(Received for publication, September 30, 1996)
From the Department of Biological Regulation, The Weizmann Institute of Science, Rehovot, 76100, Israel
The kinase splitting membranal proteinase (KSMP)
is a metalloendopeptidase that inactivates the catalytic (C) subunit of
protein kinase A (PKA) by clipping off its carboxyl terminal tail. Here we show that this cleavage occurs at
Glu332-Glu333, within the cluster of acidic
amino acids (Asp328-Glu334) of the kinase. The
Km values of KSMP and of meprin (which
reproduces KSMP activity) for the C-subunit are below 1 µM. The Km for peptides containing a
stretch of four Glu residues are in the micromolar range, illustrating
the significant contribution of this cluster to the substrate
recognition of meprin
. This conclusion is supported by a systematic
study using a series of the C-subunit mutants with deletions and
mutations in the cluster of acidics. Hydrophobic amino acids vicinal to
the cleavage site increase the Kcat of the
proteinase. These studies unveil a new specificity for meprin
,
suggesting new substrates that are 1-2 orders of magnitude better in
their Km and Kcat than
those commonly used for meprin assay. A search for substrates having
such a cluster of acidics and hydrophobics, which are accessible to
meprin under physiological conditions, point at gastrin as a potential
target. Indeed, meprin
is shown to cleave gastrin at its cluster of
five glutamic acid residues and also at the M-D bond within its
WMDF-NH2 sequence, which is indispensable for all the known
biological activities of gastrins. The latter meprin cleavage will lead
to the inactivation of gastrin and thus to the control of its
activity.
The presence of a kinase splitting membranal proteinase
(KSMP)1 in the brush-border membranes of
the rat small intestine was demonstrated as early as 1979 (1). This
proteinase was shown to clip the catalytic (C) subunit of PKA, yielding
a distinct cleavage product (C) that was found to be devoid of the
kinase activity. The biochemical characterization of KSMP as a
proteinase revealed that it is an intriguing enzyme with a combination
of the following unique features. (a) KSMP cleaves the C-subunit when
it is free but not when inhibited by its regulatory (R) subunits, as in
the R2C2 complex (1, 2). (b) This cleavage
could not be simulated by other proteinases (trypsin, chymotrypsin,
clostripain, and papain (2)), suggesting that its specificity is not
due merely to an interdomain exposure in C. (c) The proteinase was found to single out and selectively cleave the C-subunit in the presence of the large number of other proteins found in crude extracts
of different tissues (brain, liver, or muscle) (3). (d) KSMP was found
to cleave the C-subunit in its native conformation but not if the
kinase is pre-denatured (2). (e) It distinguishes between the
"open" and "closed" conformations of the C-subunit (4) that
were recently identified by x-ray crystallography of this kinase
(5).
The cleavage of the C-subunit by KSMP leads to the removal of the
carboxyl terminus tail of this kinase and seemed to occur at a distinct
site (6-8). Interestingly, two other kinases, the EGF- and the
insulin-receptor kinases (which share certain sequence homology with
the C-subunit (9)) were also shown to undergo a specific and
conformation-dependent cleavage by KSMP (6, 10-12). In
both receptor kinases, it was shown that the KSMP cleavage occurs at
the carboxyl-terminal part of the molecules. The specific and
restricted character of the KSMP cleavage of C, as well as the EGF- and
the insulin-receptor kinases, suggested the existence of a common
structural motif that is recognized by the proteinase. Indeed,
inspection of the primary sequences of the three kinases revealed that
they share a stretch of acidic amino acid downstream from their common
protein kinase core, raising the possibility that this stretch is an
important biorecognition element for KSMP. This suggestion was
supported by three additional findings. (i) The polyglutamic acid
effectively inhibits the KSMP cleavage of the C-subunit
(Ki = 6 µM) (7). (ii) The monoclonal antibodies against a branched polyamino acid with exposed clusters of
Glu cross react with the C-subunit but not with its KSMP cleavage product (C) while a monoclonal anti-idiotype of these antibodies specifically binds to the active site of KSMP and inhibits it (7).
