable insights into this process. Little is known about fucosyltransferases in molluscs. For this study, a sequence of the Pacific oyster, Crassostrea gigas, based on amino acid sequence homologies with rabbit and human α-1,2-fucosyltransferases, was chosen. The recombinant enzyme (350 amino acids) was able to transfer fucose from GDP-fucose to the galactose residue of type II disaccharides, terminal galactoses in complex N-glycan structures and several linear and branched galactans which were tested using a glycan microarray. The α-1,2-linkage formed was confirmed by NMR analysis. The enzyme was active in a broad pH-range, it was relatively stable upon storage conditions and its activity was not dependent on the presence of divalent cations. In this study, we were able to clone, express and characterise a novel α-1,2-fucosyltrasferase from Crassostrea gigas (CgFUT2).

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.

The stem region of α1,6-fucosyltransferase FUT8 is required for multimer formation but not catalytic activity.

Alpha-1,6-fucosyltransferase (FUT8) synthesizes core fucose in N-glycans, which plays critical roles in various physiological processes. FUT8, as with many other glycosyltransferases, is a type-II membrane protein, and its large C-terminal catalytic domain is linked to the FUT8 stem region, which comprises two α-helices. Although the stem regions of several glycosyltransferases are involved in the regulation of Golgi localization, the functions of the FUT8 stem region have not been clarified as yet. Here, we found that the FUT8 stem region is essential for enzyme oligomerization. We expressed FUT8Δstem mutants, in which the stem region was replaced with glycine/serine linkers, in FUT8-KO HEK293 cells. Our immunoprecipitation and native-PAGE analysis showed that FUT8 WT formed a multimer but FUT8Δstem impaired multimer formation in the cells, although the mutants retained specific activity. In addition, the mutant protein had lower steady-state levels, increased endoplasmic reticulum localization, and a shorter half-life than FUT8 WT, suggesting that loss of the stem region destabilized the FUT8 protein. Furthermore, immunoprecipitation analysis of another mutant lacking a part of the stem region revealed that the first helix in the FUT8 stem region is critical for multimer formation. Our findings demonstrated that the FUT8 stem region is essential for multimer formation but not for catalytic activity, providing insights into how the FUT8 protein matures and functions in mammalian cells.

The role of FUT8-catalyzed core fucosylation in Alzheimer's amyloid-ß oligomer-induced activation of human microglia.

Fucosylation, especially core fucosylation of N-glycans catalyzed by α1-6 fucosyltransferase (fucosyltransferase 8 or FUT8), plays an important role in regulating the peripheral immune system and inflammation. However, its role in microglial activation is poorly understood. Here we used human induced pluripotent stem cells-derived microglia (hiMG) as a model to study the role of FUT8-catalyzed core fucosylation in amyloid-ß oligomer (AßO)-induced microglial activation, in view of its significant relevance to the pathogenesis of Alzheimer's disease (AD). HiMG responded to AßO and lipopolysaccharides (LPS) with a pattern of pro-inflammatory activation as well as enhanced core fucosylation and FUT8 expression within 24 h. Furthermore, we found increased FUT8 expression in both human AD brains and microglia isolated from 5xFAD mice, a model of AD-like cerebral amyloidosis. Inhibition of fucosylation in AßO-stimulated hiMG reduced the induction of pro-inflammatory cytokines, suppressed the activation of p38MAPK, and rectified phagocytic deficits. Specific inhibition of FUT8 by siRNA-mediated knockdown also reduced AßO-induced pro-inflammatory cytokines. We further showed that p53 binds to the two consensus binding sites in the Fut8 promoter, and that p53 knockdown abolished FUT8 overexpression in AßO-activated hiMG. Taken together, our evidence supports that FUT8-catalyzed core fucosylation is a signaling pathway required for AßO-induced microglia activation and that FUT8 is a component of the p53 signaling cascade regulating microglial behavior. Because microglia are a key driver of AD pathogenesis, our results suggest that microglial FUT8 could be an anti-inflammatory therapeutic target for AD.

Spindly is a nucleocytosolic O-fucosyltransferase in Dictyostelium and related proteins are widespread in protists and bacteria.

