(Received for publication, March 14, 1995; and in revised form, June 7, 1995)
From the
Cathepsin E (EC 3.4.23.34), an intracellular aspartic proteinase, was purified from monkey intestine by simple procedures that included affinity chromatography and fast protein liquid chromatography. Cathepsin E was very active at weakly acidic pH in the processing of chemically synthesized precursors such as the precursor to neurotensin/neuromedin, proopiomelanocortin, the precursor to xenopsin, and angiotensinogen. The processing sites were adjacent to a dibasic motif in the former two precursors and at hydrophobic recognition sites in the latter two. The common structural features that specified the processing sites were found in the carboxyl-terminal sequences of the active peptide moieties of these precursors; namely, the sequence Pro-Xaa-X`aa-hydrophobic amino acid was found at positions P4 through P1. Pro at the P4 position is thought to be important for directing the processing sites of the various precursor molecules to the active site of cathepsin E. Although the positions of Xaa and X`aa were occupied by various amino acids, including hydrophobic and aromatic amino acids, some of these had a negative effect, as typically observed when Glu/Arg and Pro were present at the P3 and P2 positions, respectively. Cathepsin D was much less active or was almost inactive in the processing of the precursors to neurotensin and related peptides as a result of the inability of the Pro-directed conformation of the precursor molecules to gain access to the active site of cathepsin D. Thus, the consensus sequence of precursors, Pro-Xaa-X`aa-hydrophobic amino acid, might not only generate the best conformation for cleavage by cathepsin E but might be responsible for the difference in specificities between cathepsins E and D.
Many biologically active peptides are first synthesized as
precursor molecules that must be processed correctly to yield the
active peptides (reviewed in (1, 2, 3, 4) ). The bioactive
peptide moiety in the precursor molecule often lies between paired
dibasic motifs (Lys/Arg-Arg), occasionally between single basic
residues, and rarely between hydrophobic recognition sites. Several
endoproteolytic processing enzymes have been identified and
characterized as being specified for these cleavage sites. Proteinases
that are specific for the carboxyl side of pairs of basic amino acids
include furin and prohormone convertases and they belong to the family
of serine proteinases(2, 3, 4) . Precursors
to some bioactive peptides are known to be processed by different types
of proteinase. The involvement of aspartic proteinases has been
demonstrated in some cases, for example the processing of
proopiomelanocortin and the precursors to NT ()and related
peptides. Although an aspartic proteinase,
proopiomelanocortin-converting enzyme, has been isolated and
characterized(5) , the proteinase(s) that processes the
precursors to NT and related peptides has not been identified since
pepsin has been used to mimic the processing proteinase(s) in most
studies of the processing of these
precursors(6, 7, 8, 9, 10, 11) .
To date, three types of intracellular aspartic proteinase, apart from proopiomelanocortin-converting enzyme, have been identified, namely cathepsin D, cathepsin E, and renin (reviewed in (12, 13, 14, 15, 16, 17) ). Cathepsins D and E have general proteolytic activities against protein substrates, such as hemoglobin and albumin, at acidic pH(12, 17) , while renin has a strict specificity for angiotensinogen at neutral pH (13) . Therefore, cathepsins D and E are thought to be able to process certain types of precursor including the NT/NMN precursor. Indeed, such processing activity was reported for cathepsin D, although the rate of hydrolysis was low(10) . Nonetheless, some important differences have been found between cathepsins D and E. While cathepsin D is a typical lysosomal enzyme, cathepsin E has been shown to be localized in the endoplasmic reticulum, in endosomes, or in other compartments where the processing of precursors is thought to occur(18, 19, 20, 21) . Moreover, as we have reported recently, cathepsin E has a unique structure among aspartic proteinases (22) and it is very active against certain types of peptide at weakly acidic pH, such as pH 5, with rates of hydrolysis several hundred-fold higher than those of reactions catalyzed by cathepsin D(23) . Since these results suggested that cathepsin E might be a more plausible candidate for a processing proteinase in some cases, we thought it appropriate to investigate the catalytic activity of cathepsin E with various precursor molecules.
