Equine Β-lactoglobulin Research Articles

Genes encoding equine β-lactoglobulin (LGB1 and LGB2): Polymorphism, expression, and impact on milk composition.

β-lactoglobulin is one of the most abundant milk whey proteins in many mammal species, including the domestic horse. The aim of this study was to screen for polymorphism in the equine LGB1 and LGB2 gene sequences (all exons, introns, and 5’-flanking region) and to assess potential relationship of particular genotypes with gene expression levels (measured in milk somatic cells) and milk composition traits (protein, fat, lactose, and total β-lactoglobulin content). Direct DNA sequencing analysis was performed for twelve horse breeds: Polish Primitive Horse (PPH), Polish Coldblood Horse (PCH), Polish Warmblood Horse (PWH), Silesian, Hucul, Fjording, Haflinger, Shetland Pony, Welsh Pony, Arabian, Thoroughbred, and Percheron—and revealed the presence of 83 polymorphic sites (47 and 36 for LGB1 and LGB2 genes, respectively), including eight that were previously unknown. Association analysis of the selected polymorphisms, gene expression, and milk composition traits (conducted for the PPH, PCH, and PWH breeds) showed several statistically significant relationships; for example, the two linked LGB1 SNPs (rs1143515669 and rs1144647991) were associated with total milk protein content (p < 0.01). Our study also confirmed that horse breed had significant impact on both gene transcript levels (p < 0.01) and on milk LGB content (p < 0.05), whereas an influence of lactation period was seen only for gene relative mRNA abundances (p < 0.01).

DNA-based analysis of protein variants reveals different genetic variability of the paralogous equine ß-lactoglobulin genes LGB1 and LGB2

The genetic variability of milk protein genes may influence the nutritive value or processing and functional properties of the milk. While numerous protein variants are known in ruminants, knowledge about milk protein variability in horses is still limited. Mare's milk is, however, produced for human consumption in many countries. Beta-lactoglobulin belonging to the protein family of lipocalins, which are known as common food- and airborne allergens, is a major whey protein. It is absent from human milk and thus a key agent in provoking cow's milk protein allergy. Mare's milk is, however, usually better tolerated by most affected people. Several functions of β-lactoglobulin have been discussed, but its ultimate physiological role remains unclear. In the current study, the open reading frames of the two equine β-lactoglobulin paralogues LGB1 and LGB2 were re-sequenced in 249 horses belonging to 14 different breeds in order to predict the existence of protein variants at the DNA-level. Thereby, only a single signal peptide variant of LGB1, but 10 different putative protein variants of LGB2 were identified. In horses, both genes are expressed and in such this is a striking previously unknown difference in genetic variability between the two genes. It can be assumed that LGB1 is the ancestral paralogue, which has an essential function causing a high selection pressure. As horses have very low milk fat content this unknown function might well be related to vitamin-uptake. Further studies are, however, needed, to elucidate the properties of the different gene products.

Effect of non-native helix destabilization on the folding of equine β-lactoglobulin.

β-lactoglobulin forms a non-native α-helix during an early stage of folding. To address the role of the non-native structure in the folding process, we designed several mutants of equine β-lactoglobulin with reduced helical propensity in the non-native helix region. One of them, A123T, showed a similar structure to that of the wild-type protein; its folding kinetics was investigated by stopped-flow circular dichroism (CD) and fluorescence. Although A123T showed a reduced burst-phase CD intensity, its folding rate was similar to that of the wild-type protein, which indicated that the formation of the non-native helix does not accelerate or decelerate the folding reaction.

Structural study of hNck2 SH3 domain protein in solution by circular dichroism and X-ray solution scattering

We have done conformational study of hNck2 SH3 domain by means of far-ultraviolet (far-UV) circular dichroism (CD) and X-ray solution scattering (XSS). The results indicated that the following: (1) hNck2 SH3 domain protein exhibited concentration dependent monomer–dimer transition at neutral pH, while the secondary structure of this protein was independent of the protein concentration. (2) The hNck2 SH3 domain also exhibited pH dependent monomer–dimer transition. This monomer–dimer transition was accompanied with helix-β transition of the secondary structural change. Moreover, the acid-induced conformation, which was previously studied by Liu and Song by CD and nuclear magnetic resonance (NMR), was found to be not compact, but the conformation of the protein at acidic pH was similar to the cold denatured state (C-state) reported by Yamada et al. for equine β-lactoglobulin. We calculated that a structure of the equilibrium helix-rich intermediate of the hNck2 SH3 domain by DAMMIF program.

