Epitope mapping and tight-binding inhibition with monoclonal antibodies directed against Escherichia coli glucosamine 6-phosphate synthase.
In the present work, we attempt to identify inhibitory monoclonal antibodies directed against Escherichia coli glucosamine-6P synthase (GlmS) and to localize the corresponding epitopes to better understand the topology of the enzyme during catalysis. Four of the 15 monoclonal antibodies have been shown to be specific for the native form of the enzyme and 2 of them, 505.1 and 522.2, strongly inhibit the glucosamine synthase activity. Kinetic analysis of 505.1 antibody behavior revealed a tight-binding inhibition with a Ki = 40 +/- 20 pM, a value which is four orders of magnitude lower than the best active site-directed inhibitor reported so far. The reactivity of all the monoclonal antibodies with 601 overlapping octapeptides covering the entire sequence of GlmS was tested by enzyme-linked immunosorbent assay for precise epitope mapping. Four linear epitopes specific for the denatured protein and one present in both native and denatured enzyme were defined by this approach. Neither 505.1 nor 522.2 was directed against linear epitopes. However, evidence for the binding of 505.1 at the glutamine catalytic site was shown by using site-directed mutants of GlmS as well as by competition experiments with an irreversible inhibitor. The mAb 105.1, which recognizes the octapeptide containing the sequence RWATHG conserved among the six glucosamine-6P synthases reported so far, allowed the detection of the human enzyme.
Cochet
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Laboratoire de Biotechnologie des Anticorps, Institut Curie, Paris, France.
Badet-Denisot
M A
MA
Teillaud
J L
JL
Badet
B
B
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D003429:Cross Reactions; D004791:Enzyme Inhibitors; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D004926:Escherichia coli / Q000201:enzymology* / Q000276:immunology; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology; D011485:Protein Binding; D011489:Protein Denaturation; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D009842:Oligopeptides; D010446:Peptide Fragments; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)