GMPPA defects cause a neuromuscular disorder with α-dystroglycan hyperglycosylation.

GDP-mannose-pyrophosphorylase-B (GMPPB) facilitates the generation of GDP-mannose, a sugar donor required for glycosylation. GMPPB defects cause muscle disease due to hypoglycosylation of α-dystroglycan (α-DG). Alpha-DG is part of a protein complex, which links the extracellular matrix with the cytoskeleton, thus stabilizing myofibers. Mutations of the catalytically inactive homolog GMPPA cause alacrima, achalasia, and mental retardation syndrome (AAMR syndrome), which also involves muscle weakness. Here, we showed that Gmppa-KO mice recapitulated cognitive and motor deficits. As structural correlates, we found cortical layering defects, progressive neuron loss, and myopathic alterations. Increased GDP-mannose levels in skeletal muscle and in vitro assays identified GMPPA as an allosteric feedback inhibitor of GMPPB. Thus, its disruption enhanced mannose incorporation into glycoproteins, including α-DG in mice and humans. This increased α-DG turnover and thereby lowered α-DG abundance. In mice, dietary mannose restriction beginning after weaning corrected α-DG hyperglycosylation and abundance, normalized skeletal muscle morphology, and prevented neuron degeneration and the development of motor deficits. Cortical layering and cognitive performance, however, were not improved. We thus identified GMPPA defects as the first congenital disorder of glycosylation characterized by α-DG hyperglycosylation, to our knowledge, and we have unraveled underlying disease mechanisms and identified potential dietary treatment options.

A homozygous mutation in GMPPB leads to centronuclear myopathy with combined pre- and postsynaptic defects of neuromuscular transmission.

Mutations in GMPPB cause a wide spectrum of neuromuscular syndromes, including muscular dystrophies and congenital myasthenic syndrome. The mechanisms by which GMPPB mutations impair neuromuscular transmission however remain incompletely understood. We expand here upon a previous report of one such patient presenting with a myopathy-congenital myasthenic syndrome overlap phenotype. Fatigable proximal muscle weakness developed gradually between 13 and 25 years of age, with subsequent stabilization. Low-frequency repetitive nerve stimulation showed a decrement, while a muscle biopsy demonstrated the presence of a centronuclear myopathy. Genetic testing identified a homozygous c.458C > T (p.Thr153Ile) variant in GMPPB. In-vitro microelectrode recordings and ultrastructural studies showed impairment of both pre- and postsynaptic neuromuscular transmission, thus demonstrating the presence of not only postsynaptic, but also presynaptic pathology in GMPPB-related disorders.

Pathogenic Variants in Fucokinase Cause a Congenital Disorder of Glycosylation.

FUK encodes fucokinase, the only enzyme capable of converting L-fucose to fucose-1-phosphate, which will ultimately be used for synthesizing GDP-fucose, the donor substrate for all fucosyltransferases. Although it is essential for fucose salvage, this pathway is thought to make only a minor contribution to the total amount of GDP-fucose. A second pathway, the major de novo pathway, involves conversion of GDP-mannose to GDP-fucose. Here we describe two unrelated individuals who have pathogenic variants in FUK and who presented with severe developmental delays, encephalopathy, intractable seizures, and hypotonia. The first individual was compound heterozygous for c.667T>C (p.Ser223Pro) and c.2047C>T (p.Arg683Cys), and the second individual was homozygous for c.2980A>C (p.Lys994Gln). Skin fibroblasts from the first individual confirmed the variants as loss of function and showed significant decreases in total GDP-[3H] fucose and [3H] fucose-1-phosphate. There was also a decrease in the incorporation of [5,6-3H]-fucose into fucosylated glycoproteins. Lys994 has previously been shown to be an important site for ubiquitin conjugation. Here, we show that loss-of-function variants in FUK cause a congenital glycosylation disorder characterized by a defective fucose-salvage pathway.

Mobility shift of beta-dystroglycan as a marker of GMPPB gene-related muscular dystrophy.

