(Received for publication, October 11, 1994; and in revised form, November 11, 1994)
From the
We have isolated a metallopeptidase from rat liver. The
peptidase is primarily located in the mitochondrial intermembrane
space, where it interacts non-covalently with the inner membrane. The
enzyme hydrolyzes oligopeptides, the largest substrate molecule found
being dynorphin A; it has no action on proteins,
and does not interact with
-macroglobulin, and can
therefore be classified as an oligopeptidase. We term the enzyme
oligopeptidase M. Oligopeptidase M acts similarly to thimet
oligopeptidase (EC 3.4.24.15) on bradykinin and several other peptides,
but hydrolyzes neurotensin exclusively at the -Pro+Tyr- bond (the
symbol + is used to indicate a scissile peptide bond) rather than
the -Arg+Arg- bond. The enzyme is inhibited by chelating agents
and some thiol-blocking compounds, but differs from thimet
oligopeptidase in not being activated by thiol compounds. The peptidase
is inhibited by Pro-Ile, unlike thimet oligopeptidase, and the two
enzymes are separable in chromatography on hydroxyapatite. The
N-terminal amino acid sequence of rat mitochondrial oligopeptidase M
contains 19 out of 20 residues identical with a segment of rabbit
microsomal endopeptidase and 17 matching the corresponding segment of
pig-soluble angiotensin II-binding protein. Moreover, the rat protein
is recognized by a monoclonal antibody against rabbit soluble
angiotensin II-binding protein, all of which is consistent with these
proteins being species variants of a single protein that is a homologue
of thimet oligopeptidase. The biochemical properties of the
mitochondrial oligopeptidase leave us in no doubt that it is neurolysin
(EC 3.4.24.16), for which no sequence has previously been reported, and
which has not been thought to be mitochondrial.
Wünsch and Heidrich (3) designed the
synthetic substrate, Pz()-Pro-Leu-Gly-Pro-D-Arg,
containing the collagen-like sequence -Pro-Leu-Gly-Pro-, as a substrate
for clostridial collagenase. Avian and mammalian tissues contain a
Pz-peptide-hydrolyzing enzyme that is not a collagenase, and was for a
time simply termed Pz-peptidase, but is now known as thimet
oligopeptidase (EC 3.4.24.15)(4) . Thimet oligopeptidase is a
primarily cytosolic, thiol-activated metalloendopeptidase, which is
confined to action on oligopeptides of up to about 17 amino acid
residues(5) . It cleaves neurotensin at the -Arg+Arg- bond
and is potently inhibited by a series of Cpp-tripeptidyl-pAb
compounds(6, 7) . Since the determination of the amino
acid sequence of thimet oligopeptidase (8, 9) a family
of homologous metalloendopeptidases has been discovered (for review (10) ). The family contains exopeptidases, oligopeptidases, and
endopeptidases and is thus of exceptional diversity. Two homologues,
oligopeptidase A and peptidyl-dipeptidase A, have been identified in
both Escherichia coli and Salmonella
typhimurium(11) , and peptidase F of Lactococcus
lactis is also related(12) . Eukaryotic members in
addition to thimet oligopeptidase include mitochondrial intermediate
peptidase (EC 3.4.24.59), which is a matrix protein in the mitochondria
of rat and yeast(13, 14) , and saccharolysin (EC
3.4.24.37; formerly peptidase yscD), also from
yeast(15) .
Two additional homologues of thimet oligopeptidase that are of particular importance here are pig-soluble angiotensin II-binding protein and rabbit microsomal endopeptidase. Soluble angiotensin II-binding protein was discovered in the course of work directed at the identification of receptors for angiotensin II. This protein is clearly a homologue of thimet oligopeptidase, but is a separate gene product(9, 16) . Kato et al.(16) demonstrated that pig-soluble angiotensin II-binding protein has the capacity to degrade radiolabeled angiotensin. A monoclonal antibody that was raised against rabbit-soluble angiotensin II-binding protein by Soffer et al.(17) has been valuable in the present study.
It was
also noted by McKie et al.(9) and Kato et al.(16) that the deduced sequence of pig-soluble
angiotensin II-binding protein is so similar to that of the rabbit
enzyme termed microsomal endopeptidase (18, 19) as to
indicate that the proteins are species variants of a single enzyme. The
microsomal metalloendopeptidase has been suggested to be responsible
for the post-translational processing of -carboxyglutamic
acid-containing blood coagulation factors.
