Β-casein Variants Research Articles

A new bovine $\beta$-casein genetic variant characterized by a Met$_{93} \rightarrow$ Leu$_{93}$ substitution in the sequence A$^2$

A new variant of bovine β-casein was isolated from individual milk and characterized. It is proposed for nomenclature as β-CN H. The variant H differed from the variant A 2 by the substitu- tion of the residue methionine at position 93 by a leucine residue, by the substitution of the residue glutamine at position 72 by a glutamic acid residue and by another equivalent (Gln→ Glu) substitu- tion within the sequence 114-169. The leucine residue at position 93 was identified in the plasmin-in- duced peptide (f49-99) by electrospray ionization tandem mass spectrometry (ESI-MS 2 ), and confirmed by the amino acid composition of this peptide C-terminus. This mutation would corre- spond to the substitution of the codon ATG by CTG, originally reported in cDNA by Jimenez- Flores et al. (11). The molecular mass of β-casein H was measured as 23 969.1 ± 2.4 g.mol -1 .

Enzymatic release of neocasomorphin and β-casomorphin from bovine β-casein

Conditions for the release of β-casomorphin-7 from bovine β-casein by gastrointestinal proteases in vitro were investigated. β-Casomorphin-7 was released only from a genetic variant of β-casein containing a His residue at the 67th position of the peptide chain. Elastase cleaved the peptide bond between Ile 66 and His 67, releasing the carboxyl terminus of β-casomorphin-7. Pepsin and leucine aminopeptidase were required to release the amino terminus of this peptide. β-Casomorphin-9, -13, and -21 also were isolated, and their opioid activities were measured. In this study, we also isolated a novel opioid peptide neocasomorphin-6 (Tyr-Pro-Val-Glu-Pro-Phe), which was released by action of trypsin or pepsin and chymotrypsin.

Characterization of a Non-electrophoretic Genetic Variant of β-Casein by Peptide Mapping and Mass Spectrometric Analysis

Abstract From a total of 634 Holstein milk samples, 158 were identified as homozygous β -casein A 1 according to PAGE. All the β -casein A 1 samples were isolated in pure form by anion exchange chromatography followed by tryptic hydrolysis and peptide mapping of the hydrolysates by reversed-phase HPLC. A comparison of the chromatogram revealed that the β -casein A 1 samples could be classified into two groups based on differences in elution times of fragment 114–169 due to a change in hydrophobicity caused by amino acid substitution. Mass spectrometric analysis showed that 7 β -casein samples with more hydrophobic 114–169 fragment had a higher molecular mass (difference of 16 Da) than the remaining 151 samples with less hydrophobic fragment. Amino acid composition and sequence analysis confirmed the difference in mass of peptide 114–169 by a Pro→Leu substitution at position 137. It is proposed to identify the newly found variant as β -casein G.

The primary structure of water buffalo alpha(s1)- and beta-casein identification of phosphorylation sites and characterization of a novel beta-casein variant.

The primary structure of water buffalo alpha(s1)-casein and of beta-casein A and B variants has been determined using a combination of mass spectrometry and Edman degradation procedures. The phosphorylated residues were localized on the tryptic phosphopeptides after performing a beta-elimination/thiol derivatization. Water buffalo alpha(s1)-casein, resolved in three discrete bands by isoelectric focusing, was found to consist of a single protein containing eight, seven, or six phosphate groups. Compared to bovine alpha(s1)-casein C variant, the water buffalo alpha(s1)-casein presented ten amino acid substitutions, seven of which involved charged amino acid residues. With respect to bovine betaA2-casein variant, the two water buffalo beta-casein variants A and B presented four and five amino acid substitutions, respectively. In addition to the phosphoserines, a phosphothreonine residue was identified in variant A. From the phylogenetic point of view, both water buffalo beta-casein variants seem to be homologous to bovine betaA2-casein.

