Soybean Cell Wall Research Articles

Discovery of new functions of food proteins and their structural development for multifunctional applications.

Proteins and peptides derived from various food sources are used in a variety of applications, including functional foods, pharmaceuticals, and cosmetics. The three-dimensional structure of proteins provides useful insights into their functions and essential information for the creation of proteins with new functions. In this review, a series of functional conversion technologies based on protein structural information derived from foods traditionally consumed in Japan, such as natto (fermented soybeans) and rice, are introduced. For natto, we first identified two types of Bacillus subtilis-derived endolytic and exolytic enzymes with different modes of action on soybean cell wall polysaccharides and then focused on the technology used to create an endolytic enzyme from an exolytic enzyme. By applying this technology, a method for creating novel bioactive peptides from rice seed proteins was established. The modified peptides created could provide diverse options for the production of substances such as pharmaceuticals and cosmetic materials.

Cleavage of macromolecule (protein/polysaccharide)-phenolic bond in soybean cell wall through Lactobacillus casei and Lactobacillus helviticus mixed culture solid-state fermentation for chlorogenic acid extraction

Out of the available dietary sources use for chlorogenic acid extraction, soybean is new and cheap that could offer cost-effective chlorogenic acid production under solid-state fermentation (SSF). Consequently, the study aimed at investigating the cleavage of protein/carbohydrate-phenolic bond in heilong48 soybean (HS) variety via solid-state fermentation by Lactobacillus casei and Lactobacillus helviticus mixed culture for chlorogenic acid extraction with high yield and increased biological activity. A simplistic two-pot bioproduct optimization approach was used; first, screening (Plackett-Burman design) of SSF conditions and second, optimizing (Box-Behnken design) the significant SSF conditions obtained from the former design. The liquid-solid (liquid:solid) ratio of 32:68 (0.47), incubation time of 48 h, pH of 5, and temperature of 50 °C were the best-optimized SSF conditions obtained from the chronological combinatory screening and optimization methods. These conditions gave a satisfactory SSF model, and correspondingly acceptable chlorogenic acid yield (9.20 ± 0.17 mg/g), fermentation efficiency (37.26 ± 0.73%), and 2,2-diphenyl-1-picrylhydrazyl free radical-scavenging activity (DPPH) (65.21 ± 2.05 μmol AA eq/g dry sample) results. The L. casei and L. helviticus mixed culture fermented heilong48 soybean (MCFHS) sample (under optimized SSF conditions) had high (p < 0.05) chlorogenic acid yield and 2,2-diphenyl-1-picrylhydrazyl free radical-scavenging activity than the raw heilong48 soybean flour (RHSF) sample. The effectiveness of the optimized solid-state fermentation conditions on the structural modifications of heilong48 soybean sample was confirmed by scanning electron microscopy, atomic force microscopy, and Fourier transform infrared results.

Confirmation of Cage Effect and Prebiotic Production Potential of a β-Mannanase, with SBM as Substrate Using Microscopy and Wet Chemistry

In vitro assays were carried out to investigate the solubilization of cell walls and generation of mannan oligosaccharides of a b-mannanase-containing commercial product on SBM. Using commercial dosages of the b-mannanase (500 g per ton of feed) cell wall degradation of mannan in SBM cell walls was visualized and an increase in reducing ends (0.12&amp;plusmn;0.02 mg/mL) and the generation of mannan oligosaccharides of degree of polymerization 2 and 4 (22.9&amp;plusmn;3.2 mg/L and 398.8&amp;plusmn;25.4 mg/L) were also measured using HPLC. Mannan, which is H-bonded to cellulose and xyloglucan, was solubilized using a single monocomponent enzyme, allowing for visualization of the disintegration of the entire SBM cell wall structure. This work is the first of its kind using strictly commercial dosage levels of enzyme for evaluating efficacy of the same microscopically. These data confirm the hypothesis that there most likely is a need for only a single relevant NSP enzyme targeting its specific substrate, independent of the concentration of the latter within the complex polysaccharide matrix in the plant cell wall to experience the beneficial effects of the enzyme both in vitro and in vivo. An analogy to compare our data would be destruction of the foundation (mannan) of a building or a bridge (soybean cell wall) which would inevitably lead to dismantling or demolition the entire building or bridge.

