(Received for publication, November 18, 1994; and in revised form, January 13, 1995)
From the
A neutral endoprotease was isolated from porcine antral mucosa
and purified to homogeneity as examined by SDS-polyacrylamide gel
electrophoresis (PAGE). Throughout the purification, t-butyloxycarbonyl-Arg-Val-Arg-Arg-4-methylcoumaryl-7-amide
(MCA) was used as a substrate, which was found to be hydrolyzed
specifically by the enzyme at the Arg-Arg bond. Unexpectedly, however,
the enzyme was also found to hydrolyze vasoacive intestinal polypeptide
(VIP) fairly specifically and more efficiently when various
neuropeptides and related peptides were examined as substrates. It
could degrade VIP by cleaving three peptide bonds not containing an
arginine residue(s) with K = 7.7
10
M and k
/K
= 7.4
10
M
s
(at pH 7.6 in the presence of 0.1% Lubrol
PX), whereas only secretin, substance P, and a few others were
hydrolyzed at much slower rates among the various peptides examined.
Both activities toward the MCA substrate and VIP behaved in parallel
throughout the purification procedures and showed essentially the same
pH optimum and susceptibility toward various inhibitors and detergents.
Therefore, both activities are thought to be due to the same enzyme.
This endoprotease required 0.001% or a higher concentration of a
detergent such as Lubrol PX or Triton X-100 for its maximal activity.
Its optimum pH was about 7.5 and the molecular weight was estimated to
be approximately 37,000 by SDS-PAGE. This enzyme was strongly inhibited
by serine protease inhibitors such as diisopropylfluorophosphate and
phenylmethanesulfonyl fluoride. It was also inhibited by p-chloromercuribenzoic acid, but not by some other cysteine
protease inhibitors. Therefore, the enzyme appears to be most likely a
kind of serine protease although its possibility as a cysteine protease
cannot be completely excluded. Analysis of its cleavage specificity
toward various oligopeptides indicated the possibility that the
protease might recognize a specific amino acid sequence(s) and/or
conformation in the vicinity of the cleavage site of the target
peptide. Various characteristics of the endoprotease suggest that it is
a novel membrane-bound neuropeptide-degrading endoprotease fairly
specific for VIP.
Vasoactive intestinal polypeptide (VIP) ()is a
28-amino-acid residue neuropeptide, which plays many physiological
roles in the gut and nervous
systems(1, 2, 3, 4) . VIP is found
in all layers of the gut including stomach mucosa and membranes and is
known to be a physiological mediator for relaxation of gastric smooth
muscle and for pepsinogen release in stomach
mucosa(4, 5, 6, 7, 8) . The
physiological mechanism of inactivation of VIP has not been well
clarified, but the primary pathway is thought to involve proteolytic
degradation. Several peptidases which were thought to be related to
inactivation of VIP have been studied including mast cell tryptase and
chymase(9) , enkephalinase(10) , and gastric muscle
membrane-associated peptidase (5) . However, there has been no
report so far of a neuropeptide-degrading protease which can
specifically degrade VIP.
The present study was initiated in an attempt to isolate and characterize such a protease(s) that might specifically degrade VIP and/or related neuropeptides. This type of protease is thought to be present in minute quantity in the tissue, and often in the membrane-bound form. Therefore, special care should be taken to minimize proteolytic degradation that is apt to occur during the isolation procedures from gastric mucosa which contains lysosomal and a variety of other proteases(11, 12, 13, 14, 15, 16) . Thus, we used density gradient fractionation in the initial stage of the isolation procedure to minimize proteolytic degradation of the target protease(s), especially by lysosomal proteases. Further, we first chose t-butyloxycarbonyl(Boc)-Arg-Val-Arg-Arg-4-methylcoumaryl-7-amide (MCA) as a routine substrate since VIP and related neuropeptides generally contain several basic residues, often including basic amino acid pairs, at which sites cleavages were expected to occur, and assay was done by HPLC analysis of the cleavage products of the synthetic peptide MCA which is resistant to the action of aminopeptidase unlike simple oligopeptides like VIP.
Thus, we could finally isolate a novel membrane-bound neutral endoprotease from porcine antral mucosa which can fairly specifically degrade VIP as well as Boc-Arg-Val-Arg-Arg-MCA, and here we report its enzymatic characteristics.
Aminopeptidase B activity was assayed by the method of Usui et al.(17) with modifications in 200 µl of a reaction mixture containing 0.1 mM Arg-MCA and 50 mM potassium phosphate (pH 6.5), and the amount of AMC produced was measured fluorometrically.
Acid phosphatase activity was assayed by the modified Lowry method (18) with 10 mMp-nitrophenyl phosphate in 500 µl of 50 mM sodium acetate buffer (pH 5.4) at 37 °C for 1 h. After the reaction, 500 µl of 0.2 N NaOH was added, and the absorbance at 410 nm was measured.