Capillary electrophoretic analysis of genetic variants of milk proteins from different species

Polymorphism of bovine, ovine and caprine milk proteins was studied by CE. Identification of some rare bovine variants was carried out by isoelectric focussing (IEF) using PhastSystem. Genetic variants A and D of bovine α s2-casein, β-casein variants A 1, A 2, A 3, B and C and α s1-casein variants B and C were determined by CE. In addition, the different casein fractions including some genetic variants of ovine and caprine milk were identified by CE. In order to carry out this identification, collected fractions from a cation-exchange FPLC separation were injected by CE.

Hydrolysis of αs- and β-caseins during ripening of Serra cheese

Hydrolysis of the major caseins in Serra cheese manufactured from raw sheep's milk coagulated with a plant rennet ( Cynara cardunculus, L.) was monitored by urea-PAGE electrophoresis throughout a 35 day ripening period (with sampling at 0, 7, 21 and 35 days) and throughout the cheesemaking season (with sampling at November, February and May). The α s - and β-caseins were degraded up to 82 and 76%, respectively, by 35 days of ripening. The α s -casein variants ( α s2 - and α s3 -) displayed similar degradation patterns to one another, but different from those of β-casein variants ( β 1- and β 2-). Although the α s -caseins were broken down more slowly than β-caseins at early stages of ripening (97, 95, 80, and 60% of α s2 -, α s3 -, β 1-, and β 2-caseins, respectively, were still intact by 7 days), this observation was reversed for later stages of ripening (18, 18, 30, and 20% of α s2 -, α s3 -, β 1− and β 2-caseins, respectively, were still intact by 35 days of ripening). The position along the cheese-making season significantly affected the hydrolysis of only the β 2- and α s3 -caseins. Degradation of α s3 -casein was slower in February than in November or May for 21-day old cheeses; cheeses ripened for 7 days or 21 days showed more intact β 2-casein when manufactured in May than in November or February. The magnitude of the correlation coefficients pertaining to concentrations of intact α s - and β-caseins indicated that the products of proteolytic breakdown with higher mobility than α s -caseins (tentatively termed α 1- I, α 2- I, and α 3- I) were preferentially correlated with α s -caseins, the products of proteolytic breakdown with mobility between β-caseins and α s -caseins (tentatively termed β 1 and β 1- I) were preferentially correlated with β 1 and β 2-caseins rather than with α s -caseins, and the products of proteolytic breakdown with the highest mobility (tentatively termed α β 1 -II and α β 2 -II ) were preferentially correlated with β-caseins.

ミクロカラム液体クロマトグラフィー／エレクトロスプレーおよび高速原子衝撃イオン化質量分析法による天然および遺伝子組み換えタンパク質の微小構造変異の解析

A home-made electrospray ionization (ESI) source, which generated multiply charged gas-phase ions of proteins under atmospheric pressure, was coupled with a tandem quadrupole mass spectrometer. A detection limit of 18 fmol (1.8 × 10-14 mol) and a precision of 0.01% in the molecular weight measurement were attained using hen-egg lysozyme as a typical protein.ESI mass spectrometry (MS) was applied to the prompt identification of the amino acid substitutions in recombinant human lysozymes (HLY). However, the direct analysis of HLY was prevented by the co-existing inorganic salts added to retain its biological activities. On-line coupling of ESI-MS with micro-column liquid chromatography (ESI-LC/MS) was developed to perform direct molecular weight measurements of salt-containing proteins at the picomole level. LC/MS interfacing by ESI required to alleviate the sensitivity losses brought about by the high electric conductivity and the high surface tension of the LC eluent.ESI-MS and ESI-LC/MS were successfully applied to the characterization of the post-translational modifications found in recombinant wheat germ agglutinin (WGA2). These methods along with fast atom bombardment (FAB) MS and FAB LC/MS were also applied to the characterization of natural variants of β-casein (β-CA). The possibility and limitation of the present methods for the analysis of complex proteins were discussed.

