The phosphorylation pattern of human alphas1-casein is markedly different from the ruminant species.

Caseins are highly phosphorylated milk proteins assembled in large colloidal structures termed micelles. In the milk of ruminants, alphas1-casein has been shown to be extensively phosphorylated. In this report we have determined the phosphorylation pattern of human alphas1-casein by a combination of matrix-assisted laser desorption mass spectrometry and amino acid sequence analysis. Three phosphorylation variants were identified. A nonphosphorylated form, a variant phosphorylated at Ser18 and a variant phosphorylated at Ser18 and Ser26. Both phosphorylation sites are located in the amino acid recognition sequence of the mammary gland casein kinase. Notably, no phosphorylations were observed in the conserved region covering residues Ser70-Glu78, which is extensively phosphorylated in the ruminant alphas1-caseins.     

Variation in the lack of polyadenylation of the rat milk protein mRNAs during the lactation cycle

Translationally active milk protein mRNAs were found as nonpolyadenylated mRNAs in the rat mammary gland during pregnancy, lactation and involution. Analyses of whey protein mRNA and casein mRNA with the corresponding cDNAs showed that the lack of polyadenylation of these mRNAs at different time points of the lactation cycle is not consistent with the hypothesis that polyadenylation may be incomplete in the mammary gland when large amounts of mRNA are synthesized. The fraction of whey protein mRNA and casein mRNA that lacked polyadenylation was inversely proportional to the concentration of each sequence in the tissue during pregnancy, lactation and involution. A model is proposed to explain the finding that in each animal the ratio of casein mRNA to whey protein mRNA was similar in polyadenylated RNA and in nonpolyadenylated RNA at all stages of the lactation cycle.     

Essential functions of p21-activated kinase 1 in morphogenesis and differentiation of mammary glands

Although growth factors have been shown to influence mammary gland development, the nature of downstream effectors remains elusive. In this study, we show that the expression of p21-activated kinase (Pak)1, a serine/threonine protein kinase, is activated in mammary glands during pregnancy and lactation. By targeting an ectopic expression of a kinase-dead Pak1 mutant under the control of ovine beta-lactoglobulin promoter, we found that the mammary glands of female mice expressing kinase-dead Pak1 transgene revealed incomplete lobuloalveolar development and impaired functional differentiation. The expression of whey acidic protein and beta-casein and the amount of activated Stat5 in the nuclei of epithelial cells in transgenic mice were drastically reduced. Further analysis of the underlying mechanisms revealed that Pak1 stimulated beta-casein promoter activity in normal mouse mammary epithelial cells and also cooperated with Stat5a. Pak1 directly interacted with and phosphorylated Stat5a at Ser 779, and both COOH-terminal deletion containing Ser 779 of Stat5a and the Ser 779 to Ala mutation completely prevented the ability of Pak1 to stimulate beta-casein promoter. Mammary glands expressing inactive Pak1 exhibited a reduction of Stat5a Ser 779 phosphorylation. These findings suggest that Pak1 is required for alveolar morphogenesis and lactation function, and thus, identify novel functions of Pak1 in the mammary gland development.     

Casein kinase activity in rat mammary gland Golgi vesicles. Phosphorylation of endogenous caseins

A Golgi vesicle preparation isolated from the mammary tissue of rats in mid-lactation has been shown to contain the caseins of rat milk. These proteins were phosphorylated when the Golgi vesicles were incubated in the presence of [gamma-32P]ATP. Although this phosphorylation occurred when the physical integrity of the vesicles was maintained, it was markedly increased when the membrane structure was disrupted by hypoosmotic conditions or by use of detergents. The kinase responsible has been shown to be responsive to the intravesicular concentration of Ca2+ and to the extravesicular concentration of Mg2+. These results have been interpreted in terms of a model suggesting a transmembrane location for the enzyme with binding sites on the cytosolic membrane face for Mg2+ and possibly also for ATP and on the luminal surface for Ca2+ and the caseins. Others have postulated that the assembly of caseins into micelles occurs in Golgi vesicles and requires both prior phosphorylation of the proteins and the presence of Ca2+. In this investigation we demonstrate that treatments which increase the intravesicular casein phosphorylation also alter the Ca2+ balance within the vesicle lumen. These results are discussed in relation to the ATP-dependent accumulation of Ca2+ by the mammary gland Golgi vesicles.     

