Antigenic Reactivity of Dephosphorylated αs1-Casein,Phosphopeptide from β-Casein and O-Phospho-l-serine towards the Antibody to Native αs1 -Casein

To study whether the phosphoserine residue is associated with the antigenicity of bovine α_s1- casein, we examined the antigenic reactivity of dephosphorylated α_s1-casein, peptide 1~25 from bovine β-casein and three chemical reagents with IgG antibody specific to native α_s1-casein by an enzyme-linked immunosorbent assay.

The reaction between native α_s1-casein and its IgG antibody was inhibited more strongly by native α_s1-casein than by dephosphorylated α_s1-casein. Peptide 1~25, having a phosphoserine residue-concentrated region from bovine β-casein, noticeably inhibited the reaction between native α_s1 -casein and its antibody. Furthermore, the O-phospho-l-serine residue inhibited the reaction of peptide 61~123 with anti-native α_s1-casein antibody, although l-serine and sodium phosphate showed no measurable inhibition.

These results suggest that the phosphoserine residue associated with part of an antigenic site in bovine α_sl-casein.     

Reconstituted Micelle Formation Using Reduced, Carboxymethylated Bovine κ-Casein and Human β-Casein

In milk, κ-casein, a mixture of disulfide-bonded polymers, stabilizes and regulates the size of the unique colloidal complex of protein, Ca²⁺ and inorganic phosphate (P_i) termed the casein (CN) micelle. However, reduced, carboxymethylated bovine κ-CN (RCM-κ) forms fibrils at 37°C and its micelle-forming ability is in question. Here, the doubly- and quadruply-phosphorylated human β-CN forms and 1:1 (wt:wt) mixtures were combined with RCM-κ at different β/κ weight ratios. Turbidity (OD_{400 nm}) and a lack of precipitation up to 37°C were used as an index of micelle formation. Studies were with 0, 5 and 10 mM Ca²⁺ and 4 and 8 mM P_i. The RCM-κ does form concentration-dependent micelles. Also, β-CN phosphorylation level influences micelle formation. Complexes were low-temperature reversible and RCM-κ fibrils were seen. There appears to be equilibrium between fibrillar and soluble forms since the solution still stabilized after fibril removal. The RCM-κ stabilized better than native bovine κ-CN.     

Opioid Peptides from Human β-Casein

A bacterium that assimilates 2,3-dichloro-1-propanol was isolated from soil by enrichment culture. The strain was identified as Pseudomonas sp. by the taxonomic studies. The strain converted 2,3-dichloro-1-propanol to 3-chloro-1,2-propanediol, releasing chloride ion. The conversion was stereospecific because the residual 2,3-dichloro-1-propanol and formed 3-chloro-1,2-propanediol gave optical rotation. The resting cells converted various halohydrins to the dehalogenated alcohols, and cell-free extracts had strong epoxyhydrolase activity. These results indicated that the strain assimilated 2,3-dichloro-1-propanol via 3-chloro-1,2-propanediol, glycidol, and glycerol. The possibility to manufacture optically active 2,3-dichloro-1-propanol is discussed.     

Studies on κ-Casein of Bovine Milk

κ-Caseins were prepared by the calciurn-ethanol method, the Sephadex method and the urea-sulfuric acid method. Some important properties of κ-caseins were investigated using isoelectric focusing, starch gel electrophoresis, ultracentrifugation, chemical analysis, stabilization test of α_s-casein, and rennin treatment. Isoelectric focusing established that κ-casein had its isoelectric point near pH 6.0 in 6 m urea, usually accompanied by a second peak around pH 5.6. Ultracentrifugation, however, showed a single peak having a s_20,w value of 2.6 ~ 3.8 in the presence of 6 m urea and of 14.4 in the absence of such dispersing reagents. Normal contents of hexose, sialic acid, phosphorus, and nitrogen were about 1.5, 0.8, 0.2, and 14%, respectively. Relative patterns of amino acid composition were similar in all of the κ-caseins. In addition, amino acid composition in intact κ-casein and in the further purified κ-casein which formed the second peak in DEAE cellulose chromatography were almost identical, indicating that the κ-casein of the first peak is not an impurity but is one of the components which formed the original κ-casein complexes. The ability of κ-caseins to stabilize α_s-casein in the presence of calcium increased when purified by DEAE cellulose chromatography.     

