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PubMed2002

Towards the understanding of molecular mechanisms in the early stages of heat-induced aggregation of beta-lactoglobulin AB.

Heat-induced aggregation of bovine beta-lactoglobulin AB (10 mg/ml) was studied at 68.5 degrees C at two different pH values (6.7, 4.9) using gel electrophoresis techniques and matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis under non-reducing and reducing conditions showed that in the early stages of the aggregation of beta-lactoglobulin disulfide linked aggregates were formed on heating at pH 6.7, but not at pH 4.9. We related this result to the pH-dependent activity of the free thiol group at C121. Mass spectrometric analyses were conducted in two steps. The first involved the analysis of intact non-native monomers and dimers following their ultrasonic passive elution into a suitable solvent mixture in order to confirm the identity of the different gel bands. The second step comprises the analysis of in-gel digests for the determination of disulfide patterns in non-native monomers, covalent dimers and trimers. The results of in-gel digestions analyzed by mass spectrometry suggest that non-native dimers could result from the formation of inter-molecular disulfide bonds C121-C66, C160-C160, or C121-C160. Moreover, two inter-molecular bonds C121-C66 and C160-C160 between two and the same monomer units have been detected, which may play an important role in limiting the process of covalent beta-lactoglobulin network formation. The combination of SDS-PAGE and MALDI-TOF MS enables us to understand the mechanism of beta-lactoglobulin aggregation at the macromolecular level.

Amino Acid Sequence↗

PubMed2003

Detection and quantification of bovine, ovine and caprine milk percentages in protected denomination of origin cheeses by reversed-phase high-performance liquid chromatography of beta-lactoglobulins.

A method for detecting and quantifying bovine, ovine and caprine milk mixtures in milk and cheeses by means of reversed-phase high-performance liquid chromatography (RP-HPLC) of beta-lactoglobulins is described. Gradient elution was carried out with a flow rate of 0.5 ml/min and a temperature of 45 degrees C, using a mixture of two solvents: solvent A (0.1% TFA in water) and solvent B (0.09% TFA in 80% aqueous acetonitrile, v/v). The effluent was monitored at 215 nm. Under the conditions used different chromatographic patterns were obtained for bovine, ovine and caprine whey proteins. Each milk type presented different retention times for beta-lactoglobulin peaks. Binary mixtures of bovine and ovine or bovine and caprine raw milks containing 1, 2, 5, 10, 20, 30, 50, 75 and 95% (v/v) of bovine milk, as well as ovine and caprine milk mixtures containing 1, 2, 5, 10, 20, 30, 50, 75 and 95% (v/v) of ovine milk were used for cheese making. Cheeses were prepared and ripened, according to traditional methods. Milk mixtures, fresh and ripened cheeses were analyzed. A linear relationship was established between log 10 of beta-lactoglobulin peaks ratio (calculated as peak area values ratio) and log 10 of the relative percentage of bovine or ovine milk. The ratio between beta-lactoglobulin peaks was not affected by the degree of ripening. Thus, enabling the quantification of milk type percentage, with a detection limit of 2%. This technique allowed quantification of milk species within the concentration range of 5-95%. The method was successfully applied for authenticity evaluation and quantitative determination of ovine and caprine milk percentages of commercial protected denomination of origin (PDO) cheeses.

Animals↗

PubMed1999

The mucosal adhesion receptor alpha4beta7 integrin is selectively increased in lymphocytes stimulated with beta-lactoglobulin in children allergic to cow's milk.

