Biosynthesis Of Oligosaccharides Research Articles

Abstract 251: Investigating the Role of the Hexosamine Biosynthesis Pathway in Diabetic Atherosclerosis

Background: Diabetes mellitus dramatically increases the risk for atherosclerotic cardiovascular disease. We have established that chronic hyperglycemia promotes an increase in glucose flux through the hexosamine biosynthesis pathway (HBP). Central to this pathway is glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme controlling the conversion of glucose to glucosamine. We have shown that glucosamine is a potent inducer of endoplasmic reticulum (ER) stress, which is characterized by the accumulation of misfolded proteins in the ER. Chronic ER stress can initiate a multifaceted, cell-specific, response that results in lipid accumulation, inflammation and apoptosis: the hallmark features of atherosclerosis. We hypothesize that conditions of chronic hyperglycemia, associated with diabetes mellitus, can accelerate the development of atherosclerosis by a mechanism that involves increased HBP flux resulting in glucosamine-induced ER stress and the subsequent activation of pro-atherogenic pathways. Results: STZ-induced hyperglycemic and glucosamine supplemented apoE-/- mice had significantly larger and more advanced atherosclerotic lesions compared to the control mice. Indications of ER stress were increased in atherosclerotic lesions of hyperglycemic and glucosamine-supplemented mice but not in the controls. In glucosamine-supplemented mice the accelerated atherosclerosis was independent of detectable changes in blood glucose concentration, glucose tolerance, plasma insulin, or plasma lipid levels. Treatment with either valproate or 4-phenylbutyrate, two small molecules that have been shown to block ER stress signaling, attenuated hyperglycemic and glucosamine-induced atherogenesis. Our data suggest that elevated concentrations of glucose/glucosamine induce ER stress by interfering with lipid-linked oligosaccharide biosynthesis and N-linked glycosylation. Conclusions: Similar to hyperglycemia, glucosamine-supplementation promotes ER stress and accelerated atherosclerosis in apoE-/- mice. These findings support a model by which conditions of hyperglycemia promote vascular complications via a glucosamine-intermediate.

A putative de-N-acetylase of the PIG-L superfamily affects fluoroquinolone tolerance inPseudomonas aeruginosa

A major cause of treatment failure of infections caused by Pseudomonas aeruginosa is the presence of antibiotic-insensitive persister cells. The mechanism of persister formation in P. aeruginosa is largely unknown, and so far, only few genetic determinants have been linked to P. aeruginosa persistence. Based on a previous high-throughput screening, we here present dnpA (de-N-acetylase involved in persistence; gene locus PA14_66140/PA5002) as a new gene involved in noninherited fluoroquinolone tolerance in P. aeruginosa. Fluoroquinolone tolerance of a dnpA mutant is strongly reduced both in planktonic culture and in a biofilm model, whereas overexpression of dnpA in the wild-type strain increases the persister fraction. In addition, the susceptibility of the dnpA mutant to different classes of antibiotics is not affected. dnpA is part of the conserved LPS core oligosaccharide biosynthesis gene cluster. Based on primary sequence analysis, we predict that DnpA is a de-N-acetylase, acting on an unidentified substrate. Site-directed mutagenesis suggests that this enzymatic activity is essential for DnpA-mediated persistence. A transcriptome analysis indicates that DnpA primarily affects the expression of genes involved in surface-associated processes. We discuss the implications of these findings for future antipersister therapies targeted at chronic P. aeruginosa infections.

