Nucleotide Sugar Biosynthesis Research Articles

Exploring protein N-glycosylation in the green microalga Dunaliella salina

N-glycosylation is a major post-translational modification of proteins that has a crucial influence on cell targeting, activity, and half-life. This process starts in the endoplasmic reticulum where an oligosaccharide precursor is added to the newly synthesized protein and continues in the Golgi apparatus where the N-linked carbohydrate sequences are processed. Importantly, the most approved recombinant pharmaceutical proteins (so-called biologics) are glycoproteins mainly currently produced in mammalian cells which is a lengthy, costly, and complex process. Today, several microalgae such as the diatom Phaeodactylum tricornutum, and the green microalgae Chlamydomonas reinhardtii, Chlorella vulgaris, and Dunaliella salina are considered as efficient and eco-friendly alternative platforms for the production of biologics. However, unlike for C. reinhardtii, C. vulgaris, and P. tricornutum, there is to date no data reported regarding the protein N-glycosylation pathway in D. salina. Here, we first investigated the protein N-glycosylation in this green microalga by MALDI-TOF mass spectrometry. These analyses showed that proteins from D. salina are N-glycosylated with Man5GlcNAc2 oligomannoside. Using genome mining approaches, we then identified genes encoding proteins involved in the N-glycosylation pathways in D. salina. Genetic similarities and phylogenetic relationships of the putative sequences with homologues from C. reinhardtii, P. tricornutum, and humans were investigated. These data revealed that in D. salina the biosynthesis of nucleotide sugars and N-glycan biosynthesis share mainly similarities with the GnT I-independent pathway of C. reinhardtii that gives rise to the synthesis of a non-canonical oligomannoside Man5GlcNAc2. Although an α(1,3)-fucosyltransferase is identified in the D. salina genome, impairment of the cytosolic GDP-Fuc biosynthesis prevents the Golgi fucosylation of N-glycans. Taken together, these data demonstrated that proteins from D. salina are homogeneously N-glycosylated with a non-canonical Man5GlcNAc2.

A pilot study on the characterization and correlation of oropharyngeal and intestinal microbiota in children with type 1 diabetes mellitus

BackgroundType 1 Diabetes Mellitus (T1DM) is one of the most common endocrine disorders of childhood and adolescence, showing a rapidly increasing prevalence worldwide. A study indicated that the composition of the oropharyngeal and gut microbiota changed in T1DM. However, no studies have yet associated the changes between the microbiomes of the oropharyngeal and intestinal sites, nor between the flora and clinical indicators. In this study, we examined the composition and characteristics of oropharyngeal and intestinal flora in patients with T1DM in compared to healthy children. We identified correlations between oropharyngeal and intestinal flora and evaluated their association with clinical laboratory tests in patients with T1DM.MethodsThe oropharyngeal and fecal samples from 13 T1DM and 20 healthy children were analyzed by high-throughput sequencing of the V3–V4 region of 16S rRNA. The associations between microbes and microorganisms in oropharyngeal and fecal ecological niches, as well as the correlation between these and clinical indicators were further analyzed.ResultsIt was revealed that T1DM children had distinct microbiological characteristics, and the dominant oropharyngeal microbiota genus included Streptococcus, Prevotella, Leptotrichia, and Neisseria; that of intestinal microbiota included Blautia, Fusicatenibacter, Bacteroides, and Eubacterium_hallii_group. Furthermore, oropharyngeal Staphylococcus was significantly positively correlated with intestinal norank_f__Ruminococcaceae and Ruminococcus_torques_group in TIDM children. Moreover, in these children, differential genes in oropharyngeal and intestinal samples were enriched in metabolic pathways such as amino acid generation, fatty acid metabolism, and nucleotide sugar biosynthesis. Additionally, correlation analysis between the oropharyngeal/intestinal microbiome with laboratory tests showed significant correlations between several bacterial taxa in the oropharynx and intestines and glycated hemoglobin and C-peptide.ConclusionUnique microbial characteristics were found in the oropharynx and intestine in children with T1DM compared to healthy children. Positive correlations were found between changes in the relative abundance of oropharyngeal and gut microbiota in children with T1DM. Associations between the oropharyngeal/intestinal microbiota and laboratory investigations in children with T1DM suggest that the composition of the oropharyngeal and intestinal flora in children with T1DM may have some impact on glycemic control.

