Interactions in human casein systems: self-association of fully phosphorylated human beta-casein

Human beta-casein was separated according to the extent of phosphorylation and the fully phosphorylated moiety was characterized. Fully phosphorylated human beta-casein makes up to 13-15% of the beta-casein fraction. It has a partial specific volume, v, of 0.754 +/- 0.008 and an absorbancy, E1(1%)cm,280 nm of 6.4 +/- 0.2. Sedimentation and viscosity data yield a solvation of 2.9 g H2O/g protein and an axial ratio of about 5 for the monomer. This would be consistent with a prolate ellipsoid of 10 nm length and 2 nm width. There is one strong binding site for Ca2+ for each organic phosphate ester in the molecule. The protein will precipitate at room temperature upon the addition of either 10 mM Ca2+ or greater than 1 M NaCl. Increasing the temperature from 4 to 37 degrees C causes an apparent conformational change and an increase in protein aggregation which is further increased by the addition of NaCl at this temperature until a limiting size is reached at about 0.25 M NaCl. This limiting size polymer contains 95-105 monomers and is nearly spherical with a radius of about 15 nm and a solvation of 3 g H2O/g protein. If this polymer were the submicelle of human casein, it could account for the abnormally high solvation of human casein micelles but their small average size would be more difficult to reconcile without additional information concerning K-casein association. The addition of Ca2+ to the system introduces association patterns which are more complex and not easily assessed.     

C-terminal labelling of beta-casein

This paper is the first to report specific labelling of a native protein at its C-terminal end by carboxypeptidase Y-catalyzed transpeptidation between beta-casein and tritiated Phe amide. A tryptic digest of the radiolabelled protein was resolved by reversed-phase HPLC and a single labelled peptide was isolated therefrom. Sequence determination and FAB mass spectrometry showed that the last 2 residues (Val-209, Ile-208) of beta-casein had been deleted and Ile 207 substituted by Phe, deamidation presumably occurring after transpeptidation. Identical results were obtained by transpeptidating the isolated C-terminal tryptic heptapeptide (203-209) of native beta-casein.     

A new strategy for primary structure determination of proteins: application to bovine beta-casein

A new approach has been developed for sequencing proteins. A radioactive label is attached specifically to the C-terminus of the protein. The labelled molecule is subjected to varying proteolysis conditions. From the electrophoretic patterns (SDS-PAGE) of the hydrolysates, appropriate cleavage conditions are selected, giving labelled peptides of different lengths which are purified. The labelled peptides are sequenced in order of increasing size (from 1 to n), peptide (i) being sequenced until the N-terminal sequence of peptide (i-1) is encountered. This approach allows the determination of a complete protein sequence with a minimal number of Edman cycles. The method was successfully applied to bovine beta-casein (209 residues) which was completely resequenced with only 239 Edman cycles.     

Chaperone-like activity of beta-casein

The caseins are major components of milk for most mammals and are secreted as large colloidal aggregates termed micelles. They have less ordered secondary and tertiary structures in comparison with typical globular proteins. In this work, beta-casein, a member of the casein family, has been demonstrated to exhibit chaperone-like activity, being able to suppress the thermal and chemical aggregation of such substrate proteins as insulin, lysozyme, alcohol dehydrogenase, and catalase by forming stable complexes with the denaturing substrate proteins. Meanwhile, beta-casein was found to not only prevent aggregation of the substrate proteins, but also solubilize the protein aggregates already formed. Data also show that beta-casein exhibits a higher chaperone-like activity than alpha-casein, likely due to the difference in the number of proline residues present and/or in the extent of exposed hydrophobic surfaces. The implications for their in vivo functions of the caseins, based on their exhibiting such in vitro chaperone-like activities, are discussed.     

L-cell growth stimulation by a beta-casein peptide: the importance of its phosphorylation site for activity.

