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PubMed2003

Establishment of T cell lines to bovine beta-casein and beta-casein-derived epitopes in patients with type 1 diabetes.

Enhanced cellular immune response to bovine beta-casein has been reported in patients with type 1 diabetes. In this study we aimed to establish beta-casein-specific T cell lines from newly diagnosed type 1 diabetic patients and to characterise these cell lines in terms of phenotype and epitope specificity. Furthermore, since sequence homologies exist between beta-casein and putative beta-cell autoantigens, reactivity to the latter was also investigated. T cell lines were generated from the peripheral blood of nine recent onset type 1 diabetic patients with different HLA-DQ and -DR genotypes, after stimulation with antigen pulsed autologous irradiated antigen presenting cells (APCs) and recombinant human interleukin-2 (rhIL-2). T cell line reactivity was evaluated in response to bovine beta-casein, to 18 overlapping peptides encompassing the whole sequence of beta-casein and to beta-cell antigens, including the human insulinoma cell line, CM, and a peptide from the beta-cell glucose transporter, GLUT-2. T cell lines specific to beta-casein could not be isolated from HLA-matched and -unmatched control subjects. beta-Casein T cell lines reacted to different sequences of the protein, however a higher frequency of T cell reactivity was observed towards the C-terminal portion (peptides B05-14, and B05-17 in 5/9 and 4/9 T cell lines respectively). Furthermore, we found that 1 out of 9 beta-casein-specific T cell lines reacted also to the homologous peptide from GLUT-2, and that 3 out of 4 of tested cell lines reacted also to extracts of the human insulinoma cell line, CM. We conclude that T cell lines specific to bovine beta-casein can be isolated from the peripheral blood of patients with type 1 diabetes; these cell lines react with multiple and different sequences of the protein particularly towards the C-terminal portion. In addition, reactivity of beta-casein T cell lines to human insulinoma extracts and GLUT-2 peptide was detected, suggesting that the potential cross-reactivity with beta-cell antigens deserves further investigation.

Adolescent↗
PubMed1988

Phosphorylation of beta-casein and alpha-lactalbumin by casein kinase from lactating bovine mammary gland.

Two milk proteins, beta-casein and alpha-lactalbumin, were compared as substrates for casein kinase from bovine mammary gland. beta-Casein could be rephosphorylated after removal of its phosphate groups, whereas alpha-lactalbumin was an effective substrate after the protein had been reduced and carboxymethylated. The native proteins could not be phosphorylated. Magnesium2+, Ca2+, and Mn2+ stimulated phosphorylation of the modified proteins. Calcium2+ was the most effective cation for alpha-lactalbumin and Mn2+ for beta-casein. Michaelis constants were 144 microM for alpha-lactalbumin in the presence of Ca2+ and 142 microM for beta-casein in the presence of Mn2+; however, the maximum velocity for alpha-lactalbumin was three times that of beta-casein. After phosphorylation with [gamma-32P] ATP, partial hydrolysis showed that only serine residues were phosphorylated in both proteins. Chymotryptic peptides of phosphorylated alpha-lactalbumin and tryptic peptides of phosphorylated beta-casein were examined by HPLC and selected peptides were analyzed for amino acid content. Comparison of the analyses with sequence data showed that serine at position 47 in alpha-lactalbumin is the major site of phosphate incorporation. Dephosphorylated beta-casein was only partially rephosphorylated. However, the sites identified correspond to the phosphorylated residues in native beta-casein, namely, serine at position 35 and the cluster of four serines between residues 15 and 20.

Animals↗
PubMed1994

Kappa-casein and beta-caseins in human milk micelles: structural studies.

