POFUT1-mediated O-fucosylation of glycoproteins expressed in the baculovirus Sf9 insect cell expression system.

The baculovirus-insect cell expression system allows addition of O-fucose to EGF-like domains of glycoproteins, following the action of the protein O-fucosyltransferase 1 named POFUT1. In this study, recombinant Spodoptera frugiperda POFUT1 from baculovirus-infected Sf9 cells was compared to recombinant Mus musculus POFUT1 produced by CHO cells. Contrary to recombinant murine POFUT1 carrying two hybrid and/or complex type N-glycans, Spodoptera frugiperda POFUT1 exhibited paucimannose N-glycans, at least on its highly evolutionary conserved across Metazoa NRT site. The abilities of both recombinant enzymes to add in vitro O -fucose to EGF-like domains of three different recombinant mammalian glycoproteins were then explored. In vitro POFUT1-mediated O-fucosylation experiments, followed by click chemistry and blot analyses, showed that Spodoptera frugiperda POFUT1 was able to add O-fucose to mouse NOTCH1 EGF-like 26 and WIF1 EGF-like 3 domains, similarly to the murine counterpart. As proved by mass spectrometry, full-length human WNT Inhibitor Factor 1 expressed by Sf9 cells was also modified with O-fucose. However, Spodoptera frugiperda POFUT1 was unable to modify the single EGF-like domain of mouse PAMR1 with O-fucose, contrary to murine POFUT1. Absence of orthologous proteins such as PAMR1 in insects may explain the enzyme's difficulty in adding O-fucose to a domain that it never encounters naturally.

Structural basis for Lewis antigen synthesis by the α1,3-fucosyltransferase FUT9.

Mammalian cell surface and secreted glycoproteins exhibit remarkable glycan structural diversity that contributes to numerous physiological and pathogenic interactions. Terminal glycan structures include Lewis antigens synthesized by a collection of α1,3/4-fucosyltransferases (CAZy GT10 family). At present, the only available crystallographic structure of a GT10 member is that of the Helicobacter pylori α1,3-fucosyltransferase, but mammalian GT10 fucosyltransferases are distinct in sequence and substrate specificity compared with the bacterial enzyme. Here, we determined crystal structures of human FUT9, an α1,3-fucosyltransferase that generates Lewisx and Lewisy antigens, in complex with GDP, acceptor glycans, and as a FUT9-donor analog-acceptor Michaelis complex. The structures reveal substrate specificity determinants and allow prediction of a catalytic model supported by kinetic analyses of numerous active site mutants. Comparisons with other GT10 fucosyltransferases and GT-B fold glycosyltransferases provide evidence for modular evolution of donor- and acceptor-binding sites and specificity for Lewis antigen synthesis among mammalian GT10 fucosyltransferases.

Therapeutic potential of fucosyltransferases in cancer and recent development of targeted inhibitors.

Fucosyltransferases (FUTs) have significant roles in various pathophysiological events. Their high expression is a signature of malignant cell transformation, contributing to many abnormal events during cancer development, such as uncontrolled cell proliferation, tumor cell invasion, angiogenesis, metastasis, immune evasion, and therapy resistance. Therefore, FUTs have evolved as an attractive therapeutic target for treating solid cancers, and many substrate analogs have been discovered with potential as FUT inhibitors for cancer therapy. Meanwhile, the development of FUT protein structures represents a significant advance in the design of FUT inhibitors with nonsubstrate structures. In this review, we summarize the role of FUTs in cancers, the resolved protein crystal structures and progress in the development of FUT inhibitors as cancer therapeutics.

O-fucosylation of thrombospondin type 1 repeats is essential for ECM remodeling and signaling during bone development.

