Identification and expression analysis of arabinogalactan protein genes in cotton reveal the function of GhAGP15 in Verticillium dahliae resistance.

Verticillium wilt, primarily caused by the fungal pathogen Verticillium dahliae, is a serious disease in cotton. Arabinogalactan proteins (AGPs), a class of hydroxyproline-rich glycoproteins, have been widely implicated in plant growth and environmental adaptation. The purpose of this study is to identify and characterize AGP members in cotton plants and explore their roles in responding to environmental stressors. In total, 65 GhAGP members were identified in upland cotton (Gossypium hirsutum), along with 43, 35, and 37 AGP members that were also identified in G. barbadense, G. arboreum, and G. raimondii, respectively. According to gene structure and protein domains analysis, GhAGP genes in upland cotton are highly conserved. Meanwhile, tandem duplication events have occurred frequently throughout cotton's evolutionary history. Expression analysis showed that GhAGP genes were widely expressed during growth and development and in response to abiotic stressors. Many cis-elements related to hormonal responses and environmental stressors were detected in GhAGP promoter regions. GhAGP genes participate in responding to cold, drought, and salt stress, and were sensitive to ET signaling. Furthermore, the expression level of GhAGP15 was elevated during V. dahliae infection and resistance against V. dahliae in upland cotton was significantly weakened by silencing GhAGP15 using a virus-induced gene silencing (VIGS) approach. Our results further suggest that the function of GhAGP15 in V. dahliae resistance might be involved in regulation of the JA, SA, and reactive oxygen species (ROS) pathways. The comprehensive analysis of AGP genes in cotton performed in this study provides a basic framework for further functional research of these genes.

Search for evolutionary roots of land plant arabinogalactan-proteins in charophytes: presence of a rhamnogalactan-protein in Spirogyra pratensis (Zygnematophyceae).

Charophyte green algae (CGA) are assigned to be the closest relatives of land plants and therefore enlighten processes in the colonization of terrestrial habitats. For the transition from water to land, plants needed significant physiological and structural changes, as well as with regard to cell wall composition. Sequential extraction of cell walls of Nitellopsis obtusa (Charophyceae) and Spirogyra pratensis (Zygnematophyceae) offered a comparative overview on cell wall composition of late branching CGA. Because arabinogalactan-proteins (AGPs) are considered common for all land plant cell walls, we were interested in whether these special glycoproteins are present in CGA. Therefore, we investigated both species with regard to characteristic features of AGPs. In the cell wall of Nitellopsis, no hydroxyproline was present and no AGP was precipitable with the ß-glucosyl Yariv's reagent (ßGlcY). By contrast, ßGlcY precipitation of the water-soluble cell wall fraction of Spirogyra yielded a glycoprotein fraction rich in hydroxyproline, indicating the presence of AGPs. Putative AGPs in the cell walls of non-conjugating Spirogyra filaments, especially in the area of transverse walls, were detected by staining with ßGlcY. Labelling increased strongly in generative growth stages, especially during zygospore development. Investigations of the fine structure of the glycan part of ßGlcY-precipitated molecules revealed that the galactan backbone resembled that of AGPs with 1,3- 1,6- and 1,3,6-linked Galp moieties. Araf was present only in small amounts and the terminating sugars consisted predominantly of pyranosidic terminal and 1,3-linked rhamnose residues. We introduce the term 'rhamnogalactan-protein' for this special AGP-modification present in S. pratensis.

Human AGR2 Deficiency Causes Mucus Barrier Dysfunction and Infantile Inflammatory Bowel Disease.

