The chaperone action of bovine milk αS1- and αS2-caseins and their associated form αS-casein

α_S-Casein, the major milk protein, comprises α_S1- and α_S2-casein and acts as a molecular chaperone, stabilizing an array of stressed target proteins against precipitation. Here, we report that α_S-casein acts in a similar manner to the unrelated small heat-shock proteins (sHsps) and clusterin in that it does not preserve the activity of stressed target enzymes. However, in contrast to sHsps and clusterin, α-casein does not bind target proteins in a state that facilitates refolding by Hsp70. α_S-Casein was also separated into α- and α-casein, and the chaperone abilities of each of these proteins were assessed with amorphously aggregating and fibril-forming target proteins. Under reduction stress, all α-casein species exhibited similar chaperone ability, whereas under heat stress, α-casein was a poorer chaperone. Conversely, α_S2-casein was less effective at preventing fibril formation by modified κ-casein, whereas α- and α_S1-casein were comparably potent inhibitors. In the presence of added salt and heat stress, α_S1-, α- and α_S-casein were all significantly less effective. We conclude that α_S1- and α-casein stabilise each other to facilitate optimal chaperone activity of α_S-casein. This work highlights the interdependency of casein proteins for their structural stability.     

Aggregation and structural changes of α(S1)-, β- and κ-caseins induced by homocysteinylation

Elevated homocysteine levels are resulting in N-homocysteinylation of lysyl residues in proteins and they correlate with a number of human pathologies. However, the role of homocysteinylation of lysyl residues is still poorly known. In order to study the features of homocysteinylation of intrinsically unstructured proteins (IUP) bovine caseins were used as a model. α(S1)-, β- and κ-caseins, showing different aggregations and micelle formation, were modified with homocysteine-thiolactone and their physico-chemical properties were studied. Efficiency of homocysteine incorporation was estimated to be about 1.5, 2.1 and 1.3 homocysteyl residues per one β-, α(S1)-, and κ-casein molecule, respectively. Use of intrinsic and extrinsic fluorescent markers such as Trp, thioflavin T and ANS, reveal structural changes of casein structures after homocysteinylation reflected by an increase in beta-sheet content, which in some cases may be characteristic of amyloid-like transformations. CD spectra also show an increase in beta-sheet content of homocysteinylated caseins. Casein homocysteinylation leads in all cases to aggregation. The sizes of aggregates and aggregation rates were dependent on homocysteine thiolactone concentration and temperature. DLS and microscopic studies have revealed the formation of large aggregates of about 1-3μm. Homocysteinylation of α(S1)- and β-caseins results in formation of regular spheres. Homocysteinylated κ-casein forms thin unbranched fibrils about 400-800nm long. In case of κ-casein amyloidogenic effect of homocysteinylation was confirmed by Congo red spectra. Taken together, data indicate that N-homocysteinylation provokes significant changes in properties of native caseins. A comparison of amyloidogenic transformation of 3 different casein types, belonging to the IUP protein family, shows that the efficiency of amyloidogenic transformation upon homocysteinylation depends on micellization capacity, additional disulphide bonds and other structural features.     

Genetic polymorphism of αs1- and αs2-caseins in Hungarian Milking Goats

The aim of this study was to perform an initial characterization of milk quality and to determine genetic polymorphism at the CSN1S1 and CSN1S2 locus in two herds of local dairy goats (Hungarian Milking). The fat, protein and lactose level of milk samples in Hungarian Milking Goats were compared to other local goat breeds worldwide and it was concluded that the mean milk production of the Hungarian goats should be improved. The presence of the A, B, C + D, E, F and O alleles of CSN1S1 locus and A + B + C + E, D, F and O alleles of CSN1S2 locus were genotyped for by PCR-AS and PCR-RFLP methods in 103 goats. The strong B allele of CSN1S1 is more frequent in the local Hungarian Milking than in the imported Alpine and Saanen goats. The relatively high incidence of the O allele of CSN1S2 gene is also characteristic for the Hungarian Milking Goats and those special allele distribution patterns could be used to develop selection strategies to breed specialised lines of Hungarian local breeds.     