Aluminum oxide nanoparticles affect the cell wall structure and lignin composition slightly altering the soybean growth

Aluminum oxide (Al2O3) nanoparticles (NPs) are among the nanoparticles most used industrially, but their impacts on living organisms are widely unknown. We evaluated the effects of 50–1000 mg L−1 Al2O3 NPs on the growth, metabolism of lignin and its monomeric composition in soybean plants. Al2O3 NPs did not affect the length of roots and stems. However, at the microscopic level, Al2O3 NPs altered the root surface inducing the formation of cracks near to root apexes and damage to the root cap. The results suggest that Al2O3 NPs were internalized and accumulated into the cytosol and cell wall of roots, probably interacting with organelles such as mitochondria. At the metabolic level, Al2O3 NPs increased soluble and cell wall-bound peroxidase activities in roots and stems but reduced phenylalanine ammonia-lyase activity in stems. Increased lignin contents were also detected in roots and stems. The Al2O3 NPs increased the p-hydroxyphenyl monomer levels in stems but reduced them in roots. The total phenolic content increased in roots and stems; cell wall-esterified p-coumaric and ferulic acids increased in roots, while the content of p-coumaric acid decreased in stems. In roots, the content of ionic aluminum (Al+3) was extremely low, corresponding to 0.0000252% of the aluminum applied in the nanoparticulate form. This finding suggests that all adverse effects observed were due to the Al2O3 NPs only. Altogether, these findings suggest that the structure and properties of the soybean cell wall were altered by the Al2O3 NPs, probably to reduce its uptake and phytotoxicity.

Effect of soybean processing on cell wall porosity and protein digestibility.

Apart from the presence of antinutritional factors, digestibility of soybean proteins is limited in intact cells by cell wall permeability to proteolitic enzymes. Food processing may modulate cell wall permeability and hence the accessibility of protease enzymes to intracellular proteins. In this study, soybeans were processed in various ways, e.g. cooking applied alone or with either germination or fermentation processes, and the modification in cell wall permeability was investigated using confocal microscopy to visualize the penetration of FITC-dextran probes into isolated cells/cell clusters. Diffusion of fluorescently labelled trypsin into cells and cell clusters was also monitored. Microscopy observations showed that fermentation and germination as well as proteolitic enzymes increase the permeability of boiled soybean cotyledon cells. The diffusion of trypsin into all the isolated cells was observed at an early stage of simulated in vitro digestion, whereas diffusion into cell clusters was delayed due to a bigger size and limited permeability of cell clusters. A modest, although significant, increase in protein digestibility was observed when boiling was combined with fermentation or germination likely due to pre-digestion of storage proteins and inactivation of trypsin inhibitors. This study highlights the positive role of fermentation and germination in improving protein digestibility in soybeans but overall suggests that cell wall permeability to trypsin plays a minor role in the extent of protein digestion of intact soybean cells.

GmIDL2a and GmIDL4a, Encoding the Inflorescence Deficient in Abscission-Like Protein, Are Involved in Soybean Cell Wall Degradation during Lateral Root Emergence.

The number of lateral roots (LRs) of a plant determines the efficiency of water and nutrient uptake. Soybean is a typical taproot crop which is deficient in LRs. The number of LRs is therefore an important agronomic trait in soybean breeding. It is reported that the inflorescence deficient in abscission (IDA) protein plays an important role in the emergence of Arabidopsis LRs. Previously, the genes which encode IDA-like (IDL) proteins have been identified in the soybean genome. However, the functions of these genes in LR development are unknown. Therefore, it is of great value to investigate the function of IDL genes in soybean. In the present study, the functions of two root-specific expressed IDL genes, GmIDL2a and GmIDL4a, are investigated. The expressions of GmIDL2a and GmIDL4a, induced by auxin, are located in the overlaying tissue, where LRs are initiated. Overexpression of GmIDL2a and GmIDL4a increases the LR densities of the primary roots, but not in the elder root. Abnormal cell layer separation has also been observed in GmIDL2a- and GmIDL4a-overexpressing roots. These results suggest that the overlaying tissues of GmIDL2a- and GmIDL4a-overexpressing roots are looser and are suitable for the emergence of the LR primordium. Further investigation shows that the expression of some of the cell wall remodeling (CWR) genes, such as xyloglucan endotransglucosylase/hydrolases, expansins, and polygalacturonases, are increased when GmIDL2a and GmIDL4a are overexpressed in hairy roots. Here, we conclude that GmIDL2a and GmIDL4a function in LR emergence through regulating soybean CWR gene expression.

