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Protein behavior at interfaces : relevance to food allergens
No full textProtein-interface interaction, allergenicity, betalactoglobuli
PROTEIN UNFOLDING ON INTERFACES: A STRUCTURAL AND FUNCTIONAL STUDY
Full text linkThe spontaneous adsorption of protein molecules on interfaces is an ubiquitous phenomenon in natural and man-made systems. The structural rearrangement caused by the direct contact with the sorbent phase may affect protein biological activity, including bioavailability, and ability to bind micro- and macromolecular ligands. Moreover, protein immunoreactivity has been assessed to change if protein molecules interact with an hydrophobic phase; indeed adjuvant are hydrophobic substances that act as enhancers in antibodies production.
Whether proteins unfold randomly or through subsequent ordered and eventually reversible steps remains often unknown, and information about the molecular determinants of the “gain of function” or the “loss of function” observed upon adsorption is scarce. The aim of this work is to understand the structural and functional changes that soy storage proteins (beta-conglycinin and glycinin) and bovine betalactoglobulin (BLG) undergo after adsorption on hydrophobic nanostructured surfaces.
Protein conformational changes after adsorption on interfaces were evaluated by using different techniques, including fluorescence and solid-state fluorescence spectroscopy, CD spectroscopy, along with limited proteolysis followed by recognition of released peptides by MS. Moreover, changes in biological behavior were evaluated by measuring changes in immunoreacivity that may be relevant from the standpoint of immune response or immunomodulation. Experiment aimed to evaluate the influences of interface denaturated protein on live cells were carried out. For this purpose BLG and BLG-stabilized emulsions, both labeled with FITC, were incubated with monocyte and differences in protein uptake were evaluated by citofluorimetry. In order to have a model of BLG denaturation on the polystyrene interface, an in silico study was performed. The simulation was carried out using the computational suite MOE (Molecular Operating System).
In this work structural changes of -conglycinin and glycinin in solution were compared to those occurring when the proteins are adsorbed at the oil-water interface. Both proteins undergo structural modifications after adsorption on the oil droplet surface. From the standpoint of protein chemistry, the modifications occurring at the interface with the proteins investigated here have some peculiar traits, in what both these proteins expose their tryptophan-containing extension regions to the aqueous phase rather than to the droplet interior, as observed for other proteins. It is very important to note that, in beta-conglycinin, tryptophans are present in the extension domains of alpha and alpha’ subunits, and the present fluorescence data confirm previous results demonstrating that the polar extension regions in these proteins are important for their emulsifying ability. These results support the hypothesis that while the a and a’ core domains interact with oil phase, the extension regions protrude into the aqueous phase and stabilize the emulsion droplets by providing the necessary polar regions. Also glycinin’s tryptophans containing regions are exposed to the aqueous phase. However, the multiplicity of glycinin’s genetic variants makes it much more challenging to derive definite answers from the hydrophobicity profiles of this protein, and some more detailed proteomic work is needed to better understand which portion of the protein anchors to the interface. It is also interesting to note that heat treatment does not affect the structural features of either protein once they are adsorbed at the oil-water interface. In other words, the modifications occurring upon adsorption at the interface appear to “lock” the protein structure in a conformation that is insensitive to further physical denaturation, at least under the temperature/time regimes employed in this work. As a matter of fact, it is somewhat expected that, in emulsions, the structural regions more sensitive to the entropic changes ensuing from alteration in the water structure (i.e., the protein hydrophobic core) are at least partially buried into the non-polar lipid phase, and thus are insensitive to temperature-dependent changes in the colligative properties of the solvent. The various peculiarities of these systems and their practical relevance seem worth further investigation. We are currently addressing the molecular details of the observed events, in an attempt to identify specific molecular determinants of the different behaviour of these proteins, as well as the changes occurring during heating, and to assess whether the conformational changes reported here result in biologically relevant modifications when emulsions are consumed as food.
Also BLG structure changes after interaction with an hydrophobic interface. The intrinsic fluorescence spectrum of adsorbed BLG is red-shifted compared with the free-protein one thus indicating that the adsorbed protein assumes a new structure in which Trp19, usually buried inside the hydrophobic core, is exposed to water. Moreover, adsorbed BLG increases ≈2 folds its global quantum yield. The only free thiol in BLG is on Cys121, which is buried in the native structure, but becomes readily and almost completely accessible after adsorption. The overall BLG surface hydrophobicity seems to increase after interaction with the hydrophobic surface, confirming the occurrence of major rearrangements. BLG sensitivity towards trypsin – and therefore the resulting peptidic pattern - is modified as a function of the hydrophobic support where the protein is adsorbed. In fact, in the case of NP-adsorbed BLG trypsin resistance is similar to the one of free BLG, whereas it dramatically decreases for emulsion-BLG. All these data demonstrate an extended stretch of the native structure after adsorption on hydrophobic surfaces with the exposure of new protein regions usually buried from the aqueous media.
