Native beta-LG Research Articles

Ethanol Effect on the Structure of β-Lactoglobulin B and Its Ligand Binding

The changes of structure and ligand binding properties of beta-LG B have been studied by fluorescence and circular dichroism spectroscopy in ethanolic solutions. Fluorescence measurements of retinol/beta-LG interactions at 480 nm in various ethanol concentrations show that the maximal fluorescence intensity induced by this interaction between retinol and beta-LG is observed around 20% v/v of ethanol. It is reduced to zero at 40% and 50% of ethanol. These results suggest that there are two distinct structural changes in beta-LG occurring between 20% and 30% and around 40% of ethanol. The first transition, which increases affinity and the apparent number of binding sites for retinol, may be related or similar to the Tanford transition. The strong quenching of retinol emission at 480 nm in 40% of ethanol indicates the radical transformation of beta-LG tertiary structure and the release of retinol. CD spectra at the aromatic region show that secondary and tertiary structures of beta-LG are not significantly affected between 0% and 20% of ethanol. In 30% of ethanol, beta-sheet percentage of beta-LG decreases with respect to native beta-LG (from 55% to 46%). beta-Sheet percentage in beta-LG increases in 40% and 50% alcohol (51% and 53%) relative to 30% of ethanol, which also indicates the strong rearrangement of the secondary structure of beta-LG, while its tertiary structure and beta-LG interactions are radically changed.

Changes in Structure and in Interactions of Heat-Treated Bovine β-Lactoglobulin

Heat stress on structure and ligand binding of beta-LG has been studied by fluorescence, circular dichroism and gel electrophoresis at pH 6.5. Native PAGE gel electrophoresis shows that denaturation of beta-LG is reversible up to 75 degrees C then it becomes irreversible due to aggregation of beta-LG. Formation of aggregated beta-LG is completed at 95 degrees C. Circular dichroism results indicate that formation of aggregated beta-LG is accompanied by the scrambling of disulfide bonds (creation of new intramolecular and intermolecular disulfide bridges and rearrangement of old intramolecular disulfide bridges). Addition of ethanolic retinol causes a change in polarity of the solution and favors transformation of the beta<-->alpha structure. In the presence of retinol, the alpha-helix content of the secondary structure of heat-treated beta-LG is increased and the major portion of its secondary structure is helical. Fluorescence results show that heat-treated beta-LG at 95 degrees C can still bind retinol. The refolding of the tertiary structure of beta-LG heat-denatured at 95 degrees C may recreate a retinol binding site. Surprisingly, the affinity of the new site for retinol is higher than that of native beta-LG; however, the apparent molar ratio is lower than one. The binding properties of beta-LG for terpenoids have been measured after its heat treatment at 20, 75 and 95 degrees C. The intensity of tryptophan emission at 330 nm was changed only in the case of the interaction with beta-ionone. Other ligands probably cannot bind to beta-LG or they bind in a binding site far from the tryptophan residues, hence not affecting its fluorescence.

Capillary electrophoresis immunoassay using magnetic beads

Protein A-coated magnetic beads (0.3 mum) have been trapped in a small portion of a neutrally coated capillary (50 mum id). Anti-beta-lactoglobulin (beta-LG) antibodies have then been immobilized on the beads through strong affinity with protein A to subsequently capture beta-LG from model or real samples. Once the immunocomplexes formed at physiological pH, a discontinuous buffer system has been used to release the partners and preconcentrate them by transient ITP. The antigens and antibodies have finally been separated by CZE and detected by UV absorbance. An LOQ of 55 nM has been achieved. This methodology has been applied to quantify native beta-LG in pasteurized and ultra-high-temperature-treated bovine milk. All the described procedures, including immunosorbent preparation, sample extraction, cleanup, preconcentration, and separation are completely automated on a commercial CE instrument. As this CE immunoassay method is simple, rapid, selective, and sensitive, it should be a practical and attractive technology for the analysis of complicated biological samples.

Reduced T Cell Response to β-Lactoglobulin by Conjugation with Acidic Oligosaccharides

We have previously reported that the conjugation of beta-lactoglobulin (beta-LG) with alginic acid oligosaccharide (ALGO) and phosphoryl oligosaccharides reduced the immunogenicity of beta-LG. In addition, those conjugates showed higher thermal stability and improved emulsifying properties than those of native beta-LG. We examine in this study the effect of conjugation on the T cell response. Our results demonstrate that the T cell response was reduced when mice were immunized with the conjugates. The findings obtained from an experiment using overlapping synthetic peptides show that novel epitopes were not generated by conjugation. One of the mechanisms for the reduced T cell response to the conjugates was found to be the reduced susceptibility of the conjugates to processing enzymes for antigen presentation. We further clarify that the beta-LG-ALGO conjugate modulated the immune response to Th1 dominance. We consider that this property of the beta-LG-ALGO conjugate would be effective for preventing food allergy as well as by its reduced immunogenicity. Our observations indicate that the method used in this study could be applied to various protein allergens to achieve reduced allergenicity with multiple improvements in their properties.