Insight into Enzymatic Degradation of Corn, Wheat, and Soybean Cell Wall Cellulose Using Quantitative Secretome Analysis of Aspergillus fumigatus.

Lignocelluloses contained in animal forage cannot be digested by pigs or poultry with 100% efficiency. On contrary, Aspergillus fumigatus, a saprophytic filamentous fungus, is known to harbor 263 glycoside hydrolase encoding genes, suggesting that A. fumigatus is an efficient lignocellulose degrader. Hence the present study uses corn, wheat, or soybean as a sole carbon source to culture A. fumigatus under animal physiological condition to understand how cellulolytic enzymes work together to achieve an efficient degradation of lignocellulose. Our results showed that A. fumigatus produced different sets of enzymes to degrade lignocelluloses derived from corn, wheat, or soybean cell wall. In addition, the cellulolytic enzymes produced by A. fumigatus were stable under acidic condition or at higher temperatures. Using isobaric tags for a relative and absolute quantification (iTRAQ) approach, a total of ∼600 extracellular proteins were identified and quantified, in which ∼50 proteins were involved in lignocellulolysis, including cellulases, hemicellulases, lignin-degrading enzymes, and some hypothetical proteins. Data are available via ProteomeXchange with identifier PXD004670. On the basis of quantitative iTRAQ results, 14 genes were selected for further confirmation by RT-PCR. Taken together, our results indicated that the expression and regulation of lignocellulolytic proteins in the secretome of A. fumigatus were dependent on both nature and complexity of cellulose, thus suggesting that a different enzyme system is required for degradation of different lignocelluloses derived from plant cells. Although A. fumigatus is a pathogenic fungus and cannot be directly used as an enzyme source, as an efficient lignocellulose degrader its strategy to synergistically degrade various lignocelluloses with different enzymes can be used to design enzyme combination for optimal digestion and absorption of corn, wheat, or soybean that are used as forage of pig and poultry.

Enzymatic Solubilisation and Degradation of Soybean Fibre Demonstrated by Viscosity, Fibre Analysis and Microscopy

The effect of a commercial multienzyme product obtained by fermentation from Aspergillus aculeatus on soybean and soybean meal was investigated using viscosity measurements, dietary fibre component analysis and different microscopy techniques utilizing histochemical dyes and antibody labelling. The results obtained demonstrated a strong viscosity reducing effect of the enzyme preparation on soluble galactomannan and xyloglucan polysaccharides and in addition non-starch polysaccharide analysis demonstrated a notable solubilisation of all polysaccharide constituents. The degradation of these components as native integral parts of cell walls upon exposure to the enzyme was visualized with microscopy. Two histochemical dyes, coriphosphine O and alcian blue were successfully used to follow pectin solubilisation after enzyme treatment. Commercial antibodies recognizing specific components of pectin and hemicellulose components of soybean cell wall were also used to visualize several enzyme activities in the commercial enzyme preparation The challenges of using commercial antibodies elicited from a given plant source to detect similar epitiopes on another plant source are also discussed. Non-starch polysaccharide analysis of the insoluble dietary fibre constituents before and after enzyme treatment corroborated the visualized mode of action demonstrated by microscopy. The combination of techniques provided visual and quantitative measurements of the solubilisation and degradation of hemicellulose pectic soybean cell wall components as part of the undesirable antinutrients in animal feed.

