Molecular Basis for Galactosylation of Core Fucose Residues in Invertebrates: IDENTIFICATION OF CAENORHABDITIS ELEGANS N-GLYCAN CORE ��1,6-FUCOSIDE ��1,4-GALACTOSYLTRANSFERASE GALT-1 AS A MEMBER OF A NOVEL GLYCOSYLTRANSFERASE FAMILY*

Galectin CGL2 from the ink cap mushroom Coprinopsis cinerea displays toxicity toward the model nematode Caenorhabditis elegans. A mutation in a putative glycosyltransferase-encoding gene resulted in a CGL2-resistant C. elegans strain characterized by N-glycans lacking the β1,4-galactoside linked to the α1,6-linked core fucose. Expression of the corresponding GALT-1 protein in insect cells was used to demonstrate a manganese-dependent galactosyltransferase activity. In vitro, the GALT-1 enzyme showed strong selectivity for acceptors with α1,6-linked N-glycan core fucosides and required Golgi- dependent modifications on the oligosaccharide antennae for optimal synthesis of the Gal-β1,4-fucose structure. Phylogenetic analysis of the GALT-1 protein sequence identified a novel glycosyltransferase family (GT92) with members widespread among eukarya but absent in mammals.     

Unusual β1-4-galactosidase activity of an α1-6-mannosidase from Xanthomonas manihotis in the processing of branched hybrid and complex glycans

Mannosidases are a diverse group of glycoside hydrolases that play crucial roles in mannose trimming of oligomannose glycans, glycoconjugates, and glycoproteins involved in numerous cellular processes, such as glycan biosynthesis and metabolism, structure regulation, cellular recognition, and cell–pathogen interactions. Exomannosidases and endomannosidases cleave specific glycosidic bonds of mannoside linkages in glycans and can be used in enzyme-based methods for sequencing of isomeric glycan structures. α1-6-mannosidase from Xanthomonas manihotis is known as a highly specific exoglycosidase that removes unbranched α1-6 linked mannose residues from oligosaccharides. However, we discovered that this α1-6-mannosidase also possesses an unexpected β1-4-galactosidase activity in the processing of branched hybrid and complex glycans through our use of enzymatic reactions, high performance anion-exchange chromatography, and liquid chromatography mass spectrometric sequencing. Our docking simulation of the α1-6-mannosidase with glycan substrates reveals potential interacting residues in a relatively shallow pocket slightly differing from its homologous enzymes in the glycoside hydrolase 125 family, which may be responsible for the observed higher promiscuity in substrate binding and subsequent terminal glycan hydrolysis. This observation of novel β1-4-galactosidase activity of the α1-6-mannosidase provides unique insights into its bifunctional activity on the substrate structure-dependent processing of terminal α1-6-mannose of unbranched glycans and terminal β1-4-galactose of hybrid and complex glycans. The finding thus suggests the dual glycosidase specificity of this α1-6-mannosidase and the need for careful consideration when used for the structural elucidation of glycan isomers.     

A Gene of the β3-Glycosyltransferase Family Encodes N-Acetylglucosaminyltransferase II Function in Trypanosoma brucei

The bloodstream form of the human pathogen Trypanosoma brucei expresses oligomannose, paucimannose, and complex N-linked glycans, including some exceptionally large poly-N-acetyllactosamine-containing structures. Despite the presence of complex N-glycans in this organism, no homologues of the canonical N-acetylglucosaminyltransferase I or II genes can be found in the T. brucei genome. These genes encode the activities that initiate the elaboration of the Manα1–3 and Manα1–6 arms, respectively, of the conserved trimannosyl-N-acetylchitobiosyl core of N-linked glycans. Previously, we identified a highly divergent T. brucei N-acetylglucosaminyltransferase I (TbGnTI) among a set of putative T. brucei glycosyltransferase genes belonging to the β3-glycosyltransferase superfamily (Damerow, M., Rodrigues, J. A., Wu, D., Güther, M. L., Mehlert, A., and Ferguson, M. A. (2014) J. Biol. Chem. 289, 9328–9339). Here, we demonstrate that TbGT15, another member of the same β3-glycosyltransferase family, encodes an equally divergent N-acetylglucosaminyltransferase II (TbGnTII) activity. In contrast to multicellular organisms, where GnTII activity is essential, TbGnTII null mutants of T. brucei grow in culture and are still infectious to animals. Characterization of the large poly-N-acetyllactosamine containing N-glycans of the TbGnTII null mutants by methylation linkage analysis suggests that, in wild-type parasites, the Manα1–6 arm of the conserved trimannosyl core may carry predominantly linear poly-N-acetyllactosamine chains, whereas the Manα1–3 arm may carry predominantly branched poly-N-acetyllactosamine chains. These results provide further detail on the structure and biosynthesis of complex N-glycans in an important human pathogen and provide a second example of the adaptation by trypanosomes of β3-glycosyltransferase family members to catalyze β1–2 glycosidic linkages.     

