Glucosylceramide synthase maintains influenza virus entry and infection.

Influenza virus is an enveloped virus wrapped in a lipid bilayer derived from the host cell plasma membrane. Infection by influenza virus is dependent on these host cell lipids, which include sphingolipids. Here we examined the role of the sphingolipid, glucosylceramide, in influenza virus infection by knocking out the enzyme responsible for its synthesis, glucosylceramide synthase (UGCG). We observed diminished influenza virus infection in HEK 293 and A549 UGCG knockout cells and demonstrated that this is attributed to impaired viral entry. We also observed that entry mediated by the glycoproteins of other enveloped viruses that enter cells by endocytosis is also impaired in UGCG knockout cells, suggesting a broader role for UGCG in viral entry by endocytosis.

Functional characterization of GYG1 variants in two patients with myopathy and glycogenin-1 deficiency.

Glycogen storage disease XV is caused by variants in the glycogenin-1 gene, GYG1, and presents as a predominant skeletal myopathy or cardiomyopathy. We describe two patients with late-onset myopathy and biallelic GYG1 variants. In patient 1, the novel c.144-2A>G splice acceptor variant and the novel frameshift variant c.631delG (p.Val211Cysfs*30) were identified, and in patient 2, the previously described c.304G>C (p.Asp102His) and c.487delG (p.Asp163Thrfs*5) variants were found. Protein analysis showed total absence of glycogenin-1 expression in patient 1, whereas in patient 2 there was reduced expression of glycogenin-1, with the residual protein being non-functional. Both patients showed glycogen and polyglucosan storage in their muscle fibers, as revealed by PAS staining and electron microscopy. Age at onset of the myopathy phenotype was 53 years and 70 years respectively, with the selective pattern of muscle involvement on MRI corroborating the pattern of weakness. Cardiac evaluation of patient 1 and 2 did not show any specific abnormalities linked to the glycogenin-1 deficiency. In patient 2, who was shown to express the p.Asp102His mutated glycogenin-1, cardiac evaluation was still normal at age 77 years. This contrasts with the association of the p.Asp102His variant in homozygosity with a severe cardiomyopathy in several cases with an onset age between 30 and 50 years. This finding might indicate that the level of p.Asp102His mutated glycogenin-1 determines if a patient will develop a cardiomyopathy.

Identification of New Antifungal Agents Targeting Chitin Synthesis by a Chemical-Genetic Method.

Fungal infection is a leading cause of mortality in immunocompromised population; thus, it is urgent to develop new and safe antifungal agents. Different from human cells, fungi have a cell wall, which is composed mainly of polysaccharide glucan and chitin. The unique cell wall structure is an ideal target for antifungal drugs. In this research, a chemical-genetic method was used to isolate antifungal agents that target chitin synthesis in yeast cells. From a compound library, we isolated two benzothiazole compounds that showed greater toxicity to yeast mutants lacking glucan synthase Fks1 compared to wild-type yeast cells and mutants lacking chitin synthase Chs3. Both of them inhibited the activity of chitin synthase in vitro and reduced chitin level in yeast cells. Besides, these compounds showed clear synergistic antifungal effect with a glucan synthase inhibitors caspofungin. Furthermore, these compounds inhibited the growth of Saccharomyces cerevisiae and opportunistic pathogen Candida albicans. Surprisingly, the genome-wide mass-spectrometry analysis showed decreased protein level of chitin synthases in cells treated with one of these drugs, and this decrease was not a result of downregulation of gene transcription. Therefore, we successfully identified two new antifungal agents that inhibit chitin synthesis using a chemical-genetic method.

Blocking ß-1,6-glucan synthesis by deleting KRE6 and SKN1 attenuates the virulence of Candida albicans.

