beta-Lactoglobulin identified in marsupial milk. The primary structure, binding site and possible function of beta-lactoglobulin from eastern grey kangaroo (Macropus giganteus)

beta-Lactoglobulin has been isolated in the milk of the Eastern Grey Kangaroo (Macropus giganteus). This is the first time this protein has been reported to be in the milk of marsupials. The complete amino-acid sequence has been determined by spinning cup and pulsed liquid phase microsequencing of the protein and peptides after enzymatic or cyanogen bromide cleavages. The 155-residue protein is the shortest beta-lactoglobulin so far sequenced. When the kangaroo protein is included in a comparison of the members of the beta-lactoglobulin family, the percentage of residues common to all members is reduced from 33% to 13%. Despite the large number of accumulated amino-acid exchanges the protein exists as a dimer and shows higher homology to the usually very conservative dimeric, ruminant beta-lactoglobulins than to the monomeric protein from monogastrics. Half-cystine residues that form disulphide bridges are conserved. The Eastern Grey Kangaroo beta-lactoglobulin possesses significant homology in several characteristic segments thought to be important for a functional trait common to the beta-lactoglobulin family and retinol-binding proteins. Structural similarity to the retinol-binding protein is indicated by 22% of identical residues. Homology to the beta-lactoglobulins and retinol-binding proteins, the binding site and possible function based on comparative structural studies are discussed.     

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.     

Isolation and complete primary sequence of a new ovine wild-type beta-lactoglobulin C

A new wild type of beta-lactoglobulin has been identified in the milk of sheep. It has been designated as ovine beta-lactoglobulin C. Its primary structure has been determined by direct protein microsequencing of intact protein and RP-HPLC-derived tryptic peptides. The new beta-lactoglobulin C is a subtype of ovine beta-lactoglobulin A with a single exchange Arg-Gln at position 148. This exchange may influence polymerisation of beta-lactoglobulin since in the crystal structure of orthorhombic bovine beta-lactoglobulin, residues 145-150 constitute a short beta-sheet region involved in dimer formation by pairing of dyad-related strands.     

Dimerization, stability and electrostatic properties of porcine beta-lactoglobulin.

The study of homologous proteins belonging to the same family can provide a rationale for important molecular properties such as oligomer formation, folding mechanism and mode of binding. We report here a physico-chemical characterization of porcine beta-lactoglobulin, purified from pooled milk: size-exclusion chromatography, CD and NMR measurements were used to study the aggregation and stability of this protein. In spite of the high sequence identity and homology of porcine beta-lactoglobulin with the widely studied bovine species, the two proteins exhibit very different behaviours. The porcine protein shows a monomer-dimer equilibrium with a pH dependence opposite to that observed for the bovine species. Unfolding experiments revealed the presence of an intermediate that probably has excess alpha helices, as reported for equine species. Modelling studies were performed on bovine, porcine and equine proteins, and, interestingly, electrostatic surface potential calculations led to results consistent with the different dimer interface found for porcine beta-lactoglobulin in the crystal structure. Interaction studies revealed that porcine beta-lactoglobulin is unable to bind fatty acids at any pH, thus questioning the main functional role proposed for lactoglobulins as fatty acid transporters or solubilizers.     

Bioactive properties of milk proteins in humans: A review

Many studies have demonstrated that milk protein consumption has benefits in terms of promoting human health. This review assesses the intervention studies which have evaluated potential health enhancing effects in humans following the ingestion of milk proteins. The impact of milk protein ingestion has been studied to asses their satiating, hypotensive, antimicrobial, anti-inflammatory, anticancer, antioxidant and insulinotropic properties as well as their impact on morphological modifications (e.g., muscle and fat mass) in humans. Consistent health promoting effects appear to have been observed in certain instances (i.e., muscle protein synthesis, insulinotropic and hypotensive activity). However, controversial outcomes have also been reported (i.e., antimicrobial, anti-inflammatory, anticancer and antioxidant properties). Several factors including interindividual differences, the timing of protein ingestion as well as the potency of the active components may explain these differences. In addition, processing conditions have been reported, in certain instances, to affect milk protein structure and therefore modify their bioactive potential. It is thought that the health promoting properties of milk proteins are linked to the release of bioactive peptides (BAPs) during gastrointestinal digestion. There is a need for further research to develop a more in-depth understanding on the possible mechanisms involved in the observed physiological effects. In addition, more carefully controlled and appropriately powered human intervention studies are required to demonstrate the health enhancing properties of milk proteins in humans.     

