Search

Lupin species provide essential nutrients and bioactive compounds. Within pulses, they have one of the highest contents of proteins and fibers and are among the poorest in carbohydrates. The Mediterranean region is an important cradle area of the origin and domestication of cultivated white lupin (Lupinus albus L.). In this work, we present the characterization of 19 white lupin landraces collected from several sites in southern Italy, characterized by different pedoclimatic conditions. The protein contents and electrophoretic patterns, total polyphenols, phytic acid, lipids and phosphorous content, and reducing and anti-tryptic activities have been determined for each landrace. The relationships of the compositional characteristics, the area of origin of landraces and between compositional characteristics and thermo-pluviometric trends that occurred in the genotype comparison field during the two-year period between 2019 and 2020 are compared and discussed. From a nutritional point of view, some of the analyzed landraces differ from the commercial reference. The panel of molecular analyses performed can help in building an identity card for the grain to rapidly identify those varieties with the desired characteristics. [ABSTRACT FROM AUTHOR]

Lupin γ-conglutin and soybean BG7S are two legume seed proteins strongly similar to plant endo-β-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-β-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: γ-Conglutin, a glycoprotein from Lupinus albus seed, has been characterized at molecular level but its physiological function is still unknown. γ-Conglutin shares a high structural similarity with xyloglucan-specific endo-β-1,4-glucanase inhibitor proteins (XEGIPs) and Triticum aestivum xylanase inhibitor (TAXI-I), which act specifically against fungal glycosyl hydrolase belonging to families 12 and 11, respectively. To assess the possible involvement of γ-conglutin in plant defense, germinating lupin seeds were incubated with chitosan. The relative quantification of γ-conglutin mRNA extracted from cotyledons was then carried out by RT-qPCR and indicated that chitosan strongly elicited the expression of γ-conglutin. Moreover, biochemical trials aimed to test the inhibitory capacity of the protein have been also carried out. γ-Conglutin failed to inhibit representative fungal endo-glucanases and other cell wall-degrading enzymes. To explain the lack of inhibitory capacity we investigated the possible structural differences between γ-conglutin and XEGIPs and TAXI-I, including the construction of a predictive 3D model of the protein. Bioinformatic analysis suggests that the lack of inhibitory activity of γ-conglutin can be attributed to sequence differences in the inhibitor interaction domains, and in particular to a sequence deletion in one of the functional loops. [Copyright &y& Elsevier]

Abstract: The paper describes the purification, structural characterization and inhibitory properties of a trypsin inhibitor from Lupinus albus L., a leguminous plant believed to be devoid of any protease inhibitor. The protein has been isolated by a newly set-up procedure and characterized by direct amino acid sequencing, MALDI-TOF mass spectroscopy and circular dichroism. Inhibitory properties toward bovine trypsin and chymotrypsin, as well as its thermal and pH stabilities, have been also assessed. The inhibitor is 63 amino acid long (M r 6858; pI 8.22) and it is capable to inhibit two trypsin molecules simultaneously, with a Kd of 4.2±0.4nM, but not chymotrypsin. BLAST search against UniProtKB/TrEMBL database indicates that the inhibitor belongs to the Bowman–Birk inhibitor (BBI) family. The interest in these serine-protease inhibitors arises from the ability to prevent or suppress carcinogen-induced transformation, as shown in various in vitro and in vivo model systems. [Copyright &y& Elsevier]

Abstract: Seed proteome analysis by 2D IEF/SDS–PAGE techniques is challenging for the intrinsic difficulties related to quantitative disparity of the seed proteins, i.e. storage and non-storage proteins, their polymorphic nature, the extensive post-translational modifications and the paucity of deposited primary structures available. Conversely, 2D maps of seed proteomes can be extremely useful for a number of fundamental and applied investigations. In this work, we have used a combination of two experimental approaches to identify the main protein components of an emerging protein-rich legume seed, that is white lupin seed (Lupinus albus, L.). One is the canonical proteomic approach including 2D electrophoretic separation and mass spectrometry of selected trypsin-digested polypeptides; the other approach is a group comparative 2D electrophoretic analysis of cotyledonary protein families. To this second purpose, the three main families of lupin seed proteins, namely α-conglutins, the 11S globulin fraction, β-conglutins, the 7S globulin fraction, and γ-conglutin, a basic 7S protein, were isolated by conventional biochemical techniques and their 2D reference maps were compared with the total protein map. With the first approach 37 out of 40 spots, making up about 35% of total spot volumes in the 2D map, were found to belong to the main seed protein families. Thanks to cDNA-deduced lupin storage protein sequences, determined on purpose and deposited, most of the identification statistical parameters were very good. Moreover, it was possible to identify several endogenously proteolysed subunits in the map. The second comparative approach, beside confirming these attributions, allowed to allocate 124 polypeptides within the three main lupin protein families. These two approaches proved to be mutually validating and their combined use was effective for the establishment of a seed proteome map even in the case of sequence and protein post-translational processing lack of information. The results obtained also extend our knowledge of the seed storage protein polymorphism of white lupin. [Copyright &y& Elsevier]

