Influence of carbon source and surface hydrophobicity on the aggregation of the yeast Kluyveromyces bulgaricus

Aggregation of the yeast Kluyveromyces bulgaricus is mediated by the galactose-specific lectin KbCWL1. This lectin contains hydrophobic amino acids and its activity is calcium dependent. A specific fluorescent probe, 1-anilinonaphthalene-8-sulfonic acid in the free acid form (ANS; Sigma Chemical Co., St. Louis, Missouri), was used to study the hydrophobic areas on the cellular surface of K. bulgaricus. Changes in surface hydrophobicity during the growth and aggregation of yeast cells were studied. Surface hydrophobicity increased during growth and depended on the amount of yeast cells in the culture medium. During growth, the size of the hydrophobic areas on the cell surface was measured using ANS and was found to increase with the percentage of flocculating yeasts. Our results strongly suggest that the hydrophobic areas of the cell walls of yeast cells are involved in the aggregation of K. bulgaricus.     

Role of the second immunoglobulin-like loop of nectin in cell-cell adhesion

We investigated whether and how rat liver thioredoxin reductase spares alpha-tocopherol in biomembranes. Purified hydroperoxides of beta-linoleoyl-gamma-palmitoylphosphatidylcholine were decreased 35% by treatment with thioredoxin reductase and 54% by thioredoxin reductase plus E. coli thioredoxin. Thioredoxin reductase also halved the amount of hydroperoxides that had been formed during photoperoxidation of liposomes composed of beta-linoleoyl-gamma-palmitoylphosphatidylcholine, and of emulsions of both cholesterol and cholesteryl linolenate. In erythrocyte ghosts, thioredoxin reductase spared alpha-tocopherol from oxidation by both soybean lipoxygenase and ferricyanide. Thioredoxin reductase also decreased F(2)-isoprostanes in ghosts oxidized by ferricyanide, suggesting that its ability to spare alpha-tocopherol relates to reduction of lipid hydroperoxides.     

cis-Dimer formation of E-cadherin is independent of cell-cell adhesion assembly in vivo

Cadherin-based cell-cell adhesions play important roles in embryonic development and in the maintenance of normal tissue architecture. Little is known, however, about the mechanisms of controlling cadherin dynamics at the cell surface. We previously demonstrated that E-cadherin functions as a cis (lateral)-dimer on the cell surface by chemical cross-linking. In this study, we examined the dynamics of E-cadherin cis-dimer formation during cell-cell adhesion assembly by using chemical cross-linking. Although treatment with cytochalasin D, a potent inhibitor of actin polymerization, was shown to inhibit the formation of cell-cell contacts, the dynamics of E-cadherin cis-dimer formation was not affected. This result indicated that the cis-dimer formation procedure is independent of cell-cell adhesion assembly in vivo. However, the cell aggregation and dissociation assays showed that the cytochalasin D treatment shifted the cadherin-based cell adhesion from a strong to a weak state. Taken together, these results strongly support the possibility that the E-cadherin cis-dimer is a minimal functional unit in cadherin-mediated cell-cell adhesion in vivo.     

Isolation and biochemical characterization of cell wall tight protein complex involved in self-flocculation of Kluyveromyces bulgaricus