Many extracellular matrix (ECM) associated proteins that influence ECM properties have Thrombospondin type 1 repeats (TSRs) which are modified with O-linked fucose. The O-fucose is added in the endoplasmic reticulum to folded TSRs by the enzyme Protein O-fucosyltransferase-2 (POFUT2) and is proposed to promote efficient trafficking of substrates. The importance of this modification for function of TSR-proteins is underscored by the early embryonic lethality of mouse embryos lacking Pofut2. To overcome early lethality and investigate the impact of the Pofut2 knockout on the secretion of POFUT2 substrates and on extracellular matrix properties in vivo, we deleted Pofut2 in the developing limb mesenchyme using Prrx1-Cre recombinase. Loss of Pofut2 in the limb mesenchyme caused significant shortening of the limbs, long bones and tendons and stiff joint resembling the musculoskeletal dysplasias in human and in mice with mutations in ADAMTS or ADAMTSL proteins. Limb shortening was evident at embryonic day 14.5 where loss of O-fucosylation led to an accumulation of fibrillin 2 (FBN2), decreased BMP and IHH signaling, and increased TGF-ß signaling. Consistent with these changes we saw a decrease in the size of the hypertrophic zone with lower levels of Collagen-X. Unexpectedly, we observed minimal effects of the Pofut2 knockout on secretion of two POFUT2 substrates, CCN2 or ADAMTS17, in the developing bone. In contrast, CCN2 and two other POFUT2 substrates important for bone development, ADAMTS6 and 10, showed a decrease in secretion from POFUT2-null HEK293T cells in vitro. These combined results suggest that the impact of the Pofut2 mutation is cell-type specific. In addition, these observations raise the possibility that the O-fucose modification on TSRs extends beyond promoting efficient trafficking of POFUT2 substrates and has the potential to influence their function in the extracellular environment.

A Combination of Structural, Genetic, Phenotypic and Enzymatic Analyses Reveals the Importance of a Predicted Fucosyltransferase to Protein O-Glycosylation in the Bacteroidetes.

Diverse members of the Bacteroidetes phylum have general protein O-glycosylation systems that are essential for processes such as host colonization and pathogenesis. Here, we analyzed the function of a putative fucosyltransferase (FucT) family that is widely encoded in Bacteroidetes protein O-glycosylation genetic loci. We studied the FucT orthologs of three Bacteroidetes species-Tannerella forsythia, Bacteroides fragilis, and Pedobacter heparinus. To identify the linkage created by the FucT of B. fragilis, we elucidated the full structure of its nine-sugar O-glycan and found that l-fucose is linked ß1,4 to glucose. Of the two fucose residues in the T. forsythia O-glycan, the fucose linked to the reducing-end galactose was shown by mutational analysis to be l-fucose. Despite the transfer of l-fucose to distinct hexose sugars in the B. fragilis and T. forsythia O-glycans, the FucT orthologs from B. fragilis, T. forsythia, and P. heparinus each cross-complement the B. fragilis ΔBF4306 and T. forsythia ΔTanf_01305 FucT mutants. In vitro enzymatic analyses showed relaxed acceptor specificity of the three enzymes, transferring l-fucose to various pNP-α-hexoses. Further, glycan structural analysis together with fucosidase assays indicated that the T. forsythia FucT links l-fucose α1,6 to galactose. Given the biological importance of fucosylated carbohydrates, these FucTs are promising candidates for synthetic glycobiology.

Fucosyltransferase 2 inhibitors: Identification via docking and STD-NMR studies.

Fucosyltransferase 2 (FUT2) catalyzes the biosynthesis of A, B, and H antigens and other important glycans, such as (Sialyl Lewisx) sLex, and (Sialyl Lewisy) sLey. The production of these glycans is increased in various cancers, hence to design and develop specific inhibitors of FUT2 is a therapeutic strategy. The current study was designed to identify the inhibitors for FUT2. In silico screening of 300 synthetic compounds was performed. Molecular docking studies highlighted the interactions of ligands with critical amino acid residues, present in the active site of FUT2. The epitope mapping in ligands was performed using the STD-NMR experiments to identify the interactions between ligands, and receptor protein. Finally, we have identified 5 lead compounds 4, 5, 26, 27, and 28 that can be studied for further development as cancer therapeutic agents.

