UDP-glucose dehydrogenase from bovine liver: primary structure and relationship to other dehydrogenases.

The primary structure of bovine liver UDP-glucose dehydrogenase (UDPGDH), a hexameric, NAD(+)-linked enzyme, has been determined at the protein level. The 52-kDa subunits are composed of 468 amino acid residues, with a free N-terminus and a Ser/Asn microhetergeneity at one position. The sequence shares 29.6% positional identity with GDP-mannose dehydrogenase from Pseudomonas, confirming a similarity earlier noted between active site peptides. This degree of similarity is comparable to the 31.1% identity vs. the UDPGDH from type A Streptococcus. Database searching also revealed similarities to a hypothetical sequence from Salmonella typhimurium and to "UDP-N-acetyl-mannosaminuronic acid dehydrogenase" from Escherichia coli. Pairwise identities between bovine UDPGDH and each of these sequences were all in the range of approximately 26-34%. Multiple alignment of all 5 sequences indicates common ancestry for these 4-electron-transferring enzymes. There are 27 strictly conserved residues, including a cysteine residue at position 275, earlier identified by chemical modification as the expected catalytic residue of the second half-reaction (conversion of UDP-aldehydoglucose to UDP-glucuronic acid), and 2 lysine residues, at positions 219 and 338, one of which may be the expected catalytic residue for the first half-reaction (conversion of UDP-glucose to UDP-aldehydoglucose). A GXGXXG pattern characteristic of the coenzyme-binding fold is found at positions 11-16, close to the N-terminus as with "short-chain" alcohol dehydrogenases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Streptomyces Pepsin Inhibitor-Insensitive Carboxyl Proteinase from Lentinus edodes

A Streptomyces-pepsin inhibitor (S-PI or Pepstatin Ac), and DAN-insensitive carboxyl proteinase was found in a still culture filtrate of Lentinus edodes. The new carboxyl proteinase was purified, and about 9 mg purified enzyme was obtained from 19 liters of culture filtrate, with 11% recovery. The enzyme showed a single band on polyacrylamide gel electrophoresis. The molecular weight and isoelectric point were 40,000 and pH 4.2, respectively. The enzyme did not contain histidine and was composed of 387 amino acid residues. The enzyme was most active between pH 2.7 ~ 2.9, and stable over a pH of 3.2 ~ 5.2 for 3 hr at 37°C. The enzyme was not inhibited by S-PI or synthetic pepsin inhibitors such as DAN and EPNP. The physicochemical and enzymological properties were very similar to those of Scytalidium lignicolum carboxyl proteinase A, which was reported to be an S-PI-, and DAN-insensitive carboxyl proteinase.     

Reduced mannose incorporation into GDP-mannose and dolichol-linked intermediates of N-glycosylation in hamster liver during vitamin A deficiency

Summary The molecular mechanism of reduced incorporation of radioactively labeled mannose into hamster liver glycoconjugates during the progression of vitamin A deficiency was investigated. In particular the in vivo incorporation of [2-³H]mannose into GDP-mannose, dolichyl phosphate mannose (Dol-P-Man), lipid-linked oligosaccharides, and glycopeptides of hamster liver was examined. Hamsters maintained on a vitamin A-free diet showed a reduction in the incorporation of mannose into GDP-mannose about 10 days before clinical signs of vitamin A deficiency could be observed. The decrease in [2-³H]mannose incorporated into GDP-mannose was accompanied by a reduction in label incorporated into Dol-P-Man, lipid linked oligosaccharides and glycopeptides, which became more severe with the progression of vitamin A deficiency. By the time they reached a plateau stage of growth, hamsters fed the vitamin A-free diet showed a 50% reduction in the amount of [2-³H]mannose converted to GDP-mannose, and the radioactivity associated with Dol-P-Man and glycopeptides was reduced by approximately 60% as compared to retinoic acid-supplemented controls. These results strongly indicate that the reduced incorporation of mannose into lipidic intermediates and glycoproteins observed during vitamin A deficiency is due to impaired GDP-mannose synthesis.Abbreviations Dol-P-Man Dolichyl Phosphate Mannose - Dol-P Dolichyl Phosphate     

Characterization of AtNST-KT1, a novel UDP-galactose transporter from Arabidopsis thaliana