Flocculation of yeasts is a cell–cell aggregation phenomenon which is driven by interactions between cell wall lectins and cell wall heteropolysaccharides. In Sabouraud medium, Kluyveromyces bulgaricus was highly flocculent. Incubation of flocculent K. bulgaricus cells with EDTA or Hecameg® led to extracts showing hemagglutinating and flocculating properties. Purification of the extracts by native PAGE gave two bands which allowed flocculation of deflocculated K. bulgaricus. Both bands with specific reflocculating activity were composed of five subunits, of which only three possessed weak reflocculating activity upon deflocculated yeast. The mixture of these three proteins allow the recovery of initial specific reflocculating activity of the complex. These three proteins, denoted p28, p36 and p48, presented, in their first 15 amino acids, homologies with glycolysis enzymes, i.e., 3-phosphoglycerate mutase, glyceraldehyde-3-phosphate dehydrogenase and enolase, respectively. However, no such enzymatic activity could be detected in the crude extract issued from treatment with EDTA and Hecameg® of flocculent yeast cells. When yeasts had grown in glucose poor medium, flocculation was drastically affected. The EDTA and Hecameg® crude extracts showed weak reflocculating activity. After PAGE, the protein complexes did not appear in the EDTA extract, but they did appear in the Hecameg® crude extract. These results suggest that: (i) self-flocculation of K. bulgaricus depends on the expression of different floc-forming protein complex, (ii) these proteins are galactose specific lectins showing homologies in their primary structure with glycolysis enzymes.     

Studies on the glycosylation of wild-type and mutant forms of Aspergillus niger pectin methylesterase

Pectin methylesterase (PME) is one of a number of enzymes released by the fungus Aspergillus niger that are involved in the degradation of specific plant cell-wall structures. PME is a glycoprotein with three potential sites for N-linked glycosylation. The glycosylation may affect the hydrolytic activity or the substrate specificity of PME. In this work, we investigate first the structures and the attachment sites of the glycans present on recombinant wild-type PME. Further, a series of PME mutants was created in which the three potential N-linked glycosylation sites were eliminated in all possible combinations. The glycosylation of the mutants and their activities were then studied. Mass spectrometric techniques tailored for carbohydrate analysis were applied to both characterize the glycan structures and to determine the specific sites of attachment. High mannose structures with variable numbers of mannose were found on the wild-type, as well as the mutant forms. Studies using the mutants suggest that glycosylation does not strongly influence the activity. Whether it may affect the substrate specify of the enzyme is unknown, and that aspect will be explored in future work.     

Characterization of the second external alternative dehydrogenase from mitochondria of the respiratory yeast Kluyveromyces lactis

The mitochondria of the respiratory yeast Kluyveromyces lactis are able to reoxidize cytosolic NADPH. Previously, we characterized an external alternative dehydrogenase, KlNde1p, having this activity. We now characterize the second external alternative dehydrogenase of K. lactis mitochondria, KlNde2p. We examined its role in cytosolic NADPH reoxidation by studying heterologous expression of KlNDE2 in Saccharomyces cerevisiae mutants and by constructing Δklnde1 and Δklnde2 mutants. KlNde2p uses NADH or NADPH as substrates, its activity in isolated mitochondria is not regulated by exogenously added calcium and it is not down-regulated when the cells grow in glucose versus lactate. KlNde2p shows lower affinity for NADPH than KlNde1p. Both enzymes show similar pH optimum.     

Influence of sorbitol on protein production and glycosylation and cell wall formation in Trichoderma reesei

Sorbitol is often used at 1 mol/liter as an osmotic stabilizer for cultivation of fungi with a fragile cell wall phenotype. On the other hand, at this concentration sorbitol causes an osmotic stress in fungal cells resulting in intensive production of intracellular glycerol. The highly increased consumption of glucose for glycerol synthesis may lead to changes in processes requiring carbohydrate residues. This study provides new information on the consequences of osmotic stress to the cell wall composition, protein production and glycosylation, and cell morphology of Trichoderma reesei. We observed that high osmolarity conditions enhanced biomass production and strongly limited synthesis of cell wall glucans and chitin. Moreover, in these conditions the amount of secreted protein decreased nearly ten-fold and expression of cbh1 and cbh2 genes coding for cellobiohydrolase I and cellobiohydrolase II, the main secretory proteins in T. reesei, was inhibited resulting in a lack of the proteins in the cell and cultivation medium. The activity of DPM synthase, enzyme engaged in both N- and O-glycosylation pathways, was reduced two-fold, suggesting an overall inhibition of protein glycosylation. However, the two modes of glycosylation were affected divergently: O-glycosylation of secreted proteins decreased in the early stages of growth while N-glycosylation significantly increased in the stationary phase.     

