- Email: [email protected]
Involvement of metallo-endopeptidase in degradation of luteinizing hormone-releasing hormone by neuronal and glial cells cultured from rat fetal brain
Neuropeptides(1991) l&77-82 @ Longmaa Group UK Ltd 1991
Involvement of Metallo-Endopeptidase in Degradation of Luteinizing Hormone-Releasing Hormone by Neuronal and Glial Cells Cultured From Rat Fetal Brain C. SAKURADA,
S. ISHII and H. YOKOSAWA
Department of Biochemistry, Faculty of Pharmaceutical Japan (Reprint requests to HY)
Sciences, Hokkaido University,
Sapporo 060,
Abstract-Luteinizing hormone-releasing hormone (LHRH) was degraded by neuronal and glial cells cultured from fetal rat brain. The degradation of LHRH by neuronal cells was strongly inhibited by a metal chelator. Captopril only inhibited the generation of fragment (l-3) from fragment (l-5). In the presence of captopril, fragment (l-5) accumulated in the highest amount among the N-terminal fragments identified. The initial cleavage of LHRH, as determined by following the loss of the LHRH peak, was strongly inhibited by thiol-blocking reagents, as well as metal chelators. The results with glial cells were almost the same as those seen with neuronal cells. Thus, we propose that a thiol-dependent membrane-bound metallo-endopeptidase plays a major role in the initial stage of degradation of LHRH at the Tyr5-Gly6 bond in both neurons and glia. Angiotensin-converting enzyme is involved in the secondary process of the LHRH degradation in both cells.
Introduction
neurons, by analogy with acetylcholinesterase, a membrane-bound protease which is well known to hydrolyze the neurotransmitter acetylcholine rapidly at the cholinergic synapse to terminate impulse transmission. In previous studies using the neuroblastoma cells in tissue culture (3) and synaptic membranes (4) as possible models for understanding the inactivation of LHRH in neurons, we have proposed that LHRH is initially cleaved at the TyrSGly6 bond by the action of a thiol-dependent metallo-endopeptidase, followed by the action of angiotensin-converting enzyme (ACE, EC 3.4.15.11). Furthermore, we have succeeded in
Immunocytochemical and electrophysiological studies of luteinizing hormone-releasing hormone (LHRH, pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-ProGlyNH;?) indicate that this peptide functions as a central neurotransmitter and/or neuromodulator, as well as a hypothalamic hormone (1, 2). The former action of LHRH is probably terminated by enzymatic degradation by a membrane-bound protease at the synapse or plasma membrane of
Date received 7 August 1990 Date accepted 13 August 1990
77
78 isolating from neuroblastoma cell membranes and synaptic membranes a putative enzyme, which is called LHRH fragment (l-5) generating endopeptidase, functioning in the initial cleavage of LHRH (4). The enzyme seems to be similar to endopeptidase-24.15 (EC 3.4.24.15) (5), the enzyme in the membranes of hypothalamus and pituitary (6) which degrades LHRH. In this paper, we describe the degradation of LHRH by primary cultures enriched either in the neuronal cells or glial cells. Our goal was to obtain further evidence for the involvement of the metallo-endopeptidase in the degradation of LHRH in the nervous system and to compare inactivating mechanisms in neurons and glia. We expected that these cell cultures were more typical of the brain than the established cell lines used in our previous studies and also were suitable models for studying the roles of neurons and glia in the nervous system. Materials LHRH, phosphoramidon, chymostatin, and E-64 were purchased from the Peptide Institute Inc., Osaka, Japan. p-Chloromercuribenzenesulfonic diisopropylfluorophosphate acid (PCBMS), (DFP), and poly-D-Lys HBr (M.W. 30000-70000) were obtained from Sigma Chemical Co., St Louis, MO, USA. Methanesulfonic acid (4M) containing 0.2% 3-(2-aminoethyl)indole was purchased from Pierce Chemical Co., Rockford, IL, USA. Captopril, propioxatin A, and Z-Gly-ProCHzCl were kindly donated by Dr A. Awaya of Mitsui Pharmaceuticals, Inc., Tokyo, Japan, Dr Y. Inaoka of Sankyo Co., Ltd., Tokyo, Japan, and Dr T. Yoshimoto of Nagasaki University, Japan, respectively. Leupeptin and bestatin were generous gifts of Dr W. Tanaka of Nippon Kayaku Co., Ltd., Tokyo, Japan. Methods Cell culture
Cell suspensions were prepared from forebrains of 15-18-day rat fetuses according to the method of Arimatsu and Hatanaka (7). The cells were cultured at 37°C in 10% COz-90% air in poly-D-Lys coated plastic dishes (Falcon) with Dulbecco’s modified Eagle’s medium (Sigma) supplemented
NEUROPEFTIDES