Carbonylation of milk powder proteins as a consequence of processing conditions

During industrial treatments, milk proteins could be oxidatively modified, thus leading to the formation of modified/oxidised amino acid residues. The apparition of such modified residues may contribute to the formation of new immunologically reactive structures. Some of these adducts could, in an advanced stage, lead to cross-linked protein species whose proteolytic susceptibility would be drastically decreased. Such protein species, that are resistant to digestion, could also constitute major food allergens. Therefore, these oxidative protein modifications tend to increase the natural allergenicity of milk proteins. For these reasons, monitoring milk protein oxidative modifications could be very useful regarding both product quality and allergenicity issues. In the present paper, we highlight, using different analytical approaches, the preferential carbonylation of beta-lactoglobulin (beta-Lg) during industrial treatments of milk. This result is particularly interesting since native beta-Lg represents one of the major milk allergens.

Influence of Free Protein on Flocculation Stability of β-Lactoglobulin Stabilized Oil-in-Water Emulsions at Neutral pH and Ambient Temperature

The influence of protein concentration and order of addition relative to homogenization (before or after) on the extent of droplet flocculation in oil-in-water emulsions stabilized by a globular protein was examined using laser diffraction. n-Hexadecane (10 wt%) oil-in-water emulsions (pH 7, 150 mM NaCl) stabilized by beta-lactoglobulin (beta-Lg) were prepared by three methods: (1) 4 mg/mL beta-Lg added before homogenization; (2)10 mg/mL beta-Lg added before homogenization; (3) 4 mg/mL beta-Lg added before homogenization and 6 mg/mL beta-Lg added after homogenization. Emulsion 1 contained little nonadsorbed protein (<3%) and underwent extremely rapid and extensive droplet flocculation immediately after homogenization. Emulsion 2 contained a significant fraction of nonadsorbed beta-Lg and exhibited relatively slow droplet flocculation for some hours after homogenization. Measurements on Emulsion 3 showed that the extremely rapid particle growth observed in Emulsion 1 could be arrested by adding native beta-Lg immediately after homogenization. The extent of particle growth in the three types of emulsions was highly dependent on the time that the salt was added to the emulsions, i.e., after 0 or 24 h aging. We postulate that the observed differences are due to changes in droplet surface hydrophobicity caused by differences in the packing or conformation of adsorbed proteins. Our data suggest that history effects have a strong influence on the flocculation stability of protein-stabilized emulsions, which has important implications for the formulation and production of protein stabilized oil-in-water emulsions.

Modification of β-Lactoglobulin by Oligofructose: Impact on Protein Adsorption at the Air−Water Interface

Maillard products of beta-lactoglobulin (betaLg) and fructose oligosaccharide (FOS) were obtained in different degrees of modification depending on incubation time and pH. By use of a variety of biochemical and spectroscopic tools, it was demonstrated that the modification at limited degrees does not significantly affect the secondary, tertiary, and quaternary structure of betaLg. The consequence of the modification on the thermodynamics of the protein was studied using differential scanning calorimetry, circular dichroism, and by monitoring the fluorescence intensity of protein samples with different concentrations of guanidine-HCl. The modification leads to lowering of the denaturation temperature by 5 degrees C and a reduction of the free energy of stabilization of about 30%. Ellipsometry and drop tensiometry demonstrated that upon adsorption to air-water interfaces in equilibrium modified betaLg exerts a lower surface pressure than native betaLg (16 versus 22 mN/m). Moreover, the surface elastic modulus increased with increasing surface pressure but reached significantly smaller values in the case of FOS-betaLg. Compared to native betaLg, modification of the protein with oligofructose moieties results in higher surface loads and thicker surface layers. The consequences of these altered surface rheological properties are discussed in view of the functional behavior in technological applications.

Spectroscopic characterization of heat-induced nonnative beta-lactoglobulin monomers.