Bacitracin Methylene Disalicylate (BMD) Treatment Affects Spleen Proteome in Broiler Chicks Infected with Salmonella enteritidis.

Bacitracin Methylene Disalicylate (BMD), as a feed additive to poultry diets, enhances digestion, prevents Salmonella enteritidis (SE) colonization, and treats current infections. The objective of this study was to utilize a quantitative proteomic approach to determine the effect of BMD feed additive on broiler chickens challenged with SE in the spleen proteome. At 1 d of age, chicks were randomly allocated into four groups: control with and without SE challenge (CON, n = 60; CON-SE, n = 60), BMD with and without SE challenge (BMD, n = 60; BMD-SE, n = 60). Birds in the CON-SE and BMD-SE treatment were administered SE inoculum by oral gavage. On day three and day seven post-gavage, the spleen was collected aseptically from birds in each treatment group (CON, n = 4/day; CON-SE, n = 4/day; BMD, n = 4/day; BMD-SE, n = 4/day). Proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) showed an increased abundance of 115 proteins and decreased of 77 due to the BMD. Proteins that decreased in abundance were enriched for fibrinogen complex and extracellular space, whereas proteins that increased in abundance were enriched for proteasome-mediated ubiquitin-dependent protein catabolic process and mitochondrion. Analysis of the interaction between BMD and the Salmonella challenge found 230 differentially abundant proteins including proteins associated with RNA binding, spliceosome, protein transport, and cell adhesion among the upregulated proteins, and those associated with protein folding, carbon metabolism, biosynthesis of nucleotide sugars, response to oxidative stress, positive regulation of NIK/NF-kappaB signaling, and inflammatory response among the downregulated proteins. The impact of BMD treatment on spleen proteome indicates an anti-apoptotic effect. BMD also modified the response of the spleen to the SE challenge with a marked decrease in proteins that prompt cytokine synthesis and an increase in proteins involved in the selective removal of unfolded proteins.

Genome-wide expression analysis of novel heat-responsive microRNAs and their targets in contrasting wheat genotypes at reproductive stage under terminal heat stress.

Heat stress at terminal stage of wheat is critical and leads to huge yield losses worldwide. microRNAs (miRNAs) play significant regulatory roles in gene expression associated with abiotic and biotic stress at the post-transcriptional level. In the present study, we carried out a comparative analysis of miRNAs and their targets in flag leaves as well as developing seeds of heat tolerant (RAJ3765) and heat susceptible (HUW510) wheat genotypes under heat stress and normal conditions using small RNA and degradome sequencing. A total of 84 conserved miRNAs belonging to 35 miRNA families and 93 novel miRNAs were identified in the 8 libraries. Tae-miR9672a-3p, tae-miR9774, tae-miR9669-5p, and tae-miR5048-5p showed the highest expression under heat stress. Tae-miR9775, tae-miR9662b-3p, tae-miR1120a, tae-miR5084, tae-miR1122a, tae-miR5085, tae-miR1118, tae-miR1130a, tae-miR9678-3p, tae-miR7757-5p, tae-miR9668-5p, tae-miR5050, tae-miR9652-5p, and tae-miR9679-5p were expressed only in the tolerant genotype, indicating their role in heat tolerance. Comparison between heat-treated and control groups revealed that 146 known and 57 novel miRNAs were differentially expressed in the various tissues. Eight degradome libraries sequence identified 457 targets of the differentially expressed miRNAs. Functional analysis of the targets indicated their involvement in photosynthesis, spliceosome, biosynthesis of nucleotide sugars and protein processing in the endoplasmic reticulum, arginine and proline metabolism and endocytosis. This study increases the number of identified and novel miRNAs along with their roles involved in heat stress response in contrasting genotypes at two developing stages of wheat.

