INHIBITORY ANTI-CYP 3 A 4 PEPTIDE ANTIBODY : MAPPING OF INHIBITORY EPITOPE AND SPECIFICITY TOWARD OTHER CYP 3 A ISOFORMS

An antipeptide antibody has been produced that recognizes CYP3A4 and exhibits greater than 90–95% inhibition on CYP3A4mediated reactions [Wang RW and Lu AYH (1997) Drug Metab Dispos 25:762–767]. The inhibitory epitope of the 21-amino acid peptide, corresponding to residues 253 to 273 of CYP3A4, has been identified to reside in a 7-amino acid sequence (LEDTQKH: residues 261–267 of CYP3A4). This conclusion was based on the reversal of antibody inhibition of testosterone 6b-hydroxylation when peptides with overlapping sequence in this region were preincubated with the antibody. In immunoblotting analysis, this antibody did not recognize CYP3A5 or CYP3A7 in microsomes prepared from baculovirus-infected cells containing these two expressed isoforms. In addition, the antipeptide antibody did not inhibit testosterone 6b-hydroxylation or midazolam 1*and 4-hydroxylation in microsomes containing expressed CYP3A5 and CYP3A7. Because the corresponding sequence in CYP3A5 (LNDKQKH) and CYP3A7 (LKETQKH) differs from CYP3A4 by only two amino acids, six peptides with either one or two amino acid changes were used to determine which amino acid is essential for antibody-antigen interaction. Our data indicate that Glu, Asp, and Thr in the 7-amino acid sequence of CYP3A4 are critical determinants of selectivity among CYP3A isoforms. At least three structurally related cytochrome P-450 isoforms in the CYP3A gene subfamily are known to exist in human liver (Wrighton and Stevens, 1992; Guengerich, 1995). CYP3A4, the predominant form, is expressed in virtually all adult human livers, whereas CYP3A5 is polymorphically expressed in about 20 to 30% of adult human livers. CYP3A7, on the other hand, is fetal liver specific. All three enzymes metabolize a variety of therapeutic agents and endogenous substrates, although it is uncertain whether these three isoforms exhibit identical substrate specificities. Because of their structural relatedness, no chemical probes are available to distinguish these enzymes. In addition, antibodies produced against CYP3A4 generally recognize other CYP3A isoforms (Gelboin et al., 1995; Belloc et al., 1996). To establish the contribution of each one of the CYP3A isoforms in the metabolism of a specific substrate in human liver microsomes, it is desirable to have antibodies inhibiting only one of the three CYP3A isoforms. One way to achieve this goal is to use a peptide with a unique amino acid sequence as antigen, so that antibodies can discriminate minor differences in the amino acid sequence among structurally related isoforms. In a previous paper (Wang and Lu, 1997), we described the production of both inhibitory and noninhibitory antiCYP3A4 antibodies against a 21-amino acid peptide (residues 253– 273 of CYP3A4). Preliminary immunoblotting data indicated that these antibodies recognize only CYP3A4 and not CYP3A5. In the present study, we examined further the specificity of this antipeptide antibody toward CYP3A5 and CYP3A7. The inhibitory epitope on this 21-amino acid peptide has been mapped to reside in a 7-amino cid sequence. Moreover, we established that three amino acids, Glu, Asp, and Thr, in this short sequence play a critical role in recognizing CYP3A4 isoforms by this unique antipeptide antibody. Materials and Methods Materials. Testosterone, 6 b-hydroxytestosterone, glucose 6-phosphate, NADP, and glucose 6-phosphate dehydrogenase were purchased from Sigma (St. Louis, MO). Midazolam, 1 9-hydroxy midazolam, and 4-hydroxy midazolam were gifts from Hoffmann La Roche (Nutley, NJ). All other reagents and solvents were of high analytical grade supplied by Fisher Scientific (Fair Lawn, NJ). Vectors (pCRII and pBlueBac) and insect cells (sf21) were obtained from Invitrogen Corp. (Carlsbad, CA). The peptides were synthesized on an Applied Biosystems 430A peptide synthesizer using solid-phase chemistry by PeptidoGenic Research & Co, Inc. (Livermore, CA). The purity of synthesized peptides was greater than 90% by high-performance liquid chromatography (HPLC) 1 and IonSpray mass spectrometry analysis. Human liver microsomal preparations were kindly provided by Dr. Judy Raucy (Agouron Institute, La Jolla, CA). Microsomes prepared from baculovirus-infected cells containing CYP3A5 were obtained from Gentest Corp. (Woburn, MA). Antibody against purified CYP3A4 protein was kindly provided by Dr. Jerome Lasker (Mount Sinai Medical Center, New York, NY). Inhibitory antipeptide antibody against a 21-amino acid peptide (VKRMKESRLEDTQKHRVDFLQ: residues 253–273 of CYP3A4) was prepared in this laboratory as described (Wang and Lu, 1997). Cloning and Expression of CYP3A4 and CYP3A7 in Baculovirus System. The coding sequence for CYP3A7 was amplified from a fetal human liver Quick-Clone cDNA library (Clontech Laboratories, Inc., Palo Alto, CA) using a forward primer, 59-GATCTAGATGGATCTCATCCCAAACTTGGCCG-39, and a reverse primer, 5 9-GTCTCTAGAGCAAACCAGAAGTCCTTAGG-39. The PCR fragment was cloned into pCRII, and the plasmid was transformed in Escherichia coli. The entire cDNA fragment was sequenced from both direcPart of this work was presented previously at the Experimental Biology 98 in