Xyloglucan, galactomannan, glucuronoxylan, and rhamnogalacturonan I do not have identical structures in soybean root and root hair cell walls.

Chemical analyses and glycome profiling demonstrate differences in the structures of the xyloglucan, galactomannan, glucuronoxylan, and rhamnogalacturonan I isolated from soybean ( Glycine max ) roots and root hair cell walls. The root hair is a plant cell that extends only at its tip. All other root cells have the ability to grow in different directions (diffuse growth). Although both growth modes require controlled expansion of the cell wall, the types and structures of polysaccharides in the walls of diffuse and tip-growing cells from the same plant have not been determined. Soybean (Glycine max) is one of the few plants whose root hairs can be isolated in amounts sufficient for cell wall chemical characterization. Here, we describe the structural features of rhamnogalacturonan I, rhamnogalacturonan II, xyloglucan, glucomannan, and 4-O-methyl glucuronoxylan present in the cell walls of soybean root hairs and roots stripped of root hairs. Irrespective of cell type, rhamnogalacturonan II exists as a dimer that is cross-linked by a borate ester. Root hair rhamnogalacturonan I contains more neutral oligosaccharide side chains than its root counterpart. At least 90% of the glucuronic acid is 4-O-methylated in root glucuronoxylan. Only 50% of this glycose is 4-O-methylated in the root hair counterpart. Mono O-acetylated fucose-containing subunits account for at least 60% of the neutral xyloglucan from root and root hair walls. By contrast, a galacturonic acid-containing xyloglucan was detected only in root hair cell walls. Soybean homologs of the Arabidopsis xyloglucan-specific galacturonosyltransferase are highly expressed only in root hairs. A mannose-rich polysaccharide was also detected only in root hair cell walls. Our data demonstrate that the walls of tip-growing root hairs cells have structural features that distinguish them from the walls of other roots cells.

Hydrolyzed Okara in Low-Fat Pork Burgers under Pilot Scale

Okara (soybean curd refuse) is the major byproduct in the manufacture of soymilk and tofu. Okara cannot be used in foods due to its high moisture and poor mouth feel. Relatively high insoluble dietary fibers are the main reason for the poor sensory quality. In this study we used the ViscozymeTMand PectinexTMto degrade the first and second layers of soybean cell wall and transform okara as an ingredient for meat products. Pork burgers incorporated with six different levels of hydrolyzed wet okara (5, 10, 15, 20, 25 and 30%) were prepared and compared with controls (without okara). Cook loss (%), cholesterol content, water holding capacity (WHC), color attributes and sensory evaluation were conducted to evaluate the feasibility of hydrolyzed okara as meat product ingredient. While the moisture contents of raw and cooked pork burger increased with the addition of various percentage okara, the addition of okara reduced the cholesterol content by about 6~56% for raw pork burger and 14~47% for cooked burger. The WHC and cook loss (%) of pork burger were improved by the addition of okara. The addition of okara also increased the pH, L* and, b* values of raw pork burger but did not affect a* value. Significant effects of okara on overall acceptability were observed when the addition of okara was above 25%.

Plant Cell-Wall Cross-Links by REDOR NMR Spectroscopy

We present a new method that integrates selective biosynthetic labeling and solid-state NMR detection to identify in situ important protein cross-links in plant cell walls. We have labeled soybean cells by growth in media containing l-[ring-d(4)]tyrosine and l-[ring-4-(13)C]tyrosine, compared whole-cell and cell-wall (13)C CPMAS spectra, and examined intact cell walls using (13)C{(2)H} rotational echo double-resonance (REDOR) solid-state NMR. The proximity of (13)C and (2)H labels shows that 25% of the tyrosines in soybean cell walls are part of isodityrosine cross-links between protein chains. We also used (15)N{(13)C} REDOR of intact cell walls labeled by l-[ε-(15)N,6-(13)C]lysine and depleted in natural-abundance (15)N to establish that the side chains of lysine are not significantly involved in covalent cross-links to proteins or sugars.