Posttranslational modifications of bovine osteopontin: identification of twenty-eight phosphorylation and three O-glycosylation sites.

Osteopontin (OPN) is a multiphosphorylated glycoprotein found in bone and other normal and malignant tissues, as well as in the physiological fluids urine and milk. The present study demonstrates that bovine milk osteopontin is phosphorylated at 27 serine residues and 1 threonine residue. Phosphoamino acids were identified by a combination of amino acid analysis, sequence analysis of S-ethylcysteine-derivatized phosphopeptides, and mass spectrometric analysis. Twenty-five phosphoserines and one phosphothreonine were located in Ser/Thr-X-Glu/Ser(P)/Asp motifs, and two phosphoserines were found in the sequence Ser-X-X-Glu/Ser(P). These sequence motifs are identical with the recognition sequences of mammary gland casein kinase and casein kinase II, respectively. Examination of the phosphorylation pattern revealed that the phosphorylations were clustered in groups of approximately three spanned by unphosphorylated regions of 11-32 amino acids. This pattern is probably of importance in the multiple functions of OPN involving interaction with Ca2+ and inorganic calcium salts. Furthermore, three O-glycosylated threonines (Thr 115, Thr 124, and Thr 129) have been identified in a threonine- and proline-rich region of the protein. Three putative N-glycosylation sites (Asn 63, Asn 85, and Asn 193) are present in bovine osteopontin, but sequence and mass spectrometric analysis showed that none of these asparagines were glycosylated in bovine mammary gland osteopontin. Alignment analysis showed that the majority of the phosphorylation sites in bovine osteopontin as well as all three O-glycosylation sites were conserved in other mammalian sequences. This conservation of serines, even in otherwise less well-conserved regions of the protein, indicates that the phosphorylation of osteopontin at specific sites is essential for the function of the protein.     

High pressure-induced changes in bovine milk proteins: a review

High pressure (HP)-induced changes in the proteins of bovine milk have become an area of considerable research interest in recent years; as a result, there is now a detailed understanding of the effects of HP on casein micelles and whey proteins. HP treatment at pressures >400 or >100 MPa denatures the two most abundant whey proteins, alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg), respectively. The majority of denatured beta-lg in HP-treated milk associates with the casein micelles, although some denatured beta-lg remains in the serum phase or is attached to the milk fat globule membrane; HP-denatured alpha-la is also associated with the milk fat globules. Casein micelles are disrupted on treatment at pressures >200 MPa; the rate and extent of micellar disruption increases with pressure and is probably due to the increased solubility of calcium phosphate with increasing pressure. On prolonged treatment at 250-300 MPa, reassociation of micellar fragments occurs through hydrophobic bonding; this process does not occur at a pressure >300 MPa, leading to considerably smaller micelles in such milk. As a result of HP-induced changes, the size, number, hydration, composition and light-scattering properties of casein micelles in HP-treated milk differ considerably from those in untreated milk.     

INHIBITION OF LACTOGENIC ACTIVITIES OF BOVINE MAMMARY GLAND EXPLANTS BY THE WHEY FRACTION OF BOVINE MILK

The regulation of milk constituents, synthesis and secretion in tissue cultures of the bovine mammary gland was altered by a whey fraction of bovine milk. α-Casein gene expression, casein secretion and fatty acid synthesis were inhibited by the whey fraction in a dose-dependent manner. The whey fraction inhibited the enhancement activity of prolactin on α-casein gene expression and fatty acid synthesis, and also inhibited casein secretion to the medium, in explants cultured in a medium with or without prolactin. No effect on the expression of the β-lactoglobulin gene was found.     

The molecular manipulation of milk composition

The introduction of cloned genes into the mouse germ line is now routine. Although more difficult technically, gene transfer has been accomplished in farm animals and offers the potential for genetic improvement. In this regard, we have been investigating the use of transgenic animals as production vehicles for high value proteins in milk. We have shown that DNA sequences derived from the gene encoding sheep beta-lactoglobulin mediate efficient and specific expression in the mammary gland. A fusion gene comprising beta-lactoglobulin sequences and those encoding antihemophilic human factor IX has been constructed. This construct has been introduced into sheep; it is expressed in the mammary gland, and the corresponding protein is secreted into milk.     

