Studies on human alpha(s)- and kappa-casein fractions and human caseinoglycomacropeptide

1. Fractions have been obtained from human whole casein closely resembling the α_s- and κ-fractions of cow casein. 2. The α_s-fraction (human α_s-casein) is calcium-sensitive, heterogeneous in zone analysis and inert towards rennin. 3. The κ-fraction (human κ-casein) is calcium-insensitive, heterogeneous in zone analysis, and forms a soluble glycopeptide when acted upon by rennin. 4. Human κ-casein stabilizes human α_s-casein in the presence of Ca²⁺ ions. 5. The glycopeptides released by rennin from human casein and from cow casein have been compared. There are important differences in both the peptide and non-peptide structures of the two compounds. 6. In both human and bovine glycopeptides some of the carbohydrate residues are joined to the peptide by O-glycosidic links with threonine, and possibly with serine.     

Partial Isolation and Degradation of Caseins by Cell Wall Proteinase(s) of Streptococcus cremoris HP

The cell wall proteinase fraction of Streptococcus cremoris HP has been isolated. This preparation did not exhibit any activity due to either specific peptidases known to be located near the outside surface of and in the membrane or intracellular proteolytic enzymes. By using thin-layer chromatography for the detection of relatively small hydrolysis products which remain soluble at pH 4.6, it was shown that β-casein is preferentially attacked by the cell wall proteinase. This was also the case when whole casein or micelles were used as the substrate. κ-casein hydrolysis is a relatively slow process, and α_s-casein degradation appeared to proceed at an extremely low rate. These results could be confirmed by using ¹⁴CH₃-labeled caseins. A relatively fast and linear initial progress of ¹⁴CH₃-labeled β-casein degradation is not inhibited by α_s-casein and only slightly by κ-casein at concentrations of these components which reflect their stoichiometry in the micelles. Possible implications of β-casein degradation for growth of the organism in milk are discussed.     

Mapping and Analysis of Dairy Cattle Quantitative Trait Loci by Maximum Likelihood Methodology Using Milk Protein Genes as Genetic Markers

Maximum likelihood methodology was used to estimate effects of both a marker gene and a linked quantitative trait locus (QTL) on quantitative traits in a segregating population. Two alleles were assumed for the QTL. In addition to the effects of genotypes at both loci on the mean of the quantitative trait, recombination frequency between the loci, frequency of the QTL alleles and the residual standard deviation were also estimated. Thus six parameters were estimated in addition to the marker genotype means. The statistical model was tested on simulated data, and used to estimate direct and linked effects of the milk protein genes, β-lactoglobulin, κcasein, and β-casein, on milk, fat, and protein production and fat and protein percent in the Dutch dairy cattle population. β-Lactoglobulin had significant direct effects on milk yield and fat percent. κ-Casein had significant direct effects on milk yield, protein percent and fat yield. β-Casein had significant direct effects on milk yield, fat and protein percent and fat and protein yield. Linked QTL with significant effects on fat percent were found for κ-casein and β-casein. Since the β-casein and κ-casein genes are closely linked, it is likely that the same QTL was detected for those two markers. Further, a QTL with a significant effect on fat yield was found to be linked to κ-casein and a QTL with a significant effect on protein yield was linked to β-lactoglobulin.     

The separation of major components of the casein of bovine milk by electrophoresis in a density gradient

1. Zonal electrophoresis in a column stabilized by a density gradient has been applied to the small-scale fractionation of the proteins of the casein complex of cow's milk. 2. The α_s- and β-fractions from the milk of individual Ayrshire cows have been shown to behave as single homogeneous proteins on electrophoresis at two pH values in starch gels. 3. The α_s-fraction has been found to be indistinguishable from α_s-casein prepared by Ca²⁺ fractionation of the same milk samples. 4. On the evidence of their electrophoretic behaviour in starch gels and their elementary analyses, α₁- and α_s-casein are concluded to be substantially the same protein.     

