Functional expression of Escherichia coli enzymes synthesizing GDP-L-fucose from inherent GDP-D-mannose in Saccharomyces cerevisiae

Fucosylation of glycans on glycoproteins and -lipids requires the enzymatic activity of relevant fucosyltransferases and GDP-L-fucose as the donor. Due to the biological importance of fucosylated glycans, a readily accessible source of GDP-L-fucose would be required. Here we describe the construction of a stable recombinant S.cerevisiae strain expressing the E.coli genes gmd and wcaG encoding the two enzymes, GDP-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/4-reductase (GMER(FX)) respectively, needed to convert GDP-mannose to GDP-fucose via the de novo pathway. Taking advantage of the rich inherent cytosolic GDP-mannose pool in S.cerevisiae cells we could easily produce 0.2 mg/l of GDP-L-fucose with this recombinant yeast strain without addition of any external GDP-mannose. The GDP-L-fucose product could be used as the fucose donor for alpha1,3fucosyltransferase to synthesize sialyl Lewis x (sLex), a glycan crucial for the selectin-dependent leukocyte traffic.     

Genetic model organisms in the study of N-glycans

Recently the genomic sequences of three multicellular eukaryotes, Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis thaliana, have been elucidated. A number of cDNAs encoding glycosyltransferases demonstrated to have a role in N-linked glycosylation have already been cloned from these organisms, e.g., GlcNAc transferases and alpha 1,3-fucosyltransferases. However, many more homologues of glycosyltransferases and other glycan modifying enzymes have been predicted by analysis of the genome sequences, but the predictions of full length open reading frames appear to be particularly poor in Caenorhabditis. The use of these organisms as models in glycobiology may be hampered since they all have N-linked glycosylation repertoires unlike those of mammals. Arabidopsis and Drosophila have glycosylation similar to that of other plants or insects, while our new data from MALDI-TOF analysis of PNGase A-released neutral N-glycans of Caenorhabditis indicate that there exists a range of pauci- and oligomannosidic structures, with up to four fucose residues and up to two O-methyl groups. With all these three 'genetic model organisms', however, much more work is required for a full understanding of their glycobiology.     

Composition of Drosophila melanogaster proteome involved in fucosylated glycan metabolism.

The whole genome approach enables the characterization of all components of any given biological pathway. Moreover, it can help to uncover all the metabolic routes for any molecule. Here we have used the genome of Drosophila melanogaster to search for enzymes involved in the metabolism of fucosylated glycans. Our results suggest that in the fruit fly GDP-fucose, the donor for fucosyltransferase reactions, is formed exclusively via the de novo pathway from GDP-mannose through enzymatic reactions catalyzed by GDP-D-mannose 4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase/4-reductase (GMER, also known as FX in man). The Drosophila genome does not have orthologs for the salvage pathway enzymes, i.e. fucokinase and GDP-fucose pyrophosphorylase synthesizing GDP-fucose from fucose. In addition we identified two novel fucosyltransferases predicted to catalyze alpha1,3- and alpha1,6-specific linkages to the GlcNAc residues on glycans. No genes with the capacity to encode alpha1,2-specific fucosyltransferases were found. We also identified two novel genes coding for O-fucosyltransferases and a gene responsible for a fucosidase enzyme in the Drosophila genome. Finally, using the Drosophila CG4435 gene, we identified two novel human genes putatively coding for fucosyltransferases. This work can serve as a basis for further whole-genome approaches in mapping all possible glycosylation pathways and as a basic analysis leading to subsequent experimental studies to verify the predictions made in this work.     

An Arabidopsis thaliana cDNA complements the N-acetylglucosaminyltransferase I deficiency of CHO Lec1 cells.

