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Fucoidan-degrading enzymes are glycoside hydrolases that hydrolyze fucoidan into low molecular mass fucosan with biological activity or other products. The enzymes have great application value in the preparation of active fucoidan oligosaccharides, biopharmaceutics and disease diagnosis. In this article, the sources, properties, modes of action, structures and applications of fucoidan-degrading enzymes are reviewed, revealing that fucoidan-degrading enzymes are mainly derived from marine microorganisms and marine invertebrates and that the enzymatic properties, substrate specificity, molecular structures and catalytic mechanisms of fucoidan-degrading enzymes vary significantly depending on the species source. Different types of fucoidan-degrading enzymes catalyze different sites of complex fucoidans producing fucoidan oligosaccharides with antioxidant, anti-tumor, anti-thrombotic or other special activities. Therefore, the utilization of fucoidan-degrading enzymes in the preparation of drugs and functional foods has gradually become a research focus in the application field of polysaccharides. This article provides theoretical support for further research on fucoidan-degrading enzymes, and also provides references for the effective development of fucoidan oligosaccharides with special biological functions.

In recent years, the synthesis of human milk oligosaccharides, particularly fucooligosaccharides, has been investigated. These oligosaccharides provide protection against gastrointestinal diseases for newborns, among other functionalities. α-L-fucosidase from Thermotoga maritima of GH29 family has been reported to produce fucooligosaccharides by means of a transfucosylation reaction. However, the interaction between acceptor and donor substrates, and the enzyme has been little studied. For that reason, and due to the importance of fucooligosaccharides, the present study proposes a theoretical synthesis route employing α-L-fucosidase from T. maritima, lactose as acceptor substrate and pNP-fucose as fucosyl donor. A molecular docking study was carried out using crystallographic structure of α-L-fucosidase from Protein Data Bank. By simulating the theoretical synthesis with T. maritima fucosidase, it was observed that lactose interacts with the enzyme in regions other than the active site. However, it is approaching the active site when the fucose is in the position to form the enzyme-substrate complex. This finding may explain the transfucosylation yield documented in the literature when the proportion of fucosyl acceptor substrate increases concerning that of the donor substrate. [ABSTRACT FROM AUTHOR]

Glycoside hydrolase family 29 (GH29) encompasses α-L-fucosidases, i.e. enzymes that catalyze the hydrolytic release of fucose from fucosylated glycans, including N - and O -linked glycans on proteins, and these α-L-fucosidases clearly play important roles in biology. GH29 enzymes work via a retaining exo-action mechanism, and some can catalyze transfucosylation. There is no formal subfamily division of GH29 α-L-fucosidases, but they are nonetheless divided into two subfamilies: GH29A having a range of substrate specificities and GH29B having narrower substrate specificity. However, the sequence traits that determine the substrate specificity and transglycosylation ability of GH29 enzymes are not well characterized. Here, we present a new functional map of family GH29 members based on peptide-motif clustering via CUPP (conserved unique peptide patterns) and compare the substrate specificity and transglycosylation activity of 21 representative α-L-fucosidases across the 53 CUPP groups identified. The 21 enzymes exhibited different enzymatic rates on 8 test substrates, CNP-Fuc, 2'FL, 3FL, Lewisa, Lewisx, Fuc-α1,6-GlcNAc, Fuc-α1,3-GlcNAc, and Fuc-α1,4-GlcNAc. Certain CUPP groups clearly harbored a particular type of enzymes, e.g. the majority of the enzymes having activity on Lewisa or Lewisx categorized in the same CUPP clusters. In general, CUPP was useful for resolving GH29 into functional diversity subgroups when considering hydrolytic activity. In contrast, the transglycosylation capacity of GH29 α-L-fucosidases was distributed across a range of CUPP groups. Transglycosylation thus appears to be a common trait among these enzymes and not readily predicted from sequence comparison. [ABSTRACT FROM AUTHOR]

Bacterial vaginosis (BV) is a polymicrobial infection of the female reproductive tract. BV is characterized by replacement of health-associated Lactobacillus species by diverse anerobic bacteria, including the well-known Gardnerella vaginalis. Prevotella timonensis, and Prevotella bivia are anerobes that are found in a significant number of BV patients, but their contributions to the disease process remain to be determined. Defining characteristics of anerobic overgrowth in BV are adherence to the mucosal surface and the increased activity of mucin-degrading enzymes such as sialidases in vaginal secretions. We demonstrate that P. timonensis, but not P. bivia, strongly adheres to vaginal and endocervical cells to a similar level as G. vaginalis but did not elicit a comparable proinflammatory epithelial response. The P. timonensis genome uniquely encodes a large set of mucus-degrading enzymes, including four putative fucosidases and two putative sialidases, PtNanH1 and PtNanH2. Enzyme assays demonstrated that fucosidase and sialidase activities in P. timonensis cell-bound and secreted fractions were significantly higher than for other vaginal anerobes. In infection assays, P. timonensis efficiently removed fucose and α2,3- and α2,6-linked sialic acid moieties from the epithelial glycocalyx. Recombinantly expressed P. timonensis NanH1 and NanH2 cleaved α2,3 and α2,6-linked sialic acids from the epithelial surface, and sialic acid removal by P. timonensis could be blocked using inhibitors. This study demonstrates that P. timonensis has distinct virulence-related properties that include initial adhesion and a high capacity for mucin degradation at the vaginal epithelial mucosal surface. Our results underline the importance of understanding the role of different anerobic bacteria in BV., Importance: Bacterial vaginosis (BV) is a common vaginal infection that affects a significant proportion of women and is associated with reduced fertility and increased risk of secondary infections. Gardnerella vaginalis is the most well-known BV-associated bacterium, but Prevotella species including P. timonensis and P. bivia may also play an important role. We showed that, similar to G. vaginalis , P. timonensis adhered well to the vaginal epithelium, suggesting that both bacteria could be important in the first stage of infection. Compared to the other bacteria, P. timonensis was unique in efficiently removing the protective mucin sugars that cover the vaginal epithelium. These results underscore that vaginal bacteria play different roles in the initiation and development of BV., Competing Interests: The authors declare no conflict of interest.

