Primary structure of ovine alpha s1-caseins: localization of phosphorylation sites and characterization of genetic variants A, C and D.

The primary structures of ovine alpha s1-casein variants A, C and D (formerly called Welsh variant) were determined. Separation of variants from whole casein was achieved using a fast and reliable reversed-phase HPLC method. Extended structural characterization of the purified proteins using electrospray mass spectrometry, automated Edman degradation and peptide mapping by means of HPLC-fast atom bombardment-mass spectrometry demonstrated that the mature protein was a mixture of two molecular species that differed in the deletion of residues 141-148 and were therefore 199 and 191 residues long respectively. The 199 residue peptide chain, which accounted for approximately 80% of the entire translated alpha s1-casein, was as long as its caprine and bovine counterparts, and had a 98 and 89% degree of identity with those two proteins respectively. Nine serine residues (positions 12, 44, 46, 64 to 68 and 75) were fully phosphorylated in alpha s1-casein A, whereas Ser115 and Ser41 were phosphorylated by approximately 50 and approximately 20% respectively. The differences between the three genetic variants A, C and D were simple silent substitutions, which however involved the degree to which the protein was phosphorylated. Variant C differed from variant A in the substitution Ser13-->Pro13 which determined the loss of the phosphate group on site 12 of the protein chain, SerP12-->Ser12. A further substitution, SerP68-->Asn68 caused the disappearance of both phosphate groups in the phosphorylated residues Ser64 and Ser66 in variant D; in this last casein variant there was no evidence of phosphorylation at Ser41.