In silico prediction of proteins related to xyloglucan fucosyltransferases in Solanaceae genomes

Two independent studies have shown that the cell wall of pollen tubes from tobacco and tomato species contained fucosylated xyloglucan (XyG). These findings are intriguing as many reports have shown that XyG of somatic cells of these species is not fucosylated but instead is arabinosylated. In order to produce fucosylated XyG, plants must express a functional galactoside α-2-fucosyltransferase. Here, using a bioinformatics approach, we show that several candidate genes coding for XyG fucosyltransferases are present in the genome of coffee and several Solanaceae species including tomato, tobacco, potato, eggplant and pepper. BLAST and protein alignments with the 2 well-characterized XyG fucosyltransferases from Arabidopsis thaliana and Pisum sativum revealed that at least 6 proteins from different Solanaceae species and from coffee displayed the 3 conserved motifs required for XyG fucosyltransferase activity.     

Bisecting Galactose as a Feature of N-Glycans of Wild-type and Mutant Caenorhabditis elegans

The N-glycosylation of the model nematode Caenorhabditis elegans has proven to be highly variable and rather complex; it is an example to contradict the existing impression that “simple” organisms possess also a rather simple glycomic capacity. In previous studies in a number of laboratories, N-glycans with up to four fucose residues have been detected. However, although the linkage of three fucose residues to the N,N′-diacetylchitobiosyl core has been proven by structural and enzymatic analyses, the nature of the fourth fucose has remained uncertain. By constructing a triple mutant with deletions in the three genes responsible for core fucosylation (fut-1, fut-6 and fut-8), we have produced a nematode strain lacking products of these enzymes, but still retaining maximally one fucose residue on its N-glycans. Using mass spectrometry and HPLC in conjunction with chemical and enzymatic treatments as well as NMR, we examined a set of α-mannosidase-resistant N-glycans. Within this glycomic subpool, we can reveal that the core β-mannose can be trisubstituted and so carries not only the ubiquitous α1,3- and α1,6-mannose residues, but also a “bisecting” β-galactose, which is substoichiometrically modified with fucose or methylfucose. In addition, the α1,3-mannose can also be α-galactosylated. Our data, showing the presence of novel N-glycan modifications, will enable more targeted studies to understand the biological functions and interactions of nematode glycans.Nematodes represent, along with arthropods, one of the largest groups of animals to exist on the planet; 25.000 species are described, but the existence of up to one million has been estimated (1, 2). They have various ecological niches and include free-living “worms” in the soil, fungivorous, entomopathogenic, and necromenic species as well as parasites of plants and mammals, which share the basic conserved body plan (more-or-less a digestive tube surrounded with muscle, whether larger or smaller). There are five major clades (Rhabditina, Enoplia, Spirurina, Tylenchina, and Dorylaimia) (2), yet the glycosylation of only a few nematode species has been studied with an inevitable focus on the model nematode Caenorhabditis elegans and parasitic species (3). Thereby, the use of C. elegans mutants has been highly valuable in dissecting aspects of nematode N-glycan biosynthesis and revealing the in vivo substrates for certain glycosyltransferases (4).As many nematodes are parasites, their interactions with the immune systems of their hosts have attracted attention; particularly, there are relationships between autoimmunity, allergy, vaccination, and helminth infections. The “old friends” hypothesis seeks to understand the evolutionary factors that have shaped the immune system and to explain correlations between lifestyles in the developed world and modern “epidemics,” which are due to immunological misbalance (5–7). Promising data have suggested that “worm therapy” may bring advantages to some patients with Crohn''s disease or allergies (8, 9); however, such approaches are controversial. Nevertheless, crude extracts even of Caenorhabditis elegans were shown to induce a glycan-dependent Th2 response (10), whereas the excretory-secretory products of some nematodes also have immunomodulatory activity (11). Furthermore, the native glycoproteins of some nematodes have proven effective in vaccination trials, whereas recombinant forms are not, which is suggestive that post-translational modifications may have a role in an efficacious immune response (12).As at least some of the molecules relevant to nematode immunomodulation or vaccination are glycoproteins, a proper understanding of nematode glycosylation is of biomedical and veterinary relevance. Over the years, it has become apparent that the core chitobiosyl region of nematode N-glycans is subject to a range of modifications, with up to three core fucose residues being present (α1,3- and α1,6-linked on the reducing-terminal “proximal” GlcNAc and α1,3-linked on the second “distal” GlcNAc). However, up to four fucose residues have been detected on C. elegans N-glycans and the exact nature of the linkage of the fourth fucose has remained obscure despite work in our own and other laboratories (3, 13–15). Combined with the latest knowledge regarding the specificity of C. elegans core fucosyltransferases (13, 16, 17) as well as our recent data regarding the exact structures of N-glycans from the C. elegans double hexosaminidase mutant and other nematodes (18–20), we concluded that some models for the tri- and tetrafucosylated N-glycans were incorrect. By preparing a triple mutant unable to core fucosylate its N-glycans, we generated a C. elegans strain containing maximally one fucose residue on the N-linked oligosaccharides. Thereby a pool of unusual mannosidase-resistant N-glycans was identified and, using mass spectrometry (MS) and NMR, we reveal their modification with bisecting galactose frequently capped with fucose or methylfucose.     

