Influence of pectin fine structure on the mechanical properties of calcium-pectin and acid-pectin gels

The in vitro and in vivo functionality of the anionic plant polysaccharide pectin depends not only on the amount of ion-binding groups attached to the polymer but also on the distribution of such groups along the backbone. It has been proposed recently that information regarding this intramolecular distribution can be quantified by defining a degree of blockiness (DB or DB(abs)), and the usefulness of such measures in discriminating qualitatively between pectins originating from different sources has been demonstrated. Despite this, the value of these parameters in predicting the pseudoequilibrium elastic modulus of gels remains untested. This study seeks to address this problem through the sourcing and in-house modification of a variety of pectins in order to produce a library of distinct representative fine structures. These were subsequently characterized in terms of their relevant properties, including the determination of the proposed DB and DB(abs), and the formation of gels of these samples was monitored using small deformation mechanical spectroscopy. In addition to ionotropic calcium gels the effect of the fine structure on acid gelation was also studied.     

Down-regulation of an Auxin Response Factor in the tomato induces modification of fine pectin structure and tissue architecture

It has previously been shown that down-regulation of an auxin response factor gene (DR12) results in pleiotropic phenotypes including enhanced fruit firmness in antisense transgenic tomato (AS-DR12). To uncover the nature of the ripening-associated modifications affecting fruit texture, comparative analyses were performed of pectin composition and structure in cell wall pericarp tissue of wild-type and AS-DR12 fruit at mature green (MG) and red-ripe (RR) stages. Throughout ripening, pectin showed a decrease in methyl esterification and in the content of galactan side chains in both genotypes. At mature green stage, pectin content in methyl ester groups was slightly higher in AS-DR12 fruit than in wild type, but this ratio was reversed at the red-ripe stage. The amount of water- and oxalate-soluble pectins increased at the red-ripe stage in the wild type, but decreased in AS-DR12. The distribution of methyl ester groups on the homogalaturonan backbone differed between the two genotypes. There was no evidence of more calcium cross-linked homogalacturan involved in cell-to-cell adhesion in AS-DR12 compared with wild-type fruit. Furthermore, the outer pericarp contains higher proportion of small cells in AS-DR12 fruit than in wild type and higher occurrence of (1-->5) alpha-L-arabinan epitope at the RR stage. It is concluded that the increased firmness of transgenic fruit does not result from a major impairment of ripening-related pectin metabolism, but rather involves differences in pectin fine structure associated with changes in tissue architecture.     

Mass spectrometry for pectin structure analysis

Pectin are extremely complex biopolymers made up of different structural domains. Enzymatic degradation followed by purification and structural analysis of the degradation products proved to be efficient tools for the understanding of pectin fine structure, including covalent interactions between pectic structural domains or with other cell wall polysaccharides. Due to its high sensitivity, high throughput and capacity to analyze mixtures, mass spectrometry has gained more and more importance as a tool for oligosaccharides structural characterization in the past 10 years. This review will focus on the combined use of mass spectrometry and enzymatic digestion for pectins structural characterization.     

The modeled structure of the IgG Gar VL region and its implications for anti-flavin and anti-DNP fine specificities

The interaction of the purified C1q component of human C with synchronized, quiescent human gingival fibroblasts was investigated, and the presence of a specific binding site was demonstrated. Quantitative binding studies with radioiodinated C1q showed that binding was specific, saturable, and reversible upon addition of unlabeled C1q or by increasing the salt concentration. Scatchard plot analysis of the data yielded an affinity constant of 2 X 10(7) M-1 for all cell strains examined. The capacity for C1q binding varied among the eight cell strains examined. The number of binding sites per cell ranged from 2.6 to 17.7 X 10(6) with an average of 8.4 X 10(6). The receptor was insensitive to trypsin digestion, and it bound the collagen-like portion of the C1q molecule. Specific immunofluorescence staining showed that virtually all the viable cultured fibroblasts were able to bind added C1q. Flow cytometric analysis demonstrated a spectrum of fluorescence intensity among the cell strains, and there was a positive correlation between fluorescence intensity and the number of binding sites detected by using radiolabeled C1q.     