Antibodies raised against bovine beta-lactoglobulin react with beta 2-microglobulin. A possible antigenic region at beta-LG positions 124-140.

beta-Lactoglobulin is a major whey protein in bovine milk. Its presence has been demonstrated in all mammals examined except rodents and humans. However a positive cross reaction was observed for human milk proteins with antibodies raised against bovine beta-lactoglobulin. We isolated and characterized a protein fraction of ca. 12 kDa responsible for the positive cross reaction. N-terminal sequencing and amino-acid analysis indicated that this protein is identical with human beta 2-microglobulin. We observed structural homology between beta-lactoglobulin region 124-140 and beta 2-microglobulin 71-86, which suggested that positions 124-140 could be the antigenic region for beta-lactoglobulin. Six highly conserved amino-acid residues are suggested as candidates for the antigenic site.     

Effect of beta-lactoglobulin on the activity of pregastric lipase. A possible role for this protein in ruminant milk.

The interaction of bovine beta-lactoglobulin with palmitic and oleic acids has been studied by a partition equilibrium method. Bovine beta-lactoglobulin displays only one high affinity binding site for fatty acids whose association constants for palmitic and oleic acids are 4.2 x 10(6) and 2.3 x 10(6) M-1, respectively. However, other binding sites with low affinity are also present. The existence of one high affinity binding site is in accordance with the amount of fatty acids naturally bound to beta-lactoglobulin isolated from milk. The effect of beta-lactoglobulin on ruminant pregastric lipases from a pharyngeal extract has been assayed. The activity of pharyngeal lipase on a triglyceride emulsion is increased about 200%, 250% and 190% in the presence of 10 mg/ml, 20 mg/ml and 40 mg/ml of beta-lactoglobulin, respectively, the last concentration representing that found physiologically in colostrum. Albumin, another ligand-binding protein, increases the activity of this enzyme to a lesser extent and high levels tend to inhibit enzyme action. These results indicate that beta-lactoglobulin could participate in the digestion of milk lipids during the neonatal period by enhancing the activity of pregastric lipase through removal of the fatty acids that inhibit this enzyme.     

Insertion of a casein kinase recognition sequence induces phosphorylation of ovine beta-lactoglobulin in transgenic mice

We have shown that the cellular mechanisms of the mammary gland can be used to produce a phosphorylated form of a normally unphosphorylated milk protein. This was achieved by the insertion of a beta-casein DNA sequence coding for a group of mammary gland casein kinase recognition sites into ovine beta-lactoglobulin. Transgenic mice carrying this modified gene were generated and lactating females were shown to produce a novel beta-lactoglobulin in their milk. The infrared spectrum, reactivity to antiphosphoserine antibody and reduction of electrophoretic mobility on treatment with alkaline phosphatase showed that the novel protein recovered from the milk whey (serum) was phosphorylated and molecular mass determination by mass spectrometry was consistent with the phosphorylation of one or two residues. A similar level of phosphorylation was measured by quantitative infrared spectroscopy. Centrifugation of the milk to pellet the casein micelles showed that most of the phosphorylated beta-lactoglobulin was in the whey and hence not incorporated into casein micelles.     

The complete amino-acid sequence of dimeric beta-lactoglobulin from mouflon (Ovis ammon musimon) milk

beta-Lactoglobulin from Mouflon (Ovis ammon musimon) milk has been isolated and its complete primary structure determined. This protein has been isolated in dimeric form and has a molecular mass of 37 kDa. The amino-acid sequence has been determined by microsequencing of the native protein and the peptides were obtained after tryptic cleavage. The tryptic peptides were isolated by reversed phase high-performance liquid chromatography. The primary structure of mouflon beta-lactoglobulin shows close similarity to ruminant beta-lactoglobulins. The presence of His at position 20 indicates that this protein belongs to the B-type of dimeric ovine beta-lactoglobulins. Mouflon beta-lactoglobulin is a 162 amino acid long polypeptide chain with two disulphide bridges and one free thiol group. Structural similarities to the bilin-binding protein, BG protein from olfactory epithelium and retinol-binding protein are discussed.     

The amino acid sequence of goat beta-lactoglobulin

The isolation of beta-lactoglobulin from milk of the goat is described. The purified protein was checked for purity and has been characterized by its gross composition and end groups. The native or the modified protein was then degraded by tryptic and cyanogen bromide cleavage. The cleavage products were isolated and sequenced in the sequenator using a Quadrol and propyne program. These data provide the complete sequence of beta-lactoglobulin of the goat. The results are discussed and compared particularly with bovine beta-lactoglobulin components AB. Some biological aspects are described.     