Kinetics of peptide bond demasking in enzymatic hydrolysis of casein substrates

Proteolysis of casein substrates includes demasking stage, the transition of masked bonds to the demasked stage, where peptide bonds become accessible to the enzyme attack. Therefore, proteolysis was regarded as a two-stage process with consequent demasking and hydrolysis stages. When demasking process is kinetically significant, the peptide bonds are hydrolysed with some lag. It was shown both by theoretical simulations and experimentally that the increase of amino nitrogen can be a non-monotonous function of the hydrolysis degree or proteolysis time. The non-monotonously dependence was found for chymotryptic proteolysis of β-casein, while for α-casein the monotonous dependence was obtained. This was treated as an indication of the prevalence of the hydrophobically induced masking effect for β-casein. For the proteolysis of β-casein by wild-type and engineered trypsins, the kinetic analysis allowed us to conclude that demasking stage was initiated by the splitting of the main peptide chain, which compact conformation was initially stabilized by the interaction of hydrophobic regions of peptide chain.     

Nucleotide Sequence Evolution at the κ-Casein Locus: Evidence for Positive Selection within the Family Bovidae

κ-Casein is a mammalian milk protein involved in a number of important physiological processes. In the gut, the ingested protein is split into an insoluble peptide (para κ-casein) and a soluble hydrophilic glycopeptide (caseinomacropeptide). Caseinomacropeptide is responsible for increased efficiency of digestion, prevention of neonate hypersensitivity to ingested proteins, and inhibition of gastric pathogens. Variation within this peptide has significant effects associated with important traits such as milk production. The nucleotide sequences for regions of κ-casein exon and intron four were determined for representatives of the artiodactyl family Bovidae. The pattern of nucleotide substitution in κ-casein sequences for distantly related bovid taxa demonstrates that positive selection has accelerated their divergence at the amino acid sequence level. This selection has differentially influenced the molecular evolution of the two κ-casein split peptides and is focused within a 34-codon region of caseinomacropeptide.     

Phosphorylation of casein by cyclic AMP-dependent protein kinase.

The catalytic subunit of rabbit muscle cyclic AMP-dependent protein kinase (EC 2.7.1.37; ATP:protein transferase) has been tested on a variety of caseins. The B variant of β-casein was phosphorylated at a much greater rate than other β-caseins, α_s1-caseins, and κ-caseins. Whole casein homozygous for β-casein B was phosphorylated at 2.5 times the rate of commercial whole casein. Gel electrophoresis experiments indicate that β-casein is the predominant component phosphorylated in commerical casein. It is therefore suggested that phosphorylation of whole casein depends on its content of the specific genetic variant, β-casein B.     

Cellulose acetate electrophoresis of casein proteins.

Samples of the milk proteins α_s1-casein and β-casein partially dephosphorylated by means of bovine spleen phosphoprotein phosphatase have been electrophoretically analysed using cellulose acetate as the supporting medium and Procion blue as the protein dye. Sufficient resolution was obtained in 1 hr to allow quantification of the proteins present. Skimmed-milk samples and acid-precipitated whole casein samples have been analysed by the same technique. The advantages of the method are discussed in relation to the more conventional electrophoretic techniques normally used to analyse these milk proteins.     

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.     

In vitro and in vivo growth and casein gene expression of mouse mammary tumor epithelial cells in response to hormones

Cells from autochthonous mouse mammary carcinomas which display estrogen-independent growth _vivo were studied for their hormonal responses in primary culture. A culture system employing insulin-supplemented, serum-free medium and basement membrane Matrigel as a substratum was used to cultivate tumor cells. The cells did not exhibit in vitro estrogenor prolactin-dependent growth. Primary tumors still displayed a constitutional expression of α-, β-, and γ-casein mRNAs. These messages were dramatically reduced during the culture period. However, seven to eightfold increases in α- and β-casein mRNAs were inducible in the 5-day cultures by treatment with prolactin and hydrocortisone. If the hormones were present through a 2-week culture period, the levels of α-, β-, and γ-casein mRNAs in the cells were maintained and displayed in a time-dependent increase with a peak at 10–14 days. The accumulation of β-casein mRNA in vitro did not require DNA synthesis. Administration of prolactin directly into the growing tumors in vivo could also enhance β-casein mRNA levels in the tumor cells. Morphological studies of the cells cultured in the presence of prolactin and hydrocortisone did not reveal visible changes compared with those without hormonal treatment. Transplantation of tumor cells cultured in the presence or absence of hormones resulted in the development of tumors in mice at approximately the same time. The current studies suggest that the autochthonous mammary tumor cells, independent of estrogen for cell growth, were still inducible for casein gene expression in vitro and in vivo by appropriate hormones. The induction and maintenance of casein messages by a single hormonal treatment did not appear to correlate with morphology and DNA synthesis of cells in vitro or with tumor-producing capacities in vivo.     

