Molecular Human Reproduction, Vol. 6, No. 2, 113-126,
February 2000
© 2000 European Society of Human Reproduction and Embryology
Endocrinology |
Human placental gonadotrophin-releasing hormone-like factors: an artefact of human placental peptidases?
The University of Edinburgh Department of Obstetrics and Gynaecology, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, UK
Abstract
Non-denatured human placental cytosol fractions displaced tracer binding in parallel with gonadotrophin-releasing hormone (GnRH) isoform and agonist peptides in GnRH-specific radioimmunoassays and radioreceptor assays. However, placental immuno- and receptor binding-GnRH-like activity was highly correlated with inactivation of GnRH tracers, suggesting that placental GnRH-like factors may be an artefact of ligand degradation during assay. The properties and inhibitor sensitivities of the major 125I-labelled GnRH-degrading enzymes of term placental cytosol were studied using a dextran-coated charcoal (DCC) adsorption assay as a rapid screen for GnRH tracer inactivation. Three different activities were demonstrable: (i) a cathepsin D-like enzyme (Mr 55 kDa), active against all radiolabelled GnRH isoforms and agonists tested, optimal at acid pH, and inhibited specifically by pepstatin; (ii) a metallo-thiol endopeptidase activity (Mr 70 kDa) optimal at alkaline pH (7–9) which degraded GnRH isoforms to a greater extent than GnRH analogues, inhibited dose-dependently by low concentrations of thiol reagents (N-ethylmaleimide, thimerosal), chelating agents (o-phenanthroline, EDTA), and by tosyl-phenylalanyl-chloromethyl ketone but not by other serine protease inhibitors; and (iii) a bacitracin-sensitive enzyme optimal at physiological pH. These observations permitted the development of a robust radioreceptor assay which minimized GnRH tracer degradation. Under these assay conditions, the GnRH-like radioreceptor assay activity of human placental cytosol fractions was markedly reduced.
GnRH receptor/peptidases/placenta
Introduction
The secretion of human chorionic gonadotrophin (HCG) is stimulated by gonadotrophin-releasing hormone (GnRH) in vivo (Iwashita et al., 1993
) and in vitro (Siler-Khodr, 1987; Currie and Leung, 1993; Li et al., 1994; Leung and Peng, 1996). GnRH acts as a component of a complex local regulatory system involving inhibin peptides (Petraglia et al., 1996, 1998), interleukins (Nishino et al., 1990), prostaglandins (Kang et al., 1991), and steroid hormones (Siler-Khodr et al., 1986a; Petraglia et al., 1995; Chen et al., 1998). Human placenta expresses the gene for mammalian (m)GnRH (Seeburg and Adelman, 1984; Radovick et al., 1990; Duello et al., 1993; Wolfahrt et al., 1998), although the upstream start site differs from that used in the hypothalamus (Seeburg et al., 1987; Dong et al., 1993). GnRH stimulation of HCG secretion can be blocked by GnRH antagonists (Siler-Khodr et al., 1983, 1986b; Barnea et al., 1991; Szilagyi et al., 1992), and the GnRH-receptor gene is expressed in cytotrophoblast and syncytiotrophoblast cells (Lin et al., 1995; Wolfahrt et al., 1998). The receptor gene is similar to that expressed in human pituitary (Kakar and Jennes, 1995; Leung and Peng, 1996; Boyle et al., 1998): however, the affinity and specificity of pituitary and extrapituitary GnRH receptors differ (Bramley et al., 1992).
Most species express more than one isoform of GnRH (Sherwood et al., 1993; King and Millar, 1995). Other isoforms of GnRH have been described in mammals (Rissman et al., 1995; Kasten et al., 1996; Jimenez-Linan et al., 1997; Lescheid et al., 1997; Quanbeck et al., 1997) including the human (White et al., 1998), and are expressed in bovine placenta (Duello and Boyle, 1997). Furthermore, other GnRH-like factors are present in human reproductive tissues (Li et al., 1987), and post-translationally modified forms of GnRH are active in the human placenta (Gautron, Pattou and Kordon, 1981; Gautron et al., 1992; Currie et al., 1993), suggesting that ligands other than mGnRH play a role in the human placenta. Both authentic GnRH (Osathanondh and Elkind-Hirsch, 1981; Tan and Rousseau, 1982; Zhuang et al., 1991; Raga et al., 1999) and larger GnRH-like peptides (Mathialagan and Rao, 1986a,b; Zhuang et al., 1991) have been reported to be present in human placental tissue. However, many extraction procedures utilize treatments (boiling acid, organic solvents) which may allow the recovery of GnRH, but which will denature larger molecular weight forms. On the other hand, attempts to isolate GnRH-like factors from undenatured human placental extracts may be confounded by degradation of GnRH tracer during the radioimmunoassays and radioreceptor assays used to detect activity (Bramley et al., 1999). This was well demonstrated for human placenta by Siler-Khodr et al. (1989) who showed that a large molecular weight `GnRH-like factor' purified from human placenta was a post-proline peptidase (C-ase 1; Kang and Siler-Khodr, 1992) which degraded GnRH and other peptide hormones.
