A quantitative cytochemical study of UDP-D-glucose: NAD-oxidoreductase (E.C. 1.1.1.22) activity during stelar differentiation in Pisum sativum L. cv Meteor

A quantitative cytochemical assay for UDP-D-glucose dehydrogenase (UDPGD) activity employing scanning and integrating microdensitometry has been revised and applied to a study of this enzyme during the initiation of secondary cell wall biosynthesis during the formation of primary vascular tissues in roots of Pisum sativum L. cv Meteor. The reaction involves the use of NBT as final electron acceptor and is inhibited 10-fold by either 10 mM UDP-D-xylose or 25 mM UDP-D-glucuronic acid, two molecules involved in feed-back inhibition of UDPGD activity in vivo. UDPGD is a far-from equilibrium enzyme representing a flux-generating step in the biosynthesis of precursors for hemicelluloses involved in secondary cell wall construction, and can be demonstrated to increase sharply in activity in cells of the developing primary vascular elements. This changed activity occurs 18-20 cells back from the root cap junction and coincides with the first cells containing the activated programme for secondary cell wall synthesis.     

Functional analysis of Arabidopsis thaliana RHM2/MUM4, a multidomain protein involved in UDP-D-glucose to UDP-L-rhamnose conversion

UDP-L-rhamnose is required for the biosynthesis of cell wall rhamnogalacturonan-I, rhamnogalacturonan-II, and natural compounds in plants. It has been suggested that the RHM2/MUM4 gene is involved in conversion of UDP-D-glucose to UDP-L-rhamnose on the basis of its effect on rhamnogalacturonan-I-directed development in Arabidopsis thaliana. RHM2/MUM4-related genes, RHM1 and RHM3, can be found in the A. thaliana genome. Here we present direct evidence that all three RHM proteins have UDP-D-glucose 4,6-dehydratase, UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase, and UDP-4-keto-L-rhamnose 4-keto-reductase activities in the cytoplasm when expressed in the yeast Saccharomyces cerevisiae. Functional domain analysis revealed that the N-terminal region of RHM2 (RHM2-N; amino acids 1-370) has the first activity and the C-terminal region of RHM2 (RHM2-C; amino acids 371-667) has the two following activities. This suggests that RHM2 converts UDP-d-glucose to UDP-L-rhamnose via an UDP-4-keto-6-deoxy-D-glucose intermediate. Site-directed mutagenesis of RHM2 revealed that mucilage defects in MUM4-1 and MUM4-2 mutant seeds of A. thaliana are caused by abolishment of RHM2 enzymatic activity in the mutant strains and furthermore, that the GXXGXX(G/A) and YXXXK motifs are important for enzymatic activity. Moreover, a kinetic analysis of purified His(6)-tagged RHM2-N protein revealed 5.9-fold higher affinity of RHM2 for UDP-D-glucose than for dTDP-D-glucose, the preferred substrate for dTDP-D-glucose 4,6-dehydratase from bacteria. RHM2-N activity is strongly inhibited by UDP-L-rhamnose, UDP-D-xylose, and UDP but not by other sugar nucleotides, suggesting that RHM2 maintains cytoplasmic levels of UDP-D-glucose and UDP-L-rhamnose via feedback inhibition by UDP-L-rhamnose and UDP-D-xylose.     

Properties of a soluble polyprenyl phosphate: UDP-D-glucose glucosyltransferase.

A soluble enzyme that catalyzes the transfer of D-glucose from UDP-D-glucose to dolichyl phosphate has been prepared by sonic oscillation of Acanthamoeba castellani cysts. The product of catalysis is dolichyl beta-D-glucosyl phosphate. The enzyme requires a divalent cation, either magnesium or manganese, and the presence of a reducing agent for maximum activity. Solanesyl phosphate and ficaprenyl phosphate are alternative substrates, apparently at lower rates, but GDP-D-glucose, UDP-D-glucuronic acid, UDP-N-acetyl-D-glucosamine, and UDP-D-xylose are not substrates. The temperature optimum is 30 degrees C, the pH optimum is pH 7.0, the Km for UDP-Glc is 9.1 microM and for dolichyl phosphate it is 4.5 microM. Uridine monophosphate and UDP are inhibitors of the reaction, UDP causing reversal and UMP being a competitive inhibitor of UDP-Glc with a Ki of 62 microM. The enzyme can be stored indefinitely below -20 degrees C, is stable for several days at 4 degrees C, but is half-inactivated within 2 h at 30 degrees C and completely inactivated within 10 min at 52 degrees C.     

