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.     

A lactational study of the composition and integrity of casein micelles from the milk of the tammar wallaby (Macropus eugenii)

The amount of casein found in the milk of the tammar wallaby increases as lactation progresses. The increase is due to increasing amounts of β-casein; the α-casein remains largely constant. The α-casein is the more highly phosphorylated; the most abundant form is the 10-P, throughout lactation. The level of phosphorylation of β-casein shifts to lower average values in late lactation, possibly indicating the enzymatic reaction is overloaded by the increasing amounts of β-casein. Unlike bovine casein micelles, the wallaby micelles are not completely disrupted at pH 7.0 by sequestration of their calcium content with ethylene diamine tetraacetic acid (EDTA). Complete disruption only follows the addition of sodium dodecyl sulphate, indicating considerably greater importance for hydrophobic bonds in maintaining their integrity. This micellar behaviour indicates that, despite the evolutionary divergence of marsupials millennia ago, the caseins of wallaby milk assemble into micelles in much the same fashion as in bovine milk.     

Photolysis of Aryl Glycosides with Ultraviolet Irradiation

Bovine casein components (α_sl-, β-, and κ-caseins) were chemically phosphorylated and the properties of the modified components were compared with those of the native to clarify the function of the intrinsic phosphate groups of casein components in casein micelle formation. The calcium binding ability of casein components increased after chemical phosphorylation. The concentrations of calcium chloride required to precipitate modified α_sl- and β-caseins were higher than those for native components. However, phosphorylation of α_sl- and β-caseins did not affect their properties of forming micelles through interaction with κ-casein. The stabilizing ability of κ-casein for α_sl- and β caseins was impaired by its phosphorylation, but the stability was recovered by treating phosphorylated κ-casein with phosphoprotein phosphatase. The results show that the phosphate content of κ-casein must be low to form a stable casein micelle. The results also explain why the specific phosphorylation of casein components in the mammary gland is required.     

How to squeeze a sponge: casein micelles under osmotic stress, a SAXS study

By combining the osmotic stress technique with small-angle x-ray scattering measurements, we followed the structural response of the casein micelle to an overall increase in concentration. When the aqueous phase that separates the micelles is extracted, they behave as polydisperse repelling spheres and their internal structure is not affected. When they are compressed, the micelles lose water and shrink to a smaller volume. Our results indicate that this compression is nonaffine, i.e., some parts of the micelle collapse, whereas other parts resist deformation. We suggest that this behavior is consistent with a spongelike casein micelle having a triple hierarchical structure. The lowest level of the structure consists of the CaP nanoclusters that serve as anchors for the casein molecules. The intermediate level consists of 10- to 40-nm hard regions that resist compression and contain the nanoclusters. Those regions are connected and/or partially merged with each other, thus forming a continuous and porous material. The third level of structure is the casein micelle itself, with an average size of 100 nm. In our view, such a structure is consistent with the observation of 10- to 20-nm casein particles in the Golgi vesicles of lactating cells: upon aggregation, those particles would rearrange, fuse, and/or swell to form the spongelike micelle.     

Composition and size distribution of bovine casein micelles

Chromatography of glutaraldehyde-fixed skim-milk on controlled-pore glass (CPG-10, 300 nm) gave three micellar fractions whose averaged diameters, measured by electron microscopy, decreased progressively with increasing elution volume. Casein micelles with diameters up to 680 nm were detected. The casein composition of the same fractions from unfixed skim-milk was determined. As the fraction elution volume increased, κ-casein varied from 7.7 to 11.4% of total casein, giving α_s/κ ratios of 6.1, 4.7 and 3.3.A plot of κ-casein content versus micelle surface-to-volume ratio for skim-milk and the column fractions approximated to a straight line. Re-calculation of the published results from two other studies also gave linear relationships between κ-casein content and surface area for artificial micelles. The three regression lines thus obtained had small intercepts. It was concluded that the data indicated the same fundamental structure for casein micelles, with a pre-dominant surface location for κ-casein, whether the micelles are natural or artificial and whether they are aggregated or by Ca²⁺ alone oy Ca²⁺ together with calcium phosphate-citrate complex.     