The function of human milk micelles is to deliver nutrients, particularly insoluble minerals such as the necessary calcium phosphate, to the child in a readily ingested suspension that meets the special requirements of the human infant digestive system. The micelle structure which fulfills that function is not known. However, the development of ion-exchange and reverse-phase HPLC methods for the purification and quantitation of the kappa- and beta-caseins, along with tritium labeling of the carbohydrate of the kappa-casein in human milk to aid in its detection, provided the tools for probing micelle structure by examining the composition of micelles fractionated according to size by differential centrifugation. The relative amount of kappa-casein increased as micelle size decreased, and thus as surface area/volume increased. Since kappa-casein also stabilizes the micelles against precipitation by Ca2+ ions, a surface position for most of the kappa-casein is implied. The relative amounts of the various phosphorylation levels of the human beta-caseins remained essentially constant except for the nonphosphorylated (O-P) form, which apparently decreased as micelle size decreased. These data suggest that the beta-caseins are linked in the micelles partially through electrostatic interactions involving the organic phosphoryl groups, lacking in the O-P, so that O-P can dissociate into the whey. That further implies that the complete surface is not protected by the kappa -casein but that the beta-caseins are also accessible to the solution.

Caseins↗
PubMed2000

Sugar-casein interaction in deuterated solutions of bovine and caprine casein as determined by oxygen-17 and carbon-13 nuclear magnetic resonance: a case of preferential interactions.

17O NMR spectroscopy and (13)C NMR spectroscopy have been used to study the mechanism of interaction of sugars with bovine and caprine caseins in D(2)O. The (17)O NMR relaxation results showed in all cases an increase in water of hydration, as a result of added sugar; this was predominantly associated with "trapped" water in the caseins. Analysis of the vir al coefficients, obtained from the (17)O relaxation data, suggested that preferential interactions occur in the sugar-protein solutions. This could be the result of either sugar binding or a solute-solute thermodynamic effect, preferential hydration. The addition of sugars to deuterated solutions of bovine casein and caprine casein high in alpha(s1)-casein had little or no effect on either line width or chemical shifts of the (13)C NMR spectra of these milk proteins. (13)C NMR studies of sucrose, at various concentrations (100-300 mM) in the presence of caprine casein high in alpha(s1)-casein, showed no changes in either chemical shifts or T(1) values. This indicates that the sugar molecules tumble isotropically and therefore neither bind to the protein nor affect viscosity in the protein-sugar studies. All of these data suggest that the preferential exclusion of the sugar from the domain of the caseins results in preferential hydration of the caseins.

Animals↗
PubMed1998

The primary structure of water buffalo alpha(s1)- and beta-casein identification of phosphorylation sites and characterization of a novel beta-casein variant.

The primary structure of water buffalo alpha(s1)-casein and of beta-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 beta-elimination/thiol derivatization. Water buffalo alpha(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 alpha(s1)-casein C variant, the water buffalo alpha(s1)-casein presented ten amino acid substitutions, seven of which involved charged amino acid residues. With respect to bovine betaA2-casein variant, the two water buffalo beta-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 beta-casein variants seem to be homologous to bovine betaA2-casein.

Amino Acid Sequence↗
PubMed1990

Human-milk proteins: analysis of casein and casein subunits by anion-exchange chromatography, gel electrophoresis, and specific staining methods.

Casein in human milk is believed to serve several biological functions in newborns. However, the content and subunit composition of human casein has so far received little attention. We recently developed a method to separate human-milk whey and casein by adjustment of whole human milk to pH 4.3 and addition of calcium followed by ultracentrifugation. In this study we analyzed and evaluated human casein prepared by different methods. We used fast protein liquid chromatography (FPLC) with an anion-exchange column (Mono-Q) and polyacrylamide gradient gel electrophoresis techniques to analyze the casein subunit composition. Total casein in human milk, as determined by the Kjeldahl method, varies during lactation; the casein content is approximately 20% of the total protein content in early lactation and 45% in late lactation. We found differences in both glycosylation and phosphorylation patterns of kappa-caseins and beta-caseins from premature and term milk samples.

Caseins↗
PubMed1976

Purification of the mRNAs for Ewe alphaS-casein and beta-casein by immunoprecipitation of polysomes.

Specific polysomes synthesizing alphas-casein and beta-casein were immunoprecipitated from total polysomes of the lactating ewe mammary gland. The polysome - anti-casein complex was immunoprecipitated by anti-immunoglobulins. 22%, 32% and 10% of polysomes were immunoprecipitated with saturating amounts of anti-alphas-casein, anti-beta-casein and anti-chi-casein respectively. Poly(U)-Sepharose chromatography of the immunoprecipitated RNAs permitted the isolation of the corresponding poly(A)-containing RNA which migrated as one major band in polyacrylamide gel electrophoresis. As judged by the contamination with the messenger activity for one of the other caseins, the purity of the mRNA for alphas-casein as well as for beta-casein was estimated to be between 75% and 80%.