A further metallopeptidase that we need to introduce is one that has been termed neurotensin-degrading enzyme, and for which the recommended name is now neurolysin (EC 3.4.24.16). The enzyme is present in the cytosol and membrane fractions of brain, kidney, and other tissues, and it has been proposed to have the physiological function of destroying neurotensin and other bioactive peptides(20, 21) . In many respects, the activity of neurolysin resembles that of thimet oligopeptidase, but neurolysin is distinguished by its cleavage of neurotensin at the -Pro+Tyr- bond(22) , its sensitivity to inhibition by Pro-Ile(23) , and lack of thiol activation. Despite much work on the enzyme, no amino acid sequence data have been reported for neurolysin.
Heidrich et al.(24, 25) described Pz-peptidase activity in rat liver mitochondria, and Tisljar and Barrett (26) established that the mitochondria enzyme is distinct from thimet oligopeptidase, although there are similarities in substrate specificity. In particular, both enzymes hydrolyze a quenched fluorescence substrate related in structure to the Pz-peptide, QF02.
In the present paper, we describe the isolation and characterization of the mitochondrial peptidase, which we term oligopeptidase M. Our study of oligopeptidase M leads us to conclude that oligopeptidase M, soluble angiotensin II-binding protein, microsomal endopeptidase, and neurolysin are all one. In consequence of this, we find ourselves in possession of detailed knowledge of the primary structure and enzymology of a new member of the thimet oligopeptidase family of metallopeptidases.
Antiserum against recombinant rat thimet oligopeptidase was raised in three rabbits, each of which was injected subcutaneously with 50 µg of the recombinant enzyme at 0, 1, 2, and 7 months. The immunogen was emulsified with complete Freund's adjuvant for the first injection and incomplete adjuvant subsequently. Sera from bleeds taken 8 days after the third and fourth injections were combined. Immunoglobulin G was prepared by triple precipitation from 2.7 M ammonium sulfate, and the final precipitate was dissolved in phosphate-buffered saline and dialyzed against the same. Specific antibody was affinity purified by use of a column in which the recombinant enzyme was immobilized on activated CH-Sepharose 4B (Pharmacia). The antibody eluted with 0.1 M glycine-HCl, pH 2.3, was termed R646/P.
Antiserum against oligopeptidase M was raised in a sheep by two intramuscular injections of 50 µg emulsified in Freund's complete adjuvant, 1 month apart. Sera from bleeds taken at 7, 10, and 14 days after the second injection were combined. Immunoglobulin G was prepared by triple precipitation with ammonium sulfate as above and termed K118.
Selective assays for either oligopeptidase M or thimet oligopeptidase were made on the basis that mammalian tissues contain three enzymes that hydrolyze QF02, prolyl oligopeptidase (a serine peptidase), thimet oligopeptidase, and oligopeptidase M (see ``Results''). The activity of prolyl oligopeptidase was eliminated by use of its inhibitor, Z-thioprolylthiazolidine (0.05 µM) (31) , and immunoinhibition allowed discrimination between the two metallopeptidases (see below).
Selective immunoinhibition of the respective enzymes was achieved by preincubation of the sample with either (a) an equal volume of a 240 µg/ml solution of antibody R646/P for 10 min at room temperature, for thimet oligopeptidase, or with 10 mg/ml of antibody K118, for oligopeptidase M. Tests with the pure enzymes established that these conditions produced 100% inhibition of thimet oligopeptidase with 0% inhibition of oligopeptidase M (R646/P) and 5% inhibition of thimet oligopeptidase with 100% inhibition of oligopeptidase M (K118), respectively.
Values of K for the hydrolysis of
quenched fluorescence substrates by oligopeptidase M were determined in
continuous fluorimetric assays controlled by the FLUSYS
software(32, 5) . The method included
restandardization of the fluorimeter at each substrate concentration to
compensate for quenching by the substrates at high
concentrations(33) , and comparative measurements were made
with recombinant rat testis thimet oligopeptidase. Values for V were converted to k
on the basis that the
highest specific activities determined during a number of preparations
of the homogeneous enzymes correspond to those of the fully active
enzymes. These values (in the standard assay with QF02) were 4.0
unit/mg for oligopeptidase M and 6.0 unit/mg for thimet oligopeptidase.
Values for k
/K
were determined directly when activity could be determined
at substrate concentrations far below K
,
by use of the equation: k
/K
= v/E
S
.
Values
for K were determined with correction for
the effect of substrate on the assumption of simple
competition(7) .
