Galactosyl Residues Research Articles

Frost Grape Polysaccharide (FGP), an Emulsion-Forming Arabinogalactan Gum from the Stems of Native North American Grape Species Vitis riparia Michx.

A new arabinogalactan is described that is produced in large quantity from the cut stems of the North American grape species Vitis riparia (Frost grape). The sugar composition consists of l-arabinofuranose (l-Araf, 55.2%) and d-galactopyranose (d-Galp 30.1%), with smaller components of d-xylose (11.2%), d-mannose (3.5%), and glucuronic acid (GlcA, ∼2%), the latter linked via a galactosyl residue. Permethylation identified 3-linked Galp residues, some substituted at the 2-position with Galp or Manp, terminal Araf and Xylp, and an internal 3-substituted Araf. NMR (HSQC, TOCSY, HMBC, DOSY) identified βGalp and three αAraf spin systems, in an Araf-α1,3-Araf-α1,2-Araf-α1,2-Galp structural motif. Diffusion-ordered NMR showed that the FGP has a molecular weight of 1-10 MDa. Unlike gum arabic, the FGP does not contain a hydroxyproline-rich protein (HPRP). FGP forms stable gels at >15% w/v and at 1-12% solutions are viscous and are excellent emulsifiers of flavoring oils (grapefruit, clove, and lemongrass), giving stable emulsions for ≥72 h. Lower concentrations (0.1% w/v) were less viscous, yet still gave stable grapefruit oil/water emulsions. Hence, FGP is a β1,3-linked arabinogalactan with potential as a gum arabic replacement in the food and beverage industries.

Molecular changes in the cell surface of differentiating epidermal keratinocytes.

The specific binding of lectins to the cell surfaces in particular epidermal strata indicates that oligosaccharide moieties of the glycoconjugates on the cell membrane are altered as the keratinocyte differentiates. The disappearance of binding by the Bandeiraea simplicifolia isolectin I-B4 (BS I-B4) and the appearance of binding by the Ulex europeus agglutinin I (UEA), which occur as the basal cell moves up into the spinous layer of the cutaneous epidermis of the newborn rat, could result from (a) redistribution of existing glycoconjugates in the cell membrane, (b) hydrolysis of galactosyl residues exposing fucosyl binding sites, or (c) addition of fucosyl residues to pre-existing oligosaccharides with specificity for the galactoside. Isolation of glycoproteins from the cell membrane using lectin-affinity column chromatography on Sepharose-4B showed a decrease in the amount of protein which bound to the BS I-B4 and an increase in the protein with affinity for the UEA as the ratio of spinous to basal cells increased in the population of cells from which the glycoproteins were obtained. These data indicate the existence of two different classes of glycoproteins on the surfaces of cells in the lower epidermis and argue against a redistribution of glycoconjugates as the explanation for the selective binding of specific lectins observed in tissue sections. Exposure of sections of skin to alpha-galactosidase completely eliminated the binding of fluorescein isothiocyanate-labeled (FITC) BS I-B4 in the lower epidermis but did not result in additional binding of UEA. Exposure to fucosidase eliminated binding by UEA and increased the binding of BS I-B4 in the spinous layer.(ABSTRACT TRUNCATED AT 250 WORDS)

Design, synthesis, and cytotoxicity of perbutyrylated glycosides of 4β-triazolopodophyllotoxin derivatives.

A series of novel perbutyrylated glycosides of 4β-triazolopodophyllotoxin derivatives were synthesized by utilizing the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Evaluation of cytotoxicity against a panel of five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, SW480) using the MTT assay shows that some of these glycosylated derivatives have good anticancer activity. Among the synthesized compounds, compound 21a shows the highest activity, with IC50 values ranging from 0.49 to 6.70 μM, which is more potent than the control drugs etoposide and cisplatin. Compound 21a is characterized by a perbutyrylated α-D(+)-galactosyl residue, the absence of an additional linking spacer between the sugar residue and the triazole ring, as well as a 4'-OH group on the E ring of the podophyllotoxin scaffold.

