Epitope mapping of a monoclonal antibody directed against the alpha-subunit of phosphofructokinase-1 from Saccharomyces cerevisiae by screening phage display libraries.

Abstract

Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of two types of subunits, alpha and beta. Subunit-specific monoclonal antibodies were raised to elucidate structural and functional properties of both subunits. One monoclonal antibody, alpha-F3, binds to an epitope either at the C-terminal or at the N-terminal part of the alpha-polypeptide chain. By screening a heptapeptide library with this monoclonal antibody, a set of heptapeptides was selected, which contained the consensus sequences D-A-F and D-S-F. Two heptapeptides with these motifs were synthesized in order assess their capacity to inhibit the binding of antibody alpha-F3 to native phosphofructokinase-1. The peptide G-I-K-D-A-F-L inhibited the binding more strongly (IC50 = 1.5 microM) than the peptide A-P-W-H-D-S-F (IC50 = 33.3 microM). Sequence matching revealed the presence of the D-A-F motif in the polypeptide chain of phosphofructokinase-1 at amino acid position 172-174. As a control, the nonapeptide A-P-T-S-K-D-A-F-L which corresponds to the sequence of the putative epitope was tested in the inhibition assay. In view of the high inhibitory capacity (IC50 = 0.3 microM) it was concluded that this nonapeptide represents the continuous epitope of phosphofructokinase-1 that is recognized by antibody alpha-F3.