Vol. October
172,
No. 30.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1990
ANALYSIS OF BIG ENDOTHELIN-1
DIGESTION
BY CATHEPSIN
883-889
D
Tatsuya Sawamura, Osamu Shinmi, Naoya Kishi, Yoshiki Sugita, Masashi Yanagisawa*, Katsutoshi Got?*, Tomoh Masaki* and Sadao Kimura Department Institute Received
September
of Biochemistry and *Department of Pharmacology, of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan 22,
1990
SUMMARY: Digestion of big endothelin(ET)-1 by cathepsin D, which is the only substantially identified protease showing ET converting enzyme activity, was characterized. Increased doses of cathepsin D showed decrease of immunoreactive (ir-) ET produced from big ET-l. Time course of big ET-1 conversion showed marked increase of ir-ET in a relatively short period, but further incubation resulted in the decrease of ir-ET. Incubation at various pHs with different doses of cathepsin D revealed that low doses of cathepsin D yielded the maximum production of ir-ET at pH 354.0, but higher doses of cathepsin D showed a bimodal curve of ir-ET production, which may be due to degradation of ir-ET. HPLC anal sis revealed that cathepsin D cleaves Asn1S-Ile19 bond in addition to TrpA-Va122 bond of big ET-l. These data suggests cathepsin D is not a physiological endothelin converting enzyme. ‘J Acadamlc Presb,IrlC. 1990
Endothelin(ET) is a potent vasoconstrictor peptide purified from the culture medium of porcine aortic endothelial cells (1). At present three isopeptides named ET-l, ET-2 and ET-3 are identified and known to be expressing in various tissues (2, M. Y. et al. under submission). By the analysis of cDNA of ET-l, it was suggested that ET-1 is generated from an intermediate form, big ET-l, by the unusual proteolysis between Trp21-Va122. Since the C-terminal fragments of big ET-l including ET-1(22-39) co-exist with ET-1 at the almost same content in the supernatant of cultured porcine endothelial cells, the existence of a specific protease, calIed endothelin converting enzyme (ECE), which cleaves between Trp21 and Va122, was strongly suggested (3). 1To whom correspondence should be addressed.
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As big ET-1 has considerably less contractile activity than ET-1 on isolated porcine coronary artery, ECE is an activating enzyme by converting big ET-1 to ET-1 (4). Recently, we reported the purification of an cathepsin D like aspartic protease with ECE activity from bovine adrenal chromaffin granules as a most abundant enzyme showing ECE activity (5). Cathepsin D was also shown to be a most abundant protease having ECE activity in the soluble fraction of cultured endothelial cells (6). At present cathepsin D is the only protease with ECE activity substantially identified from ET producing cells. In this study, we characterized the digestion of big ET-1 by cathepsin D, in order to examine the possibility of cathepsin D as a physiological ECE. MATERIALS
AND METHODS
Chemicuts: Human big ET-1 and ET-1 were obtained from Peptide Institute Inc. (Osaka, Japan). Bovine spleen cathepsin D was obtained from Sigma Chemical Co.. Human ET-1(22-38) was synthesized by a solid phase peptide synthesizer (Model 430A, Applied Biosystems). ET-l(l-18) was obtained by digestion of ET-l(l-21) by carboxypeptidase Y. ECE u.ssuy: ECE activity was measured by radioimmunoassay (RIA) with the antiserum (As-ETC) recognizing the C-terminal of ET-1 as described previously (7). Briefly, 100 pmol of big ET-1 was incubated with various amounts of cathepsin D in 350 ~1 of a buffered solution at 37°C. The enzyme reaction was stopped by boiling for 5 min. ET-1 produced in the reaction mixture was measured by RIA. In the time course experiments, 800 pmol of big ET-1 was incubated with various amounts (8 pg, 2.4 pg or 0.8 ,ug) of cathepsin D in 2.8 ml of 0.1 M glycine-HCl buffer at 37°C. 350 ,ul of the reaction mixture was taken out at the time point (0, 0.5, 1, 2, 5, 8, 12 and 24 h) and boiled for 5 min to stop the reaction. Then ET-1 produced was measured by RIA. Analysis of ET related peptides in the cathepsin D digest by high peqfkrmance liquid chromatography (HPLC): 1 nmol of big ET-1 was incubated
with 0.1 pg of cathepsin D in 0.1 M glycine-HCl at 37°C for various periods. The reaction mixture was directly applied on the pre-equilibrated column (Cosmosil SODS-AR, 4.6 x 2.50 mm, Nacalai tesque, Kyoto, Japan) with 0.1 % trifluoroacetate (TFA). Then the column was eluted at 1 ml/min with three steps of linear acetonitril gradients: 0 % to 1.5 % with 1 min, 15 % to 37.5 % with 45 min and 37.5 % to 50 % with 5 min. The elution profiles were monitored by absorbance at 210 nm. The purified peptides were subjected to a gas phase peptide sequencer (Model 477A, Applied Biosystems). RESULTS
AND DISCUSSION
In investigating ECE activity in endothelial ceils at acidic pHs, it was observed that large amounts of the enzyme solution seemed to produce less 884
Vol.