BACKGROUND: Defects in glycosylation of alpha-dystroglycan (α-DG) cause autosomal-recessive disorders with wide clinical and genetic heterogeneity, with phenotypes ranging from congenital muscular dystrophies to milder limb girdle muscular dystrophies. Patients show variable reduction of immunoreactivity to antibodies specific for glycoepitopes of α-DG on a muscle biopsy. Recessive mutations in 18 genes, including guanosine diphosphate mannose pyrophosphorylase B (GMPPB), have been reported to date. With no specific clinical and pathological handles, diagnosis requires parallel or sequential analysis of all known genes. METHODS: We describe clinical, genetic and biochemical findings of 21 patients with GMPPB-associated dystroglycanopathy. RESULTS: We report eight novel mutations and further expand current knowledge on clinical and muscle MRI features of this condition. In addition, we report a consistent shift in the mobility of beta-dystroglycan (ß-DG) on Western blot analysis of all patients analysed by this mean. This was only observed in patients with GMPPB in our large dystroglycanopathy cohort. We further demonstrate that this mobility shift in patients with GMPPB was due to abnormal N-linked glycosylation of ß-DG. CONCLUSIONS: Our data demonstrate that a change in ß-DG electrophoretic mobility in patients with dystroglycanopathy is a distinctive marker of the molecular defect in GMPPB.

Mutations in GMPPA cause a glycosylation disorder characterized by intellectual disability and autonomic dysfunction.

In guanosine diphosphate (GDP)-mannose pyrophosphorylase A (GMPPA), we identified a homozygous nonsense mutation that segregated with achalasia and alacrima, delayed developmental milestones, and gait abnormalities in a consanguineous Pakistani pedigree. Mutations in GMPPA were subsequently found in ten additional individuals from eight independent families affected by the combination of achalasia, alacrima, and neurological deficits. This autosomal-recessive disorder shows many similarities with triple A syndrome, which is characterized by achalasia, alacrima, and variable neurological deficits in combination with adrenal insufficiency. GMPPA is a largely uncharacterized homolog of GMPPB. GMPPB catalyzes the formation of GDP-mannose, which is an essential precursor of glycan moieties of glycoproteins and glycolipids and is associated with congenital and limb-girdle muscular dystrophies with hypoglycosylation of α-dystroglycan. Surprisingly, GDP-mannose pyrophosphorylase activity was unchanged and GDP-mannose levels were strongly increased in lymphoblasts of individuals with GMPPA mutations. This suggests that GMPPA might serve as a GMPPB regulatory subunit mediating feedback inhibition of GMPPB instead of displaying catalytic enzyme activity itself. Thus, a triple-A-like syndrome can be added to the growing list of congenital disorders of glycosylation, in which dysregulation rather than mere enzyme deficiency is the basal pathophysiological mechanism.

Biosynthesis of GDP-fucose and other sugar nucleotides in the blood stages of Plasmodium falciparum.

Carbohydrate structures play important roles in many biological processes, including cell adhesion, cell-cell communication, and host-pathogen interactions. Sugar nucleotides are activated forms of sugars used by the cell as donors for most glycosylation reactions. Using a liquid chromatography-tandem mass spectrometry-based method, we identified and quantified the pools of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, GDP-mannose, and GDP-fucose in Plasmodium falciparum intraerythrocytic life stages. We assembled these data with the in silico functional reconstruction of the parasite metabolic pathways obtained from the P. falciparum annotated genome, exposing new active biosynthetic routes crucial for further glycosylation reactions. Fucose is a sugar present in glycoconjugates often associated with recognition and adhesion events. Thus, the GDP-fucose precursor is essential in a wide variety of organisms. P. falciparum presents homologues of GDP-mannose 4,6-dehydratase and GDP-L-fucose synthase enzymes that are active in vitro, indicating that most GDP-fucose is formed by a de novo pathway that involves the bioconversion of GDP-mannose. Homologues for enzymes involved in a fucose salvage pathway are apparently absent in the P. falciparum genome. This is in agreement with in vivo metabolic labeling experiments showing that fucose is not significantly incorporated by the parasite. Fluorescence microscopy of epitope-tagged versions of P. falciparum GDP-mannose 4,6-dehydratase and GDP-L-fucose synthase expressed in transgenic 3D7 parasites shows that these enzymes localize in the cytoplasm of P. falciparum during the intraerythrocytic developmental cycle. Although the function of fucose in the parasite is not known, the presence of GDP-fucose suggests that the metabolite may be used for further fucosylation reactions.

The balance between GMD and OFUT1 regulates Notch signaling pathway activity by modulating Notch stability.

