1,721,037 research outputs found
Intestinal epithelial Caco-2 cells responses to lupin seed protein fractions
No full textState of the art and aim: In the recent years biological active proteins have been found in lupin seeds,
such as bowman-birk inhibitor and gamma-conglutin, which showed anticarcinogenic and hypoglycemic
activity respectively (Duranti et al, 2008). Beside, a certain number of bioactive proteins and
peptides have been described in soybean and other legumes. However, little is known about the
responses at immunological level after the interactions of seed proteins with intestinal mucosae. The
relationship between the consumption of certain foods and the reduction or the establishment of
immunological diseases has been demonstrated. For example, from bovine milk can originate a number
of casein hydrolysates with positive immunomodulatory properties (Sandré et al, 2001). Therefore, the
aim of this study was to investigate the immune response of intestinal epithelial Caco-2 cells to
different lupin seed protein fractions and assess possible other cellular effects.
Results and discussion: Three protein fractions, namely alpha, beta and gamma -conglutin, were
purified from lupin seeds through a well-established procedure (Magni et al, 2007). Stable transfected
Caco-2 cell monolayers were incubated with the protein fractions (0,5 mg/mL) and interleukin 1b (2
ng/mL) for 4 hours. This cell line, containing the plasmid pNiFty2-Luc, expresses the nuclear factor
(NF)-kB-binding sites on membrane, whose activation by pro-inflammatory molecules can be
monitored measuring light emission. The results showed that gamma-conglutin, by interacting with NFkB
receptor, activated significantly the inflammatory response of the transfected Caco-2 cells. A lower
response was measured with alpha and beta -conglutins. The effects of gamma-conglutin on cell protein
expression were also investigated in hepatic cells, a possible target of gamma-conglutin. HepG2 cells
were incubated for 4 hours with or without the integer and the pepsin/pancreatin hydrolyzed gammaconglutin.
The 2D electrophoretic maps of cell protein extracts did not show differences in the protein
patterns. These preliminary results show that only gamma-conglutin negatively modulated the immune
response of transfected Caco-2 cells after 4 hours of incubation but this protein did not induced major
changes in the protein pattern of HepG2 cells as monitored in the 2D maps. Further researches will be
necessary to investigate the cellular and immune responses of cell models to gamma-conglutin
Uptake and phosphorylation of gamma-conglutin, a hypoglycaemic lupin seed protein, by HepG2 cells
No full textgamma-Conglutin, a lupin seed glycoprotein, positively influences glucose uptake in model cells and reduces glycaemia in animal models and human subjects. In particular, gamma-conglutin can activate different signaling pathways, in differentiating myocyte, that closely resembled those of insulin.
gamma-Conglutin was transcytosed through a CaCo2 cell monolayer and crossed an ex-vivo intestinal barrier in an intact form. Beside, the protein displays strong resistance to endogenous and exogenous proteolytic enzymes.
These findings encouraged us to further study the molecular mechanisms of action of this bioactive protein, which are still not completely elucidated
Relazione I anno di dottorato : Biological activity and metabolic fate of conglutin gamma, a lupin seed protein, involved in hypoglycemic effects in animal models
No full textThe effects of ochratoxin A on liver metabolism
No full textThis review summarizes the main toxic effect of ochratoxin A (OTA) on liver metabolism. This contaminant is a mycotoxin that can be found in raw materials (cereals, coffee, cocoa, spices or grapewine), in processed foods (bread and other bakery products) and, if animals are fed with contaminated feedstuffs, in pork meat. Kidney is a well-known target of OTA, although several findings suggest that liver metabolism can be affected too. OTA intake reduces, in a dose-dependent manner, the synthesis of albumin, while the concomitant increase in transaminases (ALT, ASP) and alkaline phosphatase is in agreement with the hypothesis of liver damage induced by OTA. Feeding animals with OTA-contaminated feeds has significant pro-oxidative effects that cause a reduction in anti-oxidative defences and an increase in malondialdehyde formation. Experiments on human liver cells support the hypothesis of an inflammatory effect of OTA mediated by TNF-α. An up-regulation of apoptosis has also been detected in hepatic cells after OTA treatment, which leads to a higher rate of cell death and to a reduction of liver activity. All these findings suggest that OTA can have a toxic effect on the liver too and for this reason we should pay attention to liver toxicity of OTA in the risk assessment for this mycotoxin
INVESTIGATIONS ON STRUCTURAL STABILITY, BIOLOGICAL ACTIVITY AND METABOLIC FATE OF GAMMA-CONGLUTIN, A BLOOD GLUCOSE-LOWERING LUPIN SEED PROTEIN
No full textgamma-Conglutin is a glycoprotein representing about 5% of lupin seed proteins. gamma-Conglutin is oligomeric at neutral pH, while at acidic pH it dissociates into monomers of 50 kDa. Each monomer is composed of two disulphide linked subunits of 17 and 29 kDa, originating from the endogenous proteolysis of the pro-polypeptide. Other 5 interchain disulphide bridges are also present. gamma-Conglutin sequence does not match any canonical legume storage proteins sequence; this protein is not degraded during seed germination and it was found to be extracellularly secreted during germination. These findings suggest a functional role for gamma-conglutin in the seed other than a mere storage one. gamma-Conglutin shows a great similarity (64%) with the basic globulin (BG7S) of soybean seed, which has been attributed an in vitro kinase activity and a binding activity towards some small regulatory proteins, such as a 4 kDa plant hormone-like peptide and mammalian insulin. -Conglutin shows resistance to in vitro proteolysis unless it was previously fully denatured. It can bind various divalent ions, such as Zn++ and Ni++, and protein such as acid phosphatase and insulin with a Kd of 10-5 M. Finally, gamma-conglutin was found to decrease glycemia in glucose overloading experiments on rats.