(iii) The immunochemical mapping of the C-subunit with epitope-specific
antibodies narrowed down the cleavage site location to the short region
accommodating the cluster of acidic residues in C, i.e.
Asp328-Glu334 (8).
We have recently shown that C-degrading activity of KSMP can be
reproduced by the -subunit of rat meprin (13). Meprin is a membranal
metalloendoproteinase found in the intestinal and renal brush-border
membranes of the mouse (14, 15), rat (16-18), and man (19, 20).
Meprins belong to the astacin family of endopeptidases (21-24) and are
usually composed of two types of subunits,
and
, that exist as
homo- and heterotetramers bound to each other through disulfide bridges
(25, 26). In spite of a large number of studies on meprins, the
substrate specificity and the physiological assignment are not
established yet even though several suggestions have been set forth.
The demonstration that meprin possesses a KSMP activity (13) raised
the possibility that the specificity of this enzyme might be different
from the one currently accepted. Furthermore, the protein and peptide
substrates commonly used for meprins (27-30) may mislead the search
for its physiological substrates and inhibitors, which have not yet
been established (for an excellent recent review, see Ref. 24). For
example, systematic studies on the cleavage of peptide substrates by
rat meprin did not point to any consensus motif but indicated that the
hydrolysis may occur at peptide bonds adjacent to a hydrophobic (27),
aromatic (19), or hydrophilic (31) amino acid residue. None of the
substrates previously used for the systematic analysis of the meprin
activity contained stretches of acidic amino acids.
This paper reports the identification of the KSMP cleavage site in the
C-subunit of PKA and in peptides derived from the sequence of the
C-subunit around that site. It illustrates in quantitative terms
(Km and Vmax values) the
importance of clustered acidic amino acid residues (Glu and Asp) for
optimal KSMP and meprin cleavage. This conclusion is complemented
using a series of C-subunit mutants with deletions and single-,
double-, and triple-site mutations in the acidic residues within this
cluster. On the basis of a search for proteins and peptide hormones
that have a cluster of acidics and that may come in contact with
meprin, we suggest gastrin as a potential physiological substrate for meprin
. Meprin
is shown to cleave gastrin at its cluster of 5 glutamic acid residues and also its Met-Asp bond within the carboxyl-terminal sequence (WMDF-NH2), which has been
exceedingly well preserved during evolution (32) and is claimed to be
indispensable for all the known biological activities of gastrins (33,
34). The latter meprin cleavage will, therefore, lead to an
inactivation of gastrin and thus to the control of its activity.
The purification and assay of KSMP (from rat kidney) (13) and of the catalytic subunit of PKA (35) (from bovine heart) were carried out as described earlier.
Purification of MeprinThe meprin precursor was expressed in the 293 human embryo kidney cell line as
reported earlier (13). The KSMP purification procedure described before
(13) was applied for the purification of the expressed meprin
precursor, with some modifications. The meprin
expressing clone was
grown on the selective medium until a confluent monolayer was formed,
collected with a rubber scraper, and washed three times by
phosphate-buffered saline. The cells were resuspended in 10 mM Tris-HCl buffer, pH 7.1, containing 10 mM
mannitol, and they were ruptured by ultrasound at 4 °C. The membrane
fraction (i.e. the pellet obtained by centrifugation of the
lysis suspension at 50,000 × g for 30 min) was
resuspended in the same buffer to adjust the protein concentration to 1 mg/ml, and the membranes were then solubilized by adding
octyl-
-D-glucopyranoside to a final concentration of
1%. The subsequent purification steps were essentially the same as
described for the purification of KSMP from rat kidney brush-border
membranes (13) except that a Mono Q anion-exchange chromatography step
was applied instead of DEAE-Sephacell chromatography. The Mono Q column
(HR 5 × 5, Pharmacia, Sweden) was equilibrated with a 10 mM Tris-HCl buffer, pH 7.1, containing 1.5 mM
MgCl2, 1 µM ZnCl2, and 0.5%
octyl-
-D-glucopyranoside. The meprin
preparation
purified by Cu2+-chelating agarose was extensively dialyzed
against the Mono Q equilibration buffer and then loaded on the column,
which was developed with a 0-500 mM NaCl gradient in the
same equilibration buffer. Fractions containing the pure meprin
precursor (identified by the Coomassie Blue staining of a band with a
molecular mass of 105 kDa in SDS-PAGE) were found to be eluted from the
column at ~300 mM NaCl and were pooled and used for the
experiments described here.
