Caseins of various origins and biologically active casein peptides and oligosaccharides: Structural and physiological aspects

Summary The first part of the present review is focused on structural aspects concerning the so far studied casein fractions of various origins: they are compared to the four classical major bovine caseins (_sl-, _s2- - and ). The calcium-sensitive casein fractions are always phosphorylated whereas -caseins are glycosylated. The study of the casein genes showed that the calcium-sensitive caseins diverged from a common ancestral gene and during the evolution, intergenic and intragenic duplications occurred. The considerable conservation of the phosphorylation sites emphasizes the importance of phosphorylated residues for the function of caseins, i.e. the formation of micelles and the binding of Ca²⁺. In -caseins all the prosthetic sugar groups are linked by O-glycosidic linkages: their number varies from 0 to 5 in bovine -casein and up to 10 in human -casein. The structures of the known -casein carbohydate moieties are described. Finally the milk clotting process (interaction -casein/chymosin) is compared to the blood clotting process (interaction fibrinogen/thrombin): a large number of similarities could be noted between both clotting phenomena.The second part of the review is devoted to the study of short casein peptides endowed with various biological activities. Some of them behaved as immunomodulators or casomorphins or angiotensin I converting enzyme inhibitors; others demonstrated an effect on platelet functions. A strategic zone containing immunostimulating and opioid peptides could be located in cow and human -caseins. Furthermore bitter peptides, emulsifying peptides, calcium absorption enhancing peptides, chymosin-inhibiting peptides, have also been described and several further properties have been attributed to the -caseinoglycopeptide; two tetrasaccharides isolated from the latter possess blood group activities.In conclusion caseins, the main milk proteins, should not only be considered as a nutriment but as a possible source of biologically active components.If, in the future, some of the discussed active peptides cannot be characterized in vivo, they can all, nevertheless, be synthesized and used either as food additives or in pharmacology.     

Reexamination of the polymeric distributions of κ-casein isolated from bovine milk

-Casein the stabilizing protein of the colloidal milk protein complex was purified from bovine skim milk by the method of McKenzie and Wake (Biochim. Biophys. Acta. 47, 240, 1961). The preparations were examined by sodium dodecyl sulfate gel electrophoresis in the presence and absence of a reducing agent. In the presence of a reducing agent, the -casein migrates as a single low molecular weight band. However, in the absence of a reducing agent, a characteristic pattern of aggregates of varying molecular weight was observed with components ranging from monomer to octamer in integer steps. Densitometry of the Coomassie blue stained gels showed an almost equal distribution of components in each band; carbohydrate staining showed preferential location of sugar residues in lower molecular weight components. Treatment with chymosin (rennin) caused a downward shift in apparent molecular weight for each band with no change in the relative intensity of the Coomassie blue stained bands. Similar gel patterns were observed in whole caseins and partially purified -caseins, indicating that this size distribution is a natural disulfide-linked reporter for the distribution of -casein in casein colloids (micelles).     

Stimulation of proliferation and immunoglobulin production of human-human hybridoma by various types of caseins and their protease digests

We have studied the effect of such milk proteins as caseins, lactalbumin, and lactoglobulin, on proliferation and immunoglobulin production of human-human hybridoma HB4C5 cells. It was found that -, -, and -caseins stimulated both proliferation and IgM product ion of human-human hybridoma HB4C5 cells, while the activities of -lactalbumin and -lactoglobulin were negligible. To localize the active sites of these caseins, effect of protease treatments on the activities were examined. When caseins were digested with trypsin, casein digests stimulated proliferation of the hybridoma, but not their IgM production. When -casein was digested with chymosin and fractionated top--casein and glycomacropeptide, both fragments stimulated proliferation of the cells, but onlyp--casein fragment stimulated IgM production. These results indicate that -casein has at least two proliferation stimulating sites and an IgM production stimulating site in thep--casein region.     