Establishment of bovine mammary epithelial cells (MAC-T): An in vitro model for bovine lactation

The hallmark of differentiated mammary epithelial cells is a copious secretion of milk-specific components regulated by lactogenic hormones. We describe an established clonal cell line produced from primary bovine mammary alveolar cells (MAC-T) by stable transfection with SV-40 large T-antigen. MAC-T cells show a population doubling time of approximately 17 h and have been cultured more than 350 passages without showing any sign of senescence. They show the characteristic “cobblestone” morphology of epithelial cells when grown on plastic substratum. Differentiation was induced by augmenting cell-cell interaction on a floating collagen gel in the presence of prolactin. The differentiated phenotype was characterized to include (1) increased abundance in β-casein mRNA, (2) increased number and size of indirect immunofluorescent casein secretory vesicles in each cell and (3) α_s- and β-casein protein secretion. The clonal nature of the cells, their immortality, and their ability to uniformly differentiate and secrete casein proteins make this cell line unique.     

A type-1 casein kinase from yeast phosphorylates both serine and threonine residues of casein. Identification of the phosphorylation sites

A protein kinase (casein kinase 1A) active on casein and phosvitin but not on histones has been purified to near homogeneity from yeast cytosol and meets most criteria for being considered a type-1 casein kinase: it is a monomeric enzyme exhibiting an Mr of about 27 kDa by sucrose gradient centrifugation: it is not affected by inhibitors of type-2 casein kinases, such as heparin and polyglutamate, and shows negligible affinity for GTP. It also readily phosphorylates the residue Ser-22 of beta-casein located within the sequence -Ser(P)-Ser(P)-Ser(P)-Glu-Glu-Ser22-Ile-Thr-Arg- which is typically affected by casein kinases of the first class. On the other hand, casein kinase 1A displays the unusual property of phosphorylating threonine residue(s) in both whole casein and alpha s1-casein. The threonine residue phosphorylated in alpha s1-casein and accounting for most of the 32P incorporated into this protein by casein kinase 1A has been identified as Thr-49, which occurs in the sequence -Ser(P)-Glu-Ser(P)-Thr(P*)49-Glu-Asp-Gln-, whose two Ser(P) residues are already phosphorylated in the native protein. It is concluded that some type-1 casein kinases can also phosphorylate threonine residues provided they fulfil definite structural requirements, probably an acidic cluster near their N-terminal side.     

Analysis of the human casein phosphoproteome by 2-D electrophoresis and MALDI-TOF/TOF MS reveals new phosphoforms

Mammalian breast milk contains an array of proteins and other nutrients essential for the development of the newborn. In human milk, the caseins (alpha S1, beta and kappa) are a major class of proteins; however, the dynamic range of concentrations in which the various isoforms of each casein exist presents challenges in their characterization. To study human milk casein phosphoforms, we applied traditional two-dimensional polyacrylamide gel electrophoretic (2-DE) separation combined with matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) tandem mass spectroscopic analysis. The abundant beta-casein was resolved as a train of 6 spots differing in phosphorylation level with 0-5 phosphates attached. To study the less abundant alpha S1-casein, a cysteine-tagging enrichment treatment was used prior to 2-DE. A train of 9 spots with 4.4 < p I < 5.3 were identified as alpha S1-casein. This included five previously uncharacterized phosphoforms with up to 8 phosphate groups located in two serine-rich tryptic phosphopeptides ( (27)L-R (51), (69)N-K (98)) consistent with alpha-caseins from various ruminant species. MS/MS analysis of the phosphopeptides released by tryptic digestion enabled identification of the residue-specific order of phosphorylation among the 6 beta-casein and 9 alpha S1-casein phosphoforms. Deamidation of N (47) of alpha S1-casein was also a feature of the MS analysis. This study represents the first comprehensive analysis of the human casein phosphoproteome and reveals a much higher level of phosphorylation than previously recognized. It also highlights the advantages of 2-DE for examining the global pattern of protein phosphoforms and the limitations of attempting to estimate phosphorylation site occupancies from "bottom-up" studies.     