Variation in Milk Protein Concentrations Associated with Genetic Polymorphism and Environmental Factors

Concentrations of αs-casein, β-casein, κ-casein, β-lactoglobulin, α-lactalbumin, serum albumin, and immunoglobulin in milk from 1888 Holstein cows were determined monthly over the lactation period. Cows were phenotyped for genetic variants of αs1-casein, β-casein, κ-casein, and β-lactoglobulin. Least squares analyses showed variations in individual proteins due to parity number, month of test, stage of lactation, somatic cell count, fat content, milk yield, and phenotypes of cows for milk proteins. β-Casein declined and serum proteins increased with advancing age of cows. Concentration of individual proteins decreased during the first 2 to 3 mo in lactation and then increased as lactation progressed. αs1-Casein variants significantly affected concentrations of αs-casein (BC>BB>AB) and β-lactoglobulin (AB>BB>BC). Variant B for β-casein is associated with lower αs-casein, β-lactoglobulin, immunoglobulins, and higher β-casein and α-lactalbumin concentrations than variant A1, A2, or A3. Milk from BB κ-casein, and BB β-lactoglobulin cows contained more αs-casein, κ-casein, and less κ-lactoglobulin than milk from AA cows for the two proteins. Concentrations of all proteins were negatively correlated with milk production. Increased somatic cell counts were associated with lower β-casein and higher concentrations of other proteins. Fat content of milk was positively correlated with the three casein fractions and β-lactoglobulin.

Effects of milk protein genetic variants on milk yield and composition.

Effects of genetic variants of the milk proteins, alpha S1-casein, beta-casein, kappa-casein and beta-lactoglobulin (beta-lg), on milk yield and composition, particularly the protein composition, were investigated in milk samples from 289 Jersey and 249 Friesian cows in eight commercial herds. Milk protein genotypes had no significant effect on yields over a complete lactation of milk and fat, but significant differences in fat content were detected for beta-casein (B, A1B, A2 greater than A1A2) and beta-lg (B, AB greater than A) variants. Significant differences between beta-lg variants were also found with total solids (B, AB greater than A), casein (B, AB greater than A), whey protein (A greater than AB greater than B) and beta-lg (A greater than AB, AC greater than B greater than BC) concentrations. Casein genotypes were not significantly different in total protein and casein concentrations but many differences were found in casein composition. alpha S1-Casein variants significantly affected alpha S1-casein (BC greater than B) and kappa-casein (B greater than BC) concentrations. beta-Casein variants affected concentration and proportion of beta-casein (A1B, A2B greater than A1, A1A2, A2, B), alpha S1-casein (A1, A2 greater than B) and kappa-casein (B greater than A2) and concentration of whey protein (A1 greater than most other beta-casein variants). kappa-Casein variants affected concentration and proportion of kappa-casein (B greater than AB greater than A), proportion of alpha S1-casein (A greater than AB greater than B) and concentrations of beta-lg (A greater than AB, B) and alpha-lactalbumin (A, AB greater than B). Differences in milk composition were found between breeds, herds and ages, and with stage of lactation. The potential use of milk protein genotypes as an aid in dairy cattle breeding is discussed.

Phosphorylation of casein by cyclic AMP-dependent protein kinase

The catalytic subunit of rabbit muscle cyclic AMP-dependent protein kinase (EC 2.7.1.37; ATP:protein transferase) has been tested on a variety of caseins. The B variant of β-casein was phosphorylated at a much greater rate than other β-caseins, α s1-caseins, and κ-caseins. Whole casein homozygous for β-casein B was phosphorylated at 2.5 times the rate of commercial whole casein. Gel electrophoresis experiments indicate that β-casein is the predominant component phosphorylated in commerical casein. It is therefore suggested that phosphorylation of whole casein depends on its content of the specific genetic variant, β-casein B.