Therefore, it seemed worth to investigate the molecular bases of this biological activity, as well as the properties related to gamma-conglutin metabolic fate and interaction with target cells. Indeed, aim of this work was to unravel the structural bases of gamma-conglutin assembly and stability to proteolysis and to develop suitable models to monitor its pathway after assumption and the cellular mechanisms involved in gamma-conglutin stimulation.
By using dynamic light scattering, we investigated the assembly state of gamma-conglutin at different pH conditions. The results indicated a transition from monomer to oligomer, likely hexamer, via the transient formation of a dimer while pH was raised from 4.5 to neutral values. A model of these pH transitions based on the role of charge attraction/repulsions between acidic and basic amino acid residues in driving gamma-conglutin aggregation state was proposed. By using far- and near-UV circular dichroism, and intrinsic/ANS fluorescence analyses, the conformational changes as a function of pH were measured. All results suggested that the protein secondary and tertiary structures were unmodified by pH change above 3.5, while for lower values the structural collapse of the protein took place. Nevertheless, indirect evidence obtained with a set of proteolytic enzymes showed that gamma-conglutin became susceptible to proteolysis at pH values lower than 4.25, thus confirming its overall stability at neutral to slightly acidic pHs and suggesting also that a partial opening of the protein, without loss of overall three dimensional conformation, already occurred. Although γ-conglutin 3D structure is not available, it can be argued that its native conformation, which is stabilized also by a number of intrachain disulphide bonds, is extremely compact, especially if compared to the canonical seed storage proteins.
The survival of intact gamma-conglutin into the intestinal lumen as well as its presence in the blood of suitable animal models were not proved with our assays in this work. However, the transit of gamma-conglutin through the intestinal barrier, by using in vitro and ex vivo models, namely Caco-2 cell monolayers and intestinal everted sacs, was demonstrated. Both experimental approaches showed that the transit through a transcellular way was possible for the intact gamma-conglutin, though limited to a small percentage of the applied protein. Although we were not able to detect gamma-conglutin in the sera samples of animals fed gamma-conglutin, the hypothesis of an at least partial preservation of the protein into the gastro-intestinal tract is also consistent with the observed immunogenic activity as measured in orally-treated mice in a parallel study.
Eventually, we showed that the full length protein in its native conformation was capable to activate the insulin-signaling pathway in miocyte models in an insulin-like pattern, despite the dramatic structural differences between the two proteins. Moreover, the cellular absorption of gamma-conglutin by miocytes was shown by using a 2D electrophoretic approach. This finding, obtained with a cellular model not specifically involved in protein transport, confirmed the transcellular transport for gamma-conglutin observed with Caco2 cells.
Very recent unpublished data reporting the results of the first human trial with healthy human subjects confirmed gamma-conglutin biological activity. Therefore, the present set of data is relevant to design further approaches aimed at exploiting lupin gamma-conglutin as a preventing and therapeutic agent in the cases of impaired glucose tolerance and possibly pre-diabetic and diabetic conditions
Transport of lupin conglutin gamma across human intestinal epithelial Caco-2 cell monolayers
No full textPROTEOMIC APPLICATIONS TO PROTEIN TRACEABILITY AND SAFETY STUDIES OF LUPIN-BASED FOOD PRODUCTS
No full textLupin seed is one of the richest in proteins, among grain legumes, thus representing an excellent source for the new
needs of plant proteins in food products.
