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.

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.

A case in variant in cow's milk is atherogenic

Casein is a major protein in cow's milk that occurs in several variant forms, two of which are beta-casein A(1) and beta-casein A(2). The levels of these two proteins vary considerably in milk dependent on the breed of cow, and epidemiology studies suggest that there is a relationship between their consumption and the degree of atherosclerosis. In the present study, the direct effect of consumption of beta-casein A(1) vs beta-casein A(2) on atherosclerosis development was examined in a rabbit model. Sixty rabbits had their right carotid artery balloon de-endothelialised at t = 0, divided randomly into 10 groups (n = 6 per group), then for 6 weeks fed a diet containing 0, 5, 10 or 20% casein isolate, either beta-casein variant A(1) or A(2) made up to 20% milk protein with whey. Some groups had their diets supplemented with 0.5% cholesterol. Blood samples were collected at t = 0, 3 and 6 weeks and rabbits were sacrificed at t = 6 weeks. In the absence of dietary cholesterol, beta-casein A(1) produced significantly higher (P < 0.05) serum cholesterol, LDL, HDL and triglyceride levels than whey diet alone, which in turn produced higher levels than beta-casein A(2). Rabbits fed beta-casein A(1) had a higher percent surface area of aorta covered by fatty streaks than those fed beta-casein A(2) (5.2+/-0.81 vs 1.1+/-0.39, P < 0.05) and the thickness of the fatty streak lesions in the aortic arch was significantly higher (0.04+/-0.010 vs 0.00, P < 0.05). Similarly, the intima to media ratio (I:M) of the balloon injured carotid arteries in A(1) fed animals (0.77+/-0.07) was higher than in those that consumed A(2) (0.57+/-0.04) or whey (0.58+/-0.04), but this did not reach significance. In the presence of 0.5% dietary cholesterol, the thickness of the aortic arch lesions was higher (P < 0.05) in 5, 10 and 20% casein A(1) fed animals compared with their A(2) counterparts, while other parameters were not significantly different. It is concluded that beta-casein A(1)is atherogenic compared with beta-casein A(2). (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

Endogenous amino acid flow in the avian ileum: quantification using three techniques

The aim of the present study was to compare the protein-free diet, guanidinated casein (GuC) and enzyme hydrolysed casein (EHC) methods for the quantification of endogenous amino acid (AA) flow in the avian ileum. Growing broiler chickens (5 weeks old) were used. All three assay diets were based on dextrose, and in the GuC and EHC diets GuC or EHC were the sole source of N. Endogenous AA flows determined with the use of protein-free diet were considerably lower (P < 0.05) than those determined by the GuC and EHC methods. The, total endogenous AA flows determined by the GuC and EHC methods were almost 3-fold greater (P < 0.05) than those determined by the protein-free diet. The endogenous AA values obtained from GuC and EHC methods were similar (P >0.05), except for the flow of arginine, which was lower (P < 0.05) in the EHC method. Glutamic acid, aspartic acid, threonine and glycine were the predominant endogenous AA present in digesta from the distal ileum. The contents of methionine, histidine and cystine were lower compared with other AA. The method of determination had no effect on the AA composition of endogenous protein, except for threonine, glutamic acid, lysine, arginine and cystine. The concentrations of threonine and arginine were lower (P < 0.05) and that of lysine was higher (P < 0.05) with the EHC method compared with the other two methods. The concentration of glutamic acid was greater (P < 0.05) and that of cystine was lower (P < 0.05) in the EHC and GuC methods compared with the protein-free diet method. The results showed that the ileal endogenous flows of N and AA are markedly enhanced by the presence of protein and peptides, above those determined following feeding of a protein-free diet. It is concluded that the use of EHC and GuC methods enables the measurement of ileal endogenous losses in chickens under normal physiological conditions.

Ileal digestibility of amino acids in feed ingredients for broilers.

