The unfolded protein response has a protective role in yeast models of classic galactosemia

Classic galactosemia is a human autosomal recessive disorder caused by mutations in the GALT gene (GAL7 in yeast), which encodes the enzyme galactose-1-phosphate uridyltransferase. Here we show that the unfolded protein response pathway is triggered by galactose in two yeast models of galactosemia: lithium-treated cells and the gal7Δ mutant. The synthesis of galactose-1-phosphate is essential to trigger the unfolded protein response under these conditions because the deletion of the galactokinase-encoding gene GAL1 completely abolishes unfolded protein response activation and galactose toxicity. Impairment of the unfolded protein response in both yeast models makes cells even more sensitive to galactose, unmasking its cytotoxic effect. These results indicate that endoplasmic reticulum stress is induced under galactosemic conditions and underscores the importance of the unfolded protein response pathway to cellular adaptation in these models of classic galactosemia.KEY WORDS: Yeast, Galactosemia, UPR, Lithium, Galactose     

Frequency Distribution of Q188R, N314D, Duarte 1, and Duarte 2 GALT Variant Alleles in an Indian Galactosemia Population

Classical galactosemia is a genetic disorder caused by mutations in the galactose-1-phosphate uridyltransferase (GALT) gene. The Q188R and N314D mutations are the most frequently cited GALT gene mutations. N314D is further associated with two variants, Duarte 1 and Duarte 2. Nevertheless, no reports are available on the clinical and molecular spectrum of galactosemia from the Indian population. The present study was designed to establish the frequency of these two most common mutations and their variants in Indian galactosemia patients so as to determine a single most common mutation/polymorphism for establishing the DNA-based diagnosis of galactosemia. Three alleles were found to be present at a frequency of 0.036 (Q188R), 0.40 (N314D), and 0.39 (D2); no D1 alleles were found. A significantly higher frequency of the Duarte 2 allele in our population suggests the presence of a milder form of galactosemia, which can be well managed by early diagnosis and dietary management.     

Hypoglycosylation with increased fucosylation and branching of serum transferrin N-glycans in untreated galactosemia

Untreated classic galactosemia (galactose-1-phosphate uridyltransferase [GALT] deficiency) is known as a secondary congenital disorders of glycosylation (CDG) characterized by galactose deficiency of glycoproteins and glycolipids (processing defect or CDG-II). The mechanism of this undergalactosylation has not been established. Here we show that in untreated galactosemia, there is also a partial deficiency of whole glycans of serum transferrin associated with increased fucosylation and branching as seen in genetic glycosylation assembly defects (CDG-I). Thus galactosemia seems to be a secondary "dual" CDG causing a processing as well as an assembly N-glycosylation defect. We also demonstrated that in galactosemia patients, transferrin N-glycan biosynthesis is restored upon dietary treatment.     

ARHI: A new target of galactose toxicity in Classic Galactosemia

In humans, deficiency of galactose-1-phosphate uridyltransferase (GALT) activity can lead to a potentially lethal disease called Classic Galactosemia. Although a galactose-restricted diet can prevent the acute lethality associated with the disorder, chronic complications persist in many well-treated patients. Approximately 85% of young women with Classic Galactosemia experience hypergonadotropic hypogonadism and premature ovarian failure (POF). Others suffer from mental retardation, growth restriction, speech dyspraxia, and ataxia. Despite decades of intense biochemical characterization, little is known about the molecular etiology, as well as the chronology of the pathological events leading to the poor outcomes. Several hypotheses have been proposed, most of which involved the accumulation of the intermediates and/or the deficit of the products, of the blocked GALT pathway. However, none of these hypotheses satisfactorily explained the absence of patient phenotypes in the GALT-knockout mice. Here we propose that the gene encoded the human tumor suppressor gene aplysia ras homolog I (ARHI) is a target of toxicity in Classic Galactosemia, and because ARHI gene is lost in rodents through evolution, it thus accounts for the lack of clinical phenotypes in the GALT-knockout mice.     