In this study, we have purified cathepsin E from monkey intestine and examined its processing activity with several chemically synthesized peptides that included paired basic residues and other processing sites. Cathepsin D, renin, and pepsin were used for comparison. Cathepsin E processed precursors to NT and several other peptides much more rapidly and more specifically than did cathepsin D and other aspartic proteinases. An essential sequence in precursors that are processed by cathepsin E is proposed.
Figure 1: SDS-PAGE of purified cathepsin E (E) and cathepsin D (D) from monkey. Proteins were subjected to electrophoresis under reducing (lanes1 and 2) and non-reducing (lanes3 and 4) conditions as described by Laemmli (51) and stained with Coomassie Brilliant Blue R-250. Since most cathepsin D exists in a two-chain form that consists of one heavy (30 kDa) and one light (15 kDa) chain, two bands were detected. In lane4, monkey pepsin A (35 kDa) (52) was added to the preparation of cathepsin E as a standard protein. The band of pepsin is shown by an arrow with a letterP.
Figure 2:
The sequences and sites of cleavage by
cathepsins E and D of various biologically active peptides. Residues at
P1 and P1` positions are boxed. NH, shown in letters of reducedsize, indicates an amide
group at the carboxyl terminus of a peptide. An arrow shows
the cleavage sites. The rates of hydrolysis are given in
nmol/min/µg protein at pH 5.0, except for the case of
-endorphin, in which hydrolysis occurred maximally at pH 4.0; the
values at pH 4.0 are given. The major cleavage site of
-endorphin
by cathepsin E was the Leu
-Phe
bond,
and the minor cleavage (at about 25% of the rate of the major cleavage)
occurred at the Thr
-Leu
bond. The rate
of hydrolysis given is that of the major cleavage. E,
cathepsin E; D, cathepsin D; FGF, fibroblast growth
factor.
Figure 3: The carboxyl-terminal amino acid sequence of the precursor to rat NT/NMN and sites of cleavage by cathepsin E. The numbering is based on the results of Kislauskis et al.(27) . NT and NMN are boxed, and dibasic motifs on either side of the active moieties are shaded. The sequence of NT 6-13 is also shown. Four kinds of synthetic peptide and their sites of cleavage by cathepsin E are shown in the lower part of the Fig.
Figure 4:
Time
course of the hydrolysis of NT/NMN precursor peptides by cathepsin E. A dashedline indicates the change in the amount of the
intact peptide 142-165. The sequence of the peptides and the
cleavage sites are shown in Fig.3. , NT/NMN precursor
peptide 142-162;
, peptide 142-147;
, peptide
148-162;
, peptide
152-162.
From the results of hydrolysis of the precursors to NT and related
peptides, it was clear that cathepsin E had a generally high ability to
process these precursors in the weakly acidic pH region. Cathepsin E
appeared to recognize the structures of NT and related peptides that
are specified by the occurrence of Pro and a hydrophobic amino acid at
the P4 and P1 positions, respectively (Fig.5). Such structural
features were also found in substance P, the best peptide substrate for
cathepsin E. Thus, the Pro-Xaa-X`aa-HBaa sequence seemed to be
essential for cleavage by cathepsin E of precursors. We searched a
protein data base and found some additional precursors that have the
consensus sequence. One of these was proopiomelanocortin. In this
precursor, the sequence that includes the processing site is
Pro-Leu-Glu-Phe-Lys-Arg. This precursor was correctly processed by
cathepsin E, a result that supports the present hypothesis (Table1). Cathepsin D and pepsin also processed this precursor.
However, the cleavage of proopiomelanocortin 34-43 by cathepsin D
and pepsin occurred at two sites, namely at the
Phe-Lys
and
Glu
-Phe
bonds, while the cleavage by
cathepsin E was restricted to the Phe
-Lys
bond. Since the Glu
-Phe
bond was
not an appropriate processing site, the specificity of cathepsin D and
pepsin was low as compared to that of cathepsin E. Processing of some
other precursors with the consensus sequence, such as precursors to
atrial natriuretic factor and vasoactive intestinal polypeptide, was
not catalyzed by cathepsin E or by the other aspartic proteinases
tested.