L-cell proliferation was markedly enhanced by addition to the medium of a synthetic peptide corresponding to residues 1-18 of human beta-casein. Experiments using several synthetic peptides of decreasing length demonstrated that L-S-S-S-E-E (residues 7-12), a major phosphorylation site in beta-casein, appeared to be important for the activity. The phosphorylated beta-casein peptide showed no activity. Recent findings have demonstrated that a similar sequence, S-E-E-E or S-D-D-E, is commonly present in many oncoproteins derived from nuclear oncogenes such as myc, myb and E1A, and plays an important role in transformation functions. The beta-casein peptide may affect mammalian cell proliferation through a modification of of the oncoprotein functions.     

The association of bovine beta-casein. The importance of the C-terminal region.

Bovine beta-casein exists in the monomer form in solution (pH 6.5, 0.1 M NaCl, 0.5% w/v) at low temperatures, but associates to form polymers at higher temperatures. Gel filtration chromatography at 36 degrees showed that the polymer is large with a hydrodynamic size greater than that of a globular protein with a mol wt of 1.34 times 10(6). Removal of two C-terminal amino acids per molecule decreased the proportion of polymer in the solution, although the chromatographic behavior of the modified beta-casein monomers and polymers was retained. Removal of a 20 amino acid peptide from the C terminus of the beta-casein completely destroyed its ability to form polymers and removed the 8-anilino-1-naphthalenesulfonate binding site. However, deletion of segments of the protein from the N terminus did not decrease the ability of the modified beta-casein to associate, nor did it affect the 8-anilino-1-naphthalenesulfonate binding site greatly. It seems likely that all, or some, of the 20 amino acids at the C terminus are responsible for the associative behavior of beta-casein, possibly by the direct participation of their side chains in hydrophobic bond formation. However, removal of the C-terminal peptides may have disrupted the spatial structure of the native protein so that it could no longer associate normally.     

Studies of the composition of purified Torpedo californica acetylcholine receptor and of its subunits.

Under conditions that limit proteolytic degradation, the detergent-solubilized purified receptor protein from Torpedo californica exists in monomeric and dimeric forms. The purified receptor complex is composed of four different polypeptide subunits of apparent molecular weights 40 000, 50 000, 60 000, and 65 000. The individual polypeptides have been purified and their amino acid compositions have shown them to be relatively hydrophobic. In addition, the carbohydrate composition of the intact receptor complex and of the individual polypeptides has been determined. Amino acid analysis provided evidence for the occurrence of a component with chromatographic properties similar to those of phosphoserine. Treatment of receptor with CH3NH2 in base, a condition which provided quantitative modification of O-phosphoserine residues in beta-casein, completely eliminated the peak corresponding to phosphoserine following mild acid hydrolysis. We conclude that the receptor contains O-phosphoserine residues to the extent of approximately seven residues per molecule and these residues occur in all constituent polypeptides. Other forms of O-substituted serine and threonine were also shown to occur, most likely as glycosylated residues.     

Nuclear magnetic resonance study of the conformation and dynamics of beta-casein at the oil/water interface in emulsions.

A (13)C and (31)P nuclear magnetic resonance (NMR) study has been carried out on beta-casein adsorbed at the interface of a tetradecane/water emulsion. (13)C NMR spectra show signals from the carbonyl, carboxyl, aromatic, and C alpha carbons in beta-casein, well resolved from solvent resonances. Only a small fraction of all carbon atoms in beta-casein contribute to detectable signals; intensity measurements show that the observable spectrum is derived from about 30 to 40 amino acid residues.(31)P NMR spectra show signals from the five phosphoserines on the hydrophilic N-terminal part of the protein. Analysis of T(1) relaxation times of these nuclei, using the model free approach for the spectral density function and the line shape of the alpha-carbon region, indicates that a large part of the protein is in a random coil conformation with restricted motion and a relatively long internal correlation time. The NMR results show that the conformation and dynamics of the N-terminal part of beta-casein are not strongly altered at the oil/water interface, as compared to beta-casein in micelle-like aggregates in aqueous solution.     