Structural Insights in Mammalian Sialyltransferases and Fucosyltransferases: We Have Come a Long Way, but It Is Still a Long Way Down.

Mammalian cell surfaces are modified with complex arrays of glycans that play major roles in health and disease. Abnormal glycosylation is a hallmark of cancer; terminal sialic acid and fucose in particular have high levels in tumor cells, with positive implications for malignancy. Increased sialylation and fucosylation are due to the upregulation of a set of sialyltransferases (STs) and fucosyltransferases (FUTs), which are potential drug targets in cancer. In the past, several advances in glycostructural biology have been made with the determination of crystal structures of several important STs and FUTs in mammals. Additionally, how the independent evolution of STs and FUTs occurred with a limited set of global folds and the diverse modular ability of catalytic domains toward substrates has been elucidated. This review highlights advances in the understanding of the structural architecture, substrate binding interactions, and catalysis of STs and FUTs in mammals. While this general understanding is emerging, use of this information to design inhibitors of STs and FUTs will be helpful in providing further insights into their role in the manifestation of cancer and developing targeted therapeutics in cancer.

Protein O-Fucosyltransferase 1 Undergoes Interdomain Flexibility in Solution.

Protein O-fucosyltransferase 1 (PoFUT1) is a GT-B fold enzyme that fucosylates proteins containing EGF-like repeats. GT-B glycosyltransferases have shown a remarkable grade of plasticity adopting closed and open conformations as a way of tuning their catalytic cycle, a feature that has not been observed for PoFUT1. Here, we analyzed Caenorhabditis elegans PoFUT1 (CePoFUT1) conformational behavior in solution by atomic force microscopy (AFM) and single-molecule fluorescence resonance energy transfer (SMF-FRET). Our results show that this enzyme is very flexible and adopts mainly compact conformations and to a lesser extend a highly dynamic population that oscillates between compact and highly extended conformations. Overall, our experiments illustrate the inherent complexity of CePoFUT1 dynamics, which might play a role during its catalytic cycle.

Appropriate aglycone modification significantly expands the glycan substrate acceptability of α1,6-fucosyltransferase (FUT8).

The α1,6-fucosyltransferase, FUT8, is the sole enzyme catalyzing the core-fucosylation of N-glycoproteins in mammalian systems. Previous studies using free N-glycans as acceptor substrates indicated that a terminal ß1,2-GlcNAc moiety on the Man-α1,3-Man arm of N-glycan substrates is required for efficient FUT8-catalyzed core-fucosylation. In contrast, we recently demonstrated that, in a proper protein context, FUT8 could also fucosylate Man5GlcNAc2 without a GlcNAc at the non-reducing end. We describe here a further study of the substrate specificity of FUT8 using a range of N-glycans containing different aglycones. We found that FUT8 could fucosylate most of high-mannose and complex-type N-glycans, including highly branched N-glycans from chicken ovalbumin, when the aglycone moiety is modified with a 9-fluorenylmethyloxycarbonyl (Fmoc) moiety or in a suitable peptide/protein context, even if they lack the terminal GlcNAc moiety on the Man-α1,3-Man arm. FUT8 could also fucosylate paucimannose structures when they are on glycoprotein substrates. Such core-fucosylated paucimannosylation is a prominent feature of lysosomal proteins of human neutrophils and several types of cancers. We also found that sialylation of N-glycans significantly reduced their activity as a substrate of FUT8. Kinetic analysis demonstrated that Fmoc aglycone modification could either improve the turnover rate or decrease the KM value depending on the nature of the substrates, thus significantly enhancing the overall efficiency of FUT8 catalyzed fucosylation. Our results indicate that an appropriate aglycone context of N-glycans could significantly broaden the acceptor substrate specificity of FUT8 beyond what has previously been thought.

Biotechnological Production of 2'-Fucosyllactose: A Prevalent Fucosylated Human Milk Oligosaccharide.

