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Epitope analysis of the multiphosphorylated peptide α s1 ‐casein(59–79)
Author(s) -
Perich John W.,
Black Catherine L.,
Huq N. Laila,
Reynolds Eric C.
Publication year - 1999
Publication title -
journal of peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 66
eISSN - 1099-1387
pISSN - 1075-2617
DOI - 10.1002/(sici)1099-1387(199905)5:5<221::aid-psc182>3.0.co;2-o
Subject(s) - epitope , peptide , chemistry , computational biology , microbiology and biotechnology , biochemistry , biology , antibody , genetics
The multiphosphorylated tryptic peptide α 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 α 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 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 α s1 ‐casein(59–79) by 29.5±7.4%. Substitution of Ser( P ) 75 with Ser 75 in the second inhibitory peptide Ile‐Val‐Pro‐Asn‐Ser( P ) 75 ‐Val‐Glu‐Glu also abolished inhibition of antibody binding to α 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 ‐Pro 73 ‐Asn‐Ser( P )‐Val‐Glu 77 ‐ are critical for antibody binding to α s1 ‐casein(59–79) and further demonstrate that a highly phosphorylated segment of a protein can be antigenic. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.