Construction of immobilized enzyme cascades for the biosynthesis of nucleotide sugars UDP-N-acetylglucosamine and UDP-glucuronic acid

Construction of immobilized enzyme cascades for the biosynthesis of nucleotide sugars UDP-N-acetylglucosamine and UDP-glucuronic acid

Transcriptome variation in banded newt (Ommatotriton vittatus) during its life cycle and habitat transition

Israel represents the southern limit of the distribution of the banded newt (Ommatotriton vittatus). The life cycle of O. vittatus includes several distinct phases: eggs, aquatic larvae, a terrestrial phase and an aquatic reproductive phase. We investigated differences in gene expression during the life cycle and transition of banded newts between terrestrial and aquatic habitats using mRNA-seq. We identified ∼10 k genes that were differentially expressed (DE) in one of the pairwise comparisons between 3 groups: 1 - terrestrial newts (males and females), 2 - aquatic newts (males and females), 3 - aquatic larvae before metamorphosis. The groups were clearly defined by Principal Components Analysis (PCA). The greatest difference was between aquatic newts (males and females) and aquatic larvae: ∼7.4 k DE genes. Of special interest were the ∼2.4 k genes DE between the aquatic and terrestrial phenotypes. These included prominent candidates with known roles in kidney function (uromodulin homologs were strongly associated with aquatic lifestyle), tissue structure (keratins), and the thyroid hormone signaling modulator DUOXA1. Additional developmental and metabolic pathways overrepresented among the identified DE genes included “epidermis development”, “nervous system development”, “nucleotide-sugar biosynthesis”. Overall, both metamorphosis and environmental transition of banded newts involve extensive transcriptomic remodeling involving developmental, metabolic, and cellular pathways. Understanding the roles of these pathways and individual genes is instrumental for studies of transition between habitats, especially those affected by climate change. Furthermore, the phenotypic flexibility of the newt and the underlying regulation of gene expression can shed light on the evolution of terrestrial vertebrates.

Phenotypic and genomic characterizations of Klebsiella pneumoniae ssp. pneumoniae and Rahnella inusitata strains reveal no clear association between genetic content and ropy phenotype

Ropy defect of pasteurized fluid milk is a type of spoilage which manifests itself by an increased viscosity, slimy body, and string-like flow during pouring. This defect has, among other causes, been attributed to the growth, proliferation and exopolysaccharide production by coliform bacteria, which are most commonly introduced in milk as post-pasteurization contaminants. As we identified both Klebsiella pneumoniae ssp. pneumoniae and Rahnella inusitata that were linked to a ropy defect, the goal of this study was to characterize 3 K. pneumoniae ssp. pneumoniae strains and 2 R. inusitata for (i) their ability to grow and cause ropy defect in milk at 6 and 21°C and to (ii) probe the genetic basis for observed ropy phenotype. While all K. pneumoniae ssp. pneumoniae and R. inusitata strains showed net growth of > 4 log10 over 48 h in UHT milk at 21°C, only R. inusitata strains displayed growth during 28-d incubation period at 6°C (>6 log10). Two out of 3 K. pneumoniae ssp. pneumoniae strains were capable of causing the ropy defect in milk at 21°C, as supported by an increase in the viscosity of milk and string-like flow during pouring; these 2 strains were originally isolated from raw milk. Only one R. inusitata strains was able to cause the ropy defect in milk; this strain was able to cause the defect at both 6 and 21°C, and was originally isolated from a pasteurized milk. These findings suggest that the potential of K. pneumoniae ssp. pneumoniae and R. inusitata to cause ropy defect in milk is a strain-dependent characteristic. Comparative genomics provided no definitive answer on genetic basis for the ropy phenotype. However, for K. pneumoniae ssp. pneumoniae, genes rffG, rffH, rfbD, and rfbC involved in biosynthesis and secretion of enterobacterial common antigen (ECA) could only be found in the 2 strains that produced ropy defect, and for R. inusitata a set of 2 glycosyltransferase- and flippase genes involved in nucleotide sugar biosynthesis and export could only be identified in the ropy strain. While these results provide some initial information for potential markers for strains that can cause ropy milk, the relationship between genetic content and ropiness in milk remains poorly understood and merits further investigation.

