Jessica Capraro

Spectroscopic studies on the pH-dependent structural dynamics of γ-conglutin, the blood glucose-lowering protein of lupin seeds

γ-Conglutin is a blood glucose-lowering protein purified from lupin (Lupinus albus, L.) seed. Despite various features of this protein have already been studied, no function in the seed nor any mechanism of action as a hypoglycemic nutraceutical compound have been identified so far. The lupin protein was shown to exist both in monomeric and multimeric forms as a function of pH. However, a detailed description of the pH-dependent structural dynamics of this protein, as the basis to investigate the reason/s of its functional behaviour, is not available yet. In this study, multiple and independent spectroscopic approaches, including light scattering associated to size exclusion chromatography of both untreated and covalently cross-linked protein, near and far UV circular dichroism, intrinsic and extrinsic fluorescence measurements, have been used to monitor oligomeric and conformational modifications caused by pH changes. Altogether, the results revealed a tetramer-dimer-monomer transition between neutral to slightly acidic pH and a dramatic and abrupt conformational change below pH 3.5. According to these findings, a model depicting γ-conglutin structural dynamics was drawn. This model highlights the primary role of amino acid side group electrostatic interactions in the oligomer association/dissociation equilibria and in the pH-driven collapse of the native conformation.

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.

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.

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

Abstract Cowpea seed β-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 β-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. Using a shorted protocol, -vignin with a high degree of purity was obtain. A glycolylated gene product, never identified before, and its metal binding capacity were identified.

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

Abstract 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.

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.

COMPARATIVE 1D- AND 2D-ELECTROPHORETIC PROTEIN PROFILES OF ANCESTRAL AND MODERN BUCKWHEAT SEEDS GROWN IN THE ITALIAN ALPINE REGION

Buckwheat is an old crop whose seeds are under-utilized. The protein composition of these seeds, however, makes them suitable as much needed ingredients for the production of gluten-free products. Several buckwheat species and local cultivars are known worldwide. In this work, 1D and 2D electrophoresis were used to characterize and compare the seed protein profiles of two buckwheat species (Fagopyrum esculentum and Fagopyrum tataricum). The two analyzed cultivars of F. esculentum represent authentic landraces of an Italian Alpine valley, named Valtellina. The protein profiles of F. tataricum and the two F. esculentum cultivars did not show major differences. However, narrow but significant differences were present between these two landraces, allowing their discrimination at protein level. This work represents a molecular-based approach to the designation of origin and authenticity of local buckwheat varieties and their tracing in flours for human food.

Effect of omnivorous and vegan diets with different protein and carbohydrate content on growth and metabolism of growing rats

Abstract The purpose of this study was to observe, in a rat animal model, the short and medium term effects of vegan (VEG) or omnivorous (OMNI) diets with different energy partition between nutrients (zone or classic). Six different diets were administered, for 72 days to 120 growing male Sprague-Dawley rats: (i) VEG zone diet; (ii) VEG classic diet; (iii) OMNI zone diet; (iv) OMNI classic diet; (v) OMNI zone diet with added fibre and (vi) OMNI classic diet with added fibre. Zone diets (high protein and low carbohydrates), resulted in better growth , feed efficiency, lower blood glucose and insulin responses. VEG diets have lowered cholesterol blood level. Histopathological analysis evidenced no damage to liver and kidney tissue by the intake of any of the diet types. Further longer animal and human duration studies should be performed to exclude detrimental effect of higher protein diet.

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 p2Hs 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.

Cysteine-containing peptides are produced by sequential clipping, but not released, from lupin 11S storage globulin during early germination

Abstract The digestion of the seed storage proteins is a finely regulated process operated by several proteases whose action is influenced by the exposure of specific regions, which became progressively available upon their action. We focused our study on the initial stages of germination, where more subtle modifications to the storage proteins are expected. Small-size peptides containing cysteine residues and other possible metalbinding regions are de facto produced but are not released from the “parental” protein since they remain bound trough disulphide bridges. The meaning of these findings is discussed.