2D electrophoretic and mass spectrometry analyses can be of great impact to study lupin seed proteome, to evaluate
the effects of technological treatments during food production and to monitor desirable/undesirable protein components
such as biologically active proteins or allergens.
The lupin seed 2D map (1) and its following updating have been used to identify the main protein components. Lupin
maps show the complex pattern of heterogenous storage proteins. Of 357 spots detected, 70 of them were analyzed.
Moreover, a 2D comparative approach between the total protein extract map and the purified major protein seed fractions
maps, allowed to allocate 124 polypeptides within these fractions.
Taking advantage of the availability of lupin protein 2-D map we also studied the presence, integrity and constancy
of proteins throughout the industrial processing of a lupin-based pasta product. Samples, including seeds, raw materials,
i.e. flour and protein concentrate, half-processed products and dry pasta were used to generate the corresponding 2-D
electrophoretic maps. Some differences in the protein profiles between the raw materials were attributed to the different
varieties which they arose from; on the other hand, no alteration of the covalent continuity of the main polypeptide
backbones among the samples during the industrial processing were observed.
In parallel, it was possible to trace a lupin putative dominant allergen (2), i.e.
-conglutin, which is also considered
the candidate molecule to the hypoglycemic activity of lupin seed extracts (3).
This work shows that lupin proteome and related 2D electrophoretic maps can be very useful to both quality control
strategies and traceability of specific protein components in food matrices.
References
(1) Magni et al. (2007). Phytochemistry 68: 997-1007.
(2) Magni et al. (2005). J. Agric. Food Chem. 53: 4567-4571.
(3) Magni et al. (2004). J. Nutr. Biochem. 15: 646-650
Multifunctionality of plant proteins
No full textPlant seeds proteins (SP) are traditionally considered the nitrogen reserve that supports the seedling growth during the first steps of germination. This exclusive role has recently been ruled out and emerging findings indicated that several biological activities became manifests upon the proteolytic breakdown. Protein cleavage at germination is believed to occur through a regulated mechanism involving the selective breakdown of specific peptide bonds which, before the native conformation is critically altered to allow the complete hydrolysis, origins large polypeptide fragments. During germination, several preformed and de novo-synthesized peptidases act di concerto sequentially. Some of the transiently formed intermediate peptides have been shown to possess specific bioactivities.
In legume and pseudo-cereal seeds, proteins represent from 20% to 45%. The majority of them are SP, globulins usually classified according to their sedimentation coefficients as 7S (vicilin-like) and 11S (legumin-like). SPs are synthesized during seed developing and deposited inside the cells in specific membrane-bound organelles, called protein bodies (PBs). Many bibliographic data suggest that vicilins have active role in plant defence processes. Few examples. A fragment of 45 amino acids generated during germination by proteolysis of Macadamia integrifolia vicilin has been shown to in vitro inhibit several plant pathogenic fungi. BLAD is a stable intermediary product of vicilin breakdown, which accumulate in cotyledons of Lupinus species during germination and shows defense and antibiotic properties. Unlike vicilins, no specific biological activities have been described for legumins or legumin-derived fragments. These polypeptides were found inside cotyledons and selectively secreted outside the seeds.
From an applicative point of view some biological properties have already been ascribed to specific seed storage proteins, such as radical scavenger capacity, anti-inflammatory, anti-obesity, ACE-inhibitory, hypoglycaemic, α-amylase inhibitor, trypsin-inhibitor, hypocholesterolaemic and immuno-modulation activities; thought the involved molecular mechanisms are not completely elucidated. Bioactive cryptic peptides, originated from protein hydrolysis, have also been identified. These protein fragments, inactive in the integer parent protein and released by enzymatic hydrolysis, once produced act as modulators of many biological processes in living organisms. Indeed, it has been proved that some of them are able to pass the intestine barrier, enter the blood stream, arrive to the target site and, depending on their amino acids composition and structure, play different roles.
We have shown that SP-derived peptides from quinoa and other crops are able to interact with as Toll-like receptors, activating an immune-response pathway including NF-kB, and modulate cell inflammatory responses. A suitable transfected Caco-2 cell model is currently used. These cells contain the plasmid pNiFty2-Luc, whose promoter couples nuclear factor (NF)-kB-binding sites and the luciferase reporter gene luc. The immune and inflammatory responses of cells incubated with the tested bioactive molecules and IL1β as control elicitor, can be evaluated by measuring the bioluminescence emitted from cellular extracts after cell lysis.
