ß-Lactoglobulin variants as potential carriers of pramoxine: Comprehensive structural and biophysical studies.

ß-Lactoglobulin (BLG) is a member of the lipocalin family. As other proteins from this group, BLG can be modified to bind specifically compounds of medical interests. The aim of this study was to evaluate the role of two mutations, L39Y and L58F, in the binding of topical anesthetic pramoxine (PRM) to ß-lactoglobulin. Circular dichroism spectroscopy, isothermal titration calorimetry (ITC), and X-ray crystallography were used to understand the mechanisms of BLG-PRM interactions. Studies were performed for three new BLG mutants: L39Y, L58F, and L39Y/L58F. ITC measurements indicated a significant increase in the affinity to the PRM of variants L58F and L39Y. Measurements taken for the double mutant L39Y/L58F showed the additivity of two mutations leading to about 80-fold increase in the affinity to PRM in comparison to natural protein BLG from bovine milk. The determined crystal structures revealed that pramoxine is accommodated in the ß-barrel interior of BLG mutants and stabilized by hydrophobic interactions. The observed additive effect of two mutations on drug binding opens the possibility for further designing of new BLG variants with high affinity to selected drugs.

The effect of the bgs13 mutation on the structure of the reporter protein beta-lactoglobulin: Influence on folding and aggregation in Pichia pastoris.

Pichia pastoris, a methylotrophic yeast used for recombinant protein expression, has the capability of performing many eukaryotic post-translational modifications, growing to high cell densities, and producing proteins in a cost-effective manner. However, P. pastoris's secretion properties are not always efficient, and its secretory pathway mechanisms have not been thoroughly elucidated. A previously identified mutant strain, bgs13, was found to efficiently secrete most recombinant proteins tested, raising the possibility that this bgs13 mutant is a universal super secreter. In this study, we used a reporter protein, ß-lactoglobulin (b-LG), to perform structural analysis of the protein secreted from wild type and mutant bgs13 strains to investigate the secretory mechanism. Primary, secondary, and tertiary structures of b-LG were examined using Edman sequencing, circular dichroism, tryptophan fluorescence, and temperature induced aggregation analysis. Our results demonstrate that the bgs13 produced more b-LG than the wt strain and that this protein was functionally folded similar to the wt. Surprisingly, we also found that the bgs13 b-LG was more resistant to aggregation, providing another example of the superior qualities of this strain for enhanced secreted protein production.

Site-specific glycosylation and single amino acid substitution dramatically reduced the immunogenicity of ß-lactoglobulin.

To reduce the immunogenicity of ß-lactoglobulin (BLG), we prepared recombinant BLG which has both site-specific glycosylation and single amino acid substitution (D28N/P126A), and expressed it in the methylotrophic yeast Pichia pastoris by fusion of the cDNA to the sequence coding for the α-factor signal peptide from Saccharomyces cerevisiae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the D28N/P126A was conjugated with a ∼4 kDa high-mannose chain. D28N/P126A retained ∼61% of the retinol-binding activity of BLG. Structural analyses by circular dichroism (CD) spectra, intrinsic fluorescence, and Enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies indicated that the surface structure of BLG was slightly changed by using protein engineering techniques, but D28N/P126A was covered by high-mannose chains and substituted amino acid without substantial disruption of native conformation. Antibody responses to the D28N/P126A considerably reduced in C57BL/6 mice. We conclude that inducing both site-specific glycosylation and single amino acid substitution simultaneously is an effective method to reduce the immunogenicity of BLG.

Detection of the ß-lactoglobulin genotype in zebu cattle (Gangatiri) milk using high-resolution accurate mass spectroscopy.

