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PubMed2005

Aggregation across the length-scales in beta-lactoglobulin.

The protein beta-lactoglobulin (BLG) has been widely studied, in large part because of its importance to the food industry. Following denaturation during heating, under different conditions of pH it has been found to form either particulate (around the isoelectric point at pH 5.1) or fibrillar gels. The nature of the fibrils has recently been suggested to be the same as that identified with amyloid fibrils known for a wide-range of different proteins and implicated in many disease states. We confirm that the BLG fibrils show all the classical signatures of amyloid fibrils. In addition, the fibrils are capable themselves of aggregating further to form large-scale (many microns in size) spherulites. Polarized light microscopy and Environmental scanning electron microscopy (ESEM) have been used to explore the internal structure of these spherulites under conditions in which the solvent has not been dried off. The factors which determine whether or not the spherulites form have also been considered, together with implications for other amyloid-containing systems.

Amyloid↗
PubMed1988

Enhanced thermodynamic stability of beta-lactoglobulin at low pH. A possible mechanism.

The thermodynamic stability of beta-lactoglobulin (beta-Lg) was studied at acidic and near-neutral pH values using equilibrium thermal-unfolding measurements. Transition temperature increased with a decrease in pH from 7.5 to 6.5 and 3.0 to 1.5, suggesting an increase in the net protein stability. Determination of the change in free energy of unfolding and extrapolation into the nontransition region revealed that beta-Lg increases its stability by increasing the magnitude of the change in free energy of unfolding at the temperature of maximum stability, as well as by increasing the temperature of maximum stability. The relative difference in the change in free energy of unfolding at 70 degrees C (with a reference pH of 7.5) was positive and its magnitude increased with a decrease in pH from 7.0 to 1.5 van't Hoff plots of thermal unfolding of beta-Lg at all pH values studied were non-linear and the measured changes in the enthalpy and entropy of unfolding for beta-Lg were high and positive. The relative magnitude of change of both enthalpy and entropy at 70 degrees C (compared with pH 7.5) increased with a decrease in pH up to 1.5. A possible mechanism for the increased stability of beta-Lg at low pH is discussed.

Animals↗
PubMed1992

Position-independent expression of the ovine beta-lactoglobulin gene in transgenic mice.

The major milk whey protein of sheep, beta-lactoglobulin (BLG), is expressed specifically in the mammary gland in a developmentally regulated pattern. To identify the cis-acting DNA regions involved in the regulation of BLG expression, resected gene constructs were analysed in transgenic mice. BLG transgenes which contain at least the proximal 406 bp of the 5' flanking region were expressed in all mice analysed, at levels related to transgene copy number, and thus were expressed in a position-independent manner. Expression was restricted to the mammary gland, except in a few lines where low-level expression was also detected in the salivary gland. In these mice, BLG transgenes were expressed during pregnancy and lactation in the appropriate temporal pattern. Further resection of the 5' proximal region to -146 bp resulted in a dramatically reduced frequency of expression, without affecting tissue specificity, while a construct which retained only 79 bp of 5' flanking region was not expressed. Chromatin analysis of isolated sheep nuclei showed that the promoter resides within a DNAaseI-hypersensitive region in the mammary gland but not in the liver. A BLG transgene displayed a similar tissue-specific pattern of DNAaseI hypersensitivity in mice. These data demonstrate an essential role of the proximal DNAaseI-hypersensitive sequences for position-independent expression of the BLG gene.

Animals↗
PubMed1997

Effect of temperature on the secondary structure of beta-lactoglobulin at pH 6.7, as determined by CD and IR spectroscopy: a test of the molten globule hypothesis.

Previous CD measurements of changes in the conformation of beta-lactoglobulin at neutral pH as a function of temperature indicated the formation of a molten globule state above approx. 70 degrees C. New CD measurements are reported at temperatures up to 80 degrees C with an instrument on the Daresbury synchrotron radiation source which gives spectra of good signal-to-noise ratio down to 170 nm. IR spectra were recorded up to 94.8 degrees C with a ZnSe circle cell and a single simplified model of the substructure of the amide I' band was used to give the fractional contents of beta-sheet structure unambiguously and independently of the CD spectroscopy. The results of both techniques, however, were in agreement in showing a progressive loss of beta-sheet structure with increasing temperature, beginning below the denaturation temperature. Nevertheless, the CD spectroscopy showed a fairly abrupt loss of virtually all the helical conformation at approx. 65 degrees C. Comparison of the present results with other studies on the molten globule formed at acid pH in the lipocalin family suggests that above 65 degrees C a partly unfolded state is formed, possibly by destabilization of the intermolecular beta-strand I and the loss of the main helix, but it is not a classical molten globule transition.

