Lead complexation in wines with the dimers of the grape pectic polysaccharide rhamnogalacturonan II

OENO One ◽  
1997 ◽  
Vol 31 (1) ◽  
pp. 33 ◽  
Author(s):  
Patrice Pellerin ◽  
Malcolm A. O'Neill ◽  
Cécile Pierre ◽  
Marie-Thérèse Cabanis ◽  
Alan G. Darvill ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Red Wine ◽  
Lead Content ◽  
Size Exclusion ◽  
Human Diet ◽  
Pectic Polysaccharide ◽  
Rhamnogalacturonan Ii ◽  
Grape Varieties ◽  
Lead Complex

<p style="text-align: justify;">Wine is believed to be a significant source of lead in the human diet even though the lead content of wines has decreased considerably over the last thirty years. Nevertheless, the lead content of wines must be reduced to a minimum since this heavy metal is highly toxic.</p><p style="text-align: justify;">The plant cell wall pectic polysaccharide rhamnogalacturonan Il (RG-II) is a predominant anionic molecule in red wine. RG-Il exists as a dimer (dRG-Il-B) that is cross-linked by a 1:2 borate-diol ester and forms complexes <em>in vitro</em> with lead and other selected di- and tri-valent cations. One mole of dimer binds at least 1 mole of Pb<sup>2+</sup>. We have now determined the amount of lead in wines that is bound to dRG-II-B since previous studies have suggested that most of the lead in wine is bound to an anionic macromolecule.</p><p style="text-align: justify;">Seven wines, with lead concentrations between 30 and 110 μg/l, were obtained from different grape varieties harvested at different vintages and vinified by different procedures. Two chromatography steps, adsorption on a polystyrene- divinylbenzene resin and size-exclusion on a Superdex® 75-HR column, have been used to purify a d-RG-II-B-lead complex which contained at least 85 p. cent of the total lead of each wine. The dRG-II-B-Pb complex is stable at the pH of wine and is present in a wine that was procluced in 1988. The dRG-II-B present in red (~ 100 mg/l) and white (~ 20 mg/l) wines can bind at least ten-fold more Pb<sup>2+</sup> than is typically present in wine.</p><p style="text-align: justify;">Our study is the first to show that in wine most of the lead is complexed with a pectic polysaccharide that is not degraded during vinification. dRG-II-B is also known to form complexes with other cations, including strontium and barium. However, it is not known what role dRG-II-B has in determining the metabolic fate in humans of toxic cations present in wine.</p>

scholarly journals Locating Methyl-Etherified and Methyl-Esterified Uronic Acids in the Plant Cell Wall Pectic Polysaccharide Rhamnogalacturonan II

2020 ◽  
Vol 25 (4) ◽  
pp. 329-344
Author(s):  
Malcolm A. O’Neill ◽  
Ian Black ◽  
Breeanna Urbanowicz ◽  
Vivek Bharadwaj ◽  
Mike Crowley ◽  
...  
Keyword(s):  
Plant Species ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Pectic Polysaccharide ◽  
Ionization Time ◽  
Methyl Esterification ◽  
Flight Mass Spectrometry ◽  
Borate Ester ◽  
Rhamnogalacturonan Ii

Rhamnogalacturonan II (RG-II) is a structurally complex pectic polysaccharide that exists as a borate ester cross-linked dimer in the cell walls of all vascular plants. The glycosyl sequence of RG-II is largely conserved, but there is evidence that galacturonic acid (GalA) methyl etherification and glucuronic acid (GlcA) methyl esterification vary in the A sidechain across plant species. Methyl esterification of the galacturonan backbone has also been reported but not confirmed. Here we describe a new procedure, utilizing aq. sodium borodeuteride (NaBD4)-reduced RG-II, to identify the methyl esterification status of backbone GalAs. Our data suggest that up to two different GalAs are esterified in the RG-II backbone. We also adapted a procedure based on methanolysis and NaBD4 reduction to identify 3-, 4-, and 3,4- O-methyl GalA in RG-II. These data, together with matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF) MS analysis of sidechain A generated from selected RG-IIs and their NaBD4-reduced counterparts, suggest that methyl etherification of the β-linked GalA and methyl esterification of the GlcA are widespread. Nevertheless, the extent of these modifications varies between plant species. Our analysis of the sidechain B glycoforms in RG-II from different dicots and nonpoalean monocots suggests that this sidechain has a minimum structure of an O-acetylated hexasaccharide (Ara-[MeFuc]-Gal-AceA-Rha-Api-). To complement these studies, we provide further evidence showing that dimer formation and stability in vitro is cation and borate dependent. Taken together, our data further refine the primary sequence and sequence variation of RG-II and provide additional insight into dimer stability and factors controlling dimer self-assembly.