172,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
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amount of ir-ET (6). We examined, at first, the relationship between dose of cathepsin D and increase of ir-ET (Fig. 1). 100 pmol of big ET-1 was used as substrate, and incubated for 2 h with various amounts of cathepsin D at pH 3.5. 0.2 pg of cathepsin D showed the maximum increase of ir-ET, and the larger amounts of cathepsin D was used, the less increment of ir-ET was seen. This result is consistent with our previous report on cathepsin D like ECE activity in endothelial cells (6). Time course experiments of ir-ET production by cathepsin D were performed (Fig. 2a). Using 8 pg of cathepsin D, the increment of ir-ET has reached to the maximum at 0.5 h, and the further incubation resulted in decrease of ir-ET. Using 2.4 pugor 0.8 pugof cathepsin D, the time giving the maximum increase of ir-ET sifted to 1 h or 2 h, respectively. As shown in Fig. 2b, pH dependency of ir-ET production by different doses of cathepsin D was examined. When 1 ,ug of cathepsin D was used, two peaks at pH 3.0-3.5 and pH 4.5-5.0 as the most efficient pHs were observed. When 0.1 or 0.3 pg of cathepsin D was used, however, a single optimal pH was observed. These observations in which the incubation for longer time or with larger amounts of cathepsin D resulted in the decrease of ir-ET, suggesting degradation of ET-l itself by cathepsin D. In order to know how cathepsin D decreases ir-ET, we analyzed the digested products of big ET-l, ET-l(l-21) and ET-1(22-38) by cathepsin D by reverse phase HPLC. As shown in Fig. 3a, from big ET-l, four main products (peak A, B, C and D) were observed after 2 h incubation. At 24 h, big ET-l was completely digested and the fifth peak (peak E in Fig. 3b) was appeared
Fig. 1. Dose dependency of k-ET production
by cathepsin D.
100 pmol of big ET-1 was incubated with various amounts 350 ~1 of 0.1 M glycine-HCl (pH 3.5) for 2 h at 37°C. 885
of cathepsin
D in
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172,
No.
2, 1990
BIOCHEMICAL
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PH
Fig. 2.(a) Time course of k-ET
production
by cathepsin
D.
800 pmol of big ET-1 was incubated with 8 pg ( o---o ), 2.4 pg ( o---o ) or 0.8 pg ( A---A ) of cathepsin D in 2.8 ml of 0.1 M glycine-HCl (pH 3.5) at 37°C as described in “MATERIALS AND METHODS”. (b) pH dependency
of ir-ET production
by cathepsin
D.
100 pmol of big ET-1 was incubated for 2 h at 37°C with 1 pg ( o---o ), 0.3 ,ug ( A---A ) or 0.1 ,ug ( v---v ) of cathepsin D in 350 ~1 of a buffered solution; pH 2.0-3.5: 0.1 M glycine-HCI, pH 4.0-5.5: 0.1 M sodium acetate, pH 6.0-7.5: 0.1 M potassium phosphate, pH 7.5-9.0: Tris-HCl.
