Kappa-casein gene study with molecular markers in female buffaloes (Bubalus bubalis)

Caseins comprise make up about 80% of the total protein content of milk and present polymorphism with change in the amino acid sequence. Within this abundance of proteins, kappa-casein is noteworthy, since it has been associated with differences in milk yield, composition and processing. The objective of this study was to observe the existence of polymorphism in the kappa-casein gene in female buffaloes. For this purpose, blood samples from 115 female buffaloes, collected with vacutainer by needle punctionure of the jugular vein, were used. for genomic DNA extraction was done from blood samples. The PCR-RFLP and SSCP techniques demonstrated that the studied animals were monomorphic for the kappa-casein gene. Only allele B was observed in these animals, which was present in homozygosis. Therefore, it was not possible to quantify the gene action on milk yield and its constituents. The monomorphism observed in the population studied would allow the development of a method to identify mixtures of cow and buffalo milk in mozzarella cheese production, especially because, in cattle, the kappa-casein gene is polymorphic. Copyright by the Brazilian Society of Genetics.

Single Nucleotide Variations in the Buffalo Kappa-Casein Gene (CSN3)

Caseins are the major milk proteins associated with lactation performance, milk composition and cheese yield efficiency, representing around 80% of the total amount of proteins found in milk. Among the caseins, kappa-casein is the protein that stabilizes micelle structure during milk coagulation process and being used during the cheese production. The kappa-casein gene (CSN3) has been previously mapped to buffalo chromosome 7 using a radiation hybrid panel and a comparative map was established using the sequence from bovine chromosome 6. The molecular structure of this gene has also been established in river buffalo, with a total length of 13,191 bp (GenBank: AM900443.1) and containing five exons. In this study we searched for single nucleotide variations in specific regions of the CSN3 gene in three animals representing the Murrah breed. Sequencing reactions were performed using ABI3730xl sequencer. The primer walking method was used to span the 5'-UTR and intron 2 regions of the gene, for which ten primer pairs were designed using Oligo 6 software. BLAST tool was used to verify the primers specificity. DNA sequences assemblies from all three animals were performed with Sequencher (R) software 4.1, while multiple alignments were performed using Clustal W software to identify single nucleotide variations. The sequencing revealed a total of 19 single nucleotide variations with 13 located in the upstream regulatory region of the gene (5'-UTR) and six on intron 2. These variations can be validated using commercial populations segregating specific economic traits.

Analysis of O-glycosylation site occupancy in bovine kappa-casein glycoforms separated by two-dimensional gel electrophoresis

The ability of two-dimensional gel electrophoresis (2-DE) to separate glycoproteins was exploited to separate distinct glycoforms of kappa-casein that differed only in the number of O-glycans that were attached. To determine where the glycans were attached, the individual glycoforms were digested in-gel with pepsin and the released glycopeptides were identified from characteristic sugar ions in the tandem mass spectrometry (MS) spectra. The O-glycosylation sites were identified by tandem MS after replacement of the glycans with ammonia/aminoethanethiol. The results showed that glycans were not randomly distributed among the five potential glycosylation sites in kappa-casein. Rather, glycosylation of the monoglycoform could only be detected at a single site, T-152. Similarly the diglycoform appeared to be modified exclusively at T-152 and T-163, while the triglycoform was modified at T-152, T-163 and T-154. While low levels of glycosylation at other sites cannot be excluded the hierarchy of site occupation between glycoforms was clearly evident and argues for an ordered addition of glycans to the protein. Since all five potential O-glycosylation sites can be glycosylated in vivo, it would appear that certain sites remain latent until other sites are occupied. The determination of glycosylation site occupancy in individual glycoforms separated by 2-DE revealed a distinct pattern of in vivo glycosylation that has not been recognized previously.

Chemical synthesis and structure elucidation of bovine kappa-casein (1-44)

The caseins (alpha(s1), alpha(s2), beta, and kappa) are phosphoproteins present in bovine milk that have been studied for over a century and whose structures remain obscure. Here we describe the chemical synthesis and structure elucidation of the N-terminal segment (1-44) of bovine K-casein, the protein which maintains the micellar structure of the caseins. K-Casein (1-44) was synthesised by highly optimised Boc solid-phase peptide chemistry and characterised by mass spectrometry. Structure elucidation was carried out by circular dichroism and nuclear magnetic resonance spectroscopy. CD analysis demonstrated that the segment was ill defined in aqueous medium but in 30% trifluoroethanol it exhibited considerable helical structure. Further, NMR analysis showed the presence of a helical segment containing 26 residues which extends from Pro(8) to Arg(34). This is the first report which demonstrates extensive secondary structure within the casein class of proteins. (c) 2006 Elsevier Inc. All rights reserved.