N-Acetylglucosaminyltransferase I (GlcNAcT-I, EC 2.4.1.101) is the enzyme which initiates the formation of complex N-linked glycans in eukaryotes by transforming GlcNAc to the oligo-mannosyl acceptor Man(5)GlcNAc(2)-Asn. The enzymatic activity and the structure that is synthesised by this enzyme are found in animals and plants but not in yeast. cDNAs encoding the enzyme have already been cloned from several mammals and the nematode Caenorhabditis elegans. In this article the cloning of an Arabidopsis thaliana GlcNAcT-I cDNA with homology to animal cDNAs is described. By expression of the plant cDNA in CHO Lec1 cells, a mammalian cell line deficient in GlcNAcT-I, it was shown that it encodes an active enzyme with the same enzymatic activity as the animal homologue. It has already been shown that a human GlcNAcT-I can complement an A. thaliana mutant (cgl-1). Here it is shown that the reverse is also true, the plant glycosyltransferase is able to complement a mammalian mutant (Lec1) deficient in GlcNAcT-I.     

Fucosyltransferase substrate specificity and the order of fucosylation in invertebrates

Core alpha1,6-fucosylation is a conserved feature of animal N-linked oligosaccharides being present in both invertebrates and vertebrates. To prove that the enzymatic basis for this modification is also evolutionarily conserved, cDNAs encoding the catalytic regions of the predicted Caenorhabditis elegans and Drosophila melanogaster homologs of vertebrate alpha1,6-fucosyltransferases (E.C. 2.4.1.68) were engineered for expression in the yeast Pichia pastoris. Recombinant forms of both enzymes were found to display core fucosyltransferase activity as shown by a variety of methods. Unsubstituted nonreducing terminal GlcNAc residues appeared to be an obligatory feature of the substrate for the recombinant Caenorhabditis and Drosophila alpha1,6-fucosyltransferases, as well as for native Caenorhabditis and Schistosoma mansoni core alpha1,6-fucosyltransferases. On the other hand, these alpha1,6-fucosyltransferases could not act on N-glycopeptides already carrying core alpha1,3-fucose residues, whereas recombinant Drosophila and native Schistosoma core alpha1,3-fucosyltransferases were able to use core alpha1,6-fucosylated glycans as substrates. Lewis-type fucosylation was observed with native Schistosoma extracts and could take place after core alpha1,3-fucosylation, whereas prior Lewis-type fucosylation precluded the action of the Schistosoma core alpha1,3-fucosyltransferase. Overall, we conclude that the strict order of fucosylation events, previously determined for fucosyltransferases in crude extracts from insect cell lines (core alpha1,6 before core alpha1,3), also applies for recombinant Drosophila core alpha1,3- and alpha1,6-fucosyltransferases as well as for core fucosyltransferases in schistosomal egg extracts.     

Cloning and expression of Helicobacter pylori GDP-l-fucose synthesizing enzymes (GMD and GMER) in Saccharomyces cerevisiae.

Helicobacter pylori is a Gram-negative gastric pathogen causing diseases from mild gastric infections to gastric cancer. The difference in clinical outcome has been suggested to be due to strain differences. H. pylori undergoes phase variation by changing its lipopolysaccharide structure according to the environmental conditions. The O-antigen of H. pylori contains fucosylated glycans, similar to Lewis structures found in human gastric epithelium. These Lewis glycans of H. pylori have been suggested to play a role in pathogenesis in the adhesion of the bacterium to gastric epithelium. In the synthesis of fucosylated structures, GDP-l-fucose is needed as a fucose donor. Here, we cloned the two key enzymes of GDP-l-fucose synthesis, H. pylori gmd coding for GDP-d-mannose dehydratase (GMD), and gmer coding for GDP-4-keto-6-deoxy-d-mannose-3,5-epimerase/4-reductase (GMER) and expressed them in an enzymatically active form in Saccharomyces cerevisiae. The end product of these enzymes, GDP-l-fucose was used as a fucose donor in a fucosyltransferase assay converting sialyl-N-acetyllactosamine to sialyl Lewis X.     

