Involvement of endopeptidase-24.11 in degradation of substance P by glioma cells

0143-4179/89/0014-0177/$10.00

Neuropeptides (1989) 14,177-W 0 Longman Group UK Ltd 1989

Involvement of Endopeptidase-24.11 of Substance P by Glioma Cells S. ENDO, H. YOKOSAWA

in Degradation

and S. ISHII

Department of Biochemistry, Faculty of Pharmaceutical Japan (reprint requests to HYJ

Sciences,

Hokkaido

University,

Sapporo

060,

Abstract-C6 of rat glioma cells and their plasma membranes degrade substance P (SP). The degradation, occurring mainly through the cleavage of the Gin’-Phe’, Phe’-Phe*, and GlygLeu” bonds, was strongly inhibited by phosphoramidon. Endopeptidase-24.11 (EC 3.4.24.11) purified from C6 cell membranes also cleaved SP at the same three peptide bonds in a manner sensitive to phosphoramidon. Thus, the degradation of SP by glioma cells and their membranes seems to be mediated by the action of endopeptidase-24.11.

Introduction Substance P (SP),

an undecapeptide

with the

sequence Arg-Pro-Lys-Pro-Gln-Gln-Phe-PheGly-Leu-MetNHz is present in the central and peripheral nervous systems. It is thought to function as a neurotransmitter after secretion into the synapse and rapidly to be degraded by enzymes in the synaptic region (1, 2). Membrane-bound proteases seem to be involved in the degradation of SP just as acetylcholinesterase breaks down acetylcholine in the synapse. In a previous report (3), we used neuroblastoma N-18 cells as a possible model of the neuron and investigated the degradation of SP by the cells and their plasma membranes. Based on the action of inhibitors of SP degradation, we proposed that metalloendopeptidase(s) distinct from both endopeptidase-24.11 (EC 3.4.24.11, Date received 28 April 1989 Date accepted 2 May 1989

enkephalinase) and angiotensin-converting enzyme (ACE, EC 3.4.15.1) are involved in the degradation of SP by the plasma membranes of neuroblastoma cells. We succeeded in purifying from rat brain membranes an enzyme designated as the substance P-degrading endopeptidase, which was found to be distinct from all of the other neuropeptidases that had been reported to cleave SP (4). Furthermore, results of studies of the degradation of SP by cultured fetal rat brain neurons (5) fit well with the above suggestion that substance P-degrading endopeptidase which we purified plays a major role in the initial stage of cleavage of SP by neurons. On the other hand, the results with primary cultures of glial cells (5) indicated that endopeptidase-24.11 rather than the neuronal SP-degrading peptidase is responsible for the degradation of SP by glia. In the present study, we examined the degradation of SP by C6 glioma cells, and also by endopeptidase-24.11 purified from C6 cells to 177

178 obtain definitive evidence for the involvement of endopeptidase-24.11 in the SP degradation by glia and to evaluate the contribution of the glia system to the inactivation of SP in the synaptic region. To our knowledge, this is the first report on endopeptidase-24.11 highly purified from cells of glial origin.

NEUROPEPTIDES

0.01

Materials SP, hippuryl-His-Leu, phosphoramidon, and bestatin were purchased from the Peptide Institute Inc., Osaka, Japan. SP fragments, p-chloromercuribenzenesulfonic acid (PCMBS), succinyl-AlaAla-Phe-4-methylcoumaryl-7-amide (MCA), and diisopropyl fluorophosphate (DFP) were obtained from Sigma Chemical Co., St. Louis, MO. Methanesulfonic acid (4M) containing 0.2% 3-(2aminoethyl)indole and n-octyl-p-D-thioglucoside (OTG) were purchased from Pierce Chemical Co., Rockford, IL and Dojin Chemical Co., Kumamoto, Japan, respectively. Captopril was kindly donated by Dr. A. Awaya of Mitsui Pharmaceuticals, Inc., Tokyo, Japan. Methods Cell culture An established rat glioma cell line, C6, (6) was cultured in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 2% (v/v) newborn calf serum (Boehringer-Mannheim) and 2% horse serum (GIBCO) at 37°C under 10% COz-90% air in plastic petri dishes (Falcon) for 5 to 7 days. The cells were used when they became confluent. Preparation of the plasma membrane Plasma membranes of C6 glioma cells were prepared by discontinuous density gradient centrifugation according to the method of Jones and Matus (7). Degradation of SP by glioma cells and their plasma membrane Cells were washed five times with normal solution (5mM HEPES-NaOH buffer (pH 7.4) containing 155mM NaCl, 5.4mM KCl, 1.8mM CaCl*, and 0.8mM MgC12) without detaching them from the