Reconstitution of Human Casein Micelle and Its Properties

Human casein micelles were reconstituted from isolated κ- and β-caseins and calcium ions. Micelle formation was recognized in the presence of calcium chloride even at the low concentration of 5mM. At pH levels ranging from 5.5 to 8.0, the re-formed micelles were quite stable so that precipitation of β-casein was not observed. The large micelles were constituted by a higher ratio of β-casein to κ-casein (16:1 by weight) than the small micelles (3: 1). The κ-casein in the small micelles contained carbohydrates to about 43% (w/w) in the molecule, whereas that in the large micelles was only about 25%. When the casein micelles were re-formed from κ-easein and fractionated β-casein components, the extent of phosphorylation of the β-casein component was found to influence the micelle formation; i.e., the β-casein component with no phosphate (the 0-P form) was disadvantageous to form micelles, but the component with 5 phosphates (the 5-P form) formed micelles most easily.     

Alpha casein micelles show not only molecular chaperone-like aggregation inhibition properties but also protein refolding activity from the denatured state

Casein micelles are a major component of milk proteins. It is well known that casein micelles show chaperone-like activity such as inhibition of protein aggregation and stabilization of proteins. In this study, it was revealed that casein micelles also possess a high refolding activity for denatured proteins. A buffer containing caseins exhibited higher refolding activity for denatured bovine carbonic anhydrase than buffers including other proteins. In particular, a buffer containing α-casein showed about a twofold higher refolding activity compared with absence of α-casein. Casein properties of surface hydrophobicity, a flexible structure and assembly formation are thought to contribute to this high refolding activity. Our results indicate that casein micelles stabilize milk proteins by both chaperone-like activity and refolding properties.     

A mechanism for the chymosin-induced flocculation of casein micelles

The chymosin-induced flocculation of casein micelles of bovine milk can be explained and calculated in terms of three relationships, which are (i) the action of chymosin upon the κ-casein of the micelles; (ii) the probability that a micelle, with a given proportion of its κ-casein destroyed, will aggregate, and (iii) the aggregation of micelles by a Smoluchowski mechanism. Details of the calculations are given, and the theory is shown to be in good agreement with experimental observations of the dependence of the clotting time with variations in enzyme and substrate concentrations.     

Crosslinking of casein by microbial transglutaminase and its resulting influence on the stability of micelle structure

The influence of enzymatic crosslinking by microbial transglutaminase (mTG) on the stability of casein micelles of ultrahigh temperature (UHT)-treated milk in the presence of EDTA (0-0.45 mM) or ethanol (0-74 vol%) as well as under high hydrostatic pressures up to 400 MPa was investigated. Disintegration of micelles and changes in micelle size were monitored by the measurement of turbidity as well as by dynamic light scattering. The results show that the incubation of UHTtreated milk with mTG resulted in an improved micelle stability toward disintegration on addition of EDTA, ethanol, or pressure treatment. Intramicellar formed isopetides significantly enhanced the stability of casein micelles. It is supposed that net-like crosslinks are formed within the external region of the micelles and they adopt the stabilizing role of colloidal calcium phosphate within the micelles, thus making the micelles less contestable for disrupting influences.     

Influence of Molecular Weight of Chitosan on Interaction with Casein

The process of complex formation of casein from skimmed milk and purified casein with chitosan of different molecular weights was studied. It was shown that at pH 6.3 casein micelles and parts of whey proteins coagulated with positively charged chitosan molecules with molecular weights of 45.3, 25.4, 7.7 and 1.5 kDa. As a result of ionic interaction of chitosan with skimmed milk proteins the yield of target product reached 90–92%. It consisted of all forms of casein: α-casein, β-casein, κ-casein and small amount of whey proteins.     

Aggregation‐Induced Emission of 1,8‐Naphthalimide?Casein Micelle: Investigation by Synchronous Spectrographic Method

A novel 1,8‐naphthalimide probe 1 , bearing two acetic‐acid moieties was synthesized. The acetic‐acid groups, docked into the sub‐domains of casein micelle and bound with tryptophan residues, and the 1,8‐naphthalimide chromophore adsorbed on the surface of casein micelle, forming a supermolecule, 1 ? casein micelle, which exhibited the aggregation‐induced synchronous emission (AISE) characters. The effect of pH on the intensity of supermolecule was investigated, and the result indicated that the emission enhancement was mainly due to the 1,8‐naphthalimide chromophore aggregated onto the casein micelle. Based on AISE, a novel casein quantification method was developed, which exhibited a good linear range of 0.05–10.0 μg ml^?1 and 0.07–9.5 μg ml^?1 with the detection limits of 2.8 and 3.0 ng ml^?1. The effects of metal ions and pH on the system of 1 ? casein micelle were investigated. The proposed method was applied to determine casein in milk samples, and the results were in good agreement with the result of the Biuret method.     