Biofilm formation and associated gene expression changes in Cronobacter from cereal related samples in China

Cronobacter is an important foodborne pathogen that can cause severe neonatal meningitis, necrotizing enterocolitis, and bacteremia. Currently, there is limited knowledge of biofilm formation in Cronobacter. In the present study, biofilm formation ability and associated gene expression changes in Cronobacter from cereal related samples was carried out systematically. Our results from 307 Cronobacter isolates analyzed for 48 h showed strong biofilm-forming ability in 14 strains (4.6%), moderate in 47 strains (15.3%), weak in 142 strains (46.2%), and no such ability in the remaining 104 strains (33.9%). Further studies on five strains with strong biofilm-forming ability showed that maximum biofilm formation in Cronobacter occurred after 24 h of cultivation, reaching a peak around 48 h–72 h, reducing gradually thereafter. Kyoto encyclopedia of genes and genomes (KEGG) analysis revealed that differentially expressed genes (DEGs) involved in flagellar assembly, oxidative phosphorylation, ribosome, photosynthesis, O-Antigen nucleotide sugar biosynthesis, citrate cycle (tricarboxylic acid cycle, TCA) and bacterial chemotaxis were enriched in biofilm forming cells. The genes involved these enrichment pathways were mostly downregulated when compared to planktonic cells. Several transcriptional regulator genes such as csrA and bolA, and the cell surface composition regulator gene glgS were significantly upregulated. 12 of 13 (92.3%) selected genes was found to be in agreement with the RNA-Seq of planktonic and biofilm cells by Quantitative real-time PCR analysis, thus increasing confidence in our data. Our research lays a sound theoretical basis for further studies on mechanisms regulating biofilm formation and provides a foundation for development of new food safety measures, clinical disease prevention and control.

An insight into the functional alterations in the gut microbiome of healthy adults in response to a multi-strain probiotic intake: a single arm open label trial.

Probiotic supplements, by definition, provide a benefit to the host, but few studies have investigated the effect of probiotic supplements in healthy adult populations. The present, single arm, open label clinical trial, evaluated compositional and functional changes in the fecal microbiome of healthy adults after supplementation with a 14-strain probiotic. We analysed the effect of a 14-strain probiotic blend (Bacillus subtilis NCIMB 30223, Bifidobacterium bifidum NCIMB 30179, B. breve NCIMB 30180, B. infantis NCIMB 30181, B. longum NCIMB 30182, Lactobacillus helveticus NCIMB 30184, L. delbrueckii subsp. bulgaricus NCIMB 30186, Lacticaseibacillus paracasei NCIMB 30185, Lactiplantibacillus plantarum NCIMB 30187, Lacticaseibacillus rhamnosus NCIMB 30188, L. helveticus NCIMB 30224, Lactobacillus salivarius NCIMB 30225, Lactococcus lactis subsp. lactis NCIMB 30222, and Streptococcus thermophilus NCIMB 30189), on the faecal microbiota of healthy young adults (n=41) in a single arm study. The adults consumed 4 capsules daily of the 14 strain blend(8 billion colony forming units/day) for 8 weeks. Compositional and functional changes in faecal microbiota before and after supplementation were assessed using shotgun metagenomic sequencing. Fasting breath analysis, faecal biochemistry and bowel habits were also assessed. In healthy adult participants, no significant changes to the overall alpha- or beta-diversity was observed after 8 weeks of multi-strain probiotic supplementation. However, in a simplified model that considered only time and individual differences, significant decreases (p < 0.05) in family Odoribacteraceae and Bacteroidaceae abundance and a significant increase (p < 0.05) in genus Megamonas abundance were observed. At a functional level, there were significant changes in functional gene abundance related to several functional pathways, including phenylalanine metabolism, O-antigen nucleotide sugar biosynthesis, bacterial chemotaxis, and flagellar assembly. No significant changes in stool form or frequency, fecal biochemistry, or methane and hydrogen breath tests were observed. In healthy young adults, overall alpha- and beta-diversity did not change in response to probiotic intake even though modest compositional changes at the family and genus level were observed. However, at functional level, results identified changes in gene abundance for several functional pathways.