Utilization of agro-industrial waste for β-galactosidase production under solid state fermentation using halotolerant Aspergillus tubingensis GR1 isolate

A halotolerant fungal isolate Aspergillus tubingensis GR1 was isolated from the man-made solar saltern located at Khambhat, Gujarat, India, and identified using 28S rDNA partial genome sequencing. This isolate was studied for β-galactosidase production under solid state fermentation using wheat bran and deproteinized acid cheese whey. The influence of various agro-industrial wastes, nitrogen source and other growth conditions on β-galactosidase production was investigated using ‘one-factor-at-a-time’ approach. Among various variables screened along with wheat bran and deproteinized acid cheese whey as major growth substrate, corn steep liquor and MgSO4 were found to be most significant. The optimum concentrations of these significant parameters were determined employing the response surface central composite design, revealing corn steep liquor concentration (2 mL) and magnesium sulphate (50 mg) per 5 g of wheat bran and 20 mL of deproteinized acid cheese whey for highest enzyme production (15,936 U/gds). These results suggest the feasibility of industrial large-scale production of β-galactosidase known to be valuable in whey hydrolysis and removal of galactosyl residue from polysaccharide.

New antitumor 6-chloropurine nucleosides inducing apoptosis and G2/M cell cycle arrest.

Treating cancer has been challenging for decades, following countless approaches and attempts. Nucleosides, alone or as part of nucleotides, are vital elements of living systems and have shown pharmacological effects, e.g. as antibiotic or antiviral agents. We investigated the antitumor potential on human melanoma, lung and ovarian carcinomas, and on colon adenocarcinoma of a new series of purine nucleosides based on a 6-chloropurine or a 2-acetamido-6-chloropurine scaffold linked to perbenzylated hexosyl (glucosyl, galactosyl and mannosyl) residues. All compounds were tested in a sulforhodamine B (SRB) assay for their cytotoxicity and provided micromolar GI50 values with order of magnitude comparable to structurally similar chemotherapeutics, namely 2-chloro-2'-deoxyadenosine (cladribine). Furthermore, the induction of apoptosis was established and cell cycle analysis was accomplished demonstrating a G2/M cell cycle arrest.

Water-holding capacity of soluble and insoluble polysaccharides in pressed potato fibre

Pressed potato fibres (PPF), a by-product of starch production, has a high water-holding capacity (WHC). In this study, it is shown that the WHC is caused by a network of mainly insoluble, non-cellulosic cell wall polysaccharides (CWPs). Despite the solubilization of one-fourth of the CWPs from PPF, representing 40–60 w/w% of pectic CWPs (rhamnosyl, uronyl, galactosyl and arabinosyl residues) present in PPF, the insoluble residues still had similar WHCs as PPF. Only after enzymatic hydrolysis of mainly non-cellulosic CWPs, the WHC decreased substantially (by 61%). Combining the cellulose-rich residue obtained after enzyme hydrolysis with a polymeric homogalacturonan (HG)-rhamnogalacturonan-I (RG-I)-arabinogalactan (AG) extract increased the WHC. This increased hydration is suggested to result from the observed adsorption of the soluble HG-RG-I-AG to the insoluble cellulose-rich residue. No adsorption was observed of the HG-RG-I-AG to an insoluble residue enriched in non-cellulosic CWPs.

High-quality production of human α-2,6-sialyltransferase in Pichia pastoris requires control over N-terminal truncations by host-inherent protease activities.

