Equine Beta-lactoglobulin Research Articles

The Identification of Novel Single-Nucleotide Polymorphisms of Equine Beta-Lactoglobulin and Lactotransferrin Genes

Lactoferrin (LTF), also called lactotransferrin, is an iron-binding protein and member of transferrin family, whereas β-LG is an important milk protein and belongs to the ligand-binding protein family of lipocalins and binds retinol. Beta-lactoglobulin (β-LG) is a major whey protein and it is a member of lipocalins family. β-LG is also known as a major allergen reason of cow milk protein allergy. Yet, no information has been reported about β-LG and LTF genes in donkeys. In this study, the genetic variation of β-LG and LTF genes were analyzed via DNA sequencing in 77 donkeys, which were individuals reared in Thrace region of Turkey. Four novel SNPs are identified as g.12887G>A, g.12973G>A, g.12654G>A, and g.13057T>C in the third intron region of β-LG gene and a genetic variant g.272719G>A in the 14th intron of LTF gene. The DNA sequences of β-LG and LTF genes in donkeys are reported for the first time in this study and these sequences were deposited to NCBI GenBank database, with the accession number MK125015 and MK211165, respectively. These variations may have an effect on milk yield and content in donkeys, so further investigations are needed to prove this hypothesis and these may be candidate SNPs for donkey breeding.

The Stabilization Mechanism of the Intermediate Structure of Equine Beta-Lactoglobulin

A single-disulfide mutant of equine β-lactoglobulin, C66A/C160A, forms an expanded and helical conformation at an acidic pH and a low anion concentration (C state). The C state is a model of an early folding intermediate of βlactoglobulin. The secondary structures in the C state are in the regions corresponding to F, G, H strands and the major α-helix, and they assume the native-like or nonnative helices. Peptides encompassing those helices did not form stable helices. A longer fragment, CHIBL, which encompasses the structured region in the C states, has a helical structure similar to the corresponding region of the full-length protein in the C state. This result indicates that non-local interactions responsible for the helix formation in the C state reside in the sequence of CHIBL. The disulfide bond, Cys106-Cys119, links two nonnative helices in the C state. This is one of the possible long-range interactions stabilizing the helical structures. The CD spectrum of disulfide-reduced C66A/C160A shows decreased helical content. This indicates that the helices are stabilized by this disulfide bond. The detailed structural analysis of the reduced C66A/C160A by using proline mutants suggests that the nonnative helix in the G strand region can form without the disulfide bond despite Cys106 is contained in that region. It also suggests that the disulfide bond is crucial to the formation of the nonnative helix in the H strand region. We synthesized a peptide encompassing two helices formed in the C state. The GssH peptide is disulfide-linked G-peptide and H-peptide. This peptide did not form stable helical structures. Therefore G and H strands and major helix regions must interact with each other simultaneously to form stable helices in the C state.

Structural and thermodynamic consequences of removal of a conserved disulfide bond from equine β‐lactoglobulin

A disulfide bond between cysteine 66 and cysteine 160 of equine beta-lactoglobulin was removed by substituting cysteine residues with alanine. This disulfide bond is conserved across the lipocalin family. The conformation and stability of the disulfide-deleted mutant protein was investigated by circular dichroism. The mutant protein assumes a native-like structure under physiological conditions and assumes a helix-rich molten globule structure at acid pH or at moderate concentrations of urea as the wild-type protein does. The urea-induced unfolding experiment shows that the stability of the native conformation was reduced but that of the molten globule intermediate is not significantly changed at pH 4 by removal of the disulfide bond. On the other hand, the molten globule at acid pH was destabilized by removal of the disulfide bond. This difference in the stabilizing effect of the disulfide bond was interpreted by the effect of the disulfide in keeping the molecule compact against the electrostatic repulsion at acid pH. In contrast to the wild-type protein, the circular dichroism spectrum in the molten globule state at acid pH depends on anion concentration, suggesting that the expansion of the molecule through electrostatic repulsion induces alpha-helices as observed in the cold denatured state of the wild-type protein.