2
big ET- 1
(a)
$40 z 220 4"
/y-----___---
I ___----
B
A
c
D
A
cm e B
Cc)
ET-l
172,
No. 30.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1990
ANALYSIS OF BIG ENDOTHELIN-1
DIGESTION
BY CATHEPSIN
883-889
D
Tatsuya Sawamura, Osamu Shinmi, Naoya Kishi, Yoshiki Sugita, Masashi Yanagisawa*, Katsutoshi Got?*, Tomoh Masaki* and Sadao Kimura Department Institute Received
September
of Biochemistry and *Department of Pharmacology, of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan 22,
1990
SUMMARY: Digestion of big endothelin(ET)-1 by cathepsin D, which is the only substantially identified protease showing ET converting enzyme activity, was characterized. Increased doses of cathepsin D showed decrease of immunoreactive (ir-) ET produced from big ET-l. Time course of big ET-1 conversion showed marked increase of ir-ET in a relatively short period, but further incubation resulted in the decrease of ir-ET. Incubation at various pHs with different doses of cathepsin D revealed that low doses of cathepsin D yielded the maximum production of ir-ET at pH 354.0, but higher doses of cathepsin D showed a bimodal curve of ir-ET production, which may be due to degradation of ir-ET. HPLC anal sis revealed that cathepsin D cleaves Asn1S-Ile19 bond in addition to TrpA-Va122 bond of big ET-l. These data suggests cathepsin D is not a physiological endothelin converting enzyme. ‘J Acadamlc Presb,IrlC. 1990
Endothelin(ET) is a potent vasoconstrictor peptide purified from the culture medium of porcine aortic endothelial cells (1). At present three isopeptides named ET-l, ET-2 and ET-3 are identified and known to be expressing in various tissues (2, M. Y. et al. under submission). By the analysis of cDNA of ET-l, it was suggested that ET-1 is generated from an intermediate form, big ET-l, by the unusual proteolysis between Trp21-Va122. Since the C-terminal fragments of big ET-l including ET-1(22-39) co-exist with ET-1 at the almost same content in the supernatant of cultured porcine endothelial cells, the existence of a specific protease, calIed endothelin converting enzyme (ECE), which cleaves between Trp21 and Va122, was strongly suggested (3). 1To whom correspondence should be addressed.
Vol.
172,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
As big ET-1 has considerably less contractile activity than ET-1 on isolated porcine coronary artery, ECE is an activating enzyme by converting big ET-1 to ET-1 (4). Recently, we reported the purification of an cathepsin D like aspartic protease with ECE activity from bovine adrenal chromaffin granules as a most abundant enzyme showing ECE activity (5). Cathepsin D was also shown to be a most abundant protease having ECE activity in the soluble fraction of cultured endothelial cells (6). At present cathepsin D is the only protease with ECE activity substantially identified from ET producing cells. In this study, we characterized the digestion of big ET-1 by cathepsin D, in order to examine the possibility of cathepsin D as a physiological ECE. MATERIALS
AND METHODS
Chemicuts: Human big ET-1 and ET-1 were obtained from Peptide Institute Inc. (Osaka, Japan). Bovine spleen cathepsin D was obtained from Sigma Chemical Co.. Human ET-1(22-38) was synthesized by a solid phase peptide synthesizer (Model 430A, Applied Biosystems). ET-l(l-18) was obtained by digestion of ET-l(l-21) by carboxypeptidase Y. ECE u.ssuy: ECE activity was measured by radioimmunoassay (RIA) with the antiserum (As-ETC) recognizing the C-terminal of ET-1 as described previously (7). Briefly, 100 pmol of big ET-1 was incubated with various amounts of cathepsin D in 350 ~1 of a buffered solution at 37°C. The enzyme reaction was stopped by boiling for 5 min. ET-1 produced in the reaction mixture was measured by RIA. In the time course experiments, 800 pmol of big ET-1 was incubated with various amounts (8 pg, 2.4 pg or 0.8 ,ug) of cathepsin D in 2.8 ml of 0.1 M glycine-HCl buffer at 37°C. 350 ,ul of the reaction mixture was taken out at the time point (0, 0.5, 1, 2, 5, 8, 12 and 24 h) and boiled for 5 min to stop the reaction. Then ET-1 produced was measured by RIA. Analysis of ET related peptides in the cathepsin D digest by high peqfkrmance liquid chromatography (HPLC): 1 nmol of big ET-1 was incubated
with 0.1 pg of cathepsin D in 0.1 M glycine-HCl at 37°C for various periods. The reaction mixture was directly applied on the pre-equilibrated column (Cosmosil SODS-AR, 4.6 x 2.50 mm, Nacalai tesque, Kyoto, Japan) with 0.1 % trifluoroacetate (TFA). Then the column was eluted at 1 ml/min with three steps of linear acetonitril gradients: 0 % to 1.5 % with 1 min, 15 % to 37.5 % with 45 min and 37.5 % to 50 % with 5 min. The elution profiles were monitored by absorbance at 210 nm. The purified peptides were subjected to a gas phase peptide sequencer (Model 477A, Applied Biosystems). RESULTS
AND DISCUSSION
In investigating ECE activity in endothelial ceils at acidic pHs, it was observed that large amounts of the enzyme solution seemed to produce less 884
Vol.
