Role of cytochrome P450 IA2 in acetanilide 4-hydroxylation as determined with cDNA expression and monoclonal antibodies

The role of P450 IA2 in the hydroxylation of acetanilide was examined using an inhibitory monoclonal antibody (MAb) 1-7-1 and vaccinia cDNA expression producing murine P450 IA1 (mIA1), murine P450 IA2 (mIA2), or human P450 IA2 (hIA2). Acetanilide hydroxylase (AcOH) activity was measured using an HPLC method with more than 500-fold greater sensitivity than previously described procedures. This method, which does not require the use of radioactive acetanilide, was achieved by optimizing both the gradient system and the amount of enzyme needed to achieve detection by uv light. MAb 1-7-1 inhibits up to 80% of the AcOH activity in both rat liver microsomes and cDNA expressed mouse and human P450 IA2. MAb 1-7-1, which recognizes both P450 IA1 and P450 IA2, completely inhibits the aryl hydrocarbon hydroxylase (AHH) activity of cDNA expressed in IA1. The inhibition of only 80% of the AHH activity present in MC liver microsomes by MAb 1-7-1 suggests that additional P450 forms are contributing to the overall AHH activity present in methylcholanthrene (MC)-liver microsomes as MAb 1-7-1 almost completely inhibits the AHH activity of expressed mIA1. Maximal inhibition of IA2 by 1-7-1 results in an 80% decrease in acetanilide hydroxylase activity in both liver microsomes and expressed mouse and human IA2. The capacity of MAb 1-7-1 to produce identical levels of inhibition of acetanilide hydroxylase activity in rat MC microsomes (80%) and in expressed mouse (81%) and human P450 IA2 (80%) strongly suggests that P450 IA2 is the major and perhaps the only enzyme responsible for the metabolism of acetanilide. These results demonstrate the complementary utility of monoclonal antibodies and cDNA expression for defining the contribution of specific P450 enzymes to the metabolism of a given substrate. This complementary approach allows for a more precise determination of the inhibitory capacity of MAb with respect to the metabolic capacity of the target P450.     

Immunohistochemical studies on the distribution of cellular myosin II isoforms in brain and aorta.

The distribution of nonmuscle myosin isoforms in brain and aorta was studied by using polyclonal antibodies against two synthetic peptides selected from a region near the carboxyl terminus of bovine brain (peptide IIB) and human macrophage (peptide IIA) myosin. Immunoblots of brain homogenates and purified myosin showed two major bands stained by anti-peptide IIB (MIIB1 and MIIB2) and a minor band stained by anti-peptide IIA (MIIA2). Polyclonal anti-human platelet myosin antibodies did not react with MIIB isoforms. In cryosections from bovine, rat, and mouse brains, anti-peptide IIB stained most neuronal cells. In bovine cryosections, glial staining was also observed. In contrast, anti-peptide IIA and anti-platelet myosin antibodies primarily stained blood vessels. In bovine aorta, the anti-peptide antibodies recognized four bands, MIIB3, MIIB4, MIIA1, and MIIA2. Only MIIA2 was recognized by anti-human platelet myosin antibodies. In bovine aorta cryosections, anti-peptide IIB stained smooth muscle cells in tunica intima and tunica media but did not stain endothelial cells. Anti-peptide IIA stained smooth muscle cells in the tunica media, and endothelial cells of vaso vasorum but not of aorta. Only polyclonal anti-platelet myosin antibodies stained the endothelial cells of aorta tunica intima. These results indicate that multiple isoforms of cellular myosins exist in mammals, that these isoforms are expressed in a cell specific manner, and that the major myosin isoforms isolated from whole brain originate from neurons and, at least in bovine brain, from glia, but not from blood vessels.     