Thermodynamics of the Interaction between αs1- and κ-Caseins

Pyrenebutyrate-conjugated α_s1-casein was prepared and the complex formation between α_s1- and κ-casein polymers was investigated by fluorescence polarization. The complex formation was also investigated by a microcalorimetric technique. The positive enthalpy and entropy changes and endothermic nature suggested the hydrophobic interaction between α_s1- and κ-casein polymers.

The degree of polarization of κ-casein polymer decreased with the addition of 1-anilino-8-naphthalenesulfonate (ANS), while that of α_s1-casein polymer and α_s1-κ-casein complex was invariant. Moreover the reaction of κ-casein polymer and ANS was exothermic. These facts suggested that the intermolecular hydrophobic regions in κ-casein polymer were disrupted by the adsorption of ANS. The rotational relaxation time of pyrenebutyrate conjugated complex between cyanoethyl-κ-casein and α_s1-casein polymer was smaller than that of cyanoethyl-κ-casein alone. From these results, it was postulated that the dissociation of κ-casein polymer by the complex formation with α_s1-casein polymer might be caused by the disruption of the intermolecular hydrophobic bonds in κ-casein polymer.     

A study of genetic polymorphisms of milk β-lactoglobulin, αS1-casein, β-casein,and κ-casein in five dairy breeds

Gene frequencies of the milk -lactoglobulin, _S1-casein, -casein, and -casein loci have been estimated from 1663 cows of five dairy breeds. Departure from Hardy-Weinberg equilibrium was found only in the -casein system in Jerseys. However, chance alone could have accounted for this single significant finding. Results of pairwise comparisons among the five breeds of allele frequencies at these milk protein loci indicate that of the 40 possible tests, only six comparisons are not significant at the 5% probability level. It would appear that these breeds are characterizable in terms of the gene frequencies of these milk protein loci. Nonindependent assortment of genotypes among these milk protein loci was also studied. The closely linked casein loci were not independent in almost all the breeds where tests could be carried out. The only exception was between the _S1-casein and -casein loci in Holsteins. -Lactoglobulin was independent of the casein loci in all breeds except Brown Swiss, where it was found to be significantly associated with -casein. Close linkage is proposed as an important factor for maintaining the observed milk protein polymorphisms.This paper represents a portion of a doctoral dissertation submitted by the first author as partial fulfillment of the requirements for the degree of Doctor of Philosophy at the University of Massachusetts at Amherst.     

Variants within the 5′-flanking regions of bovine milk-protein-encoding genes. III. Genes encoding the Ca-sensitive caseins αs1, αs2 and β

The 5-flanking regions of the Ca-sensitive casein-encoding gene family were analysed for DNA variants by automated DNA sequencing of 13 cows belonging to seven breeds. About 1 kbp of each 5-flanking region, including non-coding exon I, was amplified by PCR and sequenced bidirectionally. A total number of 34 variable sites (17 for the _s1, 10 for the _s2, and 7 for the casein encoding gene) was identified. Variants were computer-analysed for location in putative regulatory sites in order to predict potential influences on gene expression.     

The evolution of the α- and β-globin gene clusters in human populations

Summary DNA analysis of the - and -globin gene clusters has revealed substantial variability between individuals and populations. As well as restriction enzyme site and length polymorphisms, variation in gene copy number and type is observed. Because of this extensive polymorphism DNA analysis offers a highly informative method of studying genetic affinities between human populations. Haplotypes, consisting of a set of restriction enzyme polymorphisms distributed along the cluster, have been developed for both loci. Analysis of the molecular basis of numerous -thalassaemia alleles has revealed, in general, different sets of mutations in different populations, indicating that these postdate the racial divergence. Recent microepidemiological studies on the distribution of -thalassaemia support the hypothesis that this condition, like the {ie16-1}, has been selected because it confers protection against malaria. Population-specific DNA polymorphisms at these and other loci promise to be of considerable value to genetic anthropology.     