Animals↗
PubMed1989

Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I.

Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, we investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing [gamma-32P]ATP. The same glycoprotein was phosphorylated when [32P]GTP was substituted for [32P]ATP in the protein kinase assay. We also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. Immediately upstream from each of the casein kinase II sites was a potential casein kinase I phosphorylation site. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.

Amino Acids↗
PubMed2004

Hydrolysis of casein accelerates gastrointestinal transit via reduction of opioid receptor agonists released from casein in rats.

BACKGROUND: Protein hydrolysate accelerates gastrointestinal transit (GIT) and feeding advancement in preterm infants compared to native protein. In rat pups, opioid receptor agonists released from casein during digestion such as beta-casomorphins slow down GIT. We hypothesized that hydrolysis of casein reduces the opioid activity released during digestion thereby accelerating GIT compared to native casein. OBJECTIVE: The aim of the present study was to investigate whether casein hydrolysate accelerates GIT compared to native casein and whether pretreatment with naloxone, an opioid receptor blocker, abolishes this difference in rat pups. METHODS: In a randomized controlled trial following a 2 x 2 factorial design, 216 female Wistar rat pups were fed with pellets based on hydrolyzed or native casein. After pretreatment with naloxone or normal saline, carmine red was administered by oro-gastric gavage as a tracer for GIT velocity measurement. Four hours later the animals were sacrificed, their intestine was removed and the length of the colon from the cecocolonic junction to the anus was measured. GIT was recorded as percentage of the total colonic length (percentage of colonic transit) passed by carmine red. Data were given as mean +/- SD. RESULTS: GIT was significantly higher with hydrolyzed casein compared to native casein formula (77.4 +/- 17 and 51.2 +/- 20%), but there was no difference after naloxone pretreatment (77.1 +/- 16 and 76.5 +/- 17%). DISCUSSION: The present data suggest that hydrolysis of casein accelerates GIT via reduction of opioid activity released during digestion. Further studies are required to investigate to which extent these rat pub data apply to preterm infants.

Animals↗
PubMed1991

Exaggerated intestinal histamine release by casein and casein hydrolysate but not whey hydrolysate.

Loops of rabbit distal small intestine received luminal acetic acid (pH 4.0) alone or in combination with bovine casein, casein hydrolysate, or whey hydrolysate. Blood-to-lumen movement of 51Cr-labeled ethylenediaminetetraacetic acid (EDTA) (an index of epithelial permeability) and loop fluid histamine levels were quantified after 45 min. Luminal acetic acid caused a marked increase in 51Cr-EDTA accumulation which was not modified by the addition of bovine casein or hydrolysates by of casein or whey. However, acetic acid-induced histamine release was potentiated by casein and casein hydrolysate (six- and four-fold respectively) but was not altered by whey hydrolysate. Casein hydrolysate-dependent histamine release was evident in naloxone-pretreated rabbits, suggesting that beta-casomorphins were not solely responsible. We conclude that luminal casein or casein hydrolysate, but not whey hydrolysate. can activate intestinal mast cells under conditions of enhanced epithelial permeability. This effect appears to involve components other than beta-casomorphins.

Caseins↗
PubMed1983

The rat casein multigene family. I. Fine structure of the gamma-casein gene.

A region approximately 35 kilobase pairs (kb) in length containing the hormonally regulated rat gamma-casein gene has been characterized by examining overlapping clones of genomic rat DNA obtained from two Charon 4A libraries. The entire gamma-casein structural gene is contained in a single 17-kb phage clone. R-loop and restriction enzyme mapping analyses revealed that the gamma-casein gene is approximately 15 kb long and is, therefore, 17.4 times larger than the mature gamma-casein mRNA. The coding regions of the gamma-casein gene are split into at least nine small segments, interspersed with long intervening sequences. Sequence analysis of the 5' end of the gamma-casein gene revealed the presence of a TATA sequence which may play a role in the initiation of gene transcription. The first exon is 44 nucleotides long and encodes part of the 5' noncoding sequences of the gamma-casein mRNA. The first intron was found to contain a short interspersed repeated DNA sequences which shares a 92% homology with a cloned rat repeated DNA sequence found at the 3' end of several other rat genes. In addition, the gamma-casein gene contains several families of highly repeated sequences interspersed throughout the intervening and flanking regions, including a family of evolutionary conserved repeats. Thus, the gamma-casein gene represents an unusually large and complex split mammalian gene.

