PRIMARY STRUCTURE OF OVINE ALPHA(S1)-CASEINS - LOCALIZATION OF PHOSPHORYLATION SITES AND CHARACTERIZATION OF GENETIC-VARIANT-A, GENETIC-VARIANT-C AND GENETIC-VARIANT-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 similar to 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 Ser(115) and Ser(41) were phosphorylated by similar to 50 and similar to 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 Ser(13) --> Pro(13) which determined the loss of the phosphate group on site 12 of the protein chain, SerP(12) --> Ser(12). A further substitution, SerP(68) --> Asn(68) caused the disappearance of both phosphate groups in the phosphorylated residues Ser(64) and Ser(66) in variant D; in this last casein variant there was no evidence of phosphorylation at Ser(41).