Three dimensional fine structure of cultured cells: possible implications for subcellular motility.

To determine the three dimensional fine structure of whole motile cells, rat embryo cells, cultured on Formvar-coated cover-glasses, were glutaraldehyde/osmium-fixed, mounted on grids, dehydrated, critical point dried and examined by transmission electron microscopy using stereoscopic techniques. Three dimensional arrays of organelles occurred in a filament-rich cytoplasmic matrix. Here, besides microtubules and elongate filaments, inter-connected filaments formed a widespread fine-mesh space network which attached to the plasma membrane and closely surrounded all organelles. Negative staining revealed a similar newtork in unfixed cells. It is concluded that this network represents part of the force-generating mechanism for various subcellular movements.     

Developmental changes in guard cell wall structure and pectin composition in the moss Funaria: implications for function and evolution of stomata

Background and Aims

In seed plants, the ability of guard cell walls to move is imparted by pectins. Arabinan rhamnogalacturonan I (RG1) pectins confer flexibility while unesterified homogalacturonan (HG) pectins impart rigidity. Recognized as the first extant plants with stomata, mosses are key to understanding guard cell function and evolution. Moss stomata open and close for only a short period during capsule expansion. This study examines the ultrastructure and pectin composition of guard cell walls during development in Funaria hygrometrica and relates these features to the limited movement of stomata.

Methods

Developing stomata were examined and immunogold-labelled in transmission electron microscopy using monoclonal antibodies to five pectin epitopes: LM19 (unesterified HG), LM20 (esterified HG), LM5 (galactan RG1), LM6 (arabinan RG1) and LM13 (linear arabinan RG1). Labels for pectin type were quantitated and compared across walls and stages on replicated, independent samples.

Key Results

Walls were four times thinner before pore formation than in mature stomata. When stomata opened and closed, guard cell walls were thin and pectinaceous before the striated internal and thickest layer was deposited. Unesterified HG localized strongly in early layers but weakly in the thick internal layer. Labelling was weak for esterified HG, absent for galactan RG1 and strong for arabinan RG1. Linear arabinan RG1 is the only pectin that exclusively labelled guard cell walls. Pectin content decreased but the proportion of HG to arabinans changed only slightly.

Conclusions

This is the first study to demonstrate changes in pectin composition during stomatal development in any plant. Movement of Funaria stomata coincides with capsule expansion before layering of guard cell walls is complete. Changes in wall architecture coupled with a decrease in total pectin may be responsible for the inability of mature stomata to move. Specialization of guard cells in mosses involves the addition of linear arabinans.     

Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination

The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines (ε- and ζ-SAIL Phe) and tyrosine (ε-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic ring NMR signals. In addition to these SAIL aromatic amino acids, we recently synthesized δ-SAIL Phe and δ-SAIL Tyr, which allow us to observe and assign δ-¹³C/¹H signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the δ-, ε- or ζ-¹³C/¹H signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the δ-, ε-, and ζ-proton signals of Phe/Tyr residues give rise to complementary NOE constraints, the concomitant use of various types of SAIL-Phe and SAIL-Tyr would generate more accurate protein structures, as compared to those obtained by using conventional uniformly ¹³C, ¹⁵N-double labeled proteins. We illustrated this with the case of an 18.2 kDa protein, Escherichia coli peptidyl-prolyl cis-trans isomerase b (EPPIb), and concluded that the combined use of ζ-SAIL Phe and ε-SAIL Tyr would be practically the best choice for protein structural determinations.     

Crystal structure of plant pectin methylesterase

Pectin is a principal component in the primary cell wall of plants. During cell development, pectin is modified by pectin methylesterases to give different properties to the cell wall. This report describes the first crystal structure of a plant pectin methylesterase. The beta-helical structure embodies a central cleft, lined by several aromatic residues, that has been deduced to be suitable for pectin binding. The active site is found at the center of this cleft where Asp157 is suggested to act as the nucleophile, Asp136 as an acid/base and Gln113/Gln135 to form an anion hole to stabilize the transition state.     

