Isolation and characterization of neutralizing single-chain antibodies against Xenopus mitogen-activated protein kinase kinase from phage display libraries.
MAP kinase kinase (MAPKK) is a dual specificity protein kinase that phosphorylates and activates MAP kinase in vivo. In this study, four mouse monoclonal single-chain Fv (scFv) antibodies (Y1-6, Y1-7, Y3-6, and Y3-11) that can specifically bind to Xenopus MAPKK were isolated from combinatorial scFv-displaying phage libraries. Three scFv clones (Y1-6, Y1-7, and Y3-6) were shown to efficiently inhibit MAPKK activity in vitro. Point mutation (D98K) at VH-CDR3 of one (Y1-6) of these three clones markedly reduced its neutralizing activity. The wild-type scFv (Y1-6) inhibited the Mos-induced MAP kinase activation and germinal vesicle breakdown when injected into immature Xenopus oocytes, whereas the mutant scFv, Y1-6 (D98K), did not. The three neutralizing scFv clones (Y1-6, Y1-7, and Y3-6) were shown to bind to NH2-terminal residues 1-23 of Xenopus MAPKK, whereas the epitope of a Y3-11 clone with no neutralizing activity was shown to lie between residues 33 and 67 of MAPKK. Furthermore, a synthetic peptide (the N16 peptide) corresponding to residues 2-17 of MAPKK suppressed the neutralizing activity of the wild-type Y1-6, and a rabbit polyclonal antibody against the N16 peptide was found to possess a strong neutralizing activity against MAPKK. These results demonstrate that the neutralizing antibodies characterized here inhibit the kinase activity of MAPKK by binding to the NH2-terminal segment of MAPKK.
Kosako
H
H
Department of Genetics and Molecular Biology, Kyoto University, Japan.
Akamatsu
Y
Y
Tsurushita
N
N
Lee
K K
KK
Gotoh
Y
Y
Nishida
E
E
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping; D015151:Immunoblotting; D007128:Immunoglobulin Fragments / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D016200:Maturation-Promoting Factor / Q000378:metabolism; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D009865:Oocytes; D019151:Peptide Library; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins; D014981:Xenopus
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D011494:Protein Kinases; D016200:Maturation-Promoting Factor; D020929:Mitogen-Activated Protein Kinase Kinases