Interaction of γ-conglutin from Lupinus albus with model phospholipid membranes: Investigations on structure, thermal stability and oligomerization status.

Interaction with model phospholipid membranes of lupin seed γ-conglutin, a glycaemia-lowering protein from Lupinus albus seeds, has been studied by means of Fourier-Transform infrared spectroscopy at p2H 7.0 and at p2H 4.5. The protein maintains the same secondary structure both at p2H 7.0 and at p2H 4.5, but at p2H 7.0 a higher 1H/2H exchange was observed, indicating a greater solvent accessibility. The difference in Tm and TD1/2 of the protein at the abovementioned p2H's has been calculated around 20 °C. Infrared measurements have been then performed in the presence of DMPG and DOPA at p2H 4.5. DMPG showed a little destabilizing effect while DOPA exerted a great stabilizing effect, increasing the Tm of γ-conglutin at p2H 4.5 of more than 20 °C. Since γ-conglutin at p2H 4.5 is in the monomeric form, the interaction with DOPA likely promotes the oligomerization even at p2H 4.5. Interaction between DMPG or DOPA and γ-conglutin has been confirmed by turbidity experiments with DMPC:DMPG or DOPC:DOPA SUVs. Turbidity data also showed high-affinity binding of γ-conglutin to anionic SUVs made up with DOPA. The molecular features outlined in this study are relevant to address the applicative exploitation and to delineate a deeper comprehension of the natural functional role of γ-conglutin.

New molecular features of cowpea bean (Vigna unguiculata, l. Walp) ß-vignin.

Cowpea seed ß-vignin, a vicilin-like globulin, proved to exert various health favourable effects, including blood cholesterol reduction in animal models. The need of a simple scalable enrichment procedure for further studies for tailored applications of this seed protein is crucial. A chromatography-independent fractionation method allowing to obtain a protein preparation with a high degree of homogeneity was used. Further purification was pursued to deep the molecular characterisation of ß-vignin. The results showed: (i) differing glycosylation patterns of the two constituent polypeptides, in agreement with amino acid sequence features; (ii) the seed accumulation of a gene product never identified before; (iii) metal binding capacity of native protein, a property observed only in few other legume seed vicilins.

Structural and functional insights into the basic globulin 7S of soybean seeds by using trypsin as a molecular probe.

The basic 7S globulin (Bg7S) is one of the major globulins of soybean seeds. Despite its dual subunit composition and oligomeric assembly, Bg7S has a compact 3D structure (PDB: 3AUP) which is stabilized by a network of inter- and intra-chain disulphide bridges. Bg7S shares several structural elements with a number of homologous proteins from other seeds, whose function is still uncertain. In this work, Bg7S native conformation was probed by using the proteolytic enzyme trypsin. In spite of the presence of many arginine and lysine residues, the protein resulted extremely recalcitrant to in vitro enzymatic cleavage. Indeed, only two scissile bonds located near the C- and N-termini of the large and small subunits, respectively, were cleaved. The partially cleaved products were stable even at prolonged incubation times. Although the generated small peptide fragments were not covalently bound to the remnant of the main chains, they were held in place, as assessed by denaturing and non-denaturing chromatographic approaches. Moreover, both the already observed pH-dependent association/dissociation behaviour of the protein and its insulin binding capacity were preserved both at neutral and acidic pH values. These results are in line with the growing view that the degradation of seed proteins, either storage and non-storage, may be a controlled process related to specific functionalities.

Enhanced vitamin B12 production in an innovative lupin tempeh is due to synergic effects of Rhizopus and Propionibacterium in cofermentation.

Fermentation represents a valuable and cost-effective approach for food stabilisation and nutritional improvement. Tempeh is an example of soybean solid-state fermentation. In this work, we investigated the possibility of producing a tempeh analogue containing high amounts of vitamin B12 using seeds of three different species of the legume lupin, namely Lupinus albus, L. angustifolius and L. mutabilis, with Rhizopus oligosporus and Propionibacterium freudenreichii cofermentation. Synergic effects of Rhizopus and Propionibacterium in increasing vitamin B12 up to 1230 ng/g dw was observed. These findings indicate that this cofermentation can improve lupin nutritional quality and safety to provide a tempeh analogue with added value for vegan and vegetarian communities and low-income populations. The level of potentially toxic lupin alkaloids was also monitored during the tempeh preparation.

