Effect of different oligomerization assemblies of γ-conglutin on its interaction behavior with vitexin.

BACKGROUND: Several different factors underlie the molecular mechanisms of phenolic compound-protein interactions. They include the environmental conditions. In the case of γ-conglutin, pH conditions translate directly into the adoption of two distinct oligomeric assemblies, i.e. hexameric (pH 7.5) or monomeric (pH 4.5). This paper reports research on the pH-dependent oligomerization of γ-conglutin in terms of its ability to form complexes with a model flavonoid (vitexin). RESULTS: Fluorescence-quenching thermodynamic measurements indicate that hydrogen bonds, electrostatic forces, and van der Waals interactions are the main driving forces involved in the complex formation. The interaction turned out to be a spontaneous and exothermic process. Assessment of structural composition (secondary structure changes and arrangement/dynamics of aromatic amino acids), molecular size, and the thermal stability of the different oligomeric forms showed that γ-conglutin in a monomeric state was less affected by vitexin during the interaction. CONCLUSION: The data show precisely how environmental conditions might influence phenolic compound-protein complex formation directly. This knowledge is essential for the preparation of food products containing γ-conglutin. The results can contribute to a better understanding of the detailed fate of this unique health-promoting lupin seed protein after its intake. © 2023 Society of Chemical Industry.

Characteristics of N-Glycosylation and Its Impact on the Molecular Behavior of Lupinus angustifolius γ-Conglutin.

γ-Conglutin, a lupin seed protein, is an intriguing protein both in terms of the complexity of its molecular structure and a broad spectrum of unique health-promoting properties manifested in animal and human trials. Moreover, this protein is an evolutionary cornerstone whose physiological significance for the plant has not been determined yet. Herein, a comprehensive characterization of γ-conglutin glycosylation is presented and includes (i) the identification of the N-glycan-bearing site, (ii) the qualitative and quantitative composition of glycan-building saccharides, as well as (iii) the effect of oligosaccharide removal on structural and thermal stability. The obtained results indicate the presence of glycans belonging to different classes attached to the Asn98 residue. In addition, the detachment of the oligosaccharide significantly affects secondary structure composition, which disturbs the oligomerization process. The structural changes were also reflected in biophysical parameters, i.e., at a pH value of 4.5, an increase in γ-conglutin thermal stability was observed for the deglycosylated monomeric form. Collectively, the presented results provide evidence of the high complexity of the post-translational maturation and suggest the possibility of a functional effect that glycosylation might have on γ-conglutin structure integrity.

Influence of temperature on secretion and functionality of lupin seed γ-conglutin.

BACKGROUND: Basic 7S globulins, a group of proteins commonly found in legumes, undergo the intriguing phenomenon of release from the seeds into hot water. γ-Conglutin is a representative of this group of proteins found in lupin seeds. The physiological significance and the molecular mechanism of the selective release of γ-conglutin from lupin seeds remain unknown. Therefore, the presented study aimed to determine changes in the functionality of this protein in response to the high temperature occurring during lupin seed incubation. RESULTS: It was confirmed that the main protein fraction released from the seeds during high-temperature incubation was γ-conglutin. The incubation condition favours the occurrence of this protein in a monomeric form, and the temperature used corresponds to its midpoint unfolding temperature. Subsequent analysis carried out on the γ-conglutin monomer revealed changes in its functionality after heat shock. The thermally treated protein shows a considerable increase in its interaction strength with flavonoids. Moreover, the inhibitory activity against glycoside hydrolases was enhanced when γ-conglutin monomer was exposed to specific temperatures. CONCLUSION: The results of the present study provide a potential explanation of the physiological relevance of γ-conglutin and shed new light on a possible mechanism of its activation upon specific heat treatment. This knowledge will help characterise homologous proteins, which are commonly found in other legumes and undergo a similar heat-induced secretion phenomenon. © 2021 Society of Chemical Industry.

Heat-induced changes in lupin seed γ-conglutin structure promote its interaction with model phospholipid membranes.

A number of scientific data indicate that γ-conglutin can be internalised by different human cells and undergoes secretion from the seed in response to high temperature. In both of these cases, the protein must interact in some manner with biological membranes, however, the mechanisms underlying this phenomenon remain unknown. Herein, we found that the remarkable change of total surface hydrophobicity after appropriate heat treatment of γ-conglutin monomer led to its interaction with model membranes (liposomes). Before the interaction, the protein undergoes an intriguing thermal unfolding pattern which was studied based on a spectroscopic approach. Insight into the interaction mechanism with liposomes was possible thanks to applying two molecular probes that were differentially localised in the lipid bilayer. The results show that the thermal rearranged γ-coglutin monomer affects hydrocarbon chains in model membranes leading to their morphology change and disruption. The main driving force of this phenomenon is based on hydrophobic interaction.

