Yoichi Tsumuraya

Molecular Cloning of a β-Galactosidase from Radish That Specifically Hydrolyzes β-(1→3)- and β-(1→6)-Galactosyl Residues of Arabinogalactan Protein

A basic β-galactosidase with high specificity toward β-(1→3)- and β-(1→6)-galactosyl residues was cloned from radish (Raphanus sativus) plants by reverse transcription-PCR. The gene, designated RsBGAL1, contained an open reading frame consisting of 2,532 bp (851 amino acids). It is expressed in hypocotyls and young leaves. RsBGAL1 was highly similar to β-galactosidases having exo-β-(1→4)-galactanase activity found in higher plants and belongs to family 35 of the glycosyl hydrolases. Recombinant RsBGAL1 was expressed in Pichia pastoris and purified to homogeneity. The recombinant enzyme specifically hydrolyzed β-(1→3)- and β-(1→6)-galactooligosaccharides, the same substrates as the native enzyme isolated from radish seeds (Sekimata et al., 1989). It split off about 90% of the carbohydrate moieties of an arabinogalactan protein extracted from radish roots in concerted action with microbial α-l-arabinofuranosidase and β-glucuronidase. These results suggest that RsBGAL1 is a new kind of β-galactosidase with different substrate specificity than other β-galactosidases that exhibit exo-β-(1→4)-galactanase activity. The C-terminal region (9.6 kD) of RsBGAL1 is significantly similar to the Gal lectin-like domain, but this region is not retained in the native enzyme. Assuming posttranslational processing of RsBGAL1 with elimination of the Gal lectin-like domain results in a protein consisting of two subunits with molecular masses of 46 and 34 kD (calculated from the RsBGAL1 gene sequence). This is in good agreement with the SDS-PAGE and matrix-assisted laser desorption/ionization-time-of flight mass spectrometry measurements for subunits of the native enzyme (45 and 34 kD) and may thus partially explain the formation process of the native enzyme.

Characterization of pectin methyltransferase from soybean hypocotyls

Abstract. Pectin methyltransferase (PMT) catalyzing the transfer of the methyl group from S-adenosyl-L-methionine (SAM) to the C-6 carboxyl group of galactosyluronic acid residues in pectin was found in a membrane preparation of etiolated hypocotyls from 6-d-old soybean (Glycinemax Merr.). The enzyme was maximally active at pH 6.8 and 35–40 °C, and required 0.5% (w/v) Triton X-100. The incorporation of the methyl group was significantly enhanced by addition of a pectin with a low (22%) degree of methyl-esterification (DE) as exogenous acceptor substrate. The apparent Michaelis constants for SAM and the pectin (DE22) were 0.23 mM and 66 μg · ml−1, respectively. Attachment of the methyl group to the carboxyl group of the pectin via ester linkage was confirmed by analyzing radiolabeled product from incubation of the enzyme with [14C]methyl SAM and the acceptor pectin. Size-exclusion chromatography showed that both enzymatic hydrolysis with a pectin methylesterase and a mild alkali treatment (saponification) led to the release of radioactive methanol from the product. Enzymatic hydrolysis of the product with an endopolygalacturonase degraded it into small pectic fragments with low relative molecular mass, which also supports the idea that the methyl group is incorporated into the pectin. The soybean hypocotyls were fractionated into their cell wall components by successive extraction with water, EDTA, and alkali treatment. Among the resulting polysaccharide fractions, high PMT activity was observed when a de-esterified polysaccharide derived from the EDTA-soluble fraction (the pectic fraction) was added as an alternative acceptor substrate, indicating that the enzyme may be responsible for producing methyl-esterified pectin in vivo.

