Major Storage Protein Research Articles

7S protein is more effective than total soybean protein isolate in reducing plasma cholesterol

7S protein is one of four major storage proteins in soybean. The present study compared the relative cholesterol-lowering activity of the 7S protein with that of total soybean protein isolate (SPI). Forty-eight hamsters were divided into six groups fed non-cholesterol diet or one of five high-cholesterol diets containing 24.2% casein, 12.1–24.2% SPI, 12.1–24.2% 7S protein, respectively. Results showed the 7S protein reduced plasma total cholesterol (TC) by 20–41% while SPI reduced it by 9–16%. 7S protein increased the fecal excretion of neutral sterols by 43–116%, while SPI only had 18–68% increase. 7S protein increased the excretion of total bile acids by 208–367%, while SPI only increased it by 41–86%. 7S protein could not only down regulate gene expression of HMG-CoA reductase but also up regulate gene expression of CYP7A1. It was concluded that dietary 7S protein was more hypocholesterolemic than SPI, mediated by increasing the sterol excretion and decreasing the cholesterol synthesis.

A STUDY ON THE LEVEL OF SERUM FERRITIN IN WOMEN OF CHILDBEARING AGE IN EL-BEYDA CITY

Ferritin, a major iron storage protein, is essential to iron homeostasis and is involved in a wide range of physiologic and pathologic processes, and can hold up to 4 000 atoms of iron. The aim of our study was to determine the levels of Ferritin among different age groups in El-Beyda city in women of childbearing age. This study included 217 women in childbearing age (18- 45 years) who were visiting women clinic and in a period of 24 months from the first of January 2016 to December 2017in El-Beyda city. Reliable serum ferritin level was measured by using an enzyme immunoassay ELISA method. This study showed that there is a close relationship between age and level of serum ferritin, where we found that the age groups 25-31 and 32-38 is the lowest in the level of serum ferritin.

Receptor mediated endocytosis of vicilin in Callosobruchus maculatus (Coleoptera: Chrysomelidae) larval midgut epithelial cells.

The transport of proteins across the intestinal epithelium of insects is still not well understood. There is evidence that vicilin, a major storage protein of cowpea seeds (Vigna unguiculata), is internalized in larvae of the seed-beetle Callosobruchus maculatus. It has been reported that this vicilin interacts with proteins present in the microvillar membranes of columnar cells along the digestive tract of the larvae. In the present work, we studied the cellular pathway involved in endocytosis of vicilin in larval C. maculatus by employing ex vivo experiments. In the ex vivo approach, we incubated FITC-labelled vicilin with isolated midgut wholemounts in the absence or in the presence of endocytosis inhibitors. The fate of labelled or non-labelled globulins was monitored by confocal microscopy and fluorescence measurement. Our results suggest that the internalization of vicilins is due to receptor-mediated endocytosis. Here we report the identity of a microvillar vicilin-binding protein that was purified using affinity chromatography on a vicilin-sepharose column. The putative vicilin receptor showed high homology to proteins with the CRAL-TRIO domain, specifically the Sec14 superfamily member α-tocopherol transfer protein. The precise mechanism involved in vicilin internalization was defined through the use of specific inhibitors of the endocytosis pathway. The inhibitors filipin III and nystatin significantly inhibited the endocytosis of vicilin, while chlorpromazine and phenylarsine oxide had a much lower effect on endocytosis, suggesting that the endocytic pathway is predominantly mediated by caveolin.