0

Timrtmln) Fig. 1 Degradation of SP by glioma C6 cells. The cells in petri dishes were incubated at 37°C for 0 (a) and 8hr (b) in the absence of phosphoramidon, and for 8hr in the presence of O.OlmM of phosphoramidon (c). Aliquots in a volume of 0.05ml of the sample were subjected to HPLC using a reversed-phase column (4 X 150mm) of Nucleosil 5C1s (Marchery, Nagel and Co.) with a 32-min linear concentration gradient of l-65% acetonitrile in 0.1% trifluoroacetic acid (TFA) at a flow rate of 1 ml/min. The absorbance at 210nm was monitored. The peaks of P and i indicate those derived from the starting material, SP, and the inhibitor, phosphoramidon, respectively. The arrows in (c) indicate the peaks whose generation was ‘suppressed by the presence of phosphoramidon.

petri dish. The following were then added to the cells in a petri dish: 1.6ml of normal solution, 0.2ml of inhibitor solution, and finally 0.2ml of 0.5mM SP dissolved in normal solution. The reaction was allowed to proceed at 37°C under 10% CO*-90% air. At various times, aliquots of the reaction mixture were withdrawn and incubated at 100°C for 5 min to stop the reaction. Then, the reaction mixture was centrifuged, filtered through a Millipore membrane filter (Millex-HV; pore size, 450nm) and subjected to high-performance liquid chromatography (HPLC) for analysis of the cleavage products. The HPLC was carried out under the conditions in the legend to Figure 1. The peptide fragments were identified by comparing their retention times with those of authentic SP fragments and also by determining

INVOLVEMENT OF ENDOPEPTIDASE-24.11

IN DEGRADATION

their amino acid compositions with a Hitachi 835 amino acid analyzer after hydrolysis at 115°C for 24hr with 4M methanesulfonic acid containing 0.2% 3-(Zaminoethyl)indole (8). The degradation of SP by the plasma membranes of C6 glioma cells was carried out at 37 “C in a mixture (OSml) consisting of 5mM HEPESNaOH buffer (pH 7.4), 155mM NaCl, 5.4mM KCl, 1.8mM CaCl*, 0.8mM MgC12,0.05mM SP, and 0.05-0.3mg membrane protein. The reaction was terminated by incubating at 100°C for 5 min. The reaction mixture was treated and subjected to HPLC as described above. Protein determination

Protein was determined by the method of Bradford (9) using bovine serum albumin (Sigma) as a standard. Purification of endopeptidase-24.11 C6 cells

from glioma

Endopeptidase-24.11 activity was assayed with 0.01 mM succinyl-Ala-Ala-Phe-MCA as a substrate (10). ACE activity was assayed with 1 mM hippuryl-His-Leu as a substrate (11). C6 glioma cells were washed with normal solution and detached from petri dishes. After washing with normal solution, the cells (11 g, wet weight) were homogenized with a Teflon homogenizer in 50ml of 20mM Tris-HCl buffer (pH 7.0) containing 0.25M sucrose. The homogenate was centrifuged at 76000 x g for 1 hr, and the resulting pellet was suspended in 50ml of 20mM Tris-HCl buffer (pH 7.0) containing 50mM OTG and stirred for 3 hr. The product was centrifuged (76000 x , 1 hr) and the supernatant was applied to a column (1.6 X 5cm) of DEAE-cellulose (Whatman) previously equilibrated with 20mM Tris-HCl buffer (pH 7.0) containing 10mM OTG. After washing with the equilibration buffer, adsorbed proteins were eluted with 30ml of a linear gradient (0 to 0.3 M NaCl) in the equilibration buffer. Endopeptidase-24.11 activity was detected in both the break-through fraction (about 40% of total activity) and the adsorbed fraction (60%) as previously reported with rat brain enzyme (12). The break-through fraction was not purified further. The adsorbed fraction (122ml) was dialyzed