Determination of the phosphorylation level and deamidation susceptibility of equine beta-casein

beta-Casein was isolated from Haflinger mare's milk by RP-HPLC, and displayed microheterogeneity by urea-electrophoresis and 2-DE probably due to a variable degree of phosphorylation. To investigate the degree of phosphorylation, the primary structure of equine beta-casein was determined by tryptic hydrolysis and MS of peptides released and by MS of the protein treated by alkaline phosphatase. The molecular mass found for the apo-form of Haflinger mare's beta-casein (25 514 +/- 3 Da) was close to the theoretical mass of the reported sequence (GenBank AAG43954) modified by insertion of a region (residues 27-34) encoded by an exon sometimes out-spliced (25 511.40 Da). Hence, the beta-casein isolated from Haflinger mare's milk corresponded to a variant of 226 amino acid residues. The latter was composed by highly multi-phosphorylated isoforms with three to seven phosphate groups, and pIs, determined by 2-DE, ranging from 4.74 to 5.30. Moreover, the equine beta-casein was able to deamidate spontaneously, at the level of Asn in the potential deamidation motif (135)Asn-Gly(136). Approximately 80% of the protein was deamidated after 96 h of incubation under physiological conditions.     

Phosphoprotein phosphatase activity of human prostate acid phosphatase

Human prostate acid phosphatase (EC 3.1.3.2) has been shown to dephosphorylate different phosphoproteins with the maximum rate at pH 4.0-4.5. The activity with phosvitin is distinctly higher than with beta-casein, casein and most of all than with riboflavin-binding protein. The native phosvitin is homogeneous on isoelectric focusing with pI value of 2.1, whereas phosvitin partially dephosphorylated (in about 15%) by the prostate acid phosphatase shows multiple bands with pI values of 3.5 - 6.8 or higher. The phosphate groups bound to serine residues are removed enzymatically twice as fast as phosphothreonine residues. The apparent Km value for phosvitin was 2.4 X 10(-7) M, and is by three orders of magnitude lower than Km of p-nitrophenyl phosphate (2.9 X 10(-4) M). The competitive inhibitors of prostate acid phosphatase, fluoride and L(+)-tartrate, show the same Ki values for phosvitin and p-nitrophenyl phosphate.     

A tryptic peptide from beta-casein depresses protein synthesis and degradation and enhances ureogenesis in primary cultures of rat hepatocytes

1. A peptide which enhances ureogenesis in primary cultured hepatocytes of rats was isolated from a tryptic digest of bovine beta-casein. 2. The structure of the peptide was Ala-Val-Pro-Tyr-Pro-Gln-Arg which is located from 177th to 183rd residues from N-terminal of beta-casein. 3. The peptide also showed the activity to inhibit protein synthesis and protein degradation. 4. It also inhibited DNA synthesis of hepatocytes induced by insulin and/or epidermal growth factor.     

Characterization of a camel milk protein rich in proline identifies a new beta-casein fragment

A camel milk whey protein has been isolated by reverse-phase high performance liquid chromatography. The protein is, like caseins, rich in proline (25% of the whole protein). The N-terminal amino acid sequence shows that the protein is homologous with a C-terminal region of beta-caseins analyzed from other species. The protein is concluded to be a fragment of beta-casein, derived from a non-tryptic type of cleavage of the parent molecule, and increasing the multiplicity of known casein products.     

