Cloning and Characterization of AtRGP1 : A Reversibly Autoglycosylated Arabidopsis Protein Implicated in Cell Wall Biosynthesis

A reversibly glycosylated polypeptide from pea (Pisum sativum) is thought to have a role in the biosynthesis of hemicellulosic polysaccharides. We have investigated this hypothesis by isolating a cDNA clone encoding a homolog of Arabidopsis thaliana, Reversibly Glycosylated Polypeptide-1 (AtRGP1), and preparing antibodies against the protein encoded by this gene. Polyclonal antibodies detect homologs in both dicot and monocot species. The patterns of expression and intracellular localization of the protein were examined. AtRGP1 protein and RNA concentration are highest in roots and suspension-cultured cells. Localization of the protein shows it to be mostly soluble but also peripherally associated with membranes. We confirmed that AtRGP1 produced in Escherichia coli could be reversibly glycosylated using UDP-glucose and UDP-galactose as substrates. Possible sites for UDP-sugar binding and glycosylation are discussed. Our results are consistent with a role for this reversibly glycosylated polypeptide in cell wall biosynthesis, although its precise role is still unknown.The primary cell wall of dicot plants is laid down by young cells prior to the cessation of elongation and secondary wall deposition. Making up to 90% of the cell''s dry weight, the extracellular matrix is important for many processes, including morphogenesis, growth, disease resistance, recognition, signaling, digestibility, nutrition, and decay. The composition of the cell wall has been extensively described (Bacic et al., 1988; Levy and Staehelin, 1992; Zablackis et al., 1995), and yet many questions remain unanswered regarding the synthesis and interaction of these components to provide cells with a functional wall (Carpita and Gibeaut, 1993; Carpita et al., 1996).Heteropolysaccharide biosynthesis can be divided into four steps: (a) chain or backbone initiation, (b) elongation, (c) side-chain addition, and (d) termination and extracellular deposition (Waldron and Brett, 1985). The similarity between various polysaccharide backbones leads to the prediction that the synthesizing machinery would be conserved between them. For example, the backbone of xyloglucan polymers, β-1,4 glucan, can be synthesized independently of or concurrently with side-chain addition (Campbell et al., 1988; White et al., 1993), and this polymer and the chains that make up cellulose are identical. The later addition of side chains to xyloglucan are catalyzed by specific transferases (Kleene and Berger, 1993) such as xylosyltransferase (Campbell et al., 1988), galactosyltransferase, and fucosyltransferase (Faïk et al., 1997), all of which are localized to the Golgi compartment (Brummell et al., 1990; Driouich et al., 1993; Staehelin and Moore, 1995).The enzymes involved in wall biosynthesis have been recalcitrant to isolation (Carpita et al., 1996; Albersheim et al., 1997). Only recently has the first gene encoding putative cellulose biosynthetic enzymes, celA, been isolated from cotton (Gossypium hirsutum) and rice (Oryza sativa; Pear et al., 1996).During studies of polysaccharide synthesis in pea (Pisum sativum) Golgi membranes, Dhugga et al. (1991) identified a 41-kD protein doublet that they suggested was involved in polysaccharide synthesis. The authors showed that this protein could be glycosylated by radiolabeled UDP-Glc but that this labeling could be reversibly competed with by unlabeled UDP-Glc, UDP-Xyl, and UDP-Gal, the sugars that make up xyloglucan (Hayashi, 1989). The 41-kD protein was named PsRGP1 (P. sativum Reversibly Glycosylated Polypeptide-1; Dhugga et al., 1997). Furthermore, the conditions that stimulate or inhibit Golgi-localized β-glucan synthase activity are the same conditions that stimulate or inhibit the glycosylation of PsRGP1 (Dhugga et al., 1991). To address the role of this protein in polysaccharide synthesis, the authors purified the polypeptides and obtained the sequences from tryptic peptides (Dhugga and Ray, 1994). Antibodies raised against PsRGP1 showed that it is soluble and localized to the plasma membrane (Dhugga et al., 1991) and Golgi compartment (Dhugga et al., 1997). In addition to its Golgi localization, the steady-state glycosylation of PsRGP1 is approximately 10:7:3 (UDP-Glc:-Xyl:-Gal), which is similar to the typical sugar composition of xyloglucan (1.0:0.75:0.25; Dhugga et al., 1997).We were interested in studying various aspects of cell wall metabolism, including the synthesis of polysaccharides and their delivery to the cell wall. Studies in pea have shown that a 41-kD protein may be involved in cell wall polysaccharide synthesis, possibly that of xyloglucan (Dhugga et al., 1997). Here we report the characterization of AtRGP1 (Arabidopsis thaliana Reversibly Glycosylated Polypeptide-1), a soluble protein that can also be found weakly associated with membrane fractions, most likely the Golgi fraction. The reversible nature of the glycosylation of this Arabidopsis homolog by the substrates used to make polysaccharides (nucleotide sugars) suggests a possible role for AtRGP1 in polysaccharide biosynthesis.     