Le variant βE et le code de phosphorylation des caséines bovines

The E variant of bovine β-casein differs from the reference βA 2 variant by the substitution 36 Glu (βA 2) → Lys (βE). Seryl residue 35 is phosphorylated in βE as in βA 2. The variant βE can be compared to the variant βC which also differs from βA 2 by a Glu → Lys substitution. However, this substitution then affects the position 37, and, in this case, the seryl residue 35 is not phosphorylated. These observations tend to support the existence of the phosphorylation code of bovine caseins as postulated by Mercier et al. [1].

Thoracentesis

Crossbred Karan Fries (KF) cows, among the best yielders of milk in India are carriers of A1 and A2 alleles. These genetic variants have been established as the source of β-casomorphins (BCMs) bioactive peptides that are implicated with various physiological and health issues. Therefore, the present study was aimed to investigate the release of BCM-7/5 from β-casein variants of KF by simulated gastrointestinal digestion (SGID) performed with proteolytic enzymes, in vitro. β-Casein variants (A1A1, A1A2 and A2A2) were isolated from milk samples of genotyped Karan Fries animals and subjected to hydrolysis by SGID using proteolytic enzymes (pepsin, trypsin, chymotrypsin and pancreatin), in vitro. Detection of BCMs were carried out in two peptide fractions (A and B) of RP-HPLC collected at retention time (RT) 24 and 28 min respectively corresponding to standard BCM-5 and BCM-7 by MS–MS and competitive ELISA. One of the RP-HPLC fractions (B) showed the presence of 14 amino acid peptide (VYPFPGPIHNSLPQ) having encrypted internal BCMs sequence while no such peptide or precursor was observed in fraction A by MS–MS analysis. Further hydrolysis of fraction B of A1A1 and A1A2 variants of β-casein with elastase and leucine aminopeptidase revealed the release of BCM-7 by competitive ELISA. The yield of BCM-7 (0.20 ± 0.02 mg/g β-casein) from A1A1 variant was observed to be almost 3.2 times more than A1A2 variant of β-casein. However, release of BCM-7/5 could not be detected from A2A2 variant of β-casein. The biological activity of released peptides on rat ileum by isolated organ bath from A1A1 (IC50 = 0.534–0.595 μM) and A1A2 (IC50 = 0.410–0.420 μM) hydrolysates further confirmed the presence of opioid peptide BCM-7.

Polymorphisms in caseins of sheep milk.

Starch gel electrophoresis of 592 sheep milk samples including six breeds or crosses between these breeds revealed two variant casein types, one a-s1 and one β casein. The number of variants found was low being, .036 and .017 for as1-casein and β-casein types, respectively. The predominant type for as1-casein had three brands, whereas, the variant type had six bands. A previously reported as1-casein variant was found to be one of the whey proteins as it was absent in reconstituted casein samples subjected to electrophoresis. There were three bands in the β-casein region of the predominant type. The variant β-casein type appeared to have a lighter fourth band. The frequency of the β-casein variant allele was .017. Sheep milk could be typed on gels of low pH with precision equal to that of alkaline gels. Acid gels did not reveal additional heterogeneity.

A new β‐casein variant in Piedmont cattle

A new β‐casein variant in Piedmont cattle

Primary structure of bovine beta casein. Isolation and amino acid composition of trypsin and cyanogen bromide peptides