The class I α1,2-mannosidases of Caenorhabditis elegans

During the biosynthesis of N-glycans in multicellular eukaryotes, glycans with the compositions Man(5)GlcNAc(2-3) are key intermediates. However, to reach this 'decision point', these N-glycans are first processed from Glc(3)Man(9)GlcNAc(2) through to Man(5)GlcNAc(2) by a number of glycosidases, whereby up to four α1-2-linked mannose residues are removed by class I mannosidases (glycohydrolase family 47). Whereas in the yeast Saccharomyces cerevisiae there are maximally three members of this protein family, in higher organisms there are multiple class I mannosidases residing in the endoplasmic reticulum and Golgi apparatus. The genome of the model nematode Caenorhabditis elegans encodes seven members of this protein family, whereby four are predicted to be classical processing mannosidases and three are related proteins with roles in quality control. In this study, cDNAs encoding the four predicted mannosidases were cloned and expressed in Pichia pastoris and the activity of these enzymes, designated MANS-1, MANS-2, MANS-3 and MANS-4, was verified. The first two can, dependent on the incubation time, remove three to four residues from Man(9)GlcNAc(2), whereas the action of the other two results in the appearance of the B isomer of Man(8)GlcNAc(2); together the complementary activities of these enzymes result in processing to Man(5)GlcNAc(2). With these data, another gap is closed in our understanding of the N-glycan biosynthesis pathway of the nematode worm.     

Biochemical characterization of GDP-l-fucose de novo synthesis pathway in fungus Mortierella alpina

Mortierella alpina is a filamentous fungus commonly found in soil, which is able to produce large amount of polyunsaturated fatty acids. l-Fucose is an important sugar found in a diverse range of organisms, playing a variety of biological roles. In this study, we characterized the de novo biosynthetic pathway of GDP-l-fucose (the nucleotide-activated form of l-fucose) in M. alpina. Genes encoding GDP-d-mannose 4,6-dehydratase (GMD) and GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase (GMER) were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins. Conversion of GDP-mannose to GDP-4-keto-6-deoxy mannose by GMD and GDP-4-keto-6-deoxy mannose to GDP-l-fucose by GMER were analyzed by capillary electrophoresis, electro-spray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy. The k_m values of GMD for GDP-mannose and GMER for GDP-4-keto-6-deoxy mannose were determined to be 0.77 mM and 1.047 mM, respectively. Both NADH and NADPH may be used by GMER as the coenzyme. The optimum temperature and pH were determined to be 37 °C and pH 9.0 (GMD) or pH 7.0 (GMER). Divalent cations are not required for GMD and GMER activity, and the activities of both enzymes may be enhanced by DTT. To our knowledge this is the first report on the characterization of GDP-l-fucose biosynthetic pathway in fungi.     

Biosynthesis of truncated N-linked oligosaccharides results from non-orthologous hexosaminidase-mediated mechanisms in nematodes, plants, and insects

In many invertebrates and plants, the N-glycosylation profile is dominated by truncated paucimannosidic N-glycans, i.e. glycans consisting of a simple trimannosylchitobiosyl core often modified by core fucose residues. Even though they lack antennal N-acetylglucosamine residues, the biosynthesis of these glycans requires the sequential action of GlcNAc transferase I, Golgi mannosidase II, and, finally, beta-N-acetylglucosaminidases. In Drosophila, the recently characterized enzyme encoded by the fused lobes (fdl) gene specifically removes the non-reducing N-acetylglucosamine residue from the alpha1,3-antenna of N-glycans. In the present study, we examined the products of five beta-N-acetylhexosaminidase genes from Caenorhabditis elegans (hex-1 to hex-5, corresponding to reading frames T14F9.3, C14C11.3, Y39A1C.4, Y51F10.5, and Y70D2A.2) in addition to three from Arabidopsis thaliana (AtHEX1, AtHEX2, and AtHEX3, corresponding to reading frames At1g65590, At3g55260, and At1g05590). Based on homology, the Caenorhabditis HEX-1 and all three Arabidopsis enzymes are members of the same sub-family as the aforementioned Drosophila fused lobes enzyme but either act as chitotriosidases or non-specifically remove N-acetylglucosamine from both N-glycan antennae. The other four Caenorhabditis enzymes are members of a distinct sub-family; nevertheless, two of these enzymes displayed the same alpha1,3-antennal specificity as the fused lobes enzyme. Furthermore, a deletion of part of the Caenorhabditis hex-2 gene drastically reduces the native N-glycan-specific hexosaminidase activity in mutant worm extracts and results in a shift in the N-glycan profile, which is a demonstration of its in vivo enzymatic relevance. Based on these data, it is hypothesized that the genetic origin of paucimannosidic glycans in nematodes, plants, and insects involves highly divergent members of the same hexosaminidase gene family.     