Individual and binary exposure of embryonic zebrafish (Danio rerio) to single-walled and multi-walled carbon nanotubes in the absence and presence of dissolved organic matter

The available toxicological information was inadequate to assess the potential ecological risk of a mixture of different nanostructured carbon nanotubes (CNTs) to aquatic organisms, especially for the co-existence of mixed CNTs with dissolved organic matter (DOM). Herein, we investigated individual and binary exposure of zebrafish (Danio rerio) embryos to single-walled (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) in the absence and presence of DOM. Results indicated that embryonic chorions were more resistant to mixed-type CNTs than to single-type CNTs, yet the addition of DOM decreased this resistance. The mixed-type CNTs increased the antioxidant capacity of zebrafish embryos by increasing superoxide dismutase activity in comparison to the single-type CNTs. Furthermore, the mixed-type CNTs caused oxidative damage to the zebrafish embryos, characterized by an increase in malondialdehyde level. Nevertheless, the activation of the antioxidant defense system was modulated by the presence of DOM. Transcriptome sequencing analysis showed that the number of unique genes (UGs) and differentially expressed genes (DEGs) between the mixed-type CNTs and control groups was significantly enhanced compared to the single-type CNTs. DOM increased the number of UGs and up-regulated DEGs, but decreased the number of down-regulated DEGs. GO classification analysis revealed that the mixed-type CNTs mainly altered the cellular component process of single-type CNTs to induce joint effects. DOM generally enhanced the GO enrichment of DEGs in D. rerio embryos exposed to the mixed-type CNTs during the biological process. KEGG pathway enrichment analysis for the mixed-type CNTs showed enrichment of DEGs encoding ether lipid metabolism, glycerophospholipid metabolism, glycerolipid metabolism, citrate cycle, and biosynthesis of nucleotide sugars. However, DOM allowed more specific KEGG pathways towards the mixed-type CNTs to be identified. Despite the mixed-type CNTs exhibiting differential expression of functional genes compared to the control and single-type CNTs, DOM could regulate the expression of these functional genes associated with oxidative stress response, carbohydrate metabolism, endoplasmic reticulum stress, neuroendocrine, osmotic stress, and DNA damage and repair. Our study thus paves a solid way for exploring the molecular mechanism of aquatic toxicity of multiple nanomaterials under field-relevant conditions.

Whole genome sequence of Lactiplantibacillus plantarum MC5 and comparative analysis of eps gene clusters.

Probiotic Lactiplantibacillus plantarum MC5 produces large amounts of exopolysaccharides (EPS), and its use as a compound fermentor can greatly improve the quality of fermented milk. To gain insight into the genomic characteristics of probiotic MC5 and reveal the relationship between its EPS biosynthetic phenotype and genotype, we analyzed the carbohydrate metabolic capacity, nucleotide sugar formation pathways, and EPS biosynthesis-related gene clusters of strain MC5 based on its whole genome sequence. Finally, we performed validation tests on the monosaccharides and disaccharides that strain MC5 may metabolize. Genomic analysis showed that MC5 has seven nucleotide sugar biosynthesis pathways and 11 sugar-specific phosphate transport systems, suggesting that the strain can metabolize mannose, fructose, sucrose, cellobiose, glucose, lactose, and galactose. Validation results showed that strain MC5 can metabolize these seven sugars and produce significant amounts of EPS (> 250 mg/L). In addition, strain MC5 possesses two typical eps biosynthesis gene clusters, which include the conserved genes epsABCDE, wzx, and wzy, six key genes for polysaccharide biosynthesis, and one MC5-specific epsG gene. These insights into the mechanism of EPS-MC5 biosynthesis can be used to promote the production of EPS through genetic engineering.

Identification and analysis of hub genes of hypoxia-immunity in type 2 diabetes mellitus.