Backgroundα-2,6-sialyltransferase catalyzes the terminal step of complex N-glycan biosynthesis on human glycoproteins, attaching sialic acid to outermost galactosyl residues on otherwise fully assembled branched glycans. This “capping” of N-glycans is critical for therapeutic efficacy of pharmaceutical glycoproteins, making the degree of sialylation an important parameter of glycoprotein quality control. Expression of recombinant glycoproteins in mammalian cells usually delivers heterogeneous N-glycans, with a minor degree of sialylation. In-vitro chemo-enzymatic glycoengineering of the N-glycans provides an elegant solution to increase the degree of sialylation for analytical purposes but also possibly for modification of therapeutic proteins.ResultsHuman α-2,6-sialyltransferase (ST6Gal-I) was secretory expressed in P.pastoris KM71H. ST6Gal-I featuring complete deletion of both the N-terminal cytoplasmic tail and the transmembrane domain, and also partial truncation of the stem region up to residue 108 were expressed N-terminally fused to a His or FLAG-Tag. FLAG-tagged proteins proved much more resistant to proteolysis during production than the corresponding His-tagged proteins. Because volumetric transferase activity measured on small-molecule and native glycoprotein acceptor substrates did not correlate to ST6Gal-I in the supernatant, enzymes were purified and characterized in their action on non-sialylated protein-linked and released N-glycans, and the respective N-terminal sequences were determined by automated Edman degradation. Irrespective of deletion construct used (Δ27, Δ48, Δ62, Δ89), isolated proteins showed N-terminal processing to a highly similar degree, with prominent truncations at residue 108 - 114, whereby only Δ108ST6Gal-I retained activity. FLAG-tagged Δ108ST6Gal-I was therefore produced and obtained with a yield of 4.5 mg protein/L medium. The protein was isolated and shown by MS to be intact. Purified enzyme exhibited useful activity (0.18 U/mg) for sialylation of different substrates.ConclusionsFunctional expression of human ST6Gal-I as secretory protein in P.pastoris necessitates that N-terminal truncations promoted by host-inherent proteases be tightly controlled. N-terminal FLAG-Tag contributes extra stability to the N-terminal region as compared to N-terminal His-Tag. Proteolytic degradation proceeds up to residues 108 – 114 and of the resulting short-form variants, only Δ108ST6Gal-I seems to be active. FLAG-Δ108ST6Gal-I transfers sialic acids to monoclonal antibody substrate with sufficient yields, and because it is stably produced in P.pastoris, it is identified here as an interesting glycoengineering catalyst.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0138-8) contains supplementary material, which is available to authorized users.

Improved starch recovery from potatoes by enzymes and reduced water holding of the residual fibres

During the industrial extraction of starch from potatoes (Seresta), some starch remains within undisrupted potato cells in the fibrous side-stream. The aim of this study was to investigate if enzymatic degradation of cell wall polysaccharides (CWPs) can enhance starch recovery and lower the water holding capacity (WHC) of the “fibre” fraction. The use of a pectinase-rich preparation recovered 58% of the starch present in the “fibre” fraction. Also, the “fibre” fraction retained only 40% of the water present in the non-enzyme treated “fibre”. This was caused by the degradation of pectins, in particular arabinogalactan side chains calculated as the sum of galactosyl and arabinosyl residues.

PEGylated galactosylated cationic liposomes for hepatocytic gene delivery

The efficiency of liposome-mediated gene delivery is greatly enhanced by appropriate decoration of vehicles with cell-specific targeting ligands. However, liposome–DNA complexes may still be opsonized in serum thus ablating any advantage gained. A stealth aspect may therefore be conferred on complexes by poly(ethylene glycol) (PEG) grafting. Here, we examined the effect that degree of PEGylation has on physicochemical properties, cytotoxicity and transfection activity of lipoplexes containing the cytofectin 3β-[N-(N′, N′-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T), the neutral co-lipid dioleoylphosphatidylethanolamine (DOPE), the asialoglycoprotein receptor (ASGP-R) targeted cholesteryl-β-d-galactopyranoside (Chol-β-Gal) ligand, and plasmid DNA in ASGP-R-negative (HEK293) and receptor-positive (HepG2) human cell lines. Lipoplexes were characterized by hydrodynamic sizing, electron microscopy, band shift, ethidium bromide (EtBr) intercalation and nuclease digestion assays. Cryo-TEM and DLS studies revealed that PEGylation generated smaller and more densely aggregated lipoplexes than their non-PEGylated counterparts. MTT and AB reduction studies showed that the lipoplexes elicited a dose-dependent cytotoxic effect in both cell lines, with cell viability remaining above 65% (MTT) and 50% (AB). The Ricinus communis (RCA120) agglutination test confirmed that the galactosyl residues on the targeted lipoplexes were well exposed and accessible. Transgene activity increased by 63% and 77% when HepG2 was confronted by the 2 and 5mole% PEGylated lipoplexes, respectively, compared to their non-PEGylated counterparts. Furthermore, Chol-T Chol-β-Gal 5% PEG complexes were able to achieve a 164% increase in transfection level in the ASGP-R positive cell line (HepG2) compared to HEK293 (ASGP-R negative). Results strongly indicate that PEGylation potentiates the activity of ASGP-R-targeted lipoplexes, highlighting their gene delivery potential.