BACKGROUND & AIMS: The gastrointestinal epithelium plays a crucial role in maintaining homeostasis with the gut microbiome. Mucins are essential for intestinal barrier function and serve as a scaffold for antimicrobial factors. Mucin 2 (MUC2) is the major intestinal gel-forming mucin produced predominantly by goblet cells. Goblet cells express anterior gradient 2 (AGR2), a protein disulfide isomerase that is crucial for proper processing of gel-forming mucins. Here, we investigated 2 siblings who presented with severe infantile-onset inflammatory bowel disease. METHODS: We performed whole-genome sequencing to identify candidate variants. We quantified goblet cell numbers using H&E histology and investigated the expression of gel-forming mucins, stress markers, and goblet cell markers using immunohistochemistry. AGR2-MUC2 binding was evaluated using co-immunoprecipitation. Endoplasmic reticulum (ER) stress regulatory function of mutant AGR2 was examined by expression studies in Human Embryonic Kidney 293T (HEK293T) using tunicamycin to induce ER stress. RESULTS: Both affected siblings were homozygous for a missense variant in AGR2. Patient biopsy specimens showed reduced goblet cells; depletion of MUC2, MUC5AC, and MUC6; up-regulation of AGR2; and increased ER stress. The mutant AGR2 showed reduced capacity to bind MUC2 and alleviate tunicamycin-induced ER stress. CONCLUSIONS: Phenotype-genotype segregation, functional experiments, and the striking similarity of the human phenotype to AGR2-/- mouse models suggest that the AGR2 missense variant is pathogenic. The Mendelian deficiency of AGR2, termed "Enteropathy caused by AGR2 deficiency, Goblet cell Loss, and ER Stress" (EAGLES), results in a mucus barrier defect, the inability to mitigate ER stress, and causes infantile-onset inflammatory bowel disease.

AGR2-AGR3 hetero-oligomeric complexes: Identification and characterization.

In this paper we compare electrochemical behavior of two homolog proteins, namely anterior gradient 2 (AGR2) and anterior gradient 3 (AGR3), playing an important role in cancer cell biology. The slight variation in their protein structures has an impact on protein adsorption and orientation at charged surface and also enables AGR2 and AGR3 to form heterocomplexes. We confirm interaction between AGR2 and AGR3 (i) in vitro by immunochemical and constant current chronopotentiometric stripping (CPS) analysis and (ii) in vivo by bioluminescence resonance energy transfer (BRET) assay. Mutation of AGR2 in dimerization domain (E60A) prevents development of wild type AGR2 dimers and also negatively affects interaction with wild type AGR3 as shown by CPS analysis. Beside new information about AGR2 and AGR3 protein including their joint interaction, our work introduces possible applications of CPS in bioanalysis of protein complexes, including those relatively unstable, but important in the cancer research.

Arabinogalactan-proteins from non-coniferous gymnosperms have unusual structural features.

Arabinogalactan-proteins (AGPs), important signalling molecules of the plant cell wall, are structurally extensively investigated in angiosperms, but information on AGPs in gymnosperms is still limited. We characterized AGPs from the gymnosperms Ginkgo biloba, Ephedra distachya, Encephalartos longifolius and Cycas revoluta. The protein contents are comparable to that of angiosperm AGPs. Hydroxyproline is the site of linking the carbohydrate part and was detected in all AGPs with highest concentration in Cycas AGP (1.1 % of the AGP). Interestingly, with the exception of Cycas, all AGPs contained the monosaccharide 3-O-methylrhamnose not present in angiosperm polysaccharides. The carbohydrate moieties of Cycas and Ephredra showed the main components 1,3,6-linked galactose and terminal arabinose typical of angiosperm AGPs, whereas that of Ginkgo AGP was unique with 1,4-linked galactose as dominant structural element. Bioinformatic search for glycosyltransferases in Ginkgo genome also revealed a lower number of galactosyltransferases responsible for biosynthesis of the 1,3-Gal/1,6-Gal AGP backbone.

Characterization of pectin-xylan-glucan-arabinogalactan proteins complex from Siberian fir Abies sibirica Ledeb.

Polysaccharide ASK was isolated from the Abies sibirica foliage by extraction with an aqueous KOH solution. ASK was shown to contain structurally different polymers such as arabinoglucuronoxylans, xyloglucans, glucomannans, arabinogalactan-proteins (AGPs). The pectic polysaccharides were also found in the alkaline extract of ASK and were represented by regions of homogalactorunan and rhamnogalactouronan-I whose side sugar chains were made up chiefly of highly branched 1,5-α-l-arabinan. The potential couplings between those polysaccharides were examined. Our studies showed simultaneous elution of pectin, xyloglucans, arabinoglucuronoxylans and AGPs, indicating that pectins can be covalently bound to the other cell-wall polysaccharides. NMR spectroscopy results revealed that the polysaccharides obtained by ion-exchange chromatography almost had no free reducing ends. These findings corroborate the conclusion that pectin, AGPs, glucan and xylan are bound together. The existence of the covalently bound complex of pectin-xylan-xyloglucan-AGP is suggested herein. Pectin and xylan are hypothesized to be covalently linked through RG-I regions.