The aims of this study were: (i) to establish whether placental GnRH-like activity was due to an endogenous GnRH-like factor(s), or simply to peptidase interference in the GnRH assays employed and (ii) to develop radioimmunoassay and radioreceptor assays for placental GnRH that were insensitive to peptidase interference. The studies were therefore focused primarily on those GnRH isoforms and analogues that are good receptor-binding ligands for human placental GnRH receptors (Bramley et al., 1992).
Previous studies of GnRH degradation have mostly utilized tritiated mGnRH or high concentrations of peptide, coupled with high performance liquid chromatography (HPLC; Carone et al., 1987; Siler-Khodr et al., 1989; Maggi et al., 1993, 1995; Lew et al., 1994a,b; Kerschler et al., 1995; Leibovitz et al., 1995) or thin layer chromatography (TLC; Kuhl and Baumann, 1981; Leibovitz et al., 1994, 1995). HPLC is a powerful technique which can resolve the major GnRH degradation products and, together with mass spectroscopy (Brudel et al., 1994) or aminoacid analysis of the purified fragments produced (Elkabes et al., 1981; Sandow et al., 1982), allows definitive assignment of the cleavage site(s) in GnRH. However, HPLC is costly, requires relatively large amounts of peptides, and cannot screen more than a few samples in a working day. Moreover, a single HPLC system cannot resolve all the many possible degradation products that could be formed from GnRH isoforms and analogues, and, in the absence of mass spectroscopy, HPLC requires a detailed knowledge of the chromatographic behaviour of all possible GnRH peptide fragments for each particular HPLC resolution system. Although some information is available for (non-iodinated) mGnRH and a few agonist analogues, such data are not yet available for other (iodinated) GnRH isoforms and analogues. Furthermore, the pattern of degradation products formed may vary at different GnRH concentrations (Elkabes et al., 1981).
Because of these limitations, a number of studies have used indirect measurements of gross inactivation of GnRH or GnRH tracers, relying on techniques to distinguish intact from degraded hormone such as fluorescamine fluorescence (Horsthemke et al., 1981), radioimmunoassay of (intact) GnRH (Kuhl et al., 1979; O'Connor and Mahesh, 1988; Bramley and Menzies, 1996), loss of 125I-labelled GnRH re-binding to human receptors (Clayton et al., 1979; Menzies and Bramley, 1992; Bramley and Menzies, 1996), or differential adsorption of intact and degraded GnRH peptides (Horsthemke and Bauer, 1981; Berger et al., 1988; Bramley et al., 1999). However, binding assays require large amounts of receptor, and are feasible only for those GnRH tracers, isoforms or analogues which bind to the particular receptor employed. Thus, GnRH agonists mGnRH, salmon GnRH (sGnRH) and chicken GnRH II (cGnRH II) can be used as binding ligands for human placental membranes (but not other isoforms or GnRH antagonists; Bramley et al., 1992), whereas only mGnRH, GnRH agonists and GnRH antagonists (but not other GnRH isoforms) can be used as tracers to study binding to rat pituitary receptors. Finally, most anti-GnRH antibodies are intentionally specific for a particular GnRH isoform(s).
In a search for a simple, rapid and inexpensive screening method for estimating GnRH-tracer inactivation which: (i) could measure degradation not only of 125I-labelled GnRH, but also of other GnRH-receptor-active isoforms and analogues, (ii) was independent of GnRH receptor or antibody specificity, (iii) was unaffected by incubation conditions, (iv) did not require large amounts of receptor or antiserum, and (v) could screen large numbers of samples simultaneously, an assay was developed based on the adsorption of intact (but not degraded) GnRH tracers to dextran-coated charcoal (DCC; Bramley et al., 1999). With appropriate controls, the method was found to resolve degraded forms of GnRH and its analogues from intact tracers, and could measure inactivation of GnRH tracers under conditions where tracer degradation was difficult to demonstrate by TLC and HPLC.
The different properties and inhibitor sensitivities of the major placental cytosol GnRH-degrading enzymes were exploited to develop a radioreceptor assay in which GnRH tracer degradation was minimal. Under these conditions, the GnRH-like activity of human placental cytosol fractions was dramatically reduced.