Variants of galactose-1-phosphate uridyl transferase in the Greek populations

Summary The frequency of variants of galactose-1-phosphate uridyl transferase was determined among the nine Greek populations by studying a sample of 1570 unselected individuals. Average frequency of normal allele GALT=0.942, galactosemia gene GALT ^G=0.0021 and the Duarte variant gene GALT ^D=0.0548 were observed. Frequency of galactosemia heterozygotes among Greeks was similar to that in other Caucasian populations, but the frequency of the Duarte variant was considerably higher. With the exception of two populations, one with low (Epirus) and one with high (Thrace) frequencies, the polymorphism of the Duarte variant displays very similar frequencies in the various Greek population groups.Supported by Grants HD 01 974 and GM 15 253 from the U.S. Public Health Service     

Detection of common mutations in the GALT gene through ARMS

Type I galactosemia is an inborn error resulting from mutations on both alleles of the GALT gene, which leads to the absence or deficiency of galactose-1-phosphate uridyltranseferase (GALT), the second of three enzymes catalyzing the conversion of galactose into glucose. On the basis of residual GALT activity, Type I galactosemia is classified into severe “Classical” and mild “Duarte” phenotypes. Classical galactosemia is frequently associated with S135L, Q188R and K285N mutations in the GALT gene. The functionally neutral N314D variation in the GALT gene is associated with Duarte galactosemia and is widespread among various worldwide populations. The present study aimed at detecting S135L, Q188R and K285N mutations and the N314D variant in the GALT gene by PCR using amplification refractory mutation system (ARMS). ARMS assays were established using standard DNA samples and were used for 8 galactosemia patients and 190 unrelated normal subjects all of Pakistani origin. S135L and K285N mutations were present neither in galactosemia patients nor in normal subjects. Only one galactosemia patient carried Q188R mutation that was in homozygous state. However, the N314D variant was frequently found both in affected (7 out of 16 alleles) and normal subjects (55 out of 380 alleles). This finding indicates that Duarte allele D314 might be far more common in Pakistani population than in European and North American ones.     

Studies on the metabolism of galactose-6-phosphate in rat heart and brain.

The subcellular distribution of NADP⁺ and NAD⁺-dependent glucose-6-phosphate and galactose-6-phosphate dehydrogenases were studied in rat liver, heart, brain, and chick brain. Only liver particulate fractions oxidized glucose-6-phosphate and galactose-6-phosphate with either NADP⁺ or NAD⁺ as cofactor. While all of the tissues examined had NADP⁺-dependent glucose-6-phosphate dehydrogenase activity, only rat liver and rat brain soluble fractions had NADP⁺-dependent galactose-6-phosphate dehydrogenase activity. Rat liver microsomal and rat brain soluble galactose-6-phosphate dehydrogenase activities were kinetically different (K_m's 0.5 mm and 10 mm, respectively, for galactose-6-phosphate), although their reaction products were both 6-phosphogalactonate. Rat brain subcellular fractions did not oxidize 6-phosphogalactonate with either NADP⁺ or NAD⁺ cofactors but phosphatase activities hydrolyzing 6-phosphogalactonate, galactose-6-phosphate and galactose-1-phosphate were found in crude brain homogenates. In addition, galactose-6-phosphate and 6-phosphogalactonate were tested as inhibitors of various enzymes, with largely negative results, except that 6-phosphogalactonate was a competitive inhibitor (K_i = 0.5 mM) of rat brain 6-phosphogluconate dehydrogenase.     

Phosphomannose isomerase inhibitors improve N-glycosylation in selected phosphomannomutase-deficient fibroblasts