BACKGROUND: It has been shown in mice that the integrin alpha4beta7 directs the migration of memory T cells into the gut-associated lymphoid tissue. However, little is known about T-cell homing mechanisms in children with food allergies. OBJECTIVE: We investigated the expression of this and other integrins in children with different manifestations of cow's milk allergy (urticaria, atopic dermatitis, and wheezing). METHODS: PBMCs were stimulated with beta-lactoglobulin, 1 of the major allergenic proteins in cow's milk, and tetanus toxoid. Integrin expression was studied by flow cytometric analysis after 1 week of culture. RESULTS: We found significantly higher expression of the alpha4beta7 integrin in cells from patients compared with control subjects with no allergies (P =. 005) when beta-lactoglobulin was used to stimulate the cells. alpha4beta7 integrin was also expressed at significantly higher levels in beta-lactoglobulin-stimulated cells than in tetanus toxoid-stimulated cells (P =.005). The alphaEbeta7 and the alpha4beta1 integrins were not upregulated by allergen stimulation. Most alpha4beta7 integrin-expressing cells were identified as CD4(+) T cells. CONCLUSION: These results show that alpha4beta7 integrin expression after stimulation with beta-lactoglobulin correlates with the presumptive site of cow's milk sensitization (ie, the gut-associated lymphoid tissue but not with the site of symptoms of cow's milk allergy).

CD4-Positive T-Lymphocytes↗

PubMed1998

Reversibility of heat-induced conformational changes and surface exposed hydrophobic clusters of beta-lactoglobulin: their role in heat-induced sol-gel state transition.

The effects of heat-treatment on the conformational changes of beta-lactoglobulin were monitored by differential scanning calorimetry (DSC), binding properties to 1-anilino-8-naphthalenesulphonic acid (ANS) and to 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB). The thermal transition of beta-lactoglobulin was 100% reversible on re-heating and its binding properties to the ANS fluorescent-dye and to the DTNB probe did not significantly change when the first heating was made to a temperature T < Tmax, that of the DSC maximal peak deviation of unheated solutions. When the solutions were heated to higher temperatures, the degree of reversibility of the thermal transition decreased, while the beta-lactoglobulin surface hydrophobicity increased. Furthermore, the time (tg) needed for the sol-gel state transitions was highly temperature-dependent for the solutions showing no significant reactivity with the DTNB probe, a high percentage of residual tertiary structure but a low surface hydrophobicity. For beta-lactoglobulin showing < 50% residual tertiary structure but high surface hydrophobicity, tg values were hardly temperature-dependent. The results are discussed in terms of the role of hydrophobic interactions in the aggregation process of denatured beta-lg molecules.

Anilino Naphthalenesulfonates↗

PubMed1996

High helicity of peptide fragments corresponding to beta-strand regions of beta-lactoglobulin observed by 2D-NMR spectroscopy.

BACKGROUND: Whereas protein fragments, when they are structured, adopt conformations similar to that found in the native state, the high helical propensity of beta-lactoglobulin, a predominantly beta-sheet protein, suggested that the fragments of beta-lactoglobulin can assume the non-native helical conformation. In order to assess this possibility, we synthesized four 17-18-residue peptides corresponding to three beta-strand regions and one helical region (as a control) of beta-lactoglobulin and examined their conformation. RESULTS: We observed residual helicities of up to 17% in water, by far-UV CD, for all four peptide fragments. The helices could be significantly stabilized by the addition of TFE, and the NMR analyses in a mixture of 50% water/TFE indicated that helical structures are formed in the central region whereas both termini are frayed. Thus, the very same residues that form strands in the native beta-lactoglobulin showed high helical preferences. CONCLUSIONS: These results stand out from the current general view that peptide fragments isolated from proteins either are unfolded or adopt native-like secondary structures. The implications of the results in the mechanism of protein folding and in designing proteins and peptides are significant.

Amino Acid Sequence↗

PubMed1993

Comparison of the ability to bind lipids of beta-lactoglobulin and serum albumin of milk from ruminant and non-ruminant species.

The interaction of sheep, horse, pig, human and guinea-pig whey proteins with fatty acids has been studied. Using gel filtration and autoradiography, it was found that sheep beta-lactoglobulin and serum albumin from all species had the ability to bind fatty acids in vitro. Sheep beta-lactoglobulin, isolated from milk, had approximately 0.5 mol fatty acids bound per mol monomer protein, and albumin from sheep, horse and pig contained approximately 4.5, 2.9 and 4.7 mol fatty acids/mol protein respectively. However, beta-lactoglobulin from horse and pig milk had neither fatty acids physiologically bound nor the ability to bind them in vitro. Albumin was the only whey protein detected with bound fatty acids in these species as well as in human and guinea pig. This suggests that the ability of ruminant beta-lactoglobulin to bind fatty acids was not shared by the same protein of non-ruminants.