Carbohydrate structural analysis of wheat flour arabinogalactan protein

The water-extractable arabinogalactan protein (AGP) was isolated from bread wheat flour (Triticum aestivum L. variety Cadenza) and the structure of the arabinogalactan (AG) carbohydrate component was studied. Oligosaccharides, released by hydrolysis of the AG with a range of AGP-specific enzymes, were characterised by Matrix Assisted Laser Desorption Ionisation (MALDI)-Time of Flight (ToF)-Mass Spectrometry (MS), MALDI-ToF/ToF high energy collision induced dissociation (CID) and Polysaccharide Analysis by Carbohydrate gel Electrophoresis (PACE). The AG is composed of a β-(1→3)-D-galactan backbone with β-(1→6)-D-galactan side chains. These side chains are highly variable in length, from one to at least 20 Gal residues and are highly substituted with α-L-Araf. Single GlcA residues are also present at the non-reducing termini of some short β-(1→6)-galactan side chains. In addition, the β-(1→6)-galactan side chains are also substituted with β-L-Arap. We propose a polysaccharide structure of the wheat flour AGP that is substantially revised from earlier models.

β-Galactosyl Yariv reagent binds to the β-1,3-galactan of arabinogalactan-proteins

Yariv phenylglycosides specifically bind to β-1,3-galactan main chains of arabinogalactan proteins. Yariv phenylglycosides [1,3,5-tri(p-glycosyloxyphenylazo)-2,4,6-trihydroxybenzene] are a group of chemical compounds that selectively bind to arabinogalactan proteins (AGPs), a type of plant proteoglycan. Yariv phenylglycosides are widely used as cytochemical reagents to perturb the molecular functions of AGPs as well as for the detection, quantification, purification, and staining of AGPs. However, the target structure in AGPs to which Yariv phenylglycosides bind has not been determined. Here, we identify the structural element of AGPs required for the interaction with Yariv phenylglycosides by stepwise trimming of the arabinogalactan moieties using combinations of specific glycoside hydrolases. Whereas the precipitation with Yariv phenylglycosides (Yariv reactivity) of radish (Raphanus sativus) root AGP was not reduced after enzyme treatment to remove α-l-arabinofuranosyl and β-glucuronosyl residues and β-1,6-galactan side chains, it was completely lost after degradation of the β-1,3-galactan main chains. In addition, Yariv reactivity of gum arabic, a commercial product of acacia (Acacia senegal) AGPs, increased rather than decreased during the repeated degradation of β-1,6-galactan side chains by Smith degradation. Among various oligosaccharides corresponding to partial structures of AGPs, β-1,3-galactooligosaccharides longer than β-1,3-galactoheptaose exhibited significant precipitation with Yariv in a radial diffusion assay on agar. A pull-down assay using oligosaccharides cross linked to hydrazine beads detected an interaction of β-1,3-galactooligosaccharides longer than β-1,3-galactopentaose with Yariv phenylglycoside. To the contrary, no interaction with Yariv was detected for β-1,6-galactooligosaccharides of any length. Therefore, we conclude that Yariv phenylglycosides should be considered specific binding reagents for β-1,3-galactan chains longer than five residues, and seven residues are sufficient for cross linking, leading to precipitation of the Yariv phenylglycosides.

Structural Characterization of Arabidopsis Leaf Arabinogalactan Polysaccharides

Proteins decorated with arabinogalactan (AG) have important roles in cell wall structure and plant development, yet the structure and biosynthesis of this polysaccharide are poorly understood. To facilitate the analysis of biosynthetic mutants, water-extractable arabinogalactan proteins (AGPs) were isolated from the leaves of Arabidopsis (Arabidopsis thaliana) plants and the structure of the AG carbohydrate component was studied. Enzymes able to hydrolyze specifically AG were utilized to release AG oligosaccharides. The released oligosaccharides were characterized by high-energy matrix-assisted laser desorption ionization-collision-induced dissociation mass spectrometry and polysaccharide analysis by carbohydrate gel electrophoresis. The Arabidopsis AG is composed of a β-(1→3)-galactan backbone with β-(1→6)-d-galactan side chains. The β-(1→6)-galactan side chains vary in length from one to over 20 galactosyl residues, and they are partly substituted with single α-(1→3)-l-arabinofuranosyl residues. Additionally, a substantial proportion of the β-(1→6)-galactan side chain oligosaccharides are substituted at the nonreducing termini with single 4-O-methyl-glucuronosyl residues via β-(1→6)-linkages. The β-(1→6)-galactan side chains are occasionally substituted with α-l-fucosyl. In the fucose-deficient murus1 mutant, AGPs lack these fucose modifications. This work demonstrates that Arabidopsis mutants in AGP structure can be identified and characterized. The detailed structural elucidation of the AG polysaccharides from the leaves of Arabidopsis is essential for insights into the structure-function relationships of these molecules and will assist studies on their biosynthesis.