Congenital disorders of glycosylation (CDG) are rare genetic disorders due to impaired glycosylation. The patients with subtypes CDG-Ia and CDG-Ib have mutations in the genes encoding phosphomannomutase 2 (PMM2) and phosphomannose isomerase (MPI or PMI), respectively. PMM2 (mannose 6-phosphate → mannose 1-phosphate) and MPI (mannose 6-phosphate ⇔ fructose 6-phosphate) deficiencies reduce the metabolic flux of mannose 6-phosphate (Man-6-P) into glycosylation, resulting in unoccupied N-glycosylation sites. Both PMM2 and MPI compete for the same substrate, Man-6-P. Daily mannose doses reverse most of the symptoms of MPI-deficient CDG-Ib patients. However, CDG-Ia patients do not benefit from mannose supplementation because >95% Man-6-P is catabolized by MPI. We hypothesized that inhibiting MPI enzymatic activity would provide more Man-6-P for glycosylation and possibly benefit CDG-Ia patients with residual PMM2 activity. Here we show that MLS0315771, a potent MPI inhibitor from the benzoisothiazolone series, diverts Man-6-P toward glycosylation in various cell lines including fibroblasts from CDG-Ia patients and improves N-glycosylation. Finally, we show that MLS0315771 increases mannose metabolic flux toward glycosylation in zebrafish embryos.     

Glycosylated synaptomatrix regulation of trans-synaptic signaling

Synapse formation is driven by precisely orchestrated intercellular communication between the presynaptic and the postsynaptic cell, involving a cascade of anterograde and retrograde signals. At the neuromuscular junction (NMJ), both neuron and muscle secrete signals into the heavily glycosylated synaptic cleft matrix sandwiched between the two synapsing cells. These signals must necessarily traverse and interact with the extracellular environment, for the ligand-receptor interactions mediating communication to occur. This complex synaptomatrix, rich in glycoproteins and proteoglycans, comprises heterogeneous, compartmentalized domains where specialized glycans modulate trans-synaptic signaling during synaptogenesis and subsequent synapse modulation. The general importance of glycans during development, homeostasis and disease is well established, but this important molecular class has received less study in the nervous system. Glycan modifications are now understood to play functional and modulatory roles as ligands and co-receptors in numerous tissues; however, roles at the synapse are relatively unexplored. We highlight here properties of synaptomatrix glycans and glycan-interacting proteins with key roles in synaptogenesis, with a particular focus on recent advances made in the Drosophila NMJ genetic system. We discuss open questions and interesting new findings driving this investigation of complex, diverse, and largely understudied glycan mechanisms at the synapse.     

Deficiency of Dol-P-Man Synthase Subunit DPM3 Bridges the Congenital Disorders of Glycosylation with the Dystroglycanopathies

Alpha-dystroglycanopathies such as Walker Warburg syndrome represent an important subgroup of the muscular dystrophies that have been related to defective O-mannosylation of alpha-dystroglycan. In many patients, the underlying genetic etiology remains unsolved. Isolated muscular dystrophy has not been described in the congenital disorders of glycosylation (CDG) caused by N-linked protein glycosylation defects. Here, we present a genetic N-glycosylation disorder with muscular dystrophy in the group of CDG type I. Extensive biochemical investigations revealed a strongly reduced dolichol-phosphate-mannose (Dol-P-Man) synthase activity. Sequencing of the three DPM subunits and complementation of DPM3-deficient CHO2.38 cells showed a pathogenic p.L85S missense mutation in the strongly conserved coiled-coil domain of DPM3 that tethers catalytic DPM1 to the ER membrane. Cotransfection experiments in CHO cells showed a reduced binding capacity of DPM3(L85S) for DPM1. Investigation of the four Dol-P-Man-dependent glycosylation pathways in the ER revealed strongly reduced O-mannosylation of alpha-dystroglycan in a muscle biopsy, thereby explaining the clinical phenotype of muscular dystrophy. This mild Dol-P-Man biosynthesis defect due to DPM3 mutations is a cause for alpha-dystroglycanopathy, thereby bridging the congenital disorders of glycosylation with the dystroglycanopathies.     

Uridine and dolichyl diphosphate activated oligosaccharides are intermediates in the biosynthesis of the S-layer glycoprotein of Methanothermus fervidus