Difference Spectra of Bovine κ-Casein with Urea

Bovine κ-casein showed a typical CD spectrum for an aperiodic conformation in the far UV region. The comparison of the near UV absorption spectrum of native κ-casein with that of a model compound mixture (Ac-tyr-OEt+Ac-trp-OEt, molar ratio=9: 1) showed a red shift of the former by 2 nm to the longer wavelength. The difference spectra produced by the addition of urea to κ-casein solution showed three peaks at 280, 287, and 292 nm, of which the sign was negative (denaturation blue shift). The magnitude of the blue shift of the trypto- phyl group was found to be ?2,200. It is concluded from these results that some tyrosyl groups are exposed to the solvent, and the other tyrosyl groups and a tryptophyl group are buried in the hydrophobic regions which is very susceptible to the action of a denaturing agent, urea. All the chromophores in κ-casein was exposed to the solvent in the presence of 4 m urea. κ-Casein in the native state was proved to have an aperiodic structure but not a flexible random coil.     

Thermal and Alkaline Denaturation of Bovine β-Casein

The secondary structure of bovine beta-casein was characterized using circular dichroism (CD) and FTIR spectroscopies under physiologically relevant conditions. Analytical ultracentrifugation technique was used to follow the highly temperature, pH and concentration dependent self-association behavior. CD measurements provide convincing evidence for short segments of polyproline II-like structures in beta-casein in addition to a wide range of secondary structure elements, such as 10-20% alpha-helix, approximately 30% turns, 32-35% extended sheet. Results obtained at extreme pH (10.5) revealed structural destabilization in the monomeric form of the protein. At least four distinct structural transitions at 10, 33, 40 and 78 degrees C were observed at pH 6.75 by CD analysis, compared to only two transitions, 26 and 40 degrees C, at pH 10.5. Calculations from analytical ultracentrifugation suggest that the transitions at lower temperature (< or = 30 degrees C) occur primarily in the monomer. It is hypothesized that the transition at 10 degrees C and neutral pH may represent a general conformational change or cold denaturation. Those middle ranged transitions, i.e. 33 and 40 degrees C are more likely the reflection of hydrophobic changes in the core of beta-casein. As beta-casein undergoes self-association and increases in size, the transition at higher temperature (78 degrees C) is perhaps caused by the apparent conformational change within the micelle-like polymers. It has been shown that beta-casein binds the hydrophobic fluorescent probe ANS with high affinity in much similar fashion to molten globular proteins. The effect of urea denaturation on the bound complex effectively supports this observation.     

Neurite Outgrowth-Stimulating Peptide Derived from Bovine κ-Casein

We found a novel peptide that stimulates neurite outgrowth in Neuro-2a mouse neuroblastoma cells, from the pepsin-pancreatin digest of bovine κ-casein. The amino acid sequence of this peptide is Phe–Leu–Pro–Tyr–Pro–Tyr (FLPYPY), corresponding to peptidic sequence 76–81 of bovine κ-casein. The neurite outgrowth-stimulating activity of FLPYPY was seen over 10^?9 M. On the other hand, FLPYP and FLPYPYY, which corresponded to sequences 76–80 and 76–82 of bovine κ-casein respectively, were ineffective.     