|$\text{L} $| -Fucose is the most widely distributed |$\text{L} $| -hexose in marine and terrestrial environments and presents a variety of functional roles. |$\text{L} $| -Fucose is the major monosaccharide in the polysaccharide fucoidan from cell walls of brown algae and is found in human milk oligosaccharides (HMOs) and the Lewis blood group system, where it is important in cell signaling and immune response stimulation. Removal of fucose from these biomolecules is catalyzed by fucosidases belonging to different carbohydrate-active enzyme (CAZy) families. Fucosidases of glycoside hydrolase family 29 (GH29) release α- |$\text{L} $| -fucose from non-reducing ends of glycans and display activities targeting different substrate compositions and linkage types. While several GH29 fucosidases from terrestrial environments have been characterized, much less is known about marine members of GH29 and their substrate specificities, as only four marine GH29 enzymes were previously characterized. Here, five GH29 fucosidases originating from an uncultured fucoidan-degrading marine bacterium (Paraglaciecola sp.) were cloned and produced recombinantly in Escherichia coli. All five enzymes (Fp231, Fp239, Fp240, Fp251 and Fp284) hydrolyzed the synthetic substrate CNP-α- |$\text{L} $| -fucose. Assayed against up to 17 fucose-containing oligosaccharides, Fp239 showed activity against the Lewis Y antigen, 2′- and 3-fucosyllactose, while Fp284 degraded 2′-fucosyllactose and Fuc(α 1,6)GlcNAc. Furthermore, Fp231 displayed strict specificity against Fuc(α 1,4)GlcNAc, a previously unreported specificity in GH29. Fp231 is a monomeric enzyme with pH and temperature optima at pH 5.6–6.0 and 25°C, hydrolyzing Fuc(α 1,4)GlcNAc with k cat = 1.3 s−1 and K m = 660 μM. Altogether, the findings extend our knowledge about GH29 family members from the marine environment, which are so far largely unexplored. [ABSTRACT FROM AUTHOR]

The elucidation of the physicochemical properties of glycosidases is essential for their subsequent technological application, which may include saccharide hydrolysis processes and oligosaccharide synthesis. As the application of cloning, purification and enzymatic immobilization methods can be time consuming and require a heavy financial investment, this study has validated the recombinant production of the set of Lacticaseibacillus rhamnosus fucosidases fused with Usp45 and SpaX anchored to the cell wall of Lacticaseibacillus cremoris subsp cremoris MG1363, with the aim of avoiding the purification and stabilization steps. The cell debris harboring the anchored AlfA, AlfB and AlfC fucosidases showed activity against p -nitrophenyl α-L-fucopyranoside of 6.11 ± 0.36, 5.81 ± 0.29 and 9.90 ± 0.58 U/mL, respectively, and exhibited better thermal stability at 50 °C than the same enzymes in their soluble state. Furthermore, the anchored AlfC fucosidase transfucosylated different acceptor sugars, achieving fucose equivalent concentrations of 0.94 ± 0.09 mg/mL, 4.11 ± 0.21 mg/mL, and 4.08 ± 0.15 mg/mL of fucosylgalatose, fucosylglucose and fucosylsucrose, respectively. • The anchoring of enzymes as an alternative to enzyme immobilization is unexplored. • L. rhamnosus fucosidases fused to Usp45 and SpaX were anchored to the host cell wall. • Anchored fucosidases showed better activity and stability than in soluble state. • Anchored AlfC fucosidase transfucosylated galactose, glucose and sucrose. • Enzymes fused with Usp45 and SpaX do not require purification and stabilization steps. [ABSTRACT FROM AUTHOR]

Human milk oligosaccharides (HMOs), which are natural bifidogenic prebiotics, were recently commercialized to fortify formula milk. However, HMO assimilation phenotypes of bifidobacteria vary by species and strain, which has not been fully linked to strain genotype. We have recently shown that specialized uptake systems, particularly for the internalization of major HMOs (fucosyllactose [FL]), are associated with the formation of a Bifidobacterium-rich gut microbial community. Phylogenetic analysis revealed that FL transporters have diversified into two clades harboring four clusters within the Bifidobacterium genus, but the underpinning functional diversity associated with this divergence remains underexplored. In this study, we examined the HMO consumption phenotypes of two bifidobacterial species, Bifidobacterium catenulatum subsp. kashiwanohense and Bifidobacterium pseudocatenulatum, both of which possess FL-binding proteins that belong to phylogenetic clusters with unknown specificities. Growth assays, heterologous gene expression experiments, and HMO consumption analyses showed that the FL transporter type from B. catenulatum subsp. kashiwanohense JCM 15439T conferred a novel HMO uptake pattern that includes complex fucosylated HMOs (lacto-N-fucopentaose II and lacto-N-difucohexaose I/II). Further genomic landscape analyses of FL transporterpositive bifidobacterial strains revealed that the H-antigen- or Lewis antigen-specific fucosidase gene(s) and FL transporter specificities were largely aligned. These results suggest that bifidobacteria have acquired FL transporters along with the corresponding gene sets necessary to utilize the imported HMOs. Our results provide insight into the species- and strain-dependent adaptation strategies of bifidobacteria in HMO-rich environments. [ABSTRACT FROM AUTHOR]