We studied the genetic polymorphism of beta-lactoglobulin (ß-Lg) whey protein in Gangatiri zebu cows for this Research Communication. The polymorphic nature of milk protein fractions and their association with milk production traits, composition and quality has attracted several efforts in evaluating the allelic distribution of protein locus as a potential dairy trait marker. Genetic variants of ß-Lg have highly significant effects on casein number (B > A) and protein recovery (B > A) and also determine the yield of cheese dry matter (B > A). Molecular techniques of polyacrylamide gel electrophoresis and high-resolution accurate mass-spectroscopy were applied to characterize the ß-Lg protein obtained from the Gangatiri breed milk. Sequence analysis of ß-Lg showed the presence of variant B having UniProt database accession number P02754, coded on the PAEP gene. Our study can provide reference and guidance for the selection of superior milk (having ß-LgB) from this indigenous breed that could potentially give a good yield of ß-Lg for industrial applications.

Genetic variants associated with two major bovine milk fatty acids offer opportunities to breed for altered milk fat composition.

BACKGROUND: Although bovine milk is regarded as healthy and nutritious, its high content of saturated fatty acids (FA) may be harmful to cardiovascular health. Palmitic acid (C16:0) is the predominant saturated FA in milk with adverse health effects that could be countered by substituting it with higher levels of unsaturated FA, such as oleic acid (C18:1cis-9). In this work, we performed genome-wide association analyses for milk fatty acids predicted from FTIR spectroscopy data using 1811 Norwegian Red cattle genotyped and imputed to a high-density 777k single nucleotide polymorphism (SNP)-array. In a follow-up analysis, we used imputed whole-genome sequence data to detect genetic variants that are involved in FTIR-predicted levels of C16:0 and C18:1cis-9 and explore the transcript profile and protein level of candidate genes. RESULTS: Genome-wise significant associations were detected for C16:0 on Bos taurus (BTA) autosomes 11, 16 and 27, and for C18:1cis-9 on BTA5, 13 and 19. Closer examination of a significant locus on BTA11 identified the PAEP gene, which encodes the milk protein ß-lactoglobulin, as a particularly attractive positional candidate gene. At this locus, we discovered a tightly linked cluster of genetic variants in coding and regulatory sequences that have opposing effects on the levels of C16:0 and C18:1cis-9. The favourable haplotype, linked to reduced levels of C16:0 and increased levels of C18:1cis-9 was also associated with a marked reduction in PAEP expression and ß-lactoglobulin protein levels. ß-lactoglobulin is the most abundant whey protein in milk and lower levels are associated with important dairy production parameters such as improved cheese yield. CONCLUSIONS: The genetic variants detected in this study may be used in breeding to produce milk with an improved FA health-profile and enhanced cheese-making properties.

Meta-Analysis of Gene Polymorphism of Beta-Lactoglobulin Gene in Indian Dairy Cows.

The aim of the present paper was to summarize the gene polymorphisms of beta-lactoglobulin (BLG) gene and its effects on milk yield in 1840 genotyped Indian dairy cows reported in 17 published studies. The meta-analysis was undertaken using gene frequencies of individual studies under random effects model, whereas for association analysis of genotypes with milk yield, standardized mean differences (SMDs) along with 95% confidence interval (CI) were obtained under four genetic models such as additive (AA vs. BB), dominant (AA+AB vs. BB), completely over dominant (AA+BB vs. AB) and recessive (AA vs. AB+BB). The heterogeneity index (I2) was used to determine heterogeneity between studies. The results of meta-analysis suggested that the pooled allelic frequency of allele A was subsidiary as 0.29 (95% CI 0.24, 0.33, I2 = 88.54%) in targeted population, and also, it was non-significantly (P > 0.05) different between Bos indicus (0.28) and Bos taurus/cross cows (0.30). Egger's test indicated no risk of publication bias (P > 0.05). The results also revealed that BLG gene variants have non-significant (P > 0.05) association with milk yield under all genetic models. Although positive effects of SMDs under some models were observed, however, they failed to meet statistical significance (P > 0.05) due to high heterogeneity between studies which lead to conclusion of only uncertain influences of SNP genotypes with milk yield. It was concluded that BLG markers may not be beneficial for improving milk yield in Indian dairy cows. However, it is suggested that the revalidation of the present results should be done by using more number of studies.