The chronic metabolic disease named type 2 diabetes (T2D) accounts for over 90% of diabetes mellitus. An increasing number of evidences have revealed that hypoxia has a significantly suppressive effect on cell-mediated immunity, as well as the utilization of glucose in diabetics. Therefore, we aimed to screen and identify hypoxia-immune-related hub genes in T2D through bioinformatic analysis. The Gene Expression Omnibus (GEO) database was used to get T2D gene expression profile data in the peripheral blood samples (GSE184050), and hypoxia-related genes were acquired from Molecular Signatures Database (MSigDB). Differentially expressed mRNAs (DEGs) and lncRNAs (DELs) between T2D and normal samples were identified by DeSeq2 package. The clusterProfiler package was used to perform enrichment analyses for the overlapped genes of DEGs and hypoxia-related genes. Further, Hypoxia-related hub genes were discovered using two machine learning algorithms. Next, the compositional patterns of immune and stromal cells in T2D and healthy groups were estimated by using xCell algorithm. Moreover, we used the weighted correlation network analysis (WGCNA) to examine the connection between genes and immune cells to screen immune-related genes. Gene Set Enrichment Analysis (GSEA) was used to investigate the functions of the hypoxia-immune-related hub genes. Finally, two peripheral blood cohorts of T2D (GSE184050 and GSE95849) as well as the quantitative real-time PCR (qRT-PCR) experiments for clicinal peripheral blood samples with T2D were used for verification analyses of hub genes. And meanwhile, a lncRNA-TF-mRNA network was constructed. Following the differentially expressed analysis, 38 out of 3822 DEGs were screened as hypoxia-related DEGs, and 493 DELs were found. These hypoxia-related DEGs were mainly enriched in the GO terms of pyruvate metabolic process, cytoplasmic vesicle lumen and monosaccharide binding, and the KEGG pathways of glycolysis/gluconeogenesis, pentose phosphate pathway and biosynthesis of nucleotide sugars. Moreover, 7 out of hypoxia-related DEGs were identified as hub genes. There were six differentially expressed immune cell types between T2D and healthy samples, which were further used as the clinical traits for WGCNA to identify AMPD3 and IER3 as the hypoxia-immune-related hub genes. The results of the KEGG pathways of genes in high-expression groups of AMPD3 and IER3 were mainly concentrated in glycosaminoglycan degradation and vasopressin-regulated water reabsorption, while the low-expression groups of AMPD3 and IER3 were mainly associated with RNA degradation and nucleotide excision repair. Finally, when compared to normal samples, both the AMPD3 and IER3 were highly expressed in the T2D groups in the GSE184050 and GSE95849 datasets. The result of lncRNA-TF-mRNA regulatory network showed that lncRNAs such as BACH1-IT1 and SNHG15 might induce the expression of the corresponding TFs such as TFAM and THAP12 and upregulate the expression of AMPD3. This study identified AMPD3 and IER3 as hypoxia-immune-related hub genes and potential regulatory mechanism for T2D, which provided a new perspective for elucidating the upstream molecular regulatory mechanism of diabetes mellitus.

Application of proteomics to determine the mechanism of ozone on sweet cherries (Prunus avium L.) by time-series analysis.

This research investigated the mechanism of ozone treatment on sweet cherry (Prunus avium L.) by Lable-free quantification proteomics and physiological traits. The results showed that 4557 master proteins were identified in all the samples, and 3149 proteins were common to all groups. Mfuzz analyses revealed 3149 candidate proteins. KEGG annotation and enrichment analysis showed proteins related to carbohydrate and energy metabolism, protein, amino acids, and nucleotide sugar biosynthesis and degradation, and fruit parameters were characterized and quantified. The conclusions were supported by the fact that the qRT-PCR results agreed with the proteomics results. For the first time, this study reveals the mechanism of cherry in response to ozone treatment at a proteome level.

Proteomic changes associated with the development of açaí (Euterpe oleracea Mart.) seeds.

Açaí palm (Euterpe oleracea Mart.) seeds are a rich source of mannans, which can be used to generate bioethanol or be converted to high-value D-mannose, in addition to being a source of polyphenols with beneficial health properties. Here, we present a quantitative proteome dataset of açaí seeds at four stages of development (S1, S2, S3, and S4 stages), in which 2465 high confidence proteins were identified and 524 of them show statistically different abundance profiles during development. Several enzymes involved in the biosynthesis of nucleotide-sugars were quantified, especially those dedicated to the formation of GDP-mannose, which showed an increase in abundance between stages S1 and S3. Our data suggest that linear mannans found abundantly in endosperm cell walls are initially deposited as galactomannans, and during development lose the galactosyl groups. Two isoforms of alpha-galactosidase enzymes showed significantly increased abundances in the S3 and S4 stages. Additionally, we quantified the enzymes participating in the central pathway of flavonoid biosynthesis responsible for the formation of catechin and epicatechin, which are subunits of procyanidins, the main class of polyphenols in the açaí seeds. These proteins showed the same pattern of deposition, in which higher abundances were seen in the S1 and S2 stages.