Figure 5:
Structural characteristics of substance P,
various precursors, and their mutants that were hydrolyzed by cathepsin
E. Substance P, porcine angiotensinogen 1-14 (acetylated form),
and human angiotensinogen 1-13 were commercial products. Other
peptides were synthesized on the basis of the sequence data for the rat
NT/NMN precursor(27) , canine xenopsin precursor(53) ,
and cattle proopiomelanocortin(54) . Residues at the P4 and P1
positions are boxed, and dibasic motifs are shaded.
The sequences of the active peptide moieties are shown in normalcapitalletters, and those of the other parts
are shown in italicletters. Residues in filledboxes are substituted residues. NH at the
carboxyl terminus of substance P represents an amide moiety. An arrow shows the cleavage sites. The rate of hydrolysis of each
peptide by cathepsin E at pH 5 is given in nmol/min/µg protein. UC, uncleaved.
Precursors to some bioactive peptides are known to be processed by aspartic proteinases(5, 6, 7, 8, 9, 10, 11) . However, pepsin has been used as a model processing proteinase in most relevant studies, and the actual proteinases have not been identified. In the present study, we examined the processing activity of cathepsin E in a comparison with the activities of other aspartic proteinases, such as cathepsin D. In our experiments, we used various synthetic peptides composed of 8-24 residues that were equivalent to parts of natural precursors. Although natural precursors are thought to be processed in more complicated systems, the experiments using synthetic oligopeptides should yield results that are useful for understanding the basic mechanism of the processing of native precursor molecules.
We showed for the first time that cathepsin E processes a group of precursors to bioactive peptides, such as NT, NMN, and xenopsin, with much higher activity and specificity than do cathepsin D and other aspartic proteinases. Since these precursors are thought to be processed on their way from the endoplasmic reticulum to secretory vesicles (1) and, occasionally, in acidified vesicles(29) , the maximal activity of cathepsin E at weakly acidic pH and its localization in the endoplasmic reticulum, endosomes, or other compartments (18, 19, 20, 21) favor its reaction with precursor molecules. Cathepsin E is also able to process angiotensinogen, as well as other aspartic proteinases such as renin(13, 28) , although their optimal pH values differ significantly. Cathepsin E might be partly involved in the intracellular processing of angiotensinogen in various cells and tissues(30) , although it may be inactive in processing angiotensinogen at neutral pH in the serum. Cathepsin E processes various precursors by recognizing the structural features of active peptide moieties, which are specified by the occurrence of Pro and a hydrophobic residue at the P4 and P1 positions, respectively. The consensus sequence is Pro-Xaa-X`aa-HBaa at the P4 through P1 positions (Fig.5). Let us consider the significance of this consensus sequence. First, a hydrophobic (or aromatic) residue at the P1 position seems to be essential. To date, the proteolytic specificity of cathepsin E has been investigated by several authors using various proteins and peptides, such as the B-chain of insulin (23, 31, 32, 33, 34) and many kinds of biologically active peptides(23) . The P1 and P1` positions of the cleavage sites in most of these cases have been occupied by hydrophobic amino acids. This specificity is common to aspartic proteinases such as cathepsin D (12) and pepsin (35, 36) and was the case in the present study for the hydrolysis of various bioactive peptides that included substance P and related tachykinins (Fig.2). The P1` residue, however, was shown not to be absolutely hydrophobic since precursors such as the NT/NMN precursor having a non-hydrophobic P1` residue were efficiently processed by cathepsin E.