Animals↗
PubMed1999

Allergy to bovine beta-lactoglobulin: specificity of human IgE to tryptic peptides.

BACKGROUND: Bovine beta-lactoglobulin (Blg) is a major cow's milk allergen. It is the main whey protein, without any counterpart in human milk. Blg chemical hydrolysates appeared to retain most of the immunoreactivity of the native protein. Allergenicity of Blg has already been shown to be associated with the four peptides derived from cyanogen bromide cleavage of Blg. OBJECTIVES: To map the major allergenic epitopes (e.g. regions of the molecule able to bind IgE) on Blg using specific IgE from sera of 46 milk-allergic patients as a probe. METHODS: Direct and competitive inhibition enzyme immunoassays involving immobilized native protein or purified peptides derived from Blg tryptic cleavage. RESULTS: Several peptides capable of specifically binding human IgEs were identified and were classified according to the intensity and frequency of the responses. The major epitopes appeared to be fragments (41-60), (102-124) and (149-162) recognized by 92, 97 and 89% of sera, respectively, whilst a second group which contained the fragments (1-8) and (25-40) was recognized by 58 and 72% of the population. A third group, comprising peptides (9-14), (84-91) and (92-100), was still detected by more than 40% of sera. CONCLUSION: Three peptides were identified as major epitopes, recognized by a large majority of human IgE antibodies. Numerous other epitopes are scattered all along the Blg sequence.

Amino Acid Sequence↗
PubMed2001

Identification of beta-lactoglobulin-derived peptides and class II HLA molecules recognized by T cells from patients with milk allergy.

BACKGROUND: Cow's milk allergy impairs the health and development of many infants since it deprives them of adequate nutrition. Cow's milk fractions contain many allergens, and beta-lactoglobulin (BLG) is one of the major allergens. OBJECTIVE: The purpose of this study was to determine T cell epitopes, antigen-presenting molecules and cytokine production by T cells in relation to BLG. The results can provide new therapeutic possibilities of using analogue peptides of BLG for infants with cow's milk allergy. METHODS: Using a mixture of a panel of overlapping synthetic peptides that cover the entire BLG molecule, we established polyclonal BLG-specific short-term T cell lines and clones from peripheral blood mononuclear cells of four patients with allergy to cow's milk carrying most of the common human leucocyte antigen (HLA) haplotypes seen in the Japanese population. We then identified the T cell epitopes and antigen-presenting molecules, and measured the production of cytokines interleukin (IL)-4, IL-5 and interferon-gamma in the culture supernatants. RESULTS: The T cell lines established from the four patients responded to seven different peptides. Three of the peptides stimulated the T cells of two donors, regardless of the HLA types. The patterns of inhibition of the proliferative responses of the cell lines by anti-HLA class II antibodies were heterogeneous; three were mainly inhibited by anti-HLA-DR mAbs, and the other was inhibited by anti-HLA-DQ mAbs. High levels of IL-5 were produced by these T cell lines. CONCLUSIONS: Patients' T cells recognized BLG in association with a variety of HLA-DR or -DQ as antigen-presenting molecules. Although some peptides did have a more potent T cell stimulatory activity than others, the T cell receptor ligands formed with the BLG molecule are heterogeneous. Peptides for the desensitization of T cells of the patients with cow's milk allergy need to be designed keeping in mind the different requirements in different ethnic groups.

Amino Acid Sequence↗
PubMed2003

Elicitation of the allergic reaction in beta-lactoglobulin-sensitized Balb/c mice: biochemical and clinical manifestations differ according to the structure of the allergen used for challenge.