Changes in structure and hydrophobic surface properties of beta-lactoglobulin determined by partition in aqueous two-phase polymeric systems.

The non-polar surface properties of beta-lactoglobulin and especially its interaction with poly(ethylene glycol)-bound palmitate has been studied as a function of pH, temperature and protein concentration. The maximum interaction between beta-lactoglobulin and polymer-bound palmitate occurs at pH 4.3 and pH 7.8. The change in conformation of beta-lactoglobulin around pH 7.5 seems to involve exposure of apolar amino acids to the solvent which results in an increased affinity for hydrocarbons. This is contrary to the situation at pH 4.8--6.0 where the corresponding change in conformation does not affect the protein-hydrocarbon interaction. The results suggest that partition studies in an aqueous two-phase system is a very useful tool to detect changes in conformation and aggregation and to characterize the corresponding hydrophobic surface properties of a protein.     

Identification of alpha-lactalbumin and beta-lactoglobulin in cynomolgus monkey (Macaca fascicularis) milk.

1. An electrophoretic analysis of whey protein from cynomolgus monkey milk revealed that its constituents are more similar to bovine milk than human milk, i.e. cynomolgus monkey milk whey contains, besides alpha-lactalbumin-like protein (LaP), another predominant component similar to bovine beta-lactoglobulin (LgP), in its electrophoretic behavior on both disc- and SDS-polyacrylamide gel electrophoreses. 2. The amino acid composition of LaP shows close similarity to that of human alpha-lactalbumin, and LaP forms an immunoprecipitin line with anti-human alpha-lactalbumin rabbit antiserum. The homology between LaP and alpha-lactalbumin was further confirmed by an analysis of the N-terminal amino acid sequence. 3. LgP is not immunologically identical to bovine beta-lactoglobulin, but its amino acid composition is similar. The result of the N-terminal amino acid sequence analysis of LgP (up to the 26th residue) strongly suggests homology between this protein and beta-lactoglobulin.     

Whey proteins as a model system for chromatographic separation of proteins

Although chromatographic separation of whey proteins has been considered too expensive, whey may serve as an excellent model mixture to investigate and validate the use of simulation tools in the development and optimization of chromatographic separations and the outcome could easily be utilized since the model system has an intrinsic value. Besides, milk from transgenic animals could be an attractive source of pharmaceuticals which must be separated from the other proteins in the milk. Several whey proteins are of interest especially, alpha-lactalbumin, beta-lactoglobulins, immunoglobulins, lactoperoxidase, and lactoferrin. The scope of the project is to develop a consistent set of chromatographic data for whey proteins including isotherms, transport properties and scale-up studies and to develop the appropriate models for the anion exchangers Q-Sepharose XL, Source 30Q, Ceramic Q-HyperD F, and Merck Fractogel EMD TMAE 650 (S). In this work we have determined and correlated gradient and isocratic retention volumes in the linear range of the isotherm for alpha-lactalbumin, beta-lactoglobulin A and B, and bovine serum albumin at a pH from 6 to 9 at various NaCl concentrations.     

Thermal unfolding and refolding of beta-lactoglobulin. An intrinsic andextrinsic fluorescence study.

The conformational features of beta-lactoglobulin, refolded by cooling from a thermally perturbed state, has been characterized by intrinsic and extrinsic fluorescence measurements on the protein. It is found that even at 85-90 degrees C, beta-lactoglobulin does not completely lose its folded structure. The unfolding and refolding of beta-lactoglobulin as observed through intrinsic tryptophan fluorescence is nearly reversible because the native beta-lactoglobulin and its refolded form, following heating and cooling, show nearly identical tryptophan fluorescence properties. However, the fluorescence properties of an extrinsic probe 1-anilino 8-naphthalene sulfonic acid (ANS) for the native and refolded forms are quite different from each other. Significant increase in fluorescence intensity and blue shifts in emission maxima of ANS bound to refolded beta-lactoglobulin is observed compared to that of the native form. Our results indicate that beta-lactoglobulin, refolded after heating to above 70 degrees C, has deep hydrophobic pockets which can be accessed by ANS. These pockets are either nonexistent or inaccessible to ANS in native beta-lactoglobulin. The opening of the central cavity collapses at pH close to the isoelectric pH of the protein. This indicates that electrostatic repulsion is necessary to keep this access open.     