Trypsin-like protease activity toward Boc-Gln-Gly-Arg-MCA was assayed fluorometrically as described (11) with 0.1 mM substrate in 200 µl of 50 mM Tris-Cl (pH 8.2).
Cysteine protease activity toward Boc-Leu-Arg-Arg-MCA was assayed with 0.1 mM substrate in 200 µl of 50 mM potassium phosphate (pH 6.5) and 1 mM 2-mercaptoethanol, and the amount of AMC produced was measured fluorometrically.
The solubilized sample was
centrifuged at 20,000 g for 25 min in a Beckman 50.2
Ti rotor. The resulting clear supernatant was dialyzed against 40
mM Tris-Cl buffer (pH 7.6), 0.2% Lubrol PX. The dialyzed
sample was applied to a DE52 column (1.5
10 cm) equilibrated
with 40 mM Tris-Cl buffer (pH 7.6), 0.2% Lubrol PX, and eluted
with a linear gradient of 0-0.5 M NaCl. The active
fractions were pooled and concentrated to 1 ml, and applied to a
Sephacryl S-200 column (1
145 cm) equilibrated and eluted with
40 mM Tris-Cl buffer (pH 7.6) containing 0.2 M NaCl
and 0.05% Lubrol PX.
The pooled active fraction from the Sephacryl
S-200 was applied to a Mono-Q/FPLC HR5/5 column and eluted with a
linear gradient of 0-0.5 M NaCl in 50 mM Tris-Cl (pH 7.6), 0.02% Lubrol PX. The active fractions were
pooled and applied to a PCMB-agarose affinity column (0.9 3 cm)
and eluted first with 50 mM 2-mercaptoethanol, 50 mM Tris-Cl (pH 7.6), 0.2 M NaCl, and then with 100 mM 2-mercaptoethanol, 50 mM Tris-Cl (pH 7.6), and 1.0 M NaCl. The active fractions were pooled and dialyzed against 40
mM Tris-Cl (pH 7.6).
Figure 1:
Isolation of a new
protease activity from porcine antral mucosal vesicle fraction through
sucrose density gradient centrifugation. A, sucrose
density() and protein(
). Sucrose density gradient
centrifugation was done as described under ``Experimental
Procedures.'' B, activities of aminopeptidase B(
)
and acid phosphatase (
). C, trypsin-like protease
activity toward Boc-Gln-Gly-Arg-MCA(
) and thiol protease activity
toward Boc-Leu-Arg-Arg-MCA (
). D, activity of a novel
protease. A novel endoprotease activity was screened with 1 mM Boc-Arg-Val-Arg-Arg-MCA as a substrate in 50 mM Tris-Cl
(pH 7.2) containing 1 mM CaCl
, 0.02% Lubrol PX,
and 20 µM leupeptin with aminopeptidase B and the reaction
products were analyzed fluorometrically as described under
``Experimental Procedures.''
To detect a novel protease, we used the synthetic peptide Boc-Arg-Val-Arg-Arg-MCA which has often been used as a substrate for furin and related proteases(20) . In addition to the fluorometric determination of AMC produced directly by enzymatic reaction, we analyzed the reaction products by HPLC or determined AMC after additional reaction with aminopeptidase B because some proteases may cleave at other site(s) than the Arg-MCA bond in the substrate(21) . Through this screening method we found a new proteolytic activity unexpectedly cleaving the Arg-Arg bond, but not the Arg-MCA bond, of Boc-Arg-Val-Arg-Arg-MCA. This activity was found in fraction 7 (at density 1.17-1.18 g/ml) (Fig. 1D), was not inhibited by leupeptin, and was clearly different from the activities of the trypsin-like protease(s), cysteine protease(s), and aminopeptidase(s) which were also found in gastric mucosal vesicle fractions.
Figure 2: Purification of a VIP-degrading endoprotease. A, DE52 chromatography (fraction size, 3 ml; flow rate, 0.5 ml/min). The enzyme activity was analyzed in the Applied Biosystems 130A analyzer as described under ``Experimental Procedures.'' Fractions 20-26 were pooled. B, Sephacryl S-200 chromatography (fraction size, 2 ml; flow rate, 6 ml/h). The enzyme activity was analyzed in the Applied Biosystems 130A analyzer. Fractions 42-48 were pooled. C, Mono-Q/FPLC (fraction size, 0.5 ml; flow rate, 0.5 ml/min). The enzyme activity was analyzed in the Applied Biosystems 130A analyzer. Fractions 24-27 were pooled. D, PCMB-agarose affinity chromatography. The sample was loaded and washed in 50 mM of Tris-Cl (pH 7.6), 0.2 M NaCl, 0.02% Lubrol PX (a), and eluted first with 50 mM 2-mercaptoethanol, 50 mM of Tris-Cl (pH 7.6), 0.2 M NaCl (b), and then eluted with 100 mM 2-mercaptoethanol, 50 mM of Tris-Cl (pH 7.6), 1.0 M NaCl (c). The enzyme activity was analyzed in the Applied Biosystems 130A analyzer. Fractions 22-24 were pooled and dialyzed against 40 mM Tris-Cl (pH 7.6).