Nucleotide sugar transporters (NST) mediate the transfer of nucleotide sugars from the cytosol into the lumen of the endoplasmatic reticulum and the Golgi apparatus. Because the NSTs show similarities with the plastidic phosphate translocators (pPTs), these proteins were grouped into the TPT/NST superfamily. In this study, a member of the NST-KT family, AtNST-KT1, was functionally characterized by expression of the corresponding cDNA in yeast cells and subsequent transport experiments. The histidine-tagged protein was purified by affinity chromatography and reconstituted into proteoliposomes. The substrate specificity of AtNST-KT1 was determined by measuring the import of radiolabelled nucleotide mono phosphates into liposomes preloaded with various unlabelled nucleotide sugars. This approach has the advantage that only one substrate has to be used in a radioactively labelled form while all the nucleotide sugars can be provided unlabelled. It turned out that AtNST-KT1 represents a monospecific NST transporting UMP in counterexchange with UDP-Gal but did not transport other nucleotide sugars. The AtNST-KT1 gene is ubiquitously expressed in all tissues. AtNST-KT1 is localized to Golgi membranes. Thus, AtNST-KT1 is most probably involved in the synthesis of galactose-containing glyco-conjugates in plants.     

Characterization of the mannosyl and fucosyl transferases in the ovine anterior pituitary glands.

Ovine anterior pituitary glands contain mannosyl- and fucosyl-transferases localized in the microsomes and able to incorporate mannose or fucose as such from GDP-mannose or GDP-fucose into endogenous glycoproteins. The requirements and conditions necessary for maximum activity were investigated. The value of the Km is very similar for the two enzyme systems, 3 X 10(-7) M in the case of mannosyl-transferases and 5 X 10(-7) M in the case of fucosyl-transferases.     

<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> expresses multiple Golgi-localised nucleotide-sugar transporters related to GONST1

Transport of nucleotide-sugars across the Golgi membrane is required for the lumenal synthesis of a variety of essential cell surface components, and is mediated by nucleotide sugar transporters (NSTs) which are members of the large drug/metabolite superfamily of transporters. Despite the importance of these proteins in plants, so far only two have been described, GONST1 and AtUTr1 from Arabidopsis thaliana. In this work, our aim was to identify further Golgi nucleotide-sugar transporters from Arabidopsis. On the basis of their sequence similarity to GONST1, we found four additional proteins, which we named GONST2, 3, 4 and 5. These putative NSTs were grouped into three clades: GONST2 with GONST1; GONST3 with GONST4; and GONST5 with six further uncharacterized proteins. Transient expression in tobacco cells of a member of each clade, fused to the Green Fluorescent Protein (GFP), suggested that all these putative NSTs are localised in the Golgi. To obtain evidence for nucleotide sugar transport activity, we expressed these proteins, together with the previously characterised GONST1, in a GDP-mannose transport-defective yeast mutant ( vrg4-2). We tested the transformants for rescue of two phenotypes associated with this mutation: sensitivity to hygromycin B and reduced glycosylation of extracellular chitinase. GONST1 and GONST2 complemented both phenotypes, indicating that GONST2, like the previously characterized GONST1, is a GDP-mannose transporter. GONST3, 4 and 5 also rescued the antibiotic sensitivity, but not the chitinase glycosylation defect, suggesting that they can also transport GDP-mannose across the yeast Golgi membrane but with a lower efficiency. RT-PCR and analysis of Affymetrix data revealed partially overlapping patterns of expression of GONST1–5 in a variety of organs. Because of the differences in ability to rescue the vrg4 -2 phenotype, and the different expression patterns in plant organs, we speculate that GONST1 and GONST2 are both GDP-mannose transporters, whereas GONST3, GONST4 and GONST5 may transport other nucleotide-sugars in planta.Communicated by G. JürgensThe first two authors contributed equally to this work     

The Burkholderia cepacia bceA gene encodes a protein with phosphomannose isomerase and GDP-D-mannose pyrophosphorylase activities

The bceA gene is part of the Burkholderia cepacia IST408 exopolysaccharide (EPS) biosynthetic cluster. It encodes a 55.3-kDa bifunctional protein (type II PMI family) with phosphomannose isomerase (PMI) and GDP-mannose pyrophosphorylase (GMP) activities. GMP activity is strongly dependent on the presence of Ca(2+) or Mn(2+), while PMI activity can use a broader variety of divalent cations (Ca(2+)>Mn(2+)>Mg(2+)>Co(2+)>Ni(2+)). The lack of a functional bceA gene does not affect EPS production yield in a non-polar insertion bceA mutant. The in silico search for putative bceA homologues revealed the presence of 2-5 bceA orthologues in the Burkholderia genomes available. This suggests that in B. cepacia IST408 putative bceA functional homologues may compensate the bceA mutation. However, the viscosity of aqueous solutions prepared with the EPS produced by the bceA mutant was significantly reduced compared with wild-type biopolymer and the mutant forms biofilms with a size reduced by 6-fold.     