The effects of water activity (aw), pH, and temperature on transglycosylation activity of α-L-fucosidase from Thermotoga maritima in the synthesis of fucosylated oligosaccharides were evaluated using different water-organic cosolvent reaction systems. The optimum conditions of transglycosylation reaction were the pH range between 7 and 10 and temperature 90–95 °C. The addition of organic cosolvent decreased α-L-fucosidase transglycosylation activity in the following order: acetone > dimethyl sulfoxide (DMSO) > acetonitrile (0.51 > 0.42 > 0.18 mM/h). However, the presence of DMSO and acetone enhanced enzyme-catalyzed transglycosylation over hydrolysis as demonstrated by the obtained transglycosylation/hydrolysis rate (rT/H) values of 1.21 and 1.43, respectively. The lowest rT/H was calculated for acetonitrile (0.59), though all cosolvents tested improved the transglycosylation rate in comparison to a control assay (0.39). Overall, the study allowed the production of fucosylated oligosaccharides in water-organic cosolvent reaction media using α-L-fucosidase from T. maritima as biocatalyst. [ABSTRACT FROM AUTHOR]

Cellulosimicrobium strain SE3T was isolated from the San Elijo coastal lagoon near San Diego. A whole genome-based phylogenetic comparison shows great heterogeneity within the Cellulosimicrobium genus. Based on average nucleotide identity, whole genome-based comparison, and the presence of a unique l-fucose metabolic pathway, strain SE3T was shown to belong to a novel species within the genus, together with five other strains. The name Cellulosimicrobium fucosivorans sp. nov. is proposed, with strain SE3T as the type strain. The strain encodes a unique alpha-l-fucosidase and the l-fucose metabolic pathway is homologous to the one recently described in Campylobacter jejuni. C. fucosivorans is able to grow on l-fucose, and interestingly, the biosynthesis of the yellow carotenoid is dependent on the presence of l-fucose in the media. The ability to metabolize fucose and the linked production of carotenoids are expected to provide C. fucosivorans with a competitive advantage in the sunny coastal lagoon area. [ABSTRACT FROM AUTHOR]

Fucosylated oligosaccharides present in human milk perform various biological functions that benefit infants' health. These compounds can be also obtained by enzymatic synthesis. In this work, the effect of the immobilization of α-l-fucosidase from Thermotoga maritima on the synthesis of fucosylated oligosaccharides was studied, using lactose and 4-nitrophenyl-α-l-fucopyranoside (pNP-Fuc) as acceptor and donor substrates, respectively, and Eupergit® CM as an immobilization support. The enzyme was immobilized with 90% efficiency at pH 8 and ionic strength of 1.5 M. Immobilization decreased enzyme affinity for the donor substrate as shown by a 1.5-times higher KM value and a 22-times decrease of the kcat/KM ratio in comparison to the unbound enzyme. In contrast, no effect was observed on the synthesis/hydrolysis ratio (rs/rh) when α-l-fucosidase was immobilized. Also, the effect of initial concentration of substrates was studied. An increase of the acceptor concentration improved the yields of fucosylated oligosaccharides regardless enzyme immobilization. The synthesis yields of 38.9 and 40.6% were obtained using Eupergit® CM-bound or unbound enzyme, respectively, and 3.5 mM pNP-Fuc and 146 mM lactose. In conclusion, α-l-fucosidase from Thermotoga maritima was efficiently immobilized on Eupergit® CM support without affecting the synthesis of fucosylated oligosaccharides. [ABSTRACT FROM AUTHOR]

Fusarium graminearum produces an α-L-fucosidase, FgFCO1, which so far appears to be the only known fungal GH29 α-L-fucosidase that catalyzes the release of fucose from fucosylated xyloglucan. In our quest to synthesize bioactive glycans by enzymatic catalysis, we observed that FgFCO1 is able to catalyze a transglycosylation reaction involving transfer of fucose from citrus peel xyloglucan to lactose to produce 2'-fucosyllactose, an important human milk oligosaccharide. In addition to achieving maximal yields, control of the regioselectivity is an important issue in exploiting such a transglycosylation ability successfully for glycan synthesis. In the present study, we aimed to improve the transglycosylation efficiency of FgFCO1 through protein engineering by transferring successful mutations from other GH29 α-L-fucosidases. We investigated several such mutation transfers by structural alignment, and report that transfer of the mutation F34I from BiAfcB originating from Bifidobacterium longum subsp. infantis to Y32I in FgFCO1 and mutation of D286, near the catalytic acid/base residue in FgFCO1, especially a D286M mutation, have a positive effect on FgFCO1 transfucosylation regioselectivity. We also found that enzymatic depolymerization of the xyloglucan substrate increases substrate accessibility and in turn transglycosylation (i.e., transfucosylation) efficiency. The data include analysis of the active site amino acids and the active site topology of FgFCO1 and show that transfer of point mutations across GH29 subfamilies is a rational strategy for targeted protein engineering of a xyloglucan-active fungal α-L--fucosidase. [ABSTRACT FROM AUTHOR]