ß-1,6-glucan is an important component of the fungal cell wall. The ß-1,6-glucan synthase gene KRE6 was thought to be essential in the fungal pathogen Candida albicans because it could not be deleted in previous efforts. Also, the role of its homolog SKN1 was unclear because its deletion caused no defects. Here, we report the construction and characterization of kre6Δ/Δ, skn1Δ/Δ and kre6Δ/Δ skn1Δ/Δ mutants in C. albicans. While deleting KRE6 or SKN1 had no obvious phenotypic consequence, deleting both caused slow growth, cell separation failure, cell wall abnormalities, diminished hyphal growth, poor biofilm formation and loss of virulence in mice. Furthermore, the GPI-linked cell surface proteins Hwp1 and the invasin Als3 or Ssa1 were not detected in kre6Δ/Δ skn1Δ/Δ mutant. In GMM medium, RT-qPCR and western blotting revealed a constitutive expression of KRE6 and growth conditions-associated activation of SKN1. Like many hypha-specific genes, SKN1 is repressed by Nrg1, but its activation does not involve the transcription factor Efg1. Dysregulation of SKN1 reduces C. albicans ability to damage epithelial and endothelial cells and attenuates its virulence. Given the vital role of ß-1,6-glucan synthesis in C. albicans physiology and virulence, Kre6 and Skn1 are worthy targets for developing antifungal agents.

TALEN-Based Knockout System.

Targeted mutagenesis of genes-of-interest is a powerful method of addressing the functions of genes. Genome editing techniques, such as transcriptional activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems, have enabled this approach in various organisms because of their ease of use. In the ascidian, Ciona intestinalis, recent studies show that TALEN-based knockout can be applied to establishing both mutant lines and tissue-specific knockout for addressing gene functions. Here, we introduce recent updates to the TALEN toolkit that facilitate detailed functional analysis of genes in ascidians.

Polyglucosan myopathy and functional characterization of a novel GYG1 mutation.

OBJECTIVES: Disorders of glycogen metabolism include rare hereditary muscle glycogen storage diseases with polyglucosan, which are characterized by storage of abnormally structured glycogen in muscle in addition to exercise intolerance or muscle weakness. In this study, we investigated the etiology and pathogenesis of a late-onset myopathy associated with glycogenin-1 deficiency. MATERIALS AND METHODS: A family with two affected siblings, 64- and 66-year-olds, was studied. Clinical examination and whole-body MRI revealed weakness and wasting in the hip girdle and proximal leg muscles affecting ambulation in the brother. The sister had weakness and atrophy of hands and slight foot dorsiflexion difficulties. Muscle biopsy and whole-exome sequencing were performed in both cases to identify and characterize the pathogenesis including the functional effects of identified mutations. RESULTS: Both siblings demonstrated storage of glycogen that was partly resistant to alpha-amylase digestion. Both were heterozygous for two mutations in GYG1, one truncating 1-base deletion (c.484delG; p.Asp163Thrfs*5) and one novel missense mutation (c.403G>A; p.Gly135Arg). The mutations caused reduced expression of glycogenin-1 protein, and the missense mutation abolished the enzymatic function as analyzed by an in vitro autoglucosylation assay. CONCLUSION: We present functional evidence for the pathogenicity of a novel GYG1 missense mutation located in the substrate binding domain. Our results also demonstrate that glycogenin-1 deficiency may present with highly variable distribution of weakness and wasting also in the same family.

Muscle glycogen synthesis and breakdown are both impaired in glycogenin-1 deficiency.

OBJECTIVE: To study fat and carbohydrate metabolism during exercise in patients with glycogenin-1 (GYG1) deficiency, and to study whether IV glucose supplementation can alleviate exercise intolerance in these patients. METHODS: This is a case-control study with 4 patients with GYG1 deficiency and 4 healthy controls. Patients performed 1 hour of cycling at 50% of their maximal workload capacity, while controls cycled at the same absolute workloads as patients. Heart rate was measured continuously, and production and utilization of fat and glucose was assessed by stable isotope technique. The following day, patients repeated the exercise, this time receiving an IV 10% glucose supplement. RESULTS: Glucose utilization during exercise was similar in patients and controls, while palmitate utilization was greater in patients compared to controls. However, exercise-induced increases in lactate were attenuated to about half normal in patients. This was also the case during a handgrip exercise test. Glucose infusion improved exercise tolerance in patients, and lowered heart rate by on average 11 beats per minute during exercise. CONCLUSIONS: The findings suggest that patients with GYG1 deficiency not only have abnormal formation of glycogen, but also have impaired muscle glycogenolysis, as suggested by impaired lactate production during exercise and improved exercise tolerance with glucose infusion.