Properties and physiological functions of UDP-sugar pyrophosphorylase in Arabidopsis.

UDP-sugar pyrophosphorylase catalyzes the conversion of various monosaccharide 1-phosphates to the respective UDP-sugars in the salvage pathway. Using the genomic database, we cloned a putative gene for UDP-sugar pyrophosphorylase from Arabidopsis. Although relatively stronger expression was detected in the vascular tissue of leaves and the pollen, AtUSP is expressed in most cell types of Arabidopsis, indicating a housekeeping function in nucleotide sugar metabolism. Recombinant AtUSP expressed in Escherichia coli exhibited broad specificity toward monosaccharide 1-phosphates, resulting in the formation of various UDP-sugars such as UDP-glucose, -galactose, -glucuronic acid, -xylose and -L-arabinose. A loss-of-function mutation in the AtUSP gene caused by T-DNA insertion completely abolished male fertility. These results indicate that AtUSP functions as a UDP-sugar pyrophosphorylase in the salvage pathway, and that the generation of UDP-sugars from monosaccharide 1-phosphates catalyzed by AtUSP is essential for pollen development in Arabidopsis.

Structure of l-arabino-d-galactan-containing glycoproteins from radish leaves

Abstract Two l -arabino- d -galactan-containing glycoproteins having a potent inhibitory activity against eel anti-H agglutinin were isolated from the hot saline extracts of mature radish leaves and characterized to have a similar monosaccharide composition that consists of l -arabinose, d -galactose, l -fucose, 4-O-methyl- d -glucuronic acid, and d -glucuronic acid residues. The chemical structure features of the carbohydrate components were investigated by carboxyl group reduction, methylation, periodate oxidation, partial acid hydrolysis, and digestion with exo- and endo-glycosidases, which indicated a backbone chain of (1→3)-linked β- d -galactosyl residues, to which side chains consisting of α-(1→6)-linked d -galactosyl residues were attached. The α- l -arabinofuranosyl residues were attached as single nonreducing groups and as O-2- or O-3-linked residues to O-3 of the β- d -galactosyl residues of the side chains. Single α- l -fucopyranosyl end groups were linked to O-2 of the l -arabinofuranosyl residues, and the 4-O-methyl-β- d -glucopyranosyluronic acid end groups were linked to d -galactosyl residues. The O-α- l -fucopyranosyl-(1→2)-α- l -arabinofuranosyl end-groups were shown to be responsible for the serological, H-like activity of the l -arabino- d -galactan glycoproteins. Reductive alkaline degradation of the glycoconjugates showed that a large proportion of the polysaccharide chains is conjugated with the polypeptide backbone through a 3-O- d -galactosylserine linkage.

Activation of a limulus coagulation factor G by (1 → 3)-g⊝d-glucans

Abstract Various oligo- and poly-saccharides differing in sugar compositions and types of linkage were examined for their ability to activate a limulus coagulation factor G, the first protease in an alternative coagulation cascade of the horseshoe crab, Tachypleus tridentatus , whose amebocytes have originally been known to contain a lipopolysacchaide(LPS)-driven pathway leading to the formationof coagulin gel. Linear and branched (1 → 3)-β- d -glucans and mixed linkage (1 → 3),(1 → 4)-β- d -glucans were found to exhibit the ability to activate factor G at concentrations of 10 −8 –10 −10 g/mL as assayed by amidolytic activity of the clotting enzyme. Laminaran oligosaccharides, laminaran dextrins (number-average mol. wt. ≦ 5800), and other polysaccharides including carboxymethylcellulose, amylose, starch, d -fructans, α- l -arabinan, β- d -xylans, (1 → 3)-β- d -galactan, water-soluble chitin derivatives, chondroitin, chondroitin sulfates, hyaluronan, keratan sulfate, heparins, heparan sulfate, and LPSs were virtually inactive as activators even at a concentration of 10 −7 g/mL. The activating ability increased with increasing number-average mol. wt. (6800–216 000) of linear (1 → 3)-β- d -glucans. The apparent activating ability of gyrophoran, nigeran, and yeast α- d -mannan was largely abolished by digestion with a highly purified Arthrobacter luteus endo-(1 → 3)-β- d -glucanase, which provided supportive evidence for the activation to be ascribed to contaminating (1 → 3)-β- d -glucan(s). Possible participation of ordered structures of (1 → 3)-β- d -glucans in the activation of factor G is discussed.

