Proteolysis of bovine beta-lactoglobulin during thermal treatment in subdenaturing conditions highlights some structural features of the temperature-modified protein and yields fragments with low immunoreactivity.
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Bovine beta-lactoglobulin was hydrolyzed with trypsin or chymotrypsin in the course of heat treatment at 55, 60 and 65 degrees C at neutral pH. At these temperatures beta-lactoglobulin undergoes significant but reversible structural changes. In the conditions used in the present study, beta-lactoglobulin was virtually insensitive to proteolysis by either enzyme at room temperature, but underwent extensive proteolysis when either protease was present during the heat treatment. High-temperature proteolysis occurs in a progressive manner. Mass spectrometry analysis of some large-sized breakdown intermediates formed in the early steps of hydrolysis indicated that both enzymes effectively hydrolyzed some regions of beta-lactoglobulin that were transiently exposed during the physical treatments and that were not accessible in the native protein. The immunochemical properties of the products of beta-lactoglobulin hydrolysis were assessed by using various beta-lactoglobulin-specific antibodies, and most epitopic sites were no longer present after attack of the partially unfolded protein by the two proteases. (+info)
Multiple effects of genetic background on variegated transgene expression in mice.
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BLG/7 transgenic mice express an ovine beta-lactoglobulin transgene during lactation. Unusually, transgene expression levels in milk differ between siblings. This variable expression is due to variegated transgene expression in the mammary gland and is reminiscent of position-effect variegation. The BLG/7 line was created and maintained on a mixed CBA x C57BL/6 background. We have investigated the effect on transgene expression of backcrossing for 13 generations into these backgrounds. Variable transgene expression was observed in all populations examined, confirming that it is an inherent property of the transgene array at its site of integration. There were also strain-specific effects on transgene expression that appear to be independent of the inherent variegation. The transgene, compared to endogenous milk protein genes, is specifically susceptible to inbreeding depression. Outcrossing restored transgene expression levels to that of the parental population; thus suppression was not inherited. Finally, no generation-dependent decrease in mean expression levels was observed in the parental population. Thus, although the BLG/7 transgene is expressed in a variegated manner, there was no generation-associated accumulated silencing of transgene expression. (+info)
Interaction of proteins in solution from small-angle scattering: a perturbative approach.
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In this work an improved methodology for studying interactions of proteins in solution by small-angle scattering is presented. Unlike the most common approach, where the protein-protein correlation functions g(ij)(r) are approximated by their zero-density limit (i.e., the Boltzmann factor), we propose a more accurate representation of g(ij)(r) that takes into account terms up to the first order in the density expansion of the mean-force potential. This improvement is expected to be particularly effective in the case of strong protein-protein interactions at intermediate concentrations. The method is applied to analyze small-angle x-ray scattering data obtained as a function of the ionic strength (from 7 to 507 mM) from acidic solutions of beta-lactoglobulin at the fixed concentration of 10 gl(-1). The results are compared with those obtained using the zero-density approximation and show significant improvement, particularly in the more demanding case of low ionic strength. (+info)
Humoral and cell mediated immune response to cow's milk proteins in Behcet's disease.
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OBJECTIVE: To investigate the humoral and cellular immune response against cow's milk proteins in Behcet's disease and to distinguish any behaviour during active or inactive disease. METHODS: Peripheral blood mononuclear cells from 16 patients and from eight normal controls were cultured in the presence of phytohaemagglutinin (PHA), beta-casein, beta-lactoglobulin, or chicken egg albumin. Interferon gamma (IFNgamma) and interleukin 4 (IL4) were measured in the culture supernatants by enzyme linked immunosorbent assay (ELISA). Serum samples from 46 patients with Behcet's disease and from 37 healthy subjects were also studied for antibody detection. Antibodies to beta-casein, beta-lactoglobulin, and chicken egg albumin were determined by ELISA. RESULTS: High IFNgamma but not IL4 levels were found in the supernatants of lymphocytes from patients with active disease cultured in the presence of cow's milk proteins. Levels were comparable with those obtained in cultures stimulated with PHA. A significantly higher level of anti-beta-casein and anti-beta-lactoglobulin IgG and IgA antibodies was found in patients with active Behcet's disease. No relation was found between their occurrence and the age of the patients, the duration of disease, or the presence of gastrointestinal abnormalities. Antibodies to chicken albumin were detected at low levels and with a prevalence similar to that of healthy subjects. CONCLUSION: The results indicate that an active immune response occurs in Behcet's disease. This response involves an increased frequency of antibodies to cow's milk protein and a strong Th1 polarisation after exposure to these antigens. The occurrence of these abnormalities supports a putative role for cow's milk proteins immune response in the pathogenesis of Behcet's disease. (+info)
Manipulating monomer-dimer equilibrium of bovine Beta -lactoglobulin by amino acid substitution.