Conjugation reaction with ferulic acid boosts the antioxidant property of arabinogalactan-protein and enhances its ability to form complex with ß-lactoglobulin.

Ferulic acid was chemically grafted onto the arabinogalactan protein of Aegle marmelos fruit gum using 1,1'-carbonyldiimidazole as coupling reagent. Thus, grafted polysaccharides with different degrees of substitution were prepared and then characterized by gas chromatography/mass spectrometry, size exclusion chromatography, and ultraviolet-visible, infra-red, and nuclear magnetic resonance spectroscopic investigations. Fluorescence spectroscopic investigation showed hydrophobic microdomain formation in grafted polymers. The antioxidant activities of the derivatives, as determined by the 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl radical assay, were strong and increases with increasing the degree of feruloylation. Compared to parental arabinogalactan protein (K = 2.38 × 106 M-1), these grafted polymers bind more strongly with ß-lactoglobulin (K = 11.4 × 106 M-1 and 8.19 × 106 M-1). Given that gum polysaccharides are valuable component in functional foods, synthesis of antioxidative graft polymer possessing good compatibility with ß-lactoglobulin may have important implication.

An ultrasonic-extracted arabinoglucan from Tamarindus indica L. pulp: A study on molecular and structural characterizations.

In this study, an ultrasonic-extracted polysaccharide (nCPTP-55) was obtained with the highest yield (61.08%, w/w) from tamarind pulp, which consisted chiefly of total sugar (85.98%, w/w) with few protein (2.10%, w/w). Monosaccharide analysis showed nCPTP-55 was mainly composed of arabinose (39.19 mol%) and glucose (50.48 mol%) with negligible GlcA (2.05 mol%), indicating the neutral nature of nCPTP-55, which was further elucidated structurally via GC-MS and NMR, i.e., an arabinoglucan composed of →3)-ß-D-Glcp-(1→ backbone with only T-α-L-Araf-(1→ branched at O-4 (27.82%) and O-6 (39.99%), resulting in relatively high A/G ratio (0.68-0.70). Based on MM2 minimized energy, the 3D schematic structures of nCPTP-55 could be considered as structural basis for its conformational behavior, which was preliminarily estimated via HPSEC-MALLS as between compact sphere and loosely hyper-branched chain (ρ = 0.84). Therefore, the relationship between molecular structure and conformational behavior was basically established for nCPTP-55, which was in a bid to have a better knowledge of its structure-property and structure-bioactivity relationships potentially required for more applications in food, cosmetic and pharmaceutical fields.

Structural characteristics of oxalate-soluble polysaccharides from Norway spruce (Picea abies) foliage.

Structurally different polymers were derived from Picea abies foliage by successive extraction with water (PAW), HCl solution (PAA) and (NH4)2C2O4 solution (PAO). The P. abies foliage was found to contain basically low-methoxyl pectin extractable with an (NH4)2C2O4 solution. PAW was shown to comprise primarily arabinogalactan-proteins (AGPs); PAA was composed of mixed AGPs and pectic polysaccharides, with the latter prevailing; and polysaccharide PAO isolated in the highest yield included chiefly pectic polysaccharides. The major constituents of PAO were low-methoxyl and low-acetylated 1,4-α-d-galacturonan and partially acetylated RG-I. The sugar side chains of RG-I contained chiefly highly branched 1,5-α-l-arabinan and arabinogalactan type I as a minor constituent. RG-I whose side chains had 1,5-α-l-arabinan represented short regions alternating with non-acetylated and unmethylesterified galacturonan regions. In addition to pectins, polysaccharide PAO contained AGPs, xylanes and glucomannans, indicating that these polysaccharides are in an intimate interaction.

Contributions to Arabinogalactan Protein Analysis.