The enteropathogenic bacterium, Campylobacter jejuni, was considered to be non‐saccharolytic, but recently it emerged that l‐fucose plays a central role in C. jejuni virulence. Half of C. jejuni clinical isolates possess an operon for l‐fucose utilisation. In the intestinal tract, l‐fucose is abundantly available in mucin O‐linked glycan structures, but C. jejuni lacks a fucosidase enzyme essential to release the l‐fucose. We set out to determine how C. jejuni can gain access to these intestinal l‐fucosides. Growth of the fuc + C. jejuni strains, 129,108 and NCTC 11168, increased in the presence of l‐fucose while fucose permease knockout strains did not benefit from additional l‐fucose. With fucosidase assays and an activity‐based probe, we confirmed that Bacteriodes fragilis, an abundant member of the intestinal microbiota, secretes active fucosidases. In the presence of mucins, C. jejuni was dependent on B. fragilis fucosidase activity for increased growth. Campylobacter jejuni invaded Caco‐2 intestinal cells that express complex O‐linked glycan structures that contain l‐fucose. In infection experiments, C. jejuni was more invasive in the presence of B. fragilis and this increase is due to fucosidase activity. We conclude that C. jejuni fuc + strains are dependent on exogenous fucosidases for increased growth and invasion. [ABSTRACT FROM AUTHOR]

The deoxy sugar l -fucose is frequently found as a glycan constituent on and outside living cells, and in mammals it is involved in a wide range of biological processes including leukocyte trafficking, histo-blood group antigenicity and antibody effector functions. The manipulation of fucose levels in those biomedically important systems may provide novel insights and therapeutic leads. However, despite the large established sequence diversity of natural fucosidases, so far, very few enzymes have been characterized. We explored the diversity of the α- l -fucosidase-containing CAZY family GH29 by bio-informatic analysis, and by the recombinant production and exploration for fucosidase activity of a subset of 82 protein sequences that represent the family's large sequence diversity. After establishing that most of the corresponding proteins can be readily expressed in E. coli , more than half of the obtained recombinant proteins (57% of the entire subset) showed activity towards the simple chromogenic fucosylated substrate 4-nitrophenyl α- l -fucopyranoside. Thirty-seven of these active GH29 enzymes (and the GH29 subtaxa that they represent) had not been characterized before. With such a sequence diversity-based collection available, it can easily be used to screen for fucosidase activity towards biomedically relevant fucosylated glycoproteins. As an example, the subset was used to screen GH29 members for activity towards the naturally occurring sialyl-Lewis x-type epitope on glycoproteins, and several such enzymes were identified. Together, the results provide a significant increase in the diversity of characterized GH29 enzymes, and the recombinant enzymes constitute a resource for the further functional exploration of this enzyme family. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Synthesis of N -alkylated 1-deoxyfuconojirimycins as α-fucosidase inhibitors. • N -Decyl-1-deoxyfuconojirimycin was toxic towards various cancer cells. • Development of homology models for fucosidases from bovine and human origin. Abstract 1,5-Dideoxy-1,5-imino- l -fucitol (1-deoxyfuconojirimycin, DFJ) is an iminosugar that inhibits fucosidases. Herein, N -alkyl DFJs have been synthesised and tested against the α-fucosidases of T. maritima (bacterial origin) and B. taurus (bovine origin). The N -alkyl derivatives were inactive against the bacterial fucosidase, while inhibiting the bovine enzyme. Docking of inhibitors to homology models, generated for the bovine and human fucosidases, was carried out. N -Decyl-DFJ was toxic to cancer cell lines and was more potent than the other N -alkyl DFJs studied. [ABSTRACT FROM AUTHOR]

α- l -Fucosidases are widely occurring enzymes that remove fucose residues from N - and O-fucosylated glycoproteins. Comparison of amino acid sequences of fucosidases reveals that although the nucleophile is conserved among all α- l -fucosidases, the position of the acid/base residue is quite variable. Although several site-directed mutation studies have previously been performed on bacterial fucosidases, the only eukaryotic fucosidase so studied was the human fucosidase. Recent alignments indicate that human and Arthropoda α- l -fucosidases share at least 50% identity and the acid/base residue seems to be conserved among them suggesting a common acid/base residue in Metazoa. Here we describe the cloning and expression in Pichia pastoris of a very active α- l -fucosidase from the spider Nephilingis cruentata (NcFuc) with a K mvalue for pNPFuc of 0.4 mM. NcFuc hydrolyzed fucoidan, 2´fucosyllactose and also lacto- N -difucohexaose II. Mutants modified at the conserved residues D214N, E209A, E59A were expressed and characterized. The 500-fold lower k catof D214N than the wild type was consistent with a role in catalysis, as was the 8000-fold lower k catvalue of E59A. This was supported by the 57-fold increase in the k catof E59A upon addition of azide. A complex pH/rate profile was seen for the wild-type and mutant forms of NcFuc, similar to those measured previously for the Sulfolobus fucosidase. The non-conservative catalytic structure and distinct active site organization reinforce the necessity of structural studies of new fucosidases. [ABSTRACT FROM AUTHOR]

Fusarium graminearum produces an α-l-fucosidase, FgFCO1, which so far appears to be the only known fungal GH29 α-l-fucosidase that catalyzes the release of fucose from fucosylated xyloglucan. In our quest to synthesize bioactive glycans by enzymatic catalysis, we observed that FgFCO1 is able to catalyze a transglycosylation reaction involving transfer of fucose from citrus peel xyloglucan to lactose to produce 2′-fucosyllactose, an important human milk oligosaccharide. In addition to achieving maximal yields, control of the regioselectivity is an important issue in exploiting such a transglycosylation ability successfully for glycan synthesis. In the present study, we aimed to improve the transglycosylation efficiency of FgFCO1 through protein engineering by transferring successful mutations from other GH29 α-l-fucosidases. We investigated several such mutation transfers by structural alignment, and report that transfer of the mutation F34I from BiAfcB originating from Bifidobacterium longum subsp. infantis to Y32I in FgFCO1 and mutation of D286, near the catalytic acid/base residue in FgFCO1, especially a D286M mutation, have a positive effect on FgFCO1 transfucosylation regioselectivity. We also found that enzymatic depolymerization of the xyloglucan substrate increases substrate accessibility and in turn transglycosylation (i.e., transfucosylation) efficiency. The data include analysis of the active site amino acids and the active site topology of FgFCO1 and show that transfer of point mutations across GH29 subfamilies is a rational strategy for targeted protein engineering of a xyloglucan-active fungal α-l-fucosidase.

