Epitope analysis of the multiphosphorylated peptide alpha(s1)-casein(59-79)

The multiphosphorylated tryptic peptide alpha(s1)-casein(59-79) has been sh own to be antigenic with anti-casein antibodies. In an approach to determin e the amino acyl residues critical for antibody binding we undertook an epi tope analysis of the peptide using overlapping synthetic peptides. With alp ha(s1)-casein(59-79) as the adsorbed antigen in a competitive ELISA only tw o of five overlapping synthetic peptides at 1 mM significantly inhibited bi nding of the anti-casein antibodies. Peptides Glu-Ser(P)-Ile-Ser(P)-Ser(P)- Ser(P)-Glu-Glu and Ile-Val-Pro-Asn-Ser(P)-Val-Glu-Glu inhibited antibody bi nding by 20.0 +/- 3.6% and 60.3 +/- 7.9%, respectively. The epitope of Glu( 63)-Ser(P) -Ile-Ser(P) -Ser(P)-Ser(P)-Glu-Glu(70) was further localised to the phosphoseryl cluster as the peptide Ser(P)-Ser(P)-Ser(P) significantly inhibited binding of the anti-casein antibodies to alpha(s1)-casein(59-79) by 29.5 +/- 7.4%. Substitution of Ser(P)(75) with Ser(75) in the second inh ibitory peptide Ile-Val-Pro-Asn-Ser(P)(75)-Val-Glu-Glu also abolished inhib ition of antibody binding to alpha(s1)-casein (59-79) demonstrating that Se r(P)75 is also a critical residue for recognition by the antibodies. These data show that the phosphorylated residues in the cluster sequence -Ser(P)( 66)-Ser(P)-Ser(P)(68) and in the sequence -Pro(73)-Asn-Ser(P)-Val-Glu(77)- are critical for antibody binding to alpha(s1)-casein(59-79) and further de monstrate that a highly phosphorylated segment of a protein can be antigeni c. Copyright (C) 1999 European Peptide Society and John Wiley & Sons, Ltd.