Target Milk Proteins Heterogeneity among the Mammalian Species of Interest for Dairy Industry

Almost all the applications of proteomics analysis to milk and cheese originate from the heterogeneity of the major milk proteins among the mammalian species. This heterogeneity consists of differences in the primary structure, such as single amino acid

Advances in Dairy Products, First Edition.

Edited by Francesco Conto, Matteo A. Del Nobile, Michele Faccia, Angelo V. Zambrini, and Amalia Conte.

© 2018 John Wiley & Sons Ltd. Published 2018 by John Wiley & Sons Ltd.

Alignment of primary structure of cow, buffalo, goat and sheep p-CN. Light grey highlight

Figure 2.1.4.1 Alignment of primary structure of cow, buffalo, goat and sheep p-CN. Light grey highlight: signal peptide; underlined characters: positively charged residues; underlined bold grey characters: negatively charged peptide; dark grey highlight: phosphorylated residues.

Alignment of bovine, goat, and sheep asCN. Highlights and characters legend is reported in the caption of Figure 2.1.4.1

Figure 2.1.4.2 Alignment of bovine, goat, and sheep asrCN. Highlights and characters legend is reported in the caption of Figure 2.1.4.1.

substitutions, that are responsible for small changes of the electrical charge state of the proteins. In Figure 2.1.4.1, the aligned primary structures of cow, buffalo, goat, and sheep p-casein (-CN) are reported. p-casein has 209 amino acids (aa) in cow and buffalo, whereas in goat and sheep there are 207 amino acids due to the deletion of residues 194 and 195.

The substitution of asparagine for lysine in 83 of buffalo p-CN produces one more potential cleavage site for plasmin (Di Luccia et al., 2009). The same amino acids substitution (asparagine for lysine) in the p-CN of goat and sheep in 147 is accountable for the difference observed in y-casein electrophoretic pattern among the three species. p-CN and as1-CN together represent 70% of the whole caseins. The alignment of as1-CN is

Kappa-casein alignment, bold black characters

Figure 2.1.4.3 Kappa-casein alignment, bold black characters: glycosylation site; the legend of the other character and highlights is in the caption of Figure 2.1.4.1.

reported in Figure 2.1.4.2; this protein consists of 199 amino acids, and no deletions are observed in the three species considered. Bovine aS1-CN shows five negative extra charges in respect to the others species, for glutamic acid substitution in 76, 78, 148, 163, and 207. Furthermore, ovine aS1-CN shows two more phosphoserine residues than in the others. These differences account for the higher electrophoretic mobility of bovine aS1-CN with respect to goat and sheep aS1-CN. к-casein is the only casein fraction showing glycosylation. The amount of carbohydrates in к-CN is about 5% consisting of N-acetylneuraminic acid (sialic acid), galactose and N-acetylgalactosamine (Fox & McSweeney, 1998). к-CN shows seven N-linked carbohydrates in cow, six in goat, and five in sheep; this casein fraction is highly important for the clotting characteristics of a milk, which are responsible for casein micelles stability.

Figure 2.1.4.3 shows the primary structure of bovine, goat and sheep k-CNs. It has 171 residues but к-casein of bovine is two aa shorter due to the deletion of 154 and 155. The para к-casein is the N-terminal fragment (22-126) of к-casein obtained after the action of chymosin during the curd production process. Positions 40 and 111 are negatively charged in goat and sheep since a glutamic acid replaces a serine and an alanine respectively. Position 29 of sheep shows an arginine instead of a proline residue and finally in position 31 of bovine k-CN, arginine replaces a cysteine residue. Because of these modifications, para к-casein of bovine is slightly more alkaline than the para к- casein of goat, which in turn is more alkaline than that of sheep. Finally, among whey proteins, p-lactoglobulin and a-lactalbumin are the most abundant. Two isoforms of P-lactoglobulin are generally detectable in bovine milk, variant A and B. The substitutions G—D80 and A——V134 differentiate the two variants. Looking to the primary structure of p-lactoglobulin B in Figure 2.1.4.4 bovine shows three negatively charged residues more than sheep and goat, for the presence of aspartic or glutamic acids in 71, 148, and 176. On the other hand, sheep and goat have a positively charged residue more than bovine for the substitution of his38 for sheep and lys148 for goat. As result, P-lactoglobulin B of bovine has a pi of 4.83, whereas that of goat and sheep shows a pi of 5.29 and 5.26, respectively.

Primary structure of bovin, goat, and sheep p-lactoglobulin. Highlight and characters legend is reported in the caption of Figure 2.1.4.1

Figure 2.1.4.4 Primary structure of bovin, goat, and sheep p-lactoglobulin. Highlight and characters legend is reported in the caption of Figure 2.1.4.1.

 
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