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.
Ferranti
P
P
Dipartimento di Scienza degli Alimenti, Facoltà di Agraria, Università degli Studi di Napoli Federico II, Portici, Italia.
Malorni
A
A
Nitti
G
G
Laezza
P
P
Pizzano
R
R
Chianese
L
L
Addeo
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002364:Caseins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D014644:Genetic Variation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D016339:Spectrometry, Mass, Fast Atom Bombardment; D014357:Trypsin / Q000378:metabolism
D002364:Caseins; D010446:Peptide Fragments; D010710:Phosphates; D010768:Phosphoserine; D014357:Trypsin