The surface of the extremely thermophilic archaebacterium Methanothermus fervidus is covered by glycoprotein subunits. The carbohydrate moiety of the surface glycoprtein accounts for about 17 mol%. It is composed of mannose, 3-O-methylglucose, galactose, N-acetylglucosamine and N-acetylgalactosamine. From cell extracts the corresponding surgar-1-phosphates and nucleotide activated derivatives of Man, Gal, GlcNAc and GalNAc were isolated. Furthermore UDP-and dolichyl activated oligosaccharides were obtained. On the basis of the isolated precursors a pathway for the biosynthesis of the oligosaccharide chains is proposed.Abbreviations DNP-Glu N-2,4-dinitrophenyl-glutamic acid - Dol dolichol - Gal galactose - Gal-1-P galactose-1-phosphate - GalNAc N-acetylgalactosamine - GalNAc-1-P N-acetylgalactosamine-1-phosphate - Glc glucose - GlcNAc N-acetylglucosamine - GlcNAc-1-P N-acetylglucosamine-1-phosphate - Man mannose - Man-1-P mannose-1-phosphate - 3-O-MeGlc 3-O-methylglucose - P phosphate - TCA trichloroacetic acid - TLC thin-layer chromatography - Tris tris(hydroxymethyl)aminomethan     

Congenital disorder of glycosylation Ia: New differentially expressed proteins identified by 2-DE

Congenital disorders of glycosylation (CDG) comprise a family of inherited multisystemic disorders resulting from the deficiency of glycosylation pathways. N-glycosylation defects are classified as two biochemical and genetic established types, of which CDG-Ia is the most frequent. We performed 2-DE proteomic analysis on serum from two functional hemizygous CDG-Ia patients bearing T237M and D65Y missense changes. Comparative analysis of control/patient serum proteome allowed us to identify differential expression of 14 proteins. The most remarkable groups included proteins involved in immune response, coagulation mechanism and tissue protection against oxidative stress. The patient bearing D65Y mutation had less favourable clinical outcome and showed more abnormalities in the spot patterns, suggesting that the proteomic results might also be correlated with the phenotype of CDG patients. This study describes for the first time the differential expression of α₂-macroglobulin, afamin, fibrin and fibrinogen in CDG disorder and shows how the proteomic approach might be useful for understanding its physiopathology.     

PGM3 Mutations Cause a Congenital Disorder of Glycosylation with Severe Immunodeficiency and Skeletal Dysplasia

Human phosphoglucomutase 3 (PGM3) catalyzes the conversion of N-acetyl-glucosamine (GlcNAc)-6-phosphate into GlcNAc-1-phosphate during the synthesis of uridine diphosphate (UDP)-GlcNAc, a sugar nucleotide critical to multiple glycosylation pathways. We identified three unrelated children with recurrent infections, congenital leukopenia including neutropenia, B and T cell lymphopenia, and progression to bone marrow failure. Whole-exome sequencing demonstrated deleterious mutations in PGM3 in all three subjects, delineating their disease to be due to an unsuspected congenital disorder of glycosylation (CDG). Functional studies of the disease-associated PGM3 variants in E. coli cells demonstrated reduced PGM3 activity for all mutants tested. Two of the three children had skeletal anomalies resembling Desbuquois dysplasia: short stature, brachydactyly, dysmorphic facial features, and intellectual disability. However, these additional features were absent in the third child, showing the clinical variability of the disease. Two children received hematopoietic stem cell transplantation of cord blood and bone marrow from matched related donors; both had successful engraftment and correction of neutropenia and lymphopenia. We define PGM3-CDG as a treatable immunodeficiency, document the power of whole-exome sequencing in gene discoveries for rare disorders, and illustrate the utility of genomic analyses in studying combined and variable phenotypes.     

Mass spectrometry of transferrin and apolipoprotein C-III for diagnosis and screening of congenital disorder of glycosylation

Congenital disorder of glycosylation (CDG), formerly representing a group of diseases due to defects in the biosynthetic pathway of protein N-glycosylation, currently covers a wide range of disorders affecting glycoconjugates. Since its first application to serum transferrin from a CDG patient with phosphomannomutase-2 deficiency in 1992, mass spectrometry (MS) has been playing a key role in identification and characterization of glycosylation defects affecting glycoproteins. MS of native transferrin detects a lack of glycans characteristic to the classical CDG-I type of molecular abnormality. Electrospray ionization MS of native transferrin, especially, allows glycoforms to be analyzed precisely but requires basic knowledge regarding deconvolution of multiply-charged ions which may generate ghost signals upon transformation into a singly-charged form. MS of glycopeptides from tryptic digestion of transferrin delineates site-specific glycoforms and reveals a delicate balance of donor/acceptor substrates or the conformational effect of nascent proteins in cells. Matrix-assisted laser desorption ionization MS of apolipoprotein C-III is a simple method of elucidating the profiles of mucin-type core 1 O-glycans including site occupancy and glycoforms. In this technological review, the principle and pitfalls of MS for CDG are discussed and mass spectra of various types of CDG are presented.     