The Characterization of Rennin Action on κ-Casein Using CM-Cellulose

Rennin action on κ-Casein was studied using the CM-cellulose method which determines the amount of para-κ-casein formed during the enzymatic hydrolysis of κ-casein. The reaction rate was measured as a function of the time and enzyme concentration. A Km value of 8.9 ×10^?4m and a V value of 1.2 × 10^?4 M/min were obtained under the assay condition used in this study. The maximum initial rate of para-κ-casein formation occurred at pH 5.0 and 50°C. The present study also demonstrated that the CM-cellulose method is useful for measuring the rennin activity on κ-casein.     

Environmental Effects on Disulfide Bonding Patterns of Bovine κ-Casein

Bovine -casein, the stabilizing protein of the colloidal milk protein complex, has a unique disulfide bonding pattern. The protein exhibits varying molecular sizes on SDS-PAGE ranging from monomer to octamer and above in the absence of reducing agents. Heating the samples with SDS prior to electrophoresis caused an apparent decrease in polymeric distribution: up to 60% monomer after 30min at 90°C as estimated by densitometry of SDS-PAGE. In contrast, heating the samples without detergent at 90 or 37°C caused a significant increase in high-molecular-weight polymers as judged by electrophoresis and analytical ultracentrifugation. In 6 M urea, the protein could be completely reduced, but upon dialysis, varying degrees of polymer reformation occurred depending on the dialysis conditions. Spontaneous reoxidation to polymeric forms is favored at low pH (<5.15) and low ionic strength. The results are discussed with respect to the influence of the method of preparation on the polymer size of -caseins and on their resultant physical chemical properties.     

The Primary Structure of Water Buffalo αs1- and β-Casein: Identification of Phosphorylation Sites and Characterization of a Novel β-Casein Variant

The primary structure of water buffalo α_s1-casein and of β-casein A and B variants has been determined using a combination of mass spectrometry and Edman degradation procedures. The phosphorylated residues were localized on the tryptic phosphopeptides after performing a β-elimination/thiol derivatization. Water buffalo α_s1-casein, resolved in three discrete bands by isoelectric focusing, was found to consist of a single protein containing eight, seven, or six phosphate groups. Compared to bovine α_s1-casein C variant, the water buffalo α_s1-casein presented ten amino acid substitutions, seven of which involved charged amino acid residues. With respect to bovine βA₂-casein variant, the two water buffalo β-casein variants A and B presented four and five amino acid substitutions, respectively. In addition to the phosphoserines, a phosphothreonine residue was identified in variant A. From the phylogenetic point of view, both water buffalo β-casein variants seem to be homologous to bovine βA₂-casein.     

Alternative Nonallelic Deletion Is Constitutive of Ruminant αs1-Casein

Multiple forms of α_s1-casein were identified in the four major ruminant species by structural characterization of the protein fraction. While α_s1-casein phenotypes were constituted by a mixture of at least seven molecular forms in ovine and caprine species, there were only two forms in bovine and water buffalo species. In ovine and caprine forms the main component corresponded to the 199-residue-long form, and the deleted proteins differed from the complete one by the absence of peptides 141–148, 110–117, or Gln78, or a combination of such deletions. The deleted segments corresponded to the sequence regions encoded by exons 13 and 16, and by the first triplet of exon 11 (CAG), suggesting that the occurrence of the short protein forms is due to alternative skipping, as previously demonstrated for some caprine and ovine phenotypes. The alternative deletion of Gln78 in α_s1-casein, the only form common to the milk of all the species examined and located in a sequence region joining the polar phosphorylation cluster and the hydrophobic C-terminal domain of the protein, may play a functional role in the stabilization of the milk micelle structure.     

The Primary Structure of Water Buffalo αs1- and β-Casein: Identification of Phosphorylation Sites and Characterization of a Novel β-Casein Variant

The primary structure of water buffalo _s1-casein and of -casein A and B variants has been determined using a combination of mass spectrometry and Edman degradation procedures. The phosphorylated residues were localized on the tryptic phosphopeptides after performing a -elimination/thiol derivatization. Water buffalo _s1-casein, resolved in three discrete bands by isoelectric focusing, was found to consist of a single protein containing eight, seven, or six phosphate groups. Compared to bovine _s1-casein C variant, the water buffalo _s1-casein presented ten amino acid substitutions, seven of which involved charged amino acid residues. With respect to bovine A₂-casein variant, the two water buffalo -casein variants A and B presented four and five amino acid substitutions, respectively. In addition to the phosphoserines, a phosphothreonine residue was identified in variant A. From the phylogenetic point of view, both water buffalo -casein variants seem to be homologous to bovine A₂-casein.     