Arabinogalactan proteins (AGPs) are important plant proteoglycans involved in many development processes. In roots, AGPs occur in the cell wall of root cells and root cap-derived cells as well as in the secreted mucilage. Detection, localization , and quantification techniques are therefore essential to unravel the AGP diversity of structures and functions. This chapter details root-adapted immunocytochemical methods using monoclonal antibodies, and a collection of biochemical analysis protocols using ß-D-glucosyl Yariv reagent for comprehensive AGP characterization.

Bioinformatic Identification of Plant Hydroxyproline-Rich Glycoproteins.

Hydroxyproline-rich glycoproteins (HRGPs) are a superfamily of plant cell wall proteins that function in diverse aspects of plant growth and development. This superfamily consists of three members: arabinogalactan-proteins (AGPs), extensins (EXTs), and proline-rich proteins (PRPs). Hybrid and chimeric HRGPs also exist. A bioinformatic software program, BIO OHIO 2.0, was developed to expedite the genome-wide identification and classification of AGPs, EXTs, and PRPs based on characteristic HRGP motifs and biased amino acid compositions. This chapter explains the principles of identifying HRGPs and provides a stepwise tutorial for using the BIO OHIO 2.0 program with genomic/proteomic data. Here, as an example, the genome/proteome of the common bean (Phaseolus vulgaris) is analyzed using the BIO OHIO 2.0 program to identify and characterize its set of HRGPs.

Properties of arabinogalactan-proteins in European pear (Pyrus communis L.) fruits.

Arabinogalactans (AGs) and arabinogalactan-proteins (AGPs) were partially purified from an extract of fruits of the European pear (Pyrus communis L.) by DEAE-cellulose ion-exchange and Sepharose 6B gel-filtration chromatography. Among 7 AG(P)-containing fractions, a neutral AGP (SE-1) was confirmed to be highly purified (Mr 67,000) and rich in L-Ara and Gal; this fraction included a small amount (2.6%, w/w) of protein and showed the highest reactivity forming precipitate with ß-Glc Yariv reagent among the 7 fractions, the intensity of which was comparable to that of gum arabic, a standard AGP. Another accompanying minor low-Mr neutral AGP (SE-2; Mr approx. 7200) still contained other polysaccharide (starch fragments) and did not show Yariv reactivity. The carbohydrate moieties of SE-1 consisted of consecutive (1 → 3)-linked ß-galactosyl backbone chains substituted with side chains of (1 → 6)-linked ß-galactosyl residues at O-6, to which mainly single α-l-arabinofuranosyl residues were attached through O-3. This structural feature was also observed for SE-2. Successive digestion of SE-1 with α-l-arabinofuranosidase and exo-ß-(1 → 3)-galactanase with the aid of endo-ß-(1 → 3)-galactanase released most (more than 98%, w/w) of the carbohydrate moieties as low-Mr fragments. These consisted of free L-Ara and Gal, and a series of ß-(1 → 6)-galactooligosaccharides with degree of polymerization (dp) up to at least 17, indicative of attachment of (1 → 6)-linked ß-galactosyl side chains of varying length along the (1 → 3)-linked ß-galactosyl backbone chains.

Effect of arabinogalactan protein complex content on emulsification performance of gum arabic.

The emulsification properties of the standard (STD), matured (EM2 and EM10) and fractionated gum arabic samples via phase separation induced molecular fractionation were investigated to find out how the content of arabinogalactan protein (AGP) complex affects the resulting emulsion properties. Phase separation and the accompanying molecular fractionation were induced by mixing with different hydrocolloids including hyaluronan (HA), carboxymethyl cellulose (CMC), and maltodextrin (MD). Increase of AGP content from 11 to 28% resulted in the formation of emulsions with relatively smaller droplet sizes and better stability. Further increase in the AGP content to 41% resulted in the formation of emulsions with larger droplets. In spite of the larger droplets sizes, these emulsions were extremely stable. In addition, the emulsions prepared with GA higher AGP content better stability in the presence of ethanol. The results indicate that AGP content plays a vital role in emulsion stability and droplet size.

Molecular, rheological and physicochemical characterisation of puka gum, an arabinogalactan-protein extracted from the Meryta sinclairii tree.