Immunological characterization of constitutive isozymes of cytochrome P-450 from rainbow trout. Evidence for homology with phenobarbital-induced rat P-450s

Immunoglobulin G fractions (IgGs), isolated from rabbits immunized against hepatic cytochrome P-450 isozymes were used to investigate the immunochemical homology among trout P-450s and between trout and rat P-450s. The antigens used for immunization were five constitutive trout P-450s (LMC1 to LMC5), one beta-naphthoflavone (BNF)-inducible trout P-450 (LM4b), and one phenobarbital-induced rat P4500IIB1 (PB-B). In the enzyme-linked immunosorbent assay (ELISA), strong cross-reactivity was observed between anti-LMC2 IgG and P-450 LMC1, and between anti-LMC3 IgG and P-450 LMC4. There was little or no cross-reactivity of anti-LMC5 IgG with other trout P-450s. Trout P-450 LM4b was not recognized by any of the antibodies against constitutive trout P-450s. Antibodies to P-450 LMC1 and P450 LMC2 cross-reacted strongly with rat P450IIB1 and with proteins of PB-induced rat liver microsomes. Rat P450IA1 (BNF-B) did not cross-react with anti-LMC1 or anti-LMC2 IgG. These cross-reactions were essentially confirmed by immunoblot (Western blot) analysis. Western blots of PB-induced rat liver microsomes probed with anti LMC1 revealed two major immunoreactive proteins in the P-450 region, one of which co-migrated with rat P450IIB1. P450IIB1 itself cross-reacted strongly with anti-LMC1 IgG. In control rats, a single protein band cross-reacted poorly with anti-LMC1 IgG. Antibodies to LMC1 and LMC2 did not cross-react with rat P450IA1 in Western blots. The antigenic epitopes in rat P450IIB1 recognized by anti-LMC1 IgG and anti-LMC2 IgG are probably not located at or near the active site of the enzyme since these antibodies did not inhibit benzphetamine N-demethylase activity of P450IIB1 or of PB-induced rat liver microsomes. In general, our results demonstrate: (1) the presence of a significant homology between LMC1 and LMC2, and between constitutive trout P-450 (LMC1) and PB-induced rat P-450 (P450IIB1); and (2) distant homology between constitutive trout P-450s and constitutive rat P-450s or BNF-induced rat P-450s.     

Comparison of rainbow trout and mammalian cytochrome P450 enzymes: evidence for structural similarity between trout P450 LMC5 and human P450IIIA4

Studies were undertaken to determine the immunochemical relationship between constitutive trout cytochrome P450s and mammalian cytochrome P450IIIA enzymes. Polyclonal antibodies (IgG) generated against trout P450 LMC5 reacted strongly with P450IIIA1 in dexamethasone-induced rat liver microsomes and with P450IIIA4 in human liver microsomes in immunoblots. In contrast, rabbit anti-P450 LMC1 IgG did not recognize these proteins in rat and human liver microsomes. Reciprocal immunoblots using anti-rat P450IIIA1 showed that this antibody does not recognize trout P450 LMC1 or LMC5. However, anti-human P450IIIA4 IgG was found to cross react strongly with P450 LMC1 and LMC5. Progesterone 6 beta-hydroxylase activity of trout liver microsomes, a reaction catalyzed by P450 LMC5, was markedly inhibited by anti-P450IIIA4 and by gestodene, a mechanism-based inactivator of P450IIIA4. These results provide evidence for a close structural similarity between trout P450 LMC5 and human P450IIIA4.     