Development of TaqMan PCR assay for detection of A and B variants of the bovine ß-lactoglobulin.

ß-Lactoglobulin (BLG) is one of the prevalent whey protein in cattle. To date, several variants of bovine BLG have been found, but the most common are A and B, which differ from each other by SNPs rs109625649 and rs110066229. Numerous studies showed effects of A and B variants of BLG on milk yield, fat and protein content and cheese-making properties. To date, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), allele-specific polymerase chain reaction (ASPCR), PCR single-strand conformation polymorphism (PCR-SSCP) and high resolution melting (HRM) methods have been proposed for detection of A and B variants of bovine BLG. These methods involve multistep sample processing, which is an essential disadvantage in conducting large-scale cattle genotyping projects. This article describes a development of TaqMan PCR assay for detection of A and B variants (rs109625649) of bovine BLG. In this method a primer pair, initiating amplification of 101-bp fragment of BLG gene, and two allele-specific TaqMan probes are used. Identification of B and A variants of BLG is based on comparison of final fluorescence intensity of FAM and VIC dyes, respectively. The developed one-step method requires less time and is more suitable for large-scale genotyping of cattle compared to the commonly used PCR-RFLP.

Genotyping of κ-casein and ß-lactoglobulin genes in native cattle from Barishal region of Bangladesh.

The study was conducted to determine the genetic variants of κ-casein and ß-lactoglobulin genes in native cattle. DNA was extracted from blood samples (n = 80) collected from Babuganj, Barishal followed by PCR with gene-specific primers. Genotyping was done by RFLP with HindIII, and HaeIII restriction enzymes. Allelic and genotypic frequencies, genetic diversity, heterozygosity and Hardy-Weinberg equilibrium were estimated using the Popgen32 software. A total 80 samples were genotyped and three genotypes, namely AA, AB and BB, were detected for both the genes. In case of κ-casein gene, higher frequency was observed for AA genotype (0.73) followed by AB (0.23) and BB (0.04) genotype. A allele (0.84) was found to dominate over B allele (0.16). For ß-lactoglobulin gene, BB genotype (0.66) was found more frequently than AB (0.18) and AA (0.16) genotypes. Highest frequency was found for B (0.75) followed by A (0.25) allele. The average genetic diversity (He) was 0.38. The result indicated differences between observed (Ho) and expected (He) heterozygosity and it was out of equilibrium genetics, assumed that selection pressure was in population. To the best of our knowledge, this is the first reported study on κ-casein and ß-lactoglobulin gene variants analysis in cattle in Bangladesh.

The ß-casein (CSN2) A2 allelic variant alters milk protein profile and slightly worsens coagulation properties in Holstein cows.

Milk proteins genetic variants have long attracted interest as they are associated with important issues relating to milk composition and technological properties. An important debate has recently opened at an international level on the role of ß-casein (ß-CN) A1 and A2 polymorphisms, toward human health. For this reason, a lot of efforts has been put into the promotion of A2 milk by companies producing and selling A1-free milk, leading the farmers and breeders to switch toward A2 milk production without paying attention on the potential effect of the processability of milk into cheese. The aim of the present work was to evaluate the effects of ß-CN, specifically the A1 and A2 allelic variants, on the detailed milk protein profile and cheese-making traits in individual milk samples of 1,133 Holstein Friesian cows. The protein fractions were measured with reversed-phase (RP)-HPLC (expressed in g/L and % N), and the cheese-making traits, namely milk coagulation properties, cheese yield, and curd nutrient recoveries assessed at the individual level, with a nano-scale cheese-making procedure. The ß-CN (CSN2), κ-CN (CSN3), and ß-lactoglobulin (LGB) genetic variants were first identified through RP-HPLC and then confirmed through genotyping. Estimates of the effects of protein genotypes were obtained using a mixed inheritance model that considered, besides the standard nuisance variables (i.e., days in milk, parity, and herd-date), the milk protein genes located on chromosome 6 (CSN2, CSN3) and on chromosome 11 (LGB), and the polygenic background of the animals. Milk protein genes (CSN2, CSN3, and LGB) explained an important part of the additive genetic variance in the traits evaluated. The ß-CN A1A1 was associated with a significantly lower production of whey proteins, particularly of ß-lactoglobulin (-8.2 and -6.8% for g/L and % N, respectively) and α-lactalbumin (-4.7 and -4.4% for g/L and % N, respectively), and a higher production of ß-CN (6.8 and 6.1% for g/L and % N, respectively) with respect to the A2A2 genotype. Regarding milk cheese-making ability, the A2A2 genotype showed the worst performance compared with the other genotypes, particularly with respect to the BA1, with a higher rennet coagulation time (7.1 and 28.6% compared with A1A1 and BA1, respectively) and a lower curd firmness at 30 min. Changes in milk protein composition through an increase in the frequency of the A2 allele in the production process could lead to a worsening of the coagulation and curd firming traits.