A water-soluble polysaccharide (type II arabinogalactan-protein) extracted from the gum exudate of the native New Zealand puka tree (Meryta sinclairii), was characterised for its molecular, rheological and physicochemical properties. In 0.1 M NaCl, the weight average molecular weight (Mw) of puka gum is 5.9 × 106 Da with an RMS radius of 56 nm and z-average hydrodynamic radius of 79 nm. The intrinsic viscosity of the polysaccharide is 57 ml/g with a coil overlap concentration 15% w/w. Together, the shape factor, p, of 0.70 (exponent of RMS radius vs. hydrodynamic radius), Smidsrød-Haug's stiffness parameter B of 0.031 and Mark-Houwink exponent α of 0.375 indicate that the polysaccharide adopts a spherical conformation in solution, similar to gum arabic. The pKa is 1.8. The polysaccharide exhibits a Newtonian to shear-thinning behaviour from 0.2 to 25% w/w. Viscosity of the polysaccharide (1 s-1) decreases with decreasing concentration, increasing temperature, ionic strength, and at acidic pH.

Evolution Analysis of the Fasciclin-Like Arabinogalactan Proteins in Plants Shows Variable Fasciclin-AGP Domain Constitutions.

The fasciclin-like arabinogalactan proteins (FLAs) play important roles in plant development and adaptation to the environment. FLAs contain both fasciclin domains and arabinogalactan protein (AGP) regions, which have been identified in several plants. The evolutionary history of this gene family in plants is still undiscovered. In this study, we identified the FLA gene family in 13 plant species covering major lineages of plants using bioinformatics methods. A total of 246 FLA genes are identified with gene copy numbers ranging from one (Chondrus crispus) to 49 (Populus trichocarpa). These FLAs are classified into seven groups, mainly based on the phylogenetic analysis of plant FLAs. All FLAs in land plants contain one or two fasciclin domains, while in algae, several FLAs contain four or six fasciclin domains. It has been proposed that there was a divergence event, represented by the reduced number of fasciclin domains from algae to land plants in evolutionary history. Furthermore, introns in FLA genes are lost during plant evolution, especially from green algae to land plants. Moreover, it is found that gene duplication events, including segmental and tandem duplications are essential for the expansion of FLA gene families. The duplicated gene pairs in FLA gene family mainly evolve under purifying selection. Our findings give insight into the origin and expansion of the FLA gene family and help us understand their functions during the process of evolution.

Arabinogalactan-proteins in spore-producing land plants.

Arabinogalactan-proteins (AGPs) are proteoglycans of the extracellular matrix of plants that were first isolated and described in the 1970s. Today, the consensus is that the following features are regarded as typical for these molecules: In contrast to broad knowledge on AGPs in seed plants, insight in occurrence and structure of AGPs in spore-producing land plants (bryophytes, lycophytes and monilophytes) is very limited, although these plants are the closest living relatives to seed plants. In general, understanding of cell wall evolution is incomplete due to limited knowledge of cell wall structure of non-flowering plants. In this review, current knowledge on AGPs of mosses, clubmosses and ferns is summarized, possible functions are discussed and suggestions for future investigations are given.

Deconstructing Wine Grape Cell Walls with Enzymes During Winemaking: New Insights from Glycan Microarray Technology.

Enzyme-aid maceration is carried out in most modern winemaking industries with a range of positive impacts on wine production. However, inconsistencies in enzyme efficiency are an issue complicated by unclear targets (limited information available on berry cell wall architecture of different cultivars) and the complex wine environment (i.e., fermenting must). Recent studies have been performed to develop a clearer picture of grape cell wall structures, maceration effects, and interactions between important wine compounds and grape-derived polysaccharides. This review highlights critically important recent studies on grape berry cell wall changes during ripening, the importance of enzymes during maceration (skin contact phase) and deconstruction processes that occur during alcoholic fermentation. The novelty of the Comprehensive Microarray Polymer Profiling (CoMPP) technique using cell wall probes (e.g., antibodies) as a method for following cell wall derived polymers during different biological and biotechnological processes is discussed. Recent studies, using CoMPP together with classical analytical methods, confirmed the developmental pattern of berry cell wall changes (at the polymer level) during grape ripening. This innovative technique were also used to track enzyme-assisted depectination of grape skins during wine fermentation and determine how this influence the release of wine favourable compounds. Furthermore, polysaccharides (e.g., arabinogalactan proteins) present in the final wine could be identified. Overall, CoMPP provides a much more enriched series of datasets compared to traditional approaches. Novel insights and future studies investigating grape cell wall and polyphenol interactions, and the tailoring of enzyme cocktails for consistent, effective and "customized" winemaking is advanced and discussed.