O-Glycomic and Proteomic Signatures of Spontaneous and Butyrate-Stimulated Colorectal Cancer Cell Line Differentiation

Gut microbiota of the gastrointestinal tract provide health benefits to the human host via bacterial metabolites. Bacterial butyrate has beneficial effects on intestinal homeostasis and is the preferred energy source of intestinal epithelial cells, capable of inducing differentiation. It was previously observed that changes in the expression of specific proteins as well as protein glycosylation occur with differentiation. In this study, specific mucin O-glycans were identified that mark butyrate-induced epithelial differentiation of the intestinal cell line CaCo-2 (Cancer Coli-2), by applying porous graphitized carbon nano–liquid chromatography with electrospray ionization tandem mass spectrometry. Moreover, a quantitative proteomic approach was used to decipher changes in the cell proteome. It was found that the fully differentiated butyrate-stimulated cells are characterized by a higher expression of sialylated O-glycan structures, whereas fucosylation is downregulated with differentiation. By performing an integrative approach, we generated hypotheses about the origin of the observed O-glycome changes. These insights pave the way for future endeavors to study the dynamic O-glycosylation patterns in the gut, either produced via cellular biosynthesis or through the action of bacterial glycosidases as well as the functional role of these patterns in homeostasis and dysbiosis at the gut–microbiota interface.     

Protein glycosylation in pmt mutants of Saccharomyces cerevisiae. Influence of heterologously expressed cellobiohydrolase II of Trichoderma reesei and elevated levels of GDP-mannose and cis-prenyltransferase activity

Protein O-mannosylation has been postulated to be critical for production and secretion of glycoproteins in fungi. Therefore, understanding the regulation of this process and the influence of heterologous expression of glycoproteins on the activity of enzymes engaged in O-glycosylation are of considerable interest. In this study we expressed cellobiohydrolase II (CBHII) of T. reesei, which is normally highly O-mannosylated, in Saccharomyces cerevisiae pmt mutants partially blocked in O-mannosylation. We found that the lack of Pmt1 or Pmt2 protein O-mannosyltransferase activity limited the glycosylation of CBHII, but it did not affect its secretion. The S. cerevisiae pmt1Δ and pmt2Δ mutants expressing T. reesei cbh2 gene showed a decrease of GDP-mannose level and a very high activity of cis-prenyltransferase compared to untransformed strains. On the other hand, elevation of cis-prenyltransferase activity by overexpression of the S. cerevisiae RER2 gene in these mutants led to an increase of dolichyl phosphate mannose synthase activity, but it did not influence the activity of O-mannosyltransferases. Overexpression of the MPG1 gene increased the level of GDP-mannose and stimulated the activity of mannosyltransferases elongating O-linked sugar chains, leading to partial restoration of CBHII glycosylation.     

Enhanced secretion of heterologous proteins in Kluyveromyces lactis by overexpression of the GDP-mannose pyrophosphorylase, KlPsa1p

GDP-mannose is the mannosyl donor for the glycosylation reactions and is synthesized by GDP-mannose pyrophosphorylase from GTP and d-mannose-1-phosphate; in Saccharomyces cerevisiae this enzyme is encoded by the PSA1/VIG9/SRB1 gene. We isolated the Kluyveromyces lactis KlPSA1 gene by complementing the osmotic growth defects of S. cerevisiae srb1/psa1 mutants. KlPsa1p displayed a high degree of similarity with other GDP-mannose pyrophosphorylases and was demonstrated to be the functional homologue of S. cerevisiae Psa1p. Phenotypic analysis of a K. lactis strain overexpressing the KlPSA1 gene revealed changes in the cell wall assembly. Increasing the KlPSA1 copy number restored the defects in O-glycosylation, but not those in N-glycosylation, that occur in K. lactis cells depleted for the hexokinase Rag5p. Overexpression of GDP-mannose pyrophosphorylase also enhanced heterologous protein secretion in K. lactis as assayed by using the recombinant human serum albumin and the glucoamylase from Arxula adeninivorans.