with 5% (v/v) heat-inactivated horse serum (GIBCO), 5% (v/v) fetal calf serum (GIBCO), SOIU/ml penicillin G (Meiji Seika, Japan) and 0.1 mg/ml streptomycin sulfate (Meiji Seika, Japan). The cultures enriched in neurons, which we call neurons in this paper, were prepared by treatment of primary cultured cells with O.OlmM arabinosylcytosine (Sigma) for 24 h (8). Cultures enriched in glia, glial cells, were obtained by subculturing the confluent primary cultures without arabinosylcytosine treatment. Degradation of LHRH by cultured cells
The cells cultured in 16mm petri dishes were washed five times with 5mM HEPES-NaOH buffer (pH 7.4) containing 155mM NaCl, 5.4mM KCl, 1.8mM CaC12, and 0.8mM MgC& (HEPES Buffer) without detaching them from their dishes. Four tenths of a ml of HEPES Buffer was added to each dish plus 0.05ml of inhibitor solution or HEPES Buffer. After the mixture (0.45ml) was preincubated at 37 “C for 30 mitt, 0.05 ml of 1 .OmM LHRH dissolved in HEPES Buffer was added to the dishes, and the reaction was allowed to proceed at 37°C in 10% CO*-90% air (the final pH of the solution was 7.4). Aliquots of the reaction mixture (0.1 ml) were withdrawn at various times and incubated at 100°C for 5 min to stop the reaction. Then the sample was centrifuged, filtered (Millipore Millex-HV, pore size 450nm), and subjected to high-performance liquid chromatography (HPLC) for analysis of the cleavage products. The HPLC was carried out as described in the legend to Figure 1.
Preparation of membranes from cultured cells
All procedures were done at 4°C. The cells in the dishes were washed with HEPES Buffer. They were then detached with a cell scrapper, suspended in HEPES Buffer, and centrifuged at 100 x g for 3 min. Ten volumes of 1OmM Tris-HCl buffer (pH 7.5) containing 10% (w/v) sucrose were added to the resulting pellet, and the cells were homogenized with a Teflon homogenizer. The homogenate was centrifuged at 800 X g for 20 min and then at 9000 x g for 20 min. The resulting pellet was suspended in 20mM Tris-HC1 buffer containing
METALLO-ENDOPEF’TIDASE IN DEGRADATION OF LUTEINIZING HORMONE-RELEASING HORMONE
was carried out at 37°C in a reaction mixture (O.lml) consisting of 0.07ml of HEPES Buffer, O.Olml of l.OmM LHRH, O.Olml of inhibitor solution or water, and 0.04-o. 1 mg of membranes. The reaction was terminated by incubating at 100°C for 5 min. The reaction mixture was treated and subjected to HPLC analysis as described in the section on degradation by cells.
0.02 5 0 k 2
79
0
f 8
Protein determination
0.02
Proteins were determined by the method of Bradford (9) using bovine serum albumin (Sigma) as a standard.
s!
0 0
5
10
15
0
TIME
5
10
Results
15
(min)
Degradation of LHRH by neurons
Fig. 1 Degradation of LHRH by neurons. The neuronal cells were incubated at 37°C for 5 h in the absence of inhibitor (a) and in the presence of 1mM o-phenanthroline (b), O.OlmM phosphoramidon (c), and 0.01 mM captopril (d). Aliquots in a volume of 0.02ml were subjected to HPLC using a reversedphase column of Nucleosil 5C1s (Marchery, Nagel, and Co.) with a 32-min linear concentration gradient of l-65% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of lml/min. The absorbance at 210nm was monitored. The peaks A to H in (a) represent the fragments identified. The peak i represents that originated from the inhibitor.
The degradation of LHRH by neurons in the presence or absence of protease inhibitors was analysed by reverse-phase HPLC (Fig. 1). In the absence of inhibitors (Fig. la), the area under the LHRH peak (peak H) decreased as a function of time, whereas those of other peaks (A to G) increased. (Note that peaks within retention times of 7 and 10.5 min are not derived from LHRH.) The analytical results on amino acid compositions of the cleavage products separated by HPLC allowed the assignment of these products as shown in Table 1. Fragments (l-3), (l-4), and (l-5) were detected as major N-terminal fragments, followed by fragment (1-6). The appearance of almost all of the fragments except for fragment (l-4) and those originating from the cells themselves was strongly depressed by the addition of the metal chelator,
10% (w/v) sucrose and again centrifuged at 9000 x for 20 min. The pellet was suspended in HEPES Buffer and used as the membrane preparation. g
Inhibition of LHRH-degradation
by membranes
The degradation of LHRH by the membrane preparation in the presence or absence of inhibitor Table 1
Amino
Acid Composition
of Fragments
GlU
His
TOP
From LHRH
Degraded
by Neurons*
Tyr
Gly
Leu
Ara
Pro
Fragment identified
Yield t
Ser
1 100 0 3 1 20 18 10
28
16
26
24
6-9 or 7-10
0 40 5 5 21 1 10
0 20 2 1 0 3 10
0. 20 2 0 0 3 10
0 20 1 0 0 0 10
Tyr 6-10 1-4 l-3 l-6 l-5 complete
14 8 6 8 8 1 5
Amino acid Peak
Produced
2
0
0
2
0
0
0
0
0 25 36 20 25 11
0 22 29 15 20 10
0 19 24 6 8 10
0 22 3 18 22 10
*The extent of degradation of LHRH was 58%. tYield was determined on the basis of LHRH degraded.