Comparative proteomics analysis of differentially expressed proteins in soybean cell wall during flooding stress

Flooding is a major problem for soybean crop as it reduces the growth and grain yield. To investigate the function of the soybean cell wall in the response to flooding stress, cell wall proteins were analyzed. Cell wall proteins from roots and hypocotyls of soybeans, which were germinated for 2days and subjected to 2days of flooding, were purified, separated by two-dimensional polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. Sixteen out of 204 cell wall proteins showed responses to flooding stress. Of these, two lipoxygenases, four germin-like protein precursors, three stem 28/31kDa glycoprotein precursors, and one superoxide dismutase [Cu-Zn] were downregulated. A copper amine oxidase was found to have shifted from the basic to acidic zone following flooding stress. Based on these results, it was confirmed by the lignin staining that the lignification was suppressed in the root of soybean under the flooding stress. These results suggest that the roots and hypocotyls of soybean caused the suppression of lignification through decrease of these proteins by downregulation of reactive oxygen species and jasmonate biosynthesis under flooding stress.

Soybean (Glycine max) cell wall composition and availability to feed enzymes.

Defatted untoasted soybean cotyledons and hulls were fractionated as water solutes (WSc and WSh) and water unextractable (WUc and WUh). Further fractionation of WUc through deproteinization yielded the isolation of a water unextractable solid (WUS) fraction that was mainly composed (molar percent) of galactose (28.1%), glucose (27.8%), arabinose (13.3%), and uronic acids (17.6%), which accounted for 76% of the water insoluble polysaccharides in soybean cotyledons (WUc). The cell wall (WUS) was sequentially fractionated with chelating agents (chelating agent soluble solids, ChSS) and a gradient of agents (dilute alkali, DASS; 1 M alkali, 1MASS; and 4M alkali, 4MASS), which gave a final cellulosic residue. The ChSS and DASS extracts were characterized as pectin-rich fractions, whereas 1MASS and 4MASS were hemicellulose- and cellulose-rich fractions. Incubation in vitro of the WUc fraction with pectinase, cellulase, and xylanase resulted in the release of low amounts (not more than 5% bound basis) of monosaccharides, mostly uronic acids, xylose, and arabinose. Protein extraction hardly increased this release after enzymatic incubation (<7%). However, progressive fractionation of the cell wall matrix markedly increased the release of monosaccharides from pectin- (ChSS and DASS) and hemicellulose-rich fractions (1MASS and 4MASS). Significant degradation of cellulose (up to 20%) was achieved only after complete protein, pectin, and hemicellulose extraction.

Xyloglucan from soybean ( Glycine max) meal is composed of XXXG-type building units

Soybean cell wall material was depectinated by extraction with a hot chelating agent and a cold dilute alkali. The hemicelluloses were solubilised from the residue with 1 and 4 M KOH solutions, resulting in 1 M Alkali Soluble Solids (1 MASS) and 4 M Alkali Soluble Solids (4 MASS) fractions. The polysaccharides extracted with 1 M KOH were fractionated by ion-exchange chromatography, yielding a neutral and a pectic population. The sugar composition of the neutral population indicated the presence of xyloglucans and possibly xylans. Enzymatic degradation with endo-xylanases and endo-glucanases showed the presence of xyloglucans only. Analysis of the digest formed after incubation of the neutral population with endo-glucanase V using both HPAEC and MALDI-TOF MS showed the formation of the characteristic poly-XXXG xyloglucan oligomers (XXG, XXXG, XXFG, XLXG, and XLFG).