Materials and methods
Materials
All fine chemicals, enzyme inhibitors, peptides, anti-GnRH antibodies and other reagents were obtained from sources described previously (Bramley et al., 1999). GnRH peptides were radio-iodinated using a glucose oxidase/lactoperoxidase method, and were purified by chromatography on Sephadex G25 columns (Bramley et al., 1992). Specific activities of radiolabelled mono-iodinated GnRH isoform preparations (85–1100 Ci/g; n = 9 separate GnRH isoform preparations) were estimated by self-displacement assay (Clayton, 1983) using a conformation-dependent anti-GnRH antibody (EL14), and specific activities of GnRH agonist tracers, by self-displacement assay of binding to immature female rat pituitary homogenates (350–1105 Ci/g, n = 12 preparations).
Tissues
Rat pituitaries were obtained from adult female Sprague–Dawley rats and homogenized in ice-cold 0.3 mol/l sucrose/10 mmol/l Tris/1 mmol/l EDTA, pH 7.4 (SET medium; two glands/ml) using a loose-fitting all-glass Dounce homogenizer. Aliquots (2 ml) were snap-frozen in solid CO2, and stored at –20°C until required.
Human placentae were obtained from spontaneous vaginal deliveries (n = 20) or from elective Caesarean section at term (n = 2) from normal women. Approval for these studies was obtained from the Human Reproduction Ethical Committee of the Lothian Health Board Hospitals Trust. Placental villous tissue was dissected free of other tissues, washed extensively in ice-cold isotonic phosphate-buffered saline (PBS: Flow Laboratories, Irvine, UK) to reduce blood contamination, and villi homogenized in ice-cold SET buffer (5 ml/g) using a Polytron homogenizer (two 10 s bursts at full speed, separated by a 1 min cooling period in ice). After filtration through four layers of cheesecloth, homogenates were centrifuged at 1000 g for 10 min (4°C), and supernatants re-centrifuged at 100 000 g for 60 min in a Sorvall OTD-50 refrigerated (4°C) ultracentrifuge. The microsomal pellets obtained were gently rehomogenized (5–10 strokes in a loose Dounce homogenizer) in SET medium, and supernatants (cytosol) and membranes stored in 2 ml aliquots at –70°C or in liquid nitrogen until required.
Assays
Protein was measured by the method of Lowry et al. (1951) with crystalline bovine serum albumin as a standard.
125I-Labelled GnRH binding assays
Receptor binding
Specific binding of radiolabelled GnRH agonist and GnRH isoforms to human placental membranes was measured in triplicate as described previously (Bramley et al., 1992). Non-specific binding was measured in duplicate in the presence of 10 µg of unlabelled buserelin ([D-Ser (tBu)6]GnRH ethylamide). After incubation (usually 1 h at 20°C), bound hormone was recovered by polyethyleneglycol (PEG) precipitation, and pellets counted for 125I in a Packard `Crystal' gamma-counter at an efficiency of 75%. The difference between binding with and without unlabelled agonist represented specific binding (7–35% of total counts added). Controls without tissue, with and without unlabelled GnRH agonist, were included to correct for displacement of tracer from assay tubes by cold analogue (0.5–1% of total counts added).
Specific binding of radiolabelled GnRH agonists to rat pituitary receptors was measured as described above, except that incubation was for 4 h at 4°C.
Antibody binding
Binding of 125I-labelled GnRH tracers (20 000–30 000 c.p.m. per tube) to a conformational anti-GnRH antibody (EL14; final dilution, 1:30 000) or to anti-cGnRH II antisera (Ab 10.2 or Ab 11.3; final dilution, 1:20 000–1:50 000; Sharp et al., 1987, 1989) was performed as described previously (Bramley et al., 1992).
Measurement of GnRH tracer inactivation
A previous study of a range of different methods for estimation of degradation of GnRH tracers demonstrated that adsorption to DCC with TLC was a sensitive and rapid measure of tracer inactivation (Bramley et al., 1999).
Dextran-coated charcoal assay
Aliquots of placental cytosol (5–100µl) were incubated for 1 h at 20°C (except where stated) in a 0.5 or 1 ml system containing 40 mmol/l Tris–HCl (or 0.1 mol/l citrate or glycine buffers) of the appropriate pH, containing 0.1% (w/v) bovine serum albumin (BSA) and 30 000–100 000 c.p.m. of 125I-labelled GnRH analogue as indicated. Tubes were immediately immersed in crushed ice, and vigorously stirred ice-cold dextran-coated charcoal (DCC; 2.5 g/l Norit A activated charcoal plus 0.25 g/l Dextran T70 in Tris-BSA) was added to each tube. After vortexing vigorously and centrifuging at 2500 g for 10 min (4°C), aliquots of supernatant (500 µl) were carefully transferred to clean tubes, and unadsorbed 125I present in the DCC supernatants counted.
