Comprehensive analysis of the UDP-glucuronate decarboxylase (UXS) gene family in tobacco and functional characterization of NtUXS16 in Golgi apparatus in Arabidopsis.

BACKGROUND: UDP-glucuronate decarboxylase (also named UXS) converts UDP-glucuronic acid (UDP-GlcA) to UDP-xylose (UDP-Xyl) by decarboxylation of the C6-carboxylic acid of glucuronic acid. UDP-Xyl is an important sugar donor that is required for the synthesis of plant cell wall polysaccharides. RESULTS: In this study, we first carried out the genome-wide identification of NtUXS genes in tobacco. A total of 17 NtUXS genes were identified, which could be divided into two groups (Group I and II), and the Group II UXSs can be further divided into two subgroups (Group IIa and IIb). Furthermore, the protein structures, intrachromosomal distributions and gene structures were thoroughly analyzed. To experimentally verify the subcellular localization of NtUXS16 protein, we transformed tobacco BY-2 cells with NtUXS16 fused to the monomeric red fluorescence protein (mRFP) at the C terminus under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The fluorescent signals of NtUXS16-mRFP were localized to the medial-Golgi apparatus. Contrary to previous predictions, protease digestion analysis revealed that NtUXS16 is not a type II membrane protein. Overexpression of NtUXS16 in Arabidopsis seedling in darkness led to a significant increase in hypocotyl length and a reduction in root length compared with the wild type. In summary, these results suggest Golgi apparatus localized-NtUXS16 plays an important role in hypocotyl and root growth in the dark. CONCLUSION: Our findings facilitate our understanding of the novel functions of NtUXS16 and provide insights for further exploration of the biological roles of NtUXS genes in tobacco.

Disruption of sugar nucleotide clearance is a therapeutic vulnerability of cancer cells.

Identifying metabolic steps that are specifically required for the survival of cancer cells but are dispensable in normal cells remains a challenge1. Here we report a therapeutic vulnerability in a sugar nucleotide biosynthetic pathway that can be exploited in cancer cells with only a limited impact on normal cells. A systematic examination of conditionally essential metabolic enzymes revealed that UXS1, a Golgi enzyme that converts one sugar nucleotide (UDP-glucuronic acid, UDPGA) to another (UDP-xylose), is essential only in cells that express high levels of the enzyme immediately upstream of it, UGDH. This conditional relationship exists because UXS1 is required to prevent excess accumulation of UDPGA, which is produced by UGDH. UXS1 not only clears away UDPGA but also limits its production through negative feedback on UGDH. Excess UDPGA disrupts Golgi morphology and function, which impedes the trafficking of surface receptors such as EGFR to the plasma membrane and diminishes the signalling capacity of cells. UGDH expression is elevated in several cancers, including lung adenocarcinoma, and is further enhanced during chemoresistant selection. As a result, these cancer cells are selectively dependent on UXS1 for UDPGA detoxification, revealing a potential weakness in tumours with high levels of UGDH.

Transcriptome-guided gene isolation and functional characterization of UDP-xylose synthase and UDP-D-apiose/UDP-D-xylose synthase families from Ornithogalum caudatum Ait.

KEY MESSAGE: The present study first identified the involvement of OcUAXS2 and OcUXS1-3 in anticancer polysaccharides biosynthesis in O. caudatum. UDP-xylose synthase (UXS) and UDP-D-apiose/UDP-D-xylose synthase (UAXS), both capable of converting UDP-D-glucuronic acid to UDP-D-xylose, are believed to transfer xylosyl residue to anticancer polysaccharides biosynthesis in Ornithogalum caudatum Ait. However, the cDNA isolation and functional characterization of genes encoding the two enzymes from O. caudatum has never been documented. Previously, the transcriptome sequencing of O. caudatum was performed in our laboratory. In this study, a total of six and two unigenes encoding UXS and UAXS were first retrieved based on RNA-Seq data. The eight putative genes were then successfully isolated from transcriptome of O. caudatum by reverse transcription polymerase chain reaction (RT-PCR). Phylogenetic analysis revealed the six putative UXS isoforms can be classified into three types, one soluble and two distinct putative membrane-bound. Moreover, the two UAXS isoenzymes were predicted to be soluble forms. Subsequently, these candidate cDNAs were characterized to be bona fide genes by functional expression in Escherichia coli individually. Although UXS and UAXS catalyzed the same reaction, their biochemical properties varied significantly. It is worth noting that a ratio switch of UDP-D-xylose/UDP-D-apiose for UAXS was established, which is assumed to be helpful for its biotechnological application. Furthermore, a series of mutants were generated to test the function of NAD+ binding motif GxxGxxG. Most importantly, the present study determined the involvement of OcUAXS2 and OcUXS1-3 in xylose-containing polysaccharides biosynthesis in O. caudatum. These data provide a comprehensive knowledge for UXS and UAXS families in plants.

Characterisation and expression of the pathway from UDP-glucose to UDP-xylose in differentiating tobacco tissue.