Distinct adaptor proteins assist exit of Kre2-family proteins from the yeast ER

ABSTRACTThe Svp26 protein of S. cerevisiae is an ER- and Golgi-localized integral membrane protein with 4 potential membrane-spanning domains. It functions as an adaptor protein that facilitates the ER exit of Ktr3, a mannosyltransferase required for biosynthesis of O-linked oligosaccharides, and the ER exit of Mnn2 and Mnn5, mannosyltransferases, which participate in the biosynthesis of N-linked oligosaccharides. Ktr3 belongs to the Kre2 family, which consists of 9 members of type-II membrane proteins sharing sequence similarities. In this report, we examined all Kre2 family members and found that the Golgi localizations of two others, Kre2 and Ktr1, were dependent on Svp26 by immunofluorescence microscopy and cell fractionations in sucrose density gradients. We show that Svp26 functions in facilitating the ER exit of Kre2 and Ktr1 by an in vitro COPII budding assay. Golgi localization of Ktr4 was not dependent on Svp26. Screening null mutants of the genes encoding abundant COPII membrane proteins for those showing mislocalization of Ktr4 in the ER revealed that Erv41 and Erv46 are required for the correct Golgi localization of Ktr4. We provide biochemical evidence that the Erv41-Erv46 complex functions as an adaptor protein for ER exit of Ktr4. This is the first demonstration of the molecular function of this evolutionally conserved protein complex. The domain switching experiments show that the lumenal domain of Ktr4 is responsible for recognition by the Erv41-Erv46 complex. Thus, ER exit of Kre2-family proteins is dependent on distinct adaptor proteins and our results provide new insights into the traffic of Kre2-family mannosyltransferases.

Glycoengineering of yeasts from the perspective of glycosylation efficiency

N-linked glycosylation of proteins is one of the most common posttranslational modifications. N-glycan structures and N-glycosylation efficiency are crucial parameters in the production of N-glycosylated proteins. Yeast cells can be seen as an attractive production host for therapeutic glycoproteins and pioneering work of glycoengineering was performed in Pichia pastoris, realizing yeast strains capable of producing defined, human-type N-glycans. Most strategies used for glycoengineering rely on the modification of the lipid-linked oligosaccharide biosynthesis for the generation of the substrate for Golgi-localized glycosyltransferases. However, modifications in the lipid-linked oligosaccharide biosynthesis often result in the accumulation of intermediate structures and cause hypoglycosylation of client proteins. In order to ensure complete N-glycosylation, the flow of lipid-linked oligosaccharide through the biosynthetic pathway and the transfer of the oligosaccharide from the donor lipid onto the protein have to be optimized. A promising tool to improve site occupancy is the expression of protozoan oligosaccharyltransferases, which possess altered specificities for the oligosaccharide and also for the protein acceptor site. Furthermore, flipping of the lipid-linked oligosaccharide into the ER lumen can be improved by overexpression of an artificial flippase. Improving the glycosylation efficiency ensures that not only homogeneous N-glycan structures are generated, but also client proteins are fully glycosylated.

Genotype and Growing Environment Interaction Shows a Positive Correlation between Substrates of Raffinose Family Oligosaccharides (RFO) Biosynthesis and Their Accumulation in Chickpea (Cicer arietinum L.) Seeds

To develop genetic improvement strategies to modulate raffinose family oligosaccharides (RFO) concentration in chickpea ( Cicer arietinum L.) seeds, RFO and their precursor concentrations were analyzed in 171 chickpea genotypes from diverse geographical origins. The genotypes were grown in replicated trials over two years in the field (Patancheru, India) and in the greenhouse (Saskatoon, Canada). Analysis of variance revealed a significant impact of genotype, environment, and their interaction on RFO concentration in chickpea seeds. Total RFO concentration ranged from 1.58 to 5.31 mmol/100 g and from 2.11 to 5.83 mmol/100 g in desi and kabuli genotypes, respectively. Sucrose (0.60-3.59 g/100 g) and stachyose (0.18-2.38 g/100 g) were distinguished as the major soluble sugar and RFO, respectively. Correlation analysis revealed a significant positive correlation between substrate and product concentration in RFO biosynthesis. In chickpea seeds, raffinose, stachyose, and verbascose showed a moderate broad sense heritability (0.25-0.56), suggesting the use of a multilocation trials based approach in chickpea seed quality improvement programs.