Subcellular distribution and translocation of radionuclides in plants

AbstractThe subcellular distribution of radionuclides in Glycine max Merr. (soybean) and Cucumis sativus L. (cucumber) and translocation of plant absorbed radionuclides with growth in soybean were studied. More than 60% of cellular incorporated Rb, Sr, Mn, Co, Nb, and Se remained in the supernatant fraction; 55% and 20% of Cr was bound to soybean and cucumber cell wall fractions, respectively; 70% or more of Be, Y, and Fe was fixed in the chloroplast fraction; and approx. 10% of Sc, Fe, V, and As were fixed in the mitochondrial fraction. Translocation of nuclides within the soybean plant at different stages of growth has been determined. Vanadium, Y, Be, Se, Nb, Sc, Cr, and Zr were predominantly accumulated in the root. Although the total percentage of plant uptake of Sc, Zr, Nb, and Cr was high, because of low mobility and translocation to shoot, their accumulation in the fruit fraction was negligible. The translocation of mobile nuclides in plants was demonstrated clearly by Rb, Zn, and Fe. Data on the nuclide fraction mobilized from vegetative parts into edible parts was used to assess the percentage of accumulated radionuclides in plants that may reach humans through beans.

Seed Protein Topology: Molecular Profiles

Corn, wheat, and soybean seed proteins were examined for potentially useful regularity, size, and polarity. Volume and amphiphilicity profiles evidence analogies to spider silk and human collagen, but seed proteins are 28% larger and 3.6 times more polar per residue. Wheat proteins are more uniform than silk; corn zeins less polar than collagen. A soybean cell wall protein is the most uniform (±5 A 3 ); a corn cell wall peptide is the smallest (92±9 A 3 ) and least polar (2±18) per residue. Selected parts appear to be more useful than whole molecules

Combined effects of diffusional hindrances, electrostatic repulsion and product inhibition on the kinetic properties of a bound acid phosphatase

The kinetic properties of a soybean cell wall phosphatase were studied and compared under different environmental conditions. The native enzyme isolated from the wall exhibits classical Michaelis Menten kinetics. When buried into the cell wall, its natural environment, the enzyme mimics an apparent negative cooperativity. This deviation from hyperbolic dependence of the activity vs. the substrate concentration may result from an heterogeneous distribution of the enzyme molecules between the surface and the inside of the wall, where an electrostatic partition effect takes place. Cell wall fragments compacted on a Millipore-type membrane allow the study of the dual effects of diffusional hindrances and electrostatic interactions on the global kinetic behavior. By increasing the ionic strength, partition effects can be suppressed and therefore diffusional effects alone can be taken into account. On the one hand, diffusion and partition act individually and synergetically to decrease the apparent global affinity of phosphatase with respect to glucose 6-phosphate. On the other hand, product inhibition by inorganic phosphate is subjected to dual effects from diffusion through accumulation and partition through repulsion. A simple diffusion—partition reaction model accounts qualitatively and quantitatively for the experimental observations.

Deletion analysis and localization of SbPRP1, a soybean cell wall protein gene, in roots of transgenic tobacco and cowpea1

SbPRP1 is a member of the soybean (Glycine max L. Merr) proline-rich cell wall protein family and is expressed at high levels in root tissue. To characterize the sequences required for this expression, we have fused 1.1 kb of upstream flanking DNA sequence from an SbPRP1 genomic clone to a gene encoding beta-glucuronidase (GUS). This construct was introduced into tobacco using Agrobacterium tumefaciens-mediated transformation. Histochemical staining of GUS activity in transgenic tobacco indicated that SbPRP1 is expressed in the apical and elongating region of both primary and lateral roots, most strongly in the epidermis. A similar localization pattern was found in transformed hairy roots when this construct was introduced into cowpea (Vigna aconitifolia) using Agrobacterium rhizogenes-mediated transformation. Nested 5'-deletion analysis of the SbPRP1 promoter indicated that a minimal promoter for SbPRP1 expression in roots is located within the first 262 bases of upstream flanking DNA and that the region between -1080 and -262 is required for maximal expression of this gene. Gel retardation assays showed that nuclear factors can be detected in soybean roots which specifically bind to sequences located between -1080 and -623, a region which is needed for maximal expression of the SbPRP1 promoter. Northern hybridization analysis was also used to show that little SbPRP1 mRNA was present in roots during the first 24 h after imbibition. These studies indicate that SbPRP1 expression is localized to the actively growing region of the root and that this expression is temporally regulated during very early stages of seedling growth.