Old Man Saltbush mortality following fire challenges the resilience of post-mine rehabilitation in central Queensland,Australia

Landscape rehabilitation following mining is required to be resilient to disturbance impacts such as fire, drought and disease. As mining companies undergo the process of rehabilitation certification and mine closure, there are notable knowledge gaps on the ecological risks associated with mature rehabilitated landscapes, based largely on the assumption that rehabilitation is analogous to reference communities. However, the response to fire disturbance across a range of landscapes remains largely untested and in particular there is limited understanding of recovery traits of plant species that occur naturally or are commonly seeded into rehabilitation. In August 2018, a controlled fire was applied to 37 hectares of 12-year-old coal-mine rehabilitation in central Queensland, Australia. We used a combination of (i) ground plot surveys and (ii) drone imagery to compare the vegetation response of burnt woody species to unburnt controls prior to, and for, two years following the fire. The survival of the most dominant shrub species found on the rehabilitation site was significantly impacted by the fire. Old Man Saltbush (Atriplex nummularia Lindl. subsp. nummularia) recorded significant post-fire mortality, with ground surveys recording an average reduction of 89% of stems per hectare across the burnt site, while unburnt controls remained unchanged. The plot data analysis was supported with high spatial and temporal resolution drone imagery, classified using a Random Forest machine-learning approach. Change analysis of these maps showed a significant decline of 82% in Old Man Saltbush plant density and 92% reduction in foliage cover following the fire. In addition, the mean canopy area of individual Old Man Saltbush shrubs reduced significantly from a pre-fire mean of 11.3 to 4.8 m² two years following the fire. A spatial proximity analysis showed that those individuals that survived the fire were located significantly closer to unburnt areas and bare spoil, indicating that discontinuous ground fuel loads can greatly improve the survivability of individuals. This study provides new evidence on the contested fire sensitivity of Old Man Salt bush and demonstrates the risk that future climate-driven extreme events may have on the resilience of novel ecosystems.     

Problem-solving of the cylinder,tile and lever tasks by wild animals in Dryandra National Park,Western Australia

Problem-solving is an important ability that allows animals to overcome environmental challenges. As such, it is a useful measure of behavioural flexibility and could be beneficial for conservation work. However, there is currently little known about the solving abilities of many Australian species, despite the high threat of environmental degradation and loss that they face. We therefore measured the problem-solving abilities of native Australian species living in the Dryandra National Park, Western Australia using food-baited puzzles (cylinder task, tile task and lever task) placed in front of camera traps. We recorded 12 species on cameras, with 10 species interacting with at least one puzzle. Of these species, woylies and koomal solved all tasks across multiple sites and using multiple behaviours, suggesting that they may be capable of adapting to novel conditions or environments. We also recorded a chuditch solving the tile task at one site. Regardless of species and puzzle type, animals had a higher chance of solving puzzles with increasing interactions. Our results document the first occurrence of problem-solving in woylies and chuditch, and highlight the potential for problem-solving measures to be incorporated into conservation management.     

The “less-is-more” in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity

Therapeutic monoclonal antibodies are the fastest growing class of biological therapeutics for the treatment of various cancers and inflammatory disorders. In cancer immunotherapy, some IgG1 antibodies rely on the Fc-mediated immune effector function, antibody-dependent cellular cytotoxicity (ADCC), as the major mode of action to deplete tumor cells. It is well-known that this effector function is modulated by the N-linked glycosylation in the Fc region of the antibody. In particular, absence of core fucose on the Fc N-glycan has been shown to increase IgG1 Fc binding affinity to the FcγRIIIa present on immune effector cells such as natural killer cells and lead to enhanced ADCC activity. As such, various strategies have focused on producing afucosylated antibodies to improve therapeutic efficacy. This review discusses the relevance of antibody core fucosylation to ADCC, different strategies to produce afucosylated antibodies, and an update of afucosylated antibody drugs currently undergoing clinical trials as well as those that have been approved.     