O-GlcNAcylation is a prominent modification of nuclear and cytoplasmic proteins in animals and plants and is mediated by a single O-GlcNAc transferase (OGT). Spindly (Spy), a paralog of OGT first discovered in higher plants, has an ortholog in the apicomplexan parasite Toxoplasma gondii, and both enzymes are now recognized as O-fucosyltransferases (OFTs). Here we investigate the evolution of spy-like genes and experimentally confirm OFT activity in the social amoeba Dictyostelium-a protist that is more related to fungi and metazoa. Immunofluorescence probing with the fucose-specific Aleuria aurantia lectin (AAL) and biochemical cell fractionation combined with western blotting suggested the occurrence of nucleocytoplasmic fucosylation. The absence of reactivity in mutants deleted in spy or gmd (unable to synthesize GDP-Fuc) suggested monofucosylation mediated by Spy. Genetic ablation of the modE locus, previously predicted to encode a GDP-fucose transporter, confirmed its necessity for fucosylation in the secretory pathway but not for the nucleocytoplasmic proteins. Affinity capture of these proteins combined with mass spectrometry confirmed monofucosylation of Ser and Thr residues of several known nucleocytoplasmic proteins. As in Toxoplasma, the Spy OFT was required for optimal proliferation of Dictyostelium under laboratory conditions. These findings support a new phylogenetic analysis of OGT and OFT evolution that indicates their occurrence in the last eukaryotic common ancestor but mostly complementary presence in its eukaryotic descendants with the notable exception that both occur in red algae and plants. Their generally exclusive expression, high degree of conservation, and shared monoglycosylation targets suggest overlapping roles in physiological regulation.

Biosynthesis of the O antigen of pathogenic Escherichia coli O157:H7. Characterization of α1,4-Fuc-transferase WbdO.

The O157:H7 strain of Escherichia coli is responsible for frequent outbreaks of hemorrhagic colitis worldwide. Its lipopolysaccharide is a virulence factor and contains an O antigen having repeating units with the tetrasaccharide structure [2-D-PerNAcα1-3-L-Fucα1-4-D-Glcß1-3-D-GalNAcα1-]n. Genes encoding glycosyltransferases WbdN, WbdO, and WbdP are responsible for the biosynthesis of this repeating unit. We have previously characterized the second enzyme in the pathway, WbdN, which transfers Glc in ß1-3 linkage to GalNAcα-O-PO3-PO3-(CH2)11-O-Ph (GalNAc-PP-PhU). In this work, Fuc-transferase WbdO from E. coli O157:H7 expressed in BL21 bacteria was characterized using the product of WbdN as the acceptor substrate. We showed that WbdO is specific for GDP-ß-L-Fuc as the donor substrate. Compounds that contained terminal Glc or Glcß1-3GalNAc structures but lacked the diphosphate group did not serve as acceptor substrates. The structure of the WbdO product was identified by mass spectrometry and Nuclear magnetic resonance (NMR) as L-Fucα1-4-D-Glcß1-3-D-GalNAc PP-PhU. WbdO is an unusual bivalent metal ion-dependent Fuc-transferase classified as an inverting GT2 family enzyme that has 2 conserved sequences near the N-terminus. The Asp37 residue within the 36VDGGSTD42 sequence was found to be essential for catalysis. Mutation of Asp68 to Ala within the conserved 67YDAMNK72 sequence resulted in a 3-fold increase in activity. These studies show that WbdOO157 is a highly specific Fuc-transferase with little homology to other characterized Fuc-transferases.

Toward α-1,3/4 fucosyltransferases targeted drug discovery: In silico uncovering of promising natural inhibitors of fucosyltransferase 6.

Sialyl Lewis X (sLex ) antigen is a fucosylated cell-surface glycan that is normally involved in cell-cell interactions. The enhanced expression of sLex on cell surface glycans, which is attributed to the upregulation of fucosyltransferase 6 (FUT6), has been implicated in facilitating metastasis in human colorectal, lung, prostate, and oral cancers. The role that the upregulated FUT6 plays in the progression of tumor to malignancy, with reduced survival rates, makes it a potential target for anticancer drugs. Unfortunately, the lack of experimental structures for FUT6 has hampered the design and development of its inhibitors. In this study, we used in silico techniques to identify potential FUT6 inhibitors. We first modeled the three-dimensional structure of human FUT6 using AlphaFold. Then, we screened the natural compound libraries from the COCONUT database to sort out potential natural products (NPs) with best affinity toward the FUT6 model. As a result of these simulations, we identified three NPs for which we predicted binding affinities and interaction patterns quite similar to those we calculated for two experimentally tested FUT6 inhibitors, that is, fucose mimetic-1 and a GDP-triazole derived compound. We also performed molecular dynamics (MD) simulations for the FUT6 complexes with identified NPs, to investigate their stability. Analysis of the MD simulations showed that the identified NPs establish stable contacts with FUT6 under dynamics conditions. On these grounds, the three screened compounds appear as promising natural alternatives to experimentally tested FUT6 synthetic inhibitors, with expected comparable binding affinity. This envisages good prospects for future experimental validation toward FUT6 inhibition.

