Neurohormone gonadoliberin (GnRH), a decapeptide released from median eminence to portal blood, is the primary regulator of reproductive system activity through the stimulation of pituitary gonadotropin secretion and consequent stimulation of gametogenesis and steroidogenesis in gonads (1). Like other bioactive peptides, GnRH production can be regulated at the transcriptional, translational and post-translational levels, leading to increased or decreased synthesis and release from GnRH neurons. Since there are no known reuptake systems for peptides, the concentration of GnRH that reaches the anterior pituitary appears to be determined also by the extent of degradation by specific proteolytic enzymes (2). Indeed, GnRH was shown to be degraded both in the hypothalamus and the anterior pituitary gland by two endopeptidases acting in a stepwise manner (3). Moreover, a short half-life of the GnRH molecule demonstrated in early studies in vitro (4) seems to be a direct consequence of its low resistance to proteolytic enzymes activity. Detailed studies revealed that the splitting of GnRH AA bonds involves the activity of five endopeptidases. Pyroglutamate aminopeptidase EC 3.4.19.3 splits the pGlu1-His2 bond, endopeptidase EC 3.4. 24.11 is responsible for His2-Trp3, Ser4-Tyr5 and Gly6-Leu7 s proteolysis whereas endopeptidase EC 3.3.25.15 acts on the Tyr5-Gly6 bond. Angiotensin I (EC 3.4.15.1) splits the Trp3-Ser4, Leu7-Arg8 bonds and postproline endopeptidase EC 3.4.21.26 responds to Pro9-Gly10NH2 proteolysis. For therapeutic reasons the search for GnRH primary structure modification in terms of enhancing its stability attained special importance.

So far more than 2000 known chemically modified GnRH analogs with agonistic or antagonistic effects on GnRH receptor activity are known (5). GnRH analogs are useful tools extensively employed in the treatment of sex hormone dependent diseases such as reproductive cancers, precocious puberty, and endometriosis (6), but when combined with steroid hormone replacement they may also be considered as contraceptives for men (7) and women (8). GnRH agonists exhibit a significantly longer half-life which makes them more applicable both in basic research and clinical applications (9). For example, replacing Gly6 with the D-phenylalanine caused 2.5-fold greater stability of the obtained analog, while the substitution of Gly-NH2 with ethyloamide in D-Trp6-GnRH molecule created an analog with increased resistance to degradation by human placenta enzymes (10). In contrast, Tyr5 methylation provided [Tyr-(OME)5]-GnRH molecule characterized by either a lower affinity for GnRH receptor (11) or the reduced ability of GnRH receptor desensitization as demonstrated in fish pituitary cells (12). The substitution of D-amino acid at position 6 or by lactam ring involving residues 6 and 7, resulted in an increased GnRH receptor binding affinity (13), whereas modification at the Tyr5-Gly6 was shown to generate a “super-agonistic” GnRH analog which remained non- degraded up to 4 h of incubation with metalloendopeptidase EP.24.15 (14).

Although the therapeutic properties of GnRH analogs are well known, their peptide nature conveys rapid metabolic clearance and poor oral bioavailability. To overcome these problems alternative approaches were proposed which enabled the development of nonpeptide small molecule GnRH antagonists (15) or the conjugation of moieties to GnRH peptide antagonists such as hydroxylated progesterone to GnRH analogs (16).

Studies on GnRH analogues that contain moieties related to the cytotoxic complexes cisplatin [cis-diamminedichloroplatinum(II)] and trans-bis (salicylaldoximato)copper(II) revealed that metals may affect GnRH activity. The incorporation of PtCl2 or trans-bis (salicylaldoximato)copper(II) into the D-Lys6-GnRH molecule was reported to generate agonistic analogs being, respectively, 55 or 25 times more effective in the stimulation of LH release than GnRH (17). Moreover, metallopeptide analogs also exhibited high affinities for the membrane receptors of rat pituitary and human breast cancer cells (17). A broad spectrum of physiological properties which are affected by peptide-metal complexes raised the question of their resistance to proteolysis. In our previous study, copper-gonadoliberin (Cu-GnRH) complex originally synthesized at the Faculty of Chemistry, University of Wroclaw, was compared to non-complexed GnRH molecule in order to characterize their chromatographic properties (18). Briefly, RP-HPLC analysis demonstrated that both peptides eluted as an excellent, close to symmetrical, peaks: Cu-GnRH eluted at 30.3±0.2 min whereas GnRH at 31.5±0.2 min. Moreover, GnRH and Cu-GnRH peaks were absent from the blank when the optimized liquid chromatographic elution program was used. Photodiode and fluorescence detection also revealed molecule-dependent differences in UV and fluorescence responses indicating that the presence of copper considerably modifies spectral properties of Cu-GnRH.