Functional Coupling of a Nematode Chemoreceptor to the Yeast Pheromone Response Pathway

Sequencing of the Caenorhabditis elegans genome revealed sequences encoding more than 1,000 G-protein coupled receptors, hundreds of which may respond to volatile organic ligands. To understand how the worm''s simple olfactory system can sense its chemical environment there is a need to characterise a representative selection of these receptors but only very few receptors have been linked to a specific volatile ligand. We therefore set out to design a yeast expression system for assigning ligands to nematode chemoreceptors. We showed that while a model receptor ODR-10 binds to C. elegans Gα subunits ODR-3 and GPA-3 it cannot bind to yeast Gα. However, chimaeras between the nematode and yeast Gα subunits bound to both ODR-10 and the yeast Gβγ subunits. FIG2 was shown to be a superior MAP-dependent promoter for reporter expression. We replaced the endogenous Gα subunit (GPA1) of the Saccharomyces cerevisiae (ste2Δ sst2Δ far1Δ) triple mutant (“Cyb”) with a Gpa1/ODR-3 chimaera and introduced ODR-10 as a model nematode GPCR. This strain showed concentration-dependent activation of the yeast MAP kinase pathway in the presence of diacetyl, the first time that the native form of a nematode chemoreceptor has been functionally expressed in yeast. This is an important step towards en masse de-orphaning of C. elegans chemoreceptors.     

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.     

Neurons and Glia Modify Receptor Protein-tyrosine Phosphatase ζ (RPTPζ)/Phosphacan with Cell-specific O-Mannosyl Glycans in the Developing Brain

Protein O-mannosylation is a glycan modification that is required for normal nervous system development and function. Mutations in genes involved in protein O-mannosyl glycosylation give rise to a group of neurodevelopmental disorders known as congenital muscular dystrophies (CMDs) with associated CNS abnormalities. Our previous work demonstrated that receptor protein-tyrosine phosphatase ζ (RPTPζ)/phosphacan is hypoglycosylated in a mouse model of one of these CMDs, known as muscle-eye-brain disease, a disorder that is caused by loss of an enzyme (protein O-mannose β-1,2-N-acetylglucosaminyltransferase 1) that modifies O-mannosyl glycans. In addition, monoclonal antibodies Cat-315 and 3F8 were demonstrated to detect O-mannosyl glycan modifications on RPTPζ/phosphacan. Here, we show that O-mannosyl glycan epitopes recognized by these antibodies define biochemically distinct glycoforms of RPTPζ/phosphacan and that these glycoforms differentially decorate the surface of distinct populations of neural cells. To provide a further structural basis for immunochemically based glycoform differences, we characterized the O-linked glycan heterogeneity of RPTPζ/phosphacan in the early postnatal mouse brain by multidimensional mass spectrometry. Structural characterization of the O-linked glycans released from purified RPTPζ/phosphacan demonstrated that this protein is a significant substrate for protein O-mannosylation and led to the identification of several novel O-mannose-linked glycan structures, including sulfo-N-acetyllactosamine containing modifications. Taken together, our results suggest that specific glycan modifications may tailor the function of this protein to the unique needs of specific cells. Furthermore, their absence in CMDs suggests that hypoglycosylation of RPTPζ/phosphacan may have different functional consequences in neurons and glia.     