Site-Specific Anti-C-ERB A Antibodies Recognizing Native Thyroid Hormone Receptors: Their use to Detect the Expression and Localization of α and β C-ERB A Proteins in Rat Liver

Abstract

The cell-specific expression and tissue distribution of c-erbA proteins α and β is still unknown. To address this problem, we prepared anti-peptide antibodies directed against epitopes of human (h) c-erbA, specific for the α or β form of thyroid hormone receptors. The cDNAs coding for h c-erbA β1, α1 and α2 were transcribed and the mRNAs were translated in vitro in the presence of 35S-methionine, and then their reactivity with the antisera was evaluated. The antiserum anti-β 62–81 immunoprecipitated only the β1 receptor. The antiserum anti-α 144–162 determined precipitation of both α1 and α2 proteins but not of the β1 receptor. Anti-α2 431–451 produced a selective precipitation of α2, and had no effect on α1 or β1 receptor. In order to study the interaction of the antibodies with native T3 receptor we evaluated the binding of antibodies to rat liver T3 receptors by Sephacryl S300 chromatography: both antisera anti-β 62–81 and anti-α 144–162 caused a partial shift of the labeled T3–receptor complex to a higher molecular form, while the antibody directed against c-erbA α2 did not produce any significant shift. The anti-peptide antibodies were then immunopurified by affinity chromatography and used to immunolocalize the different forms of c-erb A proteins in adult and fetal rat liver, by a sensitive immunohistochemical technique. All 3 antibodies stained mainly the nuclei of the majority of adult liver cells. No staining was detectable when the original antiserum was deprived of anti-peptide antibodies by running through the affinity columns or when the antibodies were pre-absorbed with the homologous peptide. No significant staining was present in the liver from rat fetus.     

Purification and immunological characterization of a novel cytochrome P450 from C3H/10T1/2 cells

The major polycyclic aromatic hydrocarbon-metabolizing cytochrome P450 in the mouse embryo fibroblast-derived C3H/10T1/2 CL8 cell line (P450-EF) has been partially purified from benz[a]anthracene (BA)-induced 10T1/2 cells (40 pmol P450/mg). The purification of P450-EF was carried out by sequential chromatography of solubilized microsomes over hydrophobic aminohexyl-Sepharose 4B, anion exchange DE-52 cellulose, and cation exchange carboxymethyl trisacryl columns. The final preparation (1700 pmol/mg) appeared as a single major 55-kDa band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reconstitution of detergent-free partially purified P450-EF yielded a relatively high turnover for 7,12-dimethylbenz[a]anthracene (DMBA) metabolism (5.4 nmol/nmol/min). Polyclonal antibodies to purified P450-EF (anti-P450-EF), raised in, respectively, rabbit and chicken, detected a single 55-kDa band in 10T1/2 cell microsomes that was highly inducible by BA (approximately 20-fold) and TCDD (approximately 5-fold). Rabbit anti-P450-EF was much more effective than the corresponding chicken antibody at binding denatured P450-EF protein on Western blots. Conversely, only the chicken antibody was effective at inhibiting DMBA metabolism catalyzed by microsomal P450-EF. This antibody did not inhibit P450IA1-mediated DMBA metabolism. Rabbit anti-P450-EF recognized very weakly (less than 1% of homologous protein response) pure P450IA1, IIB1, IIC7, IIE1, and IIIA1 proteins on Western blots but exhibited substantial cross-reactivity (approximately 10%) with pure P450IIA1 and very strong cross-reactivity (approximately 75%) with a hormonally regulated rat adrenal P450. Polyclonal antibodies to several major P450 subfamilies either did not recognize P450-EF (anti-P450IA, IIB, and IIC) or recognized it very weakly (anti-P450IIA1). P450-EF is probably distantly related to the P450IIA subfamily and may belong to a new P450 subfamily.     

Differences in the cytochrome P-450 isoenzymes involved in the 2-hydroxylation of oestradiol and 17 alpha-ethinyloestradiol. Relative activities of rat and human liver enzymes.