Probing the interaction between recombinant human myelin basic protein and caseins using surface plasmon resonance and diffusing wave spectroscopy

An intrinsically unstructured human myelin basic protein (hMBP) was expressed in the milk of transgenic cows (TGmilk) and found exclusively associated with the casein micellar phase. The interaction between the recombinant protein and milk caseins was investigated using surface plasmon resonance (SPR). An anti‐human myelin basic protein antibody was covalently immobilized to the surface of the sensor chip. Subsequently the interaction between the recombinant protein (captured by this antibody) and caseins was studied in comparison to that noted with its human counterpart. Results showed a calcium‐mediated interaction between the recombinant protein and caseins. The order of magnitude of this interaction was in agreement with the number of phosphorylated residues carried by each type of casein (α_s‐ > β‐ > κ‐casein). This selective interaction was not noted between the human protein and milk caseins indicating that the recombinant protein was phosphorylated to a higher extent than the human protein. The obtained results indicated that the co‐expression of the recombinant protein and caseins by the mammary gland along with the recombinant protein's ability to form calcium bridges played a key role in the association of the recombinant human myelin basic protein (rhMBP) with the casein micelles of milk. Despite this association between the recombinant protein and milk caseins, light scattering investigations using diffusing wave spectroscopy (DWS) showed no significant differences between the milks of the transgenic and the non‐transgenic control cows, with respect to both the average micelle size and surface charges. This was attributed to the low expression levels of the recombinant protein in milk. Copyright © 2009 John Wiley & Sons, Ltd.     

Secretion of whey acidic protein and cystatin is down regulated at mid-lactation in the red kangaroo (Macropus rufus)

Milk collected from the red kangaroo (Macropus rufus) between day 100 and 260 of lactation showed major changes in milk composition at around day 200 of lactation, the time at which the pouch young begins to temporarily exit the pouch and eat herbage. The carbohydrate content of milk declined abruptly at this time and although there was only a small increase in total protein content, SDS PAGE analysis of milk revealed asynchrony in the secretory pattern of individual proteins. The levels of alpha-lactalbumin, beta-lactoglobulin, serum albumin and transferrin remain unchanged during lactation. In contrast, the protease inhibitor cystatin, and the putative protease inhibitor whey acidic protein (WAP) first appeared in milk at elevated concentrations after approximately 150 days of lactation and then ceased to be secreted at approximately 200 days. In addition, a major whey protein, late lactation protein, was first detected in milk around the time whey acidic protein and cystatin cease to be secreted and was present at least until day 260 of lactation. The co-ordinated, but asynchronous secretion of putative protease inhibitors in milk may have several roles during lactation including tissue remodelling in the mammary gland and protecting specific proteins in milk required for physiological development of the dependent young.     

Differential protein composition of bovine whey: a comparison of whey from healthy animals and from those with clinical mastitis

During clinical mastitis in dairy cows, the quantity of milk produced decreases and the composition of the milk is altered. As the severity of inflammation associated with the disease increases, the chemical composition of milk approaches that of blood as a consequence of increased permeability of the blood mammary barrier, or de novo intramammary synthesis, as has been suggested for mammary associated serum amyloid A3. A better understanding of these events may provide new approaches for the diagnosis and treatment of mastitis. The objective of this study was to document the changes in the protein composition of milk during clinical mastitis using a proteomic approach, with the objective of identifying new diagnostic markers of mastitis. Whey from dairy cows with clinical mastitis was compared to whey from healthy animals by two-dimensional gel electrophoresis (2-DE) with colloidal Coomassie staining and matrix-assisted desorption/ionization mass spectrometry. Increases in the concentrations of proteins of blood serum origin, including serotransferrin and albumin, were identified in mastitic whey compared to normal whey, while concentrations of the major whey proteins alpha-lactalbumin and beta-lactoglobulin were reduced in mastitic whey. Mass spectrometry subsequently confirmed the location of albumin, alpha-lactalbumin and beta-lactoglobulin on the 2-DE gels at M(r)/pI of 69 294/5.8, 14 200/4.5 and 19 883/4.9 respectively.     