Molecular Structure of Human-Liver Glycogen

Glycogen is a highly branched glucose polymer which is involved in maintaining blood-sugar homeostasis. Liver glycogen contains large composite α particles made up of linked β particles. Previous studies have shown that the binding which links β particles into α particles is impaired in diabetic mice. The present study reports the first molecular structural characterization of human-liver glycogen from non-diabetic patients, using transmission electron microscopy for morphology and size-exclusion chromatography for the molecular size distribution; the latter is also studied as a function of time during acid hydrolysis in vitro, which is sensitive to certain structural features, particularly glycosidic vs. proteinaceous linkages. The results are compared with those seen in mice and pigs. The molecular structural change during acid hydrolysis is similar in each case, and indicates that the linkage of β into α particles is not glycosidic. This result, and the similar morphology in each case, together imply that human liver glycogen has similar molecular structure to those of mice and pigs. This knowledge will be useful for future diabetes drug targets.     

Structural and Biochemical Insights into the Peptidoglycan Hydrolase Domain of FlgJ from Salmonella typhimurium

FlgJ is a glycoside hydrolase (GH) enzyme belonging to the Carbohydrate Active enZyme (CAZy) family GH73. It facilitates passage of the bacterial flagellum through the peptidoglycan (PG) layer by cleaving the β-1,4 glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid sugars that comprise the glycan strands of PG. Here we describe the crystal structure of the GH domain of FlgJ from bacterial pathogen Salmonella typhimurium (StFlgJ). Interestingly, the active site of StFlgJ was blocked by the C-terminal α-helix of a neighbouring symmetry mate and a β-hairpin containing the putative catalytic glutamic acid residue Glu223 was poorly resolved and could not be completely modeled into the electron density, suggesting it is flexible. Previous reports have shown that the GH73 enzyme Auto from Listeria monocytogenes is inhibited by an N-terminal α-helix that may occlude the active site in similar fashion. To investigate if the C-terminus of StFlgJ inhibits GH activity, the glycolytic activity of StFlgJ was assessed with and without the C-terminal α-helix. The GH activity of StFlgJ was unaffected by the presence or absence of the α-helix, suggesting it is not involved in regulating activity. Removal of the C-terminal α-helix did, however, allow a crystal structure of the domain to be obtained where the flexible β-hairpin containing residue Glu223 was entirely resolved. The β-hairpin was positioned such that the active site groove was fully solvent-exposed, placing Glu223 nearly 21.6 Å away from the putative general acid/base residue Glu184, which is too far apart for these two residues to coordinate glycosidic bond hydrolysis. The mobile nature of the StFlgJ β-hairpin is consistent with structural studies of related GH73 enzymes, suggesting that a dynamic active site may be common to many GH73 enzymes, in which the active site opens to capture substrate and then closes to correctly orient active site residues for catalysis.     