3P062 ウマβラクトグロブリン初期中間体における非天然ヘリックスのフォールディングキネティクスへの影響(01C.蛋白質:物性,ポスター,日本生物物理学会年会第51回(2013年度))

3P062 ウマβラクトグロブリン初期中間体における非天然ヘリックスのフォールディングキネティクスへの影響(01C.蛋白質:物性,ポスター,日本生物物理学会年会第51回(2013年度))

Importance of polypeptide chain length for the correct local folding of a β-sheet protein

Equine β-lactoglobulin is a 162-residue β-sheet protein. A partially folded form of equine β-lactoglobulin contains a β-hairpin and an α-helix. The β-hairpin converts into non-native α-helices at temperatures <0°C. CHIBL, a truncated equine β-lactoglobulin (residues 88–142), contains the low-temperature α-helical structure even at room temperature, indicating that the interactions responsible for the stability of the β-hairpin reside in non-CHIBL residues. For the study reported herein, we characterized two truncated mutants and their leucine103 → proline103 variants to identify residues that stabilize the β-hairpin. The dependence of their circular dichroism spectra on chloride ion concentration and temperature revealed that the ability to transition from the non-native α-helices to the β-hairpin depends on the polypeptide chain length and improves as the chain length increases despite the apparent absence of any ordered structure in the extended sequences.

A Native Disulfide Stabilizes Non-Native Helical Structures in Partially Folded States of Equine β-Lactoglobulin

Equine β-lactoglobulin (ELG) assumes non-native helices during refolding and in partially folded states. Previously, circular dichroism (CD) combined with site-directed mutagenesis identified helical regions in the acid- and cold-denatured states of ELG. It is also known that a fragment of ELG, CHIBL (residues 88-142), has a structure similar to that of the cold-denatured state. For the study reported herein, the structure of a shorter fragment, CHIBLΔF (residues 97-142), was investigated by CD and nuclear magnetic resonance spectroscopy. The secondary chemical shifts clearly showed that non-native α-helices are present in two different regions, residues 98-107 and 114-135, and are connected by a native disulfide bond. The CD spectra of two peptides that correspond to the helical regions are characterized by weak helical signatures, and the sum of their CD spectra is nearly the same as the spectrum of disulfide-reduced CHIBLΔF. Therefore, the non-native helices are stabilized by the disulfide, and non-native helix formation may occur only during the refolding of the disulfide-intact protein. Supporting this conclusion is the observation that tear lipocalin, a homologue of ELG that lacks the disulfide, does not form non-native helices during folding.

3P065 Folding of equine β-lactoglobulin entrapped in wet silica gels studied by circular dichroism spectroscopy(Protein: Property,The 48th Annual Meeting of the Biophysical Society of Japan)

3P065 Folding of equine β-lactoglobulin entrapped in wet silica gels studied by circular dichroism spectroscopy(Protein: Property,The 48th Annual Meeting of the Biophysical Society of Japan)

Crystal Structure of A Chimeric b-Lactoglobulin, Gyuba

β-lactoglobulin is a major whey protein of 162 residues and is composed of nine β-strands (A-I strand) and one a -helix. Eight out of the nine strands form an upand-down b -barrel structure. Under physiological conditions, equine βlactoglobulin (ELG) is monomer, whereas bovine β-lactoglobulin (BLG) exists as a dimer in which I strand forms an intermolecular β-sheet. In this study, we report the 2.0 resolution crystal structure of Gyuba which was constructed from the amino acid sequences of secondary-structured regions of BLG and those of loop regions of ELG. This structure revealed that Gyuba formed dimer in which I strand forms an intermolecular β-sheet. This dimerization is similar to BLG.

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.

Helix-rich transient and equilibrium intermediates of equine β-lactoglobulin in alkaline buffer

Acidic buffer conditions are known to stabilize helix-rich states of even those proteins with a predominantly β-sheet native secondary structure. Here we investigated whether such states also exist under alkaline buffer conditions. The guanidine hydrochloride (GuHCl)-induced unfolding transition and kinetic refolding of equine β-lactoglobulin (ELG) by GuHCl-jump were investigated at pH 8.7 by far-ultraviolet circular dichroism. We found that an equilibrium intermediate appeared in 45% ethylene glycol (EGOH) buffer with 1.5 M GuHCl. The intermediate is rich in non-native α-helix, which is similar to the helix-rich state of ELG at pH 4.0. A kinetic study was done on the folding rate of ELG and compared with bovine β-lactoglobulin (BLG). Transient intermediates, which were observed as the burst phase of the refolding reaction, were also rich in α-helix. The activation enthalpy of ELG was calculated to be c.a. 80 kJ/mol, whereas that of BLG was c.a. 70 kJ/mol in the presence of 45% EGOH. The ellipticities of the transient intermediate of ELG show temperature dependence in the presence of 45% EGOH, whereas that of BLG did not show significant dependence. This study therefore extends the existence of helix-rich equilibrium and transient intermediates of predominantly β-sheet proteins to alkaline buffer conditions.