scholarly journals Protocols for Isolating and Characterizing Polysaccharides From 1 Plant Cell Walls: A Case Study Using Rhamnogalacturonan-II

2021 ◽  
Author(s):  
Breeanna Urbanowicz ◽  
William Barnes ◽  
Sabina Koj ◽  
Ian Black ◽  
Stephanie Archer-Hartmann ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Size Exclusion ◽  
Proton Nuclear Magnetic Resonance ◽  
Wall Material ◽  
Resonance Spectroscopy ◽  
Cell Wall Polysaccharide ◽  
Pectic Polysaccharide ◽  
Rhamnogalacturonan Ii

Abstract Background: In plants, there is a large diversity of polysaccharides that comprise the cell wall. Each major type of plant cell wall polysaccharide, including cellulose, hemicellulose, and pectin, has distinct structures and functions that contribute to wall mechanics and influence plant morphogenesis. In recent years, pectin modification and valorization has attracted much attention due to its expanding roles of pectin in biomass deconstruction, food science, material science, and environmental remediation. However, pectin utilization has been limited by our incomplete knowledge of pectin structure. Herein, we present a workflow of principles relevant for the characterization of polysaccharide primary structure using nature’s most complex polysaccharide, rhamnogalacturonan-II (RG-II), as a model.Results: We outline how to isolate RG-II from celery and duckweed cell wall material and red wine using chemical or enzymatic treatments coupled with size-exclusion chromatography. From there, we demonstrate the use of mass spectrometry (MS)-based techniques to determine the glycosyl residue and linkage compositions of the intact RG II molecule and RG-II-derived oligosaccharides including special considerations for labile monosaccharides. In doing so, we demonstrated that in the duckweed Wolffiella repanda the arabinopyranosyl (Arap) residue of side chain B is substituted at O-2 with rhamnose. As RG-II is further modified by non-glycosyl modifications including methyl-ethers, methyl-esters, and acetyl-esters, we then describe ways to use electrospray-MS to identify these moieties on RG-II-derived oligosaccharides. We then explored the utility of proton nuclear magnetic resonance spectroscopy (1H-NMR) in identifying RG-II-specific sugars and non-glycosyl modifications to complement and extend MS-based approaches. Finally, we describe how to assess the factors that affect RG-35 II dimerization using liquid chromatographic and NMR spectroscopic approaches.Conclusions: The complexity of pectic polysaccharide structures has hampered efforts aimed at their valorization. In this work, we used RG-II as a model to demonstrate the steps necessary to isolate and characterize polysaccharides using chromatographic, MS, and NMR techniques. The principles can be applied to the characterization of other saccharide structures and will help inform researchers on how saccharide structure relates to functional properties in the future.

scholarly journals Ultrasonic-Assisted Extraction, Structural Characterization, Chain Conformation, and Biological Activities of a Pectic-Polysaccharide from Okra (Abelmoschus esculentus)

Molecules ◽  
2020 ◽  
Vol 25 (5) ◽  
pp. 1155 ◽  
Author(s):  
Xi-Rui Nie ◽  
Yuan Fu ◽  
Ding-Tao Wu ◽  
Ting-Ting Huang ◽  
Qin Jiang ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Light Scattering ◽  
Chain Conformation ◽  
Size Exclusion ◽  
Pectic Polysaccharide ◽  
Assisted Extraction ◽  
Ultrasonic Assisted ◽  
Pharmaceutical Industries ◽  
Ultrasonic Assisted Extraction