Effect of Dephosphorylation on the Self-association and the Precipitation of β-Casein

The pyrolyzate of the nondialyzable melanoidin prepared from glucose-ammonia reaction system (kept in pH 5.3~6.0 during the reaction) was fractionated to volatile fraction and nonvolatile fraction. Among the volatile components, two pyridines and four alkylpyrazines were identified. On the other hand, one imidazole compound and two β-hydroxypyridines isolated from the nonvolatile fraction were identified as 4(5)-methylimidazole, 3-hydroxypyridine and 2-methyl-5-hydroxypyridine, respectively. It is inferred that these compounds are not produced by the fission of the main skeleton in the melanoidin molecule, but formed by pyrolysis of the heterocyclic compounds present as a small moiety in the melanoidin.     

Genetic polymorphism detection of two α-Casein genes in three Egyptian sheep breeds

Sheep milk is an excellent raw material for the milk processing industry especially in cheese production. The protein content and composition of sheep milk are important in the cheese manufacturing. The casein fraction of ruminant milk proteins consists of four caseins, namely αs1, αs2, β and κ-Casein. Casein genetic polymorphisms are important due to their effects on quantitative traits and technological properties of milk.This study aimed to detect the genetic polymorphism of αs1- and αs2-Casein genes in three native Egyptian sheep breeds; Rahmani, Barki and Ossimi. PCR-SSCP and PCR-RFLP were used to detect the genetic polymorphism of αs1-CN and αs2-CN genes, respectively.A 223-bp fragment of αs1-CN gene was amplified by PCR and SSCP results recorded the presence of three different patterns; TT, TC and CC; in 87 tested sheep animals. The sequence analysis of two homologous patterns showed a single nucleotide polymorphism (SNP) (T → C) at position 170. The frequencies of three patterns in the tested sheep breeds were 43.33%, 50.00%, and 6.67% in Rahmani; 83.33%, 13.33%, and 3.33% in Ossimi and 74.07%, 22.22%, and 3.70% in Barki, respectively. Our nucleotide sequences of αs1-CN T and C alleles were submitted to GenBank with the accession numbers KF018339 and KF018340, respectively.The restriction digestion of αs2-CN PCR product (1300-bp) by Tru1I endonuclease revealed three different genotypes; AA, AG and GG with frequencies of 66.67%, 30.00%, and 3.33% in Rahmani; 96.67%, 3.33%, and 0.00% in Ossimi and 96.15%, 3.85%, and 0.00% in Barki, respectively. The sequence analysis revealed the presence of a single nucleotide polymorphism (A → G) in intron 6 of αs2-CN gene. Our nucleotide sequence of αs2-CN gene was submitted to GenBank with the accession number JX080380.     

κ-Casein terminates casein micelle build-up by its “soft” secondary structure

In our previous paper (Nagy et?al. in J Biol Chem 285:38811–38817, 2010) by using a multilayered model system, we showed that, from α-casein, aggregates (similar to natural casein micelles) can be built up step by step if Ca-phosphate nanocluster incorporation is ensured between the protein adsorption steps. It remained, however, an open question whether the growth of the aggregates can be terminated, similarly to in nature with casein micelles. Here, we show that, in the presence of Ca-phosphate nanoclusters, upon adsorbing onto earlier α-casein surfaces, the secondary structure of α-casein remains practically unaffected, but κ-casein exhibits considerable changes in its secondary structure as manifested by a shift toward having more β-structures. In the absence of Ca-phosphate, only κ-casein can still adsorb onto the underlying casein surface; this κ-casein also expresses considerable shift toward β-structures. In addition, this κ-casein cover terminates casein aggregation; no further adsorption of either α- or κ-casein can be achieved. These results, while obtained on a model system, may show that the Ca-insensitive κ-casein can, indeed, be the outer layer of the casein micelles, not only because of its “hairy” extrusion into the water phase, but because of its “softer” secondary structure, which can “occlude” the interacting motifs serving casein aggregation. We think that the revealed nature of the molecular interactions, and the growth mechanism found here, might be useful to understand the aggregation process of casein micelles also in?vivo.     