Expression of a bovine κ-CN cDNA in the mammary gland of transgenic mice utilizing a genomic milk protein gene as an expression cassette

Transgenic mice were produced by microinjection of a DNA construct composed of the bovine -casein (-CN) cDNA under the control of the goat -CN 5 promoter elements and 3 flanking regions into pronuclear-stage embryos. The gene construct targeted the expression of bovine -CN RNA to the mammary gland and secretion of bovine -CN in the milk. In the three lines studied (BC-7, BC-31 and BC-67) the transgene was stably integrated and propagated as a Mendelian locus. Expression of the bovine protein in lactating mice from the three transgenic lines was demonstrated by northern and western blots. In ten different tissues analysed by northern blotting, expression was confined to the mammary gland of lactating transgenic mice from line BC-7, with low-level expression also observed in the salivary gland of lines BC-31 and BC-67. Transgene expression in the mammary gland paralleled normal casein gene expression during lactation and was not observed in virgin females. The level of bovine -CN mRNA expression on day 10 of lactation in hemizygous transgenic females in relation to endogenous mRNA of whey acid protein (WAP) gene expression was 14%, 69% and 127% in lines BC-7, BC-31 and BC-67, respectively. No association between transgene copy number and expression was observed. The bovine -CN concentration in milk on day 10 of lactation ranged from 0.94 to 3.85 mg of protein per ml of milk. The bovine -CN expressed in mouse milk had the same molecular mass and immunoactivity with polyclonal antibodies as did -CN from bovine milk. A high degree of variation in the production of bovine -CN within each of the transgenic lines was observed.     

A study of genetic polymorphisms of milk β-lactoglobulin, αS1-casein, β-casein,and κ-casein in five dairy breeds

Gene frequencies of the milk -lactoglobulin, _S1-casein, -casein, and -casein loci have been estimated from 1663 cows of five dairy breeds. Departure from Hardy-Weinberg equilibrium was found only in the -casein system in Jerseys. However, chance alone could have accounted for this single significant finding. Results of pairwise comparisons among the five breeds of allele frequencies at these milk protein loci indicate that of the 40 possible tests, only six comparisons are not significant at the 5% probability level. It would appear that these breeds are characterizable in terms of the gene frequencies of these milk protein loci. Nonindependent assortment of genotypes among these milk protein loci was also studied. The closely linked casein loci were not independent in almost all the breeds where tests could be carried out. The only exception was between the _S1-casein and -casein loci in Holsteins. -Lactoglobulin was independent of the casein loci in all breeds except Brown Swiss, where it was found to be significantly associated with -casein. Close linkage is proposed as an important factor for maintaining the observed milk protein polymorphisms.This paper represents a portion of a doctoral dissertation submitted by the first author as partial fulfillment of the requirements for the degree of Doctor of Philosophy at the University of Massachusetts at Amherst.     

Calcium-induced associations of the caseins: Thermodynamic linkage of calcium binding to colloidal stability of casein micelles

The caseins occur in milk as colloidal complexes of protein aggregates, calcium, and inorganic phosphate. As determined by electron microscopy, these particles are spherical and have approximately a 650 Å radius (casein micelles). In the absence of calcium, the protein aggregates themselves (submicelles) have been shown to result from mainly hydrophobic interactions. The fractional concentration of stable colloidal casein micelles can be obtained in a calcium caseinate solution by centrifugation at 1500g. Thus, the amount of stable colloid present with varying Ca²⁺ concentrations can be determined and then analyzed by application of equations derived from Wyman's Thermodynamic Linkage Theory. Ca²⁺-induced colloid stability profiles were obtained experimentally for model micelles consisting of only _s1- (a calcium insoluble casein) and the stabilizing protein -casein, eliminating the complications arising from - and minor casein forms. Two distinct genetic variants _s1-A andB were used. Analysis of _s1-A colloid stability profiles yielded a precipitation (salting-out) constantk ₁, as well as colloid stability (salting-in) parameterk ₂. No variations ofk ₁ ork ₂ were found with increasing amounts of -casein. From the variation of the amount of colloidal casein capable of being stabilized vs. amount of added -casein an association constant of 4 L/g could be calculated for the complexation of _s1-A and -casein. For the _s1-B and -casein micelles, an additional Ca²⁺-dependent colloidal destabilization parameter,k ₃, was added to the existingk ₁ andk ₂ parameters in order to fully describe this more complex system. Furthermore, the value ofk ₃ decreased with increasing concentration of -casein. These results were analyzed with respect to the specific deletion which occurs in _s1-caseinA in order to determine the sites responsible for these Ca²⁺-induced quaternary structural effects.     