Human milk oligosaccharide (HMO) is a key component of human milk carbohydrates and is closely related to the nutrition and health benefits of breastfeeding in infants. 2'-Fucosyllactose (2'-FL) is the most abundant fucosylated HMO, which has remarkable value in nutrition and medicine, such as suppressing pathogen infection, regulating intestinal flora, and boosting immunity. However, 2'-FL production via the method of extraction or chemical synthesis cannot meet its large demand, and as a result, environmentally friendly and efficient biotechnological approaches, including in vitro enzymatic synthesis and microbial cell factory production, have been developed and applied to its commercialized production. This review introduces, summarizes, and discusses the recent advances in the biotechnological production of 2'-FL. Furthermore, future research directions for the biotechnological production of 2'-FL as well as the strategies to further improve its concentration are highlighted and discussed.

Characterizing human α-1,6-fucosyltransferase (FUT8) substrate specificity and structural similarities with related fucosyltransferases.

Mammalian Asn-linked glycans are extensively processed as they transit the secretory pathway to generate diverse glycans on cell surface and secreted glycoproteins. Additional modification of the glycan core by α-1,6-fucose addition to the innermost GlcNAc residue (core fucosylation) is catalyzed by an α-1,6-fucosyltransferase (FUT8). The importance of core fucosylation can be seen in the complex pathological phenotypes of FUT8 null mice, which display defects in cellular signaling, development, and subsequent neonatal lethality. Elevated core fucosylation has also been identified in several human cancers. However, the structural basis for FUT8 substrate specificity remains unknown.Here, using various crystal structures of FUT8 in complex with a donor substrate analog, and with four distinct glycan acceptors, we identify the molecular basis for FUT8 specificity and activity. The ordering of three active site loops corresponds to an increased occupancy for bound GDP, suggesting an induced-fit folding of the donor-binding subsite. Structures of the various acceptor complexes were compared with kinetic data on FUT8 active site mutants and with specificity data from a library of glycan acceptors to reveal how binding site complementarity and steric hindrance can tune substrate affinity. The FUT8 structure was also compared with other known fucosyltransferases to identify conserved and divergent structural features for donor and acceptor recognition and catalysis. These data provide insights into the evolution of modular templates for donor and acceptor recognition among GT-B fold glycosyltransferases in the synthesis of diverse glycan structures in biological systems.

Differential Labeling of Glycoproteins with Alkynyl Fucose Analogs.

Fucosylated glycans critically regulate the physiological functions of proteins and cells. Alterations in levels of fucosylated glycans are associated with various diseases. For detection and functional modulation of fucosylated glycans, chemical biology approaches using fucose (Fuc) analogs are useful. However, little is known about how efficiently each unnatural Fuc analog is utilized by enzymes in the biosynthetic pathway of fucosylated glycans. We show here that three clickable Fuc analogs with similar but distinct structures labeled cellular glycans with different efficiency and protein specificity. For instance, 6-alkynyl (Alk)-Fuc modified O-Fuc glycans much more efficiently than 7-Alk-Fuc. The level of GDP-6-Alk-Fuc produced in cells was also higher than that of GDP-7-Alk-Fuc. Comprehensive in vitro fucosyltransferase assays revealed that 7-Alk-Fuc is commonly tolerated by most fucosyltransferases. Surprisingly, both protein O-fucosyltransferases (POFUTs) could transfer all Fuc analogs in vitro, likely because POFUT structures have a larger space around their Fuc binding sites. These findings demonstrate that labeling and detection of fucosylated glycans with Fuc analogs depend on multiple cellular steps, including conversion to GDP form, transport into the ER or Golgi, and utilization by each fucosyltransferase, providing insights into design of novel sugar analogs for specific detection of target glycans or inhibition of their functions.

Efficient Biosynthesis of 2'-Fucosyllactose Using an In Vitro Multienzyme Cascade.