Effects of protein oxidation on gelatinization characteristics during rice storage

Gliadin and glutelin are major rice storage proteins. In this study, we evaluated changes in these proteins to determine the influence of pasting properties on rice storage. Notably, the physical and chemical properties of these proteins changed steadily. We analyzed protein oxidation indexes and determined correlations between the protein oxidation index and gelatinization property index. The results showed that significant oxidation of gliadin and glutelin occurred during the process of rice storage (P < 0.05). Protein oxidation had a significant impact on pasting properties. For glutelin, changes in the structure and function of the protein during rice storage had a significant impact on the five rice pasting property indexes, including peak viscosity (PV), holding strength (HS), breakdown, final viscosity (FV), and setback (SB; P < 0.05). In particular, the levels of carbonyl compounds and active sulfur and the surface hydrophobicity of glutelin were significantly correlated with rice pasting property indexes (P < 0.05). However, gliadin only significantly affected three indexes, i.e., PV, HS, and FV (P < 0.05). Thus, these findings suggested that the carbonyl compounds, active sulfur, and surface hydrophobicity of glutelin could be used as sensitive indexes for changes in rice quality evaluation.

Ferritin, cellular iron storage and regulation.

Ferritin is considered the major iron storage protein which maintains a large iron core in its cavity and has ferroxidase activity. There are many types of ferritin particularly in prokaryotes that include the canonical 24-mer FTN molecules, the heme-containing BFR, the smaller 12-mer DPS and the newly recognized EncFtn of encapsulin that forms a very large iron storage compartment. Recent studies show that ferritin function is more dynamic than previous depicted and new mechanisms of ferritin iron recycling are emerging. They participate to the regulation of cellular iron homeostasis as those of ferritin biosynthesis, cooperating also with the iron-dependent mechanism of cellular iron secretion. Some of these basic processes are in common between unicellular and animal cells, and this review aims at discussing the findings on the connections between iron storage, cellular iron regulation and ferritin iron recycling that have been explored in unicellular organisms and in animals. © 2017 IUBMB Life, 69(6):414-422, 2017.

Identification, characterization and modulation of ferritin-H in the sub-Antarctic Notothenioid Eleginops maclovinus challenged with Piscirickettsia salmonis

Ferritin is a major iron storage protein essential not only in the infectious process, but also in any circumstance generating oxidative stress. In this study, the cDNA coding sequence of ferritin-H was obtained from the sub-Antarctic Notothenioid fish Eleginops maclovinus through transcriptomic analysis of the head kidney. This sequence contained a 534 bp open reading frame that coded for a 177 amino acid protein with a molecular weight of 20,786.2 Da and a theoretical pI of 5.56. The protein displayed a region of iron putative response elements in the 5′UTR, two putative ferritin iron-binding region signatures, and seven characteristic amino acids with ferroxidase functions. Phylogenetic analysis related this sequence to ferritin-H sequences of other Antarctic Notothenioid fish, sharing 96.61% similarity. Constitutive gene expression analysis in different organs revealed increased ferritin-H gene expression in the gills, spleen, muscle, and liver. After infection with two bacterial strains of Piscirickettsia salmonis (LF-89 and Austral-005), ferritin-H was differentially expressed depending on bacterial strain and tissue. This study provides relevant information towards understanding the iron metabolism of a sub-Antarctic Notothenioid fish.

Dioscorea alata tuber proteome analysis shows over thirty dioscorin isoforms and novel tuber proteins

In Dioscorea, dioscorin (31 kDa) is the major storage protein constituting 85% of the total tuber proteins. An integrated proteomic and biochemical approach was used to understand the physiological role of dioscorin in the two contrasting growth stages (germinating and mature tuber). HPLC analysis showed 3 fold reduction in mannitol and 12.88 and 1.24 fold increase in sucrose and maltose in the germinating tuber. A 1.8 and 3 fold increase in sucrose phosphate synthase and mannitol dehydrogenase activity respectively was observed in the germinating tuber while a 2 fold higher invertase probably lowers the sucrose accumulation in the mature tuber. SDS-PAGE and 2-D maps of the mature and germinating tubers confirmed depletion (more than 50%) of dioscorin on germination. Dioscorin was purified using ion exchange and gel filtration chromatography with 43.32 fold purification and 38.16 yield. Out of a trail of 35 spots at 31 kDa only 12 spots (identified as dioscorin isoforms) were present in the 2D gel of the purified fraction. To search for other tuber proteins besides dioscorin, the unbound fractions of DEAE column were analysed by 2DGE. DREB 1A, caffeic acid 3-O-methyltransferase and Rab-1 small GTP binding protein were identified perhaps for the first time in the Dioscorea proteome. The interactome analysis revealed these to be involved in oxidative stress, carotenoid synthesis and vesicular transport. This is perhaps the first attempt to identify tuber proteome (although limited) and to understand the physiological significance of these proteins.