GneZ, a UDP-GlcNAc 2-epimerase, is required for S-layer assembly and vegetative growth of Bacillus anthracis.

Bacillus anthracis, the causative agent of anthrax, forms an S-layer atop its peptidoglycan envelope and displays S-layer proteins and Bacillus S-layer-associated (BSL) proteins with specific functions to support cell separation of vegetative bacilli and growth in infected mammalian hosts. S-layer and BSL proteins bind via the S-layer homology (SLH) domain to the pyruvylated secondary cell wall polysaccharide (SCWP) with the repeat structure [→4)-β-ManNAc-(1→4)-β-GlcNAc-(1→6)-α-GlcNAc-(1→]n, where α-GlcNAc and β-GlcNAc are substituted with two and one galactosyl residues, respectively. B. anthracis gneY (BAS5048) and gneZ (BAS5117) encode nearly identical UDP-GlcNAc 2-epimerase enzymes that catalyze the reversible conversion of UDP-GlcNAc and UDP-ManNAc. UDP-GlcNAc 2-epimerase enzymes have been shown to be required for the attachment of the phage lysin PlyG with the bacterial envelope and for bacterial growth. Here, we asked whether gneY and gneZ are required for the synthesis of the pyruvylated SCWP and for S-layer assembly. We show that gneZ, but not gneY, is required for B. anthracis vegetative growth, rod cell shape, S-layer assembly, and synthesis of pyruvylated SCWP. Nevertheless, inducible expression of gneY alleviated all the defects associated with the gneZ mutant. In contrast to vegetative growth, neither germination of B. anthracis spores nor the formation of spores in mother cells required UDP-GlcNAc 2-epimerase activity.

Purification and Characterization of Phosphonoglycans from Glycomyces sp. Strain NRRL B-16210 and Stackebrandtia nassauensis NRRL B-16338

Two related actinomycetes, Glycomyces sp. strain NRRL B-16210 and Stackebrandtia nassauensis NRRL B-16338, were identified as potential phosphonic acid producers by screening for the gene encoding phosphoenolpyruvate (PEP) mutase, which is required for the biosynthesis of most phosphonates. Using a variety of analytical techniques, both strains were subsequently shown to produce phosphonate-containing exopolysaccharides (EPS), also known as phosphonoglycans. The phosphonoglycans were purified by sequential organic solvent extractions, methanol precipitation, and ultrafiltration. The EPS from the Glycomyces strain has a mass of 40 to 50 kDa and is composed of galactose, xylose, and five distinct partially O-methylated galactose residues. Per-deutero-methylation analysis indicated that galactosyl residues in the polysaccharide backbone are 3,4-linked Gal, 2,4-linked 3-MeGal, 2,3-linked Gal, 3,6-linked 2-MeGal, and 4,6-linked 2,3-diMeGal. The EPS from the Stackebrandtia strain is comprised of glucose, galactose, xylose, and four partially O-methylated galactose residues. Isotopic labeling indicated that the O-methyl groups in the Stackebrandtia phosphonoglycan arise from S-adenosylmethionine. The phosphonate moiety in both phosphonoglycans was shown to be 2-hydroxyethylphosphonate (2-HEP) by (31)P nuclear magnetic resonance (NMR) and mass spectrometry following strong acid hydrolysis of the purified molecules. Partial acid hydrolysis of the purified EPS from Glycomyces yielded 2-HEP in ester linkage to the O-5 or O-6 position of a hexose and a 2-HEP mono(2,3-dihydroxypropyl)ester. Partial acid hydrolysis of Stackebrandtia EPS also revealed the presence of 2-HEP mono(2,3-dihydroxypropyl)ester. Examination of the genome sequences of the two strains revealed similar pepM-containing gene clusters that are likely to be required for phosphonoglycan synthesis.