Structural basis of the lack of endo-glucanase inhibitory activity of Lupinus albus γ-conglutin.

Lupin γ-conglutin and soybean BG7S are two legume seed proteins strongly similar to plant endo-ß-glucanases inhibitors acting against fungal GH11 and GH12 glycoside hydrolase. However these proteins lack inhibitory activity. Here we describe the conversion of lupin γ-conglutin to an active inhibitor of endo-ß-glucanases belonging to GH11 family. A set of γ-conglutin mutants was designed and expressed in Pichia pastoris, along with the wild-type protein. Unexpectedly, this latter was able to inhibit a GH11 enzyme, but not GH12, whereas the mutants were able to modulate the inhibition capacity. In lupin, γ-conglutin is naturally cleaved in two subunits, whereas in P. pastoris it is not. The lack of proteolytic cleavage is one of the reasons at the basis of the inhibitory activity of recombinant γ-conglutin. The results provide new insights about structural features at the basis of the lack of inhibitory activity of wild-type γ-conglutin and its legume homologues.

The future of lupin as a protein crop in Europe.

Europe has become heavily dependent on soya bean imports, entailing trade agreements and quality standards that do not satisfy the European citizen's expectations. White, yellow, and narrow-leafed lupins are native European legumes that can become true alternatives to soya bean, given their elevated and high-quality protein content, potential health benefits, suitability for sustainable production, and acceptability to consumers. Nevertheless, lupin cultivation in Europe remains largely insufficient to guarantee a steady supply to the food industry, which in turn must innovate to produce attractive lupin-based protein-rich foods. Here, we address different aspects of the food supply chain that should be considered for lupin exploitation as a high-value protein source. Advanced breeding techniques are needed to provide new lupin varieties for socio-economically and environmentally sustainable cultivation. Novel processes should be optimized to obtain high-quality, safe lupin protein ingredients, and marketable foods need to be developed and offered to consumers. With such an integrated strategy, lupins can be established as an alternative protein crop, capable of promoting socio-economic growth and environmental benefits in Europe.

Hypocholesterolaemic effect of rat-administered oral doses of the isolated 7S globulins from cowpeas and adzuki beans.

The role of seed proteins, especially soyabean 7S globulins, in controlling dyslipidaemia is widely acknowledged. Amino acid sequence homology among the proteins of this family could reflect similar biological functions in other species. The aim of the present study was to unveil a hypolipidaemic effect of the 7S globulins from cowpeas (7S-C) and adzuki beans (7S-A), administered orally to rats fed a hypercholesterolaemic (HC; high cholesterol and TAG) diet for 28 d. A total of forty-five rats were divided into five groups (nine rats per group): (1) standard (STD) diet; (2) HC diet; (3) HC diet + 7S-C (300 mg/kg per d); (4) HC diet + 7S-A (300 mg/kg per d); and (5) HC diet + simvastatin (SVT; 50 mg/kg per d), as a control. Significant decreases in food intake and final body weight of rats receiving HC + 7S-C and HC + 7S-A diets compared with groups fed the HC and STD diets were observed. Significant decreases in serum total and non-HDL-cholesterol of 7S-C, 7S-A and SVT groups were also observed. HDL-cholesterol levels increased in the 7S-C, 7S-A and SVT groups, while hepatic cholesterol and TAG concentrations were significantly lower than in the HC diet group for the 7S-C-supplemented group only. Faecal excretions of fat and cholesterol in HC diet groups were considerably higher in animals consuming the 7S globulins. The results show that cowpea and adzuki bean 7S globulins promote cholesterol-decreasing effects in hypercholesterolaemic rats even at low dosages, as already observed for other legume seed storage proteins of this family. This main effect is discussed in relation to the possible mechanisms of action.

Proteolytic cleavage at twin arginine residues affects structural and functional transitions of lupin seed 11S storage globulin.