Japanese Quince Seeds as a Promising Rich Source of Proteins and Essential Amino Acids.

The Japanese quince (Chaenomeles japonica) seeds (JQS) are generated in large amounts as a by-product in considerable quantities during industrial quince fruit processing. Due to economic, environmental, and nutritional reasons, the utilization of JQS as a protein source seems to be highly justified. Therefore, three cultivars were studied, 'Rasa', 'Rondo', and 'Darius' of JQS as a protein source during three harvesting years. The total protein content in JQS determined by the Kjeldahl method amounted from 24.6 to 33.2% and was significantly affected (p ≤ 0.05) by the cultivar and harvesting year. The protein isolation was most effective in the pH range of 6-9, and their profile was mainly characterized by a molecular weight of 54.1-57.7 kDa and 70.0-77.6 kDa. The amino acids profile of JQS turned out to be valuable from a nutritional point of view due to the relatively high content of essential and relatively essential amino acids such as histidine (6-9%), leucine (11-12%), lysine (10-16%), phenylalanine (12-16%), glutamine/glutamic acid (7-15%), and tyrosine (9-13%). The initial analysis of JQS indicates that they can be a good source of proteins characterized by a favorable amino acid profile.

Insight into thermally induced structural changes of lupin seed γ-conglutin.

A multidimensional analysis aimed to determine the thermal impact on γ-conglutin at the two oligomeric states was carried out. A wide range of biophysical and bioinformatic methods allowed to get insight into a thermal unfolding mechanism. The determined midpoint transition temperature (Tm) values were remarkably different, being 56.5 °C and 71.1 °C for γ-conglutin monomer and hexamer, respectively. The unfolding pattern for hexamer molecules included aggregation/precipitation, while monomers tended to form soluble aggregates after heat exposure. Interestingly, differences in the aromatic amino acid residues movements indicate that during thermal treatment of γ-conglutin hexamer red-shift occurred contrary to the monomer in the case of which blue-shift was noted. The obtained results provide an essential contribution to expand our knowledge about the molecular characterization of this intriguing lupin seed protein.

Lupin seeds storage protein composition and their interactions with native flavonoids.

BACKGROUND: Lupin-based food, due to the high content of functional proteins and phenolic compounds, are widely used in human nutrition. Unfortunately, proteins and phenolic compounds can easily interact with each other which results in formation of complexes that affect properties of both components. Therefore, in this study, composition of the seeds storage proteins isolated from Lupinus albus and L. angustifolius and their interactions with native flavonoids were investigated. RESULTS: Based on the chromatographic separations, six proteins fractions of lupin seeds storage proteins were identified. The results indicate that two dominant fractions, α-conglutin and ß-conglutin, constitute up to 80% of all proteins present in the seeds. Three flavonoids interacting with the proteins were identified as apigenin C-glycosides. The lowest flavonoids content was noted in the main storage proteins while in both lupin seeds species over 90% of flavonoids interacted with the proteins present in late-embryogenesis abundant (LEA) protein fraction. CONCLUSIONS: Protein-phenolic compound complexes can affect the digestibility of proteins and bioavailability of phenolic compounds, and thus the functional and nutritional properties of products derived from lupin seeds can be changed. Therefore, a better understanding of factors affecting the nutritional value of lupin seeds proteins and flavonoids is necessary to optimize the biological use of this plant for human nutrition. © 2019 Society of Chemical Industry.

Bioaccessibility of defatted lupin seed phenolic compounds in a standardized static in vitro digestion system.

Phenolic compounds are secondary plant metabolites whose beneficial health effects make them of intense interest to researchers. The aim of the study presented here was to evaluate the change in the phenolic compound profile of lupin seed in in vitro digestion. The most abundant phenolic compounds in undigested lupin seeds were mostly apigenin derivatives. The in vitro digestion of lupin seeds resulted in qualitatively altered phenolic compound profiles. Approximately 80% of phenolic compounds were released from lupin seeds during the in vitro digestion, which simulated gastric processes. Continued digestion, imitating the intestinal phase, additionally increased the bioaccessibility of lupin seed polyphenols by about 10%. The in vitro gastrointestinal model was also used to elucidate how the content of native phenolic compounds affects the digestion susceptibility of lupin seed proteins. An inverse correlation between protein digestibility and phenolic compound content, was also demonstrated.