Analysis of glycosylation motifs and glycosyltransferases in Bacteria and Archaea

The process of glycosylation has been studied extensively in prokaryotes but many questions still remain unanswered. Glycosyltransferase is the enzyme which mediates glycosylation and has its preference for the target glycosylation sites as well as for the type of glycosylation i.e. N-linked and O-linked glycosylation. In this study we carried out the bioinformatics analysis of one of the key enzymes of pgl locus from Campylobacter jejuni, known as PglB, which is distributed widely in bacteria and AglB from archaea. Relatively little sequence similarity was observed in the archaeal AglB(s) as compared to those of the bacterial PglB(s). In addition we tried to the answer the question of as to why not all the sequins Asp-X-Ser/Thr have an equal opportunity to be glycosylated by looking at the influence of the neighboring amino acids but no significant conserved pattern of the flanking sites could be identified. The software tool was developed to predict the potential glycosylation sites in autotransporter protein, the virulence factors of gram negative bacteria, and our results revealed that the frequency of glycosylation sites was higher in adhesins (a subclass of autotransporters) relative to the other classes of autotransporters.     

Inter- and intramolecular glycosylation of exo-glycals promoted by metallic Lewis acids

exo-Glycosyl carbonates were employed for inter- and intramolecular glycosylation reactions. A number of metallic Lewis Acids and solvents were examined to enhance the reactivity. The optimum conditions were found to be the use of AlCl3 in 1-nitropropane. The method was demonstrated to be useful for the intermolecular glycosyl transfer of several nucleophiles, including simple alcohols, sugars, and amino acid derivatives; however, intramolecular glycosylations were not successful.     

Pilin glycosylation in Neisseria meningitidis occurs by a similar pathway to wzy-dependent O-antigen biosynthesis in Escherichia coli

Pili (type IV fimbriae) of Neisseria meningitidis are glycosylated by the addition of O-linked sugars. Recent work has shown that PglF, a protein with homology to O-antigen 'flippases', is required for the biosynthesis of the pilin-linked glycan and suggests pilin glycosylation occurs in a manner analogous to the wzy-dependent addition of O-antigen to the core-LPS. O-Antigen ligases are crucial in this pathway for the transfer of undecraprenol-linked sugars to the LPS-core in Gram-negative bacteria. An O-antigen ligase homologue, pglL, was identified in N. meningitidis. PglL mutants showed no change in LPS phenotypes but did show loss of pilin glycosylation, confirming PglL is essential for pilin O-linked glycosylation in N. meningitidis.     

RNAi-mediated gene silencing of WsSGTL1 in W.somnifera affects growth and glycosylation pattern

Sterol glycosyltransferases (SGTs) belong to family 1 of glycosyltransferases (GTs) and are enzymes responsible for synthesis of sterol–glucosides (SGs) in many organisms. WsSGTL1 is a SGT of Withania somnifera that has been found associated with plasma membranes. However its biological function in W.somnifera is largely unknown. In the present study, we have demonstrated through RNAi silencing of WsSGTL1 gene that it performs glycosylation of withanolides and sterols resulting in glycowithanolides and glycosylated sterols respectively, and affects the growth and development of transgenic W.somnifera. For this, RNAi construct (pFGC1008-WsSGTL1) was made and genetic transformation was done by Agrobacterium tumefaciens. HPLC analysis depicts the reduction of withanoside V (the glycowithanolide of W.somnifera) and a large increase of withanolides (majorly withaferin A) content. Also, a significant decrease in level of glycosylated sterols has been observed. Hence, the obtained data provides an insight into the biological function of WsSGTL1 gene in W.somnifera.     