Candida albicans phosphate transport, facilitating nucleotide sugar biosynthesis, contributes to cell wall stability.

The Candida albicans high-affinity phosphate transporter Pho84 is required for normal Target of Rapamycin signaling, oxidative stress resistance and virulence of this fungal pathogen. It also contributes to C. albicans’ tolerance of two antifungal drug classes, polyenes and echinocandins. Echinocandins inhibit biosynthesis of a major cell wall component, beta-1,3-glucan. Cells lacking Pho84 were hypersensitive to other forms of cell wall stress beyond echinocandin exposure, while their cell wall integrity signaling response was weak. Metabolomics experiments showed that levels of phosphoric intermediates, including nucleotides like ATP and nucleotide sugars, were low in pho84 mutant compared to wild type cells recovering from phosphate starvation. Non-phosphoric precursors like nucleobases and nucleosides were elevated. Outer cell wall phosphomannan biosynthesis requires a nucleotide sugar,GDP-mannose. The nucleotide sugar UDP-glucose is the substrate of enzymes that synthesize two major structural cell wall polysaccharides, beta-1,3- and beta-1,6-glucan. Another nucleotide sugar, UDP-N-acetylglucosamine, is the substrate of chitin synthases which produce a stabilizing component of the intercellular septum and of lateral cell walls. Lack of Pho84 activity, and phosphate starvation, potentiated pharmacological or genetic perturbation of these enzymes. Our model is that low substrate concentrations of beta-D-glucan- and chitin synthases diminish enzymatic reaction rates and potentiate pharmacologic inhibitors to decrease the yield of their cell wall-stabilizing products. Phosphate import is not conserved between fungal and human cells, and humans do not synthesize beta-D-glucans or chitin. Hence inhibiting these processes simultaneously could yield potent antifungal effects with low toxicity to humans.

Optimization of Metabolic Oligosaccharide Engineering with Ac4GalNAlk and Ac4GlcNAlk by an Engineered Pyrophosphorylase.

Metabolic oligosaccharide engineering (MOE) has fundamentally contributed to our understanding of protein glycosylation. Efficient MOE reagents are activated into nucleotide-sugars by cellular biosynthetic machineries, introduced into glycoproteins and traceable by bioorthogonal chemistry. Despite their widespread use, the metabolic fate of many MOE reagents is only beginning to be mapped. While metabolic interconnectivity can affect probe specificity, poor uptake by biosynthetic salvage pathways may impact probe sensitivity and trigger side reactions. Here, we use metabolic engineering to turn the weak alkyne-tagged MOE reagents Ac4GalNAlk and Ac4GlcNAlk into efficient chemical tools to probe protein glycosylation. We find that bypassing a metabolic bottleneck with an engineered version of the pyrophosphorylase AGX1 boosts nucleotide-sugar biosynthesis and increases bioorthogonal cell surface labeling by up to two orders of magnitude. A comparison with known azide-tagged MOE reagents reveals major differences in glycoprotein labeling, substantially expanding the toolbox of chemical glycobiology.

Nucleotide sugar biosynthesis occurs in the glycosomes of procyclic and bloodstream form Trypanosoma brucei.

In Trypanosoma brucei, there are fourteen enzymatic biotransformations that collectively convert glucose into five essential nucleotide sugars: UDP-Glc, UDP-Gal, UDP-GlcNAc, GDP-Man and GDP-Fuc. These biotransformations are catalyzed by thirteen discrete enzymes, five of which possess putative peroxisome targeting sequences. Published experimental analyses using immunofluorescence microscopy and/or digitonin latency and/or subcellular fractionation and/or organelle proteomics have localized eight and six of these enzymes to the glycosomes of bloodstream form and procyclic form T. brucei, respectively. Here we increase these glycosome localizations to eleven in both lifecycle stages while noting that one, phospho-N-acetylglucosamine mutase, also localizes to the cytoplasm. In the course of these studies, the heterogeneity of glycosome contents was also noted. These data suggest that, unlike other eukaryotes, all of nucleotide sugar biosynthesis in T. brucei is compartmentalized to the glycosomes in both lifecycle stages. The implications are discussed.

