A proteomic study of African elephant milk: inter-species comparisons and proteome dynamics
Milk is a complex and complete food for the specific nutritional requirements of the neonate. For the dairy industry, milk is a suitable raw material for the production of other high value products. Although extensive research has been carried out on milk of economically exploited dairy animals such as cow, goat, sheep, buffalo and camel, there are properties which are not explicit in the milk of these, that are not completely understood. Research of the milk from non-dairy animals, in which these properties are explicit, may provide answers. One of the unique properties is the content of oligosaccharides, which is low in the dairy animals, but high in some species. This property points to a specialized saccharide synthesis in the latter, where the whey protein α-lactalbumin may play a role. Another unique property is the structure of casein micelles, which in the dairy animals is stabilized by the presence of a specific ratio of four casein types, as well as their specific structural properties. The most important is the κ-casein with its amphipathic nature. In some non-dairy species, stable casein micelles are formed in spite of the absence, or low content, of some of the casein types, specifically the κ- casein. The milk of the African elephant (Loxodonta africana) displays several unique properties. In this research the proteome of its milk was investigated, with a focus on α-lactalbumin and the caseins, in order to shed light on the mentioned unique properties. The proteomics approach was used, which includes gel electrophoresis and mass spectrometry. Computer modeling was also employed. The major proteins αs1-, αs2-, β-casein, α-lactalbumin, β-lactoglobulin and serum albumin of African elephant milk were identified with one-dimensional electrophoresis and mass spectrometry. Better results were obtained with two-dimensional electrophoresis and orbitrap mass spectrometry, with which α-lactalbumin, lactoferrin, β- and κ-casein were identified. The multiple sequence alignment of α-lactalbumins showed that there are six amino acid positions that are unique to that of African elephant. Most of the amino acid substitutions in this protein were found to be conserved, and the structure model of African elephant α-lactalbumin was found to be homologous to the X-ray crystallography structures of several species. Consequently the structure models of β- 1,4-galactosyltransferase 1 and the lactose synthase complex were built, again showing homology to crystallographic data from other species. It may therefore be concluded that structures of α-lactalbumin and β-1,4-galactosyltransferase 1 are highly conserved amongst species. The saccharide synthesis in the African elephant milk would probably not differ from that of other mammals, and may therefore not be the reason for high levels of oligosaccharides in its milk. The comparison of amino acid sequences and hydropathy plots of African elephant β- casein with that of other species showed that it would self-aggregate and interact via hydrophobic interactions with other caseins to form casein micelles, similar to the model proposed for cow’s milk micelles. However, the African elephant β-casein displays several more hydrophilic regions, compared to the cow’s protein. The amino acid sequence and hydropathy plots predicted that African elephant κ- casein would function in the same way as the equivalent of other species. The ratio of African elephant milk κ-casein to β-casein was calculated to be approximately 1:8.5, which is in the same order as camel and rat milk, compared to the 3:8 of cow’s milk. This means that there would be very little κ-casein on the surface to effect repulsion of the micelles. There should therefore be other protein regions protruding from the micelle surface to aid in this function. It is suggested that in African elephant milk the hydrophilic regions of β-casein carry out this role.