Structural Basis of the Function of Yariv Reagent—An Important Tool to Study Arabinogalactan Proteins

Arabinogalactan proteins are very abundant, heavily glycosylated plant cell wall proteins. They are intensively studied because of their crucial role in plant development as well as their function in plant defence. Research of these biomacromolecules is complicated by the lack of tools for their analysis and characterisation due to their extreme heterogeneity. One of the few available tools for detection, isolation, characterisation, and functional studies of arabinogalactan proteins is Yariv reagents. Yariv reagent is a synthetic aromatic glycoconjugate originally prepared as an antigen for immunization. Later, it was found that this compound can precipitate arabinogalactan proteins, namely, their ß-D-(1→3)-galactan structures. Even though this compound has been intensively used for decades, the structural basis of arabinogalactan protein precipitation by Yariv is not known. Multiple biophysical studies have been published, but none of them attempted to elucidate the three-dimensional structure of the Yariv-galactan complex. Here we use a series of molecular dynamics simulations of systems containing one or multiple molecules of ß-D-galactosyl Yariv reagent with or without oligo ß-D-(1→3)-galactan to predict the structure of the complex. According to our model of Yariv-galactan complexes, Yariv reagent forms stacked oligomers stabilized by π-π and CH/π interactions. These oligomers may contain irregularities. Galactan structures crosslink these Yariv oligomers. The results were compared with studies in literature.

Investigations of changes in the arabinogalactan proteins (AGPs) structure, size and composition during the fruit ripening process

Arabinogalactan proteins (AGPs) are ubiquitous cell wall and plasma membrane components and are characterised by extensive glycosylation and heterogeneity of their carbohydrate and protein units. The aim of the study was to evaluate the structural features of AGPs present in apple fruits at different stages of the ripening process. AGPs were extracted using the Yariv reagent and examined using SDS-PAGE, immunoblotting, FT-IR, and AFM. In situ analysis, immunofluorescence (CLSM) and immunogold-labelling (TEM), were performed. We demonstrated that AGPs were indeed present in apple fruits at the different stages of the ripening process. The changes in the amount (1.52–2.08 mg g−1), diameter (152.73–75.05 nm), molecular mass (50–250 kDa), and distribution in the cell of AGPs demonstrate their variable presence and changeable structure during the ripening process. We propose specific wavenumbers, i.e. 1265 cm−1, 1117 cm−1, and 960 cm−1, which could be assigned to AGPs. The immunofluorescence and immunogold-labelling results indicate that the JIM13 antibody is the most characteristic for AGPs in apple fruits. This study quantitatively demonstrated for the first time that AGP accumulation occurs in ripe fruits, which is supported by the highest AGPs content, the highest molecular mass, and the appearance of a specific distribution pattern at the cellular level.

The Chirality of Yariv Reagent Aggregates Correlates with AGP–binding Ability

Yariv reagents are glycosylated triphenylazo dyes, some of which bind to the polysaccharide component of arabinogalactan proteins (AGPs), proteoglycans found in plant cell walls. However, the exact reason for the selectivity in the presence/absence of AGP binding ability among Yarivs remains unknown. The Yariv reagents are known to form supramolecular aggregates in solution. We use circular dichroism to show that the Yariv reagent aggregates possess helical chirality, and the AGP binding ability of the Yariv reagents is correlated to its helical chirality.

The Chirality of Yariv Reagent Aggregates Correlates with AGP–binding Ability

Yariv reagents are glycosylated triphenylazo dyes, some of which bind to the polysaccharide component of arabinogalactan proteins (AGPs), proteoglycans found in plant cell walls. However, the exact reason for the selectivity in the presence/absence of AGP binding ability among Yarivs remains unknown. The Yariv reagents are known to form supramolecular aggregates in solution. We use circular dichroism to show that the Yariv reagent aggregates possess helical chirality, and the AGP binding ability of the Yariv reagents is correlated to its helical chirality.

Structure of the exudate gum from Meryta sinclairii

A gum that exudes from the wounded trunk of the New Zealand native tree Meryta sinclairii has been isolated. The gum was completely precipitated by the β-glucosyl Yariv reagent and was thus determined to be an arabinogalactan-protein (AGP). It contained >95% w/w carbohydrate and only 2% w/w protein with a high level of hydroxyproline. SEC-MALLS showed that the gum had a weight-average molecular weight of 4.45×106Da compared with 6.02×105Da for gum arabic. Constituent sugar and linkage analyses were consistent with polymers comprised of a highly branched backbone of 1,3-linked galactopyranosyl (Galp) residues, with side-chains made up of arabinofuranose- (Araf) containing oligosaccharides, terminated variously by rhamnopyranosyl (Rhap), arabinopyranosyl (Arap), Galp and glucuronopyranosyl (GlcpA) residues. Analysis by one-dimensional and two-dimensional 1H and 13C NMR experiments confirmed the linkage analyses. The structure of the gum is discussed in comparison with the structure of gum arabic and other AGPs. © 2003 Elsevier Science Ltd. All rights reserved.

Structure of the exudate gum from Meryta sinclairii

A gum that exudes from the wounded trunk of the New Zealand native tree Meryta sinclairii has been isolated. The gum was completely precipitated by the β-glucosyl Yariv reagent and was thus determined to be an arabinogalactan-protein (AGP). It contained >95% w/w carbohydrate and only 2% w/w protein with a high level of hydroxyproline. SEC-MALLS showed that the gum had a weight-average molecular weight of 4.45×106Da compared with 6.02×105Da for gum arabic. Constituent sugar and linkage analyses were consistent with polymers comprised of a highly branched backbone of 1,3-linked galactopyranosyl (Galp) residues, with side-chains made up of arabinofuranose- (Araf) containing oligosaccharides, terminated variously by rhamnopyranosyl (Rhap), arabinopyranosyl (Arap), Galp and glucuronopyranosyl (GlcpA) residues. Analysis by one-dimensional and two-dimensional 1H and 13C NMR experiments confirmed the linkage analyses. The structure of the gum is discussed in comparison with the structure of gum arabic and other AGPs. © 2003 Elsevier Science Ltd. All rights reserved.

An Improved Protocol for the Synthesis and Purification of Yariv Reagents

Yariv reagents are widely used tools to study plant proteoglycans known as the Arabinogalactan proteins (AGPs). Despite widespread use of the Yariv reagent in the study of AGPs and several reports of synthetic protocols, there remains a lack of a simple procedure to obtain pure Yariv reagents. We report our optimized protocols to address the purification issues faced upon synthesis of Yariv reagents. Additionally, we report challenges that make characterization difficult such as peak broadening in NMR due to Yariv-water interactions. We also show ways of processing Yariv reagents to overcome the characterization issues caused by peak broadening.

An Improved Protocol for the Synthesis and Purification of Yariv Reagents

Yariv reagents are widely used tools to study plant proteoglycans known as the Arabinogalactan proteins (AGPs). Despite widespread use of the Yariv reagent in the study of AGPs and several reports of synthetic protocols, there remains a lack of a simple procedure to obtain pure Yariv reagents. We report our optimized protocols to address the purification issues faced upon synthesis of Yariv reagents. Additionally, we report challenges that make characterization difficult such as peak broadening in NMR due to Yariv-water interactions. We also show ways of processing Yariv reagents to overcome the characterization issues caused by peak broadening.