Peptides were synthesized by solid-phase peptide synthesis in the Chemical Services at the Weizmann Institute of Science, Rehovot. All the synthetic peptides were purified by reverse-phase HPLC before use and subjected to amino acid composition and sequence analysis to confirm their structure.
Determination of the KSMP Cleavage Site in CA preparative
scale cleavage of the C-subunit was carried out in a reaction mixture
(final volume 5.5 ml) that contained 350 µg of the C-subunit and 25 µg of purified KSMP (13) in 20 mM Tris-HCl, pH 7.1, with
the addition of 1.5 mM MgCl2 and 0.15% octyl--D-glucopyranoside. The reaction was allowed to
proceed at 22 °C for 80 min and then arrested by adding 50%
trifluoroacetic acid to a final concentration of 0.5%. The resulting
fragments were loaded onto Ultrasphere ODS (150 × 4.6 mm)
reverse-phase HPLC column (Beckman Instruments) and then eluted with a
linear gradient of acetonitrile (0-80%) in 0.1% trifluoroacetic
acid. Two peptide peaks (which were not present at time zero) with
retention times of ~43 and ~41 min were collected,
rechromatographed on the same column, and sequenced in a gas-phase
sequencing apparatus (Applied Biosystems). It should be noted that the
amount of the major peptide (retention time ~43 min) was present in a
20-fold excess over the minor peptide (retention time ~41 min).
The kinetic constants Km and vmax were measured as described by Cleland (36). Assuming a single substrate mechanism for the reaction, the relationship between the velocity of the reaction and the substrate concentration is given by the equation,
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Purified
meprin precursor was activated by trypsin (sequencing grade,
Boehringer Mannheim). The preparative scale reaction mixture (500 µl
total volume) contained 0.05 mg/ml meprin
precursor, 0.2 µg/ml
trypsin (the trypsin:protein ratio was 1:250) in 20 mM
Tris-HCl, pH 7.1, supplemented with 1.5 mM
MgCl2 and 0.1% octyl-
-D-glucopyranoside. The reaction was allowed to proceed for 5 min at 37 °C, and the trypsin cleavage was then arrested by adding soybean trypsin inhibitor in a 10-fold excess (w/w) over the added trypsin. Complete trypsin inhibition was ascertained in each experiment by running an appropriate control; a substrate was incubated with the same mixture of trypsin and
its inhibitor in the absence of the meprin
precursor.
Cleavage of synthetic
peptide substrates was performed in a 20 mM Tris-HCl
buffer, pH 7.1, with an addition of 1.5 mM
MgCl2 and 0.1% octyl--D-glucopyranoside.
Analytical scale reaction mixtures were 30 µl final volume,
preparative scale reaction mixtures were 200 µl, and the minimal
amount of a peptide in the reaction mixture was not below 0.5 nmol. The
peptide concentrations in the reaction mixtures ranged from 1 to 200 µM, and the reaction was allowed to proceed for a time
interval within the linear region of dependence on time. The cleavage
was arrested by adding trifluoroacetic acid to the reaction mixture to
a final concentration of 0.5%, and then its volume was adjusted to 500 µl by 0.1% trifluoroacetic acid and injected to a reverse-phase HPLC
column (LiChroCART 125-4 LiChroSphere RP-18, Merck, Germany). A flow
rate of 1 ml/min was used, and elution of the bound material was
achieved using a linear gradient of acetonitrile. The effluent was
monitored simultaneously at 210 and 280 nm with a diode-array detector
(Hewlett-Packard). A quantitative conversion of the peak area into the
amount of peptide in it was calculated from a calibration curve. A
quantitative amino acid analysis of the eluted peaks was run along with
measuring the area of the peaks. The enzyme activity was defined as
micromole of product released by 1 mg of enzyme/1 min.