172,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
amount of ir-ET (6). We examined, at first, the relationship between dose of cathepsin D and increase of ir-ET (Fig. 1). 100 pmol of big ET-1 was used as substrate, and incubated for 2 h with various amounts of cathepsin D at pH 3.5. 0.2 pg of cathepsin D showed the maximum increase of ir-ET, and the larger amounts of cathepsin D was used, the less increment of ir-ET was seen. This result is consistent with our previous report on cathepsin D like ECE activity in endothelial cells (6). Time course experiments of ir-ET production by cathepsin D were performed (Fig. 2a). Using 8 pg of cathepsin D, the increment of ir-ET has reached to the maximum at 0.5 h, and the further incubation resulted in decrease of ir-ET. Using 2.4 pugor 0.8 pugof cathepsin D, the time giving the maximum increase of ir-ET sifted to 1 h or 2 h, respectively. As shown in Fig. 2b, pH dependency of ir-ET production by different doses of cathepsin D was examined. When 1 ,ug of cathepsin D was used, two peaks at pH 3.0-3.5 and pH 4.5-5.0 as the most efficient pHs were observed. When 0.1 or 0.3 pg of cathepsin D was used, however, a single optimal pH was observed. These observations in which the incubation for longer time or with larger amounts of cathepsin D resulted in the decrease of ir-ET, suggesting degradation of ET-l itself by cathepsin D. In order to know how cathepsin D decreases ir-ET, we analyzed the digested products of big ET-l, ET-l(l-21) and ET-1(22-38) by cathepsin D by reverse phase HPLC. As shown in Fig. 3a, from big ET-l, four main products (peak A, B, C and D) were observed after 2 h incubation. At 24 h, big ET-l was completely digested and the fifth peak (peak E in Fig. 3b) was appeared
Fig. 1. Dose dependency of k-ET production
by cathepsin D.
100 pmol of big ET-1 was incubated with various amounts 350 ~1 of 0.1 M glycine-HCl (pH 3.5) for 2 h at 37°C. 885
of cathepsin
D in
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172,
No.
2, 1990
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PH
Fig. 2.(a) Time course of k-ET
production
by cathepsin
D.
800 pmol of big ET-1 was incubated with 8 pg ( o---o ), 2.4 pg ( o---o ) or 0.8 pg ( A---A ) of cathepsin D in 2.8 ml of 0.1 M glycine-HCl (pH 3.5) at 37°C as described in “MATERIALS AND METHODS”. (b) pH dependency
of ir-ET production
by cathepsin
D.
100 pmol of big ET-1 was incubated for 2 h at 37°C with 1 pg ( o---o ), 0.3 ,ug ( A---A ) or 0.1 ,ug ( v---v ) of cathepsin D in 350 ~1 of a buffered solution; pH 2.0-3.5: 0.1 M glycine-HCI, pH 4.0-5.5: 0.1 M sodium acetate, pH 6.0-7.5: 0.1 M potassium phosphate, pH 7.5-9.0: Tris-HCl.
2
big ET- 1
(a)
$40 z 220 4"
/y-----___---
I ___----
B
A
c
D
A
cm e B
Cc)
ET-l