Marker enzymes were cytochrome oxidase for
mitochondria(34) , -galactosidase and cathepsins B and L
for lysosomes(35, 36) , catalase for
peroxisomes(34) , and glucose-6-phosphatase for endoplasmic
reticulum(37) . A mitochondrial matrix marker was
2-oxoglutarate dehydrogenase(38) . Protein was determined by
use of the Bio-Rad kit, and all results were calculated as described by
de Duve et al.(39) .
The
washes were discarded, and the pellet (purified mitochondria) was
diluted to a protein concentration of 100 mg/ml with TS buffer.
Digitonin stock solution was added to make 0.55% digitonin, and after
15 min with occasional stirring three volumes of TS buffer was added,
and the resulting mitoplasts were washed twice by centrifugation
(25,000 g, 15 min) and resuspension in TS buffer.
The mitoplast pellet was diluted to a protein concentration of 50
mg/ml in TS buffer, and W-1 stock solution (10%, w/v) was added to make
1.1%. After 30 min with occasional stirring, the suspension was diluted
with an equal volume of TS buffer, and centrifuged at 100,000 g for 1 h. The supernatant formed the source for the
chromatographic isolation of oligopeptidase M.
Fractions containing activity against QF02 were combined,
concentrated to about 40 ml by ultrafiltration over an Amicon PM-30
membrane, and dialyzed against 10 mM sodium phosphate buffer,
pH 6.8, containing 5 mM 2-mercaptoethanol, 0.05% Brij-35, and
0.1 mM ZnCl (PB buffer).
Figure 1: Purification of oligopeptidase M from rat liver mitochondria. A, SDS-polyacrylamide gel electrophoresis of samples obtained during the purification of oligopeptidase M. Samples were a, mitochondria; b, W-1 extract of mitoplasts; c, DEAE-cellulose fraction; d, hydroxyapatite fraction; e, Sephacryl S300 HR fraction; f, Hitrap Blue-Sepharose fraction; g, Mono Q fraction (final product). Lane h contained a reference sample of thimet oligopeptidase. B, immunoblot of lanes g and h from a gel duplicating A, above, developed with monoclonal antibody M3C against rabbit-soluble angiotensin II-binding protein.
When run in SDS-polyacrylamide gel electrophoresis, oligopeptidase M (Fig. 1A, lane g) appears as a single band of protein corresponding to a molecular mass of 76 kDa, about 2 kDa less than that of thimet oligopeptidase (lane h). A persistent contaminant (seen as the strongest band in lane e) was identified by N-terminal sequence analysis (data not shown) as serine-tRNA ligase and was almost completely removed by the HiTrap Blue column.
The immunoblot of a similar gel developed with monoclonal antibody M3C to soluble angiotensin II-binding protein showed a strong reaction with oligopeptidase M and none with thimet oligopeptidase (Fig. 1B).
A parallel electroblot was stained for glycoproteins by the periodate-Schiff method, with transferrin as positive control. The reaction of oligopeptidase M was completely negative.
The N-terminal sequence determined for oligopeptidase M by automated sequencing from a blot is shown in Fig. 2, aligned with those of related enzymes.
Figure 2: N-terminal sequence of oligopeptidase M. The N-terminal sequence of oligopeptidase M (a) is compared with those of rabbit microsomal endopeptidase (b), pig-soluble angiotensin II-binding protein (c), and rat thimet oligopeptidase (d). Basic residues indicative of a mitochondrial-targeting function for the N-terminal parts of sequences (b) and (c) are printed in bold type, and residues identical to those in oligopeptidase M are printed in white on black. Residue X was unidentified.
Figure 3:
Specificity of oligopeptidase M. The
pattern of cleavage of peptide bonds by oligopeptidase M is shown in
comparison to that of thimet oligopeptidase (Dando et al.,
1993). A shows bonds cleaved similarly by both enzymes. B shows differing activities of the enzymes, for thimet
oligopeptidase and
for oligopeptidase M. The symbol &cjs1229;
indicates a weak cleavage by thimet
oligopeptidase.
The dipeptide Pro-Ile was
introduced into the study because oligopeptidase M was found to have
properties in common with neurolysin, for which Pro-Ile has been
described as a rather specific inhibitor(23) . We confirmed
that the dipeptide has little effect on thimet oligopeptidase.
Oligopeptidase M was markedly inhibited, although with a K value of 0.54 mM, which was higher than
that of 0.09 mM determined for neurolysin by the previous
workers, using potentially less precise, discontinuous assays.