Proteomic analysis of whey and casein proteins in early milk from the marsupial Trichosurus vulpecula,the common brushtail possum

Whey and casein proteins representing the first and second halves of the early lactation phase in the common brushtail possum (Trichosurus vulpecula) have been compared by two dimensional gel electrophoresis. Nine components of whey were differentially expressed during early lactation, including proteins identified as cathepsin B, clusterin, late lactation protein, lysozyme, ganglioside M2 activator and neutrophil gelatinase-associated lipocalin. A major novel protein, termed very early lactation protein (VELP), was identified in whey. Partial amino acid sequence data obtained from VELP did not appear to match any other reported protein sequence. VELP was shown to be an acidic glycoprotein of 20–30 kDa which exists as a homodimer. In the casein fraction, κ-casein appeared to be differentially post-translationally modified during early lactation and fragments of β-casein were relatively more abundant at the earlier lactation stage.     

Effects of gastric digestive products from casein on CCK release by intestinal cells in rat

We investigated the ability of gastric digestive products from casein to stimulate cholecystokinin release by intestinal cells using the isolated vascularly perfused rat duodenojejunum. Casein digests were prepared with an in vitro system simulating gastric digestion and emptying.

The luminal infusion of the digesta emptied from the artificial stomach for the first 10 minutes produced a sharp rise of portal cholecystokinin-like immunoreactivity to 300% of basal, followed by a well-sustained plateau secretion until the end of the infusion. The residual casein fraction of this digest brought about a modest cholecystokinin secretion, while the peptide component was as strong a stimulant as total digest. The peptide responsible for this effect was the glycomacropeptide that is a glycosylated fragment (106–169) of κ-casein. Only the slightly glycosylated forms of the peptide originating from variant A of κ-casein were active. The carbohydrate-free peptide did not alter basal cholecystokinin. The highly glycosylated forms of the peptide and the slightly glycosylated peptide from κ-casein variant B induced only a transient and low rise of portal cholecystokinin. The removal of N-acetylneuraminic acid from the active peptide suppressed its effect, while the infusion of an N-acetylneuraminic acid solution induced only a very low response.

It is concluded that the glycomacropeptide released from dietary casein during gastric digestion can stimulate cholecystokinin release by intestinal cells in the rat. A well-defined structure is required for the peptide activity. A part of the peptide chain and some glycosidic chains containing N-acetylneuraminic acid, especially those bound to the amino acid residue threonyl 31 of caseinomacropeptide variant A, would be involved in this structure.     

Comparative Study of Action of Cell Wall Proteinases from Various Strains of Streptococcus cremoris on Bovine αs1-, β-, and κ-Casein

Partially purified cell wall proteinases of eight strains of Streptococcus cremoris were compared in their action on bovine α_s1-, β-, and κ-casein, as visualized by starch gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thin-layer chromatography. Characteristic degradation profiles could be distinguished, from which the occurrence of two proteinases, represented by strain HP and strain AM₁, was concluded. The action of the HP-type proteinase P₁ (also detectable in strains Wg₂, C₁₃, and TR) was established by electrophoretic methods to be directed preferentially towards β-casein. The AM₁-type proteinase P_III (also detectable in strain SK₁₁) was also able to degrade β-casein, but at the same time split α_s1- and κ-casein more extensively than did P_I. Strain FD₂₇ exhibited mainly P_I activity but also detectable P_III degradation characteristics. The cell wall proteinase preparation of strain E₈ showed low P_I as well as low P_III activity. All proteinase preparations produced from κ-casein positively charged degradation products with electrophoretic mobilities similar to those of degradation products released by the action of the milk-clotting enzyme chymosin. The differences between P_I and P_III in mode of action, as detected by gel electrophoresis and thin-layer chromatography, were reflected by the courses of the initial degradation of methyl-¹⁴C-labeled β-casein and by the effect of α_s1- plus κ-casein on these degradations. The results are discussed in the light of previous comparative studies of cell wall proteinases in strains of S. cremoris and with respect to the growth of this organism in milk.     