Previous studies have shown that two altered monomeric species were formed in the early steps of thermal denaturation of bovine beta-lactoglobulin (beta-lg), the well-known Cys121-exposed intermediate (Mcys121), and a new, stable monomer with exposed nonnative Cys119 (Mcys119). In this study, circular dichroism and fluorescence spectroscopies were used to characterize the structural features of these molecules. The structural characteristics of MCys121 after heating and cooling cycles are similar to those of native beta-lg. In contrast, Mcys119 monomer exhibits some characteristics of the well-known molten-globule state. Combined with other published data, these results indicate that heating induces at least two molten globule-like states of beta-lg, a highly reactive Mcys121 that returns to native state after cooling, and a less-reactive Mcys119 that is trapped and stabilized in a molten globule-like state by nonnative disulfide bond.

Effect of heat denaturation on beta-lactoglobulin-induced gastrointestinal sensitization in rats: denatured betaLG induces a more intensive local immunologic response than native betaLG.

Beta-lactoglobulin (betaLG) is one of the first foreign antigens encountered by a newborn child, and it is the major allergen causing cow's milk allergy. Heat denaturation causes changes to the protein structure, but the significance of heat-induced changes for immunogenicity or allergenicity is not known. To clarify how heat denaturation affects allergenicity and immunogenicity, we immunized Hooded-Lister rat pups with intra-peritoneal injections of native or heat-denatured betaLG at days 43 and 62 after birth. The animals were then fed native and denatured milk products twice weekly from 73 to 101 days of age with a feeding tube, after which they were allowed cheese and milk ad libitum, until they were killed on day 131. Total immunoglobulin (Ig)E and betaLG-specific IgG1 and IgG2a levels were determined from serum samples. Spontaneous interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) production was measured from duodenal specimens, and specimens of gastrointestinal mucosae were studied for the presence of inflammatory cells. The rats immunized with native betaLG had higher levels of total serum IgE than the unimmunized controls or the rats immunized with heat-denatured betaLG, while heat-denatured betaLG induced a significantly more intensive mononuclear inflammatory cell and eosinophil infiltration in the gastroduodenal mucosa. The betaLG-specific IgG antibody and IL-4 and IFN-gamma responses were similar in the two groups of immunized animals. Hence, denaturation modifies the immunogenic and allergenic properties of betaLG. Heat-denatured betaLG induces a more intensive local reaction in the gastrointestinal mucosa, while there is some evidence for enhanced systemic allergic sensitization by native betaLG.

Reversible conformational change in beta-lactoglobulin A modified with N-ethylmaleimide and resistance to molecular aggregation on heating.

beta-Lactoglobulin A (beta LG A) modified with N-ethylmaleimide (NEM-beta LG A) was purified by ion exchange chromatography, and modification of beta LG A by NEM was confirmed by time of flight mass spectrometry and 5,5'-dithiobis(2-nitrobenzoic acid) methods. The fluorescent spectrum of NEM-beta LG A was slightly different from that of native beta LG A. NEM-beta LG A gave no polymerization after heating at 80 degrees C and pH 7.5, as shown by polyacrylamide gel electrophoresis. Conformational change of NEM-beta LG A was observed at 80 degrees C by ultraviolet differential spectra, whereas after cooling it recovered to its original state as before heating, indicating apparent reversible thermal denaturation. Native beta LG A is resistant to pepsin hydrolysis, whereas heated beta LG A was easily hydrolyzed by pepsin. NEM-beta LG A before heating was also resistant to pepsin hydrolysis, and after heating NEM-beta LG A was still resistant to pepsin hydrolysis. These results indicate that NEM-beta LG A maintained a conformation similar to its native form even after heating. Addition of 0.2 M NaCl to the beta LG A heated under salt-free condition induced polymerization of heated beta LG A molecules, but not that of heated NEM-beta LG A. This seemed to indicate that the formation of inter- or intramolecular disulfide linkage made the heat-induced conformational change of beta LG A irreversible.

β-Lactoglobulin Molten Globule Induced by High Pressure

Beta-lactoglobulin (beta-LG) was treated with high hydrostatic pressure (HHP) at 600 MPa and 50 degrees C for selected times as long as 64 min. The intrinsic tryptophan fluorescence of beta-LG indicated that HHP treatment conditions induced a conformational change. HHP treatment conditions also promote a 3-fold increase in the extrinsic fluorescence of 1-anilinonaphthalene-8-sulfonate and a 2.6-fold decrease for cis-paraneric acid, suggesting an increase in accessible aromatic hydrophobicity and a decrease in aliphatic hydrophobicity. Far-ultraviolet circular dichroism (CD) spectra reveal that the secondary structure of beta-LG converts from native beta-sheets to non-native alpha-helices following HHP treatment, whereas near-ultraviolet CD spectra reveal that the native tertiary structure of beta-LG essentially disappears. Urea titrations reveal that native beta-LG unfolds cooperatively, but the pressure-treated molecule unfolds noncooperatively. The noncooperative state is stable for 3 months at 5 degrees C. The nonaccessible free thiol group of cysteine121 in native beta-LG became reactive to Ellman's reagent after adequate HHP treatment. Gel electrophoresis with and without beta-mercaptoethanol provided evidence that the exposed thiol group was lost concomitant with the formation of S-S-linked beta-LG dimers. Overall, these results suggest that HHP treatments induce beta-LG into hydrophobic molten globule structures that remain stable for at least 3 months.