Identification and characterization of yak α-lactalbumin and ß-lactoglobulin.

α-Lactalbumin (α-LA) and ß-lactoglobulin (ß-LG) were isolated from yak milk and identified by mass spectrometry. The variant of α-LA (L8IIC8) in yak milk had 123 amino acids, and the sequence differed from α-LA from bovine milk. The amino acid at site 71 was Asn (N) in domestic yak milk, but Asp (D) in bovine and wild yak milk sequences. Yak ß-LG had 2 variants, ß-LG A (P02754) and ß-LG E (L8J1Z0). Both domestic yak and wild yak milk contained ß-LG E, but it was absent in bovine milk. The amino acid at site 158 of ß-Lg E was Gly (G) in yak but Glu (E) in bovine. The yak α-LA and ß-LG secondary structures were slightly different from those in bovine milk. The denaturation temperatures of yak α-LA and ß-LG were 52.1°C and 80.9°C, respectively. This study provides insights relevant to food functionality, food safety control, and the biological properties of yak milk products.

Structure-based design approach to rational site-directed mutagenesis of ß-lactoglobulin.

Recombinant proteins play an important role in medicine and have diverse applications in industrial biotechnology. Lactoglobulin has shown great potential for use in targeted drug delivery and body fluid detoxification because of its ability to bind a variety of molecules. In order to modify the biophysical properties of ß-lactoglobulin, a series of single-site mutations were designed using a structure-based approach. A 3-dimensional structure alignment of homologous molecules led to the design of nine ß-lactoglobulin variants with mutations introduced in the binding pocket region. Seven stable and correctly folded variants (L39Y, I56F, L58F, V92F, V92Y, F105L, M107L) were thoroughly characterized by fluorescence, circular dichroism, isothermal titration calorimetry, size-exclusion chromatography, and X-ray structural investigations. The effects of the amino acid substitutions were observed as slight rearrangements of the binding pocket geometry, but they also significantly influenced the global properties of the protein. Most of the mutations increased the thermal/chemical stability without altering the dimerization constant or pH-dependent conformational behavior. The crystal structures reveal that the I56F and F105L mutations reduced the depth of the binding pocket, which is advantageous since it can reduce the affinity to endogenous fatty acids. The F105L mutant created a unique binding mode for a fatty acid, supporting the idea that lactoglobulin can be altered to bind unique molecules. Selected variants possessing a unique combination of their individual properties can be used for further, more advanced mutagenesis, and the presented results support further research using ß-lactoglobulin as a therapeutic delivery agent or a blood detoxifying molecule.

Frequencies of milk protein variants and haplotypes estimated from genotypes of more than 1 million bulls and cows of 12 French cattle breeds.