The influence of CaCl2 and NaCl in the hydrolytic activity and the influence of CaCl2 in the synthesis of fucosylated oligosaccharides using α-L-fucosidase from Thermotoga maritima were evaluated. The hydrolytic activity of α-L-fucosidase from Thermotoga maritima displayed a maximum increase of 67% in the presence of 0.8 M NaCl with water activity (aw) of 0.9672 and of 138% in the presence of 1.1 M CaCl2 (aw 0.9581). In addition, the hydrolytic activity was higher when using CaCl2 compared to NaCl at aw of 0.8956, 0.9581 and 0.9672. On the other hand, the effect of CaCl2 in the synthesis of fucosylated oligosaccharides using 4-nitrophenyl-fucose as donor substrate and lactose as acceptor was studied. In these reactions, the presence of 1.1 M CaCl2 favored the rate of transfucosylation, and improved the yield of synthesis duplicating and triplicating it with lactose concentrations of 58 and 146 mM, respectively. CaCl2 did not significatively affect hydrolysis rate in these reactions. The combination of the activating effect of CaCl2, the decrement in aw and lactose concentration had a synergistic effect favoring the synthesis of fucosylated oligosaccharides. [ABSTRACT FROM AUTHOR]

Fucosylated oligosaccharides, such as 2′-fucosyllactose in human milk, have important biological functions such as prebiotics and preventing infection. In this work, the effect of an acceptor substrate (lactose) and the donor substrate 4-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) on the synthesis of a fucosylated trisaccharide was studied in a transglycosylation reaction using α-L-fucosidase from Thermotoga maritima. Conducting a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), it was demonstrated that synthesized oligosaccharide corresponded to a fucosylated trisaccharide, and high-performance liquid chromatography (HPLC) of the hydrolyzed compound confirmed it was fucosyllactose. As the concentration of the acceptor substrate increased, the concentration and synthesis rate of the fucosylated trisaccharide also increased, and the highest concentration obtained was 0.883 mM (25.2% yield) when using the higher initial lactose concentration (584 mM). Furthermore, the lower donor/acceptor ratio had the highest synthesis, so at the molar ratio of 0.001, a concentration of 0.286 mM was obtained (32.5% yield). [ABSTRACT FROM AUTHOR]

BACKGROUND: Carcinogenesis, a multistep process involves sequential changes during neoplastic transformation. The various hallmarks of cancer aid in cell survival, proliferation, and dissemination. Aberrant glycosylation, a recently defined hallmark of cancer, is influenced by glycosylation enzymes during carcinogenesis. Therefore, the present study measured α-2,3 and α-2,6 sialyltransferase (ST), sialidase, and α-L-fucosidase activity in patients with oral precancerous conditions (OPC) and oral cancer patients. SUBJECTS: The study enrolled 100 oral cancer patients, 50 patients with OPC, 100 healthy controls, and 46 posttreatment follow-ups of oral cancer patients. Blood and saliva were collected from all the participants. MATERIALS AND METHODS: Sialidase activity was measured by spectrofluorimetric method, α-2,3 and α-2,6 ST by ELISA using biotinylated lectins, and α-L-fucosidase by spectrophotometric method. RESULTS: The results depicted increased levels of sialidase, α-2,3 and α-2,6 ST, α-L-fucosidase in patients with OPC and oral cancer patients. Receiver operating characteristic curve indicated significant discriminatory efficacy in distinguishing controls and oral cancer patients for serum and salivary sialidase and α-Lfucosidase activity, and serum α-2,6 ST. Furthermore, serum and salivary α-L-fucosidase activity and serum sialidase activity significantly distinguished controls and patients with OPC. Serum and salivary sialidase, α-L-fucosidase, and serum α-2,3 ST activity were higher in patients with metastasis as compared to nonmetastatic patients. Higher values of serum α-L-fucosidase activity were significantly associated with low-overall survival. CONCLUSION: The increased levels of enzymes correlated with tumor initiation, progression, and metastasis in oral cancer patients. The alterations in glycosyltransferases/glycosidases thus support the view of glycosylation as a hallmark of cancer. [ABSTRACT FROM AUTHOR]