Structure, composition and functional properties of storage proteins extracted from bambara groundnut (Vigna subterranea) landraces

AbstractBambara groundnut is a protein‐rich traditional legume. In this study, storage proteins were isolated from three bambara landraces. Bambara protein revealed four major protein bands: one broad band at 55 kDa, two medium bands at 62 kDa and 80 kDa and a high molecular weight (HMW) protein at 141 kDa. The vicilin (7S) subunits with molecular weight of 55 kDa and 62 kDa were major fractions in bambara storage proteins. Bambara proteins showed two endothermic peaks ranging from 64 to 69 °C and 76 to 90 °C, respectively. Bambara protein isolates had well‐defined tertiary and secondary structures, respectively, at pH 3.0, and this well‐defined structure decreased slightly at higher pH values. The isolates revealed a strong secondary structure dominated by α‐helical conformation. Foaming capacities of bambara proteins were dependent on pH with maximum percentage FC observed at pH 3.0, while the emulsion activity increased with increasing pH for all the isolates. Vicilin (7S) fraction seems to be the major storage protein fraction of bambara. Bambara proteins could serve as excellent ingredients for the formulation of food foams and emulsions.

Functional ferritin nanoparticles for biomedical applications.

Ferritin, a major iron storage protein with a hollow interior cavity, has been reported recently to play many important roles in biomedical and bioengineering applications. Owing to the unique architecture and surface properties, ferritin nanoparticles offer favorable characteristics and can be either genetically or chemically modified to impart functionalities to their surfaces, and therapeutics or probes can be encapsulated in their interiors by controlled and reversible assembly/disassembly. There has been an outburst of interest regarding the employment of functional ferritin nanoparticles in nanomedicine. This review will highlight the recent advances in ferritin nanoparticles for drug delivery, bioassay, and molecular imaging with a particular focus on their biomedical applications.

Rational identification of a novel soy-derived anxiolytic-like undecapeptide acting via gut-brain axis after oral administration

Here we found that the chymotryptic digest of soy β-conglycinin, a major storage protein, exhibited anxiolytic-like effects in mice. We then searched for anxiolytic-like peptides in the digest. Based on a comprehensive peptide analysis of the chymotryptic digest by high performance liquid chromatograph connected to an LTQ Orbitrap mass spectrometer and the structure-activity relationship of known peptides, we explored anxiolytic-like peptides present in the digest. FLSSTEAQQSY, which corresponds to 323–333 of the β-conglycinin α subunit [βCGα(323–333)] emerged as a candidate. Oral administration of synthetic βCGα(323–333) exhibited anxiolytic-like effects in the elevated plus-maze and open-field test in male mice. Orally administered βCGα(323–333) exhibited anxiolytic-like effects in sham-operated control mice but not in vagotomized mice. In addition, oral administration of βCGα(323–333) increased the expression of c-Fos, a marker of neuronal activity, in the nucleus of the solitary tract, which receives inputs from the vagus nerve. These results suggest that the anxiolytic-like effects were mediated by the vagus nerve. The anxiolytic-like effects of βCGα(323–333) were also blocked by antagonists of the serotonin 5-HT1A, dopamine D1 and GABAA receptors. However βCGα(323–333) had no affinity for these receptors, suggesting it stimulates the release of endogenous neurotransmitters to activate the receptors. Taken together, a soy-derived undecapeptide, βCGα(323–333), may exhibit anxiolytic-like effects after oral administration via the vagus nerve and 5-HT1A, D1 and GABAA systems.