The pathway from UDP-glucose to UDP-xylose has been characterised in differentiating tobacco tissue. A xylogenic suspension cell culture of tobacco has been used as a source for the purification of the enzymes responsible for the oxidation of UDP-glucose to UDP-glucuronic acid and its subsequent decarboxylation to UDP-xylose. Protein purification and transcriptional studies show that two possible candidates can contribute to the first reaction. Most of the enzyme activity in the cultured cells could be accounted for by a protein with an Mr of 43 kDa which had dual specificity for UDP-glucose and ethanol. The cognate cDNA, with similarity to alcohol dehydrogenases (NtADH2) was expressed in E. coli to confirm the dual specificity. A second UDP-glucose dehydrogenase, corresponding to the monospecific form, ubiquitous amongst plants and animals, could not be purified from the tobacco cell cultures. However, two cDNAs were cloned with high similarity to the family of UDP-glucose dehydrogenases. Transcripts of both types of dehydrogenase showed highest expression in tissues undergoing secondary wall synthesis. The UDP-glucuronate decarboxylase was purified as polypeptides of Mr 87 and 40 kDa. Peptide fingerprinting of the latter polypeptide identified it as a form of UDP-glucuronate decarboxylase and functionality was established by expressing the cognate cDNA in E. coli. Expression of 40 kDa polypeptide and its corresponding mRNA was also found to be highest in tissues associated with secondary wall formation.

Properties and kinetic analysis of UDP-glucose dehydrogenase from group A streptococci. Irreversible inhibition by UDP-chloroacetol.

UDP-glucuronic acid is used by many pathogenic bacteria in the construction of an antiphagocytic capsule that is required for virulence. The enzyme UDP-glucose dehydrogenase catalyzes the NAD+-dependent 2-fold oxidation of UDP-glucose and provides a source of the acid. In the present study the recombinant dehydrogenase from group A streptococci has been purified and found to be active as a monomer. The enzyme contains no chromophoric cofactors, and its activity is unaffected by the presence of EDTA or carbonyl-trapping reagents. Initial velocity and product inhibition kinetic patterns are consistent with a bi-uni-uni-bi ping-pong mechanism in which UDP-glucose is bound first and UDP-glucuronate is released last. UDP-xylose was found to be a competitive inhibitor (Ki, 2.7 microM) of the enzyme. The enzyme is irreversibly inactivated by uridine 5'-diphosphate-chloroacetol due to the alkylation of an active site cysteine thiol. The apparent second order rate constant for the inhibition (ki/Ki) was found to be 2 x 10(3) mM-1 min-1. Incubation with the truncated compound, chloroacetol phosphate, resulted in no detectable inactivation when tested under comparable conditions. This supports the notion that uridine 5'-diphosphate-chloroacetol is bound in the place of UDP-glucose and is not simply acting as a nonspecific alkylating agent.

Topography of glycosylation and UDP-xylose production.

In order to define the location and organization of the numerous reactions involved in polysaccharide assembly during synthesis of proteoglycans and glycoproteins, the topography of some of the glycosylation reactions in chondroitin sulfate synthesis was examined using a relatively new technique for generating permeable cells. Permeable chondrocytes were shown to directly take up nucleotide sugar precursors and incorporate them into chondroitin sulfate proteoglycan (CSPG), allowing specific labeling at each step in chondroitin sulfate synthesis. Subcellular fractionation following labeling with UDP-[14C]xylose, UDP-[14C]galactose, UDP-[14C]glucuronic acid, or [35S]PAPS localized the labeled CSPG to the compartment where each glycosylation reaction occurred. From these experiments it appears that xylose addition begins in the endoplasmic reticulum and continues in the Golgi apparatus where galactose, glucuronic acid, and sulfate are added. This conclusion was confirmed by direct visualization of xylose incorporation using electron microscopic autoradiography (Vertel, B. M., Walters, L. M., Flay, N., Kearns, A. E., and Schwartz, N. B. (1993) J. Biol. Chem. 268, 11105-11112). Further examination of xylose addition showed that permeable chondrocytes can utilize both exogenous UDP-xylose transported into the lumen and UDP-xylose generated from UDP-glucuronic acid within the lumen. The enzyme responsible for this reaction, UDP-glucuronate carboxy-lyase, co-localized with xylosyltransferase activity in subcellular fractions. Orientation toward the lumen in subcellular compartments was determined by trypsin sensitivity in the permeable chondrocytes. Therefore, we conclude that UDP-xylose can be produced in the lumen of the compartment where it is utilized in CSPG synthesis, obviating the need for a direct transport mechanism for this nucleotide sugar and providing close regulation of UDP-xylose and UDP-glucuronic acid levels.

Xylosylation is an endoplasmic reticulum to Golgi event.