Comparison of milk oligosaccharides between goats with and without the genetic ability to synthesize αs1-casein

Milk oligosaccharides (OS)—free complex carbohydrates—confer unique health benefits to the nursing neonate. Though human digestive enzymes cannot degrade these sugars, they provide nourishment to specific commensal microbes and act as decoys to prevent the adhesion of pathogenic micro-organisms to gastrointestinal cells. At present, the limited quantities of human milk oligosaccharides (HMO) impede research on these molecules and their potential applications in functional food formulations. Considerable progress has been made in the study of OS structures; however, the synthetic pathways leading to their synthesis in the mammary gland are poorly understood. Recent studies show that complex OS with fucose and N-acetyl neuraminic acid (key structural elements of HMO bioactivity) exist in goat milk. Polymorphisms in the CSN1S1 locus, which is responsible for synthesis of αs1-casein, affect lipid and casein micelle structure in goat milk. The present study sought to determine whether CSN1S1 polymorphisms also influence goat milk oligosaccharide (GMO) production and secretion. The GMO compositions of thirty-two goat milk samples, half of which were from genotype A/A (αs1-casein producers) and half from genotype O/O (αs1-casein non-producers), were determined with nanoflow liquid chromatography high-accuracy mass spectrometry. This study represents the most exhaustive characterization of GMO to date. A systematic and comprehensive GMO library was created, consolidating information available in the literature with the new findings. Nearly 30 GMO, 11 of which were novel, were confirmed via tandem mass spectrometric analyses. Six fucosylated OS were identified; 4 of these matched HMO compositions and three were identified for the first time in goat milk. Importantly, multivariate statistical analysis demonstrated that the OS profiles of the A/A and O/O genotype milks could be discriminated by the fucosylated OS. Quantitative analysis revealed that the goat milk samples contained 1.17g/L of OS; however, their concentration in milks from A/A and O/O genotypes was not different. This study provides evidence of a genetic influence on specific OS biosynthesis but not total OS production. The presence of fucosylated GMO suggests that goat milk represents a potential source of bioactive milk OS suitable as a functional food ingredient.

Aberrant glycosylation of IgA in Wiskott-Aldrich syndrome and X-linked thrombocytopenia

Aberrant glycosylation of IgA in Wiskott-Aldrich syndrome and X-linked thrombocytopenia

Chemoenzymatic synthesis of an isoprenoid phosphate tool for the analysis of complex bacterial oligosaccharide biosynthesis

Undecaprenyl Pyrophosphate Synthase (UPPS) is a key enzyme that catalyzes the production of bactoprenols, which act as membrane anchors for the assembly of complex bacterial oligosaccharides. One of the major hurdles in understanding the assembly of oligosaccharide assembly is a lack of chemical tools to study this process, since bactoprenols and the resulting isoprenoid-linked oligosaccharides lack handles or chromophores for use in pathway analysis. Here we describe the isolation of a new UPPS from the symbiotic microorganism Bacteroides fragilis, a key species in the human microbiome. The protein was purified to homogeneity and utilized to accept a chromophore containing farnesyl diphosphate analogue as a substrate. The analogue was utilized by the enzyme and resulted in a bactoprenyl diphosphate product with an easy to monitor tag associated with it. Furthermore, the diphosphate is shown to be readily converted to monophosphate using a common molecular biology reagent. This monophosphate product allowed for the investigation of complex oligosaccharide biosynthesis, and was used to probe the activity of glycosyltransferases involved in the well characterized Campylobacter jejuni N-linked protein glycosylation. Novel reagents similar to this will provide key tools for the study of uncharacterized oligosaccharide assemblies, and open the possibility for the development of rapid screening methodology for these biosynthetic systems.

Effect of CMV infection and high temperatures on the enzymes involved in raffinose family oligosaccharide biosynthesis in melon plants

Ultrastructural and molecular studies have provided experimental evidence for the classification of cucurbits as symplastic loaders, mainly translocating the raffinose family oligosaccharides (RFOs) raffinose and stachyose. Earlier studies established that cucumber mosaic virus (CMV) infection causes a significant increase in the sucrose-to-RFO ratio in the phloem sap of melon plants. The alteration in phloem sap sugar composition was associated with upregulation of CmSUT1 transcript within the vascular bundles.The current research aimed to explore the effect of CMV infection on the enzymes involved in symplastic phloem loading and RFO biosynthesis. Viral infection did not affect the activity of either raffinose or stachyose synthases in source leaves, but caused upregulation of the respective transcripts. Interestingly, activity of galactinol synthase was higher in CMV-infected leaves, associated with upregulation of CmGAS2. A significant increase in CmGAS2 expression in source leaves of melon plants exposed to high temperatures indicated that this response is common for both biotic and abiotic stresses. However, the effect of CMV or heat stress on phloem sap sugar composition is not due to alteration in RFO biosynthesis.