2'-Fucosyllactose (2-FL) is a fucose-containing oligosaccharide that is found in humans and is believed to have potential nutraceutical and pharmaceutical uses. Here, a promising in vitro multienzyme cascade catalysis system (MECCS) was designed to convert L-fucose and lactose to 2-FL. The cascade comprises L-fucokinase/GDP-L-fucose phosphorylase (FKP), α-1,2-fucosyltransferase (FucT), and pyruvate kinase (PK). This MECCS was able to efficiently regenerate ATP or GTP with 5.67-fold improvement of GDP-L-fucose. To address the rate-limiting step in the MECCS, various FucT orthologues were screened, and HpFucT from Helicobacter pylori showed the highest catalytic efficiency, with a (kcat/KM) of 39.28 min-1 mM-1, while TeFucT from Thermosynechococcus elongatus showed the highest thermostability, with a melting temperature (Tm) of 48 °C. The dissociation constant (KD) of TeFucT (1.34 ± 0.41 µM) was 15-fold lower than that of HpFucT (20.24 ± 1.81 µM), suggesting that TeFucT had much higher affinity for GDP. Structural analysis of HpFucT indicated that Arg169 is part of a unique substrate-binding site that interacts with two oxygen atoms from the phosphate group of GDP-L-fucose. The 2-FL productivities of the MECCS in fed-batch reached 0.67 and 0.73 g/L/h with TeFucT and HpFucT, respectively. This research provides an alternative pathway for efficient production of 2-FL.

Chemoenzymatic synthesis of 3-deoxy-3-fluoro-l-fucose and its enzymatic incorporation into glycoconjugates.

The first synthesis of 3-deoxy-3-fluoro-l-fucose is presented, which employs a d- to l-sugar translation strategy, and involves an enzymatic oxidation of 3-deoxy-3-fluoro-l-fucitol. Enzymatic activation (FKP) and glycosylation using an α-1,2 and an α-1,3 fucosyltransferase to obtain two fluorinated trisaccharides demonstrates its potential as a novel versatile chemical probe in glycobiology.

Detecting substrate glycans of fucosyltransferases with fluorophore-conjugated fucose and methods for glycan electrophoresis.

Like sialylation, fucose usually locates at the nonreducing ends of various glycans on glycoproteins and constitutes important glycan epitopes. Detecting the substrate glycans of fucosyltransferases is important for understanding how these glycan epitopes are regulated in response to different growth conditions and external stimuli. Here we report the detection of these glycans on glycoproteins as well as in their free forms via enzymatic incorporation of fluorophore-conjugated fucose using FUT2, FUT6, FUT7, FUT8 and FUT9. Specifically, we describe the detection of the substrate glycans of these enzymes on fetal bovine fetuin, recombinant H1N1 viral neuraminidase and therapeutic antibodies. The detected glycans include complex and high-mannose N-glycans. By establishing a series of precursors for the synthesis of Lewis X and sialyl Lewis X structures, we not only provide convenient electrophoresis methods for studying glycosylation but also demonstrate the substrate specificities and some kinetic features of these enzymes. Our results support the notion that fucosyltransferases are key targets for regulating the synthesis of Lewis X and sialyl Lewis X structures.

hFUT1-Based Live-Cell Assay To Profile α1-2-Fucoside-Enhanced Influenza Virus A Infection.

Host cell surface glycans play critical roles in influenza virus A (IVA) infection ranging from modulation of IVA attachment to membrane fusion and host tropism. Approaches for quick and sensitive profile of viral avidity toward a specific type of host cell glycan can contribute to the understanding of tropism switching among different IVA strains. Here, we developed a method based on chemoenzymatic glycan engineering to investigate the possible involvement of α1-2-fucosides in IVA infections. Using a truncated human fucosyltransferase 1 (hFUT1), we created α1-2-fucosides in situ on host cells to assess their influence on the host cell binding to IVA hemagglutinin and the susceptibility of host cells toward IVA-induced killing. We discovered that the newly created α1-2-fucosides on host cells enhanced the infection of several human pandemic IVA subtypes either directly or indirectly. These findings suggest that glycan epitopes other than sialic acid should also be considered for assessing the human pandemic risk of this viral pathogen.