Disulfide cross-links in the interaction of a cataract-linked αA-crystallin mutant with βB1-crystallin

A number of αA-crystallin mutants are associated with hereditary cataract including cysteine substitution at arginine 49. We report the formation of affinity-driven disulfide bonds in the interaction of αA-R49C with βB1-crystallin. To mimic cysteine thiolation in the lens, βB1-crystallin was modified by a bimane probe through a disulfide linkage. Our data suggest a mechanism whereby a transient disulfide bond occurs between αA- and βB1-crystallin followed by a disulfide exchange with cysteine 49 of a neighboring αA-crystallin subunit. This is the first investigation of disulfide bonds in the confine of the chaperone/substrate complex where reaction rates are favored by orders of magnitude. Covalent protein cross-links are a hallmark of age-related cataract and may be a factor in its inherited form.     

Role of zinc and copper ions in the pathogenetic mechanisms of Alzheimer’s and Parkinson’s diseases

Disbalance of zinc (Zn²⁺) and copper (Cu²⁺) ions in the central nervous system is involved in the pathogenesis of numerous neurodegenerative disorders such as multisystem atrophy, amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, Wilson-Konovalov disease, Alzheimer’s disease, and Parkinson’s disease. Among these, Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the most frequent age-related neurodegenerative pathologies with disorders in Zn²⁺ and Cu²⁺ homeostasis playing a pivotal role in the mechanisms of pathogenesis. In this review we generalized and systematized current literature data concerning this problem. The interactions of Zn²⁺ and Cu²⁺ with amyloid precursor protein (APP), β-amyloid (Abeta), tau-protein, metallothioneins, and GSK3β are considered, as well as the role of these interactions in the generation of free radicals in AD and PD. Analysis of the literature suggests that the main factors of AD and PD pathogenesis (oxidative stress, structural disorders and aggregation of proteins, mitochondrial dysfunction, energy deficiency) that initiate a cascade of events resulting finally in the dysfunction of neuronal networks are mediated by the disbalance of Zn²⁺ and Cu²⁺.     

Fluorescence Polarization of αs1, κ-Caseins and αs1-κ-Casein Complex

Conjugates of α^s1-,κ-caseins and α^s1-,κ-casein complex were prepared with dimethylaminonaphthalenesulfonate and pyrenebutyrate. Their fluorescence lifetimes and the rotational relaxation times were measured by single photon counting technique and fluorescence depolarization technique, respectively. Both dimethylaminonaphthalenesulfonate and pyrenebutyrate conjugates had more than two lifetimes and the longer lifetime of pyrenebutyrate conjugates was near 140 nsec.

The rotational relaxation time of pyrenebutyrate α^s1-,κ-casein complex was smaller than that of pyrenebutyrate κ-casein polymer, which suggested that the complex formation of α^s1- and κ-casein polymers led to dissociation of the κ-casein polymer.

Changes of the rotational relaxation time as a function of weight ratio of α^s1- and κ-casein polymers (α^s1/κ) showed the specific variation and it was suggested that 4 moles of α^s1-κ-casein complex were formed from one mole of κ-casein polymer.     

Limits and Sites of Lysinoalanine Formation in Lysozyme, α-Lactalbumin and αs1- and β-Caseins by Alkali Treatment

Lysinoalanine was determined by gas chromatography. Lysinoalanine formation in lysozyme depended on alkali concentration, pH, temperature and exposure time. The upper limits of lysinoalanine formation in lysozyme and α-lactalbumin were related to the number of lysine residues with a cystine disulfide bond in the adjacent position rather than the individual contents of these residues. In cases of α_sl- and β-caseins, the upper limits were related to the number of phosphoserine residues, regardless of their sequential relationship to lysine residues. Gel electrophoresis suggested that intermolecular cross-linking took place in the α-lactalbumin and caseins.     