The Metabolic Origins of Mannose in Glycoproteins

Mannose in N-glycans is derived from glucose through phosphomannose isomerase (MPI, Fru-6-P ↔ Man-6-P) whose deficiency causes a congenital disorder of glycosylation (CDG)-Ib (MPI-CDG). Mannose supplements improve patients'' symptoms because exogenous mannose can also directly contribute to N-glycan synthesis through Man-6-P. However, the quantitative contributions of these and other potential pathways to glycosylation are still unknown. We developed a sensitive GC-MS-based method using [1,2-¹³C]glucose and [4-¹³C]mannose to measure their contribution to N-glycans synthesized under physiological conditions (5 mm glucose and 50 μm mannose). Mannose directly provides ∼10–45% of the mannose found in N-glycans, showing up to a 100-fold preference for mannose over exogenous glucose based on their exogenous concentrations. Normal human fibroblasts normally derive 25–30% of their mannose directly from exogenous mannose, whereas MPI-deficient CDG fibroblasts with reduced glucose flux secure 80% of their mannose directly. Thus, both MPI activity and exogenous mannose concentration determine the metabolic flux into the N-glycosylation pathway. Using various stable isotopes, we found that gluconeogenesis, glycogen, and mannose salvaged from glycoprotein degradation do not contribute mannose to N-glycans in fibroblasts under physiological conditions. This quantitative assessment of mannose contribution and its metabolic fate provides information that can help bolster therapeutic strategies for treating glycosylation disorders with exogenous mannose.     

Laboratory diagnosis of congenital disorders of glycosylation type I by analysis of transferrin glycoforms

Congenital disorders of glycosylation (CDG) are being recognized as a rapidly growing and complex group of disorders. The pathophysiology results from depressed synthesis or remodeling of oligosaccharide moieties of glycoproteins. The ultimate result is the formation of abnormal glycoproteins affecting their structure and metabolic functions. The most thoroughly studied subset of CDG are the type I defects affecting N-glycosylation. Causal mutations occur in at least 12 different genes which encode primarily monosaccharide transferases necessary for N-glycosylation in the endoplasmic reticulum. The broad clinical presentation of these glycosylation defects challenge clinicians to test for these defects in a variety of clinical settings. The first described CDG was a phosphomannomutase deficiency (CDG-Ia). The original method used to define the glycosylation defect was isoelectric focusing (IEF) of transferrin. More recently, the use of other charge separation methods and electrospray-mass spectrometry (ESI-MS) has proven valuable in detecting type I CDG defects. By mass resolution, the under-glycosylation of transferrin is characterized as the total absence of one or both N-linked oligosaccharide. Beyond providing a new understanding of the structure of transferrin in type I CDG patients, it is adaptable to high throughput serum analysis. The use of transferrin under-glycosylation to detect the type I CDG provides limited insight into the specific site of the defect in oligosaccharide assembly since its value is constrained to observation of the final product of glycoprotein synthesis. New analytical targets and tools are converging with the clinical need for diagnosis of CDG. Defining the biosynthetic sites responsible for specific CDG phenotypes is in progress, and ten more type I defects have been putatively identified. This review discusses current methods, such as IEF and targeted proteomics using mass spectrometry, that are used routinely to test for type I CDG disorders, along with some newer approaches to define the defective synthetic sites responsible for the type I CDG defects. All diagnostic endeavors are followed by the quest for a reliable treatment. The isolated success of CDG-Ib treatment will be described with the hope that this may expand to other type I CDG disorders.     

