Short synthetic peptides exploited for reliable and specific targeting of antibodies to the C-termini of cytochrome P450 enzymes

doi:10.1016/0006-2952(94)00395-3

Biochemical Pharmacology

Volume 49, Issue 1, 6 January 1995, Pages 39-47

https://doi.org/10.1016/0006-2952(94)00395-3 Get rights and content

Abstract

An antibody was raised against a synthetic peptide (Ser-Glu-Asn-Tyr-Lys-Asp-Asn) corresponding to residues 290–296 of the cytochrome P450 enzyme, CYP1A2, of both rat and mouse. A cysteine residue attached to the N-terminus of the peptide during synthesis allowed coupling in a specific orientation via the thiol group to the carrier protein, keyhole limpet haemocyanin. Antiserum raised in rabbits bound specifically to CYP1A2 in the rat and mouse. To determine those amino acid residues involved in binding of the antibody, related peptides of various lengths were synthesised and the binding of the antibody was determined by an enzyme-linked immunosorbent assay. These studies show that the minimum epitope is the C-terminal tripeptide sequence, Lys-Asp-Asn. Other than in rat and mouse CYP1A2, this tripeptide is found as an internal sequence in a large number of proteins including bovine: fibronectin, chicken gizzard myosin heavy chain, and the P450 enzymes, rabbit CYP3A6 and human CYP3A4, but the antibody did not bind to any of these proteins. However, the antibody did bind to yeast glucose-6-phosphate dehydrogenase in which the tripeptide sequence is the C-terminus. Antibodies raised against a truncated peptide (Tyr-Lys-Asp-Asn), representing the C-terminal half of the peptide, also bound to glucose-6-phosphate dehydrogenase, but failed to bind to CYP1A2; thus although the C-terminal region of the peptide 290–296 is strongly immunogenic, it appears that it is not this population of antibodies that binds to CYP1A2. As antibodies were found to bind strongly to the C-terminus of glucose-6-phosphate dehydrogenase, the C-termini of proteins as targets for anti-peptide antibodies were investigated further by immunising rabbits with four 5-residue peptides which represent the C-termini of the P450 enzymes, CYP1A1, CYP1A2, CYP2E1 and CYP2A6. The peptides were coupled to keyhole limpet haemocyanin through their N-termini via cysteine residues added to the sequences. All four antisera bound specifically to their respective target proteins, as demonstrated by immunoblotting using hepatic microsomal fractions from rat, rabbit and human. It is suggested that this method of antibody production could be of general use for the reliable production of antisera against proteins where their sequence at the C-terminus is known, and such antibodies can be highly specific as they do not bind to internal sequences.

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P450 enzymes in the CYP2D subfamily have been suggested to contribute to the susceptibility of individuals in developing Parkinson's disease. We have used specific anti-peptide antisera and peroxidase immunohistochemistry to investigate the expression of CYP2D enzymes in the rat brain and some possible factors that may affect their regulation. In male Wistar rats, CYP2D1 was not detected in the basal ganglia or in any other brain region. CYP2D2 was weakly expressed within neurones of the subthalamic nucleus, substantia nigra and interpeduncular nucleus as well as in the hippocampus, dentate gyrus, red nucleus and pontine nucleus. CYP2D3 and CYP2D4 were absent from the basal ganglia, although moderate amounts of CYP2D3 were detected within fibres of the oculomotor root, and very low levels of CYP2D4 were present in white matter tracts. In contrast, CYP2D5 was extensively expressed in the basal ganglia, including neurones in the subthalamic nucleus, substantia nigra and interpeduncular nucleus, as well as other areas of the brain, including the ventral tegmental area, piriform cortex, hippocampus, dentate gyrus, medial habenular nucleus, thalamic nucleus and pontine nucleus. Lesioning of the nigro-striatal tract to cause almost a complete loss of tyrosine hydroxylase containing neurones in the substantia nigra, also reduced the number of neurones expressing CYP2D5 by 50%, indicating that CYP2D5 is expressed in dopaminergic neurones. Castration of pre-pubertal or adult Wistar rats had no effect on the number of CYP2D5-positive neurones in the substantia nigra. Although Dark Agouti rats lack hepatic CYP2D2, expression in the midbrain was similar to that of Wistar rats; furthermore, there was no difference in expression or distribution between male and female rats. In contrast to naive rats, extensive expression of CYP2D4 was found throughout the basal ganglia and in other brain nuclei in Wistar rats treated with not only clozapine, but also saline, suggesting that CYP2D4 may be induced as a result of mild stress. The function of CYP2D enzymes in the brain remains unknown, but their selective localisation suggests a physiological role in neuronal activity and in adaptation to abnormal situations.
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^†: Present address: Laboratory of Experimental Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, U.S.A.

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Research paperShort synthetic peptides exploited for reliable and specific targeting of antibodies to the C-termini of cytochrome P450 enzymes

Abstract

Methods Enzymol

J Biol Chem

Biochem Pharmacol

J Immunol Methods

Methods Enzymol

J Biol Chem

Mol Immunol

J Immunol Methods

Mol Immunol

J Immunol Methods

J Biol Chem

Biochem Pharmacol

J Biol Chem

Arch Biochem Biophys

J Immunol Methods

Biochem Biophys Res Commun

Antibodies that react with predetermined sites on proteins

Science

Cellular proteins reactive with monoclonal antibodies directed against simian virus 40 T-antigen

J Virol

HLA-D region alpha-chain monoclonal antibodies: cross-reaction between an anti-DP alpha-chain antibody and smooth muscle

J Pathol

Unexpected immunoreactivities of intermediate filament antibodies in human brain and brain tumors

Am J Pathol

The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature

DNA Cell Biol

Research paper
Short synthetic peptides exploited for reliable and specific targeting of antibodies to the C-termini of cytochrome P450 enzymes