Resolution and characterisation of multiple isoforms of bovine kappa-casein by 2-DE following a reversible cysteine-tagging enrichment strategy

Visualisation of multiple isoforms of kappa-casein on 2-D gels is restricted by the abundant alpha- and beta-caseins that not only limit gel loading but also migrate to similar regions as the more acidic kappa-casein isoforms. To overcome this problem, we took advantage of the absence of cysteine residues in alpha(S1)- and beta-casein by devising an affinity enrichment procedure based on reversible biotinylation of cysteine residues. Affinity capture of cysteine-containing proteins on avidin allowed the removal of the vast majority of alpha(S1)- and beta-casein, and on subsequent 2-D gel analysis 16 gel spots were identified as kappa-casein by PMF. Further analysis of the C-terminal tryptic peptide along with structural predictions based on mobility on the 2-D gel allowed us to assign identities to each spot in terms of genetic variant (A or B), phosphorylation status (1, 2 or 3) and glycosylation status (from 0 to 6). Eight isoforms of the A and B variants with the same PTMs were observed. When the casein fraction of milk from a single cow, homozygous for the B variant of kappa-casein, was used as the starting material, 17 isoforms from 13 gel spots were characterised. Analysis of isoforms of low abundance proved challenging due to the low amount of material that could be extracted from the gels as well as the lability of the PTMs during MS analysis. However, we were able to identify a previously unrecognised site, T-166, that could be phosphorylated or glycosylated. Despite many decades of analysis of milk proteins, the reasons for this high level of heterogeneity are still not clear.

Polimorfismos genéticos da kappa-caseína e da beta-lactoglobulina e produção de leite em bovinos

As variantes gênicas da beta-lactoglobulina (β-LG) e da kappa-caseína (κ-CN) bovinas são associadas à produção, qualidade e características de processamento do leite. O objetivo deste trabalho foi analisar as frequências dos genótipos AA, AB e BB, por meio da técnica de PCR-RFLP, da β-LG e da κ-CN bovinas, e suas associações com a produção de leite (kg leite/dia) em bovinos das raças Girolanda, Holandesa e Jersey. Para a κ-CN, a frequência do genótipo AA foi maior nos animais das raças Holandesa (37%) e Girolanda (63%). Na raça Jersey, houve predomínio do genótipo BB (60%). Para a β-LG, o genótipo AB foi o mais encontrado nas raças Girolanda (54%) e Holandesa (58%), enquanto nos animais da raça Jersey houve predomínio do genótipo BB (45%). Houve associação do alelo B da κ-CN com maior produtividade leiteira nas raças Girolanda e Holandesa, e do alelo A da β-LG com maior produtividade de leite na raça Jersey. As variantes genéticas da κ-CN podem ser usadas como marcadores na seleção para a produtividade leiteira nas raças Girolanda e Holandesa. Para a raça Jersey, as variantes da β-LG seriam mais adequadas para essa seleção.

Proteomic analysis of k-casein micro-heterogeneity

The proteome of bovine milk is dominated by just six gene products that constitute approximately 95% of milk protein. Nonetheless, over 150 protein spots can be readily detected following two-dimensional electrophoresis of whole milk. Many of these represent isoforms of the major gene products produced through extensive posttranslational modification. Peptide mass fingerprinting of in-gel tryptic digests (using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) in reflectron mode with alpha-cyano-4-hydroxycinnamic acid as the matrix) identified 10 forms of K-casein with isoelectric point (pl) values from 4.47 to 5.81, but could not distinguish between them. MALDI-TOF MS in linear mode, using sinapinic acid as the matrix, revealed a large tryptic peptide (mass > 5990 Da) derived from the C-terminus that contained all the known sites of genetic variance, phosphorylation and glycosylation. Two genetic variants present as singly or doubly phosphorylated forms could be distinguished using mass data alone. Glycoforms containing a single acidic tetrasaccharide were also identified. The differences in electrophoretic mobility of these isoforms were consistent with the addition of the acidic groups. While more extensively glycosylated forms were also observed, substantial loss of N-acetylneuraminic acid from the glycosyl group was evident in the MALDI spectra such that ions corresponding to the intact glycopeptide were not observed and assignment of the glycoforms was not possible. However, by analysing the pl shifts observed on the two-dimensional gels in conjunction with the MS data, the number of N-acetylneuraminic acid residues, and hence the glycoforms present, could be determined.