Attachment Sites of Carbohydrate Portions to Peptide Chain of κ-Casein from Bovine Colostrum

Short glycopeptides were prepared from bovine colostral κ-casein treated with cyanogen bromide and proteases (pronase P and thermolysin), followed by gel filtrations and ion exchange chromatography. It was confirmed by Edman degradation that glycopeptide I among short glycopeptides obtained was homogeneous. From the effect of alkali treatment, it was assumed that three polysaccharide chains of glycopeptide I were attached to the peptide chain through OH groups of threonines. By chemical procedures and carboxypeptidase P treatment, the amino acid sequence of glycopeptide I was established to be Ser-Gly-Glu-Pro-Thr-Ser-Thr-Pro-Thr-Thr-Glu-Ala-Val. Threonine residues of No. 5, 7 and 9 were bound to the carbohydrate chains through galactosamine. The sugar chain bound to the threonine residue at No. 7 contained glucosamine. Glycopeptide I corresponded to residues of No. 127–139 in κ-casein A from normal milk.     

Expression of a Biologically Active GFP-αS1-Casein Fusion Protein in Lactococcus lactis

In this study, we successfully developed a recombinant strain of Lactococcus lactis NZ9000 (NZ9000) that produced green fluorescent protein fused to α_S1-casein (GFP-α_S1Cas). A modified lactic acid bacterial vector (pNZ8148#2) was constructed by inserting genes for GFP and α_S1-casein, a major cow’s milk allergen, and the resulting vector, pNZ8148#2-GFP-α_S1Cas, was applied to the expression of recombinant GFP-α_S1Cas protein (rGFP-α_S1Cas) in NZ9000. After inducing expression with nisin, the production of rGFP-α_S1Cas was confirmed by confocal laser microscopic analysis, and the expression conditions were optimized based on fluorescent analysis and western blotting results. Moreover, the in vitro treatment of splenocytes isolated from α-casein (≥70 % α_S-casein)-immunized mice with rGFP-α_S1Cas resulted in increased IL-13 mRNA expression. The observed allergic activity is indicative of the Th2-cell mediated immune response and is similar to the effects induced by exposure to α-casein. Our results suggest that the expression of rGFP-α_S1Cas in NZ9000 may facilitate in vivo applications of this system aimed at improving the specificity of immunological responses to specific milk allergen.     

Bioactive Peptides from Bovine Milk α-Casein: Isolation,Characterization and Multifunctional properties

α-Casein group of proteins makes up to 65% of the total casein and consists of α_S1- casein_, α_S2- casein and other related proteins. Among all the proteases employed, chymotryptic peptides showed maximum inhibition for angiotensin converting enzyme (ACE). The degree of hydrolysis and release kinetics of the peptides during chymotrypsin hydrolysis was compared with biological activity and the potent peptides fractions were identified. The crude fraction obtained after 110 min of hydrolysis shows multifunctional activities, like ACE inhibition, antioxidant activity, prolyl endopeptidase inhibitory activity and antimicrobial activities. This fraction was further purified by HPLC and sequenced by mass spectra. This fraction constituted peptides with molecular weights of 1,205, 1,718 Da respectively. The sequencing of peptides by MALDI-TOF MS/MS shows sequences QKALNEINQF and TKKTKLTEEEKNRL from α-_S2 casein.