Structure Binding Relationship of Galactosylated Glycoclusters toward Pseudomonas aeruginosa Lectin LecA Using a DNA-Based Carbohydrate Microarray

Pseudomonas aeruginosa (PA) is a major public health issue due to its impact on nosocomial infections as well as its impact on cystic fibrosis patient mortality. One of the main concerns is its ability to develop antibiotic resistance. Therefore, inhibition of PA virulence has been proposed as an alternative strategy to tackle PA based infections. LecA (or PA-IL), a galactose binding lectin from PA, is involved in its virulence. Herein, we aimed at designing high affinity synthetic ligands toward LecA for its inhibition and at understanding the key parameters governing the binding of multivalent galactosylated clusters. Twenty-five glycoclusters were synthesized and their bindings were studied on a carbohydrate microarray. Monosaccharide centered clusters and linear comb-like clusters were synthesized with different linkers separating the core and the galactosyl residues. Their length, flexibility, and aromaticity were varied. Our results showed that the binding profile of LecA to galactosylated clusters was dependent on both the core and the linker and also that the optimal linker was different for each core. Nevertheless, an aryl group in the linker structure drastically improved the binding to LecA. Our results also suggest that optimal distances are preferred between the core and the aromatic group and the core and the galactose.

Cladribine in the treatment of acute myeloid leukemia

Treating cancer has been challenging for decades, following countless approaches and attempts. Nucleosides, alone or as part of nucleotides, are vital elements of living systems and have shown pharmacological effects, e.g. as antibiotic or antiviral agents. We investigated the antitumor potential on human melanoma, lung and ovarian carcinomas, and on colon adenocarcinoma of a new series of purine nucleosides based on a 6-chloropurine or a 2-acetamido-6-chloropurine scaffold linked to perbenzylated hexosyl (glucosyl, galactosyl and mannosyl) residues. All compounds were tested in a sulforhodamine B (SRB) assay for their cytotoxicity and provided micromolar GI50 values with order of magnitude comparable to structurally similar chemotherapeutics, namely 2-chloro-2′-deoxyadenosine (cladribine). Furthermore, the induction of apoptosis was established and cell cycle analysis was accomplished demonstrating a G2/M cell cycle arrest.

Altered dynamics of a lipid raft associated protein in a kidney model of Fabry disease

Accumulation of globotriaosylceramide (Gb3) and other neutral glycosphingolipids with galactosyl residues is the hallmark of Fabry disease, a lysosomal storage disorder caused by deficiency of the enzyme alpha-galactosidase A (α-gal A). These lipids are incorporated into the plasma membrane and intracellular membranes, with a preference for lipid rafts. Disruption of raft mediated cell processes is implicated in the pathogenesis of several human diseases, but little is known about the effects of the accumulation of glycosphingolipids on raft dynamics in the context of Fabry disease. Using siRNA technology, we have generated a polarized renal epithelial cell model of Fabry disease in Madin–Darby canine kidney cells. These cells present increased levels of Gb3 and enlarged lysosomes, and progressively accumulate zebra bodies. The polarized delivery of both raft-associated and raft-independent proteins was unaffected by α-gal A knockdown, suggesting that accumulation of Gb3 does not disrupt biosynthetic trafficking pathways. To assess the effect of α-gal A silencing on lipid raft dynamics, we employed number and brightness (N&B) analysis to measure the oligomeric status and mobility of the model glycosylphosphatidylinositol (GPI)-anchored protein GFP-GPI. We observed a significant increase in the oligomeric size of antibody-induced clusters of GFP-GPI at the plasma membrane of α-gal A silenced cells compared with control cells. Our results suggest that the interaction of GFP-GPI with lipid rafts may be altered in the presence of accumulated Gb3. The implications of our results with respect to the pathogenesis of Fabry disease are discussed.