To more precisely formulate feed and predict animal performance, it is important to base both the recommendations and feed formulations on digestible rather than total amino acid contents. Most published data on the digestibility of amino acids in feed ingredients for poultry are based on excreta digestibility. Ileal digestibility is an alternative and preferred approach to estimate amino acid availability in feed ingredients. Both methodologies are described and assessed. In addition, the differences between apparent and standardised (in which corrections are made for basal endogenous losses) digestible amino acid systems are discussed. The concept of a standardised digestibility system as a mean of overcoming the limitations of apparent digestibility estimates is proposed. In this context, different methodologies for the determination of basal endogenous amino acid losses are discussed. Although each methodology suffers from some limitations and published data on endogenous losses at the ileal level in growing poultry are limited, averaged data from repeated experiments using the 'enzymatically hydrolysed casein' method are considered as the best measure of basal losses. Standardised ileal amino acid digestibility values of 17 feed ingredients commonly used in broiler nutrition are presented including grains (barley, corn, sorghum, triticale, wheat), grain by-products (wheat middlings, rice pollard), plant protein sources (soybean meal, canola meal, corn gluten meal, cottonseed meal, lupins, peas/beans, sunflower meal), and animal by-products (feather meal, fish meal, meat and bone meal). This comprehensive set of the ileal amino acid digestibility of feed ingredients in broiler nutrition may serve as a basis for the establishment of the system in broiler feeding and for further research.

Comparison of texture of yogurt made from conventionally treated milk and UHT milk fortified with low-heat skim milk powder

The textures of yogurt made from ultra-high temperature (UHT) treated and conventionally treated milks at high total solids were investigated. The yogurt premixes, fortified with low-heat skim milk powder to 16%, 18%, and 20% total solids, were UHT processed at 143 degreesC for 6 s and heated at 85 degreesC for 30 min using the conventional method. The onset of gelation was delayed in the UHT-processed milk compared with conventionally heated milk. During fermentation, the viscosity of yogurt made, from UHT-treated milk at 20% total solids was close to that of yogurt made from conventionally treated milk with 16% total solids. However, after storage for greater than or equal to1 d, the yogurt made from UHT-treated milk had lower viscosity and gel strength than the yogurt made from conventionally treated milk. The solids level had no influence on yogurt culture growth.

Increased expression of the MBP mRNA binding protein HnRNP A2 during oligodendrocyte differentiation

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor that mediates intracellular trafficking of myelin basic protein (MBP) mRNA to the myelin compartment in oligodendrocytes, is most abundant in the nucleus, but shuttles between the nucleus and cytoplasm. In the cytoplasm, it is associated with granules that transport mRNA from the cell body to the processes of oligodendrocytes. We found that the overall level of hnRNP A2 increased in oligodendrocytes as they differentiated into MBIP-positive cells, and that this augmentation was reflected primarily in the cytoplasmic pool of hnRNP A2 present in the form of granules. The extranuclear distribution of hnRNP A2 was also observed in brain during the period of myelination in vivo. Methylation and phosphorylation have been implicated previously in the nuclear to cytoplasmic distribution of hnRNPs, so we used drugs that block methylation and phosphorylation of hnRNPs to assess their effect on hnRNP A2 distribution and mRNA trafficking. Cultures treated with adenosine dialdehyde (AdOx), an inhibitor of S-adenosyl-L-homocysteine hydrolase, or with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), a drug that inhibits casein kinase 2 (CK2), maintained the preferential nuclear distribution of hnRNP A2. Treatment with either drug affected the transport of RNA trafficking granules that remained confined to the cell body. (C) 2004 Wiley-Liss, Inc.

Impacts of feeding system and season on milk composition and Cheddar cheese yield in a subtropical environment

Milk obtained from cows on 2 subtropical dairy feeding systems were compared for their suitability for Cheddar cheese manufacture. Cheeses were made in a small-scale cheesemaking plant capable of making 2 blocks ( about 2 kg each) of Cheddar cheese concurrently. Its repeatability was tested over 10 separate cheesemaking days with no significant differences being found between the 2 vats in cheesemaking parameters or cheese characteristics. In the feeding trial, 16 pairs of Holstein - Friesian cows were used in 2 feeding systems (M1, rain-grown tropical grass pastures and oats; and M5, a feedlot, based on maize/barley silage and lucerne hay) over 2 seasons ( spring and autumn corresponding to early and late lactation, respectively). Total dry matter, crude protein (kg/cow. day) and metabolisable energy (MJ/cow.day) intakes were 17, 2.7, and 187 for M1 and 24, 4, 260 for M5, respectively. M5 cows produced higher milk yields and milk with higher protein and casein levels than the M1 cows, but the total solids and fat levels were similar (P > 0.05) for both M1 and M5 cows. The yield and yield efficiency of cheese produced from the 2 feeding systems were also not significantly different. The results suggest that intensive tropical pasture systems can produce milk suitable for Cheddar cheese manufacture when cows are supplemented with a high energy concentrate. Season and stage of lactation had a much greater effect than feeding system on milk and cheesemaking characteristics with autumn ( late lactation) milk having higher protein and fat contents and producing higher cheese yields.