Covalent structure of the minor monomeric beta-lactoglobulin II component from donkey milk

The complete primary structure of the minor beta-lactoglobulin II component from donkey milk is presented. It has been established by amino-acid sequencing and mass-spectrometry analysis of intact protein and peptides obtained after enzymatic and chemical cleavages. The molecular mass and the pI of the protein are calculated to be 18,261 Da and 4.5 respectively. Despite the close structural similarity of the donkey and horse major beta-lactoglobulin I components, their minor beta-lactoglobulin II components show substantial differences in sequence. Most observed exchanges are clustered at residues 78-106 where only 6 amino-acid residues are conserved. The primary structure of donkey beta-lactoglobulin II reveals some unusual features of minor beta-lactoglobulins II and gives new light to the evolution of beta-lactoglobulins and other lipocalins involved in retinol binding or reproductive functions.     

High pressure-low temperature processing of food proteins

High pressure-low temperature (HP-LT) processing is of interest in the food field in view of: (i) obtaining a "cold" pasteurisation effect, the level of microbial inactivation being higher after pressurisation at low or sub-zero than at ambient temperature; (ii) limiting the negative impact of atmospheric pressure freezing on food structures. The specific effects of freezing by fast pressure release on the formation of ice I crystals have been investigated on oil in water emulsions stabilized by proteins, and protein gels, showing the formation of a high number of small ice nuclei compared to the long needle-shaped crystals obtained by conventional freezing at 0.1 MPa. It was therefore of interest to study the effects of HP-LT processing on unfolding or dissociation/aggregation phenomena in food proteins, in view of minimizing or controlling structural changes and aggregation reactions, and/or of improving protein functional properties. In the present studies, the effects of HP-LT have been investigated on protein models such as (i) beta-lactoglobulin, i.e., a whey protein with a well known 3-D structure, and (ii) casein micelles, i.e., the main milk protein components, the supramolecular structure of which is not fully elucidated. The effects of HP-LT processing was studied up to 300 MPa at low or sub-zero temperatures and after pressure release, or up to 200 MPa by UV spectroscopy under pressure, allowing to follow reversible structural changes. Pressurisation of approximately 2% beta-lactoglobulin solutions up to 300 MPa at low/subzero temperatures minimizes aggregation reactions, as measured after pressure release. In parallel, such low temperature treatments enhanced the size reduction of casein micelles.     

The interrelationship between the inhibitory activity of milk with different types of beta-lactoglobulins and the resistance of cattle to mastitis]

In the course of the investigation of piebald (black-white) cattle it is found that 17,62% animals produce the AA type beta-lactoglobulin, 49,52%--the AB type and 32,86%--the BB type. The higher inhibitory activity of milk with the BB type beta-lactoglobulin was found which retained in dilution 1 : 32. The total flora of teat, cyst and parenchyma milk of animals with the BB type beta-lactoglobulin as well as Enterococcus bacteria were much lower than in milk of cows with the AA type homogenous form. The animals with the first type of milk protein had mastitis more rarely as compared to those with the second type. The animals with heterogenous form of beta-lactoglobulin had intermediate values in most of their characteristics.     

Comparison of carbon footprint and water scarcity footprint of milk protein produced by cellular agriculture and the dairy industry

The International Journal of Life Cycle Assessment - This paper studies the carbon footprint and water scarcity footprint (WSF) of a milk protein, beta-lactoglobulin, produced by cellular...     

Synthesis and evolution of concentration of beta-lactoglobulin and alpha-lactalbumin from cow and sheep colostrum and milk throughout early lactation

Synthesis of beta-lactoglobulin and alpha-lactalbumin by explants of ovine mammary gland and evolution of concentration of these proteins in cow and sheep colostrum and milk throughout early lactation have been studied. The evolution of both proteins was similar in cow and sheep species. The highest concentration was found in the first milking (19 and 2 mg/ml for beta-lactoglobulin and alpha-lactalbumin, respectively). Then, levels of beta-lactoglobulin decreased sharply and those of alpha-lactalbumin slowly during the first days of lactation, reaching stable values during the second week postpartum (4 and 1.5 mg/ml). The concentration ratio beta-lactoglobulin/alpha-lactalbumin was four times greater in colostrum than in mature milk. On the other hand, synthesis of these proteins represented about 25-30% of the synthesized total soluble proteins. The synthesis ratio beta-lactoglobulin/alpha-lactalbumin in explants obtained at 12 and 30 hours postpartum was found to be 3.5 and 1.7. These results indicate that the synthesis and secretion of beta-lactoglobulin are comparatively greater than those of alpha-lactalbumin during colostral period, suggesting that beta-lactoglobulin could have some specific function during this period.