Environmental influences on bovine kappa-casein: reduction and conversion to fibrillar (amyloid) structures

The caseins of milk form a unique calcium–phosphate transport complex that provides these necessary nutrients to the neonate. The colloidal stability of these particles is primarily the result of -casein. As purified from milk, this protein occurs as spherical particles with a weight average molecular weight of 1.18 million. The protein exhibits a unique disulfide bonding pattern, which (in the absence of reducing agents) ranges from monomer to octamers and above on SDS-PAGE. Severe heat treatment of the -casein (90°C) in the absence of SDS, before electrophoresis, caused an increase in the polymeric distribution: up to 40% randomly aggregated high–molecular weight polymers, presumably promoted by free sulfhydryl groups (J. Protein Chem. 17: 73–84, 1998). To ascertain the role of the sulfhydryl groups, the protein was reduced and carboxymethylated (RCM-). Surprisingly, at only 37°C, the RCM--casein exhibited an increase in weight average molecular weight and tendency to self-association when studied at 3000 rpm by analytical ultracentrifugation. Electron microscopy (EM) of the 37°C RCM sample showed that, in addition to the spherical particles found in the native protein, there was a high proportion of fibrillar structures. The fibrillar structures were up to 600 nm in length. Circular dichroism (CD) spectroscopy was used to investigate the temperature-induced changes in the secondary structure of the native and RCM--caseins. These studies indicate that there was little change in the distribution of secondary structural elements during this transition, with extended strand and turns predominating. On the basis of three-dimensional molecular modeling predictions, there may exist a tyrosine-rich repeated sheet-turnsheet motif in -casein (residues 15–65), which may allow for the stacking of the molecules into fibrillar structures. Previous studies on amyloid proteins have suggested that such motifs promote fibril formation, and near-ultraviolet CD and thioflavin-T binding studies on RCM--casein support this concept. The results are discussed with respect to the role that such fibrils may play in the synthesis and secretion of casein micelles in lactating mammary gland.     

Genetic polymorphism at the κ chain locus in mice: Comparisons of restriction enzyme hybridization fragments of variable and constant region genes

Variable (V) and constant (C) region genes of the mouse kappa light chain have been compared in inbred strains and in geographically isolated or genetically separated populations of mice by Southern blot analysis of endonuclease-restricted germline DNA. In most cases, the C gene is found on a single restriction fragment while the V genes of the V19 and V21 groups are each found on several (6–18) fragments. The restriction fragment (RF) patterns of V19 and V21 groups are both polymorphic when compared among inbred mouse strains. Southern blot patterns of V21 and V19 of inbred strains are also found among some geographically isolated populations of mice, suggesting that inbred strains acquired kappa loci from different subspecies. Some populations of geographical isolates show V21, V19, and C contexts similar to inbred mice while more distantly related species within the genus Mus and laboratory rats show no apparent similarity in context to inbred strains. Variable region genes determining the RF patterns of V19 and V21 appear to be linked to each other and to the C and Lyt-3 loci.     

Structural relatedness ofκ-casein and fibrinogenγ-chain

Summary -Caseins, involved in the milk clotting process, and human fibrinogen-chain, involved in the blood clotting process, show structural similarities. Several long-casein sections, together corresponding to 80% of the whole protein molecule, have their counterparts in the-chain of fibrinogen, in that 31-42% of the amino acid residues occupy identical positions. The section of-casein which contains the chymosin-sensitive bond has a counterpart not only in the but also in the B-chain of fibrinogen. Furthermore, the secondary structures of the-caseins and of the-chain predicted according to the method of Chou and Fasman present several common features.41th communication on caseins.     

