Shift in the isoelectric-point of milk proteins as a consequence of adaptive divergence between the milks of mammalian species

Abstract

Background: Milk proteins are required to proceed through a variety of conditions of radically varying pH, which are not identical across mammalian digestive systems. We wished to investigate if the shifts in these requirements have resulted in marked changes in the isoelectric point and charge of milk proteins during evolution.

Results: We investigated nine major milk proteins in 13 mammals. In comparison with a group of orthologous non-milk proteins, we found that 3 proteins κ-casein, lactadherin, and muc1 have undergone the highest change in isoelectric point during evolution. The pattern of non-synonymous substitutions indicate that selection has played a role in the isoelectric point shift, since residues that show significant evidence of positive selection are much more likely to be charged (p = 0.03 for κ-casein; p < 10-8 for muc1). However, this selection does not appear to be solely due to adaptation to the diversity of mammalian digestive systems, since striking changes are seen among species that resemble each other in terms of their digestion.

Conclusion: The changes in charge are most likely due to changes of other protein functions, rather than an adaptation to the different mammalian digestive systems. These functions may include differences in bioactive peptide releases in the gut between different mammals, which are known to be a major contributing factor in the functional and nutritional value of mammalian milk. This raises the question of whether bovine milk is optimal in terms of particular protein functions, for human nutrition and possibly disease resistance.

Figure 1

pI values for the nine major milk proteins in 13 mammalian species compared to the pI of the human proteome. The top histogram represents the pI distribution of the human proteome. The histogram's x-axis is shared with that of the major milk proteins' pI shown below. The Colors indicate the different milk proteins. The tree on the left is the mammalian species tree from Benton and Donoghue [23]. Two extra pI values are represented between brackets at the opossum level, these represent the pI of the reported extra copies of κ-casein this species possess [6]. The values are for the proteins with the accession numbers FJ548612 and FJ548626 respectively.

Figure 2

Ancestral reconstruction of κ-casein and the representation of pI, dN/dS ratio and the stomach pH values. Ancestral values are represented in grey. The ratio dN/dS was calculated only for species with a well-defined cDNA (this is not the case for guinea pig, cat, and dog). When the ratio dN/dS is undefined due to extremely small dS values we used the symbol "--". Despite the fact that the pH of other digestive compartments can show marked differences between mammals, we chose only to represent the stomach pH values, as this compartment is the main first barrier for milk proteins. A review of the pH values is reported in Table 5 of the following reference [24] (p366), we could not find well-defined values for chimp, horse, cow, and guinea pig.

Figure 3

pI values of all the orthologous proteins in human, mouse, and cow. The vertical and horizontal lines represents the neutral pH = 7. Most proteins have a similar pI between species, with some exceptions lying out on both sides of the diagonal. The red dots represent the three milk proteins that have the highest shift (κ-casein, lactadherin, and muc1).

Figure 4

Alignment of κ-casein between human, mouse, and cow. The sequences of casoxin peptides A, B, and C are in the pink colored boxes. Cleavage sites are to the right of the red residues, while green residues are the corresponding residues that are not cleavable by the same enzyme in human and mouse. Casoxin-A and C are cleaved by a pepsin-trypsin digest for the former, and a trypsin digest for the later [12]. The peptide Casoplatelin [25] that inhibits ADP-induced platelet aggregation and fibrinogen binding is also represented on the figure together with the chymosin/rennin cleavage site between the F and M residues in red (while the same positions are in green in human and mouse). Horizontal lines represent gaps. Stars indicate sites that were predicted to be under positive selection (see results). Orange residues have been shown in the literature to undergo phosphorylation. Blue residues have been shown in the literature to undergo glycosylation. One potential phosphorylation site indicated in lavender in mouse.