The purpose of this study was to better understand the chemical characteristics and chain conformation of okra polysaccharides extracted by ultrasonic-assisted extraction. A pectic-polysaccharide, named OPP-D, was obtained, which was mainly composed of rhamnose, galacturonic acid, and galactose with a molar ratio of 1.01:1.00:2.31. Combined with NMR analysis, -4)-α-d-GalAp-(1,2,4)-α-l-Rhap-(1- were identified as the backbone with galactan side chains substituted partly at O-4 of Rhap. Molecular weight and radius of gyration of OPP-D were determined as 2.19 × 105 Da and 27.0 nm, respectively. OPP-D was determined as an air-core sphere with branching chains in 0.9% NaCl solution by high-performance size-exclusion chromatography coupled with multi-angle laser light scattering and dynamic light scattering for the first time. Moreover, OPP-D exhibited typical shear-thinning behavior. In addition, OPP-D exhibited remarkable in vitro antioxidant activities and prebiotic activities, while the relatively high molecular weight, high degree of esterification, high content of uronic acids, and highly branched globular conformation of OPP-D might contribute to its in vitro anti-diabetic activities and binding capacities. Results can contribute to a better understanding of the structure–bioactivity relationship of OPPs, and OPP-D has great potential applications in the functional food and pharmaceutical industries.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Crystal structures of plant inorganic pyrophosphatase, an enzyme with a moonlighting autoproteolytic activity

2019 ◽  
Vol 476 (16) ◽  
pp. 2297-2319 ◽  
Author(s):  
Marta Grzechowiak ◽  
Milosz Ruszkowski ◽  
Joanna Sliwiak ◽  
Kamil Szpotkowski ◽  
Michal Sikorski ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Inorganic Pyrophosphatase ◽  
Prolonged Storage ◽  
Mitochondrial Targeting ◽  
Cleavage Rate ◽  
Inorganic Pyrophosphate ◽  
Putative Signal Peptide ◽  
Targeting Peptide

Abstract Inorganic pyrophosphatases (PPases, EC 3.6.1.1), which hydrolyze inorganic pyrophosphate to phosphate in the presence of divalent metal cations, play a key role in maintaining phosphorus homeostasis in cells. DNA coding inorganic pyrophosphatases from Arabidopsis thaliana (AtPPA1) and Medicago truncatula (MtPPA1) were cloned into a bacterial expression vector and the proteins were produced in Escherichia coli cells and crystallized. In terms of their subunit fold, AtPPA1 and MtPPA1 are reminiscent of other members of Family I soluble pyrophosphatases from bacteria and yeast. Like their bacterial orthologs, both plant PPases form hexamers, as confirmed in solution by multi-angle light scattering and size-exclusion chromatography. This is in contrast with the fungal counterparts, which are dimeric. Unexpectedly, the crystallized AtPPA1 and MtPPA1 proteins lack ∼30 amino acid residues at their N-termini, as independently confirmed by chemical sequencing. In vitro, self-cleavage of the recombinant proteins is observed after prolonged storage or during crystallization. The cleaved fragment corresponds to a putative signal peptide of mitochondrial targeting, with a predicted cleavage site at Val31–Ala32. Site-directed mutagenesis shows that mutations of the key active site Asp residues dramatically reduce the cleavage rate, which suggests a moonlighting proteolytic activity. Moreover, the discovery of autoproteolytic cleavage of a mitochondrial targeting peptide would change our perception of this signaling process.

CHARACTERIZATION OF A NEW LCAT MUTATION CAUSING FAMILIAL LCAT DEFICIENCY (FLD) AND THE ROLE OF APOE AS A MODIFIER GENE OF THE FLD PHENOTYPE

2009 ◽  
Vol 32 (6S) ◽  
pp. 3
Author(s):  
A Baass ◽  
H Wassef ◽  
M Tremblay ◽  
L Bernier ◽  
R Dufour ◽  
...  
Keyword(s):  
Modifier Gene ◽  
Size Exclusion ◽  
Plasma Lipoproteins ◽  
Lipoprotein Profile ◽  
Exclusion Chromatography ◽  
Low Hdl ◽  
Lcat Deficiency ◽  
Apoe Genotypes

