Thermally-Induced Lactosylation of Whey Proteins: Identification and Synthesis of Lactosylated ß-lactoglobulin Epitope.

The high temperatures used in the production of milk may induce modifications in proteins structure. Due to occurrence of the Maillard reaction, lactose binds lysine residues in proteins, affecting the nutritional value. Milk is also an important source of allergenic proteins (i.e., caseins, ß-lactoglobulin and α-lactalbumin). Thus, this modification may also affect the allergenicity of these proteins. Focusing on milk whey proteins, a screening on different Ultra High Temperatures (UHT) and pasteurized milk samples was performed to identify lactosylation sites, in particular in protein known epitopes, and to verify the correlation between lactosylation and the harshness of the treatment. Whey proteins were extracted from milk samples after caseins precipitations at pH 4.6 and, after chymotryptic and tryptic in solution digestion, peptides were analysed by UPLC-MS and LTQ-Orbitrap. Results show the presence of lactosylated lysine residues in several known epitopes. Then, a ß-lactoglobulin epitope was selected and synthesized by solid phase synthesis followed by in solution lactosylation, obtaining high reaction yields and purities. The synthesis of lactosylated allergenic epitopes, described here for the first time, is a useful tool for further studies on the technological impacts on food allergenicity.

Polymorphism at donkey ß-lactoglobulin II locus: identification and characterization of a new genetic variant with a very low expression.

In the last years, donkey milk had evidenced a renewed interest as a potential functional food and a breast milk substitute. In this light, the study of the protein composition assumes an important role. In particular, ß-lactoglobulin (ß-LG), which is considered as one of the main allergenic milk protein, in donkey species consists of two molecular forms, namely ß-LG I and ß-LG II. In the present research, a genetic analysis coupled with a proteomic approach showed the presence of a new allele, here named F, which is apparently associated with a null or a severely reduced expression of ß-LG II protein. The new ß-LG II F genetic variant shows a theoretical average mass (Mav) of 18,310.64 Da, a value practically corresponding with that of the variant D (∆mass < 0.07 Da), but differs from ß-LG II D for two amino acid substitutions: Thr100 (variant F) → Ala100 (variant D) and Thr118 (variant F) → Met118 (variant D). Proteomic investigation of the whey protein fraction of an individual milk sample, homozygous FF at ß-LG II locus, allowed to identify, as very minor component, the new ß-LG II F genetic variant. By MS/MS analysis of enzymatic digests, the sequence of the ß-LG II F was characterized, and the predicted genomic data confirmed.

Genomic architecture of bovine κ-casein and ß-lactoglobulin.

The objective of this study was to characterize the genetic architecture underlying the absolute concentrations of 2 important milk proteins, κ-casein (κ-CN) and ß-lactoglobulin (ß-LG), in a backcross population of (Holstein × Jersey) × Holstein cattle. A genome-wide association analysis was performed using a selective DNA pooling strategy and the Illumina BovineHD BeadChip assay [777,000 (777K) SNP markers; Illumina Inc., San Diego, CA]. After correction for multiple testing, 25 single nucleotide polymorphisms were found to be associated with κ-CN and 36 single nucleotide polymorphisms were associated with ß-LG. A pathway association analysis revealed 15 Gene Ontology (GO) terms associated with the κ-CN trait and 28 GO terms associated with ß-LG. In addition, several GO terms were associated with both milk proteins. Further analysis revealed that κ-CN and ß-LG production is regulated by both kinase and phosphatase activity, including mechanisms regulating the extracellular matrix. These results are in concordance with the complex multihormonal process controlling the expression of milk proteins and interactions between mammary epithelial cells and extracellular matrix components. Although κ-CN and ß-LG milk proteins are expressed by single genes, the results from this study showed that many loci are involved in the regulation of the concentration of these 2 proteins.

Differences in whey protein content between cow's milk collected in late pasture and early indoor feeding season from conventional and organic farms in Poland.