The α-1,3/4- l -fucosidases (EC 3.2.1.111; GH29) Bb AfcB from Bifidobacterium bifidum and Cp Afc2 from Clostridium perfringens can catalyse formation of the human milk oligosaccharide (HMO) lacto- N -fucopentaose II (LNFP II) through regioselective transfucosylation of lacto- N- tetraose (LNT) with 3-fucosyllactose (3FL) as donor substrate. The current work exploits structural differences between the two enzymes with the aim of engineering Bb AfcB into a more efficient transfucosidase and approaches an understanding of structure-function relations of hydrolytic activity vs. transfucosylation activity in GH29. Replacement of a 23 amino acids long α-helical loop close to the active site of Bb AfcB with the corresponding 17-aminoacid α-helical loop of Cp Afc2 resulted in almost complete abolishment of the hydrolytic activity on 3FL (6000 times lower hydrolytic activity than WT Bb AfcB), while the transfucosylation activity was lowered only one order of magnitude. In turn, the loop engineering resulted in an α-1,3/4- l -fucosidase with transfucosylation activity reaching molar yields of LNFP II of 39 ± 2% on 3FL and negligible product hydrolysis. This was almost 3 times higher than the yield obtained with WT Bb AfcB (14 ± 0.3%) and comparable to that obtained with Cp Afc2 (50 ± 8%). The obtained transfucosylation activity may expand the options for HMO production: mixtures of 3FL and LNT could be enriched with LNFP II, while mixtures of 3FL and lacto- N -neotetraose (LNnT) could be enriched with LNFP III. [ABSTRACT FROM AUTHOR]

Fucosyl-oligosaccharides are natural prebiotics that promote the growth of probiotics in human gut and stimulate the innate immune system. In this work, the release of α-lfucosidase by Lactobacillus rhamnosus GG, and the use of this enzyme for the synthesis of fucosyl-oligosaccharides were investigated. Since α-lfucosidase is a membrane-bound enzyme, its release from the cells was induced by addition of 4-nitrophenyl-α-l-fucopyranoside (pNP-Fuc). Enzyme activity associated with the cell was recovered at 78% of its total activity. Fucosyl-oligosaccharides where synthesized using α-l-fucosidase extract and pNP-Fuc as donor substrate, and D-lactose or D-lactulose as acceptor substrates, reaching a yield up to 25%. Fucosyllactose was obtained as a reaction product with D-lactose, and its composition was confirmed by mass spectrometry (MALDI-TOF MS). It is possible that the fucosyl-oligosaccharide synthesized in this study has biological functions similar to human milk oligosaccharides.

Human milk oligosaccharides (HMOs) signify a unique group of oligosaccharides in breast milk, which is of major importance for infant health and development. The functional benefits of HMOs create an enormous impetus for biosynthetic production of HMOs for use as additives in infant formula and other products. HMO molecules can be synthesized chemically, via fermentation, and by enzymatic synthesis. This treatise discusses these different techniques, with particular focus on harnessing enzymes for controlled enzymatic synthesis of HMO molecules. In order to foster precise and high-yield enzymatic synthesis, several novel protein engineering approaches have been reported, mainly concerning changing glycoside hydrolases to catalyze relevant transglycosylations. The protein engineering strategies for these enzymes range from rationally modifying specific catalytic residues, over targeted subsite −1 mutations, to unique and novel transplantations of designed peptide sequences near the active site, so-called loop engineering. These strategies have proven useful to foster enhanced transglycosylation to promote different types of HMO synthesis reactions. The rationale of subsite −1 modification, acceptor binding site matching, and loop engineering, including changes that may alter the spatial arrangement of water in the enzyme active site region, may prove useful for novel enzyme-catalyzed carbohydrate design in general.

α- l -Fucosyl residues are frequently found in oligosaccharides, polysaccharides and glycoconjugates which play fundamental roles in various biological processes. α- l -Fucosidases, glycoside hydrolases for catalyzing the removal of α- l -fucose, can serve as desirable tools in the study and the modification of fucose-containing biomolecules. In this study, an α- l -fucosidase named as Alf1_Wf was purified from a marine bacterium Wenyingzhuangia fucanilytica by using a combination of chromatographic procedures. The sequence of Alf1_Wf was identified via proteomics analysis against the predicted proteome of the bacterium. Recombinant Alf1_Wf with 6×His tag was expressed in E. coli and showed α- l -fucosidase activity. Sequence annotation revealed that Alf1_Wf contained a combination of GH29 catalytic domain and CBM35 accessory domain. Alf1_Wf was confirmed as a member of GH29-A subfamily based on the phylogenetic analysis. Furthermore, biochemical properties and kinetic characteristics of the enzyme were also determined. Substrate specificity determination showed that Alf1_Wf was capable in hydrolyzing α1,4-fucosidic linkage and synthetic substrate pNP-fucose. Besides, Alf1_Wf could act on partially degraded fucoidan. This study successfully purified, identified, cloned, expressed and characterized a novel α- l -fucosidase, and meanwhile revealed a new multidomain structure of glycoside hydrolase. The knowledge gained from this study should facilitate the further research and application of α- l -fucosidases. [ABSTRACT FROM AUTHOR]

Three novel bacterial a-L-fucosidases, which cleave terminal fucosyl residues from glycoconjugates are reported in this work. Originating from the recently discovered bacterium Emticicia oligotrophica, recombinant fucosidase isoforms designated as Eo0918, Eo3066 and Eo3812 were shown to have the highest activity between pH 6.0 and 7.0 and temperature optima between 30 and 45°C. All enzymes catalyzed the hydrolysis of the model substrate pNP-a-L-fucose and revealed significantly different regiospecificities towards fucose-containing oligosaccharides: Eo0918 liberated exclusively a1,6-linked fucose and Eo3812 released only a1,3-fucosyl residues, whereas Eo3066 showed broader substrate promiscuity. The enzymatic activity of Eo0918 and Eo3812 increased upon the addition of Ca2+, Mn2+ and Zn2+ ions, whereas the activity of Eo3066 was significantly decreased in the presence of these metal ions. In addition, Eo0918 also catalyzed the transfer of fucose from pNP-a-L-fucose to the 7-hydroxyl group of 4-methylumbelliferone with up to 15%transglycosylation yield. Facile recombinant expression in E. coli, distinct substrate specificities and the transglycosylation ability of Eo0918 presented herein make these newly discovered fucosidases valuable candidates for bioanalytical and biotechnological applications. [ABSTRACT FROM AUTHOR]