Transforming growth factor beta3 induces cell death during the first stage of mammary gland involution

Involution of the mammary gland following weaning is divided into two distinct phases. Initially, milk stasis results in the induction of local factors that cause apoptosis in the alveolar epithelium. Secondly after a prolonged absence of suckling, the consequent decline in circulating lactogenic hormone concentrations initiates remodeling of the mammary gland to the virgin-like state. We have shown that immediately following weaning TGFbeta3 mRNA and protein is rapidly induced in the mammary epithelium and that this precedes the onset of apoptosis. Unilateral inhibition of suckling and hormonal reconstitution experiments showed that TGFbeta3 induction is regulated by milk stasis and not by the circulating hormonal concentration. Directed expression of TGFbeta3 in the alveolar epithelium of lactating mice using a beta-lactoglobulin promoter mobilized SMAD4 translocation to the nucleus and caused apoptosis of these cells, but not tissue remodeling. Transplantation of neonatal mammary tissue derived from TGFbeta3 null mutant mice into syngenic hosts resulted in a significant inhibition of cell death compared to wild-type mice upon milk stasis. These results provide direct evidence that TGFbeta3 is a local mammary factor induced by milk stasis that causes apoptosis in the mammary gland epithelium during involution.     

A Novel Method for Separation of Caseins from Milk by Phosphates Precipitation

Milk protein of farm animals is difficult to isolate because of the presence of casein micelles, which are hard to separate from whey by using centrifugation or filtration. Insoluble casein micelles also create an obstacle for purification instruments to operate efficiently. The conventional method, to precipitate caseins by lowering pH to 4.6 and then recover the whey fraction for further purification using chromatography techniques, is not applicable to proteins having an isoelectric point similar to caseins. In addition, the acid condition used for casein removal usually leads to significantly poor yields and reduced biological activities. In this study, a novel method of precipitating caseins under neutral or weak acidic conditions is presented. The method employs a phosphate salt and a freeze–thaw procedure to obtain a casein-free whey protein fraction. This fraction contains more than 90% yield with little loss of bioactivity of the target protein, and is readily available for further chromatographic purification. This method was successfully applied to purify recombinant human factor IX and recombinant hirudin from the milk of transgenic pigs in the presented study. It is an efficient pretreatment approach prior to chromatographic purification of milk protein from farm animals and particularly of great value to collect those recombinants secreted from transgenic livestock.     

Characterization of the gene encoding ovine beta-lactoglobulin. Similarity to the genes for retinol binding protein and other secretory proteins

Beta-lactoglobulin is the major whey protein in the milk of ruminants and is expressed in the mammary gland during pregnancy and lactation. Here we describe the isolation and characterization of genomic clones encoding ovine beta-lactoglobulin. Two very similar but non-identical, types of beta-lactoglobulin clone were obtained. DNA sequence analysis of one of these showed that the gene is 4900 bases long and contains seven exons. It codes for a protein of 180 amino acid residues, containing an 18-residue signal peptide, within exons I to VI; exon VII is non-coding. We show that the genes encoding serum retinol binding protein, major urinary protein, alpha-1-acid glycoprotein and apolipoprotein D have a similar organization of exons and introns to beta-lactoglobulin. In particular, a comparison between beta-lactoglobulin and retinol binding protein shows that both genes encode equivalent elements of three-dimensional protein structure within analogous exons. These proteins are all members of a large, diverse family of secretory proteins, many of which function in binding small hydrophobic molecules.     

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.     

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.     

Model process for removal of caseins from milk of transgenic animals

We describe a method for selective removal of caseins from milk. The method was developed as a model for transgenic milk processing. Raw cow milk spiked with nonmilk proteins was chosen as the model to resemble transgenic animal milk containing recombinant proteins. The most important elements of the process are (1) "deconstruction" of casein micelles in milk by destroying their Ca(2+) core using a chelating agent (EDTA), thus freeing any protein that might be entrapped in casein aggregates, and (2) "reconstruction" of micelles by providing them with a new Ca(2+) core, thus precipitating them away from the whey proteins, and the protein of interest. Calcium phosphate particles (CAP) were used to reform the disrupted casein micelles. The crystal clear supernatant fraction generated by this method provided >90% recovery and 6- to 13-fold concentration of the desired protein. Product-rich supernatant contained no detectable casein residues, as silver-stained SDS-PAGE and Western blot analyses demonstrated.