Identification of epitopes on cytochrome P450 3A4/5 recognized by monoclonal antibodies.
In this study we describe the mapping of epitopes on CYP3A4/5 recognized by a panel of monoclonal antibodies (MAbs). CYP3A4 and CYP3A5 cDNAs were cloned in GST expression vectors and the fusion proteins were subjected to Western blot. Eight MAbs reacted with the full-length GST-3A4 fusion protein as well as baculovirus cDNA-expressed CYP3A4, while six of these reacted with baculovirus cDNA-expressed CYP3A5. Five (MAb 347, 351, 352, 354, and 357) out of 8 MAbs were inhibitory in a metabolic assay using quinine as substrate. MAbs 352, 354, and 357 brought about a moderate inhibition of quinine metabolism (60-70%) while MAb 347 inhibited quinine 3- hydroxylation in human liver microsomes (n=6) by more than 70%. MAb 347 was a potent inhibitor of baculovirus-expressed CYP3A5-catalyzed metabolism of quinine (95%) at </=0.20 mg IgG/nmol P450 but only moderately inhibited CYP3A4 at much higher ratios of MAb to P450. This MAb was mapped to a region of 283 to 504 amino acids on CYP3A4 protein and to an identical region on CYP3A5 protein. The region that was identified on the CYP3A5 construct was further validated based on the ability of the construct harboring the epitope to reverse the inhibition of hydroxylation of quinine by MAb 347. Our experiments clearly demonstrate that a spatial antigenic determinant is responsible for the inhibitory potency of MAb 347.
Parimoo
Bhama
B
Chemical Biology Department, Ernest Mario School of Pharmacy, Rutgers The State University of New Jersey, EOHSI Bldg., 170 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA.
Mishin
Vladimir M
VM
Busch
Christine M
CM
Thomas
Paul E
PE
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D016367:Baculoviridae / Q000378:metabolism; D015153:Blotting, Western; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008862:Microsomes, Liver / Q000378:metabolism; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011802:Quinidine / Q000031:analogs & derivatives* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D000939:Epitopes; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; C013569:3-hydroxyquinidine; D003577:Cytochrome P-450 Enzyme System; C104464:CYP3A protein, human; C510164:CYP3A5 protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D005982:Glutathione Transferase; D011802:Quinidine