Functional characterization of zebrafish orthologs of the human Beta 3-Glucosyltransferase B3GLCT gene mutated in Peters Plus Syndrome.

Peters Plus Syndrome (PPS) is a rare autosomal recessive disease characterized by ocular defects, short stature, brachydactyly, characteristic facial features, developmental delay and other highly variable systemic defects. Classic PPS is caused by loss-of-function mutations in the B3GLCT gene encoding for a ß3-glucosyltransferase that catalyzes the attachment of glucose via a ß1-3 glycosidic linkage to O-linked fucose on thrombospondin type 1 repeats (TSRs). B3GLCT was shown to participate in a non-canonical ER quality control mechanism; however, the exact molecular processes affected in PPS are not well understood. Here we report the identification and characterization of two zebrafish orthologs of the human B3GLCT gene, b3glcta and b3glctb. The b3glcta and b3glctb genes encode for 496-aa and 493-aa proteins with 65% and 57% identity to human B3GLCT, respectively. Expression studies demonstrate that both orthologs are widely expressed with strong presence in embryonic tissues affected in PPS. In vitro glucosylation assays demonstrated that extracts from wildtype embryos contain active b3glct enzyme capable of transferring glucose from UDP-glucose to an O-fucosylated TSR, indicating functional conservation with human B3GLCT. To determine the developmental role of the zebrafish genes, single and double b3glct knockouts were generated using TALEN-induced genome editing. Extracts from double homozygous b3glct-/- embryos demonstrated complete loss of in vitro b3glct activity. Surprisingly, b3glct-/- homozygous fish developed normally. Transcriptome analyses of head and trunk tissues of b3glct-/- 24-hpf embryos identified 483 shared differentially regulated transcripts that may be involved in compensation for b3glct function in these embryos. The presented data show that both sequence and function of B3GLCT/b3glct genes is conserved in vertebrates. At the same time, complete b3glct deficiency in zebrafish appears to be inconsequential and possibly compensated for by a yet unknown mechanism.

Suppression of sucrose synthase affects auxin signaling and leaf morphology in tomato.

Metabolic enzymes have been found to play roles in plant development. Sucrose synthase (SUS) is one of the two enzyme families involved in sucrose cleavage in plants. In tomato, six SUS genes have been found. We generated transgenic tomato plants with RNAi suppression of SlSUS1, SlSUS3 and SlSUS4 genes. Independent transgenic lines with RNAi suppression of more than one SUS gene exhibited morphological effects on their cotyledons and leaf structure, but there were no significant effects on their carbohydrate levels, demonstrating that SUS has a developmental function, in addition to its metabolic function. Shoot apices of the transgenic lines showed elevated expression of JAGGED (JAG) and the auxin transporter PIN1. In a PIN1-GFP fusion reporter/SUS-RNAi hybrid, PIN1-GFP patterns were altered in developing leaves (as compared to control plants), indicating that SlSUS suppression alters auxin signaling. These results suggest possible roles for SUS in the regulation of plant growth and leaf morphology, in association with the auxin-signaling pathway.

O-Glycosylation modulates the stability of epidermal growth factor-like repeats and thereby regulates Notch trafficking.

Glycosylation in the endoplasmic reticulum (ER) is closely associated with protein folding and quality control. We recently described a non-canonical ER quality control mechanism for folding of thrombospondin type 1 repeats by protein O-fucosyltransferase 2 (POFUT2). Epidermal growth factor-like (EGF) repeats are also small cysteine-rich protein motifs that can be O-glycosylated by several ER-localized enzymes, including protein O-glucosyltransferase 1 (POGLUT1) and POFUT1. Both POGLUT1 and POFUT1 modify the Notch receptor on multiple EGF repeats and are essential for full Notch function. The fact that POGLUT1 and POFUT1 can distinguish between folded and unfolded EGF repeats raised the possibility that they participate in a quality control pathway for folding of EGF repeats in proteins such as Notch. Here, we demonstrate that cell-surface expression of endogenous Notch1 in HEK293T cells is dependent on the presence of POGLUT1 and POFUT1 in an additive manner. In vitro unfolding assays reveal that addition of O-glucose or O-fucose stabilizes a single EGF repeat and that addition of both O-glucose and O-fucose enhances stability in an additive manner. Finally, we solved the crystal structure of a single EGF repeat covalently modified by a full O-glucose trisaccharide at 2.2 Å resolution. The structure reveals that the glycan fills up a surface groove of the EGF with multiple contacts with the protein, providing a chemical basis for the stabilizing effects of the glycans. Taken together, this work suggests that O-fucose and O-glucose glycans cooperatively stabilize individual EGF repeats through intramolecular interactions, thereby regulating Notch trafficking in cells.