(mol
%)
(“/a)
80
NBUROPEPTIDES
I(d)
10
150 TIME (min)
5
10
Fig. 2 Degradation of SP by glia. The glial cells were incubated at 37°C for 5 h in the absence of inhibitor (a) and in the presence of 1 mM o-phenanthroline (b), 0.01 mM phosphoramidon (c), and O.OlmM captopril (d). Aliquots in a volume of 0.05 ml were subjected to HPLC as in Fig. 1. The peaks A to I in (a) represent the fragments identified. The peak i represents that originated from the inhibitor.
o-phenanthroline, to the reaction mixture (Fig. lb). The effect of o-phenanthroline on the generation of fragment (l-4) could not be determined because the (l-4) peak and the o-phenanthroline peaks overlap. On the other hand, the addition of an inhibitor of metallophosphoramidon, endopeptidases such as endopeptidase-24.11 (EC 3.4.24.11), had little effect (Fig. lc). In the presence of captopril, an ACE inhibitor, the generation of fragment (l-3) was decreased, whereas that of fragment (l-5) increased (Fig. Id). Thus, since the generation of fragment (l-5) was completely inhibited by o-phenanthroline, we think that the degradation of LHRH begins when the TyrS-Gly6 bond is cleaved by a metallo-endopeptidase, and that fragment (l-5) is subsequently cleaved by ACE. This is almost the same as LHRH-degradation by neuroblastoma cells described previously (3).
Thiol-blocking reagents could not be added to cultured neurons because they proved too toxic. In neuroblastoma cells (3) and synaptic membranes (4), the metallo-endopeptidase functioning in the initial cleavage of LHRH is thiol-dependent. Using membranes prepared from neurons, we examined the effects of thiol-blocking reagents on the initial cleavage of LHRH as determined by the decrease of the HPLC peak of LHRH. The results including those with the other inhibitors are shown in Table 2. The initial cleavage of LHRH was strongly inhibited by thiol-blocking reagents such as PCMBS and N-ethylmaleimide, as well as o-phenanthroline and EDTA, but scarcely inhibited by the other inhibitors including phosphoramidon and captopril. Thus, the presence of sulfhydryl groups is essential for LHRH-degrading metallo-endopeptidase in neurons. Since the fragments generated by intact neurons and membranes were similar, we conclude that LHRH is broken down at the plasma membrane. Degradation of LHRH
by glia
The degradation of LHRH by glia was analysed by HPLC (Fig. 2), and the cleavage products were isolated (A to I in Fig. 2a) and characterized (Table 3). Two peaks appearing at retention times of 7 min and 10.5 min were judged to originate
Table 2 Effects of Various Protease Inhibitors on the Degradation of LHRH by the Membranes of Neurons or Glia
Inhibitor o-Phenanthroline EDTA PCMBS N-Ethylmaleimide Phosphoramidon Propioxatin A Captopril Bestatin DFP Chymostatin Leupeptin Z-Gly-Pro-CH&L E-64
Concentration (mM) 1.0 1.0 1.0 1.0 0.01 0.01 0.01 0.01 1.0 0.01 0.01 0.01 0.01
Inhibition (%) Neurons Glia 89 56 100 60 3 0 0 0 0 0 0 0 0
The extent of degradation of LHRH was determined measurine the decrease of LHRH detected bvI HPLC.
100 65 100 91 9 9 0 4 4 21 0 8 0 by
METALLO-ENDOPEPTIDASE IN DEGRADATION OF LUTEINIZING HORMONE-RELEASING HORMONE
81
Table 3 Amino Acid Compositions of Fragments Produced From LHRH Degraded by Glia* Peak
Amino acid (mol %) Gl) Tyr
Glu
His
Trp
Ser
3 6 4
0 0 2
0 0 0
2 3 2
1 68 3
A 0 C
D E F G
H 1,
0
0
0
0
0
21 36 20 21 10
21 36 16 18 10
14 28 10 17 9
18 0 16 18 9
4 0 18 20 10
30 20 29 40 8 0 20 2 20
Arg
Pro
Fragment identified
Yield t
Leu 13 0 23 20 4 0 0 2 10
25 0 21 21 4 0 0 2 10
24 0 16 19 3 0 0 0 10
6-9 or 7-10 Tyr 6-9 or 7-10 6-10 l-4 l-3 l-6 l-5 complete
29 9 2 11 8 24 2 10
(%)
*The extent of degradation of LHRH was 44%. tYield was determined on the basis of LHRH degraded.