Isolation and characterization of a novel α-glucosidase with transglycosylation activity from Arthrobacter sp. DL001

Abstract A strain of Arthrobacter sp. DL001 with high transglycosylation activity was successfully isolated from the Yellow Sea of China. To purify the extracellular enzyme responsible for transglycosylation, a four-step protocol was adopted and the enzyme with electrophoretical purity was obtained. The purified enzyme has a molecular mass of 210 kDa and displays a narrow hydrolysis specificity towards α-1,4-glucosidic bond. Its hydrolytic activity was identified as decreasing in the order of maltotriose > panose > maltose. Only 3.61% maltose activity occurs when p-nitrophenyl α- d -glycopyranoside serves as a substrate, suggesting that this enzyme belongs to the type II α-glucosidase. In addition, the enzyme was able to transfer glucosyl groups from the donors containing α-1,4-glucosidic bond specific to glucosides, xylosides and alkyl alcohols in α-1,4- or α-1,6-manners. A decreased order of activity was observed when maltose, maltotriose, panose, β-cyclodextrin and soluble starch served as glycosyl donors, respectively. When maltose was utilized as a donor and a series of p-nitrophenyl-glycosides as acceptors, the glucosidase was capable of transferring glucosyl groups to p-nitrophenyl-glucosides and p-nitrophenyl-xylosides in α-1,4- or α-1,6-manners. The yields of p-nitrophenyl-oligosaccharides could reach 42–60% in 2 h. When a series of alkyl alcohols were utilized as acceptors, the enzyme exhibited its transglycosylation activities not only to the primary alcohols but also to the secondary alcohols with carbon chain length 1–4. Therefore, all the results indicated that the purified α-glucosidase present a useful tool for the biosynthesis of oligosaccharides and alkyl glucosides.

5-thiomannosides block the biosynthesis of dolichol-linked oligosaccharides and mimic class I congenital disorders of glycosylation.

In a cell-based assay for novel inhibitors, we have discovered that two glycosides of 5-thiomannose, each containing an interglycosidic nitrogen atom, prevented the correct zymogen processing of the prohormone proopiomelanocortinin (POMC) and the transcription factor sterol-regulatory element-binding protein-2 (SREBP-2) in mouse pituitary cells and Chinese hamster ovary (CHO) cells, respectively. In the case of SREBP-2, these effects were correlated with the altered N-linked glycosylation of subtilisin/kexin-like isozyme-1 (SKI-1), the protease responsible for SREBP-2 processing under sterol-limiting conditions. Further examination of the effects of these compounds in CHO cells showed that they cause extensive protein hypoglycosylation in a manner similar to type I congenital disorders of glycosylation (CDGs) since the remaining N-glycans in treated cells were complete (normal) structures. The under-glycosylation of glycoproteins in 5-thiomannoside-treated cells is now shown to be caused by the compromised biosynthesis of the dolichol-linked oligosaccharide (DLO) N-glycosylation donor, although the nucleotide sugars required for the synthesis of DLOs were neither reduced under these conditions, nor were their effects reversed upon the addition of exogenous mannose. Analysis of DLO intermediates by fluorophore-assisted carbohydrate electrophoresis demonstrated that 5-thiomannose-containing glycosides block DLO biosynthesis most likely at a stage prior to the GlcNAc(2) Man(3) intermediate, on the cytosolic face of the endoplasmic reticulum.

