Epitope mapping of a monoclonal antibody directed against the alpha-subunit of phosphofructokinase-1 from Saccharomyces cerevisiae by screening phage display libraries.
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.
Hollborn
M
M
Institute of Biochemistry, Medical Faculty, University of Leipzig, Liebigstrasse 16, Germany.
Kirchberger
J
J
Birkenmeier
G
G
Kopperschläger
G
G
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001435:Bacteriophages / Q000235:genetics; D001667:Binding, Competitive; D016384:Consensus Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D005656:Fungal Proteins / Q000276:immunology*; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D019151:Peptide Library; D010732:Phosphofructokinase-1 / Q000276:immunology*; D012441:Saccharomyces cerevisiae / Q000276:immunology*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005656:Fungal Proteins; D010446:Peptide Fragments; D019151:Peptide Library; D010732:Phosphofructokinase-1