The Notch signaling pathway plays an important role in development and physiology. In Drosophila, Notch is activated by its Delta or Serrate ligands, depending in part on the sugar modifications present in its extracellular domain. O-fucosyltransferase-1 (OFUT1) performs the first glycosylation step in this process, O-fucosylating various EGF repeats at the Notch extracellular domain. Besides its O-fucosyltransferase activity, OFUT1 also behaves as a chaperone during Notch synthesis and is able to down regulate Notch by enhancing its endocytosis and degradation. We have reevaluated the roles that O-fucosylation and the synthesis of GDP-fucose play in the regulation of Notch protein stability. Using mutants and the UAS/Gal4 system, we modified in developing tissues the amount of GDP-mannose-deshydratase (GMD), the first enzyme in the synthesis of GDP-fucose. Our results show that GMD activity, and likely the levels of GDP-fucose and O-fucosylation, are essential to stabilize the Notch protein. Notch degradation observed under low GMD expression is absolutely dependent on OFUT1 and this is also observed in Notch Abruptex mutants, which have mutations in some potential O-fucosylated EGF domains. We propose that the GDP-fucose/OFUT1 balance determines the ability of OFUT1 to endocytose and degrade Notch in a manner that is independent of the residues affected by Abruptex mutations in Notch EGF domains.

The balance between GMD and OFUT1 regulates Notch signaling pathway activity by modulating Notch stability

The Notch signaling pathway plays an important role in development and physiology. In Drosophila, Notch is activated by its Delta or Serrate ligands, depending in part on the sugar modifications present in its extracellular domain. O-fucosyltransferase-1 (OFUT1) performs the first glycosylation step in this process, O-fucosylating various EGF repeats at the Notch extracellular domain. Besides its O-fucosyltransferase activity, OFUT1 also behaves as a chaperone during Notch synthesis and is able to down regulate Notch by enhancing its endocytosis and degradation. We have reevaluated the roles that O-fucosylation and the synthesis of GDP-fucose play in the regulation of Notch protein stability. Using mutants and the UAS/Gal4 system, we modified in developing tissues the amount of GDP-mannose-deshydratase (GMD), the first enzyme in the synthesis of GDP-fucose. Our results show that GMD activity, and likely the levels of GDP-fucose and O-fucosylation, are essential to stabilize the Notch protein. Notch degradation observed under low GMD expression is absolutely dependent on OFUT1 and this is also observed in Notch Abruptex mutants, which have mutations in some potential O-fucosylated EGF domains. We propose that the GDP-fucose/OFUT1 balance determines the ability of OFUT1 to endocytose and degrade Notch in a manner that is independent of the residues affected by Abruptex mutations in Notch EGF domains.

Biochemical characterization and membrane topology of Alg2 from Saccharomyces cerevisiae as a bifunctional alpha1,3- and 1,6-mannosyltransferase involved in lipid-linked oligosaccharide biosynthesis.

N-Linked glycosylation involves the ordered, stepwise synthesis of the unique lipid-linked oligosaccharide precursor Glc(3)Man(9) GlcNAc(2)-PP-Dol on the endoplasmic reticulum (ER), catalyzed by a series of glycosyltransferases. Here we characterize Alg2 as a bifunctional enzyme that is required for both the transfer of the alpha1,3- and the alpha1,6-mannose-linked residue from GDP-mannose to Man(1)GlcNAc(2)-PP-Dol forming the Man(3)GlcNAc(2)-PP-Dol intermediate on the cytosolic side of the ER. Alg2 has a calculated mass of 58 kDa and is predicted to contain four transmembrane-spanning helices, two at the N terminus and two at the C terminus. Contradictory to topology predictions, we prove that only the two N-terminal domains fulfill this criterion, whereas the C-terminal hydrophobic sequences contribute to ER localization in a nontransmembrane manner. Surprisingly, none of the four domains is essential for transferase activity because truncated Alg2 variants can exert their function as long as Alg2 is associated with the ER by either its N- or C-terminal hydrophobic regions. By site-directed mutagenesis we demonstrate that an EX(7)E motif, conserved in a variety of glycosyltransferases, is not important for Alg2 function in vivo and in vitro. Instead, we identify a conserved lysine residue, Lys(230), as being essential for activity, which could be involved in the binding of the phosphate of the glycosyl donor.

A Temperature-sensitive mutation in the Arabidopsis thaliana phosphomannomutase gene disrupts protein glycosylation and triggers cell death.