The tumor suppressor p53 functions by inducing the transcription of a collection of target genes. We previously attempted to identify p53 target genes by microarray expression and Ch IP-sequencing analyses. In this study, we describe a novel p53 target gene, FUCA1, which encodes a fucosidase. Although fucosidase, α- l-1 ( FUCA1) has been reported to be a lysosomal protein, we detected it outside of lysosomes and observed that its activity is highest at physiological pH. As there is a reported association between fucosylation and tumorigenesis, we investigated the potential role of FUCA1 in cancer. We found that overexpression of FUCA1, but not a mutant defective in enzyme activity, suppressed the growth of cancer cells and induced cell death. Furthermore, we showed that FUCA1 reduced fucosylation and activation of epidermal growth factor receptor, and concomitantly suppressed epidermal growth factor signaling pathways. FUCA1 loss-of-function mutations are found in several cancers, its expression is reduced in cancers of the large intestine, and low FUCA1 expression is associated with poorer prognosis in several cancers. These results show that protein defucosylation mediated by FUCA1 is involved in tumor suppression. [ABSTRACT FROM AUTHOR]

The synthesis and the biological evaluation of a new ferrocenyl-iminosugar conjugate designed for fucosidase inhibitory and anticancer activity is described. The compound showed strong affinity for fucosidase from bovine kidney ( K i = 23 nM) and from Bacteroides thetaiotaomicron ( K i = 150 nM), displaying a 10-fold tighter binding affinity for these enzymes than the previous analogs. The interaction pattern that improves binding has been evaluated through structural analysis of the inhibitor–enzyme complex. The ferrocenyl-iminosugar exhibits significant anticancer activity on MDA-MB-231 and SK-MEL28 cell lines at 100 μM. [ABSTRACT FROM AUTHOR]

Human milk oligosaccharides (HMOs) are recognized as benefiting breast-fed infants in multiple ways. As a result, there is growing interest in the synthesis of HMOs mimicking their natural diversity. Most HMOs are fucosylated oligosaccharides. α-L-Fucosidases catalyze the hydrolysis of α-L-fucose from the non-reducing end of a glucan. They fall into the glycoside hydrolase GH29 and GH95 families. The GH29 family fucosidases display a classic retaining mechanism and are good candidates for transfucosidase activity. We recently demonstrated that the α-L-fucosidase from Thermotoga maritima (TmaFuc) from the GH29 family can be evolved into an efficient transfucosidase by directed evolution (Osanjo et al. 2007). In this work, we developed semi-rational approaches to design an α-Ltransfucosidase starting with the α-L-fucosidase from commensal bacteria Bifidobacterium longum subsp. infantis (BiAfcB, Blon_2336). Efficient fucosylation was obtained with enzyme mutants (L321P-BiAfcB and F34I/L321P-BiAfcB) enabling in vitro synthesis of lactodifucotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose III and lacto-N-difucohexaose I. The enzymes also generated more complex HMOs like fucosylated para-lacto-N-neohexaose (F-p-LNnH) and mono- or difucosylated lacto-N-neohexaose (F-LNnH-I, F-LNnH-II and DF-LNnH). It is worth noting that mutation at these two positions did not result in a strong decrease in the overall activity of the enzyme, which makes these variants interesting candidates for large-scale transfucosylation reactions. For the first time, this work provides an efficient enzymatic method to synthesize the majority of fucosylated HMOs. [ABSTRACT FROM AUTHOR]

The aim of the present study was to demonstrate the presence of alpha-L-fucosidase in Trypanosoma cruzi. Immunocytochemical and biochemical techniques were used to localize and characterize a membrane-associated, neutral-pH-optimum, alpha-L-fucosidase from the parasite. Light and electron microscopy localized the alpha-L-fucosidase specifically on the surface of the parasite and on membranes in the posterior region of the epimastigote stage. Although much less intense, labeling was also detected on the surface of trypomastigotes. At least 50% of the alpha-L-fucosidase activity was associated with epimastigote membrane solubilized with 1 M NaCl or 1% Triton X-100, suggesting that alpha-L-fucosidase is peripherally associated with membranes. The enzyme from epimastigotes had a neutral pH optimum (near 7) but displayed low specific activity when p-nitrophenyl-alpha-L-fucoside was employed as substrate (0.028 U/mg protein for epimastigotes and 0.015 U/mg protein for tissue culture-derived trypomastigotes). Polyacrylamide gel electrophoresis and Western blotting analysis both showed an expected 50-kDa polypeptide which was immunoreactive with anti-alpha-L-fucosidase antibodies.