The aim of this study was to determine whether the Cu-GnRH complex differs from GnRH in its susceptibility to endopeptidase-dependent proteolysis in hypothalamic and pituitary tissue in vitro. Therefore, the time-dependent changes of exogenous GnRH and Cu-GnRH content within incubation in the presence of male rat hypothalamic/pituitary supernatant and pellet fractions were analyzed with the RIA method. Moreover, HPLC analysis was applied both to characterize the elution pattern of GnRH and Cu-GnRH degradation products as well as their AA composition.


MATERIAL AND METHODS
GnRH and bacitracin were purchased from Sigma Aldrich (St. Louis, MO) and 125INa from Hartmann Analytic (Munich, Germany). The Cu-GnRH complex was synthesized according to the method previously described by Kozlowski et al. (19). Briefly, GnRH and copper acetate (Cu(CH3COO)2x2H2O) were mixed in equimolar peptide to a metal ratio then stirred and simultaneously heated up to 40°C for 1 h and then ice-cooled. After water evaporation, stechiometry and purity of the obtained complex were determined by elementary analyses as well as an electrospray ionisation-mass spectrometry (ESI-MS).

Hypothalami and pituitaries were obtained from 35, 4-month old male Wistar rats. Immediately after dissection, hypothalmi and pituitaries were collected in, respectively, 30 ml or 10 ml ice-cold 0.01 M Tris-HCI buffer (pH 7.6) with 50 mM KC1 and 12 mM MgCl2 and then homogenized in a glass homogenizer. After centrifugation (1000 g for 10 min) the obtained supernatants (containing cytosol fraction) and pellets (containing remnants of cell membranes) were used for further analysis. 0.25 ml of supernatant (an equivalent of 0.5 mg of the respective tissue) was placed into incubation tubes with 0.30 ml of Tris-HCl buffer (with or without 125 µg/sample of bacitracin) and 2.5 µg/50 µl of GnRH or Cu-GnRH. Pellets were resuspended in 0.55 ml Tris-HCl buffer (with or without 125 µg/sample of bacitracin) and 2.5 µg/50 µl of GnRH or Cu-GnRH. All samples were incubated at 30°C for 0–90 min and afterwards all tubes were boiled for 5 min, cooled down in RT and centrifuged (1000 g for 10 min). Collected supernatants were kept in –80°C until RIA analysis.

To determine elution profiles and AA content in degradation products obtained after GnRH and Cu-GnRH proteolysis in vitro, hypothalamic/pituitary cytosols and pellets were prepared from 10 male rats (4 month old, 250-350 g) according to the protocol described above. Next, cytosol and pellet fractions were incubated (37°C, 5 h) with 100 µg/400 µl of exogenous GnRH or Cu-GnRH and then adjusted for HPLC analysis as described below.

Radioimmunoassay

GnRH and Cu-GnRH concentration in subcellular hypothalamic and pituitary fractions was determined by double-antibody RIA according to the modified Kerdelhue et al. method (20). Briefly, respective hypothalamic/pituitary samples were incubated at 4°C for 24 h with the specific anti GnRH antibody (the final tube concentration 1:14000) , normal rabbit serum (the final tube concentration 1:1200) and 125I-GnRH (10,000 cpm). Next day, an ovine anti rat antibody (final tube dilution 1:250) was added for the 24 h. After completing an incubation, all samples were centrifuged (3000 rpm/20 min) and radioactivity of precipitated complex was measured on Cobra II -counter. GnRH standard (curve range: 1.9 pg–1 ng/100 µl) was purchased from Sigma and anti-GnRH antibody was obtained in our laboratory (21). Sensitivity of detection was 30 pg/ml and intra- and interassay variation were below 10%.