Thin layer chromatography
Aliquots (5–10µl) of 125I-labelled GnRH tracer (incubated with or without placental cytosol, and with or without adsorption to DCC, as indicated) were spotted onto polyethyleneimine cellulose TLC plates, dried under a stream of cold air, and chromatographed in 0.1 mol/l ammonium bicarbonate, pH 7.8. TLC plates were dried, wrapped in clingfilm and placed in a Molecular Dynamics (Symantech, CA, USA) phosphorimager cassette for 12–72 h. Radioactivity associated with each spot was quantified using ImageQuant Software (Molecular Dynamics; IQMac, version 1.2).
Calculations and statistics
Statistical significance of differences between means was estimated by Student's t-test with Bessel's correction for small numbers, or by Wilcoxon's rank order test. P < 0.05 was considered to be statistically significant.
Results
Validation of dextran-coated charcoal adsorption assay of GnRH tracer integrity
Dextran-coated charcoal (DCC) efficiently adsorbed all radiolabelled GnRH isoforms and analogue tracers tested over a wide pH range (>90% at 50 µl DCC/ml; data not shown). Radiolabelled cGnRH II incubated in the absence of placental cytosol was adsorbed by increasing concentrations of DCC (Figure 1A). TLC confirmed the presence of a single radioactive spot, migrating at the origin of the plate, which had the same mobility as intact 125I-labelled cGnRH II (Figure 2; lane 1). The intensity of this spot decreased with increasing DCC concentrations (Figure 2; lanes 1–9). There was an excellent correlation between the intensity of this spot and the amounts of tracer recovered in the supernatant following DCC treatment (Figure 1B; r2 = 0.987).
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) or with 10 µl (•), 25 µl (
) or 50 µl (
) of a term placental cytosol preparation. Points are means of triplicate estimates for a single experiment. Similar data were obtained in a number of less complete experiments. (B) Correlation between cGnRH II tracer unadsorbed by DCC and intensity of intact cGnRH II quantified by phosphorimaging after thin layer chromatography (TLC). (C) Plot of slope of adsorption curves for intact cGnRH II tracer after incubation with different placental cytosol concentrations (data taken from Figure 1B
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Following incubation of 125I-labelled cGnRH II with human term placental cytosol (1 h at 20°C, pH 7.4), the amount of tracer recovered in the supernatant following DCC treatment increased, shifting DCC adsorption curves to the right (Figure 1A
). This was accompanied by a marked reduction in the amount of intact cGnRH II recovered at the TLC origin, whilst a new major radioactive spot (Rf 0.45) appeared (Figure 2; lanes 10–18). (Note: Rf is the mobility of the compound expressed as a fraction of the mobility of the solvent front.) Variable amounts of a minor spot (Rf 0.15) were observed in some experiments. There was an excellent inverse correlation between the amount of novel product(s) formed and intact tracer remaining (r2 = 0.96; data not shown). Unlike intact tracer (Figure 2; lanes 2–9), the Rf 0.45 spot was decreased only slightly with increasing DCC (Figure 2; lanes 11–18). The slope of curves of the adsorption of cGnRH II tracer with increasing DCC (Figure 1B) was increased by placental cytosol in a concentration-dependent manner (Figure 1C), and there was a strong linear correlation between the amount of novel products formed and radioactivity recovered in the DCC supernatants (Figure 1D; r2 = 0.922). The appearance of novel TLC products of cGnRH II and the right-shift in DCC adsorption curves were abolished following incubation of tracer: (i) in the absence of cytosol at 20°C, (ii) with cytosol at 0°C, or (iii) with boiled placental cytosol at 20°C (data not shown).
Thus (compared to intact 125I-labelled cGnRH II) degraded forms of radiolabelled cGnRH II were adsorbed poorly by DCC (Figure 2). Moreover, incubation of placental cytosol with radioiodinated cGnRH II, mGnRH buserelin and [D-Trp6]GnRH ethylamide ([D-Trp6]GnRH EtA) also right-shifted DCC adsorption curves for these tracers, and the shift induced was greater for mGnRH and cGnRH II compared to GnRH agonists (data not shown), suggesting that the latter tracers were more resistant to degradation. TLC of each tracer demonstrated that novel products were indeed generated following incubation with cytosol, though (except for cGnRH II) the products formed were often difficult to resolve from intact tracers by TLC (data not shown).
Effects of cytosol on integrity of 125I-labelled cGnRH II
Influence of temperature and duration of incubation
Incubation of radiolabelled cGnRH II with placental cytosol at 20°C led to a time- and temperature-dependent loss of intact tracer (TLC origin), with a corresponding increase in the amounts of degraded products formed (Figure 3A). No degradation of cGnRH II tracer was observed following incubation with boiled cytosol, however (Figure 3A, lane 13). Moreover, DCC treatment prior to TLC adsorbed intact cGnRH II tracer (Figure 3A, lanes 2 and 15), whereas much of the novel Rf 0.45 product formed following incubation with placental cytosol persisted even after DCC treatment (Figure 3A, lanes 10 and 14). The effects of incubation with placental cytosol on the integrity of radiolabelled cGnRH II (and [D-Trp6]GnRH EtA) were confirmed by high resolution liquid chromatography (see Bramley et al., 1999; data not shown).