The wild-type murine C-subunit gene cloned
into the pRSET-B vector (Invitrogen) under control of T7 polymerase
promoter was a generous gift from Dr. S. S. Taylor (Univerity of
California, San Diego). Site-directed mutations were introduced by
oligonucleotide-directed mutagenesis of a uracil-containing
single-stranded Kunkel template (37). The translation of the coding
sequences was carried out in the TNT-coupled transcription/translation
rabbit reticulocyte expression system (Promega), as recommended by the
manufacturer, and performed in the presence of
[35S]methionine (Amersham, UK).
The wild-type C-subunit and its mutant forms were
translated in the rabbit reticulocyte lysate TNT system and subjected
to proteolysis by KSMP. The cleavage reaction (15 µl) contained 2 µl of the standard translation reaction mixture, 0.01 µg of
purified KSMP preparation in a 20 mM Tris-HCl buffer, pH
7.1, with an addition of 1.5 mM MgCl2 and 0.1%
octyl--D-glucopyranoside. The cleavage was allowed to
proceed at 22 °C. The components in the reaction mixture were
separated by SDS-PAGE, the gel was dried, and the 35S-labeled protein bands were visualized by
autoradiography. Quantitation of the cleavage was done by densitometric
analysis of the bands corresponding to the intact and KSMP-cleaved
products. The KSMP activity was determined by quantitation of the
C-subunit to C
conversion using the equation, activity (%) = C
/(C + C
). Calculated from several
time points, the rate of cleavage was extrapolated to time zero. The
initial cleavage rate of wild-type C-subunit was taken as 100%, to
which the cleavage rates of the mutants were compared.
To get an insight into the molecular basis of the specific cleavage of the C-subunit by KSMP, it was essential to determine exactly the bonds cleaved in the kinase by this proteinase. This recently became possible upon achieving a purification of KSMP to homogeneity (13).
To identify the exact sites of the KSMP cleavage in the C-subunit, we carried out this cleavage on a preparative scale, and then separated the cleavage products by reverse phase HPLC and sequenced them. Two new peaks were shown to be formed, a major and a minor peak (HPLC data not shown). In quantitative terms, the major peak was about 20-fold more abundant than the minor one. As seen in Table I, the major peak had the sequence EEIRVSINEK*GKEFS resulting from a cleavage of the E332-E333 bond within the cluster of acidic amino acids of this kinase, i.e. residues D328-E334. This is in agreement with earlier experiments in our laboratory, which implicated the involvement of this cluster of acidic amino acids in the recognition of the C-subunit and of other kinases by KSMP (7, 10, 11) and with our more recent immunological mapping of the cleavage site with specific anti-peptide antibodies (8). The minor peak had the sequence DFLKK (Table I), which fits the stretch 25DFLKK29 in the amino-terminal region of the C-subunit (Fig. 1).
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KSMP and Meprin
In an attempt to gain
further support to our finding that the KSMP cleavage of the C-subunit
can be reproduced by meprin , we compared the Km
and Vmax values of KSMP with meprin
in the
cleavage of this kinase. A preparation of KSMP obtained from rat kidney
and recombinant meprin
purified from transfected 293 cells (13)
were used in this comparison. The cleavage was performed at different
concentrations of the C-subunit as described under "Materials and
Methods," securing a linear dependence of the proteolysis with time
and with the concentration of the proteinase. Each of the reaction
mixtures was subjected to SDS-PAGE followed by Coomassie Blue staining.
The extent of proteolysis was determined by computing densitometry of
these gels, monitoring the amount of the clipped C-subunit
(i.e. C
) formed. Plotting the KSMP (C-subunit-degrading) activity versus the concentration of the C-subunit in double
reciprocal coordinates gave straight lines (Fig. 2) from
which the Km and Vmax values
were calculated. Average values for these Km and
Vmax values were calculated from three
independent experiments and were found to be Km = 0.44 ± 0.03 µM and Vmax = 19 ± 9 nmol/min × mg for KSMP and were
Km = 0.58 ± 0.07 µM and
Vmax = 22.8 ± 2.7 nmol/min × mg for
the expressed meprin
.