Physicochemical properties of kafirin protein and its applications as building blocks of functional delivery systems.

Kafirin, the major storage protein from grain sorghum, belongs to the prolamin protein family with its characteristic properties of being soluble in aqueous alcohol solutions, high proline content and high yield of ammonia upon hydrolysis. Due to its inferior nutritional quality and poor digestive performance in the human digestive tract, research efforts in recent years have been directed to its applications as novel value-added biomaterials. Recent studies revealed its unique physical properties of high hydrophobicity, good solubility in aqueous alcohol and acetic acid, elongated conformation and the ability to self-assemble into biomaterials with controlled morphology. The present review summarized the current understanding of kafirin protein in terms of its extraction, composition, structure as well as self-assembly behavior. Recent applications utilizing kafirin as an ingredient in gluten-free formulations and as novel building block for functional delivery systems are discussed. Kafirin has shown promising potential as a gluten-free ingredient and a novel value-added biodegradable polymer. Further studies are required not only to overcome the challenges of large-scale kafirin production, but also to enrich our understanding of its structural changes in different solvents and its interactions with other components to broaden its application spectrum.

Crystal Structure of Colocasia esculenta Tuber Agglutinin at 1.74 Å Resolution and Its Quaternary Interactions

Many members of the monocot mannose-binding lectin family, characterized by specificity towards mannose have been characterized and cloned. A majority of these lectins molecules contain 1-4 polypeptides of about 110 residues each. From the previously solved crystal structures of a few such lectins, mostly from non-edible plants, these lectins are thought to possess a common β-prism II fold structure. The major tuber storage protein of Colocasia esculenta is a monocot mannose-binding, widely used, dietary lectin. This tuber agglutinin contains two polypeptides of 12.0 and 12.4 kDa by matrix assisted laser desorption ionisation time-of-flight mass spectrometry. By gel filtration at pH 7.2, the purified lectin has a α2β2 form of apparent molecular mass of 48.2 kDa in solution but at pH 3, it has the heterodimeric αβ form. Lectin crystals were obtained by hanging-drop, vapor-diffusion method at room temperature and high-resolution X-ray diffraction data were collected using a home X-ray source. Among previously solved crystal structures of this family are garlic, Solomon’s seal, snowdrop, daffodil and Spanish blue-bell lectins, but the protein sequence of the Colocasia esculenta tuber agglutinin was found to be closest to that of the Remusatia vivipara lectin having no simple mannose-binding property. Using the previously solved 2.4A crystal structure of the Remusatia vivipara lectin, that of Colocasia esculenta has been solved by molecular replacement and subsequent crystallographic refinement and root mean square deviations between various lectins are tabulated and rationalized. The asymmetric unit in our lectin crystal structure contains four β-prism II domains or two αβ heterodimers, each forming a α2β2 heterotetramer with a symmetry related unit. The tetrameric interface obtained from our crystal structure is used to explain the conversion to dimers in acidic pH. Five ordered magnesium ions were located in the asymmetric unit and the presence of magnesium verified by atomic absorption spectroscopy.

Magnetic mapping of iron in rodent spleen

Evaluation of iron distribution and density in biological tissues is important to understand the pathogenesis of a variety of diseases and the fate of exogenously administered iron-based carriers and contrast agents. Iron distribution in tissues is typically characterized via histochemical (Perl's) stains or immunohistochemistry for ferritin, the major iron storage protein. A more accurate mapping of iron can be achieved via ultrastructural transmission electron microscopy (TEM) based techniques, which involve stringent sample preparation conditions. In this study, we elucidate the capability of magnetic force microscopy (MFM) as a label-free technique to map iron at the nanoscale level in rodent spleen tissue. We complemented and compared our MFM results with those obtained using Perl's staining and TEM. Our results show how MFM mapping corresponded to sizes of iron-rich lysosomes at a resolution comparable to that of TEM. In addition MFM is compatible with tissue sections commonly prepared for routine histology.