A novel FUT1*c.889C>T allele associated with the para-Bombay AB phenotype and computational evaluation of the mutation effect.

BACKGROUND: Mutation in the FUT1 gene can impact the structure and function of α-(1,2)-fucosyltransferase 1 (α2FucT1). To explain the para-Bombay phenotype of a novel FUT1 allele, three-dimensional (3D) modeling and mutation effect analysis of α2FucT1 were performed by bioinformatic tools. MATERIALS AND METHODS: Blood and saliva samples were collected from a patient who was suspected to be a para-Bombay phenotype. H, A, and B antigens were determined with routine serologic methods for those samples. FUT1 and FUT2 coding regions were determined by Sanger sequencing. The novel heterozygous mutation was confirmed by cloning and sequencing. 3D model of mutant α2FucT1 was built by Phyre 2 and the mutation effect was evaluated by Chimera, PROVEAN, and Polyphen-2. RESULTS: Weak H, A, and B antigens were detected on RBCs of the proband and normal quantities of H, A, and B antigens were observed in his saliva. Cloning sequencing showed that the proband carried a novel FUT1 allele (c.889C>T, p.Leu297Phe) and a null FUT1*01N.06 allele. 3D model showed that the p.Leu297Phe variant in α2FucT1 reduced the number of hydrogen bonds and the mutation effect was predicted to be deleterious and possibly damaging, which suggested that the conformation and activity of the enzyme might be significantly damaged. CONCLUSION: A novel missense mutation led to an amino acid variant p.Leu297Phe in α2FucT1, which was a potential cause of the inactivation of the enzyme. Computational evaluation was a convenient and useful approach for the mutation effect analysis of the enzyme.

FUT2 inhibits the EMT and metastasis of colorectal cancer by increasing LRP1 fucosylation.

BACKGROUND: Fucosyltransferase 2(FUT2) and its induced α-1,2 fucosylation is associated with cancer metastasis. However, the role of FUT2 in colorectal cancer (CRC) metastasis remains unclear. METHODS: The expression levels and clinical analyses of FUT2 were assessed in CRC samples. Migration and invasion assays, EMT detection, nude mice peritoneal dissemination models and intestinal specific FUT2 knockout mice (FUT2△IEC mice) were used to investigate the effect of FUT2 on metastasis in colorectal cancer. Quantitative proteomics study of glycosylated protein, UEA enrichment, Co-immunoprecipitation identified the mediator of the invasive-inhibiting effects of FUT2. RESULTS: FUT2 is downregulated in CRC tissues and is positively correlated with the survival of CRC patients. FUT2 is an inhibitor of colorectal cancer metastasis which, when overexpressed, suppresses invasion and tumor dissemination in vitro and in vivo. FUT2 knock-out mice (FUT2△IEC mice) develop AMO and DSS-induced tumors and promote EMT in colorectal cancers. FUT2-induced α-1,2 fucosylation impacts the ability of low-density lipoprotein receptor-related protein 1(LRP1) to suppress colorectal cancer invasion. CONCLUSIONS: Our study demonstrated that FUT2 induces α-1,2 fucosylation and inhibits EMT and metastasis of colorectal cancer through LRP1 fucosylation, suggesting that FUT2 may serve as a therapeutic target for colorectal cancer. Video Abstract.

Golgi fucosyltransferase 1 reveals its important role in α-1,4-fucose modification of N-glycan in CRISPR/Cas9 diatom Phaeodactylum tricornutum.

Phaeodactylum tricornutum (Pt) is a critical microbial cell factory to produce a wide spectrum of marketable products including recombinant biopharmaceutical N-glycoproteins. N-glycosylation modification of proteins is important for their activity, stability, and half-life, especially some special modifications, such as fucose-modification by fucosyltransferase (FucT). Three PtFucTs were annotated in the genome of P. tricornutum, PtFucT1 was located on the medial/trans-Golgi apparatus and PtFucT2-3 in the plastid stroma. Algal growth, biomass and photosynthesis efficiency were significantly inhibited in a knockout mutant of PtFucT1 (PtFucT1-KO). PtFucT1 played a role in non-core fucose modification of N-glycans. The knockout of PtFucT1 might affect the activity of PtGnTI in the complex and change the complex N-glycan to mannose type N-glycan. The study provided critical information for understanding the mechanism of protein N-glycosylation modification and using microalgae as an alternative ecofriendly cell factory to produce biopharmaceuticals.