Lectin binding in normal donkey eyeball

In the present study, the distribution of various sugar residues in the eyeball tissues of sexually mature donkey was examined by employing fluorescein isothiocyanate-conjugated lectins. Our results revealed the presence of mannose (labeled by lectins ConA), galactose (labeled by PNA, GSAI, ECA), GalNAc (labeled by SBA, VVA), and GlcNAc (labeled by WGA) residues in the donkey ocular tissues. The epithelium and stroma of the ocular tissues were labeled with mannose (ConA) and GlcNAc (WGA) binding lectins. Binding sites for WGA and PNA to the rod and cone cells of the retina were evident. The lectins Con A, WGA and GSAI are bound strongly to the endothelium of blood vessels and to smooth muscle cells of the iris. In conclusion, the findings of the present study clearly indicate that the donkey eyeball contains a wide range of glycoconjugates (bearing mannosyl, galactosyl and glucosly residues), and it lacks fucosyl residues.

Lectin binding in normal donkey eyeball

In the present study, the distribution of various sugar residues in the eyeball tissues of sexually mature donkey was examined by employing fluorescein isothiocyanate-conjugated lectins. Our results revealed the presence of mannose (labeled by lectins ConA), galactose (labeled by PNA, GSAI, ECA), GalNAc (labeled by SBA, VVA), and GlcNAc (labeled by WGA) residues in the donkey ocular tissues. The epithelium and stroma of the ocular tissues were labeled with mannose (ConA) and GlcNAc (WGA) binding lectins. Binding sites for WGA and PNA to the rod and cone cells of the retina were evident. The lectins Con A, WGA and GSAI are bound strongly to the endothelium of blood vessels and to smooth muscle cells of the iris. In conclusion, the findings of the present study clearly indicate that the donkey eyeball contains a wide range of glycoconjugates (bearing mannosyl, galactosyl and glucosly residues), and it lacks fucosyl residues.

The activity of serum beta-galactosidase in colon cancer patients with a history of alcohol and nicotine dependence: preliminary data

Beta-galactosidase (GAL) is a lysosomal exoglycosidase involved in the catabolism of glycoconjugates through the sequential release of beta-linked terminal galactosyl residues. The stimulation of activity of exoglycosidases and other degradative enzymes has been noted in cancers as well as in alcohol and nicotine addiction separately. This is the first study to evaluate the activity of the serum senescence marker GAL in colon cancer patients with a history of alcohol and nicotine dependence, as a potential factor of worse cancer prognosis. The material was serum of 18 colon cancer patients and 10 healthy volunteers. Ten colon cancer patients met alcohol and nicotine dependence criteria. The activity of beta-galactosidase (pkat/ml) was determined by the colorimetric method. Comparisons between groups were made using the Kruskal-Wallis analysis and differences evaluated using the Mann-Whitney U test. Spearman's rank correlation coefficient was used to measure the statistical dependence between two variables. The activity of serum GAL was significantly higher in colon cancer patients with a history of alcohol and nicotine dependence, in comparison to colon cancer patients without a history of drinking/smoking (p=0.015; 46% increase), and the controls (p=0.0002; 81% increase). The activity of serum GAL in colon cancer patients without a history of alcohol/nicotine dependence was higher than the activity in the controls (p = 0.043; 24% increase). Higher activity of beta-galactosidase may potentially reflect the accelerated growth of the cancer, invasion, metastases, and maturation, when alcohol and nicotine dependence coincide with colon cancer. For a better prognosis of colon cancer, alcohol and nicotine withdrawal seems to be required.