Effective intercalation of zein into Na-montmorillonite: role of the protein components and use of the developed biointerfaces.

Biohybrid materials based on the intercalation of zein, the major storage protein in corn, into sodium-exchanged montmorillonite were prepared following two synthesis strategies. The first one made use of zein dissolved in 80% (v/v) ethanol/water solution, the usual solvent for this protein, while the second method is new and uses a sequential process that implies the previous separation of zein components in absolute ethanol. This treatment of zein with ethanol renders a soluble yellow phase and an agglomerate of insoluble components, which are able to intercalate the layered silicate when an aqueous dispersion of montmorillonite is added to the ethanol medium containing both phases. The diverse steps in this second route were investigated individually in order to understand the underlying mechanism that drives to the intercalation of this complex hydrophobic biomacromolecule into the hydrophilic interlayer space of sodium-exchanged montmorillonite. In addition to physicochemical characterization of the resulting materials, these biohybrid interfaces were also evaluated as biofillers in the preparation of diverse ecofriendly nanocomposites.

Impact of Electron Beam on Storage Protein Subunits, In Vitro Protein Digestibility and Trypsin Inhibitor Content in Soybean Seeds

The present investigation was carried out to study the effect of 4.8, 9.2, 15.3 and 21.2 kGy of electron beam (EB)-irradiation on major storage proteins viz. glycinin (11S) and β-conglycinin (7S), in vitro protein digestibility (IVPD), and trypsin inhibitor (TI) content in seeds of three soybean genotypes. Densitometry of SDS-PAGE protein profile revealed significant (P < 0.05) reduction in α’, α and β subunits of 7S fraction at all doses. This reduction was higher (P < 0.05) than the decline observed in acidic and basic subunit of 11S fraction. Basic subunit registered significant (P < 0.05) increase at specific doses in two genotypes. All the doses induced significant (P < 0.05) increase in IVPD, and the increase due to 9.2, 15.3 and 21.2 kGy was higher (P < 0.05) compared to 4.8 kGy dose. The impact on TI content was genotype-dependent. The study showed a higher (P < 0.05) decline in the concentration of 7S fraction compared to 11S fraction and improvement in IVPD of soybean seeds due to exposure to EB-irradiation, which may influence the functional and nutritional value of soy products processed from them.

Proteome rebalancing in transgenic Camelina occurs within the enlarged proteome induced by β-carotene accumulation and storage protein suppression

Oilseed crops are global commodities for their oil and protein seed content. We have engineered the oilseed Camelina sativa to exhibit increased protein content with a slight decrease in oil content. The introduction of a phytoene synthase gene with an RNAi cassette directed to suppress the storage protein 2S albumin resulted in seeds with an 11-24% elevation in overall protein. The phytoene synthase cassette alone produced enhanced β-carotene content of an average 275±6.10μg/g dry seed and an overall altered seed composition of 11% less protein and comparable nontransgenic amounts of both oil and carbohydrates. Stacking an RNAi to suppress the major 2S storage protein resulted in seeds that contain elevated protein and slight decrease in oil and carbohydrate amounts showing that Camelina rebalances its proteome within an enlarged protein content genotype. In both β-carotene enhanced seeds with/without RNAi2S suppression, the seed size was noticeably enlarged compared to nontransgenic counterpart seeds. Metabolic analysis of maturing seeds indicate that the enhanced β-carotene trait had the larger effect than the RNAi2S suppression on the seed metabolome. The use of a GRAS (generally regarded as safe) β-carotene as a visual marker in a floral dip transformation system, such as Camelina, might eliminate the need for costly regulatory and controversial antibiotic resistance markers. β-carotene enhanced RNAi2S suppressed Camelina seeds could be further developed as a rapid heterologous protein production platform in a nonfood crop leveraging its enlarged protein content and visual marker.