BACKGROUND: The aim of the study was to investigate bioactive whey protein concentrations in cow's milk collected in late pasture (LP) and early indoor feeding (EIF) season from conventional and organic farms in Poland. RESULTS: Results showed that in the LP somatic cell count (SCC) was higher under organic farming conditions. However, percentages of protein and fat were higher under conventional farming conditions. In EIF, milk from conventional dairy farms had a higher percentage of fat and lactose and a lower concentration of protein and SCC in comparison to milk from organic farms. Organic milk in LP had higher concentrations of beneficial whey proteins than conventional milk, including ß-lactoglobulin (ß-Lg, 4.12 vs. 2.68 g L⁻¹), lactoferrin (Lf, 334.99 vs. 188.02 mg L⁻¹), and lysozyme (Lz, 15.68 vs. 12.56 µg L⁻¹). However, conventional milk in EIF had higher concentrations of bovine serum albumin (146.47 vs. 118.65 mg L⁻¹), Lf (49 vs. 185.27 mg L⁻¹), and Lz (16.63 vs. 13.22 µg L⁻¹). CONCLUSIONS: The results show significant differences in the investigated parameters between organic milk and milk from conventional system during EIF and LP. Moreover, extending the pasture season during EIF in organic farms decreases concentration of bioactive compounds of milk.

Lactoferrin, an iron-binding protein in neutrophilic leukocytes.

Lactoferrin, an iron-binding protein previously shown to occur in many external secretions, is identified as one of the major proteins present in human and guinea pig neutrophilic polymorphonuclear leukocytes. The identification of this protein in leukocyte extracts was based upon a comparison of its electrophoretic, antigenic, and iron-combining properties with the corresponding properties of the same protein isolated from human and guinea pig milk. Immunochemical quantitations showed that lactoferrin occurs in human neutrophilic leukocytes at the concentration of 3 microg per 10(6) cells. Tissue cultures from guinea pig bone marrow and spleen actively synthesized the protein, as shown both by net production of lactoferrin and incorporation of labeled amino acids into the protein. Immunohistochemical data indicate that lactoferrin first appears in myeloid cells at the stage of the promyelocyte.

The production of soluble and correctly folded recombinant bovine beta-lactoglobulin variants A and B in Escherichia coli for NMR studies.

The production of soluble and correctly folded eukaryotic proteins in prokaryotic systems has always been hampered by the difference in or lack of cell machinery responsible for folding, post-translation modification and secretion of the proteins involved. In the case of bovine beta-lactoglobulin (BLG), a major cow's milk allergen and a protein widely used for protein folding studies, a eukaryotic yeast expression system has been the preferred choice of many researchers, particularly for the production of isotopically labeled protein required for NMR studies. Although this system yields high amounts of recombinant protein, the BLG produced is usually associated with extracellular polysaccharides, which is problematic for NMR analysis. In our study we show that when co-expressed with the signal-sequence-less disulfide bond isomerase (Delta ssDsbC) in the dual expression vector, pETDUET-1, both BLG A and BLG B can be reproducibly produced in a soluble form. Expression was carried out in Escherichia coli Origami(DE3), a trxB/gor mutant for thioredoxin- and glutathione reductase, which allows for proper formation of disulfide bonds in the cytoplasm. The protein was purified by anion exchange chromatography followed by salting-out at low pH and size exclusion chromatography. Our expression system is able to consistently produce milligram quantities of correctly folded BLG A and B with no additional amino acid residues at the N-terminus, except for a methionine. (15)N-labeled BLG A and B, prepared and purified using this method, produced HSQC spectra typical of native bovine BLG.

Aberrant low expression level of bovine beta-lactoglobulin is associated with a C to A transversion in the BLG promoter region.

Beta-lactoglobulin (beta-LG) is the major whey protein in cow's milk. It is well established that the predominant 2 genetic variants, beta-LG A and B, are differentially expressed. Extensive investigation of the genetic variation in the promoter region of the BLG gene revealed the existence of specific haplotypes associated with the A and B variants, respectively. However, the genetic basis for the differential expression of BLG A and B alleles is still elusive. We have previously reported a quantitative beta-LG B variant, characterized by a very low beta-LG protein expression level. Here, we report that the corresponding BLG allele (BLG B*) shows a correspondingly low mRNA expression level. Comparative DNA sequencing of 7,670 bp of the BLG B* allele and the established BLG B allele revealed a unique difference of a C to A transversion at position 215 bp upstream of the translation initiation site (g.-215C>A). This mutation segregated perfectly with the differential phenotypic expression in a paternal half-sib family and could be confirmed in 2 independent cases. The sequence of the BLG B allele in the region of the mutation is highly conserved among 4 related ruminant species. The site of the mutation corresponds to a putative consensus-binding sequence for the transcription factors c-Rel and Elk-1 as predicted by searching the TRANSFAC database. The beta-LG B* site might be relevant in the natural production of milk of low beta-LG content.