The subcellular site of xylosylation, the first carbohydrate modification of the core protein that initiates glycosaminoglycan chain synthesis, was characterized in situ. Methods were developed to combine electron microscopic (EM) autoradiography and the radiolabeling of semi-intact chondrocytes. In the accompanying paper, Kearns et al. (Kearns, A. E., Vertel, B. M., and Schwartz, N. B. (1993) J. Biol. Chem. 268, 11097-11104) presented biochemical and subcellular fractionation studies that utilized semi-intact chondrocytes and radiolabeled UDP sugars to overcome obstacles to the direct analysis of xylosylation. The results suggested that xylosylation begins in the endoplasmic reticulum (ER) and continues in the Golgi. The site of xylosylation was not specified further due to the limitations of subcellular fractionation techniques. The studies described in this report were undertaken to localize these modifications directly in situ. Semi-intact cell preparations were optimized for ultrastructural preservation by modifications of permeabilization methods utilizing nitrocellulose filter overlays. Biochemical analysis demonstrated the exclusive incorporation of UDP-xylose into the cartilage chondroitin sulfate proteoglycan (aggrecan) core protein and 3'-phosphoadenosine 5'-phosphosulfate (PAPS) into the highly modified proteoglycan monomer. Immunolocalization studies showed the equivalence of cytoplasmic subcompartments in normal and semi-intact chondrocytes at the levels of light and electron microscopy. Once the biochemical and morphological equivalence of intact and semi-intact cells was established, EM autoradiographic studies were pursued using UDP-[3H]xylose and [35S]PAPS. Based on both qualitative and quantitative data, silver grains resulting from incorporated sulfate were concentrated in the perinuclear Golgi, while those resulting from incorporated xylose were found at the cis or forming face of the Golgi and in vesicular regions of the peripheral cytoplasm associated with the late ER. These data support the view that xylose addition begins in a late ER compartment and continues in intermediate compartments, perhaps including the cis-Golgi.

Reconstitution into proteoliposomes and partial purification of the Golgi apparatus membrane UDP-galactose, UDP-xylose, and UDP-glucuronic acid transport activities.

Previous studies in vitro on proteoglycan biosynthesis from our laboratory have shown that nucleotide sugar precursors of all the sugars of the linkage oligosaccharides (xylose, galactose, and glucuronic acid) and of the glycosaminoglycans (N-acetylglucosamine, N-galactosamine, and glucuronic acid) are transported by specific carriers into the lumen of Golgi vesicles. More recently, we also reported the reconstitution in phosphatidylcholine liposomes of detergent-solubilized Golgi membrane proteins containing transport activities of CMP-sialic acid and adenosine-3'-phosphate-5'-phosphosulfate. We have now completed the successful reconstitution into liposomes of the Golgi membrane transport activities of UDP-galactose, UDP-xylose, and UDP-glucuronic acid. Transport of these nucleotide sugars into Golgi protein proteoliposomes occurred with the same affinity, temperature dependence, and sensitivity to inhibitors as observed with intact Golgi vesicles. Preloading of proteoliposomes with UMP, the putative antiporter for Golgi vesicle transport of these nucleotide sugars, stimulated transport of the nucleotide sugars by 2-3-fold. Transport of UDP-xylose into Golgi protein proteoliposomes was dependent on the presence of endogenous Golgi membrane lipids while that of UDP-galactose and UDP-glucuronic acid was not. This suggests a possible stabilizing or regulatory role for Golgi lipids on the UDP-xylose translocator. Finally, we have also shown that detergent-solubilized Golgi membrane translocator proteins can be partially purified by an ion-exchange chromatographic step before successful reconstitution into liposomes, demonstrating that this reconstitution approach can be used for the biochemical purification of these transporters.

Initiation of chondroitin sulfate biosynthesis: a kinetic analysis of UDP-D-xylose: core protein beta-D-xylosyltransferase.

The nature of the primary signals important for the addition of xylose to serines on the core protein of the cartilage chondroitin sulfate proteoglycan has been investigated. The importance of consensus sequence elements (Acidic-Acidic-Xxx-Ser-Gly-Xxx-Gly) in the natural acceptor was shown by the significant decrease in acceptor capability of peptide fragments derived by digestion of deglycosylated core protein with Staphylococcus aureus V8 protease, which cleaves within the consensus sequence, compared to the similar reactivity of trypsin-derived peptide fragments, in which consensus sequences remain intact. A comparison of the acceptor efficiencies (Vmax/Km) of synthetic peptides containing the proposed xylosylation consensus sequence and the natural acceptor (deglycosylated core protein) was then made by use of the in vitro xylosyltransferase assay. The two types of substrates were found to have nearly equivalent acceptor efficiencies and to be competitive inhibitors of each other's acceptor capability, with Km = Kiapparent. These results suggest that the artificial peptides containing the consensus sequence are analogues of individual substitution sites on the core protein and allowed the kinetic mechanism of the xylosyltransferase reaction to be investigated, with one of the artificial peptides as a model substrate. The most probable kinetic mechanism for the xylosyltransferase reaction was found to be an ordered single displacement with UDP-xylose as the leading substrate and the xylosylated peptide as the first product released. This represents the first reported formal kinetic mechanism for this glycosyltransferase and the only one reported for a nucleotide sugar:protein transferase.