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) and Rf 0.45 (•) products was expressed as percentage radioactivity at each time point, and plotted against duration of incubation at 20°C (B) and 37°C (C). (D) After removal of 5 µl aliquots for TLC, ice-cold DCC was added (30 µl/tube), tubes were vortexed, centrifuged (2500 g for 15 min) and radioactivity recovered in the DCC supernatant plotted against duration of incubation at 4°C (Quantification of TLC spot intensities demonstrated a time- and temperature-dependent loss of intact cGnRH II which was more rapid at 37°C (Figure 3C
) than at 20°C (Figure 3B) or 4°C (data not shown). Loss of intact cGnRH II was accompanied by the appearance of two novel products. The formation of the high mobility peak (Rf 0.45) was preceded at both temperatures by the formation of the lower mobility peak (Rf 0.15; Figure 3B,C), suggesting a precursor–product relationship. The recovery of 125I-labelled cGnRH II in the supernatant after DCC treatment (Figure 3D) correlated well with the combined concentrations of the degradation products formed at both temperatures (Figure 3E; r2 = 0.95).
Although DCC treatment (10–30 µl DCC per 1 ml assay incubation) adsorbed most (>80%) of the intact GnRH II tracers (by TLC), degraded products were reduced only slightly in intensity (Figure 1A; Figure 2, lanes 10–18; Figure 3A). However, since DCC did adsorb these degraded products to some extent, control incubations (tracer incubated without cytosol but treated with the same DCC concentration) were routinely included in all subsequent assays. DCC adsorption of GnRH isoform and agonist tracers was unaffected by incubation pH (between pH 4 and 10) or by metal ion concentration (up to 10 mmol/l for divalent ion and 100 mmol/l for monovalent metal ions respectively; data not shown).
Effects of pH on degradation of cGnRH II tracer
Radiolabelled cGnRH II incubated without cytosol remained at the TLC origin at all pH values (Figure 4A). However, cGnRH II tracer incubated with cytosol (1 h, 20°C) at neutral and alkaline pH showed the loss of the spot at the origin (intact cGnRH II), with the coincident appearance of the Rf 0.45 spot (Figure 4B; lanes 9–14). At acid pH (<6; Figure 4B, lanes 1–5), a low mobility product (Rf <0.05) was formed in the presence of cytosol.
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GnRH specificity of placental cytosol GnRH-degrading enzymes
At all pH values, GnRH degradation measured by the DCC assay was dependent on cytosol concentration (Figure 5
). However, the rate of degradation of different GnRH isoforms and analogue tracers varied at different incubation pH. Thus, at acid pH (pH 3–4), degradation of buserelin was more rapid than for [D-Trp6]GnRH EtA and cGnRH II (Figure 5A), whereas at physiological and alkaline pH, cGnRH II was degraded to a greater extent than buserelin or [D-Trp6]GnRH EtA (Figure 5B) and other GnRH isoforms (Figure 5C). Degradation of all three tracers was minimal at pH 6 (Figure 5D).
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The effects of incubation pH on the degradation of several GnRH analogues and isoforms were investigated in more detail. The effects of incubation pH on 125I-labelled cGnRH II degradation (see Figure 4B
) were confirmed using the DCC adsorption assay (Figure 6A). Three distinct cGnRH II-degrading activities were detected, the first with an acid pH optimum (between pH 3 and 4), a second activity optimal at alkaline pH (pH 8–9) which was the sole activity observed in citrate buffers, and a third activity optimal at pH 6–8 which was abolished in citrate buffers. 125I-Labelled buserelin (Figure 6B) and [D-Trp6]GnRH EtA (Figure 6C) were also degraded extensively at acid pH, but little inactivation was observed at neutral and alkaline pH (particularly in citrate buffers), suggesting that introduction of a C-terminal ethylamide and/or a D-amino acid residue at position 6 rendered the GnRH peptide resistant to peptidase attack at neutral/alkaline pH. (Production of degraded forms of [D-Trp6]GnRH EtA was confirmed by TLC and high performance liquid chromatography; see Bramley et al., 1999.) Radiolabelled mGnRH (Figure 6D), sGnRH (Figure 6E), cGnRH I (Figure 6F) and lamprey GnRH I (lGnRH I; data not shown) were also degraded extensively at acid pH values; however, degradation at neutral and alkaline pH was lower than for cGnRH II tracer.