Recombinant barley-produced antibody for detection and immunoprecipitation of the major bovine milk allergen, β-lactoglobulin

Recombinant allergens and antibodies are needed for diagnostic, therapeutic, food processing and quality verification purposes. The aim of this work was to develop a barley-based production system for β-lactoglobulin (BLG) specific immunoglobulin E antibody (D1 scFv). The expression level in the best barley cell clone was 0.8–1.2 mg/kg fresh weight, and was constant over an expression period of 21 days. In the case of barley grains, the highest stable productivity (followed up to T2 grains) was obtained when the D1 scFv cDNA was expressed under a seed-specific Glutelin promoter rather than under the constitutive Ubiquitin promoter. Translational fusion of ER retention signal significantly improved the accumulation of recombinant antibody. Furthermore, lines without ER retention signal lost D1 scFv accumulation in T2 grains. Pilot scale purification was performed for a T2 grain pool (51 g) containing 55.0 mg D1 scFv/kg grains. The crude extract was purified by a two-step purification protocol including IMAC and size exclusion chromatography. The purification resulted in a yield of 0.47 mg of D1 scFv (31 kD) with high purity. Enzyme-linked immunosorbent assay revealed that 29 % of the purified protein was fully functional. In immunoprecipitation assay the purified D1 scFv recognized the native 18 kD BLG in the milk sample. No binding was observed with the heat-treated milk sample, as expected. The developed barley-based expression system clearly demonstrated its potential for application in the processing of dairy milk products as well as in detecting allergens from foods possibly contaminated by bovine milk.     

Studies on the Interaction between αs1- and β-Caseins

The interaction of α_s1-casein with β-, dephosphorylated β-,γ- and R-caseins was studied. It was proved by the sedimentation velocity experiments that α_s1-casein formed a complex with each of these components at 25±C in the presence of 3 mm CaCl₂.

In the presence of 10 mm CaCl₂, β- and dephosphorylated β-casein prevented the precipitation of α_s1-casein and gave micelle-like turbid solutions. However, γ- and R-caseins, fragments of β-casein, did not stabilize α_s1-casein. It was concluded from these results that α-casein interacted with α_s1-casein through its hydropholic region corresponding to R-casein and that hydrophilic region of β-casein was responsible for the stabilization of α_s1-casein.     

Steroid hydroxylation with free and immobilized cells of Penicillium raistrickii in the presence of β-cyclodextrin

Free and Ca-alginate-immobilized cells of Penicillium raistrickii i 477 were used for 15-hydroxylation of 13-ethyl-gon-4-en-3,17-dione. The product formation in the presence of -cyclodetrin, in comparison with reactions carried out in the presence of methanol. Application of -cyclodextrin led to increasing solubility of the steroid substrate. The fungus was able to utilize -cyclodextrin as a carbon source. Correspondence to: H.-P. Schmauder     

Probing the binding sites of resveratrol,genistein, and curcumin with milk β-lactoglobulin

We determined the binding sites of curcumin (cur), resveratrol (res), and genistein (gen) with milk β-lactoglobulin (β-LG) at physiological conditions. Fourier transform infrared spectroscopy, circular dichroism, and fluorescence spectroscopic methods as well as molecular modeling were used to determine the binding of polyphenol–protein complexes. Structural analysis showed that polyphenols bind β-LG via both hydrophilic and hydrophobic contacts with overall binding constants of K_{curcumin–β-LG}?=?4.4 (±?.4)?×?10⁴ M^?1, K_{resveratrol–β-LG}?=?4.2 (±?.2)?×?10⁴ M^?1, and K_{genistein–β-LG}?=?1.2 (±?.2)?×?10⁴?M^?1. The number of polyphenol molecules bound per protein (n) was 1 (cur), 1.1 (res), and 1 (gen). Molecular modeling showed the participation of several amino acid residues in polyphenol–protein complexation with the free binding energy of ?12.67 (curcumin–β-LG), ?12.60 (resveratrol–β-LG), and ?10.68?kcal/mol (genistein–β-LG). The order of binding was cur?>?res?>?gen. Alteration of the protein conformation was observed in the presence of polyphenol with a major reduction of β-sheet and an increase in turn structure, causing a partial protein structural destabilization. β-LG might act as a carrier to transport polyphenol in vitro.     