BACKGROUND: Mouse models of allergy are used to study the mechanisms of induction and perpetuation of bronchopulmonary hyper-reactivity (BHR) as related to eosinophils and specific IgE. OBJECTIVE: Our aim was to adapt the current model for the study of bovine beta-lactoglobulin (BLG), a major cow's milk allergen, and to further analyse the mechanisms of the acute and late allergic reaction. METHODS: Female Balb/c mice were sensitized intraperitoneally with BLG and the influence of the adjuvant and of the BLG dose on the IgE response was analysed, IgE and IgG1 epitopes being characterized. Once optimized, this model was applied to the study of the active phase of allergy in the respiratory tract after a single airway challenge using native or denatured BLG, which contains only linear epitopes. RESULTS: An immediate allergic reaction was characterized by the rapid release of histamine into the bronchoalveolar lavage fluids. Prostaglandin (PG)D2 was only present when the standard histamine-releasing agent compound 48/80 or denatured BLG were used as triggers, whereas native BLG induced leukotriene release. Twenty-four hours after challenge, BHR, eosinophil influx, IL-4 and IL-5 production, plasma exudation and mucus production were very much increased, differently depending on the allergen structure, and indicated the occurrence of the late allergic reaction. Our results show that the murine model can be used to study the mechanisms of allergy to clinically relevant antigens, such as those contained in cow's milk. The acute allergic reaction, which depends on the structural feature of the allergen, is composed of two distinct pathways characterized by peptido-leukotrienes or PGD2 production, which may result from distinct activation intensities of mast cells, leading to distinct late reactions. CONCLUSION: This study thus demonstrates a clear link between the structural feature of a protein, and the physiopathology of the experimental asthmatic reaction.

Analysis of Variance↗
PubMed1998

Detection and characterization of antibodies specific to food antigens (gliadin, ovalbumin and beta-lactoglobulin) in human serum, saliva, colostrum and milk.

Antibodies against food antigens are usually produced in healthy people. This humoral response can be detected both in serum and secretions. The characterization of this response can be useful for a better understanding of food-related immunological alterations. In this study, IgA and IgG antibodies specific to ovalbumin, beta-lactoglobulin or gliadin were measured in serum, saliva, colostrum and milk from 40 healthy breast-feeding women. Specific IgA and IgG to the three antigens were measured by indirect ELISA. Specific IgG levels were highest in serum and very low in the other biological fluids. No correlation between the IgG specific to the different antigens was found. Specific IgA reactivity was found in all the samples analysed. Levels observed were higher in colostrum and milk than in serum and saliva. In spite of being three different unrelated food antigens, a correlation between the levels of specific IgA was found in saliva, colostrum and milk samples of all subjects studied. The specificity of IgA anti-gliadin antibodies from serum, saliva and colostrum was analysed by immunoblotting of SDS-PAGE-separated wheat proteins. Each sample presented a unique pattern of recognition. No common pattern of recognition was found either among the same biological fluids of the different subjects tested, or among the different samples--either serum, colostrum or saliva--of the same individual. Different degrees of specificity to wheat proteins among IgA from colostrum, saliva or serum were observed, suggesting that the local IgA-producing populations are functionally different in the different tissues of the organism.

Antibodies↗
PubMed1999

The effect of alpha-lactalbumin and beta-lactoglobulin hydrolysates on the metabolic activity of Escherichia coli JM103.

Bovine milk proteins alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg) were hydrolysed with seven different proteolytic enzymes, and the effect of various hydrolysates on a genetically modified luminous Escherichia coli JM103 was tested in vitro with a bioluminescence assay for bacterial growth and metabolism. Undigested proteins did not inhibit the activity of tested E. coli JM103 at a concentration as high as 0.1 g ml-1. At the same concentrations, alpha-la hydrolysed with pepsin or trypsin and beta-lg hydrolysed with alcalase, pepsin or trypsin, showed a lower metabolic activity during the first 8 h of growth. The activity of E. coli JM103 in the presence of 25 mg ml-1 alpha-la or beta-lg hydrolysed with pepsin and trypsin was only 21% of the control after incubation for 6 h. The preliminary results indicated that ultrafiltration through 10 kDa and 1 kDa molecular mass cut-off membranes may be used to enrich bacteriostatic properties.

Escherichia coli↗
PubMed2001

Crystal structures of bovine beta-lactoglobulin in the orthorhombic space group C222(1). Structural differences between genetic variants A and B and features of the Tanford transition.

The crystal structures of beta-lactoglobulin genetic variants A and B have been determined in the orthorhombic space group C222(1) (lattice Y) by X-ray diffraction at 2.0 A and 1.95 A resolution, respectively. The structural comparison shows that both variants exhibit the open conformation of the EF loop at the pH of crystallization (pH 7.9), in contrast to what has been reported for the same genetic variants at pH 7.1 in the trigonal space group P3221 (lattice Z) [Qin, B.Y., Bewley, M.C., Creamer, L.K., Baker, E.N. & Jameson, G.B. (1999) Protein Sci. 8, 75-83]. Furthermore, it was found that the stereochemical environment of Tyr42 changes significantly with pH variation between pH 7 and pH 8. This may provide a structural explanation for an as yet unexplained feature of the Tanford transition, namely the increase in exposure of a tyrosine residue.