Engineered ß-Lactoglobulin Produced in E. coli: Purification, Biophysical and Structural Characterisation.

Functional recombinant bovine ß-lactoglobulin has been produced by expression in E. coli using an engineered protein gene and purified to homogeneity by applying a new protocol. Mutations L1A/I2S introduced into the protein sequence greatly facilitate in vivo cleavage of the N-terminal methionine, allowing correctly folded and soluble protein suitable for biochemical, biophysical and structural studies to be obtained. The use of gel filtration on Sephadex G75 at the last purification step enables protein without endogenous ligand to be obtained. The physicochemical properties of recombinant ß-lactoglobulin such as CD spectra, ligand binding (n, K a, ΔH, TΔS, ΔG), chemical and thermal stability (ΔG D, C mid) and crystal structure confirmed that the protein obtained is almost identical to the natural one. The substitutions of N-terminal residues did not influence the binding properties of the recombinant protein so that the lactoglobulin produced and purified according to our protocol is a good candidate for further engineering and potential use in pharmacology and medicine.

Regulation of the sheep beta-lactoglobulin gene by lactogenic hormones is mediated by a transcription factor that binds an interferon-gamma activation site-related element.

Polypeptide and steroid hormones regulate the transcription of milk protein genes in the mammary gland. The promoter sequence motifs and factors through which these hormones mediate their effects in vivo are not clearly defined. Milk protein binding factor (MPBF) is a factor that has recognition sites in the promoters of many milk protein genes including three sites in the promoter of the sheep beta-lactoglobulin (BLG) gene. Mutagenesis of these sites reduced expression of the BLG gene in lactating mammary glands of transgenic mice but did not affect the tissue specificity of the transgene. Furthermore, mutation of all three sites abolished the response of the BLG gene to lactogenic hormones in HC11 mammary cells. Together these results indicate that MPBF mediates the effects of lactogenic hormones in the mammary gland but does not play a role in determining mammary specificity. The similarity between the MPBF binding site and the gamma-interferon activating site suggests that MPBF is related to the STAT family of cytokine-induced transcription factors.

Induction of lactogenesis in transgenic virgin pigs: evidence for gene and integration site-specific hormonal regulation.

Five month-old transgenic female pigs from three lines carrying the mouse whey acidic protein (WAP) gene and nontransgenic female littermates were implanted with slow-release estrogen and progesterone pellets. Histological analysis of biopsies taken at the time of implantation and 4 weeks later revealed that mammary alveolar development had occurred upon hormonal stimulation in vivo. beta-Casein and beta-lactoglobulin mRNA was found in all induced animals, and WAP mRNA was detected in two of the three transgenic pigs. Differential hormonal regulation between the transgenes in the three lines and also between endogenous milk protein genes was observed in induced mammary tissue cultured in vitro. In the presence of insulin, hydrocortisone, and PRL, beta-casein and WAP mRNA levels increased in all transgenic pigs. In contrast, beta-lactoglobulin mRNA had reached or exceeded lactational levels in response to the in vivo induction, and no further increase was observed in vitro. This suggests that the regulation of the beta-lactoglobulin gene is distinct from that of beta-casein and WAP. Differences were also observed during pregnancy; whereas beta-lactoglobulin gene expression was induced in early pregnancy, a time when PRL levels are low, WAP mRNA levels increased sharply around parturition. Finally, the observation that hormonal regulation of WAP transgenes greatly differed between the three lines suggests that chromatin surrounding the integration site can modify the response of transcription elements.

Improvement of bovine beta-lactoglobulin production and secretion by Lactococcus lactis.