Comparative genomics and proteomics of Drosophila,Brenner's nematode,and Arabidopsis: identification of functionally similar genes and proteins of meiotic chromosome synapsis

The published principles of computer analysis of genomes and protein sets in taxonomically distant eukaryotes are expounded. The authors developed a search strategy to identify in genomes of such organisms genes and proteins nonhomologous in primary structure but having similar functions in cells dividing by meiosis. This strategy based on the combined principles of genomics, proteomics, and morphometric analysis of subcellular structures was applied to a computer search for genes encoding the proteins of synaptonemal complexes in genomes of Drosophila melanogaster, the nematode Caenorhabditis elegans, and the plant Arabidopsis thaliana. These proteins proved to be functionally similar to their counterparts in yeast Saccharomyces cerevisiae (protein Zip1p) and mammals (protein SCP1).     

Caenorhabditis elegans contains genes encoding two new members of the Zn-containing alcohol dehydrogenase family

We have characterized two cDNA clones from the nematode Caenorhabditis elegans that display similarity to the alcohol dehydrogenase (ADH) gene family. The nucleotide sequences of these cDNAs predict that they encode Zn-containing long-chain ADH enzymes. Phylogenetic analysis suggests that one is most similar to dimeric class III ADHs found in diverse taxa; the other is most similar to the tetrameric forms of ADH previously described only in fungi. Correspondence to: J.J. Collins     

Isolation and characterization of cDNAs encoding wheat 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) is a key enzyme in the isoprenoid biosynthetic pathway. We have isolated partial cDNAs from wheat (Triticum aestivum) using the polymerase chain reaction. Comparison of deduced amino acid sequences of these cDNAs shows that they represent a small family of genes that share a high degree of sequence homology among themselves as well as among genes from other organisms including tomato, Arabidopsis, hamster, human, Drosophila, and yeast. Southern blot analysis reveals the presence of at least four genes. Our results concerning the tissue-specific expression as well as developmental regulation of these HMGR cDNAs highlight the important role of this enzyme in the growth and development of wheat.     

Gender and developmental specific N-glycomes of the porcine parasite Oesophagostomum dentatum

BackgroundThe porcine nodule worm Oesophagostomum dentatum is a strongylid class V nematode rather closely related to the model organism Caenorhabditis elegans. However, in contrast to the non-parasitic C. elegans, the parasitic O. dentatum is an obligate sexual organism, which makes both a gender and developmental glycomic comparison possible.MethodsDifferent enzymatic and chemical methods were used to release N-glycans from male and female O. dentatum as well as from L3 and L4 larvae. Glycans were analysed by MALDI-TOF MS after either 2D-HPLC (normal then reversed phase) or fused core RP-HPLC.ResultsWhereas the L3 N-glycome was simpler and more dominated by phosphorylcholine-modified structures, the male and female worms express a wide range of core fucosylated N-glycans with up to three fucose residues. Seemingly, simple methylated paucimannosidic structures can be considered ‘male’, while methylation of fucosylated glycans was more pronounced in females. On the other hand, while many of the fucosylated paucimannosidic glycans are identical with examples from other nematode species, but simpler than the tetrafucosylated glycans of C. elegans, there is a wide range of phosphorylcholine-modified glycans with extended HexNAc_2–4PC_2–4 motifs not observed in our previous studies on other nematodes.ConclusionThe interspecies tendency of class V nematodes to share most, but not all, N-glycans applies also to O. dentatum; furthermore, we establish, for the first time in a parasitic nematode, that glycomes vary upon development and sexual differentiation.General significanceUnusual methylated, core fucosylated and phosphorylcholine-containing N-glycans vary between stages and genders in a parasitic nematode.     