Selected Uncommon Legumes as a Source of Essential Fatty Acids, Tocopherols, Tocotrienols, Sterols, Carotenoids, and Squalene.

The seeds of nine species belonging to the Fabaceae family (Cassia alata, Cassia uniflora, Cassia obtusifolia, Cassia tora, Crotalaria albida, Crotalaria juncea, Crotalaria pallida, Indigofera tinctoria, and Tephrosia purpurea) were studied. The oil yield ranged from 2.0 to 9.6% dw, in the seeds of I. tinctoria and T. purpurea, respectively. The unsaturated fatty acids (UFA) were the predominant group of fatty acids (41-74%) in the investigated samples. The sum of tocopherol homologues α and γ constituted 90-100% of total tocochromanols identified in each species. The ß-sitosterol, campesterol, and Δ5-stigmasterol were the main sterols in most of the samples, however, in some of the seeds considerable levels of Δ5-avenasterol, Δ5,24-stigmastadienol, 24-ethylcholest-7-en-3beta-ol, and cycloartenol were also determined. Although the studied seeds were marked by relatively low yield of oil, the predomination of UFA, as well as relevant quantities of tocochromanols, carotenoids, sterols, and squalene (144.3-444.8, 4.1-43.1, 603.2-2590.2, and 0-88.1 mg/100 g oil, respectively), thus, the studied seeds can be considered as an alternative source of valuable ingredients for human nutrition.

Post-translational cleavage pattern of Lupinus angustifolius γ-conglutin.

BACKGROUND: Protein post-translational modifications are a key element for the functional diversity of the proteome. The modifications generally refer to the addition of functional groups to certain proteins; however, proteolytic cleavage is also one of the relevant events during protein maturation. γ-Conglutin is a unique protein fraction present in lupin seeds that is marked by numerous unusual properties. This protein fraction undergoes very complex post-translational maturation. Unfortunately, the precise mechanism of γ-conglutin post-translational processing is not yet fully understood. RESULTS: Two independent methods were used to study γ-conglutin post-translational cleavage processing. Edman N-terminal sequencing indicates that the signal peptide is processed at Tyr34, while α- and ß-subunit cleavage takes place between Ser295 and Ser296. High-resolution mass spectrometry revealed a great diversity of N-terminal sequences of γ-conglutin α-subunit. However, most abundant peptides also began from Tyr34. Mass spectrometric analyses additionally confirmed the subunit cleavage position between two serine residues. CONCLUSIONS: The results indicate that the proteolytic processing of γ-conglutin signal peptide is not precise. On the other hand, the post-translational cleavage between α- and ß-subunits of γ-conglutin is very conserved. Interestingly, the results also indicate that proteolytic processing leading to the formation of two subunits of γ-conglutin is incomplete, leaving a certain amount of the protein in an uncut form. © 2018 Society of Chemical Industry.

Proteomic analysis of Lupinus angustifolius (var. Zeus and Bojar) and Lupinus luteus (var. Lord and Parys) seed proteins and their hydrolysates.

BACKGROUND: Proteins enzymatic digestion is a very complex process, during which some components are degraded, whereas others remain in an unchanged form. Moreover, enzymatic hydrolysis is one of the most popular methods used to reduce the allergenicity of food proteins. In the present study, the efficiency of enzymatic hydrolysis of lupin seed proteins was assessed by proteomic analysis as performed by two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry identification. Two digestion systems were used: oriented digestion carried out by trypsin and model in vitro digestion mimicking the conditions present in the gastrointestinal tract. RESULTS: The comparisons of 2-DE maps of proteins isolated form different lupin seed species revealed that the differences in proteins expression were observed mainly in the central parts of gels (i.e. in the molecular weight range from 20 to 70 kDa, and the pH range 5-7). In total, 27 differentially expressed proteins spots were successfully identified by mass spectrometry analysis. An important reduction in the number of proteins spots on 2-DE maps was observed when trypsin and the in vitro digestion model were applied. The protein spot insensitive to digestion in both hydrolysis systems was identified as ß-conglutin. CONCLUSIONS: The results of the present study provide insight into the nature of the digestion process that may take place after lupin seed protein intake and highlight the important fact that some of the proteins are insensitive to digestive enzyme activity. Moreover, evaluation of digestion activity of trypsin towards lupin seed proteins may be used for the development of specific processes with respect to hypoallergenic food production. © 2017 Society of Chemical Industry.

Structure of γ-conglutin: insight into the quaternary structure of 7S basic globulins from legumes.