The metabolism of oestradiol and 17 alpha-ethinyloestradiol to their 2-hydroxy derivatives is an important determinant in their biological effects. In this work, we have investigated which rat or human cytochrome P-450 isoenzymes are involved in catalysing these reactions. Oestradiol 2-hydroxylation was catalysed by a wide variety of rat cytochrome P-450s from gene families P450IA, P450IIB, P450IIC and P450IIIA. Interestingly, 17 alpha-ethinyloestradiol, which only differs structurally from oestradiol at a position distant from the site of oxidation, was metabolized predominantly by members of the P450IIC gene subfamily. In order to establish which enzymes are responsible for the oxidation of these substrates in man, antibodies to rat liver cytochrome P-450 isoenzymes were used to inhibit these reactions in a panel of human liver microsomal fractions. Also, possible correlations between the proteins recognized by the antibodies and the 2-hydroxylation rate were determined. These experiments provide evidence that 2-hydroxylation of 17 alpha-ethinyloestradiol in man is catalysed by cytochromes from the P450IIC, P450IIE and P450IIIA gene families. In contrast, the major proteins involved in oestradiol metabolism are from the P450IA gene family, although members of the P450IIC and P450IIE gene families may also play a role. These data demonstrate that the differences in the capacity of rat P-450s to metabolize these substrates are also present in the comparable enzymes involved in man, and that a variety of factors will determine the rate of disposition of these compounds in man.     

Site-directed anti-peptide antibodies define the topography of the beta-adrenergic receptor

Molecular cloning has revealed the primary structure of a number of G-protein-linked receptors. The organization and topography of these proteins predicted to have seven hydrophobic membrane-spanning domains, in contrast, have not been established. Antibodies were prepared against 11 peptides corresponding to each of the hydrophilic sequences of the hamster beta 2-adrenergic receptor. Each of the anti-peptide antibodies displayed immunoreactivity for its synthetic peptide antigen and beta 2-adrenergic receptor (Mr 65,000) on blots of cell membranes and of purified receptor. All but three anti-peptide antisera also displayed immunoreactivity toward human placental and rat fat cell beta 1-adrenergic receptors, reflecting the level of sequence identity that exists between the two subtypes, Chinese hamster ovary cells stably transfected with an expression vector harboring the cDNA encoding the hamster beta 2-adrenergic receptor provided a cell type with 2 million receptors/cell, suitable for in situ localization of the sequences used as antigens. Indirect immunofluorescence of intact and permeabilized cells performed with these site-directed anti-peptide antibodies permitted the assignment of the general topography of each of the hydrophilic sequences of this G-protein-linked receptor. The results support the predictive value of hydropathy analysis for one class of membrane proteins with multiple transmembrane-spanning domains.     

Antibodies targeted against hypervariable and constant regions of cytochromes P450IIB1 and P450IIB2

Fusion proteins constructed between beta-galactosidase and six different segments of either cytochrome P450IIB1 or cytochrome P450IIB2 (ranging from 18 to 33 amino acids in length) were expressed in Escherichia coli. Rabbit antibodies raised against these fusion proteins were first adsorbed through a beta-galactosidase column and then immunopurified on a second column containing the corresponding fusion protein. With the exception of the antibodies directed against the hydrophobic amino-terminal segment of cytochrome P450IIB1, all the antipeptide antibodies recognized the major phenobarbital-inducible cytochromes P450IIB1 and -IIB2 on immunoblots of liver microsomal proteins. Two of the antibodies were raised against regions where cytochromes P450IIB1 and -IIB2 differ in primary structure, and were differentially reactive toward these two highly homologous cytochromes. Several of the antipeptide antibodies were also reactive with a third phenobarbital-inducible microsomal protein expressed in livers of some individual Sprague-Dawley rats which was shown to be more highly related to P450IIB1 than P450IIB2. This P450IIB1-related P450, designated P450IIB1*, was purified to apparent homogeneity and shown to hydroxylate the steroid hormones testosterone and androstenedione with the well-defined regiospecificity and high catalytic activity characteristic of P450IIB1. A fourth microsomal protein detected using the antipeptide antibodies appeared to be more highly related to P450IIB2. Because the segments on the P450 molecules recognized by these antipeptide antibodies are known, it is possible to predict where P450IIB1* and the P450IIB2-related protein differ from cytochromes P450IIB2 and -IIB1, respectively. These studies demonstrate the utility of site-specific anti-P450 antibodies raised to fusion peptides for studies on the expression of structurally related P450s and polymorphic variants within the cytochrome P450 gene superfamily.     