In order to determine the primary structure of the bovine β‐caseins, the A2 variant was first cleaved with trypsin and cyanogen bromide (CNBr).The tryptic hydrolyzate was separated by column chromatography on Dowex‐50. After additional purifications, 13 peptides numbered T1 to T13 were isolated. The 14th tryptic peptide, T14, not eluted from the column, was directly obtained from the hydrolyzate by preparative paper electrophoresis. Amino‐acid analyses were carried out on all these peptides. Peptide T1 contains 2 Arginyl residues, one of which is NH2‐terminal. This peptide represents the NH2‐terminal end of the β‐casein. Its composition is identical to that reported by Peterson et al. in 1958 [1] for a phosphopeptide isolated from the β‐casein. Peptide T11 contains 2 Lysyl residues, one of which is NH2‐terminal. The COOH‐terminal peptide was identified with peptide T4, devoid of basic amino‐acids. The 14 tryptic peptides account for all the residues of the β‐casein A2 when compared with the already published amino‐acid analyses of the whole protein [2,3]. Several other peptides, originating from incomplete or non‐specific cleavages, were also obtained.Similarly, 7 peptides accounting for the 6 methionyl residues of the protein were obtained from the same variant of β‐casein after cleavage with cyanogen bromide. Six of these peptides were separated on Sephadex G‐50. The seventh peptide was obtained by chromatography on Dowex‐50 of the whole CNBr‐digest. After additional purifications, the amino‐acid compositions of the 7 pure peptides were determined. Peptide CN1 contains peptide T1. It represents the NH2‐terminal end of β‐casein. The COOH‐terminal end of β‐casein was identified with peptide CN7. As in the case of the tryptic peptides, the 7 CNBr‐peptides account for all of the amino‐acid residues of β‐casein A2.β‐casein A2 has 208 amino‐acid residues. Its molecular weight is very close to 24000.

Evidence from amino acid analysis for a relationship in the biosynthesis of γ- and β-caseins

γ-Casein, like β-casein, is polymorphic as shown by gel electrophoresis. Two variants of γ-casein, A 2 and B, have been isolated from samples of bovine casein genetically typed β-casein A 2 and B. The β-caseins were also isolated from the same samples. By comparison of the composition of the proteins it was shown that γ-A 2 differs from γ-B only in content of single residues of four amino acids and two substitutions, Ser/Arg and His/Pro are postulated. β-A 2 differs from β-B in the same manner, implying a close relationship in the synthesis of these milk proteins. The γ-caseins differ considerably from the β-caseins in content of proline and phosphorus. Comparison of the composition of four polymorphs of β-casein indicated that, in addition to the Ser/Arg and His/Pro substitutions, a third, Glu/Lys, may occur in the β-casein variants.

Procedure for Simultaneous Phenotyping of β-Casein and β-Lactoglobulin Variants in Cow's Milk

Procedure for Simultaneous Phenotyping of β-Casein and β-Lactoglobulin Variants in Cow's Milk

DEAE-Cellulose-Urea Chromatography of Casein in the Presence of 2-Mercaptoethanol

κ-Casein, after reduction of the disulfide bond(s) by 2-inercaptoethanol, can be separated from αs1- and β-casein variants, as well as from whole casein, by DEAE-cellulose-urea chromatography. When κ-easein is in the reduced form it elutes from DEAE-cellulose at 0.05m NaCl. Use of a reduction step simplifies purification of the major casein components (αs1-, β) by chromatography.

Genetic Polymorphism in Caseins of Cow's Milk. V. End-Group Analysis of β-Caseins A, B, and C

The three known genetic variants of β-casein (A, B, and C) have been purified by column chromatography in 3.3m urea, and their purity verified by polyacrylamide-gel electrophoresis. Qualitative determination of the amino terminal amino acid by two independent chemical methods revealed arginine for each of the three variants. During reaction with carboxypeptidase A, valine was released most rapidly and was closely followed by isoleucine. The data indicate either an ileu-ileu-val or an ileu-val-ileu C-terminal sequence in each variant. Molecular weights of about 25,000 for β-caseins A, B, and C were calculated from the amount of valine released.

Non-independent Occurrence of αS1 and β-Casein Variants of Cow's Milk

THE β-casein fraction of cow's milk has been found to show genetic variants distinguishable by electrophoresis1. Part of the αs-casein fraction, now called αs1-casein (ref. 2), also shows genetic variation3. The αS1-casein type B and the β-casein type A are prevalent, while the other two known variants of each only occur in some breeds of cattle4,5. In Jersey cows αS1-casein type C and β-casein type B are seen in a fair proportion of cases.