Neural and synaptic defects in slytherin, a zebrafish model for human congenital disorders of glycosylation

Congenital disorder of glycosylation type IIc (CDG IIc) is characterized by mental retardation, slowed growth and severe immunodeficiency, attributed to the lack of fucosylated glycoproteins. While impaired Notch signaling has been implicated in some aspects of CDG IIc pathogenesis, the molecular and cellular mechanisms remain poorly understood. We have identified a zebrafish mutant slytherin (srn), which harbors a missense point mutation in GDP-mannose 4,6 dehydratase (GMDS), the rate-limiting enzyme in protein fucosylation, including that of Notch. Here we report that some of the mechanisms underlying the neural phenotypes in srn and in CGD IIc are Notch-dependent, while others are Notch-independent. We show, for the first time in a vertebrate in vivo, that defects in protein fucosylation leads to defects in neuronal differentiation, maintenance, axon branching, and synapse formation. Srn is thus a useful and important vertebrate model for human CDG IIc that has provided new insights into the neural phenotypes that are hallmarks of the human disorder and has also highlighted the role of protein fucosylation in neural development.     

The PDZ-GEF Gef26 regulates synapse development and function via FasII and Rap1 at the Drosophila neuromuscular junction

Guanine nucleotide exchange factors (GEFs) are essential for small G proteins to activate their downstream signaling pathways, which are involved in morphogenesis, cell adhesion, and migration. Mutants of Gef26, a PDZ-GEF (PDZ domain-containing guanine nucleotide exchange factor) in Drosophila, exhibit strong defects in wings, eyes, and the reproductive and nervous systems. However, the precise roles of Gef26 in development remain unclear. In the present study, we analyzed the role of Gef26 in synaptic development and function. We identified significant decreases in bouton number and branch length at larval neuromuscular junctions (NMJs) in Gef26 mutants, and these defects were fully rescued by restoring Gef26 expression, indicating that Gef26 plays an important role in NMJ morphogenesis. In addition to the observed defects in NMJ morphology, electrophysiological analyses revealed functional defects at NMJs, and locomotor deficiency appeared in Gef26 mutant larvae. Furthermore, Gef26 regulated NMJ morphogenesis by regulating the level of synaptic Fasciclin II (FasII), a well-studied cell adhesion molecule that functions in NMJ development and remodeling. Finally, our data demonstrate that Gef26-specific small G protein Rap1 worked downstream of Gef26 to regulate the level of FasII at NMJs, possibly through a βPS integrin-mediated signaling pathway. Taken together, our findings define a novel role of Gef26 in regulating NMJ development and function.     

N-Glycosylation at the SynCAM (Synaptic Cell Adhesion Molecule) Immunoglobulin Interface Modulates Synaptic Adhesion

Select adhesion molecules connect pre- and postsynaptic membranes and organize developing synapses. The regulation of these trans-synaptic interactions is an important neurobiological question. We have previously shown that the synaptic cell adhesion molecules (SynCAMs) 1 and 2 engage in homo- and heterophilic interactions and bridge the synaptic cleft to induce presynaptic terminals. Here, we demonstrate that site-specific N-glycosylation impacts the structure and function of adhesive SynCAM interactions. Through crystallographic analysis of SynCAM 2, we identified within the adhesive interface of its Ig1 domain an N-glycan on residue Asn⁶⁰. Structural modeling of the corresponding SynCAM 1 Ig1 domain indicates that its glycosylation sites Asn⁷⁰/Asn¹⁰⁴ flank the binding interface of this domain. Mass spectrometric and mutational studies confirm and characterize the modification of these three sites. These site-specific N-glycans affect SynCAM adhesion yet act in a differential manner. Although glycosylation of SynCAM 2 at Asn⁶⁰ reduces adhesion, N-glycans at Asn⁷⁰/Asn¹⁰⁴ of SynCAM 1 increase its interactions. The modification of SynCAM 1 with sialic acids contributes to the glycan-dependent strengthening of its binding. Functionally, N-glycosylation promotes the trans-synaptic interactions of SynCAM 1 and is required for synapse induction. These results demonstrate that N-glycosylation of SynCAM proteins differentially affects their binding interface and implicate post-translational modification as a mechanism to regulate trans-synaptic adhesion.