An anti-peptide antibody targeted to a specific region of rat cytochrome P-450IA2 inhibits enzyme activity.

An anti-peptide antibody has been produced which binds to and specifically inhibits the activity of cytochrome P-450IA2 in rat hepatic microsomes. This was achieved by raising an antibody against a synthetic peptide (Ser-Glu-Asn-Tyr-Lys-Asp-Asn), the sequence of which occurs in cytochrome P-450IA2 at positions 290-296. The selection of this region of cytochrome P-450IA2 was based on several criteria, including prediction of surface and loop areas, identification of variable regions between cytochromes P-450IA2 and P-450IA1, and consideration of a site on cytochrome P-450IA1 where chemical modification has been shown to cause substantial enzyme inactivation. The specificity of antibody binding was determined by enzyme-linked immunosorbent assay and by immunoblotting using hepatic microsomal preparations and purified cytochrome P-450 isoenzymes. This showed that the antibody binds specifically to rat and mouse cytochrome P-450IA2 and to no other cytochrome P-450, as was predicted from the amino acid sequences of the peptide and the cytochromes P-450. The effect of the antibody upon enzyme activity was studied in hepatic microsomes from rats treated with 3-methylcholanthrene. The antibody was shown to inhibit specifically the activity of reactions catalysed by cytochrome P-450IA2 (phenacetin O-de-ethylase and 2-acetylaminofluorene activation), but had no effect on aryl hydrocarbon hydroxylase activity, which is catalysed by cytochrome P-450IA1, or on aflatoxin B1 activation.     

Antibodies to synthetic antibody-combining sites. Antibodies against a surface-simulation peptide with antibody-combining activity toward lysozyme antigenic site 3 react with lysozyme antibodies

Recently, we reported the synthesis and immunochemistry of two peptides designed, by complementarity and surface-simulation synthesis, to mimic antibody-combining sites against two antigenic sites of lysozyme. In the present work antibodies were raised against one of these peptides, which is complementary to antigenic site 3 of lysozyme, to determine whether these antibodies will react with anti-lysozyme antibodies. Radioiodinated antipeptide antibodies were bound by immunoadsorbents of the immune IgG from two goats anti-lysozyme antisera but not by adsorbents of myoglobin, non-immune goat IgG or immune IgG of antisera against cytochrome c. The binding of anti-peptide antibodies to adsorbents of anti-lysozyme antibodies was fully inhibited by free lysozyme but not by bovine serum albumin, human hemoglobin A, horse cytochrome c or bovine ribonuclease A. Thus, antisera against an antibody-combining site can be raised by immunizing with a peptide which probably does not exist in the antibody but is designed by surface-simulation synthesis to mimic an antibody-combining site.     

Monoclonal antibodies against human cytochrome P-450 recognizing different pregnenolone 16 alpha-carbonitrile-inducible rat cytochromes P-450.

Six murine monoclonal antibodies against human hepatic cytochrome P-450 have been raised, using human liver microsomes (microsomal fractions) or semi-purified human cytochrome P-450 as immunogen. All six antibodies recognized the same highly purified of human liver cytochrome P-450 of molecular mass 53 kDa and gave rise to a single band at 53 kDa on immunoblots of human liver microsomes from 11 individuals. The antibodies also recognized proteins at 52 kDa and 54 kDa on immunoblots of control and induced male-rat liver microsomes, showing four different banding patterns. Antibodies HL4 and HP16 recognized a 52 kDa protein that was only weakly expressed in untreated rats and which was strongly induced by pregnenolone 16 alpha-carbonitrile (PCN) but not by phenobarbitone (PB), 3-methylcholanthrene (3MC), isosafrole (ISF), Aroclor 1254 (ARO), clofibrate or imidazole. HP10 and HL5 recognized a constitutive 52 kDa protein that was weakly induced by PCN but not by the other agents and was suppressed by 3MC and ARO. HP3 recognized a 54 kDa protein that was undetectable in control rats but was strongly induced by PB, PCN, ISF and ARO. HL3 appeared to recognize a combination of the proteins recognized by the other antibodies plus a 54 kDa protein that was weakly expressed in control rats. The constitutive proteins recognized were male-specific.     