Functional changes in beta-lactoglobulin upon conjugation with carboxymethyl cyclodextrin.

Bovine beta-lactoglobulin-carboxymethyl cyclodextrin (beta-LG-CMCyD) conjugate was prepared using water-soluble carbodiimide, in an effort to improve the functional properties of the protein. The molar ratio of beta-LG to CMCyD in the conjugate was 1:2. The isoelectric point of the conjugate was 4.1-5.8. Spectroscopic studies indicated that the global conformation of the conjugate was similar to that of native beta-LG. Structural analyses with monoclonal antibodies indicated that there was a conformational change around (15)Val-(29)Ile (beta-sheet). The denaturation temperature of the conjugate was about 77 degrees C, which is about 4 degrees C higher than that of native beta-LG. The beta-LG-CMCyD conjugate maintained retinol-binding activity as strong as that of beta-LG. The emulsifying activity of beta-LG was much improved and the antioxidative activity of beta-LG was maintained after conjugation with CMCyD.

Reduced immunogenicity of beta-lactoglobulin by conjugation with carboxymethyl dextran.

We prepared two beta-lactoglobulin (beta-LG)-carboxymethyl dextran (CMD) conjugates (Conj. 10A and Conj. 10B) by using a water-soluble carbodiimide to decrease the immunogenicity of beta-LG. The molar ratios of beta-LG to CMD in the conjugates were 5:1 (Conj. 10A) and 2:1 (Conj. 10B). The beta-LG-CMD conjugates maintained the retinol-binding activity of native beta-LG. Intrinsic fluorescence study indicated that shielding of the surface of beta-LG by CMD occurred in each conjugate, which was eminent in Conj. 10B. A local conformational change around (125)Thr-(135)Lys (alpha-helix) in each conjugate was detected by ELISA with monoclonal antibodies. The denaturation temperature of beta-LG evaluated by differential scanning calorimetry was greatly enhanced in each conjugate. The anti-beta-LG antibody response was markedly reduced after immunization with the beta-LG-CMD conjugates in BALB/c, C57BL/6, and C3H/He mice. We determined the B cell epitopes of beta-LG and each conjugate recognized in these mice and found that the linear epitope profiles of the beta-LG-CMD conjugates were similar to those of beta-LG, while the antibody response for each epitope was dramatically reduced. The reduced immunogenicity of beta-LG was most marked in the case of Conj. 10B, which contained more CMD than Conj. 10A, and was effectively shielded by CMD. We concluded that masking of epitopes by CMD is responsible for the decreased immunogenicity of the beta-LG in these conjugates.

Effect of heat treatment on bovine beta-lactoglobulin A, B, and C explored using thiol availability and fluorescence.

Dilute solutions of beta-lactoglobulin (beta-Lg) A, B, and C were heated at temperatures between about 40 and 94 degrees C for 10 min, cooled, and analyzed using Trp fluorescence and extrinsic fluorescence spectra of the probe 1,8-anilinonaphthalene sulfonate (ANS). Thiol availabilities using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were determined using a separate set of samples. The normalized ANS fluorescence emission intensity and the thiol availability results showed a 1:1 relationship with the loss of nativelike but not SDS-monomeric protein, as determined by PAGE analysis. The normalized Trp emission intensity results did not show a comparable 1:1 relationship with the loss of nativelike protein, indicating that the Trp intensity arose from consequential disulfide bond reorganization and not the initial unfolding reaction. The results were also analyzed in terms of two-state models, and the midpoint temperatures (T(mid)) for the proteins were generally beta-Lg C > beta-Lg A > beta-Lg B, and the slopes at the midpoint temperatures for the A variant were generally less than those for the B and C variants indicating that beta-Lg A may denature by a different mechanism from that of beta-Lg B or beta-Lg C. The T(mid) parameters derived from the ANS fluorescence intensity results were similar to those for thiol availability and both were lower than the T(mid) values for Trp emission intensity showing that creation of an ANS binding site on a beta-Lg molecule was linked to the irreversible exposure of a thiol group and the loss of native beta-Lg but preceded the decrease in Trp(61) fluorescence quenching. These results for the differences between the behavior of the A and B or the C variants involved the creation of a destabilizing cavity by the Val(118)Ala (A --> B) substitution and the changed charge distribution within the CD loop caused by the Asp(64)Gly (A --> B) substitution.