Animals↗

PubMed1998

Identification of C-terminally truncated forms of beta-lactoglobulin in whey from Romagnola cows' milk by two dimensional electrophoresis coupled to mass spectrometry.

Four minor protein components were detected in whey from Romagnola cows' milk by polyacrylamide gel isoelectric focusing and two dimensional gel electrophoresis. Individual protein spots were transferred by electroblotting on to a polyvinylidene difluoride membrane and isolated by cutting out the relevant area. After in situ trypsinolysis, a portion of the digest was analysed directly by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The mass profile allowed us to establish a correlation between beta-lactoglobulins A and B and the four minor whey protein components. They were identified as C-terminally truncated beta-lactoglobulin A and B variants with missing N-terminal peptides, beyond residues in the range 100-123 and 136-147 respectively. Two of the minor components were related to beta-lactoglobulin A and two to beta-lactoglobulin B.

Amino Acid Sequence↗

PubMed1988

Accessibility and mobility of lysine residues in beta-lactoglobulin.

N epsilon-[2H6]Isopropyllysyl-beta-lactoglobulin was prepared by reductive alkylation of beta-lactoglobulin with [2H6]acetone and NaBH4 to provide a 2H (NMR) probe for the study of lysine involvement in lipid-protein interactions. Amino acid analysis showed 80% of the protein's 15 lysine residues to be labeled. Unmodified lysine residues were located through peptide maps produced from CNBr, tryptic, and chymotryptic digests of the labeled protein. Lys47 was not modified; Lys135,138,141, located along an amphipathic helical rod, were each partially unmodified. All other lysine residues were at least 90% modified. Average correlation times calculated from 2H NMR spectra were 20 and 320 ps for 8.7 and 3.3 residues, respectively, in 6 M guanidine hydrochloride; in nondenaturing solution, values of 70 and 320 ps were obtained for 6.5 and 3.2 residues, respectively, with the remaining 2.3 modified residues not observed, suggesting that side chains of lysine residues in unordered or flexible regions were more mobile than those in stable periodic structures. 2H NMR spectra of the protein complexed with dipalmitoylphosphatidylcholine confirmed the extrinsic membrane protein type behavior of beta-lactoglobulin previously reported from 31P NMR studies of the phospholipids complexed with beta-lactoglobulin. Although no physiological function has yet been identified, comparison of these results with the X-ray structure [Papiz et al. (1986) Nature (London) 324, 383-385] supports the hypothesis that residues not accessible for modification may help to stabilize the cone-shaped beta-barrel thought to contain binding sites for small lipid-soluble molecules.

Amino Acid Sequence↗

PubMedDate not supplied

Effect of pH on the ternary solution behavior of beta-lactoglobulin.

Ternary phase diagrams (TPDs) were constructed for aqueous beta-lactoglobulin solutions containing ethanol and (NH4)2SO4 at pHs of 7, 5, and 3 for temperatures between 20 and 70 degrees C. The addition of (NH4)2SO4 generally led to the production of a reversible precipitate, a transformation that was not strongly influenced by temperature or pH. In contrast, at pH 7 and 20 degrees C, ethanol concentrations >12% led to the formation of a molten-globule structure, which gelled at protein concentrations >10%. Destabilization of beta-lactoglobulin structure occurred at lower ethanol concentrations as temperature was increased, until at 70 degrees C, all solutions that were previously liquid at room temperature had transformed into a gel. At pH 5.0, near beta-lactoglobulin's isoelectric point, demixing dominated, leading to the creation of either irreversible precipitates or a paste-like microgel. Elevated temperatures caused the previously liquid morphology to transform into either a reversible aggregate or microgel. Solution behavior at pH 3 had characteristics of what was observed at pHs 7 and 5. At moderated protein and ethanol concentrations, a paste-like microgel was observed, whereas at higher ethanol concentrations, beta-lactoglobulin formed a gel. This work demonstrates how small changes in protein structure at the molecular level can have a dramatic effect on macroscopic morphology.