High performance liquid chromatographic analysis

To examine the yield of GnRH and Cu-GnRH degradation products, two reversed phase (RP) high performance liquid chromatographic (HPLC) methods were used as previously described (18). Briefly, non-derivatived degradation products of GnRH and Cu-GnRH molecules were performed using an Alliance separation module (model 2690, Waters) with a Waters 474 fluorescence detector and a Waters 996 photodiode array detector (DAD) and the binary gradient elution program. The analytical column used was a Nova Pak C18-column (4 µm, 150x3.9 mm I.D., Waters) in conjunction with a guard Nova Pak column of 10x6 mm. DAD was operated in a UV range from 195 to 800 nm, while GnRH and its complex with Cu2+ were quantified at 280 nm. The fluorescence detection was at the excitation wavelength of 280 nm and emission wave length (em) of 360 nm. Retention time for GnRH was 31.5 min and for Cu-GnRH 30.4 min. Tryptophan in GnRH and Cu-GnRH degradation products in effluents was identified based on simultaneous photodiode (at 279 nm) and fluorescence (ex=280 nm/em=360 nm) detections (22).

The composition of GnRH or Cu-GnRH degradation products was determined separately in each collected fraction. Before HPLC analysis samples were lyophilized and then hydrolyzed in 6 M HCl at 104±2°C for 5 h (23). Finally, pre-column derivatization of AA in the hydrolysates was performed (22). For the derivatization of amino acids a methanolic solution of o-phthaldialdehyde (OPA) in the presence of ethanethiol was used. Derivatized amino acids (OPA-AA) were quantified using a Nova Pak C18 column (4 µm, 300x3.9 mm I.D., Waters) and a gradient elution program (22). Detection was carried out simultaneously using UV monitoring at 337 nm and fluorescence detection (ex=336 nm/em=425 nm). Unfortunately, among 10 AA present in GnRH molecule, proline was inaccessible for HPLC analyses as it did not react with the OPA reagent.

Statistical analysis

RIA data are expressed as mean ±S.E.M. Differences between means were estimated according to the Wilcoxon test (STATISTICATM, StatSoft).


RESULTS
Time-dependent changes of exogenous GnRH and Cu-GnRH content in hypothalamic and pituitary tissues in vitro revealed that the compared molecules differed in their resistance to enzymatic degradation. Moreover, the obtained results showed that in the presence of bacitracin Cu-GnRH was more effectively than GnRH protected against proteolysis.

As shown in Fig. 1A, in the hypothalamic supernatant exogenous GnRH content was diminished by 56% (p0.05) after 10 min of incubation. In contrast, Cu-GnRH content remained unchanged when compared to the respective initial value. Although bacitracin significantly delayed GnRH degradation in hypothalamic cytosol, its full protective effect was observed for Cu-GnRH complex (Fig. 1A). Similarly, in the hypothalamic pellet after the first 10 min of incubation, GnRH content was diminished by 45% whereas Cu-GnRH by only 21% (p0.05; Fig. 1B). In the presence of bacitracin, GnRH was reduced by 29% whereas Cu-GnRH by 15% as compared to the respective initial value (p0.05; Fig. 1B).

Fig. 1. RIA analysis of the time-dependent changes of exogenous GnRH and Cu-GnRH content during their 0–90 min incubation at 30°C in hypothalamic cytosol (Fig. 1A) or pellet (Fig. 1B). 2.5 µg of GnRH or Cu-GnRH were incubated without or in the presence of bacitracin (125 µg/sample). Peptide content at the beginning of incubation was taken as 100%. All values are expressed as mean ±S.E.M. Differences resulting in p0.05 were considered significant.

In the pituitary cytosol, after 30 min of incubation, exogenous GnRH and Cu-GnRH content were reduced by 79% and 52%, respectively, (p0.05; Fig. 2A). Although the protective effect of bacitracin was observed for both peptides, only Cu-GnRH content remained unchanged within the whole period of incubation (Fig. 2A). In the pituitary pellet, exogenous GnRH and Cu-GnRH content reached 61% and 10%, respectively, of reduction of their initial values after 10 min of incubation (p0.05; Fig. 2B). Although both molecules were protected in the presence of protease inhibitor, only complex remained fully preserved during the whole period of incubation (Fig. 2B).