IL1 Toll-like receptors are also present in plants. These include N protein from tobacco, which is required for resistance to the pathogen tobacco mosaic virus, and L6 from flax, which confers resistance to flax rust. Neither L6 nor N protein are transmembrane receptors but may interact with their ligands in the cytoplasm. RPP5 from Arabidopsis shares similar structural features with L6 and N proteins.
By and large RPP5 may play a key role by eliciting the plant defence responses at the beginning of germination or boosting them when the plant is attacked by pathogens
Applications of 2-D Electrophoresis and Western Blot to Analyse and Trace Proteins in Lupin based Pasta Products
No full textTwo-dimensional (2-D) IEF/SDS-PAGE is a powerful tool to get molecular “pictures” of food proteomes and monitor the processing effect(s) of a given food item on its protein profile. However, the
relatively scarce diffusion of proteomic techniques at industrial and analytical level has prevented their
application in this promising area so far. In this work, 2-D electrophoresis has been used to monitor the
main steps of lupin-based gluten-free pasta production. To the best of our knowledge, this work is one
of the rare examples of 2-D electrophoresis application to the analysis of legume seed protein components
in the whole production chain of a food product.
The optimised dietary exploitation of protein-rich plant sources, such as legume seeds, is the
target of several research and development programs. Among the legume seeds, lupin is an interesting
one for various reasons: a very high protein content (1), comparable to that of soybean, a low presence
of antinutritional compounds (2), the functional properties of its components in food matrices (3) and
the nutraceutical potentialities of some of its proteins (4).
In this work we have studied a lupin-based pasta product, taking advantage of the presence of a
single protein source in this product, being lupin used as the unique alternative to semolina, and of a
previously published lupin storage protein 2-D maps (5). To this aim, three different production lots of
lupin-based pasta were analysed. For each lot, samples at each critical production step, including
seeds, raw materials, namely the flour and the protein concentrate, half-processed products and dry
pasta, were used to generate the corresponding 2-D electrophoretic maps. The presence, integrity and
constancy of proteins throughout the industrial processing have been assessed. Indeed, some differences
in the protein profiles between the raw materials, i.e. lupin flour and lupin protein concentrate, were
attributed to the different varieties which they arose from. On the other hand, the electrophoretic analyses
showed only minor differences among the samples during the industrial processing. In particular no
alteration of the covalent continuity of the main polypeptide backbones. The disulphide pattern did not
change during the process, as well, and the constancy of the glycosylation pattern, as measured by the
lectin Concanavalin A on the blotted maps, indicated that this molecular feature was not affected by the
process too.
In conclusion, this work shows that taking 2-D electrophoretic “pictures” of the polypeptides at
each relevant step of a food production process can be very useful to both quality control strategies and
traceability of specific protein components in force of the high resolution of the technique also in
complex food matrices
Susceptibility of Lupin γ-Conglutin, the Plasma Glucose-Lowering Protein of Lupin Seeds, to Proteolytic Enzymes
No full textLupin seed γ-conglutin, orally administered to animal models, has been shown to display glucosecontrolling properties. Therefore, we have addressed the study of γ-conglutin susceptibility to proteolytic enzymes in vitro as the basis to unveil its metabolic fate in the body. Pepsin treatment at pH 2.0 and 3.0 caused extensive proteolytic breakdown, while at pH 4.0, where pepsin is
minimally active, γ-conglutin was unaffected. Aliquots of the pepsin-treated protein were further incubated with pancreatin at neutral pH. If the protein backbone was already cleaved by pepsin
action, then the breakdown by pancreatin was almost complete; alternatively, pancreatin did not
affect at all γ-conglutin polypeptide chain. This was not due to an inhibitory activity of γ-conglutin,
because co-incubation with casein showed complete breakdown of the milk protein. Furthermore,
γ-conglutin was incubated with bromelain, a proteinase effective between pH 4.0 and 7.0. A sharp
transition from the uncleavable to the fully cleavable form of γ-conglutin was observed below pH
4.25. Therefore, it was concluded that (i) γ-conglutin is resistant to proteolysis at pH greater than
4.0, likely because of a compact native conformation, (ii) an acidic pH renders the protein
susceptible to proteases, suggesting the occurrence of a trans conformation, which has also been
observed by circular dichroism spectral analysis, and (iii) the protein undergoes an “all or none”
degradation pathway, regardless of the enzyme used
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