The GH130 Family of Mannoside Phosphorylases Contains Glycoside Hydrolases That Target β-1,2-Mannosidic Linkages in Candida Mannan

The depolymerization of complex glycans is an important biological process that is of considerable interest to environmentally relevant industries. β-Mannose is a major component of plant structural polysaccharides and eukaryotic N-glycans. These linkages are primarily cleaved by glycoside hydrolases, although recently, a family of glycoside phosphorylases, GH130, have also been shown to target β-1,2- and β-1,4-mannosidic linkages. In these phosphorylases, bond cleavage was mediated by a single displacement reaction in which phosphate functions as the catalytic nucleophile. A cohort of GH130 enzymes, however, lack the conserved basic residues that bind the phosphate nucleophile, and it was proposed that these enzymes function as glycoside hydrolases. Here we show that two Bacteroides enzymes, BT3780 and BACOVA_03624, which lack the phosphate binding residues, are indeed β-mannosidases that hydrolyze β-1,2-mannosidic linkages through an inverting mechanism. Because the genes encoding these enzymes are located in genetic loci that orchestrate the depolymerization of yeast α-mannans, it is likely that the two enzymes target the β-1,2-mannose residues that cap the glycan produced by Candida albicans. The crystal structure of BT3780 in complex with mannose bound in the −1 and +1 subsites showed that a pair of glutamates, Glu²²⁷ and Glu²⁶⁸, hydrogen bond to O₁ of α-mannose, and either of these residues may function as the catalytic base. The candidate catalytic acid and the other residues that interact with the active site mannose are conserved in both GH130 mannoside phosphorylases and β-1,2-mannosidases. Functional phylogeny identified a conserved lysine, Lys¹⁹⁹ in BT3780, as a key specificity determinant for β-1,2-mannosidic linkages.     

C. elegans Expressing Human β2-Microglobulin: A Novel Model for Studying the Relationship between the Molecular Assembly and the Toxic Phenotype

Availability of living organisms to mimic key step of amyloidogenesis of human protein has become an indispensable tool for our translation approach aiming at filling the deep gap existing between the biophysical and biochemical data obtained in vitro and the pathological features observed in patients. Human β₂-microglobulin (β₂-m) causes systemic amyloidosis in haemodialysed patients. The structure, misfolding propensity, kinetics of fibrillogenesis and cytotoxicity of this protein, in vitro, have been studied more extensively than for any other globular protein. However, no suitable animal model for β₂-m amyloidosis has been so far reported. We have now established and characterized three new transgenic C. elegans strains expressing wild type human β₂-m and two highly amyloidogenic isoforms: P32G variant and the truncated form ΔN6 lacking of the 6 N-terminal residues. The expression of human β₂-m affects the larval growth of C. elegans and the severity of the damage correlates with the intrinsic propensity to self-aggregate that has been reported in previous in vitro studies. We have no evidence of the formation of amyloid deposits in the body-wall muscles of worms. However, we discovered a strict correlation between the pathological phenotype and the presence of oligomeric species recognized by the A11 antibody. The strains expressing human β₂-m exhibit a locomotory defect quantified with the body bends assay. Here we show that tetracyclines can correct this abnormality confirming that these compounds are able to protect a living organism from the proteotoxicity of human β₂-m.     

Limited Addition of the 6-Arm β1,2-linked N-Acetylglucosamine (GlcNAc) Residue Facilitates the Formation of the Largest N-Glycan in Plants

The most abundant N-glycan in plants is the paucimannosidic N-glycan with core β1,2-xylose and α1,3-fucose residues (Man₃XylFuc(GlcNAc)₂). Here, we report a mechanism in Arabidopsis thaliana that efficiently produces the largest N-glycan in plants. Genetic and biochemical evidence indicates that the addition of the 6-arm β1,2-GlcNAc residue by N-acetylglucosaminyltransferase II (GnTII) is less effective than additions of the core β1,2-xylose and α1,3-fucose residues by XylT, FucTA, and FucTB in Arabidopsis. Furthermore, analysis of gnt2 mutant and 35S:GnTII transgenic plants shows that the addition of the 6-arm non-reducing GlcNAc residue to the common N-glycan acceptor GlcNAcMan₃(GlcNAc)₂ inhibits additions of the core β1,2-xylose and α1,3-fucose residues. Our findings indicate that plants limit the rate of the addition of the 6-arm GlcNAc residue to the common N-glycan acceptor as a mechanism to facilitate formation of the prevalent N-glycans with Man₃XylFuc(GlcNAc)₂ and (GlcNAc)₂Man₃XylFuc(GlcNAc)₂ structures.     