Creation, characterization and utilization of Cryptococcus neoformans mutants sensitive to micafungin.

We constructed deletion mutants of Cryptococcus neoformans var neoformans (serotype D) genes encoding late ergosterol biosynthetic pathway enzymes and found that the mutations enhanced susceptibility to various drugs including micafungin, one of the echinocandins, to which wild-type Cryptococcus strains show no susceptibility. Furthermore, through isolation of a mutant resistant to micafungin from a micafungin-sensitive erg mutant and genetic analysis of it, we found that the responsible mutation occurred in the hotspot 2 of FKS1 encoding ß-1, 3-glucan synthase, indicating that micafungin inhibited the growth of the erg mutant via inhibiting Fks1 activity. Addition of ergosterol to the culture of the erg mutants recovered the resistance to micafungin, suggesting that the presence of ergosterol in membrane inhibits the accession of micafungin to its target. We found that a loss of one of genes encoding subunits of v-ATPase, VPH1, made Cryptococcus cells sensitive to micafungin. Our observation that the erg2 vph1 double mutant was more sensitive to micafungin than either single mutant suggests that these two genes act differently in becoming resistant to micafungin. The erg mutants allowed us to study the physiological significance of ß-1, 3-glucan synthesis in C. neoformans; the inhibition of ß-1, 3-glucan synthesis induced cell death and changes in cellular morphology. By observing the erg mutant cells recovering from the growth inhibition imposed by micafungin, we recognized ß-1, 3-glucan synthesis would suppress filamentous growth in C. neoformans.

Cardiomyopathy as presenting sign of glycogenin-1 deficiency-report of three cases and review of the literature.

We describe a new type of cardiomyopathy caused by a mutation in the glycogenin-1 gene (GYG1). Three unrelated male patients aged 34 to 52 years with cardiomyopathy and abnormal glycogen storage on endomyocardial biopsy were homozygous for the missense mutation p.Asp102His in GYG1. The mutated glycogenin-1 protein was expressed in cardiac tissue but had lost its ability to autoglucosylate as demonstrated by an in vitro assay and western blot analysis. It was therefore unable to form the primer for normal glycogen synthesis. Two of the patients showed similar patterns of heart dilatation, reduced ejection fraction and extensive late gadolinium enhancement on cardiac magnetic resonance imaging. These two patients were severely affected, necessitating cardiac transplantation. The cardiomyocyte storage material was characterized by large inclusions of periodic acid and Schiff positive material that was partly resistant to alpha-amylase treatment consistent with polyglucosan. The storage material had, unlike normal glycogen, a partly fibrillar structure by electron microscopy. None of the patients showed signs or symptoms of muscle weakness but a skeletal muscle biopsy in one case revealed muscle fibres with abnormal glycogen storage. Glycogenin-1 deficiency is known as a rare cause of skeletal muscle glycogen storage disease, usually without cardiomyopathy. We demonstrate that it may also be the cause of severe cardiomyopathy and cardiac failure without skeletal muscle weakness. GYG1 should be included in cardiomyopathy gene panels.

Properties of a glycogen like polysaccharide produced by a mutant of Escherichia coli lacking glycogen synthase and maltodextrin phosphorylase.