OF SUBSTANCE P BY GLIOMA CELLS

179

against 25mM imidazole-HCl buffer (pH 7.4) containing 1OmM OTG and subjected to chromatofocusing on a column (0.9 x 20cm) of PBE94 (Pharmacia) previously equilibrated with the same buffer containing 1OmM OTG. The column was developed with a pH gradient (pH 7.0 to 4.0) formed by adding Polybuffer 74 (Pharmacia)-HCI (pH 4.0) containing 10mM OTG. A broad peak of endopeptidase-24.11 activity was eluted at about pH 5.5. Fractions (19ml) containing endopeptidase-24.11 activity but lacking ACE activity were pooled, concentrated with an Amicon YM-10 membrane, and placed on a column (1.2 X 85cm) of Sephacryl S-300 (Pharmacia) previously equihbrated with 20mM Tris-HCl buffer (pH 7.5) containing 10mM OTG and 0.1 M NaCl. The column was developed with the equilibration buffer. A single peak of endopeptidase-24.11 activity eluted at about 66ml (the molecular weight was estimated to be approximately 80000). After rechromatography on the same Sephacryl S-300 column under the same conditions, active fractions were used as the purified endopeptidase-24.11. Hydrolysis of SP by endopeptidase-24.11 from C6 glioma cells

purified

SP was hydrolyzed by purified endopeptidase24.11 in a reaction mixture (0.5ml) consisting of O.lM Tris-HCI buffer (pH 7.5), 1OmM CaCl*, 0.05mM zinc acetate, and 0.25ng of purified enzyme at 37°C in the absence and the presence of 0.01 mM phosphoramidon. The reaction was stopped by heating to 100°C for 5 min. After centrifugation, the mixture was treated and subjected to HPLC analysis as described earlier.

Results C6 ghoma cells were found to degrade SP (Fig. I). The chromatographic peak area of the starting material, SP, decreased as a function of time, while the area of seven peaks, designated A to G, of cleavage products increased. ~TWO peaks appearing at retention times of 7 min and of 12 min were judged to originate from the cells because they were detected in the absence of SP. The amino acid compositions of the products appear in Table 1. Among the identified products except for

180 Table 1

NEUROPEPTIDES

Amino Acid Compositions of Fragments Produced from SP Degraded by Glioma C6 Cells*

Peak

Arg

Pro

LYS

A B C D E F G P

17 23 1 0 13 11 0 9

32 46 0 0 28 24 0 19

17 23 1 0 13 11 0 9

Amino acid (mol %) Glu Phe 34 0 1 0 28 21 25 18

0 8 85 50 14 21 19 18

GUY

Leu

Met

Fragment identified

Yieldt W)

0 0 3 50 2 12 33 9

0 0 7 0 2 0 10 9

0 0 2 0 0 0 13 9

l-6 1-4 Phe 8-9 1-7 l-9 n.d. Complete

14 24 80 20 7 30

* The extent of degradation of SP was 43%.

t Yield was determined on the basis of SP degraded. n.d., not determined.

free phenylalanine, fragment (1-9) accumulated in the highest amount, followed by fragments (l-4) and (8-9). C-terminal fragments of SP were not detected. The degradation of SP was strongly inhibited by the addition of phosphoramidon to the reaction mixture, and the generation of almost all the peaks including fragment (l-4) was suppressed (Fig. lc). Since fragment (l-4) has not been reported to be formed directly by the action of endopeptidase-24.11 on SP, it may arise from the action of some other protease on the initial cleavage products formed by endopeptidase24.11. Other peptide fragments were those formed by the cleavage between the Gln6-Phe’, Phe7Phe’, or Glyg-Leu” bond of SP, all of which have been reported to be the sites of cleavage by endopeptidase-24.11 from pig caudate synaptic membrane (13), pig kidney (14), and human kidney (15). Therefore, these fragments should be the initial cleavage products formed by endopeptidase-24.11 in C6 glioma cells. The plasma membranes of C6 cells also degraded SP (Fig. 2). First, we examined the effects of various protease inhibitors on the initial cleavage of SP as determined by the decrease of the HPLC peak for SP (peak P). EDTA (1mM) and phosphoramidon (0.1 mM) markedly inhibited SP degradation by the plasma membrane (90% and 80% inhibition, respectively). Other inhibitors including DFP (lOmM), PCMBS (ImM), captopril(O.1 mM) and bestatin (0.1 nM) had little effect (8%, 5%, O%, and 3% inhibition, respectively). Next, we examined the effects of