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Effects of protease inhibitors
The effects of a range of protease inhibitors with broad specificity for the major classes of proteases (Barrett, 1977
) on placental GnRH-degrading activities were examined. At acid pH (3–4), degradation of GnRH tracers (buserelin, cGnRH II and [D-Trp6]EtA) was markedly reduced in a concentration-dependent manner by low concentrations of pepstatin A, a carboxypeptidase inhibitor (Figure 7A). Inhibitors specific for other classes of protease were without effect. Incubation of [D-Trp6]GnRH EtA tracer with increasing pepsin concentrations at pH 3.5 mimicked the dose-dependent effects of placental cytosol in the DCC assay, whereas pronase, trypsin, collagenase, and proteinase K were much less effective (data not shown).
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). Antipain, tosyl-phenylalanyl-chloromethyl ketone (TPCK), aprotinin, soybean trypsin inhibitor, Thimerosal, bacitracin and tosyl-phenylalanyl-chloromethyl ketone (TPCK; an inhibitor of chymotrypsin-like serine proteases) inhibited degradation of 125I-labelled cGnRH II at low concentrations at neutral and alkaline pH (Figure 7B
). However, trypsin inhibitors [tosyl-lysyl-chloromethyl ketone (TLCK), 
1-antitrypsin, soybean trypsin inhibitor], and inhibitors of serine proteases (phenylmethylsulphyl fluoride, benzamidine, aprotinin), endopeptidase 2 (phosphoramidon, bestatin; Sakurada et al., 1990; Kerschler et al., 1995), proteasomal peptidases (3,4 dichloro-isocoumarin; Vinitsky et al., 1992) and calpains (N-acetyl Leu-Leu-norLeu and N-acetyl Leu-Leu-methional) all failed to prevent cGnRH II degradation (Figure 7A,B). Degradation of cGnRH II tracer by placental cytosol at physiological and alkaline pH was also reduced by high concentrations of the divalent metal ion-chelating agent, EDTA (Figure 7B), and was partially inhibited (50–70%) by the thiol reagent N-ethylmaleimide (N-EM), as judged by inhibition of tracer recovery in DCC supernatants (Figure 7B) and by quantification of degraded products by TLC (Figure 7D). Inhibitory concentrations of N-EM also reduced the right-shift of DCC adsorption curves induced by placental cytosol (data not shown). The specificities of pepstatin as an inhibitor of cGnRH II degradation by the acid activity, and of N-EM in (partially) inhibiting cGnRH II degradation at alkaline pH are shown in Figure 7C. Effects of inhibitors on the formation of the Rf 0.15 product were more difficult to assess due to its rapid metabolism to the Rf 0.45 product, but its appearance was prevented by bacitracin, and by high concentrations of captopril, an inhibitor of angiotensin converting enzyme, and N-EM (data not shown).
Gel exclusion chromatography of placental cytosol
Fractionation of human placental cytosol on a precalibrated Sephadex G100 column demonstrated the presence of a high molecular weight `GnRH-like' activity (Mr ~70 kDa) which inhibited specific binding of radiolabelled cGnRH II to human placental membranes (Figure 8A). Specific binding of 125I-labelled [D-Trp6]GnRH EtA to rat pituitary membrane GnRH receptors was also decreased by these same fractions, though inhibition was less marked (Figure 8B). These same fractions also had high amounts of GnRH immunoactivity, as shown by their ability to decrease binding of radiolabelled cGnRH II to a specific anti-cGnRH II antibody (Figure 8C) and a conformational anti-GnRH antibody (Figure 8D). However, assay of these fractions under conditions specific for the thiometallopeptidase [Tris buffer, pH 8; radiolabelled cGnRH II; inclusion of pepstatin and benzamidine (an inhibitor of C-ase 1; Siler-Khodr et al., 1989)] demonstrated that this activity was also maximal in these fractions (Figure 8E). In contrast, specific assay of placental cathepsin-like activity (citrate buffer, pH 4; radiolabelled buserelin; inclusion of N-EM, EDTA, TPCK and benzamidine) demonstrated a peak of this activity at Mr ~55 kDa (Figure 8F).
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Effects of inhibition of GnRH degradation on GnRH-like activity in placental cytosol
The effects of placental cytosol on the specific binding of 125I-labelled cGnRH II and two GnRH agonist tracers to placental receptors were studied under normal receptor-binding incubation conditions, and under conditions where tracer degradation was reduced or abolished by a cocktail of substances which inhibited GnRH tracer degradation by placental peptidases. In the absence of inhibitors, cytosol decreased cGnRH II binding in a dose-dependent manner (Figure 9A
), in parallel with the agonist standard (data not shown; see Bramley et al., 1999). Inhibition of cGnRH II binding by placental cytosol was reduced significantly by inclusion of pepstatin, EDTA, N-EM and TPCK at 20°C, but completely abolished when the binding assay was performed on ice with a cocktail of inhibitors including in addition benamidine and bacitracin (Figure 9A). As reported previously (Bramley et al., 1999), placental cytosol had a minimal effect on the binding of the two (degradation-resistant) GnRH agonists in the absence of inhibitors, and inclusion of inhibitor cocktail reduced this minimal activity further, suggesting that the GnRH-like activity of placental cytosol was attributable largely, if not entirely, to GnRH tracer degradation (Figure 9B).