Helical and Expanded Conformation of Equine β-Lactoglobulin in the Cold-denatured State

The thermal unfolding transition of equine beta-lactoglobulin (ELG) was investigated by circular dichroism (CD) over a temperature range of -15 degrees C to 85 degrees C. In the presence of 2 M urea, a cooperative unfolding transition was observed both with increasing and decreasing temperature. The CD spectrum indicated that the heat and cold-denatured states of ELG have substantial secondary structures but lack persistent tertiary packing of the side-chains. In order to clarify the relation between the heat or cold-denatured state and the acid-denatured (A) state characterized previously, we have attempted to observe the temperature dependence of the CD spectrum at pH 1.5. The CD spectrum in the heat-denatured state is similar to that in the A state. The CD spectrum in the A state does not change cooperatively with increasing temperature. These results indicate that the heat-denatured state and the A state are the same structural state. On the other hand, the CD intensity at acid pH cooperatively increased with decreasing temperature. The CD spectrum at low temperature and acid pH is consistent with that in the cold-denatured state. Therefore, the cold-denatured state is distinguished from the heat-denatured state or the A state, and ELG assumes a larger amount of non-native alpha-helices in the cold-denatured state. Small angle X-ray scattering and analytical ultracentrifugation have indicated that ELG assumes an expanded chain-like conformation in the cold-denatured state in contrast to the compact globular conformation in the A state. The relation between the molecular size and the helical content in the partially folded states is discussed.

3P054 ウマβラクトグロブリンの平衡中間体における非天然α-helixの安定化機構(蛋白質 C) 物性(安定性、折れたたみなど)))

3P054 ウマβラクトグロブリンの平衡中間体における非天然α-helixの安定化機構(蛋白質 C) 物性(安定性、折れたたみなど)))

Porcine beta‐lactoglobulin chemical unfolding: Identification of a non‐native α‐helical intermediate

The chemical unfolding behavior of porcine beta-lactoglobulin (PLG) has been followed at pH 2 and 6 in the presence of guanidinium hydrochloride. The PLG unfolding transition, monitored by tryptophan fluorescence, far and near UV circular dichroism and 1D-NMR, can be described by a three-state transition suggesting the presence of at least one intermediate state that appears to display an excess of non-native alpha-helical structures. The thermodynamic parameters, as determined through a global analysis fitting procedure, give estimates of the free energy differences of the transitions connecting the native, the intermediate and the unfolded state: DeltaG(NI) (0) = 2.8 +/- 0.7 kcal mol(-1) (pH 2) and 4.2 +/- 0.5 kcal mol(-1) (pH 6) and DeltaG(NU) (0) = 7.2 +/- 0.6 kcal mol(-1) (pH 2) and 6.9 +/- 0.6 kcal mol(-1) (pH 6). CD unfolding data of the bovine species (BLG) have been collected here under the same experimental conditions of PLG to allow a careful comparison of the two beta-lactoglobulins. Intermediates with different characteristics have been identified for BLG and PLG, and their nature has been discussed on a structural analysis basis. The thermodynamic data reported here for PLG and BLG and the comparative analysis with data reported for equine beta lactoglobulin, show that homologous beta-barrel proteins, belonging to the same family and displaying high sequence identity (52-64%) populate unfolding intermediates to different extents, even though a common tendency to the formation of non-native alpha-helical intermediates, can be envisaged. The present results provide a prerequisite foundation of knowledge for the design and interpretation of future folding kinetic studies.

3P038 ウマβラクトグロブリンの低温変性状態の構造解析(蛋白質 C) 物性 : 安定性、折れたたみなど)

3P038 ウマβラクトグロブリンの低温変性状態の構造解析(蛋白質 C) 物性 : 安定性、折れたたみなど)

Construction and characterization of beta-lactoglobulin chimeras.

At neutral pH, equine beta-lactoglobulin (ELG) is monomeric, whereas bovine beta-lactoglobulin (BLG) exists as a dimer. To understand the difference in the oligomerization properties between ELG and BLG, three mutants of ELG (LP, I, and LPI) were constructed by substituting amino acids responsible for important interactions at the dimer interface of BLG into ELG. The mutant LP has an AB loop mutation (S34A/E35Q), the mutant I has an I strand mutation (G145M/R146H/V147I/Q148R/I149L/V150S/P151F/D152N/L153P) and the mutant LPI includes both the LP and I mutations. The far- and near-UV CD spectra of the three mutants are similar to that of the wild-type ELG, indicating that the secondary and the tertiary structures of ELG are not significantly affected by the mutations. Ultracentrifuge analysis shows that all three mutants are monomeric at neutral pH, suggesting that the protein sequences in the AB loop and I strand of BLG alone cannot support dimerization of ELG. Thus, structural differences must exist between ELG and BLG that prevent the ELG mutants from forming the same interactions as BLG at the dimer interface.