Due to their major effects on milk composition and cheese-making properties and their putative effects on human health, there is a great deal of interest in bovine milk protein variants. The objectives of this study were to estimate frequencies of milk protein variants and haplotypes in 12 cattle breeds as well as their trends over time to assess the effect of selection on milk traits. Milk protein variants and haplotypes were identified from SNP genotype data from more than 1 million animals from 12 dairy, beef, or dual-purpose cattle breeds that had been genotyped for genomic selection. We examined a total of 15 loci in the genes that encode ß-lactoglobulin (ß-LG) and 3 caseins (αS1-CN, ß-CN, and κ-CN); genotypes were directly called from customized SNP chips (50.6%) or imputed (49.4%). Variants A and B of ß-LG were frequent in the 12 breeds. For the caseins, we found 3 variants for αS1-CN (B, C, and D), 6 for ß-CN (A1, A2, A3, B, C, and I), and 5 for κ-CN (A, B, C, D, and E). For αS1-CN, the B variant was the most frequent in all breeds except Jersey. For ß-CN, the A2 variant was the most abundant in all breeds except Tarentaise, although in Normande animals, the I variant (30.9%) was almost as common as A2 (39.7%). The C variant was very rare except in the Tarentaise sample (4.8%). The most frequent variant for κ-CN was A in 5 breeds (including Holstein), and B in the 7 other breeds. The B variant was present at a particularly high frequency in Jersey (82.6%) and Normande (85.5%) animals. The C and E variants of κ-CN appeared to be particularly frequent in the Tarentaise (12.7%) and Holstein (9%) breeds, respectively. We found 20 haplotype combinations of αS1-ß-κ CN that were present at a frequency >0.1% in at least one breed; however, only 6 to 9 haplotypes were found in any given breed, demonstrating a strong degree of linkage disequilibrium. The most frequent haplotypes were B-A1-A, B-A2-A, B-A2-B, B-I-B, C-A2-A, and C-A2-B. Some alleles were predominantly found in only one haplotype, such as the E and C variants of κ-CN and the I variant of ß-CN, which were mainly found in the B-A1-E, B-A1-C, and B-I-B haplotypes, respectively. We observed changes in the frequency of certain variants over time in several breeds, such as an increase in the frequency of variants A of ß-LG, I of ß-CN, and B of κ-CN. With these results, we update and complete frequency data that were first estimated 30 to 50 yr ago, and, for the first time in these breeds, we assess the effect of selection on milk protein variants.

Quantitative and qualitative detailed milk protein profiles of 6 cattle breeds: Sources of variation and contribution of protein genetic variants.

Different fractions of milk nitrogenous compounds (not only caseins) have different effects on the nutritional value of milk, its coagulation and curd firming properties, and its cheese-making efficiency. To assess different sources of variation, especially the cows' breed and genetic variants of the main protein fractions, milk samples were collected from 1,504 cows belonging to 3 dairy breeds (Holstein-Friesian, Brown Swiss, and Jersey) and 3 dual-purpose breeds (Simmental, Rendena, and Alpine Grey) reared in 41 multibreed herds. Beyond crude protein, casein (CN), and urea, 7 protein fractions were analyzed using HPLC, and 5 other N fraction traits were calculated. All 15 traits were measured qualitatively (% of milk N) and quantitatively (g/L of milk). The HPLC technique allowed us to discriminate between the main genetic variants of ß-CN, κ-CN, and ß-lactoglobulin and thus to genotype the cows for the CSN2, CSN3, and BLG genes, respectively. Data were analyzed using 2 mixed models, both including the effects of herd-date, breed, parity, and lactation stage, and only one also including the effects of the genotypes of the milk proteins. Breed of cow explained 2 to 36% of phenotypic variability for all the N fractions, with the exception of the urea and total casein contents of milk and the urea and ß-CN proportions of total milk N. Lactation stage had a considerable influence on the amount (g/L) of almost all the protein fractions in milk, but neither the nonprotein N fractions nor the percentage of milk N protein profile were affected. The inclusion of the CSN2, CSN3, and BLG genotypes in the model explained a large part of the total variability in all the milk protein and nonprotein fractions except urea. It also reduced the variance explained by breed and residual factors. An exception was shown by the proportion of αS1-CN variance explained by breed that moved from 13 to 28%. Similarly, for amount (g/L) of ß-CN, the effect of breed became significant (12%), whereas it was almost null before inclusion of genotypes. In terms of percentage of milk N, the genotypes of CSN3 notably affected all the casein fractions, whereas the BLG genotypes had a much greater influence on most noncasein traits. The genotypes of the CSN2 gene exerted an appreciable effect on αS2-CN and not ß-CN, as expected. Comparing the 2 models, we were also able to discriminate the effect of the breed on a milk N fraction, both quantitatively and qualitatively, in 2 quotas: the first due to the milk protein polymorphisms (major genes) and the second due to other genetic factors (polygene), after correcting for the effect of herd-date of sampling, parity, and lactation stage. The knowledge about the detailed milk protein profile of different cattle breeds provided by this study could be of great benefit for the dairy industry, providing new tools for the enhancement of milk payment systems and breeding program designs.