A Bifunctional Enzyme with L-Fucokinase and GDP-L-fucose Pyrophosphorylase Activities Salvages Free L-Fucose in Arabidopsis

Monomeric sugars generated during the metabolism of polysaccharides, glycoproteins, and glycolipids are imported to the cytoplasm and converted to respective nucleotide sugars via monosaccharide 1-phosphates, to be reutilized as activated sugars. Because l-fucose (l-Fuc) is activated mainly in the form of GDP derivatives in seed plants, the salvage reactions for l-Fuc are expected to be independent from those for Glc, Gal, l-arabinose, and glucuronic acid, which are activated as UDP-sugars. For this study we have identified, in the genomic data base of Arabidopsis, the gene (designated AtFKGP) of a bifunctional enzyme with similarity to both l-fucokinase and GDP-l-Fuc pyrophosphorylase. Recombinant AtFKGP (rAt-FKGP) expressed in Escherichia coli showed both l-fucokinase and GDP-l-Fuc pyrophosphorylase activities, generating GDP-l-Fuc from l-Fuc, ATP, and GTP as the starting substrates. Point mutations in rAtFKGPs at either Gly133 or Gly830 caused loss of GDP-l-Fuc pyrophosphorylase and l-fucokinase activity, respectively. The apparent Km values of l-fucokinase activity of rAtFKGP for l-Fuc and ATP were 1.0 and 0.45 mm, respectively, and those of GDP-l-Fuc pyrophosphorylase activity for l-Fuc 1-phosphate and GTP were 0.052 and 0.17 mm, respectively. The expression of AtFKGP was detected in most cell types of Arabidopsis, indicating that salvage reactions for free l-Fuc catalyzed by AtFKGP occur ubiquitously in Arabidopsis. Loss-of-function mutants with tDNA insertion in AtFKGP exhibited higher accumulation of free l-Fuc in the soluble fraction than the wild-type plant. These results indicate that AtFKGP is a bifunctional enzyme with l-fucokinase and GDP-l-Fuc pyrophosphorylase activities, which salvages free l-Fuc in Arabidopsis.

Molecular cloning and expression in Escherichia coli of a Trichoderma viride endo-beta-(1-->6)-galactanase gene.

The nucleotide sequence depicted in Figure 1 has been submitted to the DDBJ nucleotide sequence database under the accession no. AB104898. A gene encoding endo-beta-(1-->6)-galactanase from Trichoderma viride was cloned by reverse transcriptase-PCR and expressed in Escherichia coli. The gene contained an open reading frame consisting of 1437 bp (479 amino acids). The deduced amino acid sequence of the protein showed little similarity with other known glycoside hydrolases. A signal sequence (20 amino acids) was found at the N-terminal region of the protein and the molecular mass of the mature form was calculated to be 50.488 kDa. The gene product expressed in E. coli as a recombinant protein fused with thioredoxin and His(6) tags had almost the same substrate specificity and mode of action as native enzyme purified from a commercial cellulase preparation of T. viride, i.e. recombinant enzyme endo-hydrolysed beta-(1-->6)-galacto-oligomers with a DP (degree of polymerization) higher than 3, and it could also hydrolyse alpha-L-arabinofuranosidase-treated arabinogalactan protein from radish. It produced beta-(1-->6)-galacto-oligomers ranging from DP 2 to at least 8 at the initial hydrolysis stage and galactose and beta-(1-->6)-galactobiose as the major products at the final reaction stage. These results indicate that the cloned gene encodes an endo-beta-(1-->6)-galactanase. As far as we know, this is the first time an endo-beta-(1-->6)-galactanase has been cloned.