Ethanol↗

PubMed2004

Modification of IgE binding during heat processing of the cow's milk allergen beta-lactoglobulin.

The effect of heat treatment on the IgE binding ability of beta-lactoglobulin, as pure protein or in whole milk, was studied by inhibition of IgE antibody binding using FEIA-CAP inhibition. A slight but significant decreased IgE binding was seen between unheated and heat-treated beta-lactoglobulin solution at 74 degrees C (IC(50) = 2.03 and 3.59 microg/mL, respectively, p = 0.032). A more pronounced decrease was found at 90 degrees C with an IC(50) of 8.45 microg/mL (p = 0.014). The inhibition of IgE binding of milk after heat treatment at 90 degrees C was also significantly decreased (p = 0.007). However, at all heat treatments, a similar total amount of IgE antibodies could be inhibited at a sufficiently high concentration of beta-lactoglobulin. The inhibiting ability of beta-lactoglobulin was significantly impaired in some fermented acidified milk products such as yogurt as compared to that in nonfermented milk (p < 0.001). There was only a small difference of IgE binding between the native forms of genetic variants A and B.

Allergens↗

PubMed2005

Interactions of vitamin D3 with bovine beta-lactoglobulin A and beta-casein.

It is of nutritional significance to fortify processed dairy products (e.g., cheese, yogurt, and ice cream) with vitamin D3; however, the inherent complexity of these foods may influence the stability and bioavailability of this nutrient. In the present study, the interactions of vitamin D3 with beta-lactoglobulin A and beta-casein were investigated under various environmental conditions (i.e., pH and ionic strength) using fluorescence and circular dichroism spectroscopic techniques. The results indicated that vitamin D3 was bound to both beta-lactoglobulin A and beta-casein depending on the solution conditions. The apparent dissociation constants ranged from 0.02 to 0.29 microM for beta-lactoglobulin A, whereas the beta-casein apparent dissociation constants ranged from 0.06 to 0.26 microM. The apparent mole ratios were also comparable, i.e., 0.51-2.04 and 1.16-2.05 mol of vitamin D3 were bound per mole of beta-lactoglobulin A and beta-casein, respectively. It was concluded that these interactions may strongly influence vitamin D3 stability and, hence, bioavailability in processed dairy products.

Animals↗

PubMed2000

Steady-state kinetics and thermodynamics of the hydrolysis of beta-lactoglobulin by trypsin.

Hydrolysis of beta-lactoglobulin (in an equimolar mixture of the A and B variant) by trypsin in neutral aqueous solution [pH 7.7 at 25 degrees C, ionic strength 0.08 (NaCl)] was followed by capillary electrophoresis and thermodynamic parameters derived from a Michaelis-Menten analysis of rate data obtained at 10, 20, 30, and 40 degrees C for disappearance of beta-lactoglobulin. Enthalpy of substrate binding to the enzyme and the energy of activation for the catalytic process were found to have the values, DeltaH(bind) = -28 +/- 4 kJ mol(-)(1) and E(a) = 51 +/- 18 kJ mol(-)(1), respectively. Thus, beta-lactoglobulin shows an enthalpy of activation for free substrate reacting with free enzyme of about 21 kJ mol(-)(1), corresponding to a transition state stabilization of 60 kJ mol(-)(1) when compared to acid-catalyzed hydrolysis. The catalytic efficiency of trypsin in hydrolysis of beta-lactoglobulin is increased significantly by temperature; however, this effect is partly counteracted by a weaker substrate binding resulting in an increase by only 25%/10 degrees C in overall catalytic efficiency.