Escherichia coli mutant TBP38 lacks glycogen synthase (GlgA) and maltodextrin phosphorylase (MalP). When grown on maltose in fed-batch fermentation TBP38 accumulated more than 50-fold higher glycogen-type polysaccharide than its parental strain. The polysaccharides were extracted at different growth stages and migrated as one peak in size-exclusion chromatography. TBP38 produced polysaccharides ranging 2.6 × 10(6)-4.6 × 10(6)Da. A ratio of short side-chains (DP ≦ 12) in the polysaccharides was greater than 50%, and number-average degree of polymerization varied from 9.8 to 8.4. The polysaccharides showed 70-290 times greater water-solubility than amylopectin. Km values using porcine and human pancreatic α-amylases with polysaccharides were 2- to 4-fold larger than that of amylopectin. kcat values were similar for both α-amylases. The TBP38 polysaccharides had 40-60% lower digestibility to amyloglucosidase than amylopectin. Intriguingly, the polysaccharides showed strong immunostimulating effects on mouse macrophage cell comparable to lipopolysaccharides. The lipopolysaccharide contamination levels were too low to account for this effect.

Muscle pathology and whole-body MRI in a polyglucosan myopathy associated with a novel glycogenin-1 mutation.

We report a 46-year-old female with late-onset skeletal myopathy affecting proximal limb muscles. Muscle biopsy revealed a polyglucosan myopathy with PAS-positive inclusions predominantly in glycogen-depleted fibers, which were demonstrated as type I fibers by ATPase staining. Whole-body magnetic imaging disclosed that the paravertebral, scapular, and pelvic girdle muscles, the anterior compartment of the arms, and the posterior compartment of the thighs were preferentially involved. Genetic analysis revealed a homozygous novel mutation in exon 6 of the glycogenin-1 gene (GYG1) (c.634C>T, p.His212Tyr). Protein analysis revealed normal levels of glycogenin-1 even before alpha-amylase digestion indicating preserved protein expression but impaired glucosylation. In vitro functional assay demonstrated that this variant impaired the autoglucosylating ability resulting in a non-functional protein. We report a glycogenin-1 related myopathy with a distinct histopathology and unique muscle imaging pattern.

A new muscle glycogen storage disease associated with glycogenin-1 deficiency.

We describe a slowly progressive myopathy in 7 unrelated adult patients with storage of polyglucosan in muscle fibers. Genetic investigation revealed homozygous or compound heterozygous deleterious variants in the glycogenin-1 gene (GYG1). Most patients showed depletion of glycogenin-1 in skeletal muscle, whereas 1 showed presence of glycogenin-1 lacking the C-terminal that normally binds glycogen synthase. Our results indicate that either depletion of glycogenin-1 or impaired interaction with glycogen synthase underlies this new form of glycogen storage disease that differs from a previously reported patient with GYG1 mutations who showed profound glycogen depletion in skeletal muscle and accumulation of glycogenin-1.

Deficient sucrose synthase activity in developing wood does not specifically affect cellulose biosynthesis, but causes an overall decrease in cell wall polymers.

The biosynthesis of wood in aspen (Populus) depends on the metabolism of sucrose, which is the main transported form of carbon from source tissues. The largest fraction of the wood biomass is cellulose, which is synthesized from UDP-glucose. Sucrose synthase (SUS) has been proposed previously to interact directly with cellulose synthase complexes and specifically supply UDP-glucose for cellulose biosynthesis. To investigate the role of SUS in wood biosynthesis, we characterized transgenic lines of hybrid aspen with strongly reduced SUS activity in developing wood. No dramatic growth phenotypes in glasshouse-grown trees were observed, but chemical fingerprinting with pyrolysis-GC/MS, together with micromechanical analysis, showed notable changes in chemistry and ultrastructure of the wood in the transgenic lines. Wet chemical analysis showed that the dry weight percentage composition of wood polymers was not changed significantly. However, a decrease in wood density was observed and, consequently, the content of lignin, hemicellulose and cellulose was decreased per wood volume. The decrease in density was explained by a looser structure of fibre cell walls as shown by increased wall shrinkage on drying. The results show that SUS is not essential for cellulose biosynthesis, but plays a role in defining the total carbon incorporation to wood cell walls.

Sulfatides are required for renal adaptation to chronic metabolic acidosis.