Changes in immunoreactivity occurred after adsorption on hydrophobic surfaces. BLG adsorbed on oil droplet surface is more reactive (≈35%) than the free protein by using the 5G6 MAB, and also it is more reactive (≈110%) when using the 1E3 MAB. BLG adsorbed on latex NP is likely to increase its immunoreactivity by using both MAB, that indicates that BLG assumes different structures as a function of the interacting interface(s). Cells experiments show how the BLG internalization by monocites follows two different kinetics according to protein physical state. Moreover the absorption of adsorbed BLG seems to be not influenced by competition of free BLG, leading us to hypothesize the presence of two different pathway for the protein internalization depending on their physicals state.
The in silico denaturation simulations demonstrate that the interaction orientation is fundamental for the type and magnitude of protein structure reorganization. The system, all build by us, seems to be very stable, and the latex denaturating interface should be used with others proteins.
In conclusion in this thesis I described in deeply the structural modification that three protein, whit a huge importance for nutrition and food science, undergo after adsorption on different model hydrophobic interfaces. I also produced an in silico model for computational prediction of protein denaturation on polystyrene interface. Physiological implication regarding protein structural reorganization were also explored
Structural mechanism and physiological significance of betalactoglobulin unfolding at solid and liquid interfaces
No full textBetalactoglobulin (BLG) represents a major allergens in bovine milk. BLG is used alone or in combination with other proteins for the preparation of food emulsions, and interacts with the solid surfaces in processing equipment. From a structural standpoint, changes in the pattern of hydrophobic interaction is very diverse in these two cases. Adhesion to a hydrophobic solid surface in the absence of mechanical denaturation implies structural changes very different from those occurring when hydrophobic regions of the BLG structure are exposed by means of mechanical denaturation and end up penetrating a liquid apolar phase.
Here, spectroscopic and limited proteolysis approaches were used – together with the accessibility of “reporter” amino acid side chains - to compare structural features of BLG adsorbed on “liquid” oil microdroplets and on “solid” styrene nanoparticles. Immunoreactvity of the two systems was also studied, along with that of the products of simulated digestion of the various BLG states.
Structural changes were largest in the emulsion system, whereas the regions of BLG involved in ligand binding were only marginally affected in the solids-adsorbed protein. In both cases, there was a marked alteration in immunoreactivity (with a notable increase in emulsions), and in the pattern of proteolysis. Proteolytic products obtained from solids-adsorbed BLG remained bound to the styrene surface and retained their immunoreactivity, at contrast with those from emulsions, that were different in nature/reactivity also from those obtained from native or temperature-denatured BLG. These findings highlight the significance of structural issues in the field of food protein allergens
Rubredoxin refolding on nanostructured hydrophobic surfaces : evidence for a new type of biomimetic chaperones
No full textRubredoxins (Rds) are small proteins containing a tetrahedral Fe(SCys)4 site. Folded forms of metal free Rds (apoRds) show greatly impaired ability to incorporate iron compared with chaotropically unfolded apoRds. In this study, formation of the Rd holoprotein (holoRd) on addition of iron to a structured, but iron-uptake incompetent apoRd was investigated in the presence of polystyrene nanoparticles (NP). In our rationale, hydrophobic contacts between apoRd and the NP surface would expose protein regions (including ligand cysteines) buried in the structured apoRd, allowing iron incorporation and folding to the native holoRd. Burial of the hydrophobic regions in the folded holoRd would allow its detachment from the NP surface. We found that both rate and yield of holoRd formation increased significantly in the presence of NP and were influenced by the NP concentration and size. Rates and yields had an optimum at "catalytic" NP concentrations (0.2 g/L NP) when using relatively small NP (46 nm diameter). At these optimal conditions, only a fraction of the apoRd was bound to the NP, consistent with the occurrence of turnover events on the NP surface. Lower rates and yields at higher NP concentrations or when using larger NP (200 nm) suggest that steric effects and molecular crowding on the NP surface favor specific "iron-uptake-competent" conformations of apoRd on the NP surface. This bio-mimetic chaperone system may be applicable to other proteins requiring an unfolding step before cofactor-triggered refolding, particularly when over-expressed under limited cofactor accessibility
Structural and functional changes of bovine beta-lactoglobulin - a food allergen - after adsorption on hydrophobic surfaces
No full textAdsorption of protein molecules on interfaces occurs in natural and man-made systems, and plays a central role in many events related to human health, to food science, and to environmental issues. Structural rearrangements upon contact with the sorbent phase may affect the protein's biological activities (allergenicity, bioavailability, ability to bind micro- and macromolecular ligands).
Structural and functional changes that bovine beta-lactoglobulin (BLG) - a relevant food allergen - undergoes upon adsorption on hydrophobic surfaces (polystyrene nanoparticles or oil-in-water microemulsions) were evaluated by means of intrinsic fluorescence spectroscopy, binding of fluorescent probes, accessibility of cysteine thiols, and limited proteolysis. Changes in immunoreactivity were evaluated by competitive ELISA using epitope-specific monoclonal antibodies.