The glycomes of Caenorhabditis elegans and other model organisms

There is no doubt that the immense amount of information that is being generated by the initial sequencing and secondary interrogation of various genomes will change the face of glycobiological research. However, a major area of concern is that detailed structural knowledge of the ultimate products of genes that are identified as being involved in glycoconjugate biosynthesis is still limited. This is illustrated clearly by the nematode worm Caenorhabditis elegans, which was the first multicellular organism to have its entire genome sequenced. To date, only limited structural data on the glycosylated molecules of this organism have been reported. Our laboratory is addressing this problem by performing detailed MS structural characterization of the N-linked glycans of C. elegans; high-mannose structures dominate, with only minor amounts of complex-type structures. Novel, highly fucosylated truncated structures are also present which are difucosylated on the proximal N-acetylglucosamine of the chitobiose core as well as containing unusual Fuc alpha 1-2 Gal 1-2 Man as peripheral structures. The implications of these results in terms of the identification of ligands for genomically predicted lectins and potential glycosyltransferases are discussed in this chapter. Current knowledge on the glycomes of other model organisms such as Dictyostelium discoideum, Saccharomyces cerevisiae and Drosophila melanogaster is also discussed briefly.     

Structural analysis of glycosaminoglycans in Drosophila and Caenorhabditis elegans and demonstration that tout-velu, a Drosophila gene related to EXT tumor suppressors, affects heparan sulfate in vivo

We have devised a sensitive method for the isolation and structural analysis of glycosaminoglycans from two genetically tractable model organisms, the fruit fly, Drosophila melanogaster, and the nematode, Caenorhabditis elegans. We detected chondroitin/chondroitin sulfate- and heparan sulfate-derived disaccharides in both organisms. Chondroitinase digestion of glycosaminoglycans from adult Drosophila produced both nonsulfated and 4-O-sulfated unsaturated disaccharides, whereas only unsulfated forms were detected in C. elegans. Heparin lyases released disaccharides bearing N-, 2-O-, and 6-O-sulfated species, including mono-, di-, and trisulfated forms. We observed tissue- and stage-specific differences in both chondroitin sulfate and heparan sulfate composition in Drosophila. We have also applied these methods toward the analysis of tout-velu, an EXT-related gene in Drosophila that controls the tissue distribution of the growth factor Hedgehog. The proteins encoded by the vertebrate tumor suppressor genes EXT1 and 2, show heparan sulfate co-polymerase activity, and it has been proposed that tout-velu affects Hedgehog activity via its role in heparan sulfate biosynthesis. Analysis of total glycosaminoglycans from tout-velu mutant larvae show marked reductions in heparan sulfate but not chondroitin sulfate, consistent with its proposed function as a heparan sulfate co-polymerase.     

Structural and kinetic analysis of Escherichia coli GDP-mannose 4,6 dehydratase provides insights into the enzyme's catalytic mechanism and regulation by GDP-fucose

Background: GDP-mannose 4,6 dehydratase (GMD) catalyzes the conversion of GDP-(D)-mannose to GDP-4-keto, 6-deoxy-(D)-mannose. This is the first and regulatory step in the de novo biosynthesis of GDP-(L)-fucose. Fucose forms part of a number of glycoconjugates, including the ABO blood groups and the selectin ligand sialyl Lewis X. Defects in GDP-fucose metabolism have been linked to leukocyte adhesion deficiency type II (LADII). Results: The structure of the GDP-mannose 4,6 dehydratase apo enzyme has been determined and refined using data to 2.3 A resolution. GMD is a homodimeric protein with each monomer composed of two domains. The larger N-terminal domain binds the NADP(H) cofactor in a classical Rossmann fold and the C-terminal domain harbors the sugar-nucleotide binding site. We have determined the GMD dissociation constants for NADP, NADPH and GDP-mannose. Each GMD monomer binds one cofactor and one substrate molecule, suggesting that both subunits are catalytically competent. GDP-fucose acts as a competitive inhibitor, suggesting that it binds to the same site as GDP-mannose, providing a mechanism for the feedback inhibition of fucose biosynthesis. Conclusions: The X-ray structure of GMD reveals that it is a member of the short-chain dehydrogenase/reductase (SDR) family of proteins. We have modeled the binding of NADP and GDP-mannose to the enzyme and mutated four of the active-site residues to determine their function. The combined modeling and mutagenesis data suggests that at position 133 threonine substitutes serine as part of the serine-tyrosine-lysine catalytic triad common to the SDR family and Glu 135 functions as an active-site base.     