The mur4 mutant of arabidopsis is partially defective in the de novo synthesis of uridine diphospho L-arabinose.

To obtain information on the synthesis and function of arabinosylated glycans, the mur4 mutant of Arabidopsis was characterized. This mutation leads to a 50% reduction in the monosaccharide L-arabinose in most organs and affects arabinose-containing pectic cell wall polysaccharides and arabinogalactan proteins. Feeding L-arabinose to mur4 plants restores the cell wall composition to wild-type levels, suggesting a partial defect in the de novo synthesis of UDP-L-arabinose, the activated sugar used by arabinosyltransferases. The defect was traced to the conversion of UDP-D-xylose to UDP-L-arabinose in the microsome fraction of leaf material, indicating that mur4 plants are defective in a membrane-bound UDP-D-xylose 4-epimerase.     

The interaction of xylosyltransferase and glucuronyltransferase involved in glucuronoxylan synthesis in pea (Pisum sativum) epicotyls.

A particulate enzyme preparation from etiolated pea (Pisum sativum) epicotyls was found to incorporate xylose from UDP-D-xylose into beta-(1----4)-xylan. The ability of this xylan to act as an acceptor for incorporation of [14C]glucuronic acid from UDP-D-[14C]glucuronic acid in a subsequent incubation was very limited, even though glucuronic acid incorporation was greatly prolonged when UDP-D-xylose was present in the same incubation as UDP-D-[14C]glucuronic acid. This indicated that glucuronic acid could not be added to preformed xylan. However, the presence of UDP-D-glucuronic acid inhibited incorporation of [14C]xylose from UDP-D-[14C]xylose into beta-(1----4)-xylan, and neither S-adenosylmethionine nor acetyl-CoA stimulated either the xylosyltransferase or the glucuronyltransferase.     

Possible control sites of polysaccharide synthesis during cell growth and wall expansion of pea seedlings (Pisum sativum L.)

The activities of the enzymes of uridine diphosphate sugar interconversions (UDP-D-glucose 4-epimerase, UDP-D-glucuronate 4-epimerase, UDP-D-xylose 4-epimerase, UDP-D-glucose dehydrogenase and UDP-D-glucuronate decarboxylase) were measured by using enzymic preparations (protein precipitated between 40–65% (NH₄)₂SO₄ saturation) isolated from segments at different stages of elongation of the third internode of pea seedlings. All enzymic activities increased from dividing and non-elongated cells to fully elongated cells. At all stages of growth, the specific activity or the activity per cell of UDP-D-glucose dehydrogenase was much lower than that of UDP-D-glucuronate decarboxylase and this may represent a controlling step in the formation of UDP-D-xylose. During elongation, changes were also found in the activities of the epimerases. These could be correlated with the corresponding variations which occur in the chemical structure and physical properties of pectins during cell wall extension. However, the high levels of the epimerases present in cells which have completed elongation growth suggest that pectin synthesis is mainly controlled at the sites of the synthetase reactions.     

Characterization of soluble and putative membrane-bound UDP-glucuronic acid decarboxylase (OsUXS) isoforms in rice

Arabinoxylans in crop plants are the major sugar components of the cell walls, and UDP-xylose is a key substrate in the biosynthesis of xylans. In this study, the six putative UDP-D-glucuronic acid decarboxylase genes from rice (Oryza sativa UDP-xylose synthase; OsUXS) were cloned. Except for the soluble form of OsUXS3 (GenBank Accession No. \AB079064), the remaining five OsUXS enzymes contain a putative membrane-bound region. The six OsUXS genes were classified into three types by phylogenetic analysis and were expressed during the development of rice seeds. The HPLC retention times of the enzyme products and NMR data, indicate that the recombinant OsUXS2 enzyme catalyzes the conversion of UDP-D-glucuronic acid to UDP-D-xylose. Interestingly, the reactions catalyzed by the recombinant OsUXS2 and OsUXS3 enzymes were inhibited by NADP+, and accelerated by NADPH. The catalytic activities of the recombinant OsUXS2 and OsUXS3 enzymes were strongly inhibited by UDP, UTP, TDP, and TTP. The expression levels of OsUXS genes changed in different manners during the development of rice seeds, suggesting that each corresponding OsUXS enzyme plays a significant role in rice seed development at a certain stage. In the present study, we report that the UXS2-type enzyme of rice is not only characterized for the first time but also show significant findings involved in the gene expression of OsUXSs.     