Fig. 2. RIA analysis of the time-dependent changes of exogenous GnRH and Cu-GnRH content during their 0–90 min incubation at 30°C in pituitary cytosol (Fig. 2A) or pellet (Fig. 2B). 2.5 µg of GnRH or Cu-GnRH were incubated in supernatant without or in the presence of bacitracin (125 µg/sample). Peptide content at the beginning of incubation was taken as 100%. All values are expressed as mean ±S.E.M. Differences resulting in p0.05 were considered significant.

According to HPLC analysis, elution profiles obtained after exogenous GnRH and Cu-GnRH degradation in hypothalamic and pituitary tissue in vitro also indicated the differences between both peptides’ resistance to proteolysis. In the hypothalamic cytosol a number of identified GnRH degraded fragments reached 8 while in contrast only one degradation product was found for Cu-GnRH, molecule incubated in the same milieu (Fig. 3A). In the hypothalamic pellet, proteolysis process resulted in 9 (for GnRH) and 3 (for Cu-GnRH) HPLC identified degradation products (Fig. 3B). Similarly, GnRH remained more than Cu-GnRH susceptible for enzymatic degradation when incubated with the pituitary cytosol since HPLC analysis revealed 9 and 3 peaks, respectively, for both compared molecules (Fig. 4A). Also in the pituitary pellet degradation process was more intensive for non complexed molecule since GnRH incubated with this subcellular fraction derived 6 whereas Cu-GnRH only 2 degradation products (Fig. 4B).

Fig. 3. HPLC-derived elution patterns of GnRH and Cu-GnRH degradation products obtained after100 µg of respective peptide incubation at 37°C for 5 h in male rat hypothalamic cytosol (Fig. 3A) and pellet (Fig. 3B). Although after completing an incubation the majority of exogenous GnRH and Cu-GnRH were still undegraded (peak No 1), proteolytic Cu-GnRH degradation in both examined hypothalamic fractions occurred less intensively than it was detected for non complexed GnRH. Note 8 vs. 1 degradation products found for GnRH vs. Cu-GnRH molecule in hypothalamic cytosol, respectively.

Fig. 4. HPLC-derived elution patterns of GnRH and Cu-GnRH degradation products obtained after100 µg of respective peptide incubation at 37°C for 5 h in male rat pituitary cytosol (Fig. 4A) and pellet (Fig. 4B). Although after completing an incubation the majority of exogenous GnRH and Cu-GnRH were still undegraded (peak No 1), proteolytic Cu-GnRH degradation in both examined pituitary fractions occurred less intensively than it was detected for non complexed GnRH. Note 9 vs. 3 degradation products found for GnRH vs. Cu-GnRH molecule in pituitary cytosol, respectively.

Besides the comparison of the GnRH and Cu-GnRH degradation products’ elution profiles, HPLC analysis was also applied to identify the specific AA sequence in the all proteolysis-derived peptides (Table 1).

Table 1. AA sequences of GnRH and Cu-GnRH molecules degradation products. The AA composition was determined separately in each collected fraction. Samples were lyophilized and then hydrolyzed in 6 M HCl at 104±2°C for 5 h. Finally, pre-column derivatization of AA in the hydrolysates was performed. For derivatization of amino acids a methanolic solution of o-phthaldialdehyde (OPA) in the presence of ethanethiol was used.

DISCUSSION
To assess the GnRH and Cu-GnRH complex susceptibility for proteolytic degradation in the hypothalamic and pituitary tissue, time-dependent changes in exogenous GnRH and Cu-GnRH concentration were compared according to the RIA method. In the second experiment elution patterns obtained after GnRH or Cu-GnRH degradation in vitro were independently determined by HPLC analysis. This quantitative study was further supported by HPLC-derived AA sequence identification in degraded fragments of both peptides. The obtained results revealed that the Cu-GnRH complex was more resistant to endogenous enzymatic degradation than GnRH as well as more susceptible to the anti proteolytic protective effect exerted by bacitracin.