Morphology of the ferritin iron core by aberration corrected scanning transmission electron microscopy

As the major iron storage protein, ferritin stores and releases iron for maintaining the balance of iron in fauna, flora, and bacteria. We present an investigation of the morphology and iron loading of ferritin (from equine spleen) using aberration-corrected high angle annular dark field scanning transmission electron microscopy. Atom counting method, with size selected Au clusters as mass standards, was employed to determine the number of iron atoms in the nanoparticle core of each ferritin protein. Quantitative analysis shows that the nuclearity of iron atoms in the mineral core varies from a few hundred iron atoms to around 5000 atoms. Moreover, a relationship between the iron loading and iron core morphology is established, in which mineral core nucleates from a single nanoparticle, then grows along the protein shell before finally forming either a solid or hollow core structure.

Enhancing reducing ability of α-zein by fibrillation for synthesis of Au nanocrystals with continuous flow catalysis

Green low-cost synthesis and efficient recyclability are two major hindrances for Au nanocrystals as catalysts applying in diverse industrial reaction processes. By the use of low-cost α-zein (i.e. a major storage protein of corn) as the reductant, capping agent and stabilizer, Au nanocrystals with tunable catalytic activity were synthesized in a wet-chemical approach. Fibrillation of α-zein further enhanced its reducing ability due to larger specific surface area and more hydrophilic groups exposed on the surfaces. The obtained Au nanocrystals had biocompatibility, high stability in various solvents, unique solubility in aqueous alcohol and high catalytic ability, being able to detect ethanol composition in aqueous ethanol as well as H2O2 for diagnosis of diabetes mellitus. These advantages also enable efficient recyclability of Au nanocrystals with continuous flow catalysis in different solvents and environments. Thus, the use of α-zein offered Au nanocrystals not only with green low-cost synthesis, but also with tunable catalytic activities, ethanol-responsiveness and efficient recyclability, which may be applicable in diverse fields.

Composition of Proteins Extracted from Two Species of Leguminous Bauhinia Grains

There is a growing demand for alternative protein sources for industrial applications. Bauhinia are underutilized protein‐rich oilseed legumes primarily grown as ornamental plants. In this study, the composition and structure of proteins extracted from two Bauhinia species, B. galpinii and B. petersiana, were determined in comparison with soya bean. Albumin and globulin (75%) were the major storage proteins of Bauhinia. Albumin content of B. galpinii was substantially high (28%), about twice those of soya and B. petersiana. Bauhinia grains were good sources of lysine (approximately 4 g/100 g of protein). By SDS‐PAGE, Bauhinia proteins showed three major protein bands ranging from 17,000 to 76,000 for both species. Only one subunit (17,000) of the basic glycinin (11S) seemed to be present in Bauhinia compared with soya. Bauhinia proteins had more β‐sheet conformation (42%) than α‐helix (22%). Bauhinia grains may provide a new opportunity of protein for industrial applications.

Structural Properties of Cruciferin and Napin of Brassica napus (Canola) Show Distinct Responses to Changes in pH and Temperature.

The two major storage proteins identified in Brassica napus (canola) were isolated and studied for their molecular composition, structural characteristics and the responses of structural features to the changes in pH and temperature. Cruciferin, a complex of six monomers, has a predominantly β-sheet-containing secondary structure. This protein showed low pH unstable tertiary structure, and distinctly different solubility behaviour with pH when intact in the seed cellular matrix. Cruciferin structure unfolds at pH 3 even at ambient temperature. Temperature-induced structure unfolding was observed above the maximum denaturation temperature of cruciferin. Napin was soluble in a wider pH range than cruciferin and has α-helices dominating secondary structure. Structural features of napin showed less sensitivity to the changes in medium pH and temperature. The surface hydrophobicity (S0) and intrinsic fluorescence of tryptophan residue appear to be good indicators of cruciferin unfolding, however they were not the best to demonstrate structural changes of napin. These two storage proteins of B. napus have distinct molecular characteristics, therefore properties and functionalities they provide are contrasting rather than complementary.