β-Filagenin, a Newly Identified Protein Coassembling with Myosin and Paramyosin in Caenorhabditis elegans

Muscle thick filaments are stable assemblies of myosin and associated proteins whose dimensions are precisely regulated. The mechanisms underlying the stability and regulation of the assembly are not understood. As an approach to these problems, we have studied the core proteins that, together with paramyosin, form the core structure of the thick filament backbone in the nematode Caenorhabditis elegans. We obtained partial peptide sequences from one of the core proteins, β-filagenin, and then identified a gene that encodes a novel protein of 201–amino acid residues from databases using these sequences. β-Filagenin has a calculated isoelectric point at 10.61 and a high percentage of aromatic amino acids. Secondary structure algorithms predict that it consists of four β-strands but no α-helices. Western blotting using an affinity-purified antibody showed that β-filagenin was associated with the cores. β-Filagenin was localized by immunofluorescence microscopy to the A bands of body–wall muscles, but not the pharynx. β-filagenin assembled with the myosin homologue paramyosin into the tubular cores of wild-type nematodes at a periodicity matching the 72-nm repeats of paramyosin, as revealed by immunoelectron microscopy. In CB1214 mutants where paramyosin is absent, β-filagenin assembled with myosin to form abnormal tubular filaments with a periodicity identical to wild type. These results verify that β-filagenin is a core protein that coassembles with either myosin or paramyosin in C. elegans to form tubular filaments.     

Cell Surface Glycan Alterations in Epithelial Mesenchymal Transition Process of Huh7 Hepatocellular Carcinoma Cell

Background and Objective

Due to recurrence and metastasis, the mortality of Hepatocellular carcinoma (HCC) is high. It is well known that the epithelial mesenchymal transition (EMT) and glycan of cell surface glycoproteins play pivotal roles in tumor metastasis. The goal of this study was to identify HCC metastasis related differential glycan pattern and their enzymatic basis using a HGF induced EMT model.

Methodology

HGF was used to induce HCC EMT model. Lectin microarray was used to detect the expression of cell surface glycan and the difference was validated by lectin blot and fluorescence cell lectin-immunochemistry. The mRNA expression levels of glycotransferases were determined by qRT-PCR.

Results

After HGF treatment, the Huh7 cell lost epithelial characteristics and obtained mesenchymal markers. These changes demonstrated that HGF could induce a typical cell model of EMT. Lectin microarray analysis identified a decreased affinity in seven lectins ACL, BPL, JAC, MPL, PHA-E, SNA, and SBA to the glycan of cell surface glycoproteins. This implied that glycan containing T/Tn-antigen, NA2 and bisecting GlcNAc, Siaα2-6Gal/GalNAc, terminal α or βGalNAc structures were reduced. The binding ability of thirteen lectins, AAL, LCA, LTL, ConA, NML, NPL, DBA, HAL, PTL II, WFL, ECL, GSL II and PHA-L to glycan were elevated, and a definite indication that glycan containing terminal αFuc and ± Sia-Le, core fucose, α-man, gal-β(α) GalNAc, β1,6 GlcNAc branching and tetraantennary complex oligosaccharides structures were increased. These results were further validated by lectin blot and fluorescence cell lectin-immunochemistry. Furthermore, the mRNA expression level of Mgat3 decreased while that of Mgat5, FucT8 and β3GalT5 increased. Therefore, cell surface glycan alterations in the EMT process may coincide with the expression of glycosyltransferase.

Conclusions

The findings of this study systematically clarify the alterations of cell surface glycan in cancer EMT, and may provide novel insight for HCC metastasis.     

Structure and Substrate Specificity of a Eukaryotic Fucosidase from Fusarium graminearum