An enzyme-labeled protein polymer bearing pendent haptens

A new methodology for the preparation of enzyme-labeled protein polymers bearing pendent haptens was developed through the combination of chemical modification and posttranslational protein modification catalyzed by microbial transglutaminase (MTG). As a model hapten, trinitrobenzene (TNB) was chosen and chemically conjugated with the accessible Lys residues of beta-casein. The resultant trinitrophenylated beta-casein was further modified with formaldehyde to render the residual Lys residues inert toward self-cross-linking by MTG. Escherichia coli alkaline phosphatase (AP), comprising a specific peptide tag carrying a MTG-reactive Lys residue, was then conjugated to the Gln residues in beta-casein-TNB conjugates. The resultant AP-labeled beta-casein-bearing pendent TNB moieties (AP-betaCT) showed comparable specific activity with native AP. It was found that only the AP-betaCT with a sufficient number of pendent TNBs are capable of binding to a surface adsorbed with anti-TNP and anti-TNT antibodies, indicating the presence of polyvalent interactions. The utility of AP-betaCT was demonstrated by competitive immunoassays for trinitrophenol (TNP) and trinitrotoluene (TNT), with detection limits of 0.99 microg/L and 0.18 microg/L, respectively. The present study demonstrates the potential of dual labeling of protein scaffolds by chemical and enzymatic protein manipulation to create a new proteinaceous architecture.     

Isolation, characterization, and amino acid sequence of a ubiquitin-like protein from insect eggs

A protein with a primary structure identical to that of human and bovine ubiquitin has been purified from insect eggs. The isolation, secondary structure, and amino acid sequence of this ubiquitin-like protein are reported. The sequence was determined by automatic Edman degradation of the intact molecule as well as by the manual sequence analysis of the enzymatic cleavage products. The polypeptide has 74 amino acid residues and internal homology regions. Interactions of the protein with peptides results in protective effects against proteolysis. This paper reports for the first time the presence of the ubiquitin molecule in invertebrates.     

Insulin and prolactin synergistically stimulate beta-casein messenger ribonucleic acid translation by cytoplasmic polyadenylation

Previous studies have shown that the synthesis and stability of milk protein mRNAs are regulated by lactogenic hormones. We demonstrate here in cultured mouse mammary epithelial cells (CID 9) that insulin plus prolactin also synergistically increases the rate of milk protein mRNA translation. Insulin alone stimulates synthesis of both milk and nonmilk proteins, whereas prolactin alone has no effect, but insulin plus prolactin selectively stimulate synthesis of milk proteins more than insulin alone. The increase in beta-casein mRNA translation is also reflected in a shift to larger polysomes, indicating an effect on translational initiation. Inhibitors of the phosphatidylinositol 3-kinase, mammalian target of rapamycin, and MAPK pathways block insulin-stimulated total protein and beta-casein synthesis but not the synergistic stimulation. Conversely, cordycepin abolishes synergistic stimulation of protein synthesis without affecting insulin-stimulated translation. The poly(A) tract of beta-casein mRNA progressively increases from approximately 20 to about 200 A residues over 30 min of treatment with insulin plus prolactin. The 3'-untranslated region of beta-casein mRNA containing an unaltered cytoplasmic polyadenylation element is sufficient for the translational enhancement and mRNA-specific polyadenylation, based on transient transfection of cells with a reporter construct. Insulin and prolactin stimulate cytoplasmic polyadenylation element binding protein phosphorylation with no increase of cytoplasmic poly(A) polymerase activity.     

Polymorphism of bovine beta-casein and its potential effect on human health

Proteins in bovine milk are a common source of bioactive peptides. The peptides are released by the digestion of caseins and whey proteins. In vitro the bioactive peptide beta-casomorphin 7 (BCM-7) is yielded by the successive gastrointestinal proteolytic digestion of bovine beta-casein variants A1 and B, but this was not seen in variant A2. In hydrolysed milk with variant A1 of beta-casein, BCM-7 level is 4-fold higher than in A2 milk. Variants A1 and A2 of beta-casein are common among many dairy cattle breeds. A1 is the most frequent in Holstein-Friesian (0.310-0.660), Ayrshire (0.432-0.720) and Red (0.710) cattle. In contrast, a high frequency of A2 is observed in Guernsey (0.880-0.970) and Jersey (0.490-0.721) cattle. BCM-7 may play a role in the aetiology of human diseases. Epidemiological evidence from New Zealand claims that consumption of beta-casein A1 is associated with higher national mortality rates from ischaemic heart disease. It seems that the populations that consume milk containing high levels of beta-casein A2 have a lower incidence of cardiovascular disease and type 1 diabetes. BCM-7 has also been suggested as a possible cause of sudden infant death syndrome. In addition, neurological disorders, such as autism and schizophrenia, seem to be associated with milk consumption and a higher level of BCM-7. Therefore, careful attention should be paid to that protein polymorphism, and deeper research is needed to verify the range and nature of its interactions with the human gastrointestinal tract and whole organism.     