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.

Design of an Aptamer-Amphiphile for the Detection of ß-Lactoglobulin on a Liquid Crystal Interface.

An aptamer-amphiphile was designed that binds to ß-lactoglobulin (ß-LG), a major allergen from cow's milk. For this work, a 23-nucleotide ssDNA aptamer ß-LG-23, capable of forming antiparallel G-quadruplexes was used, and its specificity and binding affinity of 22 ± 2 nM for ß-LG were evaluated via enzyme-linked apta-sorbent assay (ELASA). The ß-LG-23 aptamer was synthesized as an amphiphile by conjugating it to a C16 double tail via different spacers, and the effect of the spacers on the binding affinity and secondary structure of the aptamer was investigated. From all amphiphiles tested, direct conjugation of the aptamer to the tail gave the lowest binding affinity to ß-LG (37 ± 2 nM), while maintaining the antiparallel G-quadruplex secondary structure of the aptamer. As a proof of concept, the ß-LG-23 aptamer-amphiphile was used to decorate the interface of a liquid crystal (LC) and effectively detected 10 nM or 0.18 ppm of ß-LG with a 20 min equilibration time, thus demonstrating that it has the potential to be used for fast and label-free detection of ß-LG.

Genome-wide association study for αS1- and αS2-casein phosphorylation in Dutch Holstein Friesian.

Phosphorylation of caseins (CN) is a crucial post-translational modification that allows caseins to form colloid particles known as casein micelles. Both αS1- and αS2-CN show varying degrees of phosphorylation (isoforms) in cow milk and were suggested to be more relevant for stabilizing internal micellar structure than ß- and κ-CN. However, little is known about the genetic background of individual αS2-CN phosphorylation isoforms and the phosphorylation degrees of αS1- and αS2-CN (αS1-CN PD and αS2-CN PD), defined as the proportion of isoforms with higher degrees of phosphorylation in total αS1- and αS2-CN, respectively. We aimed to identify genomic regions associated with these traits using 50K single nucleotide polymorphisms for 1,857 Dutch Holstein Friesian cows. A total of 10 quantitative trait loci (QTL) regions were identified for all studied traits on 10 Bos taurus autosomes (BTA1, 2, 6, 9, 11, 14, 15, 18, 24, and 28). Regions associated with multiple traits were found on BTA1, 6, 11, and 14. We showed 2 QTL regions on BTA1, one affecting αS2-CN production and the other harboring the SLC37A1 gene, which encodes a phosphorus antiporter and affects αS1- and αS2-CN PD. The QTL on BTA6 harbors the casein gene cluster and affects individual αS2-CN phosphorylation isoforms. The QTL on BTA11 harbors the PAEP gene that encodes for ß-lactoglobulin and affects relative concentrations of αS2-CN-10P and αS2-CN-11P as well as αS1-CN PD and αS2-CN PD. The QTL on BTA14 harbors the DGAT1 gene and affects relative concentrations of αS2-CN-10P and αS2-CN-11P as well as αS1-CN PD and αS2-CN PD. Our results suggest that effects of identified genomic regions on phosphorylation of αS1- and αS2-CN are related to changes in milk synthesis and phosphorus secretion in milk. The actual roles of SLC37A1, PAEP, and DGAT1 in αS1- and αS2-CN phosphorylation in Dutch Holstein Friesian require further investigation.