from the cells because they were detected in the absence of LHRH. The area of peak I (LHRH itself) decreased as a function of time in the absence of inhibitors (Fig. 2a). Among the N-terminal fragments identified, fragments (l-3), (l-4), and (l-5) were found to accumulate in high amounts. The effects of o-phenanthroline (Fig. 2b) and captopril (Fig. 2d) on breakdown of LHRH by glia were almost the same as with neurons (see Fig. 1); the former inhibitor inhibits the appearance of almost all of the fragments except for fragment (l-4), while the latter inhibits the generation of fragment (l-3) from fragment (l-5). Phosphoramidon (Fig. 2c) has little effect. In the presence of captopril, fragment (l-5) accumulated preferentially as with neurons. The initial cleavage of LHRH by glial membranes was also susceptible to metal chelators and thiol-blocking reagents (see Table 2). Other inhibitors such as phosphoramidon and captopril had little inhibitory effect (Table 2). These results suggest that a thiol-dependent metallo-endopeptidase present in the plasma membrane plays a major role in the initial stage of cleavage of LHRH by the glia as well as neurons.
Discussion We have previously proposed that a thiol-dependent membrane-bound metallo-endopeptidase performs the initial cleavage of LHRH at the TyrS-Gly6 bond in neuroblastoma cells in tissue
culture (3) and synaptic membranes prepared from whole brain (4). Our results with neuronal cells cultured from fetal brain give additional support to the above hypothesis. Our results also indicate that ACE cleaves LHRH after it has been cleaved by the above endopeptidase. In addition, we have presented evidence concerning LHRH-degradation by glia. Almost the same mechanism operates in glia as in neurons. Thus, a thiol-dependent metallo-endopeptidase in neurons may play an important role in the degradation of LHRH, a neurotransmitter and/or neuromodulator, in the synapse or in the surface of neurons, while the enzyme in glia may function as an LHRH scavenger. Alternatively, if glial cells have LHRH receptors, the enzyme may terminate the action of LHRH on glial cells. In connection with our studies on neuronal and glial inactivation of LHRH, it should be noted that Molinaux et al., have reached a similar conclusion; endopeptidase-24.15, which is a similar enzyme to our thiol-dependent metallo-endopeptidase, is the primary enzyme degrading LHRH in membranes of the hypothalamus and the pituitary (6). In a previous report (4), we have isolated the LHRHdegrading enzyme from neuronal tissues in a membrane-bound form. On the other hand, a soluble form of the enzyme has been purified from tissues outside the brain (5). Both enzymes have similar properties except for their molecular weights. Thus, our enzyme, probably endopeptidase-24.15, may be involved in the degradation of LHRH, inside or outside the brain.
82 Acknowledgements We wish to thank Dr K. Mizuno and Professor K. Kurihara of Hokkaido University for their help in preparing primary cultures and for their helpful discussion. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, from the Research Foundation for Pharmaceutical Sciences, and from Takeda Science Foundation.
References 1. Lamour, Y. and Epelbaum, J. (1988). Interactions between cholinergic and peptidergic systems in the cerebral cortex and hippocampus. Prog. Neurobiol. 31: 109-148. 2. Wong, M., Eaton, M. J. and Moss, R. L. (1990). Electrophysiological actions of luteinizing hormone-releasing hormone: Intracellular studies in the rat hippocampal slice preparation. Synapse 5: 65-70. 3. Yokosawa, H., Fujii, Y. and Ishii, S. (1987). Degradation of luteinizing hormone-releasing hormone by neuroblastoma cells and their membrane: Evidence for the involvement of a thiol protease and angiotensin-converting enzyme. J. Neurochem. 48: 293-298.
NBUROPEFTIDES 4. Sakurada, C., Ohgaki, Y., Yokosawa, H. and Ishii, S. (1990). Thiol-dependent membrane-bound metallo-endopeptidase functioning in degradation of luteinizing hormone-releasing hormone in neuroblastoma cells and rat synaptic membrane. Isolation and characterization. Neuropeptides in press. 5. Orlowski, M., Reznik, S., Ayala, J. and Pierotti, A. R. (1989). Endopeptidase 24.15 from rat testes. Isolation of the enzyme and its specificity toward synthetic and natural peptides, including enkephalin-containing peptides. Biothem J. 261: 951-958. 6. Mohneaux, C. J., Lasdun, A., Michaud, C. and Orlowski, M. (1988). Endopeptidase-24.15 is the primary enzyme that degrades luteinizing hormone releasing hormone both in vitro and in vivo. J. Neurochem. 51: 624-633. 7. Arimatsu, Y. and Hatanaka, H. (1986). Estrogen treatment enhances survival of cultured fetal rat amygdala neurons in a defined medium. Dev. Brain Res. 26: 151-159. 8. Dichter, M. A. (1978). Rat cortical neurons in cell culture: culture methods, cell morphology, electrophysiology, and synapse formation. Brain Res. 149: 279-293. 9. Badford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.