The most interesting feature of the
consensus sequence of the precursor molecules is the occurrence of Pro
at the P4 position. Pro in a peptide or protein forms an imido bond and
then restricts the conformation of a peptide or protein. The active
site of cathepsin E is thought to be configured such that it can
interact with the Pro-directed conformation of a substrate peptide. Pro
at the P4 position has been shown to be favorable for the hydrolysis of
synthetic chromogenic substrates for cathepsin
E(37, 38) . The present study showed, however, that
Pro at the P4 position has a positive effect but is not essential for
peptide hydrolysis since other amino acids, such as Ser and Val, were
shown to be able to replace Pro. This result seems reasonable since Ser
or Val can maintain the conformation of a peptide such that it will fit
into the active site as a consequence of the flexibility of the
peptide bond. Contrary to the case with cathepsin E, the Pro-directed
conformation of a substrate peptide appeared to be very unfavorable for
cleavage of a peptide by cathepsin D. Substitution of Pro by another
amino acid allowed cathepsin D to hydrolyze the peptide. The more
flexible
peptide bond of a substituted amino acid might change
the conformation of a substrate peptide to allow it to fit into the
active site of cathepsin D. Thus, Pro at the P4 position causes the
distinct difference in the specificity for peptide-bond hydrolysis
between cathepsins E and D, probably as a result of the inflexible
imido bond.
In our present hypothesis, a substrate peptide for a
cathepsin is expected to be long enough to introduce as many as nine
residues into subsites S6 through S3` of the active site of the
enzyme(23, 39) . In shorter synthetic chromogenic
peptides that can serve as substrate for cathepsin D, the effect of Pro
at the P4 position is unclear(40, 41) . Therefore, Pro
at the P4 position in native precursors may play a definitive role in
the processing of such precursors. The processing sites of native
precursors have been shown to be located in or immediately adjacent to
a turn(42) , as is also the case in the NT/NMN
precursor(11) . Pro might well be responsible for such a
characteristic structure since Pro is known to have the highest
potential for formation of a
turn(43) . Indeed, a high
frequency of Pro residues around the processing sites of precursor
molecules has been reported(44) . Therefore, in the native
precursors preferred by cathepsin E, Pro at the P4 position may be
primarily important for directing the conformation of the substrate to
the active site of cathepsin E. In such large peptides, replacement of
Pro by another amino acid might inhibit the processing of the precursor
molecules.
Although the consensus sequence is thought to be essential for the processing by cathepsin E, there are a few exceptions. For example, precursors to atrial natriuretic factor and vasoactive intestinal polypeptide, which have sequences of Pro-Arg-Ser-Leu and Pro-Val-Pro-Val, respectively, are not processed by cathepsin E. This failure might be due to steric hindrance by Arg and Pro at the P3 and P2 positions, respectively, as shown by the significant decrease in the rate of hydrolysis of Arg and Pro mutants of the NT/NMN precursor. Arg and Pro at the P3 and P2 positions, respectively, are known to hinder the hydrolysis of peptides by pepsin(35) .
We cannot ignore the diversity of systems that are involved in the processing of precursors to bioactive peptides. There are, as is well known, multiple processing proteinases. To date, precursors with processing sites that consist of paired basic residues are known to be processed by a group of serine proteinases, such as furin and prohormone convertases(1, 2, 3, 4) . These proteinases recognize a paired basic motif and a preceding basic residue(s), for example, in the sequence Arg-Xaa-Lys/Arg-Arg(2, 4, 45, 46, 47) . Since the NT/NMN precursor contains a Lys-Arg motif but lacks Arg at the P4 position, furin and prohormone convertases may be inactive or less active against this precursor. In the case of a precursor that has only a dibasic motif, a furin-like proteinase has been reported to be inactive(48) . However, different proteinases may process an identical precursor molecule. As shown in the present report, proopiomelanocortin 34-43 was processed by cathepsin E, while the dibasic motif of this precursor is known to be processed by other enzymes, such as prohormone convertases(45, 47) , and proopiomelanocortin-converting enzyme(5) . The occurrence of different types of proteinase for processing of the same precursor might guarantee the complete processing of the precursor in a living cell. Indeed, the processing of precursors has been reported to proceed via different pathways depending on environmental conditions such as pH (49) .