Chloride-ion concentration dependence of molecular dimension in the acid-denatured state of equine β-lactoglobulin

The chloride-ion concentration dependence of the molecular dimension in the acid-denatured state of equine β-lactoglobulin (ELG) was investigated by small-angle X-ray scattering. In the presence of chloride ion, ELG has a globular and compact conformation (the A state). The molecular dimension of ELG increases little with decreasing chloride-ion concentration. A remarkable dependence was observed for a mutant protein in which both Cys66 and Cys160 were replaced with Ala (C66A/C160A). In the presence of chloride ion, C66A/C160A has a globular and compact conformation, like the wild type. In the absence of chloride ion, however, the molecular dimension and shape was close to that in the urea-unfolded state. Previously, we have shown that the helix content in the acid-denatured state increases with decreasing chloride-ion concentration [Yamada et al. (2006). Proteins Struct. Funct. Bioinf. 63, 595–602]. These results suggest that the secondary structure in the A state is mainly determined by non-local interactions. When they are absent in an expanded conformation, the local interactions become predominant and the amount of non-native α-helix increases.

Interactions Responsible for Secondary Structure Formation during Folding of Equine β-Lactoglobulin

Equine β-lactoglobulin forms a compact intermediate at an acidic pH (A state). It also forms an expanded and helical conformation at low temperatures (C state). The structure of a single disulfide mutant C66A/C160A is similar to the A state in the presence of salts, while it is similar to the C state at low anion concentrations. We have investigated the temperature-dependent change in the secondary structure using circular dichroism and proline scanning mutagenesis. At low anion concentrations, the helical content increased linearly as temperature decreased. In the presence of salts, the A state was cooperatively transformed into the C state at low temperatures. This suggests the importance of hydrophobic interactions for stabilizing the A state. Peptides encompassing native-like and non-native α-helices were synthesized to investigate the interactions responsible for helix formation in the A and C states. These did not form stable helices, indicating that not only the helices in the A state but also the helices in the C state are stabilized by long-range interactions. A longer fragment, CHIBL, which encompasses the structured region in the A and C states, showed a helical structure. Proline-substituted mutants of CHIBL showed CD spectral changes similar to the corresponding mutants of the full-length protein in the C state. Therefore, CHIBL has a structure similar to the corresponding region of the full-length protein in the C state. This result indicates that interactions responsible for helix formation in the C state reside in the sequence of CHIBL, and that the sequences outside CHIBL are essential for secondary structure formation in the A state.

2P095 ジスルフィド結合により安定化されているウマβラクトグロブリンの中間体におけるヘリックス構造(蛋白質(物性(安定性、折れ畳みなど)),口頭発表,第45回日本生物物理学会年会)

2P095 ジスルフィド結合により安定化されているウマβラクトグロブリンの中間体におけるヘリックス構造(蛋白質(物性(安定性、折れ畳みなど)),口頭発表,第45回日本生物物理学会年会)

3P065 ジスルフィド結合欠損ウマβラクトグロブリンの二量体形成(蛋白質(物性(安定性、折れ畳みなど)),ポスター発表,第45回日本生物物理学会年会)

3P065 ジスルフィド結合欠損ウマβラクトグロブリンの二量体形成(蛋白質(物性(安定性、折れ畳みなど)),ポスター発表,第45回日本生物物理学会年会)

2P088 ウマβ-ラクトグロブリンの近紫外部円二色性スペクトルに対する各チロシン残基の寄与(蛋白質(物性(安定性、折れ畳みなど)),ポスター発表,第45回日本生物物理学会年会)

2P088 ウマβ-ラクトグロブリンの近紫外部円二色性スペクトルに対する各チロシン残基の寄与(蛋白質(物性(安定性、折れ畳みなど)),ポスター発表,第45回日本生物物理学会年会)

Proline Scanning Mutagenesis Reveals Non-Native Fold in the Molten Globule State of Equine β-Lactoglobulin

The secondary structure in the molten globule state (an equilibrium analogue of a burst-phase folding intermediate) of equine beta-lactoglobulin was investigated by changes in the circular dichroic spectrum induced by a series of site-directed proline substitutions. The results challenge the structural picture obtained from previous hydrogen/deuterium exchange experiments. A stable non-native alpha-helix was found to exist in the region corresponding to the eighth strand (H strand) in the native structure, where the backbone amide protons are the most strongly protected from exchange. Therefore, the backbone topology in the folding core is significantly different from that in the native structure. This indicates that the burst-phase folding intermediate of beta-lactoglobulin is a trapped species because of misfolded backbone topology.

1P128 Role of an off-barrel strand for the structural stabilization of equine β-lactoglobulin(3. Protein folding and misfolding (1),Poster Session,Abstract,Meeting Program of EABS &amp; BSJ 2006)

1P128 Role of an off-barrel strand for the structural stabilization of equine β-lactoglobulin(3. Protein folding and misfolding (1),Poster Session,Abstract,Meeting Program of EABS &amp; BSJ 2006)

1P122 Fragment analysis of the partially unfolded state of equine β-lactoglobulin(3. Protein folding and misfolding (1),Poster Session,Abstract,Meeting Program of EABS &amp; BSJ 2006)

1P122 Fragment analysis of the partially unfolded state of equine β-lactoglobulin(3. Protein folding and misfolding (1),Poster Session,Abstract,Meeting Program of EABS &amp; BSJ 2006)

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.