Involvement of Metallo-Endopeptidase in Degradation of Luteinizing Hormone-Releasing Hormone by Neuronal and Glial Cells Cultured From Rat Fetal Brain C. SAKURADA,
S. ISHII and H. YOKOSAWA
Department of Biochemistry, Faculty of Pharmaceutical Japan (Reprint requests to HY)
Sciences, Hokkaido University,
Sapporo 060,
Abstract-Luteinizing hormone-releasing hormone (LHRH) was degraded by neuronal and glial cells cultured from fetal rat brain. The degradation of LHRH by neuronal cells was strongly inhibited by a metal chelator. Captopril only inhibited the generation of fragment (l-3) from fragment (l-5). In the presence of captopril, fragment (l-5) accumulated in the highest amount among the N-terminal fragments identified. The initial cleavage of LHRH, as determined by following the loss of the LHRH peak, was strongly inhibited by thiol-blocking reagents, as well as metal chelators. The results with glial cells were almost the same as those seen with neuronal cells. Thus, we propose that a thiol-dependent membrane-bound metallo-endopeptidase plays a major role in the initial stage of degradation of LHRH at the Tyr5-Gly6 bond in both neurons and glia. Angiotensin-converting enzyme is involved in the secondary process of the LHRH degradation in both cells.
Introduction
neurons, by analogy with acetylcholinesterase, a membrane-bound protease which is well known to hydrolyze the neurotransmitter acetylcholine rapidly at the cholinergic synapse to terminate impulse transmission. In previous studies using the neuroblastoma cells in tissue culture (3) and synaptic membranes (4) as possible models for understanding the inactivation of LHRH in neurons, we have proposed that LHRH is initially cleaved at the TyrSGly6 bond by the action of a thiol-dependent metallo-endopeptidase, followed by the action of angiotensin-converting enzyme (ACE, EC 3.4.15.11). Furthermore, we have succeeded in
Immunocytochemical and electrophysiological studies of luteinizing hormone-releasing hormone (LHRH, pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-ProGlyNH;?) indicate that this peptide functions as a central neurotransmitter and/or neuromodulator, as well as a hypothalamic hormone (1, 2). The former action of LHRH is probably terminated by enzymatic degradation by a membrane-bound protease at the synapse or plasma membrane of
Date received 7 August 1990 Date accepted 13 August 1990
77
78 isolating from neuroblastoma cell membranes and synaptic membranes a putative enzyme, which is called LHRH fragment (l-5) generating endopeptidase, functioning in the initial cleavage of LHRH (4). The enzyme seems to be similar to endopeptidase-24.15 (EC 3.4.24.15) (5), the enzyme in the membranes of hypothalamus and pituitary (6) which degrades LHRH. In this paper, we describe the degradation of LHRH by primary cultures enriched either in the neuronal cells or glial cells. Our goal was to obtain further evidence for the involvement of the metallo-endopeptidase in the degradation of LHRH in the nervous system and to compare inactivating mechanisms in neurons and glia. We expected that these cell cultures were more typical of the brain than the established cell lines used in our previous studies and also were suitable models for studying the roles of neurons and glia in the nervous system. Materials LHRH, phosphoramidon, chymostatin, and E-64 were purchased from the Peptide Institute Inc., Osaka, Japan. p-Chloromercuribenzenesulfonic diisopropylfluorophosphate acid (PCBMS), (DFP), and poly-D-Lys HBr (M.W. 30000-70000) were obtained from Sigma Chemical Co., St Louis, MO, USA. Methanesulfonic acid (4M) containing 0.2% 3-(2-aminoethyl)indole was purchased from Pierce Chemical Co., Rockford, IL, USA. Captopril, propioxatin A, and Z-Gly-ProCHzCl were kindly donated by Dr A. Awaya of Mitsui Pharmaceuticals, Inc., Tokyo, Japan, Dr Y. Inaoka of Sankyo Co., Ltd., Tokyo, Japan, and Dr T. Yoshimoto of Nagasaki University, Japan, respectively. Leupeptin and bestatin were generous gifts of Dr W. Tanaka of Nippon Kayaku Co., Ltd., Tokyo, Japan. Methods Cell culture
Cell suspensions were prepared from forebrains of 15-18-day rat fetuses according to the method of Arimatsu and Hatanaka (7). The cells were cultured at 37°C in 10% COz-90% air in poly-D-Lys coated plastic dishes (Falcon) with Dulbecco’s modified Eagle’s medium (Sigma) supplemented
NEUROPEFTIDES
with 5% (v/v) heat-inactivated horse serum (GIBCO), 5% (v/v) fetal calf serum (GIBCO), SOIU/ml penicillin G (Meiji Seika, Japan) and 0.1 mg/ml streptomycin sulfate (Meiji Seika, Japan). The cultures enriched in neurons, which we call neurons in this paper, were prepared by treatment of primary cultured cells with O.OlmM arabinosylcytosine (Sigma) for 24 h (8). Cultures enriched in glia, glial cells, were obtained by subculturing the confluent primary cultures without arabinosylcytosine treatment. Degradation of LHRH by cultured cells
The cells cultured in 16mm petri dishes were washed five times with 5mM HEPES-NaOH buffer (pH 7.4) containing 155mM NaCl, 5.4mM KCl, 1.8mM CaC12, and 0.8mM MgC& (HEPES Buffer) without detaching them from their dishes. Four tenths of a ml of HEPES Buffer was added to each dish plus 0.05ml of inhibitor solution or HEPES Buffer. After the mixture (0.45ml) was preincubated at 37 “C for 30 mitt, 0.05 ml of 1 .OmM LHRH dissolved in HEPES Buffer was added to the dishes, and the reaction was allowed to proceed at 37°C in 10% CO*-90% air (the final pH of the solution was 7.4). Aliquots of the reaction mixture (0.1 ml) were withdrawn at various times and incubated at 100°C for 5 min to stop the reaction. Then the sample was centrifuged, filtered (Millipore Millex-HV, pore size 450nm), and subjected to high-performance liquid chromatography (HPLC) for analysis of the cleavage products. The HPLC was carried out as described in the legend to Figure 1.