Identification of cytochrome P450IA2 as a human autoantigen

Autoantibodies occurring in a patient with idiopathic autoimmune type chronic active hepatitis (CAH) were found to react with purified rabbit cytochrome P450IA2 and to a much lesser extent with P450IA1. Both cytochrome P450s are known to be inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the rabbit, and the expression of the microsomal protein recognized by the patient serum was induced in adult rabbit livers after treatment with TCDD. This protein is only weakly detected in liver microsomes from neonatal rabbits exposed to TCDD in utero, which is consistent with the age-dependent induction of P450IA2 by TCDD. The serum specifically reacted with a protein of similar size in microsomes prepared from COS-1 cells transfected with an expression vector containing the full length human P450IA2 cDNA. This reactivity was not detected in the cells transfected with the vector alone, indicating that the antibody recognizes human P450IA2. In addition, the serum extensively inhibited 7-ethoxyresorufin O-deethylation catalyzed by isolated human liver microsomes. This catalytic activity is associated with class IA P450s in other species. A screen of sera from patients with various hepatic and nonhepatic diseases indicates that the autoantibody to P450IA2 occurs rarely in CAH. Cytochrome P450IA2 becomes the third P450 identified as an autoantigen in inflammatory liver diseases.     

Acetaminophen activation by human liver cytochromes P450IIE1 and P450IA2

Acetaminophen (APAP), a widely used over-the-counter analgesic, is known to cause hepatotoxicity when ingested in large quantities in both animals and man, especially when administered after chronic ethanol consumption. Hepatotoxicity stems from APAP activation by microsomal P450 monooxygenases to a reactive metabolite that binds to tissue macromolecules, thereby initiating cellular necrosis. Alcohol consumption also causes the induction of P450IIE1, a liver microsomal enzyme that in reconstitution studies has proven to be an effective catalyst of APAP oxidation. Thus, elevated microsomal P450IIE1 levels could explain not only the known increase in APAP bioactivating activity of liver microsomes after prolonged ethanol ingestion but also the enhanced susceptibility to APAP toxicity. We therefore examined the role of P450IIE1 in human liver microsomal APAP activation. Liver microsomes from seven non-alcoholic subjects were found to convert 1 mM APAP to a reactive intermediate (detected as an APAP-cysteine conjugate by high-pressure liquid chromatography) at a rate of 0.25 +/- 0.1 nmol conjugate formed/min/nmol microsomal P450 (mean +/- SD), whereas at 10 mM, this rate increased to 0.73 +/- 0.2 nmol product/min/nmol P450. In a reconstituted system, purified human liver P450IIE1 catalyzed APAP activation at rates threefold higher than those obtained with microsomes whereas two other human P450s, P450IIC8 and P450IIC9, exhibited negligible APAP-oxidizing activity. Monospecific antibodies (IgG) directed against human P450IIE1 inhibited APAP activation in each of the human samples, with anti-P450IIE1 IgG-mediated inhibition averaging 52% (range = 30-78%) of the rates determined in the presence of control IgG. The ability of anti-P450IIE1 IgG to inhibit only one-half of the total APAP activation by microsomes suggests, however, that other P450 isozymes besides P450IIE1 contribute to bioactivation of this compound in human liver. Of the other purified P450 isozymes examined, a beta-naphthoflavone (BNF)-inducible hamster liver P450 promoted APAP activation at rates even higher than those obtained with human P450IIE1. The extensive APAP-oxidizing capacity of this hamster P450, designated P450IA2 based upon its similarity to rat P450d and rabbit form 4 in terms of NH2-terminal amino acid sequence, spectral characteristics, immunochemical properties, and inducibility by BNF, agrees with previous reports concerning the APAP substrate specificity of the rat and rabbit P450IA2 proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Assessment of P450 induction in the marmoset monkey using targeted anti-peptide antibodies