Prolific Origination of Eyes in Cnidaria with Co-option of Non-visual Opsins

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Reactions between ruthenium cluster carbonyls and 1,4-diphenylbuta-1,3-diyne

The complexes RuC(CCPh)=CPhC(CCPh)=CPh(CO)₃(NMe₃) (3), Ru₂μ-C(CCPh)=CPhC(CCPh)=CPh(CO)₆ (1), Ru₂μ-[C(CCPh)=CPh]₂CO(CO)₆ (2), Ru₃(μ₃-PhC₂CCPh)(μ-CO)(CO)₉ (4) and Ru₄(μ₄-PhC₂CCPh)(CO)₁₂ (5) have been isolated from reactions between PhC₂C₂Ph and Ru₃(CO)₁₂ or RU₃(CO)₁₀(NCMe)₂. The molecular structures of complexes 1, 2, 3 and 5 have been determined from single-crystal X-ray studies. All complexes have precedents in similar products obtained from reactions involving mono-ynes; in the present cases, each alkyne fragment retains a phenylethynyl (PhCC---) group as a non-coordinated substituent.     

A carboxy-terminal plant vacuolar targeting signal is not recognized by yeast

Three different classes of signals for plant vacuolar targeting have been defined. Previous work has demonstrated that the carboxyl-terminal propeptide (CTPP) of barley lectin (BL) is a vacuolar targeting signal in tobacco plants. When a mutant BL protein lacking the CTPP is expressed in tobacco, the protein is secreted. In an effort to determine the universality of this signal, the CTPP was tested for its ability to target proteins to the vacuole of Saccharomyces cerevisiae. Genes encoding fusion proteins between the yeast secreted protein invertase and BL domains were synthesized and transformed into an invertase deletion mutant of yeast. Invertase assays on intact and detergent-solubilized cells demonstrated that invertase+CTPP was secreted, while nearly 90% of the invertase::BL+CTPP (fusion protein between invertase and BL containing the CTPP) and invertase::BL-CTPP proteins (fusion between invertase and BL lacking the CTPP) were retained intracellularly. These fusions were secreted in a mutant of yeast that normally secretes proteins targeted to the vacuole. With this and previous work, proteins representing all three classes of plant vacuolar targeting signals have now been tested in yeast, and in all cases, the experiments indicate that the plant proteins are directed to the yeast vacuole using signals other than those recognized by plants.     

Regulation of the voltage-gated K(+) channels KCNQ2/3 and KCNQ3/5 by ubiquitination. Novel role for Nedd4-2

The muscarine-sensitive K(+) current (M-current) stabilizes the resting membrane potential in neurons, thus limiting neuronal excitability. The M-current is mediated by heteromeric channels consisting of KCNQ3 subunits in association with either KCNQ2 or KCNQ5 subunits. The role of KCNQ2/3/5 in the regulation of neuronal excitability is well established; however, little is known about the mechanisms that regulate the cell surface expression of these channels. Ubiquitination by the Nedd4/Nedd4-2 ubiquitin ligases is known to regulate a number of membrane ion channels and transporters. In this study, we investigated whether Nedd4/Nedd4-2 could regulate KCNQ2/3/5 channels. We found that the amplitude of the K(+) currents mediated by KCNQ2/3 and KCNQ3/5 were reduced by Nedd4-2 (but not Nedd4) in a Xenopus oocyte expression system. Deletion experiments showed that the C-terminal region of the KCNQ3 subunit is required for the Nedd4-2-mediated regulation of the heteromeric channels. Glutathione S-transferase fusion pulldowns and co-immunoprecipitations demonstrated a direct interaction between KCNQ2/3 and Nedd4-2. Furthermore, Nedd4-2 could ubiquitinate KCNQ2/3 in transfected cells. Taken together, these data suggest that Nedd4-2 is potentially an important regulator of M-current activity in the nervous system.