Preparation of membranes from cultured cells
All procedures were done at 4°C. The cells in the dishes were washed with HEPES Buffer. They were then detached with a cell scrapper, suspended in HEPES Buffer, and centrifuged at 100 x g for 3 min. Ten volumes of 1OmM Tris-HCl buffer (pH 7.5) containing 10% (w/v) sucrose were added to the resulting pellet, and the cells were homogenized with a Teflon homogenizer. The homogenate was centrifuged at 800 X g for 20 min and then at 9000 x g for 20 min. The resulting pellet was suspended in 20mM Tris-HC1 buffer containing
METALLO-ENDOPEF’TIDASE IN DEGRADATION OF LUTEINIZING HORMONE-RELEASING HORMONE
was carried out at 37°C in a reaction mixture (O.lml) consisting of 0.07ml of HEPES Buffer, O.Olml of l.OmM LHRH, O.Olml of inhibitor solution or water, and 0.04-o. 1 mg of membranes. The reaction was terminated by incubating at 100°C for 5 min. The reaction mixture was treated and subjected to HPLC analysis as described in the section on degradation by cells.
0.02 5 0 k 2
79
0
f 8
Protein determination
0.02
Proteins were determined by the method of Bradford (9) using bovine serum albumin (Sigma) as a standard.
s!
0 0
5
10
15
0
TIME
5
10
Results
15
(min)
Degradation of LHRH by neurons
Fig. 1 Degradation of LHRH by neurons. The neuronal cells were incubated at 37°C for 5 h in the absence of inhibitor (a) and in the presence of 1mM o-phenanthroline (b), O.OlmM phosphoramidon (c), and 0.01 mM captopril (d). Aliquots in a volume of 0.02ml were subjected to HPLC using a reversedphase column of Nucleosil 5C1s (Marchery, Nagel, and Co.) with a 32-min linear concentration gradient of l-65% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of lml/min. The absorbance at 210nm was monitored. The peaks A to H in (a) represent the fragments identified. The peak i represents that originated from the inhibitor.
The degradation of LHRH by neurons in the presence or absence of protease inhibitors was analysed by reverse-phase HPLC (Fig. 1). In the absence of inhibitors (Fig. la), the area under the LHRH peak (peak H) decreased as a function of time, whereas those of other peaks (A to G) increased. (Note that peaks within retention times of 7 and 10.5 min are not derived from LHRH.) The analytical results on amino acid compositions of the cleavage products separated by HPLC allowed the assignment of these products as shown in Table 1. Fragments (l-3), (l-4), and (l-5) were detected as major N-terminal fragments, followed by fragment (1-6). The appearance of almost all of the fragments except for fragment (l-4) and those originating from the cells themselves was strongly depressed by the addition of the metal chelator,
10% (w/v) sucrose and again centrifuged at 9000 x for 20 min. The pellet was suspended in HEPES Buffer and used as the membrane preparation. g
Inhibition of LHRH-degradation
by membranes
The degradation of LHRH by the membrane preparation in the presence or absence of inhibitor Table 1
Amino
Acid Composition
of Fragments
GlU
His
TOP
From LHRH
Degraded
by Neurons*
Tyr
Gly
Leu
Ara
Pro
Fragment identified
Yield t
Ser
1 100 0 3 1 20 18 10
28
16
26
24
6-9 or 7-10
0 40 5 5 21 1 10
0 20 2 1 0 3 10
0. 20 2 0 0 3 10
0 20 1 0 0 0 10
Tyr 6-10 1-4 l-3 l-6 l-5 complete
14 8 6 8 8 1 5
Amino acid Peak
Produced
2
0
0
2
0
0
0
0
0 25 36 20 25 11
0 22 29 15 20 10
0 19 24 6 8 10
0 22 3 18 22 10
*The extent of degradation of LHRH was 58%. tYield was determined on the basis of LHRH degraded.