Oligopeptidase M also was inhibited by Cpp-tripeptidyl-pAb inhibitors of thimet oligopeptidase, although much more weakly than thimet oligopeptidase (Table 3).
Figure 4: Subcellular distribution of activity against QF02. Subcellular fractionation of rat liver was as described under ``Experimental Procedures.'' Significant QF02 hydrolyzing activity was detected only in mitochondria and cytosol. Further experiments described in the text showed that the mitochondrial activity was almost entirely due to oligopeptidase M, whereas both thimet oligopeptidase and oligopeptidase M contributed to the cytosolic activity. Note that no significant activity was present in the microsomal fraction, despite the similarity in N-terminal amino acid sequence between oligopeptidase M and the endopeptidase previously described as microsomal.
Confirmation
of the distribution of oligopeptidase M and thimet oligopeptidase in
mitochondria and cytosol was sought from chromatography on
hydroxyapatite, the only method discovered so far by which the two
enzymes are readily separated. 15 ml of cytosol fraction (as above) was
run on hydroxyapatite (15 115 mm) as described above for the
purification of oligopeptidase M. Two completely separate peaks of
activity against QF02 were detected in the effluent fractions, at 75
mM and 140 mM phosphate. The first peak contained 65%
of the total activity, hydrolyzed neurotensin at the -Arg+Arg-
bond, and was inhibited by antibody R646/P; it was thus identified as
thimet oligopeptidase. The second peak comprised 35% of the activity,
hydrolyzed neurotensin at the -Pro+Tyr- bond, and was inhibited by
10 mM Pro-Ile. Accordingly, the second peak was attributed to
cytosolic oligopeptidase M. In contrast, when activity from mitoplasts
was run on hydroxyapatite during the purification of oligopeptidase M,
no peak attributable to thimet oligopeptidase was ever seen.
In a second experiment, the nature of the
association of oligopeptidase M with mitoplasts was investigated.
Mitochondria and mitoplasts were suspended (at 5 mg protein/ml) in
sodium acetate buffer, pH 5.0, at 0 °C for 30 min. Each suspension
was then recentrifuged at 15,000 g for 10 min, and the
supernatants were dialyzed and assayed for total and specific activity
of oligopeptidase M.
Activity was efficiently solubilized (77%) from mitoplasts by the acidic buffer, but there was little solubilization (6%) from mitochondria. A mitochondrial matrix marker, 2-oxoglutarate dehydrogenase, was retained in the sedimentable mitoplasts.
The mitochondrial enzyme that hydrolyzes the Pz-peptide was discovered by Heidrich et al.(24, 25) , and further studied by Tisljar and Barrett(26) . We here term this enzyme oligopeptidase M and have assayed it with QF02, a quenched fluorescence substrate, that contains the collagen-like sequence -Pro-Leu-Gly-Pro-, as does the Pz-peptide. The substrate is also hydrolyzed by thimet oligopeptidase (4, 5) and prolyl oligopeptidase(28) . QF02 has been used in the laboratory of Checler et al.(46) to assay neurolysin. We used selective inhibitors of thimet oligopeptidase, prolyl oligopeptidase, and oligopeptidase M to resolve the three enzymes and found that the activity against QF02 in rat liver and kidney could be totally blocked by the combined action of the three inhibitors.
The subcellular fractionation of rat liver showed that QF02 hydrolyzing activity was essentially confined to the mitochondrial and cytosolic compartments. The activity in mitochondria was due to oligopeptidase M, with a small contribution from thimet oligopeptidase. In contrast, more of the cytosolic activity was due to thimet oligopeptidase than to oligopeptidase M.
The N-terminal 20-residue sequence determined for
rat oligopeptidase M was used in a search of GenBank and other data
bases with the FASTA and BLAST programs(47, 48) . The
only significant scores obtained were those for the rabbit liver
microsomal endopeptidase and pig liver-soluble angiotensin II-binding
protein (Fig. 2). The microsomal endopeptidase has been studied
by Davie and co-workers (18, 19) as a possible
post-translational processing enzyme for blood coagulation factors. The
soluble angiotensin II-binding protein is one that was discovered
incidentally in the search for receptors of
angiotensin(17, 49) . The similarities throughout the
complete sequences of rabbit microsomal endopeptidase and pig-soluble
angiotensin II-binding protein are so great (91% identical residues)
that we and others have suggested that they are species variants of a
single endopeptidase(9, 16) . The exact properties of
this enzyme have been unclear, however. Hydrolysis of angiotensin II by
the pig liver-soluble angiotensin II-binding protein has been
detected(16) , and the microsomal enzyme has been shown to
cleave some synthetic peptides modeling the site of processing of
-carboxyglutamic acid-containing blood coagulation
factors(18, 19) .