Functional expression of Escherichia coli enzymes synthesizing GDP-L-fucose from inherent GDP-D-mannose in Saccharomyces cerevisiae

Fucosylation of glycans on glycoproteins and -lipids requires the enzymatic activity of relevant fucosyltransferases and GDP-L-fucose as the donor. Due to the biological importance of fucosylated glycans, a readily accessible source of GDP-L-fucose would be required. Here we describe the construction of a stable recombinant S.cerevisiae strain expressing the E.coli genes gmd and wcaG encoding the two enzymes, GDP-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/4-reductase (GMER(FX)) respectively, needed to convert GDP-mannose to GDP-fucose via the de novo pathway. Taking advantage of the rich inherent cytosolic GDP-mannose pool in S.cerevisiae cells we could easily produce 0.2 mg/l of GDP-L-fucose with this recombinant yeast strain without addition of any external GDP-mannose. The GDP-L-fucose product could be used as the fucose donor for alpha1,3fucosyltransferase to synthesize sialyl Lewis x (sLex), a glycan crucial for the selectin-dependent leukocyte traffic.     

The concerted inactivation of Escherichia coli uridine diphosphate galactose 4-epimerase by sugar nucleotide together with a free sugar.

1. The combined effect of the sugar nucleotides UDP-D-fucose or UDP-D-glucuronic acid together with the free sugars D-fucose or L-arabinose is the inactivation of the Escherichia coli enzyme UDP-galactose 4-epimerase (EC 5.1.3.2). The sugar nucleotide or the free sugar alone or the sugar nucleotide plus 5'-Ump do not inactivate the enzyme. 2. The inactivation of the enzyme by its substrate UDP-D-glucose was not affected by the presence of free sugar. 3. In all cases the inactivation observed follows pseudo-first-order kinetics. 4. A comparison of various sugar nucleotides indicates that the hydroxymethyl group at position 6 of the sugar moiety of the natural substrates is important for substrate binding.     

An N-acetylglucosaminyltransferase of the Golgi apparatus of the yeast Saccharomyces cerevisiae that can modify N-linked glycans

The yeast Saccharomyces cerevisiae is widely regarded as being only capable of producing N-linked glycans with high-mannose structures. To investigate the glycan structures made in different mutant strains, we made use of a reporter protein consisting of a version of hen egg lysozyme that contains a single site for N-linked glycosylation. Mass spectrometry analysis of the attached glycans revealed that a large proportion contained an unexpected extra mass corresponding to a single N-acetylhexosamine residue. In addition, the glycosylated lysozyme was recognized by an N-acetylglucosamine specific lectin. The genome of S. cerevisiae contains an uncharacterized open reading frame, YOR320c, that is related to a known N-acetylglucosaminyltransferase. Deletion of this ORF resulted in the disappearance of the extra mass on the N-linked glycans and loss of lectin binding. We show that the protein encoded by YOR320c (which we term Gnt1p) is localized to the Golgi apparatus and has GlcNAc-transferase activity in vitro. The physiological role of Gnt1p is unclear because mutants lacking the protein show no obvious growth or cell wall defects. Nonetheless, these results indicate that heterologous glycoproteins expressed in yeast can receive N-glycans with structures other than high mannose. In addition, they indicate that the lumen of the yeast Golgi contains UDP-GlcNAc, which may facilitate reconstitution of higher eukaryotic N-glycan processing.     

Effects of inositol starvation on phospholipid and glycan syntheses in Saccharomyces cerevisiae.