(mol
%)
(“/a)
80
NBUROPEPTIDES
I(d)
10
150 TIME (min)
5
10
Fig. 2 Degradation of SP by glia. The glial cells were incubated at 37°C for 5 h in the absence of inhibitor (a) and in the presence of 1 mM o-phenanthroline (b), 0.01 mM phosphoramidon (c), and O.OlmM captopril (d). Aliquots in a volume of 0.05 ml were subjected to HPLC as in Fig. 1. The peaks A to I in (a) represent the fragments identified. The peak i represents that originated from the inhibitor.
o-phenanthroline, to the reaction mixture (Fig. lb). The effect of o-phenanthroline on the generation of fragment (l-4) could not be determined because the (l-4) peak and the o-phenanthroline peaks overlap. On the other hand, the addition of an inhibitor of metallophosphoramidon, endopeptidases such as endopeptidase-24.11 (EC 3.4.24.11), had little effect (Fig. lc). In the presence of captopril, an ACE inhibitor, the generation of fragment (l-3) was decreased, whereas that of fragment (l-5) increased (Fig. Id). Thus, since the generation of fragment (l-5) was completely inhibited by o-phenanthroline, we think that the degradation of LHRH begins when the TyrS-Gly6 bond is cleaved by a metallo-endopeptidase, and that fragment (l-5) is subsequently cleaved by ACE. This is almost the same as LHRH-degradation by neuroblastoma cells described previously (3).
Thiol-blocking reagents could not be added to cultured neurons because they proved too toxic. In neuroblastoma cells (3) and synaptic membranes (4), the metallo-endopeptidase functioning in the initial cleavage of LHRH is thiol-dependent. Using membranes prepared from neurons, we examined the effects of thiol-blocking reagents on the initial cleavage of LHRH as determined by the decrease of the HPLC peak of LHRH. The results including those with the other inhibitors are shown in Table 2. The initial cleavage of LHRH was strongly inhibited by thiol-blocking reagents such as PCMBS and N-ethylmaleimide, as well as o-phenanthroline and EDTA, but scarcely inhibited by the other inhibitors including phosphoramidon and captopril. Thus, the presence of sulfhydryl groups is essential for LHRH-degrading metallo-endopeptidase in neurons. Since the fragments generated by intact neurons and membranes were similar, we conclude that LHRH is broken down at the plasma membrane. Degradation of LHRH
by glia
The degradation of LHRH by glia was analysed by HPLC (Fig. 2), and the cleavage products were isolated (A to I in Fig. 2a) and characterized (Table 3). Two peaks appearing at retention times of 7 min and 10.5 min were judged to originate
Table 2 Effects of Various Protease Inhibitors on the Degradation of LHRH by the Membranes of Neurons or Glia
Inhibitor o-Phenanthroline EDTA PCMBS N-Ethylmaleimide Phosphoramidon Propioxatin A Captopril Bestatin DFP Chymostatin Leupeptin Z-Gly-Pro-CH&L E-64
Concentration (mM) 1.0 1.0 1.0 1.0 0.01 0.01 0.01 0.01 1.0 0.01 0.01 0.01 0.01
Inhibition (%) Neurons Glia 89 56 100 60 3 0 0 0 0 0 0 0 0
The extent of degradation of LHRH was determined measurine the decrease of LHRH detected bvI HPLC.
100 65 100 91 9 9 0 4 4 21 0 8 0 by
METALLO-ENDOPEPTIDASE IN DEGRADATION OF LUTEINIZING HORMONE-RELEASING HORMONE
81
Table 3 Amino Acid Compositions of Fragments Produced From LHRH Degraded by Glia* Peak
Amino acid (mol %) Gl) Tyr
Glu
His
Trp
Ser
3 6 4
0 0 2
0 0 0
2 3 2
1 68 3
A 0 C
D E F G
H 1,
0
0
0
0
0
21 36 20 21 10
21 36 16 18 10
14 28 10 17 9
18 0 16 18 9
4 0 18 20 10
30 20 29 40 8 0 20 2 20
Arg
Pro
Fragment identified
Yield t
Leu 13 0 23 20 4 0 0 2 10
25 0 21 21 4 0 0 2 10
24 0 16 19 3 0 0 0 10
6-9 or 7-10 Tyr 6-9 or 7-10 6-10 l-4 l-3 l-6 l-5 complete
29 9 2 11 8 24 2 10
(%)
*The extent of degradation of LHRH was 44%. tYield was determined on the basis of LHRH degraded.