Involvement of the α-helical and Src homology 3 domains in the molecular assembly and enzymatic activity of human α1,6-fucosyltransferase, FUT8.

BACKGROUND: Previous structural analyses showed that human α1,6-fucosyltransferase, FUT8 contains a catalytic domain along with two additional domains, N-terminal α-helical domain and C-terminal Src homology 3 domain, but these domains are unique to FUT8 among glycosyltransferases. The role that these domains play in formation of the active form of FUT8 has not been investigated. This study reports on attempts to determine the involvement of these domains in the functions of FUT8. METHODS: Based on molecular modeling, the domain mutants were constructed by truncation and site-directed mutagenesis, and were heterologously expressed in Sf21 or COS-1 cells. The mutants were analyzed by SDS-PAGE and assayed for enzymatic activity. In vivo cross-linking experiments by introducing disulfide bonds were also carried out to examine the orientation of the domains in the molecular assembly. RESULTS: Mutagenesis and molecular modeling findings suggest that human FUT8 potentially forms homodimer in vivo via intermolecular hydrophobic interactions involving α-helical domains. Truncation or site-directed mutagenesis findings indicated that α-helical and SH3 domains are all required for enzymatic activity. In addition, in vivo cross-linking experiments clearly indicated that the SH3 domain located in close proximity to the α-helical domain in an intermolecular manner. CONCLUSIONS: α-Helical and SH3 domains are required for a fully active enzyme, and are also involved in homophilic dimerization, which probably results in the formation of the active form of human FUT8. GENERAL SIGNIFICANCE: α-Helical and SH3 domains, which are not commonly found in glycosyltransferases, play roles in the formation of the functional quaternary structure of human FUT8.

Structural basis of substrate recognition and catalysis by fucosyltransferase 8.

Fucosylation of the innermost GlcNAc of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, development of mAb therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor substrate preferences of FUT8 are well-characterized and provide a framework for understanding N-glycan maturation in the Golgi; however, the structural basis of these substrate preferences and the mechanism through which catalysis is achieved remain unknown. Here we describe several structures of mouse and human FUT8 in the apo state and in complex with GDP, a mimic of the donor substrate, and with a glycopeptide acceptor substrate at 1.80-2.50 Å resolution. These structures provide insights into a unique conformational change associated with donor substrate binding, common strategies employed by fucosyltransferases to coordinate GDP, features that define acceptor substrate preferences, and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also revealed how FUT8 dimerization plays an important role in defining the acceptor substrate-binding site. Collectively, this information significantly builds on our understanding of the core fucosylation process.

Structural basis for substrate specificity and catalysis of α1,6-fucosyltransferase.

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

Directed evolution of an α1,3-fucosyltransferase using a single-cell ultrahigh-throughput screening method.

Fucosylated glycoconjugates are involved in a variety of physiological and pathological processes. However, economical production of fucosylated drugs and prebiotic supplements has been hampered by the poor catalytic efficiency of fucosyltransferases. Here, we developed a fluorescence-activated cell sorting system that enables the ultrahigh-throughput screening (>107 mutants/hour) of such enzymes and designed a companion strategy to assess the screening performance of the system. After three rounds of directed evolution, a mutant M32 of the α1,3-FucT from Helicobacter pylori was identified with 6- and 14-fold increases in catalytic efficiency (k cat/K m) for the synthesis of Lewis x and 3'-fucosyllactose, respectively. The structure of the M32 mutant revealed that the S45F mutation generates a clamp-like structure that appears to improve binding of the galactopyranose ring of the acceptor substrate. Moreover, molecular dynamic simulations reveal that helix α5, is more mobile in the M32 mutant, possibly explaining its high fucosylation activity.