Thus we finally isolated 0.4 µg of the purified enzyme from 21.6 mg of protein of the active fraction obtained by sucrose density gradient centrifugation of the antral vesicle fraction (Table 1). Its molecular weight was estimated to be approximately 37,000 by 12.5% SDS-PAGE under both reducing and nonreducing conditions (Fig. 3), indicating that it is composed of a single chain polypeptide. The protease activity coincided well with the band of the purified protein on 12.5% native-PAGE (Fig. 3).
Figure 3:
PAGE
of the purified endoprotease. Slab gel electrophoresis was performed by
the method of Laemmli(35) , and the gel was stained using a
silver staining kit. A, 12.5% SDS-PAGE of the endoprotease. B, 12.5% native-PAGE of the endoprotease. a, carbonic
anhydrase; b, the endoprotease. C, analysis of the
enzyme activity in the native-PAGE. The gel was sliced into 24 pieces
and extracted overnight in 40 µl each of the assay buffer. The
enzymatic activities toward Boc-Arg-Val-Arg-Arg-MCA () and VIP
(
) were assayed as described under ``Experimental
Procedures.'' BPB, bromphenol
blue.
On the other hand, when various oligopeptides including gastrointestinal peptides were examined as substrates, the purified protease cleaved only VIP most rapidly, and secretin at a slower rate which has structural similarities to VIP (25) and no other peptides were hydrolyzed significantly except that substance P and oxidized insulin B chain were hydrolyzed slowly (Table 3). Interestingly, VIP was mainly cleaved by this protease at three sites which had not been expected from its specificity toward the synthetic peptide MCA substrates as shown in Fig. 4, and no cleavage occurred in the other part of VIP including the Thr-Arg-Leu-Arg-Lys sequence of which the Arg-Lys and Thr-Arg bonds were expected to be cleaved by the enzyme as judged from its specificity toward MCA substrates. The cleaving activity of VIP at the three sites (Fig. 4) paralleled the Boc-Arg-Val-Arg-Arg-MCA cleaving activity throughout the purification by successive column chromatography on Sephacryl S-200, Mono-Q/FPLC, and PCMB-agarose.
Figure 4: HPLC profiles of a digestion mixture of VIP and the cleavage sites. 1,000 pmol of VIP was digested with 5.4 fmol of the purified enzyme for 1 h and the peptide fragments were analyzed as described under ``Experimental Procedures.'' A. HPLC pattern. B. Cleavage sites and the peptide fragments produced. The amino acid sequence of VIP is shown in one-letter amino acid code. The values in parenthesis indicate the extents of cleavage.
Kinetic parameters with VIP and Boc-Arg-Val-Arg-Arg-MCA as substrates were analyzed as shown in Table 4. These results indicated that the enzyme has much higher affinity and catalytic efficiency toward VIP than toward Boc-Arg-Val-Arg-Arg-MCA.
The protease activity toward Boc-Arg-Val-Arg-Arg-MCA was increased about eight times by the addition of Lubrol PX or Triton X-100, whereas sodium cholate was not effective and SDS showed inhibition at above 0.001% (Fig. 5A). Likewise, the protease activity toward VIP was dependent on Lubrol PX (Fig. 5A). This indicated that membrane components are necessary for the endoprotease to have maximal enzymatic activity and that the enzyme is indeed a membrane-bound protease. This protease activity toward VIP and Boc-Arg-Val-Arg-Arg-MCA showed an optimum at pH 7.5 (Fig. 5B).
Figure 5:
Effects of various detergents and pH
values on the VIP-degrading endoprotease activity. A, effects
of various detergents. The activity was assayed as described under
``Experimental Procedures'' except for the addition of
detergents: toward VIP with Lubrol PX () and toward
Boc-Arg-Val-Arg-Arg-MCA with Lubrol PX (
), Triton X-100 (
),
sodium cholate (
), and SDS (
). B, effects of
pH. The activity was assayed as described under ``Experimental
Procedures'' except for the buffer used: toward VIP (
) and
toward Boc-Arg-Val-Arg-Arg-MCA (
). The pH was varied using the
universal buffer(36) .