The secreted glycoside hydrolase family 29 (GH29) α-l-fucosidase from plant pathogenic fungus Fusarium graminearum (FgFCO1) actively releases fucose from the xyloglucan fragment. We solved crystal structures of two active-site conformations, i.e. open and closed, of apoFgFCO1 and an open complex with product fucose at atomic resolution. The closed conformation supports catalysis by orienting the conserved general acid/base Glu-288 nearest the predicted glycosidic position, whereas the open conformation possibly represents an unreactive state with Glu-288 positioned away from the catalytic center. A flexible loop near the substrate binding site containing a non-conserved GGSFT sequence is ordered in the closed but not the open form. We also identified a novel C-terminal βγ-crystallin domain in FgFCO1 devoid of calcium binding motif whose homologous sequences are present in various glycoside hydrolase families. N-Glycosylated FgFCO1 adopts a monomeric state as verified by solution small angle x-ray scattering in contrast to reported multimeric fucosidases. Steady-state kinetics shows that FgFCO1 prefers α1,2 over α1,3/4 linkages and displays minimal activity with p-nitrophenyl fucoside with an acidic pH optimum of 4.6. Despite a retaining GH29 family fold, the overall specificity of FgFCO1 most closely resembles inverting GH95 α-fucosidase, which displays the highest specificity with two natural substrates harboring the Fucα1-2Gal glycosidic linkage, a xyloglucan-derived nonasaccharide, and 2′-fucosyllactose. Furthermore, FgFCO1 hydrolyzes H-disaccharide (lacking a +2 subsite sugar) at a rate 10³-fold slower than 2′-fucosyllactose. We demonstrated the structurally dynamic active site of FgFCO1 with flexible general acid/base Glu, a common feature shared by several bacterial GH29 fucosidases to various extents.     

Golgi N-Glycosyltransferases Form Both Homo- and Heterodimeric Enzyme Complexes in Live Cells

Glycans (i.e. oligosaccharide chains attached to cellular proteins and lipids) are crucial for nearly all aspects of life, including the development of multicellular organisms. They come in multiple forms, and much of this diversity between molecules, cells, and tissues is generated by Golgi-resident glycosidases and glycosyltransferases. However, their exact mode of functioning in glycan processing is currently unclear. Here we investigate the supramolecular organization of the N-glycosylation pathway in live cells by utilizing the bimolecular fluorescence complementation approach. We show that all four N-glycosylation enzymes tested (β-1,2-N-acetylglucosaminyltransferase I, β-1,2-N-acetylglucosaminyltransferase II, 1,4-galactosyltransferase I, and α-2,6-sialyltransferase I) form Golgi-localized homodimers. Intriguingly, the same enzymes also formed two distinct and functionally relevant heterodimers between the medial Golgi enzymes β-1,2-N-acetylglucosaminyltransferase I and β-1,2-N-acetylglucosaminyltransferase II and the trans-Golgi enzymes 1,4-galactosyltransferase I and α-2,6-sialyltransferase I. Given their strict Golgi localization and sequential order of function, the two heterodimeric complexes are probably responsible for the processing and maturation of N-glycans in live cells.     

Basis of Miscoding of the DNA Adduct N2,3-Ethenoguanine by Human Y-family DNA Polymerases

N²,3-Ethenoguanine (N²,3-ϵG) is one of the exocyclic DNA adducts produced by endogenous processes (e.g. lipid peroxidation) and exposure to bioactivated vinyl monomers such as vinyl chloride, which is a known human carcinogen. Existing studies exploring the miscoding potential of this lesion are quite indirect because of the lability of the glycosidic bond. We utilized a 2′-fluoro isostere approach to stabilize this lesion and synthesized oligonucleotides containing 2′-fluoro-N²,3-ϵ-2′-deoxyarabinoguanosine to investigate the miscoding potential of N²,3-ϵG by Y-family human DNA polymerases (pols). In primer extension assays, pol η and pol κ replicated through N²,3-ϵG, whereas pol ι and REV1 yielded only 1-base incorporation. Steady-state kinetics revealed that dCTP incorporation is preferred opposite N²,3-ϵG with relative efficiencies in the order of pol κ > REV1 > pol η ≈ pol ι, and dTTP misincorporation is the major miscoding event by all four Y-family human DNA pols. Pol ι had the highest dTTP misincorporation frequency (0.71) followed by pol η (0.63). REV1 misincorporated dTTP and dGTP with much lower frequencies. Crystal structures of pol ι with N²,3-ϵG paired to dCTP and dTTP revealed Hoogsteen-like base pairing mechanisms. Two hydrogen bonds were observed in the N²,3-ϵG:dCTP base pair, whereas only one appears to be present in the case of the N²,3-ϵG:dTTP pair. Base pairing mechanisms derived from the crystal structures explain the slightly favored dCTP insertion for pol ι in steady-state kinetic analysis. Taken together, these results provide a basis for the mutagenic potential of N²,3-ϵG.