Chemical characterization and complement modulating activities of an arabinogalactan-protein-rich fraction from an aqueous extract of avocado leaves.

The aim of this study was to chemically characterize an arabinogalactan-protein-rich fraction (FRAGP) obtained from an aqueous extract of avocado leaves and investigate its effects on the classical pathway of the complement system. The FRAGP contained 4.6% ±â€¯1.8%, 22.5% ±â€¯4.9%, and 76.7% ±â€¯8.8% of total protein, arabinogalactan-protein, and carbohydrates, respectively. Arabinose and galactose were the main monosaccharide constituents. FT-IR and NMR data, together with linkage analyses, indicated the presence of a structure that included a (1 → 3)-linked ß-D-Galp main chain, mainly substituted at O-6 by Gal and Ara residues, which was characteristic of a type II arabinogalactan. The effect of FRAGP on the classical pathway of complement system was examined by a hemolytic fixation test and comparing with heparin, which was used as a control for inhibition. With pre-incubation, the IC50 of FRAGP was 1.90 ±â€¯1.1 µg/mL, which was similar to that of heparin (IC50 = 2.90 ±â€¯0.3 µg/mL). Without pre-incubation, the IC50 values were 18.6 ±â€¯3.7 and 8.0 ±â€¯4.1 µg/mL for FRAGP and heparin, respectively. Collectively, these results suggested that FRAGP has an inhibitory effect on the classical pathway of the complement system.

Integrin α4ß7 switches its ligand specificity via distinct conformer-specific activation.

Chemokine (C-C motif) ligand 25 (CCL25) and C-X-C motif chemokine 10 (CXCL10) induce the ligand-specific activation of integrin α4ß7 to mediate the selective adhesion of lymphocytes to mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) or vascular cell adhesion molecule-1 (VCAM-1). However, the mechanism underlying the selective binding of different ligands by α4ß7 remains obscure. In this study, we demonstrate that CCL25 and CXCL10 induce distinct active conformers of α4ß7 with a high affinity for either MAdCAM-1 or VCAM-1. Single-cell force measurements show that CCL25 increases the affinity of α4ß7 for MAdCAM-1 but decreases its affinity for VCAM-1, whereas CXCL10 has the opposite effect. Structurally, CCL25 induces a more extended active conformation of α4ß7 compared with CXCL10-activated integrin. These two distinct intermediate open α4ß7 conformers selectively bind to MAdCAM-1 or VCAM-1 by distinguishing their immunoglobulin domain 2. Notably, Mn2+ fully opens α4ß7 with a high affinity for both ligands. Thus, integrin α4ß7 adopts different active conformations to switch its ligand-binding specificity.

Arabinogalactan proteins and their sugar chains: functions in plant reproduction, research methods, and biosynthesis.

The arabinogalactan protein (AGP) family is one of the most complex protein families and is ubiquitous in the plant kingdom. Moreover, it has been demonstrated to play various roles during plant reproduction. A typical AGP contains a hydroxyproline-rich core protein with high heterogeneity and varying numbers of polysaccharide side chains. However, the functions of the polysaccharide components (i.e. AG sugar chains) remain largely unknown due to the general difficulties associated with studying sugar chains in glycobiology. In recent years, methodological breakthroughs have resulted in substantial progress in AGP research. Here, we summarise the multiple roles of AGPs during plant gametophyte development and male-female communication, with a focus on recent advances. In addition, we discuss the analytical tools used in AGP research, and the biosynthesis and function of AG sugar chains. A comprehensive understanding of the AGP family will help clarify the mechanisms precisely controlling reproductive processes.