Interactions between the enteric nervous system (ENS) and intestinal epithelium are thought to play a vital role in intestinal homeostasis. How the ENS monitors the frontier with commensal and pathogenic microbes while maintaining epithelial function remains unclear. Here, by combining subdiaphragmatic vagotomy with transcriptomics, chemogenetic strategy, and coculture of enteric neuron-intestinal organoid, we show that enteric neurons expressing VIP shape the α1,2-fucosylation of intestinal epithelial cells (IECs). Mechanistically, neuropeptide VIP activates fut2 expression via the Erk1/2-c-Fos pathway through the VIPR1 receptor on IECs. We further demonstrate that perturbation of enteric neurons leads to gut dysbiosis through α1,2-fucosylation in the steady state and results in increased susceptibility to alcohol-associated liver disease (ALD). This was attributed to an imbalance between beneficial Bifidobacterium and opportunistic pathogenic Enterococcus faecalis in ALD. In addition, Bifidobacterium α1,2-fucosidase may promote Bifidobacterium adhesion to the mucosal surface, which restricts Enterococcus faecalis overgrowth and prevents ALD progression. [Display omitted] • Enteric neuron-derived VIP shapes IECs fucosylation and gut microbiota composition • VIP activates fut2 expression in IECs via the Erk1/2-c-Fos pathway • α1,2-fucosidase enhances Bifidobacterial adhesion to fucosyl-conjugated mucus • Bifidobacteria restrict cytolytic E. faecalis growth and prevent ALD progression Lei et al. show that enteric neurons expressing VIP direct a program of gut mucosal fucosylation via the Erk1/2-c-Fos pathway. The reduced abundance of enteric VIPergic neurons leads to the imbalance of abundance between beneficial Bifidobacterium and pathogenic E. faecalis and enhances the susceptibility of ALD. [ABSTRACT FROM AUTHOR]

Enzymatic hydrolysis of α-L-fucose from fucosylated glycoconjugates is consequential in bacterial infections and the neurodegenerative lysosomal storage disorder fucosidosis. Understanding human α-L-fucosidase catalysis, in an effort toward drug design, has been hindered by the absence of three-dimensional structural data for any animal fucosidase. Here, we have used cryoelectron microscopy (cryo-EM) to determine the structure of human lysosomal α-L-fucosidase (FucA1) in both an unliganded state and in complex with the inhibitor deoxyfuconojirimycin. These structures, determined at 2.49 Å resolution, reveal the homotetrameric structure of FucA1, the architecture of the catalytic center, and the location of both natural population variations and disease-causing mutations. Furthermore, this work has conclusively identified the hitherto contentious identity of the catalytic acid/base as aspartate-276, representing a shift from both the canonical glutamate acid/base residue and a previously proposed glutamate residue. These findings have furthered our understanding of how FucA1 functions in both health and disease. [Display omitted] • Cryo-EM structure of FucA1 resolves debate over the catalytic mechanism • Aspartate 276, which is conserved for all animal homologs, is the catalytic acid/base • A misfolded FucA1 disease variant (S150F) is stabilized by an iminosugar inhibitor Human lysosomal fucosidase (FucA1) recycles fucosylated glycans. Aberrations in the enzyme result in fucosidosis. Armstrong et al. use single-particle cryo-EM to resolve the contentious mechanism of this enzyme. They also demonstrate stabilization of a misfolded variant by deoxyfuconojirimycin and determine how FucA1 accommodates this inhibitor. [ABSTRACT FROM AUTHOR]

The degradation of blood group glycolipid A-6-2 (GalNAc(α1→3)[Fucα1→2]Gal(β1→4)GlcNAc(β1→3)Gal (β1→4)Glc(β1→1′)Cer, IV2-α-fucosyl-IV3-α-N-acetylgalactosaminylneolactotetraosylceramide), tritium-labeled in its ceramide moiety, was studied in situ, in skin fibroblast cultures from normal controls, from patients with defects of lysosomal α-N-acetylgalactosaminidase, and from patients with other lysosomal storage diseases. Uptake of the glycolipid with apolipoprotein E-coated liposomes was linear with time and with the amount of glycolipid added. In normal cells, the expected array of less polar products and some lipids resulting from re-using the liberated sphingosine, mainly sphingomyelin and phosphatidylcholine, were formed. In α-N-acetylgalactosaminidase-deficient cells, the glycolipid was virtually not degraded; product formation was less than 2% of the normal control rate, suggesting that blood group A-active glycolipids contribute as storage compounds to the pathogenesis of this disease. The expected accumulation of degradation intermediates was seen in fucosidosis, and in Sandhoff, Gaucher, and Farber disease cells, whereas normal turnover rates were found in Tay-Sachs disease cells, GM2 activator-deficient (variant AB of GM2 gangliosidosis) and in sulfatide activator- (sap-B-) deficient cells. In GM1 gangliosidosis and in sap precursor-deficient cells, the lysosomal glycolipid catabolism was found to be strongly retarded; accumulation of individual products could not be seen. Skin fibroblasts from patients with α-N-acetylgalactosaminidase deficiency (Schindler disease) cannot degrade the major blood group A glycolipid.—Asfaw, B., D. Schindler, J. Ledvinová, B. Černý, F. Šmíd, and E. Conzelmann. Degradation of blood group A glycolipid A-6-2 by normal and mutant human skin fibroblasts.

Bats are mammals of the order Chiroptera, which represents about 20% of all mammalian species worldwide. Bats have evolved various reproductive strategies, such as delayed sperm maturation, sperm storage, delayed fertilization, delayed implantation and delayed development. The strategies of delayed sperm maturation allow female bats, especially hibernating species, to reproduce successfully. Alpha-L-Fucosidase 1 gene (FUCA1) has been proved to be involved in the processes of sperm maturation and sperm-egg interactions in mammals. It is still not clear how the FUCA1 gene evolved in bats. Therefore, it is important to determine the evolutionary pattern of FUCA1 gene and positively selected sites which affect the reproductive activity of bats. In this work, we cloned the coding regions of FUCA1 from 7 species of bats and performed an evolutionary study of this gene. Although selection pressure varied among different lineages, FUCA1 gene underwent positive selection in the ancestral species leading to Old World fruit bats. Our results demonstrated that FUCA1 is basically conserved in hibernating bats but has evolved within the ancestral species of Old World fruit bats to adapt their survival environments. [ABSTRACT FROM AUTHOR]