Animals↗
PubMed2003

Trehalose influence on beta-lactoglobulin stability and hydration by time resolved fluorescence.

The stabilizing role of the disaccharide trehalose on beta-lactoglobulin (BLG) against its chemical denaturation both at native and acidic pH has been explored by means of time-resolved fluorescence of the probe acrylodan covalently bound to the unique free cysteine of BLG. The changes in acrylodan fluorescence lifetime with guanidinium chloride concentration reveal BLG sigmoidal denaturation profiles which depend upon the amount of trehalose in solution. When adding trehalose the transition midpoint shifts towards higher denaturant concentration. This effect has been measured by fitting the data with a two-state model whose parameters indicate that an almost 60% increase in the denaturation free energy is induced independently of trehalose concentrations and pH values. Fluorescence anisotropy measurements performed in the same conditions reveal that the internal dynamics are largely affected by the sugar, which makes the acrylodan environment more rigid, and by the denaturant that acts in the opposite way. The overall rotational diffusion of BLG suggests that trehalose affects the hydrodynamic properties of the solution in the proximity of the protein; tentative mechanisms are discussed.

2-Naphthylamine↗
PubMed1990

In vitro transport of beta-lactoglobulin across the jejunum of lactose-fed rats.

Changes in protein absorption through the intestinal mucosa occur with aging and might reflect modifications in enterocyte membrane characteristics. We have observed that bovine beta-lactoglobulin (beta-LG) was efficiently transported across the small intestine of adult rats in vitro and that 12-16% of the absorbed protein was recovered intact or as large hydrophobic peptides. Ten percent lactose-feeding resulted in decreased tissue conductance and significantly reduced (-58%, P less than 0.05) beta-LG transport across rat small intestinal mucosa. The amounts of beta-LG absorbed, either as amino acids and peptides or as intact protein, were reduced to the same extent. Therefore, the effect of lactose feeding might be related to a decrease in protein endocytosis at the brush-border level, rather than to reduced protein transport across tight junctions.

Animals↗
PubMed2000

Topology to geometry in protein folding: beta-lactoglobulin.

Evolution of protein structure from random coil to native is first represented topologically by its time-dependent sequences of discretized Ramachandran basins occupied by successive backbone residues. Introducing energetic and entropic criteria at each instant of observation transforms the description from a structurally ambiguous topological representation to an unambiguous geometric picture of the folding process. The method is applied with success to folding of beta-lactoglobulin, traditionally perplexing because of its reputed nonhierarchical folding pattern. This molecule passes through a stage, ca. 0.1 microseconds duration, of transient, "flickering" alpha-helical structure, until a bit of tertiary structure forms that stabilizes the system long enough to allow it to pass to its native beta-sheet.

Computer Simulation↗
PubMed1999

beta-lactoglobulin binds palmitate within its central cavity.

Bovine beta-lactoglobulin (beta-Lg) has been studied extensively in both the isolated and the naturally occurring states. It is a commercially important whey protein of obvious nutritional value but, so far, one that has no clearly identified biological function. In common with many of the other members of the lipocalin family to which it belongs, beta-Lg binds hydrophobic ligands, and it appears possible that there are at least two distinct binding sites per monomer for a variety of ligands. By comparison with other members of the family, there is a probable binding site in the central cavity of the molecule that is formed by the eight antiparallel beta-strands that are typical of the lipocalins. We have now cocrystallized beta-Lg with palmitic acid, and the refined structure (R = 0.204, Rfree = 0.240 for 6,888 reflections to 2.5-A resolution) reveals that the ligand binds in the central cavity in a manner similar to the binding of retinol to the related lipocalin, serum retinol-binding protein. The carboxyl group binds to both Lys-60 and Lys-69 at the entrance to the cavity. The hydrophobic tail stretches in an almost fully extended conformation into the center of the protein. This is the first direct observation of a ligand binding to beta-Lg.

Animals↗
PubMed2003

Reversible unfolding of bovine beta-lactoglobulin mutants without a free thiol group.