Animals↗
PubMed1984

Glycogen synthase kinases. Classification of a rabbit liver casein and glycogen synthase kinase (casein kinase-1) as a distinct enzyme.

A protein kinase, able to phosphorylate casein, phosvitin, and glycogen synthase, was purified approximately 9000-fold from rabbit liver, and appeared analogous to an enzyme studied by Itarte and Huang (Itarte, E., and Huang, K.-P. (1979) J. Biol. Chem. 254, 4052-4057). This enzyme, designated here casein kinase-1, was shown to be a distinct glycogen synthase kinase and in particular to be different from the protein kinase GSK-3 (Hemmings, B.A., Yellowlees, D., Kernohan, J.C., and Cohen, P. (1981) Eur. J. Biochem. 119, 443-451). Casein kinase-1 had native molecular weight of 30,000 as judged by gel filtration. The enzyme phosphorylated beta-casein A or B better than kappa-casein or alpha s1-casein, and modified only serine residues in beta-casein B and phosvitin. The apparent Km for ATP was 11 microM, and GTP was ineffective as a phosphoryl donor. The phosphorylation of glycogen synthase by casein kinase-1 was inhibited by glycogen, half-maximally at 2 mg/ml, and by heparin, half-maximally at 0.5-1.0 microgram/ml, but was unaffected by Ca2+ and/or calmodulin, or by cyclic AMP. Phosphorylation of muscle glycogen synthase proceeded to a stoichiometry of at least 6 phosphates/subunit with reduction in the +/- glucose-6-P activity ratio to less than 0.4. Phosphate was introduced into both a COOH-terminal CNBr fragment (CB-2) as well as a NH2-terminal fragment (CB-1). At a phosphorylation stoichiometry of 6 phosphates/subunit, 84% of the phosphate was associated with CB-2 and 6.5% with CB-1. The remainder of the phosphate was introduced into another CNBr fragment of apparent molecular weight 16,500. Phosphorylation by casein kinase-1 correlated with reduced electrophoretic mobilities, as analyzed on polyacrylamide gels in the presence of sodium dodecyl sulfate, of the intact glycogen synthase subunit, as well as the CNBr fragments CB-1 and CB-2.

Adenosine Triphosphate↗
PubMed1979

Phosphorylation of casein by human erythrocyte membrane-bound protein kinase: competition of casein with endogenous substrates.

The possibility that spectrin and band-3 protein are phosphorylated by the same membrane-bound protein kinase was investigated by adding casein to unsealed erythrocyte ghosts and examing competition of the three proteins for phosphorylation. The extent of spectrin and band-3 protein phosphorylation was reduced by up to approximately 55%. This indicated that casein was competing with these endogenous substrates for phosphorylation and was most probably phosphorylated by the same protein kinase(s). Furthermore, the extent of inhibition of the phosphorylation of the two endogenous substrates was indistinguishable over the range of casein concentrations tested (0.1 to 5 mg/ml). This indicates that spectrin and band-3 protein may be phosphorylated by the same protein kinase. In contrast, casein was found to have no effect on the cAMP-dependent phosphorylation of band 4.5. This result indicates that casein only competes with the endogenous proteins phosphorylated by the cAMP-independent protein kinase(s). The extent of reduction of endogenous substrate phosphorylation in the presence of casein was found to be constant over incubation periods of 1 to 15 min, indicating that this reduction was not due to consumption of ATP. Since the spectrin and band-3 protein phosphorylations were specifically and identically reduced by casein and these reductions were not due to the ATP consumption or to a general alteration of the membrane, we conclude that the two substrates are likely phosphorylated by one kinase which also phosphorylates casein.

Caseins↗