Genomic organization and chromosomal localization of the human casein gene family

Five yeast artificial chromosome (YAC) clones containing the human casein gene family were isolated and characterized to study the control mechanisms for the expression of these genes. Partial restriction analysis in conjunction with the chromosomal fragmentation method and fluorescence in situ hybridization (FISH) analysis were performed to construct a detailed physical map of the casein gene family and to determine the chromosomal localization of these genes. The isolated YAC clones 748F3, 750D11, 882G11, 886B3 and 960D2 were 1.2 Mb, 860 kb, 800 kb 1.5 Mb and 1.5 Mb in size, respectively. The clones 748F3, 882G11, 886B3 and 960D2 contained the entire casein gene family, while the κ-casein gene was absent in 750D11. The human αS1-, β- and κ-casein genes were found to be closely linked and arranged in the order αS1-β-κ. The distance between αS1 and β, and between αS1 and κ was approximately 10 and 300 kb, respectively. The β-casein gene was oriented in the opposite direction to the αS1- and κ-casein genes. The casein gene family was localized to chromosome 4q21.1 by FISH analysis. Received: 7 July 1996 / Revised: 29 October 1996     

Milk Protein Synthesis, Gene Expression, and Hormonal Responsiveness in Primary Cultures of Mammary Cells from Lactating Sheep

A ruminant mammary cell culture that accurately reproduces mammary function in vitro would be a valuable tool in studies of ruminant lactation, With this in mind, we have examined milk protein synthesis and secretion, milk protein mRNA abundance, and hormonal responsiveness in primary cultures of mammary acini from lecturing sheep. α- and β-casein protein synthesis, β-lactoglobulin synthesis, and α-casein, β-casein, and β-lactoglobulin secretion are maintained at high levels for 8 h in culture, but then decline to approximately 25% of maximal rates between 8 and 24 h in culture, whereas synthesis of other proteins remains unaltered. The relative abundance of α-S₁-casein, β-lactoglobulin, and α-lactalbumin mRNAs similarly decline between 8 and 24 h in culture. Extracellular labeled α-casein is increased fourfold in the presence of fetal calf serum (FCS). In total, FCS alters the abundance of 47 of 68 secreted proteins detected by two-dimensional electrophoresis. However, FCS and lactogenic/galactopoietic hormones had no effect on the rate of decline of mammary function and did not promote any regaining of function when present for up to 9 days in culture. These results suggest that providing its limitations are recognized, this primary cell culture system may be useful in studying some aspects of ruminant mammary function in vitro.     

Amino acids content and electrophoretic profile of camel milk casein from different camel breeds in Saudi Arabia

This study aimed to evaluate amino acids content and the electrophoretic profile of camel milk casein from different camel breeds. Milk from three different camel breeds (Majaheim, Wadah and Safrah) as well as cow milk were used in this study.Results showed that ash and moisture contents were significantly higher in camel milk casein of all breeds compared to that of cow milk. On the other hand, casein protein of cow milk was significantly higher compared to that of all camel milk breeds. Molecular weights of casein patterns of camel milk breeds were higher compared to that of cow milk.Essential (Phe, Lys and His) and non-essential amino acids content was significantly higher in cow milk casein compared to the casein of all camel milk breeds. However, there was no significant difference for the other essential amino acids between cow casein and the casein of Safrah breed and their quantities in cow and Safrah casein were significantly higher compared to the other two breeds. Non-essential amino acids except Arg and the essential amino acids (Met, Ile, Lue and Phe) were also significantly higher in cow milk α-casein compared to α-casein from all camel breeds. Moreover, essential amino acids (Val, Phe and His) and the non-essential amino acids (Gly and Ser) content was significantly higher in cow milk β-casein compared to the β-casein of all camel milk breeds and the opposite was true for Lys, Thr, Met and Ile. However, Met, Ile, Phe and His were significantly higher for β-casein of Majaheim compared to the other two milk breeds. The non-essential amino acids (Gly, Tyr, Ala and Asp) and the essential amino acids (Thr, Val and Ile) were significantly higher in cow milk κ-casein compared to that for all camel milk breeds. There was no significant difference among all camel milk breeds in their κ-casein content of most essential amino acids.Relative migration of casein bands of camel milk casein was not identical. The relative migration of α_s-, β- and κ-casein of camel casein was slower than those of cow casein. The molecular weights of α_s-, β- and κ-casein of camel caseins were 27.6, 23.8 and 22.4 KDa, respectively. More studies are needed to elucidate the structure of camel milk.