protease inhibitors on the generation of cleavage products of SP separated by HPLC. Thirteen peaks (A to M) were formed in the absence of inhibitor (Fig. 2a). Among them, the generation of six peaks of B, C, D, G, H, and M was inhibited by phosphoramidon (Fig. 2b). The presence of captopril suppressed the generation of two peaks of D and F (Fig. 2~). Bestatin had little effect (Fig. 2d). The amino acid compositions of the HPLC purified products were determined (Table 2). Among the cleavage products detected in the absence of inhibitor, fragment (9-11) accumulated in the highest amount, followed by fragments (L-4) (l-7), (L-5) and (10-11). The presence of phosphoramidon affected the generation of fragments (l-6), (l-7), (l-9) (7-ll), (8-9), and (10-11). The phosphoramidon-inhibitable cleavage sites (Gln6-Phe’, Phe’-Phe’, and Gly’-Leu”) observed in the presence of C6 plasma membranes were identical to those found earlier when SP was degraded by cells or endopeptidase-24.11(13-15). Thus, endopeptidase-24.11 in the plasma membrane seems to play a major role in the initial cleavage of SP by C6 glioma cells. We obtained a highly purified preparation of endopeptidase-24.11 from the membrane of C6 cells with a specific activity of 87.5 nmol succinylAla-Ala-Phe-MCA hydrolyzed/hr/mg protein. The overall yield was 9%) representing a 1030-fold purification. The activity toward succinyl-AlaAla-Phe-MCA was completely inhibited by phosphoramidon (95% inhibition at the concentration of O.OOlmM) but not by captopril. The purified

181

1NVOLVEMEN-TOF ENDOPEF’DDASE-24.11IN DEGRADATION OF SUBSTANCE P BY GLIOMA CELLS

(b)

15

20 Retention

0

5

10

15

20

time (min)

Fig. 2 Degradation of SP by the plasma membrane of glioma C6 cells. The reaction mixture (OSml, pH 7.4) containing 0.2mg protein of the membrane was incubated at 37°C for 15 hr in the absence (a) or in the presence of 0.01 mM phosphoramidon (b), 0.01 mM captopril (c), or O.OlmM bestatin (d). HPLC analysis was carried out as in Fig. 1. The peaks of P and i indicate those derived from the starting material, SP, and the inhibitor, respectively. The arrows in (b) and (c) indicate the peaks whose generation was inhibited by phosphoramidon and captopril, respectively.

enzyme also cleaved SP (Fig. 3). Eight newly formed peaks (A to I-I) were detected, together with peak P, that of SP (Fig. 3a). The positions of peak A, C, D, and H were coincident with those of authentic SP fragments (l-6), (l-7), (8-9), and (7-l 1)) respectively. The amino acid compositions of the cleavage products separated by HPLC are Table 2

Amino

Acid Compositions

of Fragments

shown in Table 3. The results allowed identification of these peptide fragments and indicated that the purified enzyme cleaved SP at the Gln6-Phe7, Phe7-Phea, and Gly’-Leu” bonds, as was the case for cleavage by cells and their plasma membranes. In the presence of phosphoramidon, the generation of almost all the HPLC peaks except for peak

Produced from SP Degraded

by The Plasma Membrane

Glioma C6 Cells*

Peak

Arg

Pro

LYS

A B C

20 17 22 0 0 0 0 13 8 10 0 3 0 0 9

37 34 48 0 0 28 0 28 22 22 0 8 0 0 19

21

D E F G H I J K L M P

Amino acid (mol %) GlU Phe

16 2.5 0 0 0 1 14 9 10 0 3 0 0 9

22 33 1 0 0 19 2 30 21 30 0 24 18 3 18

* The extent of degradation of SP was 50%.

t Yield was determined on the basis of SP degraded nd., not determined.