Étude du mécanisme de protection des spermatozoïdes de mammifères par le lait

Le lait écrémé est utilisé depuis plus d’un demi-siècle comme diluant protecteur des spermatozoïdes de mammifères. Depuis quelques années, il existe une demande grandissante pour des diluants exempts de produits d’origine animale. Toutefois, le mécanisme par lequel le lait protège les spermatozoïdes n’est pas connu, ce qui rend difficile de lui trouver un substitut. Les protéines majeures du plasma séminal de taureau, les protéines « Binder of SPerm » (BSP), sont néfastes lors de la conservation de la semence. Les spermatozoïdes sont en contact avec une grande concentration de protéines BSP qui stimulent une extraction continuelle de cholestérol/phospholipides de leur membrane plasmique. Les lipoprotéines de faible densité (LDL) du jaune d’oeuf, un autre composé utilisé dans les diluants, empêcheraient les protéines BSP de se lier à la membrane des spermatozoïdes de taureaux et de stimuler un efflux des lipides membranaires, ce qui les protégerait durant la conservation. Notre hypothèse était que les protéines du lait protègent les spermatozoïdes durant la conservation en séquestrant les protéines BSP. Premièrement, nous avons démontré par filtration sur gel qu’il y a une interaction entre les protéines BSP bovines et les protéines du lait. Le lait écrémé a été fractionné en trois fractions : F1 (alpha-lactalbumine, bêta-lactoglobuline et caséine kappa), F2 (toutes les protéines du lait) et F3 (sels, sucres et petits peptides). Les protéines BSP1 et BSP5 ont une affinité plus grande pour F1 que BSP3, tandis que toutes les protéines BSP ont une affinité pour F2. Le titrage calorimétrique isotherme a permis de confirmer l’interaction entre les protéines BSP et les protéines du lait. L’association entre la protéine BSP1 bovine et les micelles de caséines est caractérisée par une constante d’affinité (Ka) de 3.5 × 10^5 M-1 et un paramètre stoichiométrique (n) de 4,5 BSP1 pour une caséine. L’association entre la protéine BSP1 bovine et l’alpha-lactalbumine (une protéine du sérum principale), est caractérisée par un Ka de 2.4 × 10^5 M-1 et une valeur “n” de 0,8. Ces résultats indiquent que le lait protège les spermatozoïdes bovins en séquestrant les protéines BSP grâce à une interaction protéine : protéine, tandis que le jaune d’oeuf les protège grâce à une interaction protéine : lipoprotéine. Deuxièmement, nous avons démontré par filtration sur gel que les protéines homologues aux BSP bovines retrouvées dans le plasma séminal de porc, d’étalon et de bélier ont une affinité avec les protéines du lait, ce qui suggère que le mécanisme de protection des spermatozoïdes par le lait pourrait être le même chez ces espèces. Troisièmement, nous avons caractérisé l’interaction entre BSP1 bovine et les LDL du jaune d’oeuf qui a un Ka de 3.4 ± 0.4 × 10^6 M-1 et une valeur de « n » de 104 BSP1 pour une particule de LDL, indiquant qu’il existe des différences entre le mécanisme de protection des spermatozoïdes par le lait et le jaune d’oeuf. Nous croyons que les résultats présentés dans cette thèse aideront à créer de nouveaux diluants ne contenant pas de produits d’origine animale afin de cryoconserver les spermatozoïdes des mammifères.

Caseinoglycomacropeptide inhibits adhesion of pathogenic Escherichia coli strains to human cells in culture

Caseinoglycomacropeptide (CGMP) derived from kappa-casein was investigated for its ability to inhibit the adhesion of 3 strains of verotoxigenic Escherichia coli (VTEC) and 3 strains of enteropathogenic Escherichia coli (EPEC) to human HT29 tissue cell cultures. Effects on adhesion of Desulfovibrio desulfuricans, Lactobacillus pentosus, Lactobacillus casei, Lactobacillus acidophilus, and Lactobacillus gasseri were also investigated. Generally, CGMP exerted effective anti-adhesive properties at a dose of 2.5 mg/mL, albeit with a high degree of strain specificity. The CGMP reduced adhesion of VTEC strains to < 50% of the control and reduced adhesion of EPEC strains to between 80 and 10% of the control. The CGMP also reduced the adhesion of L. pentosus and L. casei to 44 and 42%, respectively. A slight but significant reduction of L. acidophilus, to 81%, was observed, but no significant effects were detected with either Dsv. desulfuricans or L. gasseri. Further investigation of the dose response relationships with the E. coli strains gave IC50 values ranging between 0.12 and 1.06 mg/mL.

