Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
Regular paperAn inhibitory monoclonal anti-protein antibody and an anti-peptide antibody share an epitope on rat cytochrome enzymes CYP1A1 and CYP1A2
Abstract
A monoclonal antibody, 12/2/3/2, which was raised against purified rat CYP1A1 recognises specifically rat and mouse CYP1A1 and CYP1A2, but not any cytochrome present in hepatic microsomal fractions from rabbit, guinea pig, hamster or human. By comparing the primary sequences of cytochromes to which 12/2/3/2 does and does hot bind, 10 possible locations for its epitope were found. Of these, one was extremely hydrophilic and, hence, predicted to be the most antigenic in the native protein. An antibody was produced against the synthetic peptide corresponding to this region (Gly-Arg-Asp-Arg-GlnPro-Arg-Leu: residues 356–363 and 350–357 of rat CYP1A1 and CYP1A2, respectively). The antibody bound to rat, mouse and hamster CYP1A1 and to rat and mouse CYP1A2, but did hot bind to any protein present in hepatic microsomal fractions from the rabbit, guinea pig or human. The binding of the anti-peptide antibody to CYP1A1 or CYP1A2 was partially antagonised by the monoclonal antibody. However, whereas the monoclonal antibody inhibited both CYP1A1- (aryl hydrocarbon hydroxylase) and CYP1A2-(high-affinity phenacetin ) dependent monooxygenase activity, the anti-peptide antibody was without effect on these activities. Antigen denaturation by 8 M urea or 0.05% (w/v) SDS had no effect on binding of the anti-peptide antibody to cytochrome , whilst binding of the monocional antibody was reduced by more than 1000-fold. The anti-peptide antibody partially antagonised the binding of 12/2/3/2 to urea-denatured but not native cytochrome . These data suggest that whilst the complete binding site for the monoclonal antibody is discontinuous, sufficient of the epitope is linear, so that when the antigen is denatured the monoclonal antibody is still able to bind and this binding is antagonised by the anti-peptide antibody. However, inhibition of catalytic activity by the monoclonal antibody must require binding to discontinuous residues.
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Cited by (13)
Identification of epitopes on cytochrome P450 3A4/5 recognized by monoclonal antibodies
2003, Archives of Biochemistry and BiophysicsCitation Excerpt :The complete binding site may be discontinuous, with some residues constituting a conformational epitope so that an antibody that binds may restrict the CYP to a conformation not optimal for catalytic activity [31,45,46].
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.
Epitope mapping of cytochrome P450cam (CYP101)
1997, Archives of Biochemistry and BiophysicsEighteen linear antigenically active sites were revealed in cytochrome P450 fromPseudomonas putida(P450cam) by hexapeptide scanning. These sites occupy about 31% of the protein sequence. Hexapeptide epitope sequences of P450cam are not found in other cytochromes P450. However, several cytochromes P450 contain shorter fragments of P450cam epitope sequences which may cause weak immune cross-reactions. P450cam antigenic determinants are located generally at the boundaries of secondary structure elements. Mapping of P450cam antigenic determinants on the three-dimensional structure of this protein reveals 14 highly water-accessible antigenic sites and only 1 site (No. 322–327, QMLSGL) which is inaccessible to water. Several functionally important sites and amino acid residues of P450cam are localized within revealed linear epitopes or very close to them. These sites include substrate-binding regions, residues responsible for the putidaredoxin interaction (Arg72, Arg112, Lys314, and Arg364), heme binding (Gln108, Arg112, Asp297, Arg299, and Cys357), and proton translocation (Lys178, Arg186, and Glu366).
Targeted antipeptide antibodies to cytochrome P450 2C18 based on epitope mapping of an inhibitory monoclonal antibody to P450 2C5
1997, Archives of Biochemistry and BiophysicsThe epitope recognized by the inhibitory monoclonal antibody designated 2F5, which was raised against P450 2C5, was mapped to amino acids 237–260 by immunoblotting using a combination of recombinant antigens and chimeric and partial fusion proteins constructed from rabbit P450s 2C2, 2C4, 2C5, and 2C16, which are recognized by 2F5, and from 2C1 and 2C3, which are not. When the sequence of the epitope for 2F5 (amino acids 237–260) was compared with those of other rabbit 2C P450s, a single lysine residue at position 253 appeared to be a likely determinant of 2F5 immunoreactivity. Substitution of lysine for glutamic acid 253 in P450 2C3 (2C3E253K) conferred immunoreactivity and the ability of 2F5 to inhibit progesterone metabolism catalyzed by P450 2C3E253K. Sequence alignment revealed that this epitope lies in close proximity to the epitope identified for LKM-1 autoantibodies to P450 2D6. Based on these results, an antipeptide antibody was raised to the corresponding region (amino acids 252–263) of human P450 2C18. The resulting antipeptide antiserum recognizes P450 2C18 but not P450 2C8, 2C9, or 2C19. However, the antipeptide 2C18 antiserum did not inhibit 2C18-catalyzed diazepam N-demethylation. Human 2C P450s were also quantitated by immunoblot analysis in a panel of six human liver microsomes usingEscherichia coliexpressed P450s as standards. Analysis of immunoblots indicated that, if present, P450 2C18 was expressed at very low levels (<2.5 pmol/mg), whereas P450s 2C8, 2C9, and 2C19 were easily detected.