Localization of κ-casein on thin sections of casein micelles by the gold method

Summary The ultrastructural location of -casein in bovine casein micelles was investigated by the protein A-gold method. Casein micelles, fixed in glutaraldehyde, were embedded at low temperature to enhance immunocytochemical marking of thin sections. -Casein was found distributed throughout the micelles of all sizes with a higher concentration in the smaller micelles. No peripheral location of -casein was observed, even in the larger micelles. These results do not agree with coat-core structures proposed for casein micelles. However they favor models where -casein is distributed uniformly throughout the micelles.     

Interactions between NFκB and Its Inhibitor iκB: Biophysical Characterization of a NFκB/iκB-α Complex

The N-terminal domain (1–318 amino acids) of mouse NFB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length iB- (MAD3, 1–317 amino acids) molecule was generated by binding the E. coli-derived iB- to the purified NFB and purifying the complex by sequential chromatography. The stoichiometry of NFB to iB in the complex was determined to be 2 to 1 by light scattering and SDS–polyacrylamide gel electrophoresis. The secondary structure of the NFB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFB occurs upon binding of DNA. The FTIR spectrum of the NFB/iB complex indicates that its secondary structure is composed of 17% -helix, 39% -strand, 18% irregular structures, and 26% -turns and loops. By comparing these data to the FTIR data for NFB alone, it is concluded that the iB (MAD3) in the complex contains 35% -helix, 27% -strand, 22% irregular structures, and 16% -turns and loops. Circular dichroism (CD) analysis of a shorter form of iB (pp40) indicates that it contains at least 20% -helix and that the iB subunit accounts for nearly all of the -helix present in the NFB/iB complex, consistent with the FTIR results. The stabilities of NFB, iB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of iB is enhanced upon the formation of the NFB/iB complex.     

An energy-minimized casein submicelle working model

To develop a molecular basis for structure-function relationships of the complex milk protein system, an energy-minimized, three-dimensional model of a casein submicelle was constructed consisting of-casein, four _s1-casein, and four-casein molecules. The models for the individual caseins were from previously reported energy-minimized, three-dimensional structures. Docking of one-casein and four _s1-casein molecules produced a framework structure through the interaction of two hydrophobic antiparallel sheets of-casein with two small hydrophobic antiparallel sheets (residue 163–174) of two preformed _s1-casein dimers. The resulting structure is approximately spherically symmetric, with a loose packing density; its external portion is composed of the hydrophilic domains of the four _s1-caseins, while the central portion contains two hydrophbic cavities on either side of the-casein central structure. Symmetric and asymmetric preformed dimers of-casein formed from the interactions of C-terminal-spiral regions as a hinge point could easily be docked into each of the two central cavities of the- framework. This yielded two plausible energy-minimized, three-dimensional structures for submicellar casein, one with two symmetric-casein dimers and one with two asymmetric dimers. These refined submicellar structures are in good agreement with biochemical, chemical, and solution structural information available for submicellar casein.Reference to a brand or firm name does not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.     

Quercetin suppresses proinflammatory cytokines production through MAP kinases and NF-κB pathway in lipopolysaccharide-stimulated macrophage

Quercetin is a flavonoid molecule ubiquitous in nature and functions as an anti-oxidant and anti-inflammatory agent with little toxicity in vivo and in vitro. Dose- and time-dependent effect of quercetin has been investigated on proinflammatory cytokine expression and NO production, focusing on its effects on the MAP kinases and the NF-B signal transduction pathways in LPS-stimulated RAW 264.7 cells by using RT-PCR and immunoblotting. Quercetin strongly reduced activation of phosphorylated ERK kinase and p38 MAP kinase but not JNK MAP kinase by LPS treatment. In addition, quercetin treatment inhibited NF-B activation through stabilization of the NF-B/IB complex and IB degradation and proinflammatory cytokines and NO/iNOS expression. Quercetin may exert its anti-inflammatory and immunomodulatory properties in the effect molecules such as proinflammatory cytokines and NO/iNOS by suppressing the activation of ERK and p38 MAP kinase, and NF-B/IB signal transduction pathways.     