Influence of sodium orthovanadate on the production of astaxanthin from green algae Haematococcus lacustris

Astaxanthin production is commonly induced under stress conditions such as nutrient deficiency (N or P), high light stress, and variations of temperature, high NaCl concentrations, and other factors. The objective of the present study is the analysis of the effect of oxidative stress by sodium orthovanadate (SOV), a nonspecific inhibitor of protein tyrosine phosphatases, on the cells growth and astaxanthin production of H. lacustris. In the presence of SOV (lower than 5.0 mM), maximum growth of H. lacustris obtained was 2.4 × 10⁵ cells/mL in MBBM medium at 24°C under continuous illumination (40 μE/m²/s) of white fluorescent light, with continuous aeration of CO₂ (0.2 vvm). Total carotenoids accumulated per cell biomass unit treated with 2.5 mM SOV has approximately shown 2.5 folds higher than the control after short period of SOV induction time as 2 days, despite that cells were grown under normal light. Meanwhile, maximal astaxanthin production from H. lacustris was 10.7 mg/g biomass in MBBM with 5 days of continuous illumination at 40 μE/m²/s, which has been established as optimal light intensity for the control culture of H. lacustris. Treating algae H. lacustris with sodium orthovanadate showed promoting the accumulation of astaxanthin by advancing either the inhibition of dephosphorylation or synthesis of ATP. Its potential role of PTPases in microalgae H. lacustris is discussed. The first two authors are equally contributed to this work.     

Cell surface expression of a GPI-anchored form of mouse acetylcholinesterase in Klpmr1Delta cells of Kluyveromyces lactis

The mouse acetylcholinesterase AChE(H) was expressed in the yeast Kluyveromyces lactis. The AChE(H) activity was detectable in intact cells whereas it was absent in the culture media. Glucanase treatment and immunoelectron microscopy data indicated that AChE(H) is anchored to plasma membrane and that the mouse GPI-signaling is compatible with the K. lactis targeting machinery. The AChE(H) was also expressed in a K. lactis strain carrying an inactivated allele of KlPMR1, the gene coding for a P-type Ca(2+)-ATPase of the Golgi apparatus. This mutant displays changes in protein glycosylation and cell wall structure. The AChE(H) activity detected in Klpmr1Delta cells was more than twofold higher than that observed in wild-type cells. The combination of AChE expression and anchoring with the characteristics of Klpmr1Delta strain of K. lactis resulted in yeast cells displaying high AChE activity. This could be regarded as a novel sensing unit to be employed for detecting AChE inhibitors as pesticides.     

A stereoselective 1,2-cis glycosylation toward the synthesis of a novel N-linked glycan from the Gram-negative bacterium, Campylobacter jejuni

It has been shown that certain prokaryotes, such as Campylobacter jejuni, have asparagine (Asn)-linked glycoproteins. However, the structures of their glycans are distinct from those of eukaryotic origin. They consist of a bacillosamine residue linked to Asn, an alpha-(1-->4)-GalpNAc repeat, and a branching beta-Glcp residue. In this paper, we describe a strategy for the stereoselective construction of the alpha-(1-->4)-GalpNAc repeat of a C. jejuni N-glycan, utilizing a pentafluoropropionyl (PFP) group as a temporary protective group of the C-4 OH group of the GalpN donor. The strategy was applied to the synthesis of the hexasaccharide alpha-GalpNAc-(1-->4)-alpha-GalpNAc-(1-->4)-[beta-Glcp-(1-->3)]-alpha-GalpNAc(1-->4)-alpha-GalpNAc-(1-->4)-GalpNAc.     

Infrared spectroscopy used to evaluate glycosylation of proteins

Infrared (IR) spectroscopy is used for studying the carbohydrate moieties of glycosylated proteins. IR spectra of mono- and disaccharides in the fingerprint region are specific to each sugar and to the environment of the sugar molecules (i.e., aqueous solution or anhydrous glass phase). The IR spectra of glycosylated proteins (mucin, soybean peroxidase, collagen IV, and avidin) were compared with those of the constituent sugars and cytochrome c (a protein with no glycosylation). Our results demonstrate that the IR absorption spectra of glycosylated proteins show distinct absorption bands for the sugar moiety, the protein amide group, and water. Therefore, IR can be used to detect glycosylation.     