The 11S storage globulin of white lupin seeds binds to a metal affinity chromatography matrix. Two unusual stretches of contiguous histidine residues, reminiscent of the multiple histidines forming metal binding motifs, at the C-terminal end of 11S globulin acidic chains were hypothesized as candidate elements responsible for the binding capacity. To prove this, the protein was incubated with a lupin seed endopeptidase previously shown to cleave at twin arginine motifs, recurrent in the sequence region of interest. Upon incubation with this enzyme, the loss of metal binding capacity paralleled that of the anti-his-tag reactive polypeptides. The recovered small proteolytic fragment was analyzed by mass spectrometry and N-terminal sequencing and found to correspond to the 24-mer region cleaved off at twin arginine residues and containing the natural his-tag-like region. Similarly, when lupin seeds were germinated for a few days, the his-tag containing 11S globulin chain was converted to a form devoid of such region, suggesting that this mechanism is a part of the natural degradatory process of the protein. The hypothesis that the ordered and controlled dismantling of storage proteins may generate peptide fragments with potential functional roles in plant ontogenesis is presented and discussed.

Lupin protein isolate versus casein modifies cholesterol excretion and mRNA expression of intestinal sterol transporters in a pig model.

BACKGROUND: Lupin proteins exert hypocholesterolemic effects in man and animals, although the underlying mechanism remains uncertain. Herein we investigated whether lupin proteins compared to casein modulate sterol excretion and mRNA expression of intestinal sterol transporters by use of pigs as an animal model with similar lipid metabolism as humans, and cellular cholesterol-uptake by Caco-2 cells. METHODS: Two groups of pigs were fed cholesterol-containing diets with either 230 g/kg of lupin protein isolate from L. angustifolius or 230 g/kg casein, for 4 weeks. Faeces were collected quantitatively over a 5 d period for analysis of neutral sterols and bile acids by gas chromatographically methods. The mRNA abundances of intestinal lipid transporters were analysed by real-time RT-PCR. Cholesterol-uptake studies were performed with Caco-2 cells that were incubated with lupin conglutin γ, phytate, ezetimibe or albumin in the presence of labelled [4-14C]-cholesterol. RESULTS: Pigs fed the lupin protein isolate revealed lower cholesterol concentrations in total plasma, LDL and HDL than pigs fed casein (P < 0.05). Analysis of faeces revealed a higher output of cholesterol in pigs that were fed lupin protein isolate compared to pigs that received casein (+57.1%; P < 0.05). Relative mRNA concentrations of intestinal sterol transporters involved in cholesterol absorption (Niemann-Pick C1-like 1, scavenger receptor class B, type 1) were lower in pigs fed lupin protein isolate than in those who received casein (P < 0.05). In vitro data showed that phytate was capable of reducing the uptake of labelled [4-14C]-cholesterol into the Caco-2 cells to the same extend as ezetimibe when compared to control (-20.5% vs. -21.1%; P < 0.05). CONCLUSIONS: Data reveal that the cholesterol-lowering effect of lupin protein isolate is attributable to an increased faecal output of cholesterol and a reduced intestinal uptake of cholesterol. The findings indicate phytate as a possible biofunctional ingredient of lupin protein isolate.

Pasta supplemented with isolated lupin protein fractions reduces body weight gain and food intake of rats and decreases plasma glucose concentration upon glucose overload trial.

The supplementation of foods with biologically active compounds can be a powerful approach for improving diet and well being. In this study we separately included in pasta matrices a concentrate of γ-conglutin, a glucose-lowering protein from Lupinus albus seeds, an isolate of the other main lupin storage proteins and ovalbumin, at a ratio corresponding to 125 mg of pure protein in 100 g of pasta. With these products we fed rats made hyperglycaemic, for 3 weeks. Among the most relevant changes measured in body and blood parameters were: (i) a significant reduction in food intake of rats fed γ-conglutin concentrate supplemented pasta and a significant limitation in the body weight increase in rats fed α, ß and δ-conglutin isolate supplemented pasta, while the food conversion indices were unchanged; (ii) a reduction in glycaemia upon glucose overload trial, especially in the γ-conglutin concentrate supplemented pasta fed animals, at a dose of 45 mg per kg body weight. The correlations among the measured parameters are discussed. Overall, the results evidence the potentiality of supplementing traditional foods with exogenous nutraceutical seed proteins to control body weight gain and glycaemia.

Internalisation and multiple phosphorylation of γ-Conglutin, the lupin seed glycaemia-lowering protein, in HepG2 cells.