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.

Lipofuscin Formation Catalyzed by the Milk Protein ß-Lactoglobulin: Lysine Residues in Cycloretinal Synthesis.

Lipofuscins are toxic autofluorescent byproducts of the visual cycle. The accumulation of lipofuscins such as cycloretinal in the retina is thought to play a role in the progression of age-related macular degeneration (AMD). Intriguingly, the milk protein ß-lactoglobulin (BLG) can promote the cyclodimerization of all-trans-retinal to cycloretinal both in vitro and in vivo. Here, site-directed mutagenesis of BLG and mass spectrometric analysis with substrate analogues demonstrate that lysine residues play a key role in catalysis. It is also shown that catalytic activity necessitates the presence of a physical binding site and cannot be mediated by a peptide chain. These studies provide insight into the mechanism of the cyclodimerization process and provide a model system for biocatalysis and biosynthesis of cycloretinal in vivo. In the long term, these studies may pave the way for drug development and inhibitor design as an early treatment regimen for AMD.

Characterizing a region on BTA11 affecting ß-lactoglobulin content of milk using high-density genotyping and haplotype grouping.

BACKGROUND: Milk ß-lactoglobulin (ß-LG) content is of interest as it is associated with nutritional and manufacturing properties. It is known that milk ß-LG content is strongly affected by genetic factors. In cattle, most of the genetic differences are associated with a chromosomal region on BTA11, which contains the ß-LG gene. The aim of this study was to characterize this region using 777 k SNP data (BovineHDbeadChip) and perform a haplotype-based association study. A statistical approach was developed to build haplotypes that capture the genetic variation associated with this genomic region. RESULTS: The SNP with the most significant effect on ß-lactoglobulin content was one of the 2 causal mutations responsible for the ß-lactoglobulin protein variants A/B. Haplotypes based on 2 to 5 selected lead SNP were clustered in groups with different effects on ß-lactoglobulin content. Four different groups were identified suggesting that ß-lactoglobulin variant A and B can be further refined in A1, A2, B1 and B2. CONCLUSIONS: This study showed that ß-lactoglobulin protein variants A/B do not explain all genetic variation associated with the tail part of BTA11 but this region contains more than one mutation with an effect on ß-lactoglobulin content. These findings can be used for selection of cows with higher cheese yield, which is desirable for the dairy industry.

Hormonal regulation of platypus Beta-lactoglobulin and monotreme lactation protein genes.

Endocrine regulation of milk protein gene expression in marsupials and eutherians is well studied. However, the evolution of this complex regulation that began with monotremes is unknown. Monotremes represent the oldest lineage of extant mammals and the endocrine regulation of lactation in these mammals has not been investigated. Here we characterised the proximal promoter and hormonal regulation of two platypus milk protein genes, Beta-lactoglobulin (BLG), a whey protein and monotreme lactation protein (MLP), a monotreme specific milk protein, using in vitro reporter assays and a bovine mammary epithelial cell line (BME-UV1). Insulin and dexamethasone alone provided partial induction of MLP, while the combination of insulin, dexamethasone and prolactin was required for maximal induction. Partial induction of BLG was achieved by insulin, dexamethasone and prolactin alone, with maximal induction using all three hormones. Platypus MLP and BLG core promoter regions comprised transcription factor binding sites (e.g. STAT5, NF-1 and C/EBPα) that were conserved in marsupial and eutherian lineages that regulate caseins and whey protein gene expression. Our analysis suggests that insulin, dexamethasone and/or prolactin alone can regulate the platypus MLP and BLG gene expression, unlike those of therian lineage. The induction of platypus milk protein genes by lactogenic hormones suggests they originated before the divergence of marsupial and eutherians.