Isolation of Tremerogen a-13, a Peptidal Sex Hormone of Tremella mesenterica

Bovine casein micelles were fractionated on controlled pore granule (CPG-10/3000) chromatography by size and the chemical properties of the fractionated micelles were compared. The results indicated the presence of two types of micelles distinguishable as large and small micelles. In skim milk, 72.7% of casein was calculated to be in the form of small micelles, 13.6% in the form of large micelles and 13.8% in non-micellar casein form.

The α_s1-casein content decreased, but β- and κ-casein content increased as the micelle size became smaller. κ-Casein in large micelles had a much higher sialic acid content than in small micelles. It was found that this difference in sialic acid content was due to the presence of non-glycosylated κ-casein in small micelles. In large micelles, non-glycosylated κ-casein was almost undetectable.

The addition of wheat germ lectin to micelles resulted in the formation of aggregates through intermicellar bridges between the carbohydrate chains of κ-casein located on the surface of the micelles. Both large and small micelles formed aggregates after the addition of wheat germ lectin. Large micelles were more sensitive to wheat germ lectin than small ones.     

Electrosorption of pectin onto casein micelles

Pectin, a polysaccharide derived from plant cells of fruit, is commonly used as stabilizer in acidified milk drinks. To gain a better understanding of the way that pectin stabilizes these drinks, we studied the adsorption and layer thickness of pectin on casein micelles in skim milk dispersions. Dynamic light scattering was used to measure the layer thickness of adsorbed pectin onto casein micelles in situ during acidification. The results indicate that the adsorption of pectin onto casein micelles is multilayered and takes place at and below pH 5.0. Renneting, i.e., cleaving-off kappa-casein from the casein micelles, did not alter the adsorption pH. It did, however, show that pectin arrests the rennet-induced flocculation of casein micelles below pH 5.0. From the findings we concluded the attachment of pectin onto casein micelles is driven by electrosorption. Adsorption measurements confirmed the multilayered nature of the adsorption of pectin onto casein micelles. Both the adsorbed amount and the layer thickness increased with decreasing pH in the relevant range 3.5-5.0. The phase behavior of a casein micelles/pectin mixture was determined and could be explained in terms of thermodynamic incompatibility being relevant above pH 5.0 and adsorption, leading to either stabilization and bridging, being relevant below pH 5.0. The results confirm that electrosorption is the driving force for the adsorption of pectin onto casein micelles.     

Caseins are cross-linked through their ester phosphate groups by colloidal calcium phosphate

Artificial casein micelles were prepared by adding 30 mM calcium, 22 mM phosphate and 10 mM citrate to sodium caseinate solutions, and the content of the casein aggregates cross-linked by colloidal calcium phosphate was determined by high-performance gel chromatography on a TSK-GEL G4000SW column in the presence of 6 M urea. The content of the casein aggregates cross-linked by colloidal calcium phosphate in artificial whole casein micelles was 48% of total casein, and their relative casein composition determined by high-performance ion-exchange chromatography was 53.1% for alpha s1-casein, 15.8% for alpha s2-casein, 31.1% for beta-casein and 0% for kappa-casein. The order of cross-linking by colloidal calcium phosphate agreed with that of the ester phosphate content of casein constituents. The content of the casein aggregates cross-linked by colloidal calcium phosphate was higher in alpha s1-kappa-casein micelles than in beta-kappa-casein micelles. kappa- and gamma-caseins and dephosphorylated alpha s1-casein were not cross-linked by colloidal calcium phosphate. Although kappa-casein was not cross-linked, chemically phosphorylated kappa-casein, of which the average phosphate content was 8.5 per molecule, was cross-linked. It is concluded that caseins are cross-linked through their ester phosphate groups by colloidal calcium phosphate.     

Localization of glycosylated κ-casein on thin sections of casein micelles by lectin-labelled gold markers

Summary The location of the glycosylated part of κ-casein in bovine casein micelles was investigated using gold particles (6 nm in diameter) labelled withRicinus communis lectin andLimulus polyphemus lectin. The pattern of marking of thin sections of micelles was similar with both lectins. Glycosylated κ-casein was distributed uniformly throughout most micelles of all sizes. Peripheral location of glycosylated κ-casein was observed only occasionally in some of the largest micelles. Quantitative data indicated that the concentration of the glycosylated protein was constant in micelles of increasing sizes. As larger micelles contain less total κ-casein than smaller ones, these data indicated that a greater proportion of their κ-casein is glycosylated. These results support models for casein micelle structure where κ-casein is distributed throughout the micelles. They do not agree with “coat-core” structures.     