Lupin seed γ-Conglutin is a protein capable of reducing glycaemia in mammalians and increasing glucose uptake by model cells. This work investigated whether γ-Conglutin is internalised into the target cells and undergoes any covalent change during the process, as a first step to understanding its mechanism of action. To this purpose, γ-Conglutin-treated and untreated HepG2 cells were submitted to confocal and transmission electron microscopy. Immune-revelation of γ-Conglutin at various intervals revealed its accumulation inside the cytosol. In parallel, 2D-electrophoresis of the cell lysates and antibody reaction of the blotted maps showed the presence of the protein intact subunits inside the treated cells, whilest no trace of the protein was found in the control cells. However, γ-Conglutin-related spots with an unexpectedly low pI were also observed in the maps. These spots were excised, trypsin-treated and submitted to MS/MS spectrometric analysis. The presence of phosphorylated amino acids was detected. These findings, by showing that γ-Conglutin is internalised by HepG2 cells in an intact form and is modified by multiple phosphorylation, open the way to the understanding of the lupin γ-Conglutin insulin-mimetic activity.

Characterization of the sensitization profile to lupin in peanut-allergic children and assessment of cross-reactivity risk.

BACKGROUND: Case reports of allergy to lupin, due to primary sensitization or cross-reactions with other legumes, are increasing as a consequence of the augmented use of lupin flour in bakery, pasta formulations and other food items. The main allergens that have been associated with the sensitization to lupin are α- and ß-conglutins and, to a lesser extent, γ- and δ-conglutin, but no conclusive data are available so far. The aim of this study was to characterize the sensitization pattern to lupin in a group of 12 Italian children allergic to peanut and identify the specific lupin proteins involved in the cross-reactivity with peanut. METHODS: The immunochemical cross-reactivity among peanut and lupin was evaluated by both in vitro immunoblotting and in vivo fresh food skin prick test (FFSPT). RESULTS: The results showed that ß-conglutin was recognized by cutaneous IgEs from 7/12 peanut-allergic children in FFSPT and serum IgEs from 5/12 in immunoblotting, while 4/12 and 8/12 patients tested positive to γ-conglutin in FFSPT and immunoblotting, respectively. No significant immunoreactive responses were observed to α- and δ-conglutins under non-reducing conditions, but they were bound in FFSPT by the sera of 5/12 and 3/12 patients, respectively. CONCLUSION: In this group of allergic children, ß-conglutin has been identified as the major lupin allergen involved both in vitro and in vivo cross-reactivity with peanut proteins. The role of γ-conglutin in the cross-reactivity between lupin and peanut proteins was also relevant and clear, despite the observed unspecificity of the immunoblotting responses.

The proteome of exudates from germinating Lupinus albus seeds is secreted through a selective dual-step process and contains proteins involved in plant defence.

The general knowledge of defence activity during the first steps of seed germination is still largely incomplete. The present study focused on the proteins released in the exudates of germinating white lupin seeds. During the first 24 h, a release of proteins was observed. Initially (i.e. during the first 12 h), the proteins found in exudates reflected the composition of the seed, indicating a passive extrusion of pre-formed proteins. Subsequently, when the rate of protein release was at its highest, the composition of the released proteome changed drastically. This transition occurred in a short time, indicating that more selective and regulated events, such as secretory processes, took place soon after the onset of germination. The present study considered: (a) the characterization of the proteome accumulated in the germinating medium collected after the appearance of the post-extrusion events; (b) the biosynthetic origin and the modalities that are the basis of protein release outside the seeds; and (c) an assessment of antifungal activity of these exudates. The most represented protein in the exudate was chitinase, which was synthesized de novo. The other proteins are involved in the cellular mechanisms responding to stress events, including biotic ones. This exudate was effectively able to inhibit fungal growth. The results of the present study indicate that seed exudation is a dual-step process that leads to the secretion of selected proteins and thus is not a result of passive leakage. The released proteome is involved in protecting the spermosphere environment and thus may act as first defence against pathogens.

Identification in lupin seed of a serine-endopeptidase activity cleaving between twin arginine pairs and causing limited proteolysis of seed storage proteins.