A Specific and Essential Role for Na,K-ATPase α3 in Neurons Co-expressing α1 and α3

Most neurons co-express two catalytic isoforms of Na,K-ATPase, the ubiquitous α1, and the more selectively expressed α3. Although neurological syndromes are associated with α3 mutations, the specific role of this isoform is not completely understood. Here, we used electrophysiological and Na⁺ imaging techniques to study the role of α3 in central nervous system neurons expressing both isoforms. Under basal conditions, selective inhibition of α3 using a low concentration of the cardiac glycoside, ouabain, resulted in a modest increase in intracellular Na⁺ concentration ([Na⁺]_i) accompanied by membrane potential depolarization. When neurons were challenged with a large rapid increase in [Na⁺]_i, similar to what could be expected following suprathreshold neuronal activity, selective inhibition of α3 almost completely abolished the capacity to restore [Na⁺]_i in soma and dendrite. Recordings of Na,K-ATPase specific current supported the notion that when [Na⁺]_i is elevated in the neuron, α3 is the predominant isoform responsible for rapid extrusion of Na⁺. Low concentrations of ouabain were also found to disrupt cortical network oscillations, providing further support for the importance of α3 function in the central nervous system. The α isoforms express a well conserved protein kinase A consensus site, which is structurally associated with an Na⁺ binding site. Following activation of protein kinase A, both the α3-dependent current and restoration of dendritic [Na⁺]_i were significantly attenuated, indicating that α3 is a target for phosphorylation and may participate in short term regulation of neuronal function.     

Association of α-subunits with nucleotide-modified β-subunits induces asymmetry in the catalytic sites of the F1-ATPase α3β3

The photoaffinity spin-labeled ATP analog, 2-N₃-SL-adenosine triphosphate (ATP), was used to covalently modify isolated β-subunits from F₁-ATPase of the thermophilic bacterium PS3. Approximately 1.2 mol of the nucleotide analog bound to the isolated subunit in the dark. Irradiation leads to covalent incorporation of the nucleotide into the binding site. ESR spectra of the complex show a signal that is typical for protein-immobilized radicals. Addition of isolated α-subunits to the modified β-subunits results in ESR spectra with two new signals indicative of two distinctly different environments of the spin-label, e.g., two distinctly different conformations of the catalytic sites. The relative ratio of the signals is approx 2∶1 in favor of the more closed conformation. The data show for the first time that when nucleotides are bound to isolated β-subunits, binding of α-subunits induces asymmetry in the catalytic sites even in the absence of the γ-subunit. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to PDV.     

NMR study of short β(1-3)-glucans provides insights into the structure and interaction with Dectin-1

β(1-3)-Glucans, abundant in fungi, have the potential to activate the innate immune response against various pathogens. Although part of the action is exerted through the C-type lectin-like receptor Dectin-1, details of the interaction mechanism with respect to glucan chain-length remain unclear. In this study, we investigated a set of short β(1-3)-glucans with varying degree of polymerization (DP); 3, 6, 7, 16, and laminarin (average DP; 25), analyzing the relationship between the structure and interaction with the C-type lectin-like domain (CTLD) of Dectin-1. The interaction of short β(1-3)-glucans (DP6, DP16, and laminarin) with the CTLD of Dectin-1 was systematically analyzed by ¹H-NMR titration as well as by saturation transfer difference (STD)-NMR. The domain interacted weakly with DP6, moderately with DP16 and strongly with laminarin, the latter plausibly forming oligomeric protein-laminarin complexes. To obtain structural insights of short β(1-3)-glucans, the exchange rates of hydroxy protons were analyzed by deuterium induced ¹³C-NMR isotope shifts. The hydroxy proton at C4 of laminarin has slower exchange with the solvent than those of DP7 and DP16, suggesting that laminarin has a secondary structure. Diffusion ordered spectroscopy revealed that none of the short β(1-3)-glucans including laminarin forms a double or triple helix in water. Insights into the interaction of the short β(1-3)-glucans with Dectin-1 CTLD provide a basis to understand the molecular mechanisms of β-glucan recognition and cellular activation by Dectin-1.