Comparative Genomics and Proteomics of Drosophila, Brenner's Nematode, and Arabidopsis: Identification of Functionally Similar Genes and Proteins of Meiotic Chromosome Synapsis

The published principles of computer analysis of genomes and protein sets in taxonomically distant eukaryotes are expounded. The authors developed a search strategy to identify in genomes of such organisms genes and proteins nonhomologous in primary structure but having similar functions in cells dividing by meiosis. This strategy based on the combined principles of genomics, proteomics, and morphometric analysis of subcellular structures was applied to a computer search for genes encoding the proteins of synaptonemal complexes in genomes of Drosophila melanogaster, the nematode Caenorhabditis elegans, and the plant Arabidopsis thaliana. These proteins proved to be functionally similar to their counterparts in yeast Saccharomyces cerevisiae (protein Zip1p) and mammals (protein SCP1).     

Characterization of GDP-mannose dehydrogenase from the brown alga Ectocarpus siliculosus providing the precursor for the alginate polymer

Alginate is a major cell wall polymer of brown algae. The precursor for the polymer is GDP-mannuronic acid, which is believed to be derived from a four-electron oxidation of GDP-mannose through the enzyme GDP-mannose dehydrogenase (GMD). So far no eukaryotic GMD has been biochemically characterized. We have identified a candidate gene in the Ectocarpus siliculosus genome and expressed it as a recombinant protein in Escherichia coli. The GMD from Ectocarpus differs strongly from related enzymes in bacteria and is as distant to the bacterial proteins as it is to the group of UDP-glucose dehydrogenases. It lacks the C-terminal ～120 amino acid domain present in bacterial GMDs, which is believed to be involved in catalysis. The GMD from brown algae is highly active at alkaline pH and contains a catalytic Cys residue, sensitive to heavy metals. The product GDP-mannuronic acid was analyzed by HPLC and mass spectroscopy. The K(m) for GDP-mannose was 95 μM, and 86 μM for NAD(+). No substrate other than GDP-mannose was oxidized by the enzyme. In gel filtration experiments the enzyme behaved as a dimer. The Ectocarpus GMD is stimulated by salts even at low molar concentrations as a possible adaptation to marine life. It is rapidly inactivated at temperatures above 30 °C.     

Array-assisted Characterization of a Fucosyltransferase Required for the Biosynthesis of Complex Core Modifications of Nematode N-Glycans

Fucose is a common monosaccharide component of cell surfaces and is involved in many biological recognition events. Therefore, definition and exploitation of the specificity of the enzymes (fucosyltransferases) involved in fucosylation is a recurrent theme in modern glycosciences. Despite various studies, the specificities of many fucosyltransferases are still unknown, so new approaches are required to study these. The model nematode Caenorhabditis elegans expresses a wide range of fucosylated glycans, including N-linked oligosaccharides with unusual complex core modifications. Up to three fucose residues can be present on the standard N,N′-diacetylchitobiose unit of these N-glycans, but only the fucosyltransferases responsible for transfer of two of these (the core α1,3-fucosyltransferase FUT-1 and the core α1,6-fucosyltransferase FUT-8) were previously characterized. By use of a glycan library in both array and solution formats, we were able to reveal that FUT-6, another C. elegans α1,3-fucosyltransferase, modifies nematode glycan cores, specifically the distal N-acetylglucosamine residue; this result is in accordance with glycomic analysis of fut-6 mutant worms. This core-modifying activity of FUT-6 in vitro and in vivo is in addition to its previously determined ability to synthesize Lewis X epitopes in vitro. A larger scale synthesis of a nematode N-glycan core in vitro using all three fucosyltransferases was performed, and the nature of the glycosidic linkages was determined by NMR. FUT-6 is probably the first eukaryotic glycosyltransferase whose specificity has been redefined with the aid of glycan microarrays and so is a paradigm for the study of other unusual glycosidic linkages in model and parasitic organisms.