The occurrence of twin-arginine motifs (-R-R-) in the amino acid sequences of animal pro-proteins frequently defines the cleavage site(s) for their structural/functional maturation. No information is available on the presence and possible biological meaning of these motifs in the seed storage proteins. In this work, a novel endopeptidase activity with cleavage specificity to twin-arginine pairs has been detected in mature dry Lupinus albus seeds. The endopeptidase was tested with a number of endogenous and exogenous protein substrates, which were selected according to the presence of one or more twin-arginine residue motifs in their amino acid sequences. The observed hydrolysis patterns were limited and highly specific. Partial proteolysis led to stable polypeptide fragments that were characterized by 1- and 2-D electrophoresis. Selected polypeptides were submitted to N-terminal amino acid sequencing and mass spectrometry analyses. These approaches, supported by bioinformatic analysis of the available sequences, allowed the conclusion that the polypeptide cleavage events had occurred at the peptide bonds comprised between twin-arginine residue pairs with all tested protein substrates. The endopeptidase activity was inhibited by 4-(2-AminoEthyl)Benzene-Sulphonyl Fluoride hydrochloride (AEBSF), leupeptin, and serine proteinase protein inhibitors, while it was not affected by pepstatin, trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E64), and ethylenediaminetetraacetic acid (EDTA), thus qualifying the Arg-Arg cleaving enzyme as a serine endopeptidase. The structural features of storage proteins from lupin and other legume seeds strongly support the hypothesis that the occurrence of an endopeptidase activity cleaving -R-R- bonds may be functional to facilitate their degradation at germination and possibly generate polypeptide fragments with specific biological activity.

Lupin seed γ-conglutin lowers blood glucose in hyperglycaemic rats and increases glucose consumption of HepG2 cells.

The aim of the present study was to evaluate the effect of a chronic oral γ-conglutin treatment in male Sprague-Dawley rats in which hyperglycaemia had been induced by supplying 10 % d-glucose in drinking-water. A γ-conglutin dosage of 28 mg/kg body weight was daily administered to animals for 21 d. Plasma glucose, insulin and glucose overloading were monitored. Chronic administration of glucose resulted in a statistically significant (P < 0·05) increase in fasting blood glucose (2·5-fold) and insulin (2·7-fold) v. the values recorded in control rats. Simultaneous treatment with γ-conglutin attenuated the rise in plasma glucose (1·9-fold) and insulin (1·8-fold) levels in the glucose-fed rats (P < 0·05). Fasting insulin and homeostasis model of insulin resistance were decreased by 34 and 48 % (P < 0·05), respectively, in the γ-conglutin-treated rats v. the values found in pair-fed animals. To confirm these results with a different approach, HepG2 cells, grown for 24 and 48 h in Dulbecco's minimum essential medium containing different glucose concentrations (5·5, 11·1 and 16·5 mmol/l), were exposed to 10 µmol/l γ-conglutin with or without 10 mmol/l metformin or 100 nmol/l insulin. γ-Conglutin increased glucose consumption (from 1·5- to 2·5-fold) in HepG2 cells, under all experimental conditions; this effect was more evident after 48 h incubation. Moreover, in this in vitro model, the addition of γ-conglutin potentiated the activity of insulin and metformin in cell glucose consumption. These findings extend the previous ones and suggest the potential use of lupin γ-conglutin in the control of glycaemia.

Biochemical and functional characterization of an albumin protein belonging to the hemopexin superfamily from Lens culinaris seeds.

The present paper reports the purification and biochemical characterization of an albumin identified in mature lentil seeds with high sequence similarity to pea PA2. These proteins are found in many edible seeds and are considered potentially detrimental for human health due to the potential allergenicity and lectin-like activity. Thus, the description of their possible presence in food and the assessment of the molecular properties are relevant. The M(r), pI, and N-terminal sequence of this protein have been determined. The work included the study of (i) the binding properties to hemine to assess the presence of hemopexin structural domains and (ii) the binding properties of the protein to thiamin. In addition, the structural changes induced by heating have been evaluated by means of spectroscopic techniques. Denaturation temperature has also been determined. The present work provides new insights about the structural molecular features and the ligand-binding properties and dynamics of this kind of seed albumin.

Heterologous expression and purification of the soybean 7S globulin α' subunit extension region: in vitro evidence of its involvement in cell cholesterol homeostasis.