The identity and expression of hepatic P450 enzymes in marmosets was investigated using a panel of anti-peptide antibodies originally targeted against human P450 enzymes. In immunoblotting, of 12 antibodies examined, 10 bound specifically to bands in marmoset liver microsomal fraction corresponding to P450 enzymes. It is proposed that these represent marmoset CYP1A1, CYP1A2, CYP2A, CYP2B, CYP2C forms (CYP2C-1 and CYP2C-2), CYP2D19, CYP3A21 and another CYP3A form (CYP3A-m). The antibodies, together with an anti-marmoset CYP2E1 antibody, were used to investigate the expression of 10 P450 enzymes in marmosets treated with P450-inducing chemicals. Treatment with phenobarbitone caused CYP2B, CYP2C-2 and CYP3A21 levels to increase, rifampicin caused increases in CYP2B and CYP2C-1 and a decrease in CYP3A21 levels, whereas dioxin caused CYP1A1 and CYP1A2 levels to increase and CYP2E1 levels to decrease. Clofibric acid did not induce any P450. P450 enzyme activities were assessed using 8 different substrates and increases were found after treatment with phenobarbitone, rifampicin, and dioxin. However, due to species differences in substrate selectivity, it proved difficult to ascribe these changes to individual P450 enzymes. Thus, the use of anti-peptide antibodies provides a more informative way of assessing the levels of specific P450 enzymes than enzyme activity measurements.     

Human thyroid peroxidase: mapping of autoantibodies, conformational epitopes to the enzyme surface

The enzyme, thyroid peroxidase (TPO), is a dominant antigen in thyroid autoimmune diseases. Autoantibodies recognised two major dominant conformational epitopes termed A and B. The epitopes have been defined by mAbs, but the amino acid residues which constitute these determinants remain unknown. Using a model of TPO, built from the structure of myeloperoxidase (MPO), we have synthesised peptides corresponding to exposed loops and generated rabbit antibodies to the peptides. Antisera to peptide sequence 599-617 (peptide 14) representing a highly protrusive loop on the TPO, showed the highest inhibition in 65 sera from patients positive with anti-TPO antibodies. The inhibition was by 15-80% (mean 41%), and no other antibody showed any inhibition. Binding of hFabs to the B determinant on TPO was inhibited by anti-peptide 14 antibodies more then 85%, but not Fabs to the A determinant. In conclusion, the peptide 14 defines a sequence taking part in building up the B major conformational epitope. None of generated anti-peptide antibodies alone inhibited the binding of human Fabs to the A epitope, however a combination of four anti-peptide antibodies (P1, P12, P14 and P18) inhibits Fabs binding to the A determinant by more then 60% and autoantibodies binding from 65% to 94%. Combination of antibodies reacting with peptides outside the surface defined by those four antipeptide antibodies did not give any inhibition of Fabs to TPO. The inhibition of Fabs and auto Abs to TPO by this combination of anti-peptide Abs is the result of steric hindrance as none of these Abs individually inhibited auto Abs' or Fabs' binding to TPO. The four peptides define an area on the enzyme surface where the A and B major conformational epitopes are localised.     