This protease was inhibited by serine
protease inhibitors such as DFP and PMSF. The activity was completely
inhibited by 10 mM DFP, although it was not so effective at 1
mM concentration. Interestingly, this protease was also
inhibited by PCMB, chymostatin, and iodoacetic acid, but not by E-64
and leupeptin which are also cysteine protease inhibitors (Table 5). The activity was increased about 40% by 1 mM Ca ion, whereas it was strongly inhibited by
metal ions such as Fe
, Cu
,
Zn
, and Hg
.
Rapid and specific inactivation of a neuropeptide after its function is important and is thought to depend on the action of its inactivating enzyme(s). There have been many reports of different peptidases which may be involved in neuropeptide degradation(16, 26, 27, 28, 29, 30, 31, 32) , but there is not much convincing evidence as yet for any of them that it is specific for a single peptide. In the present study, we have attempted to isolate a novel protease(s) from porcine antral mucosa which may be involved in neuropeptide processing or degradation using a synthetic peptide MCA substrate, Boc-Arg-Val-Arg-Arg-MCA, and could finally isolate a 37,000 dalton neutral endoprotease, which was found to cleave VIP highly efficiently as well as specifically. This new endoprotease activity was distributed in a narrow range in the sucrose density gradient centrifugation as compared with other protease activities (Fig. 1), and showed the optimum activity at pH 7.5 (Fig. 5B), which is higher than that of secretory granules or lysosomes (pH 5.5-6.5). These results indicated the possibility that this enzyme might be associated with some specific membranous structures other than secretory granules or lysosomes. The activity was dependent on some detergents (Fig. 5A). This protease required 0.001% or a higher concentration of a detergent for maximal activity. The enzyme could be kept very stable when Lubrol PX was added to the enzyme solution, but it was very unstable without Lubrol PX (data not shown). Therefore the presence of certain membrane components in vivo seems to be very important for its activity and stability. These results also suggest strongly that the enzyme is bound to a specific membranous structure by hydrophobic interaction.
The endoprotease was inhibited by serine protease inhibitors; it was completely inhibited by 10 mM DFP or 1 mM PMSF. It was also inhibited strongly by PCMB and iodoacetic acid and had a high affinity toward a PCMB-agarose affinity column, whereas it was not inhibited by other cysteine protease inhibitors such as E-64 and leupeptin, and it did not require any reducing agent for its activity. Therefore, the present protease appears to be most likely a serine protease although its action as a cysteine protease cannot be completely excluded. This enzyme is thought to have an SH group(s) in the vicinity of the active site, the modification of which results in an extensive inactivation of the enzyme. The present protease showed a distinct substrate specificity as shown in Table 2and Table 3. The protease cleaved Boc-Arg-Val-Arg-Arg-MCA at the Arg-Arg bond. This cleavage pattern is distinct from that of furin and related proteases (20, 33, 34) . The same type of cleavage was also observed at the Lys-Arg and Thr-Arg bonds in Pyr-Arg-Thr-Lys-Arg-MCA and Boc-Leu-Ser-Thr-Arg-MCA, respectively, but susceptibility was very low when compared with that of Boc-Arg-Val-Arg-Arg-MCA. On the other hand, when various peptides were used as substrates, VIP was found to be cleaved much more specifically than synthetic peptide MCA substrates (Table 4). Unexpectedly, VIP was cleaved at three sites (A-C sites in Fig. 4) not involving arginine residues. These results suggested the possibility that the Boc-Arg-Val-Arg-Arg-MCA cleaving activity, and the VIP-degrading activity might be due to different enzymes. However, each cleaving activity in VIP and the Boc-Arg-Val-Arg-Arg-MCA cleaving activity coincided well with each other in pH dependence (Fig. 5B) and the effects of inhibitors (Table 5). The protease activity toward VIP was strongly dependent on Lubrol PX as in the case of Boc-Arg-Val-Arg-Arg-MCA (Fig. 5A). Furthermore, upon native PAGE of the purified enzyme, both Boc-Arg-Val-Arg-Arg-MCA cleaving activity and VIP-degrading activity coincided practically completely with each other (Fig. 3). These results indicate that these activities are due to a single enzyme.
The present protease showed high substrate
specificity, hydrolyzing only a few substrates. There appears to be
some tendency that certain amino acid residues with bulky, but not
aromatic, side chains, such as Gln, Met, Lys, Leu, etc., are preferred
at the P and often at the P
` positions.
However, the amino acid residues at the cleavage sites and in their
vicinities are fairly variable, and it is difficult to define clearly
the subsite specificity of the enzyme. This enzyme might recognize a
specific amino acid sequence(s) and/or conformation in the vicinity of
the cleavage site of the target peptide. Taken together, the present
enzyme is strongly suggested to be involved in the enzymatic
inactivation of certain neuropeptides, especially VIP. However, further
studies are necessary to obtain a definite conclusion on the
physiological role and the substrate specificity of this novel
proteinase.