Characterisation of cell wall polysaccharides from rapeseed (Brassica napus) meal

To enable structural characteristics of individual cell wall polysaccharides from rapeseed (Brassica napus) meal (RSM) to be studied, polysaccharide fractions were sequentially extracted. Fractions were analysed for their carbohydrate (linkage) composition and polysaccharide structures were also studied by enzymatic fingerprinting.The RSM fractions analysed contained pectic polysaccharides: homogalacturonan in which 60% of the galacturonic acid residues are methyl-esterified, arabinan branched at the O-2 position and arabinogalactan mainly type II. This differs from characteristics previously reported for Brassica campestris meal, another rapeseed cultivar. Also, in the alkali extracts hemicelluloses were analysed as xyloglucan both of the XXGG- and XXXG-type decorated with galactosyl, fucosyl and arabinosyl residues, and as xylan with O-methyl-uronic acid attached. The final residue after extraction still contained xyloglucan and remaining (pectic) polysaccharides next to cellulose, showing that the cell wall matrix of RSM is very strongly interconnected.

Regulation of integrin αV subunit expression by sulfatide in hepatocellular carcinoma cells

Integrin is important in migration and metastasis of tumor cells. Changes of integrin expression and distribution will cause an alteration of cellular adhesion and migration behaviors. In this study, we investigated sulfatide regulation of the integrin αV subunit expression in hepatoma cells and observed that either exogenous or endogenous sulfatide elicited a robust upregulation of integrin αV subunit mRNA and protein expression in hepatoma cells. This regulatory effect occurred with a corresponding phosphorylation (T739) of the transcription factor Sp1. Based on the electrophoretic mobility shift assay, sulfatide enhanced the integrin αV promoter activity and strengthened the Sp1 complex super-shift. The results of chromatin immunoprecipitation analysis also indicated that sulfatide enhanced Sp1 binding to the integrin αV promoter in vivo. Silence of Sp1 diminished the stimulation of integrin αV expression by sulfatide. In the early stage of sulfatide stimulation, phosphorylation of Erk as well as c-Src was noted, and inhibition of Erk activation with either U0126 or PD98059 significantly suppressed Sp1 phosphorylation and integrin αV expression. We demonstrated that sulfatide regulated integrin αV expression and cell adhesion, which was associated with Erk activation.

Rice Os9BGlu31 Is a Transglucosidase with the Capacity to Equilibrate Phenylpropanoid, Flavonoid, and Phytohormone Glycoconjugates

Glycosylation is an important mechanism of controlling the reactivities and bioactivities of plant secondary metabolites and phytohormones. Rice (Oryza sativa) Os9BGlu31 is a glycoside hydrolase family GH1 transglycosidase that acts to transfer glucose between phenolic acids, phytohormones, and flavonoids. The highest activity was observed with the donors feruloyl-glucose, 4-coumaroyl-glucose, and sinapoyl-glucose, which are known to serve as donors in acyl and glucosyl transfer reactions in the vacuole, where Os9BGlu31 is localized. The free acids of these compounds also served as the best acceptors, suggesting that Os9BGlu31 may equilibrate the levels of phenolic acids and carboxylated phytohormones and their glucoconjugates. The Os9BGlu31 gene is most highly expressed in senescing flag leaf and developing seed and is induced in rice seedlings in response to drought stress and treatment with phytohormones, including abscisic acid, ethephon, methyljasmonate, 2,4-dichlorophenoxyacetic acid, and kinetin. Although site-directed mutagenesis of Os9BGlu31 indicated a function for the putative catalytic acid/base (Glu(169)), catalytic nucleophile residues (Glu(387)), and His(386), the wild type enzyme displays an unusual lack of inhibition by mechanism-based inhibitors of GH1 β-glucosidases that utilize a double displacement retaining mechanism.