Evolution of milk oligosaccharides and lactose: a hypothesis

Mammalian milk or colostrum contains up to 10% of carbohydrate, of which free lactose usually constitutes more than 80%. Lactose is synthesized within lactating mammary glands from uridine diphosphate galactose (UDP-Gal) and glucose by a transgalactosylation catalysed by a complex of β4-galactosyltransferase and α-lactalbumin (α-LA). α-LA is believed to have evolved from C-type lysozyme. Mammalian milk or colostrum usually contains a variety of oligosaccharides in addition to free lactose. Each oligosaccharide has a lactose unit at its reducing end; this unit acts as a precursor that is essential for its biosynthesis. It is generally believed that milk oligosaccharides act as prebiotics and also as receptor analogues that act as anti-infection factors. We propose the following hypothesis. The proto-lacteal secretions of the primitive mammary glands of the common ancestor of mammals contained fat and protein including lysozyme, but no lactose or oligosaccharides because of the absence of α-LA. When α-LA first appeared as a result of its evolution from lysozyme, its content within the lactating mammary glands was low and lactose was therefore synthesized at a slow rate. Because of the presence of glycosyltransferases, almost all of the nascent lactose was utilized for the biosynthesis of oligosaccharides. The predominant saccharides in the proto-lacteal secretions or primitive milk produced by this common ancestor were therefore oligosaccharides rather than free lactose. Subsequent to this initial period, the oligosaccharides began to serve as anti-infection factors. They were then recruited as a significant energy source for the neonate, which was achieved by an increase in the synthesis of α-LA. This produced a concomitant increase in the concentration of lactose in the milk, and lactose therefore became an important energy source for most eutherians, whereas oligosaccharides continued to serve mainly as anti-microbial agents. Lactose, in addition, began to act as an osmoregulatory molecule, controlling the milk volume. Studies on the chemical structures of the milk oligosaccharides of a variety of mammalian species suggest that human milk or colostrum is unique in that oligosaccharides containing lacto-N-biose I (LNB) (Gal(β1 → 3)GlcNAc, type I) predominate over those containing N-acetyllactosamine (Gal(β1 → 4)GlcNAc, type II), whereas in other species only type II oligosaccharides are found or else they predominate over type I oligosaccharides. It can be hypothesized that this feature may have a selective advantage in that it may promote the growth of beneficial colonic bacteria, Bifidobacteria, in the human infant colon.

ドリコール中間体生合成に関与するヒト糖転移酵素間の物理的相互作用

ドリコール中間体生合成に関与するヒト糖転移酵素間の物理的相互作用

Identification of three oligo‐/polysaccharide‐specific ligases in Yersinia enterocolitica

In lipopolysaccharide (LPS) biosynthesis of gram-negative bacteria the lipid A-core oligosaccharide (LA-core) and O-polysaccharide (O-PS) biosynthesis pathways proceed separately and converge in periplasmic space where the waaL-encoded ligase joins O-PS onto LA-core. Enterobacterial common antigen (ECA) biosynthesis follows that of O-PS except that ECA is usually ligated to phosphatidylglycerol (PG) and only rarely to LA-core. In Yersinia enterocolitica serotype O:3 LPS is composed of LA-inner core (IC) onto which a homopolymeric O-PS, a hexasaccharide called outer core (OC), and/or ECA are ligated. We found that an individual O:3 LPS molecule carries either OC or O-PS substitution but not both. Related to this, we identified three genes in Y. enterocolitica O:3 that all expressed O-PS ligase activity in the Escherichia coliΔwaaL mutant. The LPS phenotypes of Y. enterocolitica O:3 single, double and triple ligase mutants indicated that two of ligases, named as WaaL(os) and WaaL(ps) , had a preferred substrate specificity for OC and O-PS, respectively, although with some promiscuity between the ligases; the third ligase named as WaaL(xs) was not involved in LPS or ECA biosynthesis. In Y. enterocolitica O:8 the WaaL(os) homologue (Ye1727) ligated a single pentasaccharide O-unit to LA-IC suggesting that in both Y. enterocolitica O:3 and O:8 WaaL(os) is an oligosaccharide (OS)-specific ligase. Finally, Yersinia pestis and Y. pseudotuberculosis carry only the waaL(ps) gene, while either waaL(os) or waaL(xs) or both are additionally present in other Yersinia species. This is the first report on the presence of three different oligo-/polysaccharide-specific ligases in a single bacterium.