The Substrate Recognition of KSMP Analyzed by the Cleavability of the C-subunit Mutants
In order to evaluate the structural
requirements of KSMP for its substrate recognition and cleavage, we
constructed a series of the C-subunit mutants, with deletions or
substitutions in the cluster of acidic amino acids of the C-subunit
encompassing its KSMP cleavage site. The first step toward this
analysis was to set up an adequate expression system for monitoring the
cleavage of the C-subunit and its mutants since we found out that the
wild-type C-subunit itself, though fully active as a protein kinase
(38), is not cleaved if expressed in bacteria (data not
shown)2. The rabbit reticulocyte lysate translation system
using [35S]methionine was found to be appropriate for
that purpose. The wild-type C-subunit produced in this system was
cleaved by KSMP with the formation of the clipped product (C, ~34
kDa). However, as seen in Fig. 3A, a
mutagenic deletion of the whole cluster of acidic amino acids
(Scheme 1,
DDYEEEE) in the stretch
D328-E334 led to a complete lack of cleavage.
The same lack of cleavability occurred also upon removal of the stretch
of the four glutamic acid residues (
EEEE), i.e. the
E331-E334 segment. In contrast, the deletion of
D328-Y330 (as in the mutant denoted
DDY) did
not significantly affect the KSMP cleavage rate though it slightly
reduced this rate (Fig. 3A). These results point to the
importance of the cluster of the four glutamic acid residues for the
interaction of the proteinase with the C-subunit although a reduction
in the local negative charge, or a distortion in the conformation of
the kinase as a result of these deletions, may also be responsible for
the lack of cleavage. In addition, it should be kept in mind that in
this case, where the cluster of acidic amino acids is part of a tail with which the large lobe of the C-subunit embraces its smaller lobe
(cf. Fig. 5 in Ref. 4), the loss of cleavability may result from the
removal of a segment of the polypeptide backbone, which may well
prevent amino acid residues following the deletion from reaching their
counterparts in the core segment with which they interact in the
wild-type enzyme structure. This kind of "shortened rope" effect
may, of course, have a detrimental structural outcome in many deletion
studies except when the deletion involves a loop that does not interact
with the core of the protein.2
To avoid some of the difficulties mentioned above and to obtain a more accurate assessment of the individual contribution of each amino acid residue to the KSMP recognition and C-cleavage, we carried out an alanine scanning of the glutamic acid residues within the cluster of acidics in the C-subunit (Scheme 1). Quantitative analysis of the initial rates of cleavage of these mutants showed that there is only a ~20% reduction in the cleavage rate when a single glutamic acid residue is mutated into an alanine, even if the Glu to Ala mutation results in a discontinuity in the sequence of the negatively charged residues. A double Glu to Ala substitution as in the E332-333A mutant retained ~20% of the initial cleavage rate (Fig. 3B). However, replacement by alanine of three or of all four glutamic acid residues of the C-subunit (E331-333A and E331-334A) resulted in a complete lack of cleavage (Fig. 3B).
Use of Synthetic Peptides Derived from the C-subunit to Establish the Substrate Specificity of KSMPTo complement our results on
the specificity of KSMP that were based on the sequence of the cleavage
products and on mutations of the C-subunit, we attempted to elucidate
the recognition of KSMP by the use of synthetic peptides derived from
the segment in the C-subunit that encompasses the KSMP cleavage site.
We prepared four synthetic peptides whose sequences were derived from
the following segments in the C-subunit: 1)
K319-I335, 2)
F327-I335, 3)
S325-Y330, and 4)
Y330-I335 (Fig. 4). Each of
these peptides was cleaved by a pure KSMP preparation, and the cleavage
products were resolved by reverse-phase HPLC and analyzed by a
determination of their amino acid composition or sequence. As seen in
Fig. 4, KSMP cleaves the peptides between acidic amino acids in
positions that correspond either to the E332-E333 bond, or to the
D328-D329 bond in C. The cleavage site of the
peptides in the middle of the cluster of glutamic acid residues is
identical to the KSMP cleavage site detected in the C-subunit and
described above (E332-E333). The lack of
cleavage of the D328-D329 bond may be due to
steric hindrance, or an unfavorable juxtaposition of this D-D bond of
the C-subunit and the KSMP active site. Interestingly, the
three-dimensional structure of the C-subunit established by x-ray
crystallography showed that, while the backbone of the cluster of
acidic amino acids in the C-subunit exhibits relatively high B factors
and thus a greater flexibility and availability (5), the
D328-D329 bond may be less available for
interaction with KSMP since D329 forms a salt bridge with
K47 (39) that would neutralize the negative charge of
D329 and also restrict its flexibility.