G1.V

0 0

0 0

2 0 50 16 0 14 30 24 21 23 35 30 18

2 0 50 31 38 1 10 4

41 13 12 38 9

Leu

Met

0

0 0 0 44 0 0 28 0 0 0 17 14 18 13 9

0 0 56 0 6 31 0 0 0

21 12 17 16 9

Fragment identified

l- 5 l- 6 l- 4

IO-11 8- 9 nd. 9-11 l- 7 l- 9 I- 8 8-11 5-11 6-11 7-11 Complete

Yield f f%)

12 6 19 11 9 32 13 5 2 8 8 3 7

of

182

NEUROPEPTIDES

G or not. Because various endopeptidase-24.11 preparations purified from other tissues (10, 13-15) have been reported to cleave SP at the same three phosphoramidon-sensitive sites mentioned above, we conclude that endopeptidase-24.11 actually functions at the plasma membrane of C6 glioma cells. Discussion (b)

u

ReteSntion +id”bnin~

Fig. 3 Degradation of SP by endopeptidase-24.11 purified from gliomaC6cells. The reaction mixture (OSml, pH 7.5) was incubated at 37°C in the absence (a) and the presence (b) of 0.01 mM phosphoramidon. HPLC analysis was carried out as in Fig. 1 except for elution with a 16-min linear gradient of l-65% acetonitrile in 0.1% ‘WA. The peaks P and i indicate those derived from the starting material, SP, and the inhibitor, phosphoramidon, respectively. The arrows indicate the peaks whose generation was inhibited by phosphoramidon.

G was found to be completely suppressed (Fig. 3b). As the peak due to phosphoramidon overlapped peak G, we could not determine whether phosphoramidon affected the generation of peak Table 3 Amino Acid Compositions from Glioma C6 Cells*

Peak

Arg

Pro

LYS

A B C D E F G H P

16 0 15 0 11 0 0 0 9

33 0 30 0 20 0 0 0 19

17 0 14 0 10 0 0 0 9

Endopeptidase-24.11 has been reported to degrade a variety of neuropeptides present in the brain (13, 14). SP has been shown to be a good substrate for endopeptidase-24.11 in vitro (14), and this enzyme has been proposed to be involved in the degradation of SP in the synaptic region (13). On the other hand, this enzyme has been reported to be more abundant in glia than in neurons (16-18). We have suggested previously that a novel phosphoramidon-insensitive metalloendopeptidase rather than endopeptidase-24.11 is involved in the degradation of SP by neuroblastoma cells (3) and neuronal cells cultured from rat fetal brain (5). Endopeptidase-24.11 appeared to be responsible for degradation of SP by glial cells cultured from rat fetal brain (5). In the present study we attempted to provide further evidence that endopeptidase-24.11 in glia degrade SP. Since it is difficult to obtain the glia in a pure form from the whole brain, we used in this study C6 glioma cells. We confirmed the presence of endopeptidase-24.11 in C6 glioma cells by purifying it from their membranes. This is the first report on the

of Fragments Produced from SP Degraded by Endopeptidase-24.11

Amino acid (mol %) Glu Phe 34 0 27 0 15 0 0 0 18

* The extent of degradation of SP was 15%.

t Yield was determined on the basis of SP degraded.