Alg14 organizes the formation of a multiglycosyltransferase complex involved in initiation of lipid-linked oligosaccharide biosynthesis

Protein N-glycosylation begins with the assembly of a lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER) membrane. The first two steps of LLO biosynthesis are catalyzed by a functional multienzyme complex comprised of the Alg7 GlcNAc phosphotransferase and the heterodimeric Alg13/Alg14 UDP-GlcNAc transferase on the cytosolic face of the ER. In the Alg13/14 glycosyltransferase, Alg14 recruits cytosolic Alg13 to the ER membrane through interaction between their C-termini. Bioinformatic analysis revealed that eukaryotic Alg14 contains an evolved N-terminal region that is missing in bacterial orthologs. Here, we show that this N-terminal region of Saccharomyces cerevisiae Alg14 localize its green fluorescent protein fusion to the ER membrane. Deletion of this region causes defective growth at 38.5°C that can be partially complemented by overexpression of Alg7. Coimmunoprecipitation demonstrated that the N-terminal region of Alg14 is required for direct interaction with Alg7. Our data also show that Alg14 lacking the N-terminal region remains on the ER membrane through a nonperipheral association, suggesting the existence of another membrane-binding site. Mutational studies guided by the 3D structure of Alg14 identified a conserved α-helix involved in the second membrane association site that contributes to an integral interaction and protein stability. We propose a model in which the N- and C-termini of Alg14 coordinate recruitment of catalytic Alg7 and Alg13 to the ER membrane for initiating LLO biosynthesis.

Pasteurella multocida CMP-sialic acid synthetase and mutants of Neisseria meningitidis CMP-sialic acid synthetase with improved substrate promiscuity

Cytidine 5'-monophosphate (CMP)-sialic acid synthetases (CSSs) catalyze the formation of CMP-sialic acid from CTP and sialic acid, a key step for sialyltransferase-catalyzed biosynthesis of sialic acid-containing oligosaccharides and glycoconjugates. More than 50 different sialic acid forms have been identified in nature. To facilitate the enzymatic synthesis of sialosides with diverse naturally occurring sialic acid forms and their non-natural derivatives, CMP-sialic acid synthetases with promiscuous substrate specificity are needed. Herein we report the cloning, characterization, and substrate specificity studies of a new CSS from Pasteurella multocida strain P-1059 (PmCSS) and a CSS from Haemophillus ducreyi (HdCSS). Based on protein sequence alignment and substrate specificity studies of these two CSSs and a Neisseria meningitidis CSS (NmCSS), as well as crystal structure modeling and analysis of NmCSS, NmCSS mutants (NmCSS_S81R and NmCSS_Q163A) with improved substrate promiscuity were generated. The strategy of combining substrate specificity studies of enzymes from different sources and protein crystal structure studies can be a general approach for designing enzyme mutants with improved activity and substrate promiscuity.

Arabidopsis thaliana alpha1,2‐glucosyltransferase (ALG10) is required for efficient N‐glycosylation and leaf growth

Assembly of the dolichol-linked oligosaccharide precursor (Glc3Man9GlcNAc2) is highly conserved among eukaryotes. In contrast to yeast and mammals, little is known about the biosynthesis of dolichol-linked oligosaccharides and the transfer to asparagine residues of nascent polypeptides in plants. To understand the biological function of these processes in plants we characterized the Arabidopsis thaliana homolog of yeast ALG10, the α1,2-glucosyltransferase that transfers the terminal glucose residue to the lipid-linked precursor. Expression of an Arabidopsis ALG10–GFP fusion protein in Nicotiana benthamiana leaf epidermal cells revealed a reticular distribution pattern resembling endoplasmic reticulum (ER) localization. Analysis of lipid-linked oligosaccharides showed that Arabidopsis ALG10 can complement the yeast Δalg10 mutant strain. A homozygous Arabidopsis T-DNA insertion mutant (alg10-1) accumulated mainly lipid-linked Glc2Man9GlcNAc2 and displayed a severe protein underglycosylation defect. Phenotypic analysis of alg10-1 showed that mutant plants have altered leaf size when grown in soil. Moreover, the inactivation of ALG10 in Arabidopsis resulted in the activation of the unfolded protein response, increased salt sensitivity and suppression of the phenotype of α-glucosidase I-deficient plants. In summary, these data show that Arabidopsis ALG10 is an ER-resident α1,2-glucosyltransferase that is required for lipid-linked oligosaccharide biosynthesis and subsequently for normal leaf development and abiotic stress response.