Eukaryotic phosphomannomutases (PMMs) catalyze the interconversion of mannose 6-phosphate to mannose 1-phosphate and are essential to the biosynthesis of GDP-mannose. As such, plant PMMs are involved in ascorbic acid (AsA) biosynthesis and N-glycosylation. We report on the conditional phenotype of the temperature-sensitive Arabidopsis thaliana pmm-12 mutant. Mutant seedlings were phenotypically similar to wild type seedlings when grown at 16-18 degrees C but died within several days after transfer to 28 degrees C. This phenotype was observed throughout both vegetative and reproductive development. Protein extracts derived from pmm-12 plants had lower PMM protein and enzyme activity levels. In vitro biochemical analysis of recombinant proteins showed that the mutant PMM protein was compromised in its catalytic efficiency (K cat/K m). Despite significantly decreased AsA levels in pmm-12 plants, AsA deficiency could not account for the observed phenotype. Since, at restrictive temperature, total glycoprotein patterns were altered and glycosylation of protein-disulfide isomerase was perturbed, we propose that a deficiency in protein glycosylation is responsible for the observed cell death phenotype.

The de novo synthesis of GDP-fucose is essential for flagellar adhesion and cell growth in Trypanosoma brucei.

The protozoan parasite Trypanosoma brucei causes human African sleeping sickness in sub-Saharan Africa. The parasite makes several essential glycoproteins, which has led to the investigation of the sugar nucleotides and glycosyltransferases required to synthesize these structures. Fucose is a common sugar in glycoconjugates from many organisms; however, the sugar nucleotide donor GDP-fucose was only recently detected in T. brucei, and the importance of fucose metabolism in this organism is not known. In this paper, we identified the genes encoding functional GDP-fucose biosynthesis enzymes in T. brucei and created conditional null mutants of TbGMD, the gene encoding the first enzyme in the pathway from GDP-mannose to GDP-fucose, in both bloodstream form and procyclic form parasites. Under nonpermissive conditions, both life cycle forms of the parasite became depleted in GDP-fucose and suffered growth arrest, demonstrating that fucose metabolism is essential to both life cycle stages. In procyclic form parasites, flagellar detachment from the cell body was also observed under nonpermissive conditions, suggesting that fucose plays a significant role in flagellar adhesion. Fluorescence microscopy of epitope-tagged TbGMD revealed that this enzyme is localized in glycosomes, despite the absence of PTS-1 or PTS-2 target sequences.

Protein glycosylation in pmt mutants of Saccharomyces cerevisiae. Influence of heterologously expressed cellobiohydrolase II of Trichoderma reesei and elevated levels of GDP-mannose and cis-prenyltransferase activity.

Protein O-mannosylation has been postulated to be critical for production and secretion of glycoproteins in fungi. Therefore, understanding the regulation of this process and the influence of heterologous expression of glycoproteins on the activity of enzymes engaged in O-glycosylation are of considerable interest. In this study we expressed cellobiohydrolase II (CBHII) of T. reesei, which is normally highly O-mannosylated, in Saccharomyces cerevisiae pmt mutants partially blocked in O-mannosylation. We found that the lack of Pmt1 or Pmt2 protein O-mannosyltransferase activity limited the glycosylation of CBHII, but it did not affect its secretion. The S. cerevisiae pmt1Delta and pmt2Delta mutants expressing T. reesei cbh2 gene showed a decrease of GDP-mannose level and a very high activity of cis-prenyltransferase compared to untransformed strains. On the other hand, elevation of cis-prenyltransferase activity by overexpression of the S. cerevisiae RER2 gene in these mutants led to an increase of dolichyl phosphate mannose synthase activity, but it did not influence the activity of O-mannosyltransferases. Overexpression of the MPG1 gene increased the level of GDP-mannose and stimulated the activity of mannosyltransferases elongating O-linked sugar chains, leading to partial restoration of CBHII glycosylation.

Reconstitution in vitro of the GDP-fucose biosynthetic pathways of Caenorhabditis elegans and Drosophila melanogaster.

