Epitope analysis of the multiphosphorylated peptide alpha s1-casein (59-79).
The multiphosphorylated tryptic peptide alpha(s1)-casein(59-79) has been shown to be antigenic with anti-casein antibodies. In an approach to determine the amino acyl residues critical for antibody binding we undertook an epitope analysis of the peptide using overlapping synthetic peptides. With alpha(s1)-casein(59-79) as the adsorbed antigen in a competitive ELISA only two of five overlapping synthetic peptides at 1 mM significantly inhibited binding 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 binding by 20.0+/-3.6% and 60.3+/-7.9%, respectively. The epitope of Glu63-Ser(P)-Ile-Ser(P)-Ser(P)-Ser(P)-Glu-Glu70 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 Ser75 in the second inhibitory peptide Ile-Val-Pro-Asn-Ser(P)75-Val-Glu-Glu also abolished inhibition of antibody binding to x(s1)-casein (59-79) demonstrating that Ser(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 -Pro73-Asn-Ser(P)-Val-Glu77- are critical for antibody binding to x(s1)-casein(59-79) and further demonstrate that a highly phosphorylated segment of a protein can be antigenic.
Perich
J W
JW
Biochemistry and Molecular Biology Unit, School of Dental Science, The University of Melbourne, Victoria, Australia.
Black
C L
CL
Huq
N L
NL
Reynolds
E C
EC
D000595:Amino Acid Sequence; D002364:Caseins / Q000737:chemistry / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry*; D007106:Immune Sera; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology*; D010766:Phosphorylation; D011487:Protein Conformation; D016339:Spectrometry, Mass, Fast Atom Bombardment
D002364:Caseins; D000939:Epitopes; D007106:Immune Sera; D010446:Peptide Fragments; C092814:alpha-casein (59-79)