Chromatography, High Pressure Liquid↗

PubMed2004

Disruption of viscoelastic beta-lactoglobulin surface layers at the air-water interface by nonionic polymeric surfactants.

Nonequilibrium interfacial layers formed by competitive adsorption of beta-lactoglobulin and the nonionic triblock copolymer PEO99-PPO65-PEO99 (F127) to the air-water interface were investigated in order to explain the influence of polymeric surfactants on protein film surface rheology and foam stability. Surface dilatational and shear rheological methods, surface tension measurements, dynamic thin-film measurements, diffusion measurements (from fluorescence recovery after photo bleaching), and determinations of foam stability were used as methods. The high surface viscoelasticity, both the shear and dilatational, of the protein films was significantly reduced by coadsorption of polymeric surfactant. The drainage rate of single thin films, in the presence of beta-lactoglobulin, increased with the amount of added F127, but equilibrium F127 films were found to be thicker than beta-lactoglobulin films, even at low concentration of the polymeric surfactant. It is concluded that the effect of the nonionic triblock copolymer on the interfacial rheology of beta-lactoglobulin layers is similar to that of low molecular weight surfactants. They differ however in that F127 increases the thickness of thin liquid films. In addition, the significant destabilizing effect of low molecular weight surfactants on protein foams is not found in the investigated system. This is explained as due to long-range steric forces starting to stabilize the foam films at low concentrations of F127.

Adsorption↗

PubMed2000

Mechanisms of protein modification during model anti-viral heat-treatment bioprocessing of beta-lactoglobulin variant A in the presence of sucrose.

To ensure the safety of plasma and recombinant therapeutic proteins, heat treatment is routinely applied to these biopharmaceuticals as a means of virus inactivation. However, to maintain protein integrity during heat treatment it is necessary to use high concentrations of thermostabilizing excipients, such as sucrose, in order to prevent protein damage. In this study we describe the covalent modifications inferred to a model protein, beta-lactoglobulin A, that occur during typical and extended anti-viral heat treatments. The chemical derivation and mechanisms by which these modifications arise are addressed. Heat treatment initiated hydrolysis of sucrose to glucose and fructose, which in turn were degraded to glyoxal. Glyoxal and the free reducing sugars reacted with free amino groups in beta-lactoglobulin A to yield Maillard glycation adducts and advanced glycation end products (AGEs). The major mechanism for AGE formation was via degradation of glucose-derived Schiff-base adducts. Heat treatment and glycation of beta-lactoglobulin A resulted in thiol-disulphide interchange reactions leading to protein oligomerization. A small population of beta-lactoglobulin A non-disulphide-linked dimers were also observed with increasingly harsh heat treatments. These findings have implications for (i) improvements in the safety and efficacy of heat-treated protein biopharmaceuticals and (ii) our understanding of the mechanisms of protein glycation and AGE adduct formation.

Biotechnology↗

PubMed1992

Transglutaminase catalyses the modification of glutamine side chains in the C-terminal region of bovine beta-lactoglobulin.

The transglutaminase-catalysed incorporation of primary amines (putrescine and monodansylcadaverine) into bovine beta-lactoglobulin has been studied. In the presence of 1 mM-dithiothreitol between 1 and 2 mol of amine can be incorporated per mol of beta-lactoglobulin subunit. There is very little incorporation of amines in the absence of reducing agent. By isolating and sequencing the modified peptides, the sites of modification have been identified as Gln-159 (preferred) and Gln-155. C.d. has been used to study the structure of beta-lactoglobulin over a range of pH values and in the presence or absence of dithiothreitol. The results are discussed in terms of the X-ray-crystallographically determined structure of beta-lactoglobulin.

Amino Acid Sequence↗

PubMed1999

Allergy to bovine beta-lactoglobulin: specificity of immunoglobulin E generated in the Brown Norway rat to tryptic and synthetic peptides.