Urinary ammonium excretion by the kidney is essential for renal excretion of sufficient amounts of protons and to maintain stable blood pH. Ammonium secretion by the collecting duct epithelia accounts for the majority of urinary ammonium; it is driven by an interstitium-to-lumen NH3 gradient due to the accumulation of ammonium in the medullary and papillary interstitium. Here, we demonstrate that sulfatides, highly charged anionic glycosphingolipids, are important for maintaining high papillary ammonium concentration and increased urinary acid elimination during metabolic acidosis. We disrupted sulfatide synthesis by a genetic approach along the entire renal tubule. Renal sulfatide-deficient mice had lower urinary pH accompanied by lower ammonium excretion. Upon acid diet, they showed impaired ammonuria, decreased ammonium accumulation in the papilla, and chronic hyperchloremic metabolic acidosis. Expression levels of ammoniagenic enzymes and Na(+)-K(+)/NH4(+)-2Cl(-) cotransporter 2 were higher, and transepithelial NH3 transport, examined by in vitro microperfusion of cortical and outer medullary collecting ducts, was unaffected in mutant mice. We therefore suggest that sulfatides act as counterions for interstitial ammonium facilitating its retention in the papilla. This study points to a seminal role of sulfatides in renal ammonium handling, urinary acidification, and acid-base homeostasis.

Interruption of glycosphingolipid synthesis enhances osteoarthritis development in mice.

OBJECTIVE: Glycosphingolipids (GSLs) are ubiquitous membrane components that modulate transmembrane signaling and mediate cell-to-cell and cell-to-matrix interactions. GSL expression is decreased in the articular cartilage of humans with osteoarthritis (OA). This study was undertaken to determine the functional role of GSLs in cartilage metabolism related to OA pathogenesis in mice. METHODS: We generated mice with knockout of the chondrocyte-specific Ugcg gene, which encodes an initial enzyme of major GSL synthesis, using the Cre/loxP system (Col2-Ugcg(-/-) mice). In vivo OA and in vitro cartilage degradation models were used to evaluate the effect of GSLs on the cartilage degradation process. RESULTS: Although Col2-Ugcg(-/-) mice developed and grew normally, OA changes in these mice were dramatically enhanced with aging, through the overexpression of matrix metalloproteinase 13 and chondrocyte apoptosis, compared to their wild-type (WT) littermates. Col2-Ugcg(-/-) mice showed more severe instability-induced pathologic OA in vivo and interleukin-1α (IL-1α)-induced cartilage degradation in vitro. IL-1α stimulation of chondrocytes from WT mice significantly increased Ugcg messenger RNA expression and up-regulated GSL metabolism. CONCLUSION: Our results indicate that GSL deficiency in mouse chondrocytes enhances the development of OA. However, this deficiency does not affect the development and organization of cartilage tissue in mice at a young age. These findings indicate that GSLs maintain cartilage molecular metabolism and prevent disease progression, although GSLs are not essential for chondrogenesis of progenitor and stem cells and cartilage development in young mice. GSL metabolism in the cartilage is a potential target for developing a novel treatment for OA.

Disruption of Yarrowia lipolytica TPS1 gene encoding trehalose-6-P synthase does not affect growth in glucose but impairs growth at high temperature.

We have cloned the Yarrowia lipolytica TPS1 gene encoding trehalose-6-P synthase by complementation of the lack of growth in glucose of a Saccharomyces cerevisiae tps1 mutant. Disruption of YlTPS1 could only be achieved with a cassette placed in the 3' half of its coding region due to the overlap of its sequence with the promoter of the essential gene YlTFC1. The Yltps1 mutant grew in glucose although the Y. lipolytica hexokinase is extremely sensitive to inhibition by trehalose-6-P. The presence of a glucokinase, insensitive to trehalose-6-P, that constitutes about 80% of the glucose phosphorylating capacity during growth in glucose may account for the growth phenotype. Trehalose content was below 1 nmol/mg dry weight in Y. lipolytica, but it increased in strains expressing YlTPS1 under the control of the YlTEF1 promoter or with a disruption of YALI0D15598 encoding a putative trehalase. mRNA levels of YlTPS1 were low and did not respond to thermal stresses, but that of YlTPS2 (YALI0D14476) and YlTPS3 (YALI0E31086) increased 4 and 6 times, repectively, by heat treatment. Disruption of YlTPS1 drastically slowed growth at 35°C. Homozygous Yltps1 diploids showed a decreased sporulation frequency that was ascribed to the low level of YALI0D20966 mRNA an homolog of the S. cerevisiae MCK1 which encodes a protein kinase that activates early meiotic gene expression.