Different action patterns for apple pectin methylesterase at pH 7.0 and 4.5

The mechanism of action of purified apple pectin methylesterase on pectin (degree of methoxylation: DM 75) and methoxylated homogalacturonans (DM 70 and 90) was studied at pH 7.0 (optimal pH of the enzyme) and at pH 4.5 (close to the pH of apple juice). Different interchain distributions of the free carboxyl groups were obtained at pH 7.0 and 4.5: high-performance ion exchange chromatography indicated a typical single chain mechanism at pH 7.0, but a mechanism differing from the single and multiple chain ones at pH 4.5. However, the same intrachain distribution of the newly demethoxylated galacturonic acid residues was observed for both pHs by 1H NMR. The high content of consecutive de-esterified or consecutive esterified galacturonic acid residues suggested that apple PME acted with a multiple attack mechanism on the pectic substrate. The degree of multiple attack of the enzyme was greater than or equal to 10-11.     

Mesoscopic structure of pectin in solution

Mesoscopic structure of pectin with different molecular characteristics was investigated by means of small angle X‐ray scattering (SAXS), electrokinetic measurements and data modelling. The influence of a broad range of pH (2–7) on chain conformation in the dilute and semi‐diluted regime was investigated. Scattering data and concomitant analysis revealed two length scales at all environmental conditions studied. pH showed greater influence at acidic values (pH 2.0) enhancing the globular component of the structure due to association of galacturonic acid residues. Double logarithmic scattering intensity plots revealed fractal dimensions of 1.9 ± 0.2 in the low‐q regime and 1.5 ± 0.2 in the high q‐region, irrespectively of the specific environment. Increase in branching of RG‐I regions of the polysaccharide chains enhanced the compact conformation irrespectively of the pH or concentration. The present work shows that radical changes in pectin conformation can be induced only under strongly acidic conditions a finding that has important consequences in tailoring the technological performance of these biopolymers.     

A refined method for the determination of Saccharomyces cerevisiae cell wall composition and beta-1,6-glucan fine structure.

In yeast and other fungi, cell division, cell shape, and growth depend on the coordinated synthesis and degradation of cell wall polymers. We have developed a reliable and efficient micro method to determine Saccharomyces cerevisiae cell wall composition that distinguishes between beta1,3- and beta1,6-glucan. The method is based on the sequential treatment of cell walls with specific hydrolytic enzymes followed by dialysis. The low molecular weight (MW) products thus separated account for each particular cell wall polymer. The method can be applied to as little as 50-100 mg (wet wt) of radioactively labeled cells. A combination of chitinase and recombinant beta-1,3-glucanase is initially used, releasing all of the chitin and 60-65% of the beta1,3-glucan from the cell walls. Next, recombinant endo-beta-1,6-glucanase from Trichoderma harzianum is utilized to release all the beta-1,6-glucan present in the wall. The chromatographic pattern of endoglucanase digested beta-1,6-glucan provides a characteristic "fingerprint" of beta-1,6-glucan and the fine structure of the oligosaccharides in this pattern was determined by 1H NMR and electrospray ionization mass spectroscopy. The final enzymatic step uses laminarinase and beta-glucosidase to release the remaining beta-1,3-glucan. The cell wall mannan remains as a high MW fraction at the end of the fractionation procedure. Good sensitivity and correlation with cell wall composition determined by traditional methods were observed for wild-type and several cell wall mutants.     