The early biochemical consequences of inositol starvation in an inositol auxotroph of Saccharomyces cerevisiae were examined as a means of determining the cellular role of inositol. Upon withdrawal of inositol, the rate of incorporation of 32P-labeled inorganic phosphate into phosphatidylinositol and into the phosphoinositol-containing sphingolipids immediately dropped by 80 and 50%, respectively; however, synthesis of the other major phospholipids continued for 2 to 3 h at control rates. The incorporation of [U-14C]glucose into cell wall glycans began to decline immediately poststarvation and decreased to 50% of the initial rate by 80 min for mannan and by 140 min for alkali- and acid-insoluble glucan. These changes in the rates of synthesis of cell wall glycan and phosphatidylinositol were the earliest effects of inositol starvation, preceding inhibition of the synthesis of protein and ribonucleic acid as measured by incorporation of radioactive precursors into trichloroacetic acid-insoluble cell material. These results suggest that phosphatidylinositol may play a direct role in the synthesis or secretion of yeast glycans.     

Depletion of UDP-D-apiose/UDP-D-xylose synthases results in rhamnogalacturonan-II deficiency, cell wall thickening, and cell death in higher plants

D-apiose serves as the binding site for borate cross-linking of rhamnogalacturonan II (RG-II) in the plant cell wall, and biosynthesis of D-apiose involves UDP-D-apiose/UDP-D-xylose synthase catalyzing the conversion of UDP-D-glucuronate to a mixture of UDP-D-apiose and UDP-D-xylose. In this study we have analyzed the cellular effects of depletion of UDP-D-apiose/UDP-D-xylose synthases in plants by using virus-induced gene silencing (VIGS) of NbAXS1 in Nicotiana benthamiana. The recombinant NbAXS1 protein exhibited UDP-D-apiose/UDP-D-xylose synthase activity in vitro. The NbAXS1 gene was expressed in all major plant organs, and an NbAXS1-green fluorescent protein fusion protein was mostly localized in the cytosol. VIGS of NbAXS1 resulted in growth arrest and leaf yellowing. Microscopic studies of the leaf cells of the NbAXS1 VIGS lines revealed cell death symptoms including cell lysis and disintegration of cellular organelles and compartments. The cell death was accompanied by excessive formation of reactive oxygen species and by induction of various protease genes. Furthermore, abnormal wall structure of the affected cells was evident including excessive cell wall thickening and wall gaps. The mutant cell walls contained significantly reduced levels of D-apiose as well as 2-O-methyl-L-fucose and 2-O-methyl-D-xylose, which serve as markers for the RG-II side chains B and A, respectively. These results suggest that VIGS of NbAXS1 caused a severe deficiency in the major side chains of RG-II and that the growth defect and cell death was likely caused by structural alterations in RG-II due to a D-apiose deficiency.     

Cloning and sequencing of the inulinase gene of Kluyveromyces marxianus var. marxianus ATCC 12424.

Cell wall inulinase (EC 3.2.1.7) was purified from Kluyveromyces marxianus var. marxianus (formerly K. fragilis) and its N-terminal 33-amino acid sequence was established. PCR amplification of cDNA with 2 sets of degenerate primers yielded a genomic probe which was then used to screen a genomic library established in the YEp351 yeast shuttle vector. One of the selected recombinant plasmids allowed an invertase-negative Saccharomyces cerevisiae mutant to grow on inulin. It was shown to contain an inulinase gene (INU 1) encoding a 555-amino acid precursor protein with a typical N-terminal signal peptide. The sequence of inulinase displays a high similarity (67%) to S. cerevisiae invertase, suggesting a common evolutionary origin for yeast beta-fructosidases with different substrate preferences.     

Biosynthesis of chondroitin sulfate. Solubilization of chondroitin sulfate glycosyltransferases and partial purification of uridine diphosphate-D-galactose:D-xylose galactosyltrans.