The stabilizing effects of staphylococcal nuclease (Nuc) and of a synthetic propeptide (LEISSTCDA, hereafter called LEISS) on the production of a model food allergen, bovine beta-lactoglobulin (BLG), in Lactococcus lactis were investigated. The fusion of Nuc to BLG (Nuc-BLG) results in higher production and secretion of the hybrid protein. When LEISS was fused to BLG, the production of the resulting protein LEISS-BLG was only slightly improved compared to the one obtained with Nuc-BLG. However, the secretion of LEISS-BLG was dramatically enhanced (approximately 10- and 4-fold higher than BLG and Nuc-BLG, respectively). Finally, the fusion of LEISS to Nuc-BLG resulting in the protein LEISS-Nuc-BLG led to the highest production of the hybrid protein, estimated at approximately 8 microg/ml (approximately 2-fold higher than Nuc-BLG). In conclusion, the fusions described here led to the improvement of the production and secretion of BLG. These tools will be used to modulate the immune response against BLG via delivery of recombinant lactococci at the mucosal level, in a mouse model of cow's milk allergy.

Expression, purification and immunochemical characterization of recombinant bovine beta-lactoglobulin, a major cow milk allergen.

The immunological characteristics of a recombinant beta-lactoglobulin were studied using monoclonal antibodies, polyclonal antiserum and sera from allergic patients. Recombinant beta-lactoglobulin (rBLG) was expressed in Escherichia coli strain DH5alpha and purified as described previously [Cho et al. (1994) J. Biol. Chem. 269, 11 102-11 107]. The method has been modified by adding an immunoaffinity purification step. A quantity of 5-10mg of purified rBLG per liter of medium culture can be produced. rBLG shared the same molecular weight as the natural BLG (nBLG) and also possessed at least one intrachain disulfide bridge. In HPLC, rBLG appeared as a single peak, and the purity was estimated to be greater than 95%. All the monoclonal antibodies (mAbs) used in this study recognized different epitopes of the BLG and presented compatible binding. No differences could be detected between rBLG and nBLG when tested in a Western blot with rabbit polyclonal antiserum or with three mAbs that bound preferentially the reduced and S-carboxymethylated form of BLG. In a competitive enzyme immunoassay (EIA) using either a rabbit polyclonal antiserum or four mAbs that recognized conformational epitopes, we could not distinguish between rBLG or nBLG. In direct ELISA using nBLG or rBLG as the immobilized allergen, we measured a similar concentration of specific anti-BLG IgE in five sera from allergic patients. The results of this study indicate that we have obtained a rBLG with biochemical and immunological properties very similar to nBLG.

Lactoferrin is a marker for prolactin response in mouse mammary explants.

We recorded a bioassay for prolactin using densitometric analysis on polyacrylamide gels of caseins produced by mammary explants in vitro. A protein which also increased in the presence of increasing amounts of prolactin in the medium has now been identified. Its migration in several electrophoretic systems is the same as that of a whey protein of mouse milk, lactoferrin. It is an iron-binding glycoprotein with a molecular weight by SDS gel electrophoresis equal to mouse, human and bovine lactoferrin. It gives a precipitin reaction in an immunodiffusion system with monospecific antiserum raised in rabbits against mouse lactoferrin.

Truncated beta-lactoglobulin transgenes are expressed in the kidney.

The major milk whey protein of ruminants is beta-lactoglobulin. Ovine beta-lactoglobulin-encoding gene expression is restricted to the sheep mammary gland. This report describes the expression profile of a truncated beta-lactoglobulin transgene which, although not expressed in the mammary gland, is expressed in the kidney in the majority of lines generated. The high frequency of ectopic kidney expression may relate to the ability of the larger beta-lactoglobulin transgenes to be expressed in a position-independent manner in the mammary gland of transgenic mice.

Heterologous expression of bovine and porcine beta-lactoglobulins in Pichia pastoris: towards a comparative functional characterisation.

Bovine and porcine beta-lactoglobulins were cloned and expressed in host cells with the aim of developing the tools necessary for their structural, functional and conformational characterisation by NMR techniques. Both lipocalins were expressed in Pichia pastoris, where the use of a constitutive promoter turned out to allow the highest productivity. The yield of recombinant proteins was further improved through multiple integration of the encoding genes and by increasing aeration of the transformed cultures. Both proteins were obtained in the culture medium at the concentration of 200 microg/ml. Recombinant lipocalins were purified by ion-exchange chromatography from the culture medium. A preliminary NMR characterisation showed that both proteins were correctly folded.

[The interrelationship between the inhibitory activity of milk with different types of beta-lactoglobulins and the resistance of cattle to mastitis]. / Izuchenie vzaimosviazi ingibitornoi aktivnosti moloka s razlichnymi tipami beta-laktoglobulinov i rezistentnosti korov k mastitam

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.