The inactivation of pectin depolymerase associated with isolated tomato fruit cell wall: implications for the analysis of pectin solubility and molecular weight

An approach commonly employed to assess the potential role of the enzyme polygalacturonase (PG, EC 3.2.1.15) in tomato fruit cell-wall pectin metabolism includes correlating levels of extractable PG with changes in specific characteristics of cell wall pectins, most notably solubility and molecular weight. Since information on these features of pectins is generally derived from analyses of subfractions of isolated cell wall, assurance of inactivation of the various isoforms of wall-associated PG is imperative. In the present study, cell wall prepared from ripe tomato (Lycopersicon esculentum Mill. cv. Rutgers) fruit was examined for the presence of active PG and for the ability of phenolic solvents to inactivate the enzyme. Using pectin solubility and M_r (relative molecular mass) changes as criteria for the presence of wall-associated PG activity, pectins from phenol-treated and nonphenol-treated (enzymically active) cell wall from ripe fruit incubated in 50 mM Na-acetate, 50 mM cyclohexanetrans-1,2-diamine tetraacetic acid (CDTA), pH 6.5 (outside the catalytic range of PG), were of similar M_r and exhibited no change in size with incubation time. Wall prepared without exposure to the phenolic protein-denaturants exhibited extensive pectin solubilization and depolymerization when incubated in 50 mM Na-acetate, 50 mM CDTA at pH 4.5, indicating the presence of active PG. Based on the changes in the M_r of pectins solubilized in 50 mM Na-acetate, 50 mM CDTA, pH 4.5, active PG was also detected in wall exposed during isolation to phenolacetic acid-water (PAW, 2:1:1, w/v/v), a solvent commonly employed as an enzyme denaturant. Although the depolymerization of pectins in PAW-treated wall was extensive, oligouronides constituted minor reaction products. Interestingly, PAW-treated wall did not exhibit PG-mediated pectin release when incubated under conditions (30 mM Na-acetate, 150 mM NaCl, pH 4.5) in which nonphenol-treated cell wall exhibited high autolytic activity. In an alternative protocol designed to inactivate PG, cell wall was exposed to Tris-buffered phenol (BP). In contrast to pectins released from PAW-treated wall, pectins solubilized from BP-treated wall at pH 4.5 were indistinguishable in M_r from those recovered from BP-treated wall at pH 6.5 Even when incubated at pH 4.5 at 34°C, conditions under which pectins from PAW-treated wall underwent more rapid and extensive depolymerization, pectins from BP-treated wall exhibited no change in M_r, providing evidence that active PG was not present in these wall preparations. The implications of this study in interpreting the solubility and M_r of pectin in cell wall from ripening fruit are discussed.     

Strong sequence patterns in eukaryotic promoter regions: Potential implications for DNA structure

• 1.1. Analysis of eukaryotic sequences reveals recurring trends in upstream regions. Oligomers composed of (G/C)_n and (A/T)_m blocks are preferentially flanked by (G/C)₂ doublets on their 3' rather than on their 5′ ends, that is (G/C)_nä(A/T)_m(G/C)₂ > (G/C)_n+2(A/T)_m.
• 2.2. These trends are stronger for larger n and smaller m. Additional trends are outlined below.
• 3.3. The trends are correlated with DNA structural parameters, in particular with twist and roll angles.
• 4.4. Generally, the trends hold if the base pair step joining the 5′ (G/C)₂ doublet to the (G/C)_n (A/T)_m oligomer is not undertwisted and is not strongly rolled into the major groove.
• 5.5. Other DNA parameters crucial for DNA-protein interactions are discussed as well.

     

Sequence and structure patterns in proteins from an analysis of the shortest helices: implications for helix nucleation