Antigenic mapping of bacterial and animal cytochromes P-450
1996, BiochimieA peptide scanning (PEPSCAN) approach was used for antigenic mapping of two hepatic microsomal cytochromes P450 (rab1A2 and rab2B4) and the microbial cytochrome from Pseudomonas putida (P450 101 or P450cam). This approach includes simultaneous synthesis of pin-linked overlapping hexapeptides covering the whole sequences of three P450s and testing them by ELISA with corresponding polyclonal antisera. Microsomal cytochrome P450 maps were shown to vary depending on an antiserum used for testing the peptides, however, the most active linear B-epitopes were revealed with antisera from two animal species used. P450 linear B-epitopes were classified into individual and group-specific epitopes. While almost all P450 101 linear antigenic determinants are unique for this protein, rab1A2 and rab2B4 contain epitopes both individual for each protein, and subfamily- or even family-specific epitopes. These results point out the possibility of producing both monospecific and group-specific antipeptide antibodies against different P450s. THe antigenic map of P450 101 was superimposed on the structural-functional map of this protein. Its linear B-epitopes were shown to coincide with boundaries of secondary structure elements, with surface-located, water accessible regions and with sites responsible for intermolecular interactions in the Pseudomonas putida monooxygenase system. Several known or predicted functionally active sites in microsomal cytochrome P450 rab1A2 and rab2B4 were also shown to coincide with linear B-epitopes. The peculiarities of epitope locations in protein tertiary structure will allow to predict antigenic regions starting from protein structural information and vice versa, to structural protein models in accordance with antigenic mapping results. Antigenic regions which coincide with sites responsible for intermolecular interactions in monooxygenase systems may be synthesized as separate peptides and used as blockers of such interactions.
Functional assessment of specific amino acid residues of cytochrome P4501A1 using anti-peptide antibodies
1995, Archives of Biochemistry and BiophysicsSeveral positively charged amino acid residues in cytochrome P450 have been shown to be involved in the electrostatic association with NADPH-cytochrome P450 reductase. For cytochrome P4501A1, five regions were proposed as the putative binding sites for the reductase (Shen, S., and Strobel, H. W., (1993) Arch. Biochem, Biophys, 304, 257-265). To elucidate the specific roles of each of these amino acid residues, five anti-peptide antibodies defined as 1A, 4A, 5A, 6A, and 7A were generated against these regions containing 8-13 amino acids and were affinity-purified using a peptide-Sepharose 4B column. Analysis by enzyme-linked immunosorbent assay and protein immunoblot techniques demonstrated that three of five anti-peptide antibodies have specific binding to the peptides as well as to cytochrome P4501A1. Incubation of the various anti-peptide antibodies with cytochrome P450 followed by reconstitution with reductase, lipid, and NADPH resulted in significant inhibition of P450 activity for antibodies 5A and 6A, but not for 1A, 4A or 7A.. Antibody 5A also exhibited inhibition of P450 activity supported by cumene hydroperoxide, though the inhibition was 45 to 30% less than the inhibition of reductase-supported activity at each of the increasing concentrations of antibody. Kinetic studies with antibody 5A revealed no change in the Km for the substrate ethoxycoumarin, but rather a dramatic effect on the Vmax of the cytochrome P4501A1 system whether reconstituted with reductase or supported by cumene hydroperoxide. Characterization of the effects of antibody 5A on cytochrome P4501A1 suggested that the binding of antibody 5A to P4501A1 may change the binding of P4501A1 with reductase. Furthermore, the binding of 5A to cytochrome P4501A1 also lowered the Vmax of the P450. These results are consistent with the roles for the regions of cytochrome P4501A1 from amino acid residues 269 to 281 (peptide 5) and 454 to 463 (peptide 6) in cytochrome P4501A1 hydroxylation activity.
Identification of the epitope of an anti-peptide antibody which binds to CYP1A2 in many species including man
1993, Biochemical PharmacologyAn anti-peptide antibody was raised against the sequence Thr-Gly-Ala-Leu-Phe-Lys-His-Ser-Glu-Asn-Tyr-Lys which occurs at positions 283–294 in the rat cytochrome P450 enzyme CYP1A2. Compared with its binding to the peptide used for immunization, the antibody bound with only slightly reduced affinity to the truncated peptides Thr-Gly-Ala-Leu-Phe-Lys-His-Ser and Leu-Phe-Lys-His-Ser. However, binding to the peptide Ser-Glu-Asn-Tyr-Lys-Asp-Asn, which overlaps with the C-terminal region of the immunizing peptide, was very low. Thus, a major epitope for the anti-peptide antibody is Leu-Phe-Lys-His-Ser, which corresponds to a region of CYP1A2 that is conserved in many species. The antibody was tested by immunoblotting for its ability to bind to hepatic microsomal fractions from a number of species. Where possible animals were treated with compounds which induce CYP1A2 and the results compared with those with untreated animals. It was found that the antibody bound to rat, mouse, rabbit, hamster, guinea pig, pig, marmoset monkey and human CYP1A2. No evidence was found for binding to dog CYP1A2. The region corresponding to the major epitope at residues 286–290 of rat CYP1A2 was identical in mouse, hamster, rabbit and human CYP1A2. The sequence of marmoset and guinea pig CYP1A2 are not known but are predicted to be very similar to the sequence in the rat. The lack of binding of the antibody to dog CYP1A2 may be explained by two differences in this region compared with rat CYP1A2. Maximum inhibition of CYP1A2 activity by this antibody, as measured by high-affinity phenacetin O-deethylase activity, was 20%. This is in contrast to a previously described anti-peptide antibody directed to an adjacent region which caused 65% inhibition of this activity. Thus, the edge of an inhibitory region on the surface of cytochrome P450 has been identified. The ability of the antibody to bind to CYP1A2 from a number of animals should make this antibody of use for studying the levels of CYP1A2 apoprotein in many species.