It now seems that microsomal endopeptidase and soluble angiotensin II-binding protein represent species variants of the mitochondrial and cytosolic enzyme that we here characterized under the name oligopeptidase M. The ``microsomal'' localization of the putative coagulation factor processing enzyme may well have been artifactual, since the enzyme was isolated from frozen rabbit liver after prolonged treatment with a Waring blender(18) . Fragments of mitochondria could well have been present in the high speed pellet from such a preparation. The soluble angiotensin II-binding protein is presumably the cytosolic fraction of oligopeptidase M that we also have detected.
Assuming that the initiator Met residues are as shown on Fig. 2(discussed in (49) ), oligopeptidase M is synthesized with an N-terminal sequence of 28 residues that conforms to expectation for a mitochondrial-targeting sequence(50, 51) .
Neurolysin is abundant in rat
kidney, and hydrolyzes QF02(52, 22) , and yet we have
found that all of the QF02-hydrolyzing activity of rat kidney is
inhibited by the combination of inhibitors of prolyl oligopeptidase,
thimet oligopeptidase, and oligopeptidase M. This suggests that one of
these enzymes is neurolysin, and indeed, the properties reported in the
literature for neurolysin are so similar to those described here for
oligopeptidase M that there can be very little doubt that they are the
same. Thus, both enzymes are proteins of M about
75,000, with chromatographic properties closely similar to those of
thimet oligopeptidase except for greater retention on
hydroxyapatite(53, 54, 22) . Additionally,
neurolysin has metallopeptidase characteristics with no activation by
thiols (22) and has specificity generally similar to that of
thimet oligopeptidase, including bonds hydrolyzed in QF02 and
bradykinin(47) , but with distinctive cleavage of neurotensin
at Pro
+Tyr(56, 22) , and
neurotensin
to yield neurotensin
(55) . Oligopeptidase M and neurolysin are the only
enzymes apart from thimet oligopeptidase that have been found to be
inhibited by the Cpp-tripeptidyl-pAb compounds described by Orlowski et al.(6) , but in both cases the K
values are 10-100-fold higher than for thimet
oligopeptidase(22) . Inhibition by Pro-Ile (23) also
clearly links oligopeptidase M and neurolysin. Like oligopeptidase M in
rat liver, neurolysin has a partially cytosolic distribution in brain
and neural cells of several
species(53, 54, 55) . Neurolysin has not
previously been described as mitochondrial, but the success of Barelli et al.(22) in purifying the enzyme from a 6,000
g pellet from rat kidney homogenate that would have
been largely mitochondria is entirely consistent with our finding of
oligopeptidase M in this organelle. We conclude from all these lines of
evidence that there is no significant doubt that oligopeptidase M is
identical with neurolysin, and we propose to use the term neurolysin
for the enzyme in future.
Neurolysin represents an interesting new addition to the thimet oligopeptidase family of peptidases, termed family M3 by Rawlings and Barrett (57, 58, reviewed in (10) ). A phylogenetic tree for the family (58) suggests that neurolysin and thimet oligopeptidase diverged from a common ancestor about 500 million years ago, during the evolution of animals. Many of the similarities between neurolysin and thimet oligopeptidase, such as molecular size, oligopeptidase specificity, hydrolysis of the Pz-peptide and QF02, sensitivity to a number of inhibitors that affect no other enzymes(56) , and binding of angiotensin II with similar characteristics(9) , are attributable to the structural similarities of homologous proteins. With the present report, the recognized membership of family M3 becomes thimet oligopeptidase (EC 3.4.24.15), neurolysin (EC 3.4.24.16), saccharolysin (EC 3.4.24.37), mitochondrial intermediate peptidase (EC 3.4.24.59), oligopeptidase A, bacterial peptidyl-dipeptidase, and the peptidase F of lactococci.
Neurolysin has for some time appeared to be an enzyme with the potential for important biological activities. As a result of our study, we now know the amino acid sequence and evolutionary relationships of the enzyme. The demonstration of its primarily mitochondrial location suggests new types of experiments relating to its function. For example, it will be of interest to know whether the excellent inhibitors of neurolysin that are now available (56) will disrupt mitochondrial physiology.