from the cells because they were detected in the absence of LHRH. The area of peak I (LHRH itself) decreased as a function of time in the absence of inhibitors (Fig. 2a). Among the N-terminal fragments identified, fragments (l-3), (l-4), and (l-5) were found to accumulate in high amounts. The effects of o-phenanthroline (Fig. 2b) and captopril (Fig. 2d) on breakdown of LHRH by glia were almost the same as with neurons (see Fig. 1); the former inhibitor inhibits the appearance of almost all of the fragments except for fragment (l-4), while the latter inhibits the generation of fragment (l-3) from fragment (l-5). Phosphoramidon (Fig. 2c) has little effect. In the presence of captopril, fragment (l-5) accumulated preferentially as with neurons. The initial cleavage of LHRH by glial membranes was also susceptible to metal chelators and thiol-blocking reagents (see Table 2). Other inhibitors such as phosphoramidon and captopril had little inhibitory effect (Table 2). These results suggest that a thiol-dependent metallo-endopeptidase present in the plasma membrane plays a major role in the initial stage of cleavage of LHRH by the glia as well as neurons.
Discussion We have previously proposed that a thiol-dependent membrane-bound metallo-endopeptidase performs the initial cleavage of LHRH at the TyrS-Gly6 bond in neuroblastoma cells in tissue
culture (3) and synaptic membranes prepared from whole brain (4). Our results with neuronal cells cultured from fetal brain give additional support to the above hypothesis. Our results also indicate that ACE cleaves LHRH after it has been cleaved by the above endopeptidase. In addition, we have presented evidence concerning LHRH-degradation by glia. Almost the same mechanism operates in glia as in neurons. Thus, a thiol-dependent metallo-endopeptidase in neurons may play an important role in the degradation of LHRH, a neurotransmitter and/or neuromodulator, in the synapse or in the surface of neurons, while the enzyme in glia may function as an LHRH scavenger. Alternatively, if glial cells have LHRH receptors, the enzyme may terminate the action of LHRH on glial cells. In connection with our studies on neuronal and glial inactivation of LHRH, it should be noted that Molinaux et al., have reached a similar conclusion; endopeptidase-24.15, which is a similar enzyme to our thiol-dependent metallo-endopeptidase, is the primary enzyme degrading LHRH in membranes of the hypothalamus and the pituitary (6). In a previous report (4), we have isolated the LHRHdegrading enzyme from neuronal tissues in a membrane-bound form. On the other hand, a soluble form of the enzyme has been purified from tissues outside the brain (5). Both enzymes have similar properties except for their molecular weights. Thus, our enzyme, probably endopeptidase-24.15, may be involved in the degradation of LHRH, inside or outside the brain.
82 Acknowledgements We wish to thank Dr K. Mizuno and Professor K. Kurihara of Hokkaido University for their help in preparing primary cultures and for their helpful discussion. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, from the Research Foundation for Pharmaceutical Sciences, and from Takeda Science Foundation.
References 1. Lamour, Y. and Epelbaum, J. (1988). Interactions between cholinergic and peptidergic systems in the cerebral cortex and hippocampus. Prog. Neurobiol. 31: 109-148. 2. Wong, M., Eaton, M. J. and Moss, R. L. (1990). Electrophysiological actions of luteinizing hormone-releasing hormone: Intracellular studies in the rat hippocampal slice preparation. Synapse 5: 65-70. 3. Yokosawa, H., Fujii, Y. and Ishii, S. (1987). Degradation of luteinizing hormone-releasing hormone by neuroblastoma cells and their membrane: Evidence for the involvement of a thiol protease and angiotensin-converting enzyme. J. Neurochem. 48: 293-298.
NBUROPEFTIDES 4. Sakurada, C., Ohgaki, Y., Yokosawa, H. and Ishii, S. (1990). Thiol-dependent membrane-bound metallo-endopeptidase functioning in degradation of luteinizing hormone-releasing hormone in neuroblastoma cells and rat synaptic membrane. Isolation and characterization. Neuropeptides in press. 5. Orlowski, M., Reznik, S., Ayala, J. and Pierotti, A. R. (1989). Endopeptidase 24.15 from rat testes. Isolation of the enzyme and its specificity toward synthetic and natural peptides, including enkephalin-containing peptides. Biothem J. 261: 951-958. 6. Mohneaux, C. J., Lasdun, A., Michaud, C. and Orlowski, M. (1988). Endopeptidase-24.15 is the primary enzyme that degrades luteinizing hormone releasing hormone both in vitro and in vivo. J. Neurochem. 51: 624-633. 7. Arimatsu, Y. and Hatanaka, H. (1986). Estrogen treatment enhances survival of cultured fetal rat amygdala neurons in a defined medium. Dev. Brain Res. 26: 151-159. 8. Dichter, M. A. (1978). Rat cortical neurons in cell culture: culture methods, cell morphology, electrophysiology, and synapse formation. Brain Res. 149: 279-293. 9. Badford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.