The glutamine residues reactive in transglutaminase-catalyzed cross-linking of involucrin

The protein involucrin, synthesized by human keratinocytes, contains 585 amino acids, largely in the form of 10 amino acid repeats, each containing glutamines in 3 conserved positions. Involucrin is a substrate for the keratinocyte transglutaminase and is labeled by the cosubstrate amine, glycine ethyl ester. Study of tryptic peptides of involucrin shows that a single glutamine (residue 496), located 89 residues from the C-terminal end, is preferentially labeled by the enzyme. Additional glutamine residues become reactive when the molecule is fragmented. The C-terminal end, isolated as a cyanogen bromide fragment of 275 residues, is labeled equally at 2 glutamine residues. The polypeptide containing residues 148 to 280 accepts practically no amine while in intact involucrin but as a free fragment is labeled at multiple glutamine residues. It is concluded that the C-terminal and N-terminal ends of the protein are directive influences in that they suppress the reactivity of a number of glutamine residues in the intact molecule, leaving one glutamine highly preferred by the transglutaminase.     

Identification and characterization of the human Pgp-1 glycoprotein

Two monoclonal antibodies have been raised against human Pgp-1 by the immunization of mice with human fibroblasts. The human molecule, like the previously identified mouse counterpart, is an abundant membrane protein (M_r approximately 95 000) with a broad tissue distribution. Pgp-1 is phosphorylated, and phosphoamino acid analysis demonstrates that this occurs exclusively on serine residues. A major difference between the mouse and the human is that 50–60% of human thymocytes are Pgp-1⁺ compared to 5–10% of mouse thymocytes at an equivalent stage in development. Immunofluorescence studies of cryostat sections showed that the majority of human medullary thymocytes are strongly stained with Pgp-1-specific antibody, whereas the expression of Pgp-1 on cortical thymocytes is much more heterogenous.     

The primary structure of the ovine beta-caseins.

Ovine whole casein contains 2 multiphosphorylated beta-casein components designated as beta 1 and beta 2-caseins. The complete sequence of beta 1-casein and the partial sequence of beta 2-casein have been determined from cyanogen bromide and tryptic digests. The ovine beta 1 and beta 2-caseins have the same polypeptide chain and appear to differ only in that they contain 6 and 5 phosphates respectively. The amino acid composition of ovine beta 1-casein can be written as: Asp4, Asn4, Thr10, ThrP1, Ser9, SerP5, Glu19, Gn21, Pro34, Gly5, Ala4, Val21, Met6, Ile9, Leu22, Tyr3, Phe9, Trp1, Lys12, His5, Arg3. Compared to bovine beta-casein A2, which is made up of 209 residues, ovine beta 1-casein has a deletion of 2 residues (either Pro-179--Tyr-180 or Tyr-180--Pro-181) and 20 largely conservative amino acid substitutions. Although 20% of the substitutions involve proline residues, the proline contents of ovine beta 1 and bovine beta A2-caseins are very similar, around 16%. The average hydrophobicity, calculated according to Bigelow, is 5.51 kJ/residue, which is similar to that calculated for bovine beta-casein A2. The cluster of 4 phosphorylated serine residues and the highly charged nature of the amino terminal region observed for bovine beta-casein are conserved in the ovine beta-caseins. The substitution from Ile-12 (bovine) to Thr-12 (ovine) results in a new phosphorylation site, according to the phosphorylation code proposed for caseins. This site is only partially phosphorylated hence the occurrence of both beta 1 and beta 2-caseins in ovine milk.