Fucosyltransferase 9 promotes neuronal differentiation and functional recovery after spinal cord injury by suppressing the activation of Notch signaling.

Individuals with spinal cord injury (SCI) suffer from permanent disabilities such as severe motor, sensory and autonomic dysfunction. Neural stem cell transplantation has proven to be a potential strategy to promote regeneration of the spinal cord, since NSCs can produce neurotrophic growth factors and differentiate into mature neurons to reconstruct the injured site. However, it is necessary to optimize the differentiation of NSCs before transplantation to achieve a better regenerative outcome. Inhibition of Notch signaling leads to a transition from NSCs to neurons, while the underlying mechanism remains inadequately understood. Our results demonstrate that overexpression of fucosyltransferase 9 (Fut9), which is upregulated by Wnt4, promotes neuronal differentiation by suppressing the activation of Notch signaling through disruption of furin-like enzyme activity during S1 cleavage. In an in vivo study, Fut9-modified NSCs efficiently differentiates into neurons to promote functional and histological recovery after SCI. Our research provides insight into the mechanisms of Notch signaling and a potential treatment strategy for SCI.

The Association of the Polymorphisms in the FUT8-Related Locus with the Plasma Glycosylation in Post-Traumatic Stress Disorder.

The molecular underpinnings of post-traumatic stress disorder (PTSD) are still unclear due to the complex interactions of genetic, psychological, and environmental factors. Glycosylation is a common post-translational modification of proteins, and different pathophysiological states, such as inflammation, autoimmune diseases, and mental disorders including PTSD, show altered N-glycome. Fucosyltransferase 8 (FUT8) is the enzyme that catalyzes the addition of core fucose on glycoproteins, and mutations in the FUT8 gene are associated with defects in glycosylation and functional abnormalities. This is the first study that investigated the associations of plasma N-glycan levels with FUT8-related rs6573604, rs11621121, rs10483776, and rs4073416 polymorphisms and their haplotypes in 541 PTSD patients and control participants. The results demonstrated that the rs6573604 T allele was more frequent in the PTSD than in the control participants. Significant associations of plasma N-glycan levels with PTSD and FUT8-related polymorphisms were observed. We also detected associations of rs11621121 and rs10483776 polymorphisms and their haplotypes with plasma levels of specific N-glycan species in both the control and PTSD groups. In carriers of different rs6573604 and rs4073416 genotypes and alleles, differences in plasma N-glycan levels were only found in the control group. These molecular findings suggest a possible regulatory role of FUT8-related polymorphisms in glycosylation, the alternations of which could partially explain the development and clinical manifestation of PTSD.

Enhancing lactose recognition of a key enzyme in 2'-fucosyllactose synthesis: α-1,2-fucosyltransferase.

BACKGROUND: 2'-Fucosyllactose, a representative oligosaccharide in human milk, is an emerging and promising food and pharmaceutical ingredient due to its powerful health benefits, such as participating in immune regulation, regulation of intestinal flora, etc. To enable economically viable production of 2'-fucosyllactose, different biosynthesis strategies using precursors and pathway enzymes have been developed. The α-1,2-fucosyltransferases are an essential part involved in these strategies, but their strict substrate selectivity and unsatisfactory substrate tolerance are one of the key roadblocks limiting biosynthesis. RESULTS: To tackle this issue, a semi-rational manipulation combining computer-aided designing and screening with biochemical experiments were adopted. The mutant had a 100-fold increase in catalytic efficiency compared to the wild-type. The highest 2'-fucosyllactose yield was up to 0.65 mol mol-1 lactose with a productivity of 2.56 g mL-1  h-1 performed by enzymatic catalysis in vitro. Further analysis revealed that the interactions between the mutant and substrates were reduced. The crucial contributions of wild-type and mutant to substrate recognition ability were closely related to their distinct phylotypes in terms of amino acid preference. CONCLUSION: It is envisioned that the engineered α-1,2-fucosyltransferase could be harnessed to relieve constraints imposed on the bioproduction of 2'-fucosyllactose and lay a theoretical foundation for elucidating the substrate recognition mechanisms of fucosyltransferases. © 2022 Society of Chemical Industry.