UDP-D-Galactose:D-xylose galactosyltransferase, a membrane-bound enzyme which catalyzes the second glycosyl transfer reaction in the biosynthesis of chondroitin sulfate chains, has been solubilized and partially purified from embryonic chick cartilage. Solubilization was effected by treatment of a particulate fraction of a homogenate (sedimenting between 10,000 and 100,000 times g) with the nonionic detergent Nonidet P-40 (0.5%) and KCl (0.5 M) or by the alkali-detergent method described previously (Helting, T. (1971) J. Biol. Chem. 246, 815-822). The applicability of the salt-detergent procedure as a general method for solubilization of membrane-bound glycosyltransferases was tested by assay of four other glycosyltransferases involved in chondroitin sulfate synthesis (UDP-D-xylose:core protein xylosyltransferase, UDP-D-galactose:4-O-beta-D-galactosyl-D-xylose galactosyltransferase, UDP-D-glucuronic acid: 3-O-beta-D-galactosyl-D-galactose glucuronosyltransferase, and UDP-N-acetyl-D-galactosamine: (GlcUA-GalNAc-4-sulfate)4 N-acetylgalactosaminyltransferase). In each case, greater than 70% of the activity was solubilized and, on gel chromatography on Sephadex G-200, the enzymes appeared largely in included positions and partially separated from each other. After partial purification by gel chromatography on Sephadex G-200, UDP-D-galactose:D-xylose galactosyltransferase was purified further by chromatography on one of several affinity matrices, i.e. xylosylated core protein of cartilage proteoglycan coupled to CNBr-activated Sepharose, a core protein matrix saturated with UDP-D-xylose:core protein xylosyltransferase or UDP-D-xylose:core protein xylosyltransferase covalently bound to Sepharose. The specific activities of the enzyme preparations obtained by these procedures were approximately 1000-fold greater than that of the crude homogenate.     

Effects of the protein matrix on glycan processing in glycoproteins

In the biosynthesis of glycoproteins containing asparagine-linked glycans, a number of regulatory factors must be involved in converting the single glycan precursor into the variety of different final structures observed in different eukaryotic species. Among these factors are the kind of glycan-processing enzymes available in the Golgi apparatus of different cells, the specificity and regulatory properties of these enzymes, and the unique properties of the protein matrix in which a given glycan resides during the biosynthetic processing. In examining the role of this latter regulatory factor, we have considered a simplified model in which a few key steps are common to all cells, regardless of the nature of the processing enzymes available. The protein-bound oligomannose precursor Man8GlcNAc2-, arriving in the Golgi after the initial trimming in the endoplasmic reticulum (ER), first undergoes a series of preprocessing steps to yield Man5GlcNAc2- in animals and plants or Man13-15GlcNAc2- in yeast. At this stage the key commitment step--to process or not to process--determines whether the above intermediates will remain as unprocessed oligomannose structures or be initiated into a new series of reactions to yield processed structures characteristic of the organisms involved (complex or hybrid for vertebrates, polymannose for yeast, xylosylated glycans for plants and some invertebrates, or Man3GlcNAc2- structures for other invertebrates). It is proposed that this commitment step, along with the obligatory preprocessing steps, is regulated primarily by each glycan's unique exposure on its protein matrix. Subsequent processing steps leading to complex or hybrid structures, fucosylation, extent of branching, and specific structures at the nonreducing terminals are most likely determined primarily by the enzyme makeup of the individual processing machineries, but with the protein matrix still playing a significant role.     

Structurally related but functionally distinct yeast Sm D core small nuclear ribonucleoprotein particle proteins.

Spliceosome assembly during pre-mRNA splicing requires the correct positioning of the U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) on the precursor mRNA. The structure and integrity of these snRNPs are maintained in part by the association of the snRNAs with core snRNP (Sm) proteins. The Sm proteins also play a pivotal role in metazoan snRNP biogenesis. We have characterized a Saccharomyces cerevisiae gene, SMD3, that encodes the core snRNP protein Smd3. The Smd3 protein is required for pre-mRNA splicing in vivo. Depletion of this protein from yeast cells affects the levels of U snRNAs and their cap modification, indicating that Smd3 is required for snRNP biogenesis. Smd3 is structurally and functionally distinct from the previously described yeast core polypeptide Smd1. Although Smd3 and Smd1 are both associated with the spliceosomal snRNPs, overexpression of one cannot compensate for the loss of the other. Thus, these two proteins have distinct functions. A pool of Smd3 exists in the yeast cytoplasm. This is consistent with the possibility that snRNP assembly in S. cerevisiae, as in metazoans, is initiated in the cytoplasm from a pool of RNA-free core snRNP protein complexes.     

Synthesis of polyhydroxyalkanoate in the peroxisome of Saccharomyces cerevisiae by using intermediates of fatty acid beta-oxidation.