Phosphoinositide 3-kinase activity leads to silica-induced NF-κB activation through interacting with tyrosine-phosphorylated IκB-α and contributing to tyrosine phosphorylation of p65 NF-κB

The role of the subunits of phosphoinositide (PI) 3-kinase in NF-B activation in silica-stimulated RAW 264.7 cells was investigated. Results indicate that PI3-kinase activity was increased in response to silica. The p85 subunit of PI3-kinase interacted with tyrosine-phosphorylated IB- in silica-stimulated cells. PI3-kinase specific inhibitors, such as wortmannin and LY294003, substantially blocked both silica-induced PI3-kinase and NF-B activation. The inhibition of NF-B activation by PI3-kinase inhibitors was also observed in pervanadate-stimulated but not in LPS-stimulated cells. Furthermore, tyrosine phosphorylation of NF-B p65 was enhanced in cells stimulated with silica, pervanadate or LPS, and wortmannin substantially inhibited the phosphorylation event induced by the first two stimulants but not LPS. Antioxidants, such as superoxide dismutase (SOD), N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), blocked silica-induced PI3-kinase activation, suggesting that reactive oxygen species may be important regulatory molecules in NF-B activation by mediating PI3-kinase activation. Our data suggest that p85 and p110 subunits of PI3-kinase play a role in NF-B activation through interaction with tyrosine-phosphorylated IB- and contributing to tyrosine phosphorylation of p65 NF-B.     

Molecular phylogeny based on the κ-casein and cytochrome b sequences in the mammalian suborder Ruminantia

Nucleotide sequences for the -casein precursor proteins have been determined from the genomic DNAs or hair roots of the Ruminantia. The coding regions, exons 2, 3, and 4, were amplified separately via the three kinds of PCRs and then directly sequenced. The primers were designed from the sequence of bovine -casein gene; they were applicable for the amplification of the -casein genes from the 13 species in the Ruminantia except exon 2 of the lesser mouse deer. These results permitted an easy phylogenetic analysis based on the sequences of an autosomal gene. A phylogenetic tree was constructed from the mature K-casein sequences and compared with the tree of the cytochrome b genes which were sequenced from the same individuals. The Cervidae (sika deer, Cervus nippon) were separated from the branch of the Bovidae on the tree of -casein genes with a relatively high confidence level of the bootstrap analysis, but included in the branch of the Bovidae on the tree of cytochrome b genes. The -casein tree indicated a monophyly of the subfamily Caprinae, although the internal branches were uncertain in the Caprinae. The tree based on the nucleotide sequences of cytochrome b genes clearly showed the relationships of the closely related species in the genus Capricornis consisting of serow (C. smatorensis), Japanese serow (C. crispus), and Formosan serow (C. swinhoei). These results would be explained by the difference of resolving power between the -casein and the cytochrome b sequences. Correspondence to: K. Chikuni     

Effect of gelation temperature and gel-dissolving solution on cell viability and recovery of two Pseudomonas putida strains co-immobilized within calcium alginate or κ-carrageenan gel beads

Summary Since a lethal effect of an increased temperature (42°C) on Pseudomonas putida strains PaW8 and PaW130 was demonstrated, strictly ionotropic gels such as calcium alginate or -carrageenan type X 0909 were used for cell co-immobilization, rather than a thermoionotropic -carrageenan gel. Among the variety of gel-dissolving solutions tested, only a 0.05M Na₂CO₃/0.02M citric acid solution was able to preserve around 100 % of the cell viability. A complete cell recovery was obtained from calcium alginate gel beads, while only 6 % of viable cells was recovered from the ionotropic -carrageenan gel.     

Kappa opioid agonists inhibit transmitter release from guinea pig hippocampal mossy fiber synaptosomes