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Discussion
Many studies have described distinct GnRH-degrading activities in hypothalamus, pituitary, serum and several other tissues. Among the activities described are: (i) post-proline peptidase, (ii) carboxypeptidase C, (iii) pyroglutamyl aminopeptidase, (iv) and enzymes which attack GnRH at Tyr5-Gly6 or (v) Gly6-Leu7 (Chertow, 1981; Griffiths and McDermott, 1983; Friedman et al., 1984; Bauer and Horsthemke, 1985; Koch et al., 1985; Carone et al., 1987; Berger et al., 1988; Molineaux et al., 1988; Lasdun and Orlowski, 1990; Kang and Siler-Khodr, 1992; Wu et al., 1997; Cunningham and O'Connor, 1998). GnRH-degrading enzymes may have important physiological actions and be under hormonal control (Griffiths and McDermott, 1983; O'Connor and Mahesh, 1988; Lasdun and Orlowski, 1990; Contijoch and Advis, 1993; Maggi et al., 1993; Lew et al., 1997; Wu et al., 1997).
Degradation of GnRH also occurs in human placental tissue (Bramley and Menzies, 1996; Bramley et al., 1999), and may be an important regulator of GnRH paracrine action in the placenta. Using a rapid and cheap assay to measure 125I-labelled GnRH-degradation, it was shown that human placental cytosol fractions contained three distinct peptidase activities. (This is a minimal estimate of placental peptidases, as placentae were homogenized in EDTA-containing buffers; hence other metallopeptidases may have been inactivated.)
Enzyme (i) was optimal at acid pH, with a broad specificity for GnRH isoforms and analogues (including GnRH agonists containing D-amino acid substitutions at Gly6 and a Pro9-ethylamide group), and was inhibited specifically by pepstatin A, suggesting that it was a cathepsin D-like enzyme which probably acts at a hydrophobic–aromatic region common to all tracers tested (e.g. Trp3 or Trp/Leu7). Though the molecular weight (Mr 55kDa) resembled that of C-ase 1, a 58kDa placental post-proline peptidase (Siler-Khodr et al., 1989; Kang and Siler-Khodr, 1992), enzyme (i) was not inhibited by benzamidine and was optimal at acid pH.
Enzyme (ii) was optimal at pH 8–9, appeared to degrade cGnRH II preferentially (Figure 6A), though with some activity against other GnRH isoforms and agonists, particularly in non-citrate buffers (Figure 6B–F). It was partially inhibited in a concentration-dependent fashion by N-EM (Figure 9B, D) and by TPCK, thimerosal, and high concentrations of EDTA (Figure 9B), suggesting that it was a metallothiol peptidase. Its pH profile (alkaline pH optimum), Mr (70–80 kDa), substrate specificity (poor activity against GnRH agonists with a D-amino acid at Gly6) and inhibitor sensitivity of this activity (inhibited by N-EM, TPCK and high concentrations of EDTA) most closely resembles cytosolic enzymes in the sheep and rat hypothalamus and pituitary similar to EC 3.4.24.15 (Chertow, 1981; Horsthemke and Bauer, 1981; Horsthemke et al., 1981; Griffiths and McDermott, 1983; Lasdun and Orlowski, 1990; Lew et al., 1994a,b; Smith et al., 1994; Wu et al., 1997), which cleave between residues 5–6 (though cleavage at residues 6–7 is also possible; Elkabes et al., 1981; Koch et al., 1985). Indeed, the post-proline peptidase of bovine serum has a similar Mr (69.7 kDa) and pH optimum (pH 8.0–8.5), and is also affected by the composition of the incubation buffer (Cunningham and O'Connor, 1998). Moreover, this activity is a serine protease, but has a cysteine residue close to the active site, properties which could account for its inhibition by both TPCK and sulphydryl reagents. However, some properties of C-ase 1, the post-proline peptidase isolated from human placenta (Kang and Siler-Khodr, 1992), differ from those of enzyme (ii).