Heat-induced aggregation of beta-lactoglobulin as a function of pH.

The effect of pH in the range 6.0-8.0 on the denaturation and aggregation of beta-lactoglobulin (beta-lg) was investigated. Results were interpreted in terms of the reaction scheme for the denaturation and aggregation of beta-lg proposed by Roefs and De Kruif (Eur. J. Biochem. 1994, 226, 883-889). The rate of conversion of native beta-lg increased strongly at higher pH values, whereas the molecular mass of the aggregates decreased strongly. In the pH range 6.4-8.0 aggregates were formed mainly by intermolecular disulfide bonds, but even at pH 6.0, thiol/disulfide exchange reactions were involved, although to a lesser extent. The time course of the exposure of the thiol group in native beta-lg upon heating and the subsequent disappearance of this group through the formation of disulfide-linked aggregates was investigated by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) and varied strongly with pH. These observations could be used, in combination with the reaction steps of the reaction scheme, to describe qualitatively the strongly pH-dependent isothermal calorimetry curves, measured at 65 degrees C.

Preliminary characterization of bovine beta-lactoglobulin after its conjugation to polyethylene glycol

The major component of the whey fraction of bovine milk, beta-lactoglobulin (betaLG), has been transformed by grafting polyethylene glycol chains either on the thiol group (free and after reduction of the S-S bridges) of the cysteine residues, or on the amino group of the lysine residues and/or of the N-terminal amino acid. Acylation of the protein was achieved at a controlled pH of 7.0 using increasing ratios of activated PEG to betaLG. Transformation of the dimeric form into the monomer occurred at least for the fully pegylated adduct. The number of polymer chains fixed per mole of protein was determined by dosage of the free amino functions still present after reaction. The incidence of pegylation on the secondary structure of betaLG was evaluated using the Fourier Transform Infrared Spectroscopy (FTIR). Denaturation studies with guanidinium hydrochloride (Gu-HCl) by means of spectrofluorimetric measurements, showed an identical behavior of native as well as of pegylated betaLG.The antigenicity of the fully pegylated adduct was examined through antigenic competition towards native betaLG. The pegylated protein exhibited less than 1/100 of the native betaLG inhibition capacity, that could moreover never be complete. This is thus demonstrating the loss of accessibility for at least multiple conformational epitopes through pegylation procedure.Spectrofluorimetric measurements showed that betaLG-N-PEG(7) was still able to bind retinol while no effect on the intrinsic fluorescence could be detected by adding palmitic acid. Whether this last ligand binds or not to pegylated betaLG is discussed.

Unfolding/refolding studies on bovine beta-lactoglobulin with monoclonal antibodies as probes. Does a renatured protein completely refold?

We investigated whether any local moieties within a protein molecule could completely refold from the denatured state to regain the native conformation. Bovine beta-lactoglobulin (beta-LG) was denatured in the presence of guanidine hydrochloride (GdnHCl) as the denaturant. Renaturation of the denatured beta-LG was attempted by dialyzing to remove GdnHCl. The renatured molecules regained the same retinol binding activity as that of native beta-LG, and physicochemical studies also indicated that refolding of the denatured beta-LG had been almost completely successful. Local structural differences between the native and renatured beta-LG molecules were evaluated by using our panel of four anti-beta-LG monoclonal antibodies (anti-beta-LG mAbs). The structures of the epitope regions in native beta-LG recognized by two of these mAbs were the same as those in renatured beta-LG. However, it is notable that the binding properties of the other two mAbs to native beta-LG indicated a wide structural difference in the epitope regions between the native and renatured beta-LG. These regions unable to completely refold were the same as those that unfolded preferentially to the alpha-helix region, shown in the previous report (Kaminogawa, S., Shimizu, M., Ametani, A., Hattori, M., Ando, O., Hachimura, S., Nakamura, Y., Totsuka, M., and Yamauchi, K. (1989) Biochim. Biophys. Acta 998, 50-56). Complete refolding was never attained by several renaturation conditions such as quicker or slower removal of the denaturant, nor by additional oxidation treatment after reducing the disulfide bonds. These results suggest that some specific moiety(ies) in a protein molecule cannot return to the native conformation from a denatured state, even if the other moieties refold completely, and that such a conformational difference between renatured and native forms has no affect on the biological function of ligand binding.