Effects of lactoperoxidase and hydrogen peroxide on rheological properties of yoghurt

The effects of activation of the lactoperoxidase (LPO) system by H2O2-NaSCN and hydrogen peroxide (H2O2) on the accessibility of sulphydryl groups (SH) in skimmed milk, and on the dynamic rheological properties of the resulting yoghurt were investigated. Four different concentrations of each reagent (20-80 mg H2O2-NaSCN/kg milk and 100-400 mg H2O2/kg milk) were compared. Clear negative correlations were noted between the accessibility of SH groups and both LPO activation rate and H2O2 concentration. Also the native PAGE pattern of the heat-treated samples showed that with increase in the H2O2-NaSCN and H2O2 concentrations, the level of interaction between beta-lactoglobulin (beta-Ig) and kappa-casein (K-CN) decreased. The complex modulus (G*) of skimmed milk yoghurts declined gradually with the decrease in the concentration of accessible SH groups accordingly. Tan delta values of yoghurt samples were found to be different from the control, but close to each other, indicating that protein interaction forces taking place in the formation of gel networks of treated yoghurts were different from the control.

Effects of CSN3 and LGB gene polymorphisms on production traits in beef cattle

The objective of the present study was to estimate the allele and genotype frequencies of the CSN3/Hinfl and LGB/HaeIII gene polymorphisms in beef cattle belonging to different genetic groups, and to determine the effects of these polymorphisms on growth and carcass traits in these animals, which are submitted to an intensive production model. Genotyping was performed on 79 Nelore, 30 Canchim (5/8 Charolais + 3/8 Zebu) and 275 crossbred cattle originating from the crosses of Simmental (n = 30) and Angus (n = 245) sires with Nelore females. Body weight, weight gain, dressing percentage, longissimus dorsi area and backfat thickness were fitted using the GLM procedure, and least square means of the genotypes were compared by the F test. The results showed that the CSN3/Hinfl and LGB/HaeIII polymorphisms did not have any effect on growth or carcass traits (p > 0.05). Copyright by the Brazilian Society of genetics.

Determinação de GMP e CMP* no leite por métodos espectrofotométrico (ANSM) e cromatográfico (HPLC) - parâmetros metodológicos (*glicomacropeptídeo e caseinomacropeptídeo)

Glycomacropeptide is a glycosilated fraction of bovine kappa-casein that remains soluble when milk is clotted by rennin. Determinations of milk sialic acid content are useful because its concentration reflects the amount of free GMP of milk. In normal milk these amounts are very low, 12 to 16 times lower than in sweet whey. Therefore, its determination may be applied to verify possible frauds with whey addictions, since it works as a fingerprint. With the description of a new spectrophotometric method for determination of free GMP (ANSM) occurred a simplification of procedures, being faster than others (HPLC method), without loss of accuracy. However, due to variations of glycosilation in kappa-casein between animals, during the lactation period, due to mastitis and yet due to proteolysis on milk, it was necessary to know these variations to interpret correctly the analytical results. It was analyzed 1,703 samples of producer's raw milk and 1,189 samples of processed milk (HTST and UHT). The results showed that normal milk from herd (producer's milk) have only small amounts of free GMP, with A470nm = 0.232±0.088 or 3.89±1.25 mg of sialic acid/L. The upper limit of this distribution was A = 0.496; thus every bigger value may represent a problem, being outside of normal distribution.

Análise comparativa de genes das caseínas de búfalo

Pós-graduação em Genética e Melhoramento Animal - FCAV

Effects of nutrient restriction on mammary cell turnover and mammary gland remodeling in lactating dairy cows

The aim of this study was to investigate the effects of a severe nutrient restriction on mammary tissue morphology and remodeling, mammary epithelial cell (MEC) turnover and activity, and hormonal status in lactating dairy cows. We used 16 Holstein x Normande crossbred dairy cows, divided into 2 groups submitted to different feeding levels (basal and restricted) from 2 wk before calving to wk 11 postpartum. Restricted-diet cows had lower 11-wk average daily milk yield from calving to slaughter than did basal-diet cows (20.5 vs. 33.5 kg/d). Feed restriction decreased milk fat, protein, and lactose yields. Restriction also led to lower plasma insulin-like growth factor 1 and higher growth hormone concentrations. Restricted-diet cows had lighter mammary glands than did basal-diet cows. The total amount of DNA in the mammary gland and the size of the mammary acini were smaller in the restricted-diet group. Feed restriction had no significant effect on MEC proliferation at the time of slaughter but led to a higher level of apoptosis in the mammary gland. Gelatin zymography highlighted remodeling of the mammary extracellular matrix in restricted-diet cows. Udders from restricted-diet cows showed lower transcript expression of alpha-lactalbumin and kappa-casein. In conclusion, nutrient restriction resulted in lower milk yield in lactating dairy cows, partly due to modulation of MEC activity and a lower number of mammary cells. An association was found between feed restriction-induced changes in the growth hormone-insulin-like growth factor-1 axis and mammary epithelial cell dynamics.