The deoxyhexose sugar fucose has an important fine-tuning role in regulating the functions of glycoconjugates in disease and development in mammals. The two genetic model organisms Caenorhabditis elegans and Drosophila melanogaster also express a range of fucosylated glycans, and the nematode particularly has a number of novel forms. For the synthesis of such glycans, the formation of GDP-fucose, which is generated from GDP-mannose in three steps catalysed by two enzymes, is required. By homology we have identified and cloned cDNAs encoding these two proteins, GDP-mannose dehydratase (GMD; EC 4.2.1.47) and GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase (GER or FX protein; EC 1.1.1.271), from both Caenorhabditis and Drosophila. Whereas the nematode has two genes encoding forms of GMD (gmd-1 and gmd-2) and one GER-encoding gene (ger-1), the insect has, like mammalian species, only one homologue of each (gmd and gmer). This compares to the presence of two forms of both enzymes in Arabidopsis thaliana. All corresponding cDNAs from Caenorhabditis and Drosophila, as well as the previously uncharacterized Arabidopsis GER2, were separately expressed, and the encoded proteins found to have the predicted activity. The biochemical characterization of these enzymes is complementary to strategies aimed at manipulating the expression of fucosylated glycans in these organisms.

Characterisation of a Leishmania mexicana knockout lacking guanosine diphosphate-mannose pyrophosphorylase.

In eukaryotes, the enzyme GDP-mannose pyrophosphorylase (GDP-MP) is essential for the formation of GDP-mannose, the donor of activated mannose for all glycosylation reactions. Unlike other eukaryotes, where deletion of GDP-mannose pyrophosphorylase is lethal, deletion of this gene in Leishmania mexicana has no effect on viability, but leads to the generation of avirulent parasites. In this study, we show that the null mutants have a perturbed morphology and cytokinesis, retarded growth and increased adherence to the substratum where they form large colonies. The null mutants attach avidly to mouse macrophages, but unlike the wild type organisms, they do not bind to the complement receptor 3 and are slow to induce phagocytosis. Once internalised, they localise to the phagolysosome, but in contrast to wild type organisms which transform into the intracellular amastigote and establish in the macrophage, they are cleared by 24 h in culture and by 5 h in vivo. The null mutants are hypersensitive to human but not mouse complement and to temperature and acidic pH. Surprisingly, in view of the lack of several known host-protective antigens, injection of the mutant parasites into BALB/c mice confers significant and long lasting protection against infection, suggesting that these temperature sensitive mutants are an attractive candidate for a live attenuated vaccine.

Molecular evolution of the GDP-mannose pathway genes (manB and manC) in Salmonella enterica.

The evolutionary history of the GDP-mannose pathway in Salmonella enterica was studied via sequencing manB and manC genes from 13 representative strains for O antigens containing mannose and/or sugar derivatives of GDP-D-mannose. In addition, colanic acid (CA) manB and manC genes were sequenced from selected strains, as the basis for a detailed comparison. Interestingly, including the eight previously characterized O antigen gene clusters, 12 of the 21 S. enterica strains studied in total (each representing a different O antigen structure) possess a manB gene which displays DNA identity, ranging from 93 to 99%, to the CA manB gene of S. enterica LT2. Furthermore, the CA-like manB genes (as well as the CA manB and manC genes) display subspecies specificity, and the CA and CA-like manB genes (for individual strains) appear to be evolving in concert via gene conversion events. In comparison, the manC genes were generally not CA-like, a situation also apparent in Escherichia coli,and therefore most strongly reflected the evolutionary history of the S. enterica O antigen GDP-mannose pathway. It appears that, in relatively recent times, gene capture from a distant source has occurred infrequently, and that groups of manB and manC genes have been maintained and are continuing to evolve within S. enterica and more closely related species.

Genetics of xanthan production in Xanthomonas campestris: the xanA and xanB genes are involved in UDP-glucose and GDP-mannose biosynthesis.

The nucleotide sequence of a 3.4-kb EcoRI-PstI DNA fragment of Xanthomonas campestris pv. campestris revealed two open reading frames, which were designated xanA and xanB. The genes xanA and xanB encode proteins of 448 amino acids (molecular weight of 48,919) and 466 amino acids (molecular weight of 50,873), respectively. These genes were identified by analyzing insertion mutants which were known to be involved in xanthan production. Specific tests for the activities of enzymes involved in the biosynthesis of UDP-glucose and GDP-mannose indicated that the xanA gene product was involved in the biosynthesis of both glucose 1-phosphate and mannose 1-phosphate. The deduced amino acid sequence of xanB showed a significant degree of homology (59%) to the phosphomannose isomerase of Pseudomonas aeruginosa, a key enzyme in the biosynthesis of alginate. Moreover, biochemical analysis and complementation experiments with the Escherichia coli manA fragment revealed that xanB encoded a bifunctional enzyme, phosphomannose isomerase-GDP-mannose pyrophosphorylase.