Improved production of monoclonal antibodies through oxygen-limited cultivation of glycoengineered yeast

Glycoengineering technology can elucidate and exploit glycan related structure-function relationships for therapeutic proteins. Glycoengineered yeast has been established as a safe, robust, scalable, and economically viable expression platform. It has been found that specific productivity of antibodies in glycoengineered Pichia pastoris is a non-linear function of specific growth rate that is dictated by a limited methanol feed rate. The optimal carbon-limited cultivation requires an exponential methanol feed rate with an increasing biomass concentration and more significantly an increase in heat and mass transfer requirements that often become the limiting factor in scale-up. Both heat and mass transfer are stoichiometrically linked to the oxygen uptake rate. Consequently an oxygen-limited cultivation approach was evaluated to limit the oxygen uptake rate and ensure robust and reliable scale-up. The oxygen-limited process not only limited the maximum oxygen uptake rate (and consequently the required heat removal rate) in mut+ P. pastoris strains but also enabled extension of the induction phase leading to an increased antibody concentration (1.9 g L⁻¹ vs. 1.2 g L⁻¹), improved N-glycan composition and galactosylation, and reduced antibody fragmentation. Furthermore, the oxygen-limited process was successfully scaled to manufacturing pilot scale and thus presents a promising process option for the glycoengineered yeast protein expression platform.     

Bacillus pasteurii urease shares with plant ureases the ability to induce aggregation of blood platelets

Ureases (EC 3.5.1.5) are highly homologous enzymes found in plants, bacteria and fungi. Canatoxin, an isoform Canavalia ensiformis urease, has several biological properties unrelated to its ureolytic activity, like platelet-aggregating and pro-inflammatory effects. Here, we describe that Bacillus pasteurii urease (BPU) also induces aggregation of rabbit platelets, similar to the canatoxin-induced effect (ED(50) 0.4 and 0.015 mg/mL, respectively). BPU induced-aggregation was blocked in platelets pretreated with dexamethasone and esculetin, a phospholipase A(2) and a lipoxygenase inhibitor, respectively, while platelets treated with indomethacin, a cyclooxygenase inhibitor, showed increased response to BPU. Methoxyverapamil (Ca(2+) channel blocker) and AMP (ADP antagonist) abrogated urease-induced aggregation, whereas the PAF-acether antagonist Web2170 had no effect. We concluded that platelet aggregation induced by BPU is mediated by lipoxygenase-derived eicosanoids and secretion of ADP from the platelets through a calcium-dependent mechanism. Potential relevance of these findings for bacterium-plant interactions and pathogenesis of bacterial infections are discussed.     

Effect of bacitracin on flagellar assembly and presumed glycosylation of the flagellins of Methanococcus deltae

Methanococcus deltae is an irregularly-shaped coccoid methanogen which possesses peritrichously arranged flagella. The flagella are composed of 2 flagellins of M _r=27000 and 32000 and possess a carbohydrate component as determined by thymol-sulfuric acid staining of SDS-PAGE. Cell growth was sensitive to bacitracin at levels near 10 g/ml. Growth in the presence of 5g/ml bacitracin resulted in the appearance of presumably hypoglycosylated flagellins as detected by Western immunoblotting. Continual passage of cells from 5 g/ml bacitracin through 10, 25, and 50 g/ml bacitracin resulted in cells capable of growth at 100 g/ml bacitracin. Western immunoblot analysis revealed that cells grown in the highest concentration of bacitracin no longer possessed native flagellins but only the hypoglycosylated forms. Electron microscopy corroborated the absence of normal flagella. These studies suggest that a bacitracin-sensitive dolichol-diphosphate carrier is responsible for attachment of at least a large proportion of the carbonhydrate content of the flagellins, and that a minimum amount of glycosylation is essential for normal flagellum assembly.