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Bovine beta-lactoglobulin, a major protein in cow's milk composed of nine beta-strands (betaA-betaI) and one alpha-helix, exists as a dimer at neutral pH while it dissociates to a native monomer below pH 3.0. It is assumed that the intermolecular beta-sheet formed between I-strands and salt bridges at AB-loops play important roles in dimer formation. Several site-directed mutants in which intermolecular interactions stabilizing the dimer would be removed were expressed in the methylotrophic yeast Pichia pastoris, and their monomer-dimer equilibria were studied by analytical ultracentrifugation. Various I-strand mutants showed decreases in K(a), suggesting that the intermolecular beta-sheet is essential for dimer formation. By substituting either Asp(33) or Arg(40) on the AB-loop to oppositely charged residues (i.e. R40D, R40E, and D33R), a large decrease in K(a) was observed probably because of the charge repulsion, which is consistent with the role of electrostatic attraction between Arg(40) on one monomer and Asp(33) on the other monomer in the wild-type dimer. However, when two of these mutants, R40D and D33R, were mixed, a heterodimer was formed by the electrostatic attraction between Arg(33) and Asp(40) of different molecules. These results suggested that protein-protein interactions of bovine beta-lactoglobulin can be manipulated by redesigning the residues on the interface without affecting global folding. (+info)
Lactobacillus bulgaricus proteinase expressed in Lactococcus lactis is a powerful carrier for cell wall-associated and secreted bovine beta-lactoglobulin fusion proteins.
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Lactic acid bacteria have a good potential as agents for the delivery of heterologous proteins to the gastrointestinal mucosa and thus for the reequilibration of inappropriate immune responses to food antigens. Bovine beta-lactoglobulin (BLG) is considered a major allergen in cow's milk allergy. We have designed recombinant Lactococcus lactis expressing either full-length BLG or BLG-derived octapeptide T6 (IDALNENK) as fusions with Lactobacillus bulgaricus extracellular proteinase (PrtB). In addition to constructs encoding full-length PrtB for the targeting of heterologous proteins to the cell surface, we generated vectors aiming at the release into the medium of truncated PrtB derivatives lacking 100 (PrtB partial differential, PrtB partial differential-BLG, and PrtB partial differential-T6) or 807 (PrtBdelta) C-terminal amino acids. Expression of recombinant products was confirmed using either anti-PrtB, anti-BLG, or anti-peptide T6 antiserum. All forms of the full-length and truncated recombinant products were efficiently translocated, irrespective of the presence of eucaryotic BLG sequences in the fusion proteins. L. lactis expressing PrtB partial differential-BLG yielded up to 170 microg per 10(9) CFU in the culture supernatant and 9 microg per 10(9) CFU at the bacterial cell surface within 14 h. Therefore, protein fusions relying on the use of PrtB gene products are adequate for concomitant cell surface display and secretion by recombinant L. lactis and thus may ensure maximal bioavailability of the eucaryotic antigen in the gut-associated lymphoid tissue. (+info)
Induced chirality upon binding of cis-parinaric acid to bovine beta-lactoglobulin: spectroscopic characterization of the complex.
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Binding of the polyunsaturated cis-parinaric acid to bovine beta-lactoglobulin (BLG) was studied by circular dichroism (CD), electronic absorption spectroscopy and mass spectrometry methods. Upon protein binding, the UV absorption band of parinaric acid is red shifted by ca. 5 nm, showing hypochromism and reduced vibrational fine structure, suggesting that the ligand binds as a monomer in non-planar geometry. In the CD spectra measured at pH 7.36 and 8.5 a strong, negative Cotton band appears centered at 310 nm (Delta epsilon = -25 M(-1) cm(-1)) corresponding to the long-wavelength absorption band of cis-parinaric acid. The source of this induced optical activity is the helical distortion of the polyene chromophore caused by the chiral protein environment. From CD spectral data the value of the association constant was calculated to be 4.7 x 10(5) M(-1) at pH 7.36. CD and mass spectrometry measurements showed that parinaric acid binds weakly to BLG in acidic solution, though small peaks at mass 18,559 and 18,645 can be obtained in the reconstructed electrospray mass spectrum; these correspond to the binding of parinaric acid in 1:1 stoichiometry to both monomer variants of BLG B and A. The hydrophobic interior cavity of BLG was assigned as the primary binding site of cis-parinaric acid. (+info)
Protein isoforms observed by ultrahigh resolution capillary isoelectric focusing electrospray ionization mass spectrometry.
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On-line coupling of capillary isoelectric focusing (CIEF) to electrospray ionization mass spectrometry (ESI-MS) as a two-dimensional separation/analysis system was employed for high-resolution analysis of the protein isoforms observed during CIEF process. The analytical system was established by using neutral coated long capillary (80 cm), active capillary positioning and sheath-liquid interface. Proteins were separated and resolved in CIEF according to their differences in isoelectric point (pI), and then characterized by ESI-MS. The focused protein zones were eluted to the entrance of MS by combining cathodic mobilization with gravity. The ultrahigh resolution (difference in pI<0.04) of this technique obtained under certain conditions led to the detection of three isoforms in hemoglobin A and in sickle cell hemoglobin (with similar charge distribution and same molecular weight, but their differences in pIranging from 0.04 to 0.08) and two isoforms of beta-lactoglobulin A (difference in pI is 0.6). The isoelectric points, relative amounts, and molecular masses of these isoforms were determined simultaneously by CIEF-ESI-MS. (+info)