Medium-chain-length polyhydroxyalkanoates (PHAs) are polyesters having properties of biodegradable thermoplastics and elastomers that are naturally produced by a variety of pseudomonads. Saccharomyces cerevisiae was transformed with the Pseudomonas aeruginosa PHAC1 synthase modified for peroxisome targeting by the addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. The PHAC1 gene was put under the control of the promoter of the catalase A gene. PHA synthase expression and PHA accumulation were found in recombinant S. cerevisiae growing in media containing fatty acids. PHA containing even-chain monomers from 6 to 14 carbons was found in recombinant yeast grown on oleic acid, while odd-chain monomers from 5 to 15 carbons were found in PHA from yeast grown on heptadecenoic acid. The maximum amount of PHA accumulated was 0.45% of the dry weight. Transmission electron microscopy of recombinant yeast grown on oleic acid revealed the presence of numerous PHA inclusions found within membrane-bound organelles. Together, these data show that S. cerevisiae expressing a peroxisomal PHA synthase produces PHA in the peroxisome using the 3-hydroxyacyl coenzyme A intermediates of the beta-oxidation of fatty acids present in the media. S. cerevisiae can thus be used as a powerful model system to learn how fatty acid metabolism can be modified in order to synthesize high amounts of PHA in eukaryotes, including plants.     

Isolation of Sporothrix schenckii GDA1 and functional characterization of the encoded guanosine diphosphatase activity

Sporothrix schenckii is a fungal pathogen of humans and the etiological agent of sporotrichosis. In fungi, proper protein glycosylation is usually required for normal composition of cell wall and virulence. Upon addition of precursor oligosaccharides to nascent proteins in the endoplasmic reticulum, glycans are further modified by Golgi-glycosyl transferases. In order to add sugar residues to precursor glycans, nucleotide diphosphate sugars are imported from the cytosol to the Golgi lumen, the sugar is transferred to glycans, and the resulting nucleoside diphosphate is dephosphorylated by the nucleoside diphosphatase Gda1 before returning to cytosol. Here, we isolated the open reading frame SsGDA1 from a S. schenckii genomic DNA library. In order to confirm the function of SsGda1, we performed complementation assays in a Saccharomyces cerevisiae gda1? null mutant. Our results indicated that SsGDA1 restored the nucleotide diphosphatase activity to wild-type levels and therefore is a functional ortholog of S. cerevisiae GDA1.     

Co-expression of two mammalian glycosyltransferases in the yeast cell wall allows synthesis of sLex

Interactions between selectins and their oligosaccharide-decorated counter-receptors play an important role in the initiation of leukocyte extravasation in inflammation. L-selectin ligands are O-glycosylated with sulphated sialyl Lewis X epitopes (sulpho-sLex). Synthetic sLex oligosaccharides have been shown to inhibit adhesion of lymphocytes to endothelium at sites of inflammation. Thus, they could be used to prevent undesirable inflammatory reactions such as rejection of organ transplants. In vitro synthesis of sLex glycans is dependent on the availability of recombinant glycosyltransferases. Here we expressed the catalytic domain of human alpha-1,3-fucosyltransferase VII in the yeasts Saccharomyces cerevisiae and Pichia pastoris. To promote proper folding and secretion competence of this catalytic domain in yeast, it was fused to the Hsp150 delta carrier, which is an N-terminal fragment of a secretory glycoprotein of S. cerevisiae. In both yeasts, the catalytic domain acquired an active conformation and the fusion protein was externalised, but remained mostly attached to the cell wall in a non-covalent fashion. Incubation of intact S. cerevisiae or P. pastoris cells with GDP-[14C]fucose and sialyl-alpha-2,3-N-acetyllactosamine resulted in synthesis of radioactive sLex, which diffused to the medium. Finally, we constructed an S. cerevisiae strain co-expressing the catalytic domains of alpha-2,3-sialyltransferase and alpha-1,3-fucosyltransferase VII, which were targeted to the cell wall. When these cells were provided with N-acetyllactosamine, CMP-sialic acid and GDP-[14C]fucose, radioactive sLex was produced to the medium. These data imply that yeast cells can provide a self-perpetuating source of fucosyltransferase activity immobilized in the cell wall, useful for the in vitro synthesis of sLex.