γ-Conglutin from lupin seeds is an unusual 7S basic globulin protein. It is capable of reducing glycaemia in mammals, but the structural basis of this activity is not known. γ-Conglutin shares a high level of structural homology with glycoside hydrolase inhibitor proteins, although it lacks any kind of inhibitory activity against plant cell-wall degradation enzymes. In addition, γ-conglutin displays a less pronounced structural similarity to pepsin-like aspartic proteases, but it is proteolytically dysfunctional. Only one structural study of a legume 7S basic globulin, that isolated from soybean, has been reported to date. The quaternary assembly of soybean 7S basic globulin (Bg7S) is arranged as a cruciform-shaped tetramer comprised of two superposed dimers. Here, the crystal structure of γ-conglutin isolated from Lupinus angustifolius seeds (LangC) is presented. The polypeptide chain of LangC is post-translationally cleaved into α and ß subunits but retains its covalent integrity owing to a disulfide bridge. The protomers of LangC undergo an intricate quaternary assembly, resulting in a ring-like hexamer with noncrystallographic D3 symmetry. The twofold-related dimers are similar to those in Bg7S but their assembly is different as a consequence of mutations in a ß-strand that is involved in intermolecular ß-sheet formation in γ-conglutin. Structural elucidation of γ-conglutin will help to explain its physiological role, especially in the evolutionary context, and will guide further research into the hypoglycaemic activity of this protein in humans, with potential consequences for novel antidiabetic therapies.

Characterisation of different digestion susceptibility of lupin seed globulins.

This study describes in vitro digestion of lupin seed globulins by pancreatin, trypsin and chymotrypsin. Lupin seed globulins turned out to be almost totally susceptible to chymotrypsin digestion. When panceratin or trypsin were used for digestion of lupin seed globulins, γ-conglutin appeared to be resistant to proteolysis. Different fluorescence spectroscopic methods such as fluorescence anisotropy, fluorescence lifetimes and fluorescence quenching measurements were used for detailed characterisation of this phenomenon. A potential reason for γ-conglutin insensitivity to digestion may be related to the fact that lysine, as well as arginine, are positively charged at cell physiological pH. Simultaneously, flavonoids at this pH are partially ionised, which may lead to the occurrence of ionic interactions between these molecules at pH 7.5. The confirmation of this explanation may be the fact that γ-conglutin and vitexin form a static complex, which was observed using fluorescence quenching measurements.

The Interactions Between Rapeseed Lipoxygenase and Native Polyphenolic Compounds in a Model System.

The focus of the present research was to study inhibition of lipoxygenase activity by rapeseed native polyphenols and the interactions between those compounds and the enzyme. The enzyme and polyphenolic compounds (polyphenols, phenolic acids) were extracted from rapeseed (Brassica napus) varieties Aviso and PR45DO3. The total phenolic compounds concentration in tested rapeseed was 1,485-1,691 mg/100 g d.m. (dry matter) and the free phenolic acids content in both rapeseed varieties was about 76 µg/100 g d.m. The isolated proteins showed lipoxygenase activity. Prooxidant properties of phenolic compounds in the presence of lipoxygenase and linoleic acid were observed rather in the case of extracts containing a relatively high concentration of miscellaneous polyphenols. Antioxidant properties were recorded in the case of phenolic acid extracts which contain only 1.4-1.9% of phenolics present in raw phenolic extracts. We propose that the prooxidant effect of phenolic compounds comes from quinone and oxidized polyphenols formation. The observed antioxidant activity of phenolic acid extracts is probably due to their ability to scavenge free radicals formed from linoleic acid. However, reduction of lipoxygenase ferric to ferrous ions, which prevent the activation of the enzyme and inhibited its activity, was also observed.

Release of flavonoids from lupin globulin proteins during digestion in a model system.

Lupin seed globulin proteins form complexes with flavonoids, predominantly apigenin C-glycosides. Enzymes typical for the gastrointestinal tract were used to hydrolyze lupin seed globulins. Release of native flavonoids as a result of the proteolysis reaction was observed. Different analytical methods such as size exclusion chromatography, HPLC-MS, and fluorescence spectroscopy (steady-state fluorescence, fluorescence anisotropy, fluorescence lifetimes) were used for a detailed characterization of this phenomenon. Flavonoids liberated from lupin globulin proteins as a result of pancreatin-catalyzed digestion were bound by γ-conglutin resistant to this enzyme. Two possible mechanisms of this interaction may be suggested: hydrogen bonding between oligosaccharide chains of glycoproteins and the sugar moieties of the flavonoid glycosides or electrostatic attraction between positively charged γ-conglutin and flavonoids partially ionized at pH 7.5.