The modification of the GnRH primary structure by a substitution of specific amino acids in the decapeptide chain is one of the main approaches to generate GnRH peptide analogs (5). In this respect, a peptide which preserves an identical - as in native decapeptide - amino acid sequence but contains Cu2+ ion stably bound to the nitrogen atom at the imidazole ring of the His2 may be considered as a unique GnRH analog (24). Modified AA sequence may result in different physiological and pharmacological properties of GnRH molecule. Recently, highly selective GnRH agonist, histrelin [Des-Gly10, D-His(Bzl)6, Pro-NhET4]-LH’RH was shown to stimulate the vasopressin release from rat hypothalamo-neurohypophysial system, while native GnRH remained inactive in modifying this process (25). Similarly, the significance of copper ion binding in modifying some physiological properties of the GnRH molecule was evidenced in our previous studies. Indeed, we found that the Cu-GnRH complex not only more potently stimulated LH release than GnRH both in vivo (26), and in vitro (27), but also exhibited a greater affinity to GnRH receptor (28) and induced cAMP/PKA pathway activity in gonadotrope cells (29). Moreover, the results of this study demonstrate that resistance to proteolysis, important for post secretory regulation of peptide signals, is also affected after the formation of a complex with Cu2+ ion.

Apparently reduced Cu-GnRH susceptibility to proteolytic cleavage might be a consequence of modified complex conformation which results in rendered access of the specific endopeptidases to AA bonds. That metal ions might induce changes in peptide conformation was reported in our previous research. Ni (II) ion complexed at His2 of GnRH molecule were found to coordinate with four nitrogen atoms from histidine, tryptophan, serine and tyrosine inducing a well defined arrangement of aromatic side-chains and a rigid backbone structure of this peptide (30). In addition, a reduced number of HPLC- identified Cu-GnRH degradation products found in this study in hypothalamic as well as pituitary tissue further indirectly indicates significant changes in complex conformation. Moreover, detailed analysis of AA composition in Cu-GnRH peptide degradation fragments revealed that among five enzymes involved in splitting specific AA bonds in GnRH molecule, the complex appeared to be completely resistant to EC 3.4.19.3 proteolytic activity responsible for pGlu1-His2 bond dissociation.

Although a bacitracin-dependent anti-proteolytic effect on degradation process was observed for both peptides, only Cu-GnRH within the whole period of incubation remained fully protected in the presence of this inhibitor. As a competitive inhibitor of prolyl endopeptidase (PEP), bacitracin precludes the cleavage of the C-terminal glycinamide residue from the GnRH molecule and consequently blocks GnRH[1-9] peptide formation (31). Since PEP and metalloendopeptidase EC 3.4.24.15 acting in a stepwise manner are mainly involved in GnRH degradation in both the hypothalamus and pituitary gland, a lack of GnRH[1-9], which is the preferred substrate for EP 24.15, profoundly reduces the intensity of the second step of degradation. Thus, there is no cleavage of GnRH[1-5] through EP 24.15-dependent Tyr5-Gly6 bond disruption (32). Therefore, a more efficient protective effect of bacitracin exerted on Cu-GnRH complex stability may result from modified Cu-GnRH conformation which effectively precludes PEP access to Pro9- Gly10 NH2 bond and consequently blocks the initiation of the degradation process.

Our results also revealed that for both molecules their degradation rates differed according to the compared subcellular fraction. Indeed, GnRH and Cu-GnRH incubated in the presence of hypothalamic and pituitary pellet fractions were degraded less intensively than observed during incubation with respective cytosols derived from these tissues. Studies on subcellular EP 24.15 distribution in the rat brain have revealed that around 75% of the total amount of enzyme activity was associated with soluble fractions of tissue homogenates, whereas the remaining 25% belonged to membrane fractions (33). Moreover, electron microscopy studies on both glia and neurons further supported an abundant cytoplasmatic staining for EP 24.15 which was inversely correlated with intranuclear localization of this enzyme (34). Thus, subcellular differences in endopeptidases availability might be responsible for specific patterns of peptides degradation observed in hypothalamic and pituitary cytosol and pellet fractions.

In conclusion, the obtained data suggest that copper ion changed GnRH conformation and significantly modified physiological properties of this molecule due to hindered endopeptidase access to specific AA bonds. Therefore, Cu-GnRH complex might be considered as a GnRH analogue potentially capable of prolonged occupation of GnRH receptor at the gonadotrope cells in the pituitary gland.

Acknowledgments: We would like to thank Dr. Katarzyna Niedzwiedzka for her skillful technical assistance. Research was financed by the Scientific Network of the Ministry of Science and Higher Education: project no: 20/E183/SN0013.

Conflict of interests: None declared.


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