Our results indicate that BLG undergoes an extended stretch of the native structure after adsorption on hydrophobic surfaces, exposing regions buried from the aqueous media in the native structure. As a result, both the proteolytic resistance and the immunoreactivity of BLG are markedly altered upon absorption
Hunting for bacterial interactors: activated magnetic nanoparticles for exploring membrane proteomics in bacteria
No full textIntroduction: Proteomics studies on the envelope(s) of different type of bacterial cells are typically performed through the separation of the different envelope layers or through the use of proteases to directly remove surface-exposed protein domain from intact bacterial cells. This approach has been successfully used for Gram positive bacteria but protease shaving of Gram-negative bacteria surface appears less straightforward, as with contamination with cytoplasmic peptides is likely to occur in Gram-negative bacteria.
Results: A new and specific technique for magneto-capturing of surface-exposed proteins from intact bacterial cells was developed. This method is based on the use of carboxymethyl-dextran coated magnetic nanoparticles specifically activated to establish covalent bonds with exposed lysine amino groups in proteins. Given their chemical composition, size (average diameter = 80-90 nm), and negative charge, these NPs were expected to be atoxic for bacterial cells, as they are for eukaryotic cells.
This work investigated in detail the outer-membrane composition of P. aeruginosa, a highly adaptable Gram negative bacterium which lives in a huge number of ecosystems and can infect multiple hosts from plants to humans, where it represents an important opportunistic pathogen.
By using this technique we identified a total of 63 proteins that were captured directly by the activated nanoparticles, along with 67 proteins that were co-captured, being likely embedded in neighboring regions of the cell envelope. The envelope localization of most of the identified proteins had been only predicted, or had been unknown so far.
Conclusions: This approach developed in this study improves sensitivity and specificity of previous methods, such as surface shaving with proteases. Magneto-separation of cell envelope fragments from the soluble cytoplasmic fraction also allows the identification of the captured proteins, and that of neighboring ones. The magneto-capture procedure is simple, safe, and rapid, and appears well-suited for envelope studies in many proteomics studies, including those regarding highly pathogenic bacteria
Influence of the sorbent material and size upon structural and functional changes of bovine beta-lactoglobulin – a food allergen – after adsorption on hydrophobic surface
No full textAdsorption of protein molecules on interfaces occurs in natural and man-made systems, and plays a central role in many events related to human health, to food science, and to environmental issues. Structural rearrangements upon contact with the sorbent phase may affect the protein's biological activities (allergenicity, bioavailability, ability to bind micro- and macromolecular ligands). The surface characteristics of the sorbent material play a key role in the form and extent of these rearrangements. Knowledge of these phenomena may allow to predict or modulate the protein functional behavior after adsorption.
In this work structural and functional changes undergone by bovine beta-lactoglobulin (BLG) - a relevant food allergen - upon adsorption on different materials and size hydrophobic surfaces (46 nm and 200 nm polystyrene nanoparticles or oil-in-water microemulsions) were evaluated by means of intrinsic fluorescence spectroscopy, binding of fluorescent probes, accessibility of cysteine thiols, and limited proteolysis followed by MALDI-TOF and LC-MS identification of released peptides. Changes in immunoreactivity were evaluated by competitive ELISA using epitope-specific monoclonal antibodies.
The results indicate that BLG undergoes an extended stretch of the native structure after adsorption on hydrophobic surfaces, exposing regions buried from the aqueous media in the native structure. Hydrolysis of BLG stuck on different surfaces results into different peptide patterns, but for a single peptide – that may be a common region interacting with the sorbent phase. Immunoreactivity of BLG is markedly altered upon absorption. The amplitude of the observed differences is also depending on the nature of the sorbent material. All these results corroborate the central role of the nature and of the size of the sorbent material, as determinants of the type and extent of BLG structural rearrangements
Uptake of nanoparticle-conjugated allergens by human monocytes
No full textINTRODUCTION: Stable biocompatible magnetic nanoparticles, conveniently grafted with affinity ligands and/or transfection agents to integrate functionally and efficiently into cellular and subcellular structures, can confer cells magnetic activity, and may represent versatile and non-invasive tools for targeting specific cellular structures, and for monitoring cellular functions both in vitro and in vivo.
RESULTS: In this study, betalactoglobulin, the most abundant protein in bovine whey and a major food allergens, was covalently conjugated to biocompatible carboxymethyldextran-coated magnetic nanoparticles. The conjugated protein retained its immunoreactivity towards different monoclonal antibodies. BLG-conjugated nanoparticles were taken up by human monocytes much more efficiently than non-conjugated particles, allowing easy magnetic separation of cells that had integrated the allergen. To assess whether the interaction between monocytes and the nanoparticles was associated to their internalization rather than to mere adsorption of the particles on the cell surface, BLG was conjugated to fluorescent-labeled magnetic nanoparticles. The uptake of these materials by human monocytes was monitored through flow cytometry and confocal microscopy, in comparison with fluorescent nanoparticles devoid of the allergen or conjugated with human serum albumin.
Both approaches confirm a higher uptake of the BLG-conjugated particles, and confocal microscopy provided clear evidence of particles internalization into the cytoplasm. These results pave the way to use a combination of these approaches to improve the current understanding of the intracellular and intratissutal path of allergens that may be involved in the immune response to food
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