Opioid agonists specific for the , , and opioid receptor subtypes were tested for their ability to modulate potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity from guinea pig hippocampal mossy fiber synaptosomes. The opioid agonists U-62,066E and (–) ethylketocyclazocine, but not the agonist [D-Ala²,N-MePhe⁴,Gly⁵-ol]-enkephalin (DAGO) nor the agonist [D-Pen^2,5]enkephalin (DPDE), inhibited the potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity. U-62,066E, but not DAGO or DPDE, also inhibited the potassium-evoked rise in mossy fiber synaptosomal cytosolic Ca²⁺ levels, indicating a possible mechanism for agonist inhibition of transmitter release. DAGO and DPDE were found to be without any effect on cytosolic Ca²⁺ levels or transmitter release in this preparation. The U-62,066E inhibition of the potassium-evoked rise in synaptosomal cytosolic Ca²⁺ levels was partially attenuated by the opioid antagonist quadazocine and insensitive to the -opioid specific antagonist ICI 174,864 and the opioid-preferring antagonists naloxone and naltrexone. Quadazocine also reversed U-62,066E inhibition of the potassium-evoked release of L-glutamate, but not dynorphin B-like immunoreactivity. These results suggest that opioid agonists inhibit transmitter release from mossy fiber terminals through both opioid and non- opioid receptor mediated mechanisms.     

Anti-apoptotic action of API2-MALT1 fusion protein involved in t(11;18)(q21;q21) MALT lymphoma

At least three distinct chromosomal translocations, t(11;18)(q21;q21), t(1;14)(p22;q32) and t(14;18)(q32;q21) involving the API2 (also known as c-IAP2)-MALT1 fusion protein, BCL10, and MALT1, respectively, have been implicated in the molecular pathogenesis of mucosa associated lymphoid tissue (MALT) lymphoma. Our findings showed that several variants of the API2-MALT1 fusion protein can occur in patients with t(11;18)(q21;q21), and that API2-MALT1 can potently enfance activation of nuclear factor (NF)-B signaling, which may be relevant to the pathogenesis of MALT lymphomas. We also found that MALT1 is rapidly degraded via the ubiquitin-proteasome pathway, as is the case with API2, but upon the synthesis of fusion, API2-MALT1 becomes stable against this pathway. This stability of API2-MALT1 may thus result in inappropriate nuclear factor (NF)-B activation, thereby contributing to the pathogenesis of MALT lymphoma. Recent biochemical and genetic studies have clearly shown that BCL10 and MALT1 form a physical and functional complex and are both required for NF-B activation by antigen receptor stimulation in T and B lymphocytes. It has also been shown that CARMA1, a newly discovered member of the membrane-associated guanylate kinase (MAGUK) families, is critical for antigen receptor-stimulated NF-B activation. It can be assumed that API2-MALT1 can bypass this normal BCL10/MALT1 cellular signaling pathway linked to NF-B activation, thereby inducing antigen receptor-independent proliferation of lymphocytes. Furthermore, BCL10/MALT1- and API2-MALT1-induced NF-B activation may contribute to anti-apoptotic action probably through NF-B-mediated upregulation of apoptotic inhibitor genes. We recently provided direct evidence that API2-MALT1 indeed exerts anti-apoptotic action, in part, through its direct interaction with apoptotic regulators including Smac. Taken together, these findings prompt us to hypothesize that the anti-apoptotic action of API2-MALT1 may be mediated partly by the direct interaction with apoptotic regulators as well as partly by upregulation of apoptotic inhibitor genes. Further studies can be expected to stimulate research into the development of therapeutic drugs that specifically inhibit the antigen receptor signaling-stimulated NF-B activation pathway: such molecule targeting drugs should be useful for interfering with inappropriate proliferation of lymphocytes associated with inflammatory and neoplastic disorders.     

Endogenous abscisic acid and 2-trans-abscisic acid in alternate bearing ‘Wilking’ mandarin trees

The relationship of abscisic acid (ABA) and 2-trans-abscisic acid (t-ABA) to alternate bearing has been examined in Wilking mandarin (Citrus reticulata Blanco) trees. Leaves, stems and buds of trees loaded with fruit (on trees) had 4.3, 6.0 and 2.2 fold higher ABA levels than the corresponding organs from off trees. Leaves had higher ABA levels than stems and buds in both on and off trees. t-ABA was non-detectable in Wilking leaf, stem and bud tissue. Amounts of t-ABA not exceeding 40% of the ABA content, were found in Shamouti and Valencia orange buds and in Wilking fruit peel.The elevated levels of ABA in on tree organs may reflect a stress imposed by the fruit overload.