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 a-L-arabinofuranosyl and b-glucuronosyl residues and b-1,6-galactan side chains, it was completely lost after degradation of the b-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 b-1,6-galactan side chains by Smith degradation. Among various oligosaccharides corresponding to partial structures of AGPs, b-1,3-galactooligosaccharides longer than b-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 b-1,3-galactooligosaccharides longer than b-1,3-galactopentaose with Yariv phenylglycoside. To the contrary, no interaction with Yariv was detected for b-1,6-galactooligosaccharides of any length. Therefore, we conclude that Yariv phenylglycosides should be considered specific binding reagents for b-1,3-galactan chains longer than five residues, and seven residues are sufficient for cross linking, leading to precipitation of the Yariv phenylglycosides.Arabinogalactan proteins (AGPs) are a type of plant proteoglycans consisting of a Hyp-rich core protein and large arabinogalactan (AG) moieties (Fincher et al., 1983;Nothnagel, 1997). Although there are many molecular species of AGP differentiated by their core proteins, the AG moieties commonly comprise b-1,3-galactan main chains and b-1,6-galactan side chains, to which L-Ara and other auxiliary sugars, such as GlcA, 4-O-methyl-GlcA, L-Fuc, L-Rha, and Xyl, are attached (Fincher et al., 1983;Nothnagel, 1997;Seifert and Roberts, 2007). A commercial product of AGPs prepared from the acacia (Acacia senegal) tree is known as gum arabic and utilized as a food stabilizer. In the Japanese herbal remedy Juzen-Taiho-To, AGs from Astragalus membranaceus are the active ingredient (Majewska-Sawka and Nothnagel, 2000;Kiyohara et al., 2002). In intact plants, AGPs are implicated in various physiological events and serve as extracellular constituents and signaling molecules. For instance, an AGP from stylar transmitting tissue attracts pollen tub...

A gene encoding exo-beta-(1-->3)-galactanase from Irpex lacteus was cloned by reverse transcriptase-PCR. The deduced amino acid sequence showed high similarity with exo-beta-(1-->3)-galactanases from other sources. The molecular mass of the mature form was calculated to be 45,520 Da. The gene product expressed in Pichia pastoris specifically hydrolyzed beta-(1-->3)-galactooligosaccharides, as did other exo-beta-(1-->3)-galactanases. The recombinant enzyme showed high activity toward arabinogalactan-proteins (AGPs) from radish as well as beta-(1-->3)-galactan. Product analysis revealed that the enzyme released beta-(1-->6)-galactobiose, beta-(1-->6)-galactotriose, and alpha-L-arabinofuranosyl-(1-->3)-beta-galactosyl-(1-->6)-galactose together with Gal from beta-(1-->3)-galactans attached with and without beta-(1-->6)-galactosyl branches prepared from acacia gum. These results indicate that the exo-beta-(1-->3)-galactanase from I. lacteus efficiently hydrolyzes beta-(1-->3)-galactan main chains of AGPs by bypassing beta-(1-->6)-galactosyl side chains.

Arabinogalactan proteins are proteoglycans found on the cell surface and in the cell walls of higher plants. The carbohydrate moieties of most arabinogalactan proteins are composed of ␤-1,3-galactan main chains and ␤-1,6-galactan side chains, to which other auxiliary sugars are attached. For the present study, an endo-␤-1,3-galactanase, designated FvEn3GAL, was first purified and cloned from winter mushroom Flammulina velutipes. The enzyme specifically hydrolyzed ␤-1,3-galactan, but did not act on ␤-1,3-glucan, ␤-1,3:1,4-glucan, xyloglucan, and agarose. It released various ␤-1,3-galactooligosaccharides together with Gal from ␤-1,3-galactohexaose in the early phase of the reaction, demonstrating that it acts on ␤-1,3-galactan in an endo-fashion. Phylogenetic analysis revealed that FvEn3GAL is member of a novel subgroup distinct from known glycoside hydrolases such as endo-␤-1,3-glucanase and endo-␤-1,3:1,4-glucanase in glycoside hydrolase family 16. Point mutations replacing the putative catalytic Glu residues conserved for enzymes in this family with Asp abolished activity. These results indicate that FvEn3GAL is a highly specific glycoside hydrolase 16 endo-␤-1,3-galactanase.

Through the analysis of mutants and recombinant enzymes expressed in Pichia yeast, an Arabidopsis β-l-arabinopyranosidase and α-d-galactosidases are shown to be responsible for the hydrolysis of β-l-arabinopyranosyl residues.

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