Thermal unfolding of monomeric and dimeric beta-lactoglobulins.

The thermal stabilities of dimeric bovine beta-lactoglobulin and monomeric equine beta-lactoglobulin were investigated at neutral pH by means of differential scanning calorimetry, circular dichroism, tryptophan fluorescence, and by binding of an hydrophobic probe. Differential scanning calorimetry showed the presence of two structural domains with different thermal stabilities in both proteins. Thermodynamic analysis of the calorimetric signal revealed that the two domains unfold independently according to a mechanism where an equilibrium step is followed by an irreversible transition. The spectroscopic data supported this model and allowed recognition of the structural regions corresponding to the more thermally stable domain. The differences in thermal stability between the two proteins can be primarily ascribed to the properties of the less stable domain.

A partially unfolded state of equine beta-lactoglobulin at pH 8.7.

The urea-induced unfolding transition of equine beta-lactoglobulin was studied at pH 8.7 using circular dichroism (CD), ultracentrifugation, and gel filtration chromatography. The unfolding transition curves showed that at least one intermediate accumulates at moderate concentrations of urea. Furthermore, analytical ultracentrifugation experiments indicated that the intermediate forms a dimer. Thus, the urea-induced unfolding transition was measured by CD at various protein concentrations and was analyzed by a model assuming the four conformational states (the native, intermediate, dimeric intermediate, and unfolded states). The characteristics of the intermediate are markedly different from those of the intermediate previously observed at pH 4.0 or 1.5. The intermediate at pH 8.7 does not show the intense far-ultraviolet CD suggestive of the nonnative alpha-helix.

Folding-unfolding equilibrium and kinetics of equine beta-lactoglobulin: equivalence between the equilibrium molten globule state and a burst-phase folding intermediate.

The denaturant-induced equilibrium unfolding transition of equine beta-lactoglobulin was investigated by ultraviolet absorption, fluorescence, and circular dichroism (CD) spectra. An equilibrium intermediate populates at moderate denaturant concentrations, and its CD spectrum is similar to that of the molten globule state previously observed for this protein at acid pH [Ikeguchi, M., Kato, S., Shimizu, A., and Sugai, S. (1997) Proteins: Struct., Funct., Genet. 27, 567-575]. The unfolding and refolding kinetics were also investigated by the stopped-flow CD and fluorescence. A significant change in the CD intensity was observed within the dead time of measurements (25 ms) when the refolding reaction was initiated by diluting the urea-unfolded protein solution, indicating the transient accumulation of the folding intermediate. The CD spectrum of this burst-phase intermediate agrees well with that of the molten globule state at acid pH. The stability of the burst-phase intermediate was also estimated from the urea-concentration dependence of the burst-phase amplitude, and it shows a fair agreement with that of the equilibrium intermediate. These results indicate that the molten globule state of equine beta-lactoglobulin populates at moderate urea concentration as well as at acid pH and it is equivalent with the kinetic folding intermediate.

Human milk components cross‐reacting with antibodies against bovine β‐lactoglobulin

The human whey components cross-reacting with antibodies raised against bovine and/or equine beta-lactoglobulin were screened systematically. The milk of six women on a normal diet was collected within 72 h of confinement and whey components were fractionated by high-speed size exclusion chromatography and reversed-phase techniques. The fractions which were immunoreactive in double diffusion experiments with antisera anti-bovine and/or equine beta-lactoglobulin were subsequently purified by native PAGE and then electroblotted on Pro-blott membrane (Western blotting). Pro-blot membranes were stained in parallel with Coomassie and by immunostaining using antibodies against bovine and/or equine beta-lactoglobulin as first antibody solution. The immunoreactive bands were cut out from the membrane and N-terminally sequenced; all the immunoreactive components were clearly identified as human beta-casein or its (mainly tryptic) fragments. The strong antigenic similarity between human beta-casein and beta-lactoglobulin (bovine and equine) might be of immunological importance; it could mean that breast-fed neonates risk being sensitized to beta-lactoglobulin irrespective of the presence of cow's milk in the mother's diet.