Enzyme (iii) appeared to act specifically on cGnRH II at neutral pH (pH 6.0–7.5), though only in non-citrate buffers (Figure 6A), and had minimal activity towards other GnRH isoforms and agonists tested. Radiolabelled degradation products of other GnRH tracers did not have a similar mobility on TLC (Figure 6B and unpublished data), suggesting that the products derived from radiolabelled cGnRH II by this enzyme may be derived from cleavage before the 125I-Tyr8 residue in cGnRH II, possibly at the 6–7 or 7–8 bond. The lack of activity towards sGnRH and lGnRH I (data not shown) suggests that isoforms with a Trp7 residue are not attacked. Enzyme (iii) was inhibited by bacitracin, a post-proline peptidase inhibitor. GnRH 1–9 product formed may then be rapidly cleaved at the 6–8 positions by enzyme (ii). Indeed, though enzyme (iii) was inhibited by bacitracin, activity was also reduced by high concentrations of thimerosal and N-EM (data not shown). This would be predicted if two enzymes acted together to degrade cGnRH II. However, other peptidases cleaving mGnRH (at 1–2, 6–7 and 9–10) are also sensitive to bacitracin (Chertow, 1981). Taking into account pH optimum, size (77 kDa), inhibitor specificity (bacitracin, high N-EM), and low activity towards GnRH agonists (Horsthemke et al., 1981), it is suggested that enzyme (iii) cleaves cGnRH II to a unique radiolabelled product (Rf 0.15) which is then degraded further to the Rf 0.45 product by enzyme (ii). TLC of cGnRH II tracer degradation by cytosol at physiological pH demonstrated a clear precursor–product relationship between the Rf 0.15 and Rf 0.45 products (Figures 3B,C).
Further characterization to establish the bonds cleaved under different incubation conditions and the sequence of degradation of each (iodinated) GnRH isoform or analogue by placental cytosolic GnRH-degrading enzymes will require HPLC–mass spectroscopy analysis of the different peptides released. Such studies are beyond the scope of the present article: however, the GnRH-degradation assay described herein and the characteristics of these activities should facilitate purification and further studies of placental GnRH-inactivating enzymes. Moreover, the ratio of Rf 0.15 and Rf 0.45 products varied markedly for different placental cytosol preparations under similar incubation conditions (Figures 2, 3A and 4B). Experiments are in progress to estimate amounts of each peptidase activity in placentae from different stages of gestation.
Significant high molecular weight placental `GnRH-like' activity was demonstrated in a receptor-binding assay (incubation, 20°C) with radiolabelled cGnRH II as the binding ligand (Figure 8A) which competed in parallel with GnRH isoform and agonist standards (data not shown). These same fractions also had high GnRH immunoactivity (Figures 8C,D) and suppressed binding of GnRH agonist tracer to rat pituitary receptors (Figure 8B), though inhibition of rat pituitary binding by cytosol was less marked than for placental receptors. This was probably due to (a) the use of degradation-resistant agonist tracers and (b) the low binding incubation temperature (4°C) for measurement of pituitary GnRH binding.
125I-Labelled cGnRH II binding to placental microsomes was inhibited in a dose-dependent manner by the inclusion of placental cytosol in the binding assay (Figure 9A). In contrast, the inhibitory effects of cytosol were less marked with GnRH agonists as binding ligands, confirming our previous results (Bramley et al., 1999). Exploiting the characteristics of human placental GnRH-degrading enzymes, we measured the inhibition of GnRH tracer binding by placental cytosol was measured employing binding assay conditions (citrate buffer, pH 6; incubation temperature, 20°C; inclusion of EDTA, N-EM, TPCK and pepstatin) where tracer degradation estimated by TLC was greatly reduced (<70%; data not shown). The GnRH-like activity of placental cytosol fractions in the GnRH-binding assay was significantly reduced with cGnRH II as binding ligand (Figure 9A), and virtually abolished with the two GnRH agonist tracers (Figure 9B). Moreover, inclusion of benzamidine and bacitracin to the inhibitor cocktail, plus conducting the binding incubation on ice, completely abolished inhibition of cGnRH II binding by cytosol. The co-elution of placental GnRH-like receptor-binding, GnRH immuno-activity and alkaline GnRH-degrading activity from Sephadex G100 (Figure 8), coupled with the demonstration that the effects of placental cytosol on GnRH receptor-binding can be abolished under circumstances where ligand degradation is abolished (Figure 9), strongly suggests that the apparent `GnRH-like' activity of human term placental extracts is due solely to the degradation of GnRH tracers by placental peptidases.
Acknowledgments
We should like to thank Dr J.Sandow for the GnRH agonist, buserelin, Dr G.D.Niswender, Dr H.Urbanski and Prof. P.Sharp for their generous gifts of anti-GnRH antibodies, and the staff of the Department of Obstetrics and Gynaecology and the Simpson Memorial Maternity Pavilion, Edinburgh for the provision of placentae. Thanks are also due to Tom McFetters and Ted Pinner for photographic assistance.
Notes
1 To whom correspondence should be addressed
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Submitted on August 12, 1999; accepted on November 1, 1999.
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D. Islami, D. Chardonnens, A. Campana, and P. Bischof Comparison of the effects of GnRH-I and GnRH-II on HCG synthesis and secretion by first trimester trophoblast Mol. Hum. Reprod., January 1, 2001; 7(1): 3 - 9. [Abstract] [Full Text] [PDF]
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