In a previous paper, the biological activity of a 216-amino acid recombinant truncated form of the soybean 7S globulin α' subunit, known to control cholesterol and triglyceride homeostasis, was described. In this work, a shorter version of the polypeptide chain, spanning 142 amino acid residues from the N-terminus and thus exclusively including the so-called extension region, was cloned and overexpressed in Pichia pastoris. The yield of the recombinant polypeptide, which was termed α'E, was 8-fold greater than the previous truncated version. The α'E polypeptide was purified by simple conventional biochemical techniques to make it available for biological assays. Human hepatoma cell lines (Hep G2) were used to monitor the uptake and degradation of labeled low-density lipoproteins (LDL), according to an established procedure. The LDL uptake (+86%) and degradation (+94%) by cells tested at the highest α'E dose (2 µM) were similar to those found in cells incubated with 1 µM simvastatin, a potent inhibitor of cholesterol biosynthesis. Additionally, the cell response to α'E was found to be dose-dependent. The present findings strongly suggest that this recombinant polypeptide, or a fragment thereof, is the molecular determinant for cholesterol homeostasis and open new prospects for understanding the mechanism involved in this biological response, as a gateway to its utilization in lipid-lowering therapies.

Hypoglycemic effect of lupin seed γ-conglutin in experimental animals and healthy human subjects.

A lupin seed γ-conglutin-enriched preparation was tested in a glucose overload trial with both murine models and adult healthy volunteers. The results with rats showed a dose-dependent significant decrease of blood glucose concentration, which confirmed previous findings obtained with the purified protein. Moreover, three test-product doses equivalent to 630, 315, and 157.5 mg γ-conglutin, orally administered 30 min before the carbohydrate supply, showed a relevant hypoglycemic effect in human trials. Insulin concentrations were not significantly affected. The general hematic parameters did not change at all. This is the first report on the glucose-lowering effect of lupin γ-conglutin in human subjects.

Molecular insight into IgE-mediated reactions to sesame (Sesamum indicum L.) seed proteins.

BACKGROUND: Food allergy is becoming a major public health concern in recent times. Several sesame seed allergenic proteins have been identified. However, sensitization toward these proteins does not follow a common and unique pattern of clinical reactivity, as shown by the differential geographic recognition of single proteins. OBJECTIVE: To evaluate the sensitization profiles of 18 Italian individuals who experienced clinical symptoms after sesame seed consumption, including 4 anaphylactic reactions. METHODS: Using an in vitro approach, we adopted a 2-dimensional electrophoretic technique combined with immunoblotting analyses by using sera from 18 Italian sesame-allergic patients. RESULTS: We showed the prevalent and almost exclusive reactivity of the sesame 11S globulin. We shed light on the active role of the basic subunit of this globulin family. The limited accessibility of this polypeptide chain, unless the interchain disulphide bonds are cleaved, may be one of the reasons for its structural/functional stability and, thus, great potential for induction of IgE reactivity. CONCLUSIONS: These results confirmed previous findings on the reactivity of the basic subunit of 11S globulin in various legume species. Moreover, this experimental approach proved to be useful for the noninvasive screening of specific reactivities in sensitized patients.

IgE-mediated cross-reactivity among leguminous seed proteins in peanut allergic children.

The immunological cross-reactivity among major protein- and oil-crops, including lupin, lentil, pea, peanut, kidney bean and soybean, has been studied by a combination of in vitro and in vivo experimental approaches: SDS-PAGE separations of legume protein extracts and immuno-blot revelations with 12 peanut-sensitive subjects' sera, Immuno-CAP and Skin Prick tests on the same subjects. The immuno-blotting data showed a wide range of IgE-binding responses both displayed by one subject towards different plant extracts and among subjects. Differences were both quantitative and qualitative. The prevalent responses of most subjects' sera were seen with peanut polypeptides, as expected, as well as with various polypeptides of the other legumes, the most recurrent of which were the basic subunits of the 11S globulins. The distribution of in vivo responses generally paralleled those obtained by in vitro approaches with strong responses elicited by peanut, lentil and pea protein extracts, especially by most sensitive subjects, thus providing a consistent overall set of results. In this work, the comparison of various approaches has allowed us to get an overall broad picture of the immunological cross-reactivities among proteins of widely used different seed species and to hypothesize the role of most conserved specific polypeptides.