Studies with antibodies against the conserved cysteine region of progesterone receptor

Polyclonal antibodies were generated against two synthetic peptides corresponding to sequences from the DNA-binding domain of steroid receptors. The sequence for peptide 1 (13 amino acids) lies between the two putative metal-binding loops of the conserved cysteine region while the sequence for peptide 2 (12 amino acids) lies within one loop. Peptide antibodies were generated by injecting rabbits with peptide conjugated to bovine serum albumin. By Western blot analysis, antibodies to peptide 2 recognized chick and human progesterone receptor and human glucocorticoid receptor, but peptide 1 antibodies did not. No cross-reactivity with native chick progesterone receptor was detected with either anti-peptide. These findings suggest that the epitopes for peptide 2 antibodies, and possibly for peptide 1 antibodies, are inaccessible to antibody in the native receptor.     

Human thyroid peroxidase: mapping of autoantibodies,conformational epitopes to the enzyme surface

Abstract

The enzyme, thyroid peroxidase (TPO), is a dominant antigen in thyroid autoimmune diseases. Autoantibodies recognised two major dominant conformational epitopes termed A and B. The epitopes have been defined by mAbs, but the amino acid residues which constitute these determinants remain unknown. Using a model of TPO, built from the structure of myeloperoxidase (MPO), we have synthesised peptides corresponding to exposed loops and generated rabbit antibodies to the peptides.

Antisera to peptide sequence 599–617 (peptide 14) representing a highly protrusive loop on the TPO, showed the highest inhibition in 65 sera from patients positive with anti-TPO antibodies. The inhibition was by 15–80% (mean 41%), and no other antibody showed any inhibition. Binding of hFabs to the B determinant on TPO was inhibited by anti-peptide 14 antibodies more then 85%, but not Fabs to the A determinant. In conclusion, the peptide 14 defines a sequence taking part in building up the B major conformational epitope.

None of generated anti-peptide antibodies alone inhibited the binding of human Fabs to the A epitope, however a combination of four anti-peptide antibodies (P1, P12, P14 and P18) inhibits Fabs binding to the A determinant by more then 60% and autoantibodies binding from 65% to 94%. Combination of antibodies reacting with peptides outside the surface defined by those four anti-peptide antibodies did not give any inhibition of Fabs to TPO.

The inhibition of Fabs and auto Abs to TPO by this combination of anti-peptide Abs is the result of steric hindrance as none of these Abs individually inhibited auto Abs' or Fabs' binding to TPO.

The four peptides define an area on the enzyme surface where the A and B major conformational epitopes are localised.     

Comparison of the immunochemical properties of human placental and bovine adrenal cholesterol side-chain cleavage enzyme complex

The immunochemical relatedness between human and bovine proteins catalyzing the cholesterol side-chain cleavage reaction was investigated. In dot-immunobinding analysis, antibodies against bovine adrenocortical cytochrome P-450SCC, adrenodoxin, and adrenodoxin reductase recognized the corresponding proteins in a dose-dependent manner in mitochondrial preparations from human placenta. Limited proteolysis with trypsin cleaved bovine P-450SCC into fragments F1 and F2, which represent the NH2- and C-terminal parts of P-450SCC, respectively. Identical trypsin treatment yielded similar-size fragments from human placental P-450SCC. In Western immunoblots, anti-F1 and anti-F2 antibodies recognized the corresponding fragments in both trypsin-digested bovine and human P-450SCC. Antibodies against bovine P-450SCC, fragments F1 and F2, adrenodoxin and adrenodoxin reductase inhibited cholesterol side-chain cleavage activity in bovine adrenocortical mitochondria by 24-51%, but failed to affect the activity in human placental mitochondria. These data indicate that human and bovine P-450SCC share common antigenic determinants located outside the enzyme active site. The immunological similarity between bovine adrenodoxin and human ferredoxin allowed for a simple purification protocol of human placental P-450SCC by adrenodoxin affinity chromatography. The P-450SCC obtained by this method was electrophoretically homogeneous and showed characteristics typical to P-450SCC.