Bovine beta-lactoglobulin (beta-lg) has been used extensively as a model for studying protein folding. One of the problems preventing clarification of the folding mechanism is the incomplete reversibility from the unfolded state, probably caused by the thiol-disulfide exchange between a free thiol at Cys-121 and two disulfide bonds. We constructed and expressed three beta-lg subtype A mutants in which Cys-121 was replaced by Ala, Ser, or Val (i.e. C121A, C121S, and C121V). We studied the reversibilities of these mutants from urea denaturation using circular dichroism, tryptophan fluorescence, reversed-phase and gel-filtration high performance liquid chromatographies, and SDS-PAGE. The folded structure of each mutant was similar to that of wild-type beta-lg. Urea-induced unfolding at pH 7.0 and 3.0 showed that although the C121S mutation notably decreases the stability, the destabilizing effects of the C121A and C121V mutations are less severe. For all of the mutants, complete refolding from the unfolded state in 8 M urea at both pH 7.0 and 3.0 was observed. Kinetics of the formation of the irreversibly unfolded species of wild-type beta-lg in 8 M urea at pH 7.0 indicated that, first, an intramolecular thiol-disulfide exchange occurs to produce a mixture of species with non-native disulfide bonds followed by the intermolecular thiol-disulfide exchange producing the oligomers. These results indicate that intramolecular and intermolecular thiol-disulfide exchange reactions cause the low reversibility of wild-type beta-lg especially at neutral pH and that the mutation of Cys-121 improves the reversibility, enabling us to study the folding of beta-lg more exactly under various conditions.

Amino Acid Substitution↗
PubMed2004

Epitope mapping of a monoclonal antibody specific to bovine dry milk: involvement of residues 66-76 of strand D in thermal denatured beta-lactoglobulin.

beta-Lactoglobulin (beta-LG) is a bovine milk protein sensitive to thermal denaturation. Previously, we demonstrated that such structural change can be detected by a monoclonal antibody (mAb) specific to denatured beta-LG. In the present study, we show a dramatic increase in beta-LG immunoreactivity when heating raw milk between 70 and 80 degrees C. To map out the specific epitope of beta-LG recognized by this mAb, we used a combined strategy including tryptic and CNBr fragments, chemical modifications (acetylation and carboxymethylation), peptide array containing in situ synthesized peptides, and a synthetic soluble peptide for immunoassays. The antigenic determinant we defined was exactly located within the D strand (residues 66-76) of beta-LG. Circular dichroic spectral analysis shows that carboxymethylation on beta-LG not only resulted in a substantial loss of beta-configuration but also exerted a 10 times increase in immunoreactivity as compared with heated beta-LG. The result suggests that a further disordered structure occurred in beta-LG and thus rendered the mAb recognition. Mutations on each charged residue (three Lys and one Glu) revealed that Lys-69 and Glu-74 were extremely essential in maintaining the antigenic structure. We also show an inverse relationship between the immunoreactivity in heated beta-LG and its binding to retinol or palmitic acid. Most interestingly, pH 9-10, which neutralizes the Lys groups of beta-LG, not only reduced its immunoreactivity but also its binding to palmitic acid implicating a role of Lys-69. Taken together, we concluded that strand D of beta-LG participated in the thermal denaturation between 70 and 80 degrees C and the binding to retinol and palmitic acid. The antigenic and biochemical roles of mAb specific to D strand are discussed in detail.

Amino Acid Sequence↗
PubMed1996

Thermal unfolding of beta-lactoglobulin, alpha-lactalbumin, and bovine serum albumin. A thermodynamic approach.

Heat-treatment is one of the most commonly used processes in food preparation technology. An understanding of the thermodynamics of protein stability and of conformational changes of proteins, acquired through the measurement of the denaturation temperature, is therefore of particular importance. This paper attempts to shed light on the interpretation of recent calorimetric data on the thermal denaturation of bovine beta-lactoglobulin, alpha-lactalbumin, and bovine serum albumin by showing that thermodynamic parameters of heat-induced unfolding, measured by differential scanning calorimetry, are closely related to the prevailing chemical conditions such as pH, concentration of ions, protein purity, and protein concentration.

Hot Temperature↗
PubMed1998

Spectroscopic and thermodynamic evidence for a complex denaturation mechanism of bovine beta-lactoglobulin A.

We present a spectroscopic and calorimetric study on the thermal denaturation of bovine beta-lactoglobulin (beta-lg) variant A. Spectroscopic data allowed detection of a stable intermediate emerging from structural modifications restricted to local regions of the native molecule. It is suggested that this kind of intermediate could be a common property of lipocalins. Using the same set of parameters that has successfully related thermodynamics and structural properties of other proteins, it is shown that the thermally denatured state of beta-lg retains a significant amount of buried hydrophobic surface area. Thus, despite being a small protein composed of a single structural domain, beta-lg exhibits a complex unfolding mechanism, comprising at least two other species different from the native and completely unfolded states.

Animals↗