BACKGROUND: Animal models which reflect the induction and development of food-allergic reactions are important in the identification of allergenic potential of food proteins and peptides. A number of rat strains, including PVG, Hooded Lister and Brown Norway have been shown to produce immunoglobulin (Ig) E antibodies to food proteins as well as to inhaled allergens. Previous work in our laboratory using the Brown Norway (BN) rat has shown that specific IgE is produced following administration of ovalbumin and milk products via both enteral and parenteral route; this has allowed us to rank ovalbumin, lactoferrin and bovine serum albumin in terms of their inherent allergenic potential and has enabled us to demonstrate that milk protein allergens recognized by the systemically-sensitized animal are consistent with those recognized by sera from cow's milk-allergic patients (the most common allergens recognized were beta-lactoglobulin and the alpha, beta and kappa-caseins). OBJECTIVE: To demonstrate that the BN rat model can be used to identify the major IgE-binding peptides from beta-lactoglobulin, a known human food allergen, and that those IgE-binding peptides are similar to those recently identified using sera from cow's milk-allergic patients. METHODS: BN rats were exposed to beta-lactoglobulin or to semiskimmed milk via the intraperitoneal route in the presence of the adjuvant carrageenan. Specific IgE raised against beta-lactoglobulin was determined by a direct enzyme immunoassay using acetyl-cholinesterase substrate; specific IgG responses were also monitored. Overlapping synthetic peptides and tryptic peptides were used within the ELISA to identify the major and minor IgE-binding immunoreactive sequences. RESULTS: In terms of comparative immunogenicity, there appeared to be sequences that were predominantly IgE- or IgG-reactive. IgE-dominant regions were amino acid sequences 21-40, 41-60, 107-117 and 148-168 whereas sequences 1-24, 67-77, 82-92, 85-95 and 117-127 appeared more selective for IgG antibody recognition. An increased capacity to induce specific IgE was observed when the allergen was present in the context of whole food. CONCLUSIONS: These studies provide evidence that the immune system of the BN rat and humans - at least in the case of milk allergens - is recognizing similar protein allergens and indeed, at the molecular level, similar peptide epitopes.

Animals↗

PubMed1990

The hormonal control of ovine beta-lactoglobulin gene in cultured ewe mammary explants.

Mammary explants from pregnant ewes were cultured in the presence of insulin, cortisol and prolactin, either alone, or in combination. After 2 d of culture, total RNA was extracted from explants and the content of beta-lactoglobulin mRNA was estimated using a specific labelled cDNA probe. The mRNA for beta-lactoglobulin was only deinduced slowly in the absence of hormone during the culture. Prolactin alone induced the accumulation of the mRNA. Insulin and cortisol added together were also stimulatory, but they only moderately amplified the prolactin effect. Beta-lactoglobulin gene in ewes, is therefore, controlled by the lactogenic hormones which also induce casein gene expression. The amplitude of the stimulation was unexpectedly low. This seems due in part to the fact that the gene was only deinduced weakly. In this respect, beta-lactoglobulin gene appears to be less dependent on lactogenic hormones under these experimental conditions than casein genes.

Animals↗

PubMed1997

Consumption of cow milk and egg by lactating women and the presence of beta-lactoglobulin and ovalbumin in breast milk.

beta-Lactoglobulin and ovalbumin in mature human milk in healthy lactating Japanese women (n = 24) were determined by using an enzyme-linked immunosorbent assay. Subjects consumed > or = +200 mL cow milk/d for 1 wk before the sampling day and exactly 200 mL cow milk on the morning of the sampling day. beta-Lactoglobulin was detected (> 0.1 microgram/L) in breast milk in 15 of the 24 subjects (62.5%), with a maximum concentration of 16.5 micrograms/L. Ovalbumin was detected in only two subjects (8.3%) after the subjects followed their usual diet. beta-Lactoglobulin concentrations were low in the subjects whose cow milk consumption during the entire lactating period was low, even though all subjects consumed the same amount of cow milk before sampling. This result suggests that beta-lactoglobulin concentrations in breast milk are related to long-term consumption of cow milk. Amounts of food antigens in breast milk may be controlled by modifying the daily maternal diet.

Adult↗