γ-conglutin (γC) is a major protein of Lupinus albus seeds, but its function is still unknown. It shares high structural similarity with xyloglucan-specific endo-glucanase inhibitor proteins (XEGIPs) and, to a lesser extent, with Triticum aestivum endoxylanase inhibitors (TAXI-I), active against fungal glycoside hydrolases GH12 and GH11, respectively. However, γC lacks both these inhibitory activities. Since β-galactomannans are major components of the cell walls of endosperm in several legume plants, we tested the inhibitory activity of γC against a GH2 β-mannosidase (EC 3.2.1.25). γC was actually able to inhibit the enzyme, and this effect was enhanced by the presence of zinc ions. The stoichiometry of the γC/enzyme interaction was 1:1, and the calculated Ki was 1.55 μM. To obtain further insights into the interaction between γC and β-mannosidase, an in silico structural bioinformatic approach was followed, including some docking analyses. By and large, this work describes experimental findings that highlight new scenarios for understanding the natural role of γC. Although structural predictions can leave space for speculative interpretations, the full complexity of the data reported in this work allows one to hypothesize mechanisms of action for the basis of inhibition. At least two mechanisms seem plausible, both involving lupin-γC-peculiar structures. [ABSTRACT FROM AUTHOR]

The search for bioactivities influencing the human wellbeing of food proteins and peptides is a topic of broad and current interest. γ-Conglutin (γC) is a lupin seed protein drawing remarkable pharmacological and/or nutraceutical interest, as it is able to reduce hyperglycemia in humans and animal models. The present work deepens our investigations to understand the molecular basis of the in vitro effects of γC by testing the possible metabolic effects on cultivated Caco-2 cells. γC and its derived peptides (obtained via simulated gastrointestinal digestion) did not influence the cell viability at incubation times up to 24 h. The incubation of cells with native or digested γC caused no detectable inflammation processes mediated by Nuclear Factor kappa B (NFκB). We checked if treatment with γC or its derived peptides can elicit the expression of two peptide transporters (Pept-1 and Htp-1) by using an RT-qPCR approach. Native γC caused the halving of Pept-1 expression compared to untreated cells, but this effect disappeared when γC was digested. Either native γC or γC peptides reduced the expression levels of Hpt-1. Finally, this work also sheds light on the possible structural modifications of γC that may occur in the gastrointestinal tract, using an in vitro simulated dispersed system with polystyrene nanoparticles (NPs). [ABSTRACT FROM AUTHOR]

γ-conglutin (γ-C) is a hexameric glycoprotein accumulated in lupin seeds and has long been considered as a storage protein. Recently, it has been investigated for its possible postprandial glycaemic regulating action in human nutrition and for its physiological role in plant defence. The quaternary structure of γ-C results from the assembly of six monomers in reversible pH-dependent association/dissociation equilibrium. Our working hypothesis was that the γ-C hexamer is made up of glycosylated subunits in association with not-glycosylated isoforms, that seem to have 'escaped' the correct glycosylation process in the Golgi. Here we describe the isolation of not-glycosylated γ-C monomers in native condition by two in tandem lectin-based affinity chromatography and the characterization of their oligomerization capacity. We report, for the first time, the observation that a plant multimeric protein may be formed by identical polypeptide chains that have undergone different post-translational modifications. All obtained considered, the results strongly suggest that the not-glycosylated isoform can also take part in the oligomerization equilibrium of the protein. • A not-glycosylated γ-C monomers from the native protein has been isolated for the first time. • Native γ-C is a hexamer made up by both glycosylated and not-glycosylated monomers. • The seed glycoprotein is formed by identical polypeptide chains that have undergone different PTMs. • The not-glycosylated polypeptide is able to take part in the oligomerization equilibrium of γ-C. [ABSTRACT FROM AUTHOR]

Abstract 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 T m and T D1/2 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 T m 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. Highlights • γ-conglutin when orally administered in humans and animals significantly decrease glycaemia. • Structure, thermal stability, interaction and oligomerization status with SUVs has been studied by spectroscopic method. • DOPA exerts a great stabilizing effect on thermal stability, probably by promoting oligomerization. • High-affinity binding of γ-conglutin to anionic SUVs with DOPA has been observed. • Stabilizing and protective effects of SUVs may be useful carriers for nutraceutical applications of γ-conglutin. [ABSTRACT FROM AUTHOR]

Highlights: [•] A glycaemia-reducing lupin seed protein is internalized by HepG2 cells. [•] The protein accumulates in the cytosol in an intact form. [•] The internalized protein is multiply phosphorylated. [ABSTRACT FROM AUTHOR]

Abstract: A lupin seed glycoprotein, termed γ-conglutin, has previously been found to display insulin-mimetic activity in myocyte models and reduce plasma glucose concentration when orally administered to both rats and humans. To envisage the possible metabolic fate of this bioactive protein, we used in vitro cell and ex vivo tissue models to monitor its transit through the intestinal barrier. Caco-2 cell monolayers and rat intestinal everted sacs were treated with purified γ-conglutin and the protein was immuno-assayed by chemi-luminescence-enhanced Western blotting. The in vitro approach showed that the intact protein can transit from the apical to the basolateral side of the cell monolayers. The unmodified lupin protein was also detected inside the intestinal everted sacs. Proper controls of cell monolayer and sac integrity ruled out the possibility of protein passive leakage. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]