Glucosamine inhibits epithelial-to-mesenchymal transition and migration of retinal pigment epithelium cells in culture and morphologic changes in a mouse model of proliferative vitreoretinopathy

To explore the effect of glucosamine (GlcN) on transforming growth factor (TGF)-β signalling and several processes involved in proliferative vitreoretinopathy (PVR). We evaluated the surface levels of TGF-β receptor and its binding of TGF-β in ARPE-19 cells. Release of cytokines and collagen, and expression of signalling intermediates were quantified. Migration was qualitatively and quantitatively examined. The morphology of cells undergoing PVR in vitro and in a mouse PVR model was observed. Glucosamine reduced the surface levels of TGF-β receptor and the ability of ARPE-19 cells to bind TGF-β. In ARPE-19 cells, TGF-β1 plus epidermal growth factor (EGF) or TGF-β2 increased the expression of alpha-smooth muscle actin (α-SMA) and decreased the expression of zona occludens protein (ZO-1). Transforming growth factor-(β2) also caused the release of platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF) and type 1 collagen and increased the phosphorylation of SMAD2 and SMAD3. Platelet-derived growth factor and CTGF stimulated cell migration, and TGF-β2 stimulated wound closure, contraction of collagen and changes in cell morphology. Treatment with GlcN counteracted all of these effects, and its administration in the mouse model reduced the morphologic appearance of PVR. Glucosamine could inhibit the TGF-β signalling pathway in retinal pigment epithelium cells and several of the downstream events associated with epithelial-mesenchymal transition and PVR.

Hydrogen peroxide spraying alleviates drought stress in soybean plants

To ascertain the effect of exogenously applied hydrogen peroxide (H 2O 2) on drought stress, we examined whether the spraying of soybean leaves with H 2O 2 would alleviate the symptoms of drought stress. Pre-treatment by spraying leaves with H 2O 2 delayed foliar wilting caused by drought stress compared to leaves sprayed with distilled water (DW). Additionally, the relative water content of drought-stressed leaves pre-treated with H 2O 2 was higher than that of leaves pre-treated with DW. Therefore, we analyzed the effect of H 2O 2 spraying on photosynthetic parameters and on the biosynthesis of oligosaccharides related to water retention in leaves during drought stress. Under conditions of drought stress, the net photosynthetic rate and stomatal conductance of leaves pre-treated with H 2O 2 were higher than those of leaves pre-treated with DW. In contrast to DW spraying, H 2O 2 spraying immediately caused an increase in the mRNA levels of d-myo-inositol 3-phosphate synthase 2 ( GmMIPS2) and galactinol synthase ( GolS), which encode key enzymes for the biosynthesis of oligosaccharides known to help plants tolerate drought stress. In addition, the levels of myo-inositol and galactinol were higher in H 2O 2-treated leaves than in DW-treated leaves. These results indicated that H 2O 2 spraying enabled the soybean plant to avoid drought stress through the maintenance of leaf water content, and that this water retention was caused by the promotion of oligosaccharide biosynthesis rather than by rapid stomatal closure.

Cellular and molecular biology of glycosphingolipid glycosylation

Brain tissue is characterized by its high glycosphingolipid content, particularly those containing sialic acid (gangliosides). As a result of this observation, brain tissue was a focus for studies leading to the characterization of the enzymes participating in ganglioside biosynthesis, and their participation in driving the compositional changes that occur in glycolipid expression during brain development. Later on, this focus shifted to the study of cellular aspects of the synthesis, which lead to the identification of the site of synthesis in the neuronal soma and their axonal transport toward the periphery. In this review article, we will focus in subcellular aspects of the biosynthesis of glycosphingolipid oligosaccharides, particularly the mechanisms underlying the trafficking of glycosphingolipid glycosyltransferases from the endoplasmic reticulum to the Golgi, those that promote their retention in the Golgi and those that participate in their topological organization as part of the complex membrane bound machinery for the synthesis of glycosphingolipids.