β-Galactosyl Yariv Reagent Binds to the β-1,3-Galactan of Arabinogalactan Proteins    

Abstract

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...

Abscisic Acid- and Stress-Induced Highly Proline-Rich Glycoproteins Regulate Root Growth in Rice

“…5E; Supplemental Table S3). The differential sugar composition could be responsible for the failure of RePRPs to interact with the b-Glc Yariv reagent, which specifically binds b-1, 3-galactan chains (Kitazawa et al, 2013). Thus, RePRPs are a novel family of HRGPs but not typical AGPs, according to the distinct Pro repeat patterns, failure to interact with b-Glc Yariv, and diverse sugar components in glycan linked to Hyp residues.…”

“…In Pro-rich glycoproteins, the significant change in M r is found in the AGP subfamily that carries multiple large AG glycans linked to Hyp and specifically interacts with the b-Glc Yariv reagent (Yariv et al, 1967;Seifert and Roberts, 2007;Kitazawa et al, 2013). Due to the presence of similar discontinuous Pro repeats in RePRPs, the b-Glc Yariv reagent was used to verify whether rice RePRPs are new members of the AGP subfamily.…”

Nanospherical arabinogalactan proteins are a key component of the high-strength adhesive secreted by English ivy

“…To test whether AGPs are contained in the ivy nanoparticles, Yariv phenylglycoside dye, a reagent also called β-glucosyl Yariv (β-GlcY), which selectively binds to the AGPs via recognizing both given protein moieties and β-1,3-galactan chains with greater than five residues (24,30,(32)(33)(34)(35), was applied for identification. As shown in Fig.…”

Root exudate ofSolanum tuberosumis enriched in galactose-containing molecules and impacts the growth ofPectobacterium atrosepticum

“…On the contrary, a slight decrease in bacterial growth was observed when the bacteria were incubated with b-D-glucosyl Yariv-treated exudates. It should be noted that it is difficult to estimate the proportion of AGPs present in root exudates that are effectively bound to b-D-glucosyl Yariv (Kitazawa et al, 2013). Furthermore, we cannot rule out the possibility that b-D-glucosyl Yariv-bound AGPs remained biologically active.…”

“…To check whether AGPs present in root exudates could affect bacterial growth, exudates were incubated overnight with 5 mg mL À1 b-D-glucosyl Yariv reagent and added to the growth medium. The b-D-glucosyl Yariv reagent binds and precipitates AGPs, thus interfering with their function (Yariv et al, 1967;Sardar et al, 2006;Kitazawa et al, 2013;Marzec et al, 2015). When exudate treated with b-D-glucosyl Yariv reagent was added to the growth medium, biomass production of P. atrosepticum was only slightly reduced.…”