Introduction: LCAT (lecithin:cholesterol acyltransferase ) is an enzyme which plays an essential role in cholesterol esterification and reverse cholesterol transport. Familial LCAT deficiency (FLD) is a disease characterized by a defect in LCAT resulting in extremely low HDL-C, premature corneal opacities, anemia as well as proteinuria and renal failure. Method: We have identified two brothers presenting characteristics of familial LCAT deficiency. We sequenced the LCAT gene, measured the lipid profile as well as the LCAT activity in 15 members of this kindred. We also characterized the plasma lipoproteins by agarose gel electrophoresis and size exclusion chromatography and sequenced several candidate genes related to dysbetalipoproteinemia in this family. Results: We have identified the first French Canadian kindred with familial LCAT deficiency. Two brothers affected by FLD, were homozygous for a novel LCAT mutation. This c.102delG mutation occurs at the codon for His35 causing a frameshift that stops transcription at codon 61 abolishing LCAT enzymatic activity both in vivo and in vitro. It has a dramatic effect on the lipoprotein profile, with an important reduction of HDL-C in both heterozygotes (22%) and homozygotes (88%) and a significant decrease in LDL-C in heterozygotes (35%) as well as homozygotes (58%). Furthermore, the lipoprotein profile differed markedly between the two affected brothers who had different APOE genotypes. We propose that APOE could be an important modifier gene explaining heterogeneity in lipoprotein profiles observed among FLD patients. Our results suggest that a LCAT-/- genotype associated with an APOE ?2 allele could be a novel mechanism leading to dysbetalipoproteinemia.

scholarly journals A Bacterially-Expressed Recombinant Envelope Protein from Usutu Virus Induces Neutralizing Antibodies in Rabbits

Vaccines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 157
Author(s):  
Kinga Böszörményi ◽  
Janet Hirsch ◽  
Gwendoline Kiemenyi Kayere ◽  
Zahra Fagrouch ◽  
Nicole Heijmans ◽  
...  
Keyword(s):  
Envelope Protein ◽  
Neutralizing Antibodies ◽  
Size Exclusion ◽  
Vitro Assay ◽  
Usutu Virus ◽  
Transmission Electron ◽  
Exclusion Chromatography ◽  
Efficacious Vaccine ◽  
Human Infections

Background: Recently, an emerging flavivirus, Usutu virus (USUV), has caused an epidemic among birds in Europe, resulting in a massive die-off in Eurasian blackbirds. Currently found only in Europe and Africa, it can be envisioned that Usutu virus will follow the path of other flaviviruses, like West Nile virus and Zika virus, and will spread via its mosquito vectors and bird hosts to other parts of the world. Several cases of human infections by Usutu virus have already been published. Anticipating this spread, development of an efficacious vaccine would be highly desirable. Method: This study describes the production in E. coli, purification, and refolding of a partial USUV envelope protein. Prior to immunization, the protein was characterized using size exclusion chromatography, transmission electron microscopy and dynamic light scattering, showing the limited presence of virus-like structures, indicating that the protein solution is probably a mixture of mono and multimeric envelope proteins. Results: Immunizations of two rabbits with the refolded E-protein fraction, mixed with a strong adjuvant, resulted in the generation of neutralizing antibodies, as evidenced in an in vitro assay. Discussion: The way forward towards a subunit vaccine against Usutu virus infection is discussed.

scholarly journals Modulation of Viral Programmed Ribosomal Frameshifting and Stop Codon Readthrough by the Host Restriction Factor Shiftless

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1230
Author(s):  
Sawsan Napthine ◽  
Chris H. Hill ◽  
Holly C. M. Nugent ◽  
Ian Brierley
Keyword(s):  
Rna Binding ◽  
Mutational Analysis ◽  
Stop Codon ◽  
Leukemia Virus ◽  
Size Exclusion ◽  
Human Immunodeficiency Virus Replication ◽  
Mobility Shift ◽  
Ribosomal Frameshifting ◽  
Stop Codon Readthrough

The product of the interferon-stimulated gene C19orf66, Shiftless (SHFL), restricts human immunodeficiency virus replication through downregulation of the efficiency of the viral gag/pol frameshifting signal. In this study, we demonstrate that bacterially expressed, purified SHFL can decrease the efficiency of programmed ribosomal frameshifting in vitro at a variety of sites, including the RNA pseudoknot-dependent signals of the coronaviruses IBV, SARS-CoV and SARS-CoV-2, and the protein-dependent stimulators of the cardioviruses EMCV and TMEV. SHFL also reduced the efficiency of stop-codon readthrough at the murine leukemia virus gag/pol signal. Using size-exclusion chromatography, we confirm the binding of the purified protein to mammalian ribosomes in vitro. Finally, through electrophoretic mobility shift assays and mutational analysis, we show that expressed SHFL has strong RNA binding activity that is necessary for full activity in the inhibition of frameshifting, but shows no clear specificity for stimulatory RNA structures.

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