It should be mentioned that the peptides 319KGPGDTSNFDDYEEEEI335 and 325SNFDDY330, but not the peptide 327FDDYEEEEI335, were found to have an additional (minor) cleavage site between F327 and D328 (Fig. 4). The rate of this minor cleavage is at least ten-fold slower than the rate of cleavage between the two aspartic acid residues in these peptides.
The Cleavage of the C-subunit-derived Synthetic Peptides by KSMP Is Reproduced by MeprinOur recent demonstration that the
cleavage of the C-subunit by KSMP can be reproduced by meprin (13)
prompted us to study the specificity of the
-subunit of meprin and
to compare it with the substrate specificity of KSMP from rat kidney.
The clone of human kidney fibroblasts constitutively expressing rat
meprin
(13) was used as a source of the enzyme. The substrate
specificity of the
-subunit purified from these cells was assayed
after activation of the precursor enzyme by a limited trypsin digestion
(13), resulting in the formation of a catalytically active enzyme with an apparent molecular weight of 100 kDa (Fig. 5,
lane 2). The peptides used as substrates for the rat kidney
KSMP (see above) were also used in this study of the meprin
specificity. Again, the resulting cleavage products were
subjected to analysis by amino acid composition or sequencing and shown
to be identical to the cleavage products formed by KSMP. This finding
indicates that the
-subunit of meprin, which is present in the rat
kidney KSMP preparations (13), is not essential for either the cleavage of C or for the cleavage of these peptide substrates. While substrates for the
-subunit of meprin are known (26, 40), a systematic study
aimed at establishing its substrate specificity will have to await the
separate stable expression of this subunit.
For a more quantitative comparison between KSMP and meprin , the
kinetic parameters (Km and Kcat values)
of these two enzyme preparations were measured with a synthetic peptide (YEEEEI) containing a single cleavage site to simplify the measurements and their processing. As seen in Table II, the
Km and Kcat values of KSMP
and meprin
with this substrate were found to be quite similar,
further supporting our conclusion (13) that meprin
and KSMP are
closely related, if not identical.
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As a member of the astacin family of
metalloendopeptidases, meprin should have an arylamidase activity
(24) and have a preference for bonds flanked by neutral or hydrophobic
amino acid residues. Therefore, we assayed the meprin
expressed in
the 293 cell line (13) with some synthetic peptides derived from the
C-subunit sequence that carried alanine substitutions of the hydrophobic amino acid residues at the edges of the cluster of the four
glutamic acid residues in the C-subunit, which are recognized by both
KSMP and meprin
(Fig. 6). For the purpose of comparison, Table II
also quotes quantitative data obtained in other laboratories regarding
the substrate specificity of rat meprin (26, 27). This comparison
clearly shows that the Kcat values obtained for meprin substrates that were proposed to be physiological targets of
this proteinase (LHRH, substance P, and bradykinin, which do not
contain clusters of acidics) are of the same order of magnitude of the
Kcat values of meprin
when cleaving the
peptide YEEEI derived from the C-subunit (Table II). However, the
Km values of meprin for three synthetic peptides
containing the cluster of the four Glu residues were found to be
20-80-fold lower than the values obtained for the hormonal substrates
studied earlier in other laboratories (27). Furthermore, the catalytic
efficacy of meprin
for these acidic peptides is significantly
higher (12-100-fold, as reflected in its
Kcat/Km values) than the
comparative values obtained for the hormones LHRH, substance P, and
bradykinin (Table II).