The shortest helices (three-length 3(10) and four-length alpha), most abundant among helices of different lengths, have been analyzed from a database of protein structures. A characteristic feature of three-length 3(10)-helices is the shifted backbone conformation for the C-terminal residue (phi,psi angles: -95 degrees,0 degrees ), compared to the rest of the helix (-62 degrees,-24 degrees ). The deviation can be attributed to the release of electrostatic repulsion between the carbonyl oxygen atoms at the two C-terminal residues and further stabilization (due to a more linear geometry) of an intrahelical hydrogen bond. A consequence of this non-canonical C-terminal backbone conformation can be a potential origin of helix kinks when a 3(10)-helix is sequence-contiguous at the alpha-helix N-terminal. An analysis of hydrogen bonding, as well as hydrophobic interactions in the shortest helices shows that capping interactions, some of them not observed for longer helices, dominate at the N termini. Further, consideration of the distribution of amino acid residues indicates that the shortest helices resemble the N-terminal end of alpha-helices rather than the C terminus, implying that the folding of helices may be initiated at the N-terminal end, which does not get propagated in the case of the shortest helices. Finally, pairwise comparison of beta-turns and the shortest helices, based on correlation matrices of site-specific amino acid composition, and the relative abundance of these short secondary structural elements, leads to a helix nucleation scheme that considers the formation of an isolated beta-turn (and not an alpha-turn) as the helix nucleation step, with shortest 3(10)-helices as intermediates between the shortest alpha-helix and the beta-turn. Our results ascribe an important role played by shortest 3(10)-helices in proteins with important structural and folding implications.     

Studies on the structure of a lithium-treated soybean pectin: characteristics of the fragments and determination of the carbohydrate substituents of galacturonic acid

Two galacturonic-acid-containing polysaccharide fractions (ChSS and P) were isolated from soybean meal and subjected to lithium treatment. The fragments obtained were analyzed by using monosaccharide and methylation analyses, and NMR spectroscopy. Lithium degradation of ChSS, followed by sodium borodeuteride reduction, hydrolysis, sodium borohydride reduction, and acetylation afforded alditol acetates, of which the labeled ones reflected residues linked to GalA. As followed from quantifications of the labeled and non-labeled alditols from each constituent monosaccharide by GLC-EIMS, 6 mol% of Ara, 22 mol% of Fuc, 13 mol% of Gal, 53 mol% of Rha, and 57 mol% of Xyl are glycosidically linked to GalA. Analysis of the lithium-treated polymer revealed that it contains arabinogalactan side chains linked to Rha O-4, which consist of a beta-(1 --> 4)-linked galactan substituted with highly branched arabinan chains. On average, an arabinogalactan chain contains up to 29 Gal and 25 Ara residues. Surface plasmon resonance was used to determine conditions for affinity chromatography. Furthermore, this technique confirmed the presence of terminal alpha-Fuc residues in ChSS. Polysaccharide P turned out to be relatively resistant to lithium degradation.     

New insights into pectin methylesterase structure and function

In bacteria, fungi and plants, pectin methylesterases are ubiquitous enzymes that modify the degree of methylesterification of pectins, which are major components of plant cell walls. Such changes in pectin structure are associated with changes in cellular adhesion, plasticity, pH and ionic contents of the cell wall and influence plant development and stress responses. In plants, pectin methylesterases belong to large multigene families, are regulated in a highly specific manner, and are involved in vegetative and reproductive processes, including wood and pollen formation, in addition to plant-pathogen interactions. Although, overall, protein structures are highly conserved between isoforms, recent data indicate that structural variations might be associated with the targeting and functions of specific pectin methylesterases.     

On the fine structure of polyribosomes

Cell and Tissue Research - The ultrastructural morphology of ribosomes was studied in tissue sections of rat uterus using different fixatives (acrolein, formaldehyde, acetic acid, methanol-acetic...     

果胶甲酯酶的结构与功能研究进展

果胶甲酯酶(PME)是一种重要的果胶酶,其水解果胶中的甲酯基从而释放甲醇并降低果胶的甲酯化程度。目前在食品加工、茶饮料、造纸等生产工艺中有着广泛的应用前景。随着对PME的深入研究,已报道了几种不同来源的酶晶体结构,对这些已获得的晶体结构进行分析发现,PME属于右手平行β-螺旋结构,其催化残基为2个保守的天冬氨酸和1个谷氨酰胺残基,并且在催化过程中分别起到了一般酸碱、亲核试剂以及稳定中间体的作用。同时对其底物特异性进行分析,初步了解其底物与活性位点的识别机制。文中针对这几个相关方面进行了系统的综述。