UDP-glucose 6-dehydrogenase knockout impairs migration and decreases in vivo metastatic ability of breast cancer cells.

Dysregulated metabolism is a hallmark of cancer that supports tumor growth and metastasis. One understudied aspect of cancer metabolism is altered nucleotide sugar biosynthesis, which drives aberrant cell surface glycosylation known to support various aspects of cancer cell behavior including migration and signaling. We examined clinical association of nucleotide sugar pathway gene expression and found that UGDH, encoding UDP-glucose 6-dehydrogenase which catalyzes production of UDP-glucuronate, is associated with worse breast cancer patient survival. Knocking out the mouse homolog Ugdh in highly-metastatic 6DT1 breast cancer cells impaired migration ability without affecting in vitro proliferation. Further, Ugdh-KO resulted in significantly decreased metastatic capacity in vivo when the cells were orthotopically injected in syngeneic mice. Our experiments show that UDP-glucuronate biosynthesis is critical for metastasis in a mouse model of breast cancer.

Structural basis for substrate and product recognition in human phosphoglucomutase-1 (PGM1) isoform 2, a member of the \u03b1-d-phosphohexomutase superfamily

Human phosphoglucomutase 1 (PGM1) is an evolutionary conserved enzyme that belongs to the ubiquitous and ancient α-d-phosphohexomutases, a large enzyme superfamily with members in all three domains of life. PGM1 catalyzes the bi-directional interconversion between α-d-glucose 1-phosphate (G1P) and α-d-glucose 6-phosphate (G6P), a reaction that is essential for normal carbohydrate metabolism and also important in the cytoplasmic biosynthesis of nucleotide sugars needed for glycan biosynthesis. Clinical studies have shown that mutations in the PGM1 gene may cause PGM1 deficiency, an inborn error of metabolism previously classified as a glycogen storage disease, and PGM1 deficiency was recently also shown to be a congenital disorder of glycosylation. Here we present three crystal structures of the isoform 2 variant of PGM1, both as a free enzyme and in complex with its substrate and product. The structures show the longer N-terminal of this PGM1 variant, and the ligand complex structures reveal for the first time the detailed structural basis for both G1P substrate and G6P product recognition by human PGM1. We also show that PGM1 and the paralogous gene PGM5 are the results of a gene duplication event in a common ancestor of jawed vertebrates, and, importantly, that both PGM1 isoforms are conserved and of functional significance in all vertebrates. Our finding that PGM1 encodes two equally conserved and functionally important isoforms in the human organism should be taken into account in the evaluation of disease-related missense mutations in patients in the future.

Regulation of carbon metabolism in two maize sister lines contrasted for chilling tolerance.

Maize can grow in cool temperate climates but is often exposed to spring chilling temperatures that can affect early seedling growth. Here, we used two sister double-haploid lines displaying a contrasted tolerance to chilling to identify major determinants of long-term chilling tolerance. The chilling-sensitive (CS) and the chilling-tolerant (CT) lines were grown at 14 °C day/10 °C night for 60 d. CS plants displayed a strong reduction in growth and aerial biomass compared with CT plants. Photosynthetic efficiency was affected with an increase in energy dissipation in both lines. Chilling tolerance in CT plants was associated with higher chlorophyll content, glucose-6-phosphate dehydrogenase activity, and higher sucrose to starch ratio. Few changes in cell wall composition were observed in both genotypes. There was no obvious correlation between nucleotide sugar content and cell wall polysaccharide composition. Our findings suggest that the central starch-sucrose metabolism is one major determinant of the response to low temperature, and its modulation accounts for the ability of CT plants to cope with low temperature. This modulation seemed to be linked to a strong alteration in the biosynthesis of nucleotide sugars that, at a high level, could reflect the remobilization of carbon in response to chilling.