A Repetitive Proline-Rich Protein from the Gymnosperm Douglas Fir Is a Hydroxyproline-Rich Glycoprotein

Intact cell elution of suspension cultures derived from Douglas fir, Pseudotsuga menziesii (Mirbel) Franco, yielded two extensin monomers, the first hydroxyproline-rich glycoproteins (HRGPs) to be isolated from a gymnosperm. These HRGPs resolved on Superose-6 gel filtration. The smaller monomer was compositionally similar to angiosperm extensins like tomato P1. The larger monomer had a simple composition reminiscent of repetitive proline-rich proteins (RPRPs) from soybean cell walls and contained proline, hydroxyproline, and sugar; hence designated a proline-hydroxyproline-rich glycoprotein (PHRGP). The simple composition of the PHRGP implied a periodic structure which was confirmed by the simple chymotryptic map and 45-residue partial sequence of the major proline-hydroxyproline-rich glycoprotein chymotryptide 5: Lys-Pro-Hyp-Val-Hyp-Val-Ile-Pro-Pro-Hyp-Val-Val-Lys-Pro-Hyp-Hyp-Val- Tyr-Lys-Pro-Hyp-Val-Hyp-Val-Ile-Pro-Pro-Hyp-Val-Val-Lys-Pro-Hyp-Hyp- Val-Tyr-Lys-Ile-Pro-Pro(Hyp)-Val-Ile-Lys-Pro. Proline-hydroxyproline-rich glycoprotein chymotryptide 5 contained an 18-residue tandem repeat devoid of tetra(hydroxy)-proline or serine; it also contained two instances of the five-residue motif Hyp-Hyp-Val-Tyr-Lys and five of the general Pro-Pro-X-X-Lys motif, thereby establishing its homology with typical angiosperm RPRPs and extensins from tomato, petunia, carrot, tobacco, sugar beet, and Phaseolus. Unlike the nonglycosylated soybean RPRP, the highly purified Douglas fir PHRGP was lightly glycosylated, confirmed by a quantitative hydroxyproline glycoside profile, indicating that extensins can range from highly glycosylated hydroxyproline to little or no glycosylated hydroxyproline. Comparison of extensin sequence data strongly indicates that a major determinant of hydroxyproline glycosylation specificity is hydroxyproline contiguity: extensins with tetrahydroxyproline blocks are very highly arabinosylated (>90% hydroxyproline glycosylated), tri- and dihydroxyproline are less so, and single hydroxyproline residues perhaps not at all. Despite high yields of extensins eluted from intact cells, the Douglas fir cell wall itself was hydroxyproline poor yet remarkably rich in protein (>20%), again emphasizing the existence of other structural cell wall proteins that are neither HRGPs nor glycine-rich proteins.

Prolyl endopeptidase inhibitory activity of peptides in the repeated sequence of various proline-rich proteins

Abstract Peptides present in the repeating region in proline-rich proteins such a corn γ-zein, soybean cell wall protein (SbPRP1), carrot 33-kDa protein, collagen and mouse salivary gland protein were chemically synthesized. The prolyl endopeptidase (PEP, from Flavobacterium meningosepticum or bovine brain) inhibitory activity of these peptides was investigated. γ-Zein-related synthetic peptides such as His-Leu-Pro-Pro-Pro-Val and His-Leu-Pro-Pro-Pro-Val-His-Leu-Pro-Pro-Pro-Val inhibited PEP from both bacterial and mammalian sources. The IC 50 values of these peptides for F. meningosepticum PEP were 80 μM and 30 μM, respectively. Soybean cell wall protein-related synthetic peptides, Lys-Pro-Pro-Val and Lys-Pro-Pro-Ile also inhibited PEP to some degree. However, Pro-Pro-Pro-Pro-Gly-Gly-Pro-Gln-Pro-Arg-Pro-Pro-Glu-Gly (synthetic peptide fragment of salivary gland protein) and Gly-Pro-Hyp-Gly-Pro-Ala (fragment of collagen) showed hardly any inhibition. Therefore, we hydrolyzed γ-zein with subtilisin (Carlsberg) and confirmed the liberation of the native hexapeptide, His-Leu-Pro-Pro-Pro-Val.