Fractionation and Characterization of 9 Subcomponents of Bovine κ-Casein A

κ-casein A was fractionated into 9 subcomponents, all of which were identified as κ-casein from immunological analyses. The microheterogeneity of the subcomponents was explained by stepwise increase of their carbohydrate contents (0~4mol/mol of GalNAc, and 0~8mol/mol of NANA). The micelle-stabilizing ability of κ-casein subcomponents increased with the increase of their carbohydrate contents: the carbohydrate rich subcomponent 7 possessed twice the stabilizing ability of the carbohydrate free subcomponent 1. The sensitivity of synthetic casein micelle composed of κ-casein subcomponents and α_sl-casein to the wheat germ lectin-induced aggregation also increased with the increase of their NANA contents.     

An equilibrium thermodynamic model of the sequestration of calcium phosphate by casein phosphopeptides

Sequestration of calcium phosphate by caseins occurs in the Golgi region of mammary secretory cells during lactation, where it helps to prevent calcification of the gland and to deliver high concentrations of calcium and phosphate to the neonate in the form of milk. Calcium phosphate nanoclusters are formed when a core of amorphous calcium phosphate is sequestered within a shell of casein or casein phosphopeptides. The nanoclusters can form spontaneously from a supersaturated solution or by dispersion of a precipitate of calcium phosphate, demonstrating that they are thermodynamically stable complexes. The average size and chemical composition of the complexes are largely independent of the solution conditions (pH, temperature, peptide concentration, salt composition and rate of reaction) under which they form. Larger, metastable, colloidal particles can form if there is not enough of the phosphopeptide to sequester all the calcium phosphate, or, transiently, if the salt and peptide solutions are mixed together without sufficient care. A thermodynamic model of the sequestration process is presented which makes use of an invariant ion activity product observed in nanocluster-containing solutions. In any given solution that has thermodynamic stability, the extent of the sequestration reaction can be calculated from the empirical formula of the nanoclusters using the criterion that the solution should have the equilibrium value of the invariant ion activity product. Other members of the paralogous group of secretory calcium-binding phosphoproteins to which caseins belong may also be able to sequester calcium phosphate in biological fluids such as saliva and in the extracellular matrix of mineralizing tissues.Abbreviations -PP _s1-casein 5P (f59–79) - -PP -casein 4P (f1–25) - ACP amorphous calcium phosphate - Cit citrate - CPN calcium phosphate nanocluster - CPP commercial phosphopeptide - IAP ion activity product - MWCO molecular weight cut-off - PP phosphopeptide - SAXS small-angle X-ray scattering - SCPP secretory calcium-binding phosphoprotein - UF ultrafiltrate     

K-Casein Suppresses Melanogenesis in Cultured Pigment Cells

The effects of bovine milk proteins on melanogenesis in B16 cells were examined. Both whey protein isolate and casein exhibited depigmenting properties. Among the major protein components of milk—including β-lactoglobulin, α-lactalbumin, α-, β-, and k-casein—only K-casein exhibited the depigmenting effect. However, the carboxyl terminal peptide of K-casein, glycomacropeptide, did not show this activity. Also, K-casein promoted the proliferation of the cells and inhibited the activity of tyrosinase in the cells. These results indicate that K-casein acts as a melanogenesis-suppressing modulator.     

Appraisal of casein's inhibitory effects on aggregation accompanying carbonic anhydrase refolding and heat-induced ovalbumin fibrillogenesis

It is well accepted that whole casein and its purified major components, due to their chaperone-like activity, are able to suppress the thermal and chemical aggregation of several substrate proteins. In this study, we set out to determine whether whole and β-casein are able to prevent (or attenuate) aggregation accompanying refolding of chemically denatured carbonic anhydrase or to recover lost biological activity after its denaturation. Additionally, we showed attenuated heat-induced fibrillar aggregation of egg white ovalbumin in the presence of these commonly occurring unfolded proteins, as molecular chaperones. Also, the extent, rate and order of aggregation, in the presence and absence of aggregation suppressors, were compared. Although β-casein did not prevent aggregation as strong as whole casein, both chaperones were efficient not only in suppressing the aggregation extent of denatured carbonic anhydrase, but also in delaying elongation process of amyloid fibril formation with no effect on nucleation phase.