0 0 14 59 21 64 28 40 18

G/Y

Leu

0 0 0 41 13 36 31 27 9

0 50 0 0 0 0 21 20 9

Met

.o 50 0 0 0 0 20 13 9

Fragment identijied l- 6 10-11 l- 7 8- 9 l- 9 7- 9 8-11 7-11 Complete

Purified

Yield? (%) 34 52 8 41 43 6 12 9

INVOLVEMENT OF ENDOPEPTIDASE-24.11IN DEGRADATION OF SUBSTANCEP BY GLIOMA CELLS

purification of endopeptidase-24.11 from cells of glial origin. The fragments produced from SP by the action of this enzyme preparation (Fig. 3, Table 3) were almost identical to those produced by C6 cells themselves (Fig. 1, Table 1) and also to phosphoramidon-sensitive fragments detected in the digestion products of SP by the plasma membrane of C6 cells (Fig. 2, Table 2). The same fragments have been reported to be produced by endopeptidase-24.11 purified from tissues other than the glia (13-15). We have recently reported that a substance PLdegrading endopeptidase, which we succeeded in purifying from rat brain and found to be a metal-chelator-sensitive protease distinct from endopeptidase-24.11 and ACE, plays a major role in the degradation of SP in the synaptic membrane of rat brain, although endopeptidase-24.11 is also involved (4). Even if we would plan to adopt the synaptic membrane as a model of the synapse, it would be impossible to obtain the pure synaptic membrane completely separated from other membrane of brain origin (7). Especially, the synaptic membrane will be inevitably contaminated with the membrane of the glia whose population is much larger than that of the neuron in the brain. In order to avoid such ambiguousness, we used the established neuroblastoma (3) and glioma cells (in this series of studies) as possible ‘models of the neuron and of the glia, respectively. Though these cells were not derived from a brain region where SP is rich and the cells in culture may change their phenotype (19), they should reflect the characteristics of neuronal and glial cells (6, 20). The results with the two established cell lines have led us to propose that a metalloendopeptidase distinct from endopeptidase-24.11 is present in the neuron and may play an important role on the degradation of SP in the synapse, while endopeptidase-24.11 may function mainly in the glia as a SP scavenger. It should be noted that endopeptidase24.11 has also been reported to be present in neurons (21, 22). The relative abundance of this endopeptidase in various neuronal and glial populations remains to be determined. Acknowledgements We wish to thank Professor K. Kurihara and Dr. M. Miyake of Hokkaido University for their help for culturing cells and for

183

their helpful discussion. This work was supported in part by Grants-in-Aid for Scientific Research from Ministry of Education, Science and Culture of Japan (H.Y., SE. and S.I.), from the Research Foundation for Pharmaceutical Sciences (H.Y.), and from Japan Foundation for Applied Enzymology (H.Y.). S. Endo is a recipient of a fellowship of Japan Society for the Promotion of Sciences for Japanese Junior Scientists.

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184 15. Skidgel, R. A., Engelbrecht, S., Johnson, A. R. and Erdos, E. G. (1984). Hydrolysis of substance P and neurotensin by converting enzyme and neutral endopeptidase. Peptides 5: 769-716. 16. Horsthemke, B., Hamprecht, B. and Batter, K. (1983). Heterogeneous distribution of enkephalin-degrading peptidases between neuronal and glial cells. Biochem. Biophys. Res. Commun. 115: 423-429. 17. Lentzen, H., Monden, I., Linke, J. and Palenker, J. (1983). No evidence for enkephalinase A on neuronal cells. Life Sci. 33 (Suppl. I): 105108. 18. Lentzen, H. and Palenker, J. (1983). Localization of the thiorphan-sensitive endopeptidase, termed enkephalinase A, on glia cells. FEBS Lett. 153: 93-97. 19. Kenny, A. J. (1986). Reply from John Kenny. Trends Biochem. Sci. 11: 361.

NEUROPEPTIDES

20. Amano, T., Richelson, E. and Nirenberg, M. (1972). Neurotransmitter synthesis by neuroblastoma clones. Proc. Natl. Acad. Sci. USA 69: 258-263. 21. Matsas, R., Kenny, A. J. and Turner, A. J. (1986). An immunohistochemical study of endopeptidase-24.11 (“enkephalinase”) in the pig nervous system. Neuroscience 18: 991-1012. 22. Barnes, K., Matsas, R., Hooper, N. M., Turner, A. J. and Kenny, A. J. (1988). Endopeptidase-24.11 is striosomally ordered in pig brain and, in contrast to aminopeptidase N and peptidyl dipeptidase A (‘angiotensin converting enzyme’), is a marker for a set of striatal efferent fibres. Neuroscience 27: 799-817.