Affinity panning of peptide libraries using anti-streptokinase monoclonal antibodies: selection of an inhibitor of plasmin(ogen) active site.
To select sequences complementary to their binding sites, two anti-streptokinase (SK) monoclonal antibodies (mAbs), A4.5 and A5.5, were used in biopanning of 15-mer and hexamer phage-displayed peptide libraries, respectively. mAb A4.5 inhibits the catalytic activity of streptokinase-plasminogen activator complex (SKPAC), the binding of plasminogen to SK and the binding of human anti-SK polyclonal Abs to SK. All clones selected from the 15-mer peptide library by mAb A4.5 had identical nucleotide and amino acid sequences, RSVYRCSPFVGCWFG. An 11-mer peptide (peptide A4.5, YRCSPFVGCWF) derived from this sequence inhibited the binding of mAb A4.5 and human anti-SK polyclonal Abs to SK as well as the catalytic activity of both SKPAC and plasmin. The binding of the second mAb (mAb A5.5) to SK is lost upon interaction of SK with plasminogen, suggesting that sequences selected by this mAb are likely associated with the C-terminal cleavage site of SK. Biopanning of a hexamer peptide library with mAb A5.5 selected the sequence RYLQDY that is homologous to residues 324-328, adjacent to one possible C-terminal cleavage site in SK. A 10-mer synthetic peptide (LDFRDLYDPR) corresponding to residues 321-330 in SK specifically inhibited the binding of mAb A5.5 to SK. The selection and characterization of these two peptides enhances our understanding of SK structure, maps an antigenic epitope, and identifies a peptide inhibitor of plasminogen activation.
Parhami-Seren
Behnaz
B
Department of Biochemistry, College of Medicine, University of Vermont, Given Building, Room C444, 89 Beaumont Avenue, Burlington 05405-0068, USA. bparhami@zoo.uvm.edu
Krudysz
Jolanta
J
Tsantili
Panayota
P
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002863:Chromogenic Compounds; D018604:Epitope Mapping; D005341:Fibrinolysin / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007118:Immunoassay; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D019151:Peptide Library; D010958:Plasminogen / Q000737:chemistry / Q000378:metabolism; D013300:Streptokinase / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002863:Chromogenic Compounds; D046911:Macromolecular Substances; D010446:Peptide Fragments; D019151:Peptide Library; D010958:Plasminogen; D013300:Streptokinase; D005341:Fibrinolysin