It should be noted that substitution of either the Tyr or the Ile
residues by alanine in the peptide YEEEEI does not significantly affect
the Km values of meprin , suggesting a relatively low contribution of these hydrophobic residues in creating the affinity
between the enzyme and its peptide substrates. However, the Tyr to Ala
substitution somewhat decreases (~2.5-fold) the catalytic efficacy of
the cleavage. The possible contribution of aromatic amino acid residues
(such as Phe and Tyr) in enhancing the Kcat of
meprin
is probably reflected better in the cleavage of SNFDDY, a
peptide substrate of this proteinase derived from the cluster of acidic
amino acids in the C-subunit that is cleaved between the two aspartic
acids. As seen in Table II, the kinetic parameters of meprin
for
the YEEEEI and SNFDDY peptides are considerably distinct. The SNFDDY
peptide was found to have a substantially reduced affinity
(Km = 80 µM), which we believe is
associated with a reduction of the overall charge of the cluster of
acidics. However, due to an increasingly high rate of cleavage, the
Kcat/Km ratio for SNFDDY is
within the range determined for the high affinity substrates (Table
II).
In view
of the finding reported here that meprin has a distinct preference
for substrates containing a cluster of negatively charged amino acids,
we carried out a search in the data base for peptides and proteins
containing stretches of at least four acidic amino acid residues. This
search revealed quite a few peptides and proteins with such stretches
and, consequently, candidate substrates for meprin
. Among these,
gastrin seemed to be of particular interest since it is found in the
gastrointestinal tract and in the kidney where it can be exposed to
meprin. Discovered in 1905 as an acid-stimulating factor (41), gastrin
is now regarded also as an important growth-stimulating hormone (42).
Gastrin occurs in multiple hormonal forms that are produced as a result of proteolytic processing and may contain from 71 to 6 amino acid residues (32, 43). Its most abundant forms (G-34 and G-17) contain a
stretch of five glutamic acid residues (Fig. 7) (34). All known biological effects of gastrin reside in the conserved carboxyl-terminal tetrapeptide amide WMDF-NH2, which is
common to all gastrins and also to the cholecystokinins
(Scheme 2B).
Analysis of the fragments resulting from gastrin cleavage by either
KSMP or meprin revealed proteolysis at three distinct sites (Fig.
7). Two adjacent cleavage sites were found within the cluster of
glutamic acid residues. For the sake of simplicity, the kinetic
parameters of the cleavages were measured on the gastrin fragments
rather than on the whole molecule (Fig. 7). The first fragment,
MEEEEEAY, accommodated the two alternative cleavage sites identified in
gastrin 17. The affinity of meprin
for this peptide was found to be
very similar to that observed with the C-subunit-derived peptides
(Table II). The cleavage of the carboxyl-terminal fragment of gastrin,
EEAYGWMDF, resulted in clipping off its last two residues (cleavage at
the Met-Asp bond). Since any modification in the carboxyl-terminal
tetrapeptide amide WMDF-NH2 grossly reduces or abolishes
all its known biological effects (33, 34, 44), this cleavage will
inactivate gastrins. Notably the Km for the cleavage
at this site was found to be substantially higher than the
Km for the cleavage at the sites in the acidic cluster. However, the Kcat for this cleavage was
much higher, and thus, it had a comparable
Kcat/Km value (Table II). The
decreased affinity for the EEAYGWMDF peptide compared with the site in
the cluster of glutamic residues was actually expected in view of the
findings reported here regarding the important contribution of
clustered acidics to the affinity of meprin
for its substrates.
It should be emphasized, however, that in spite of the fact that
clusters of acidics are recognized and cleaved by meprin with a
significantly lower Km, hydrophobic amino acid residues most likely play an important role in the cleavage of physiological substrates by this proteinase. They do so with a high
Km but also with a high Kcat
and thus with a high catalytic efficacy. The evidence supporting the
importance of hydrophobic amino acid residues is summarized in the
following. (i) The protein substrate (C-subunit) with which KSMP was
originally discovered (1-3), as well as the EGF- and insulin-receptor
kinases (7, 10, 11), contain hydrophobic amino acid residues within or
adjacent to their cluster of acidic amino acids. (ii) The dye 1-anilino-8-naphthalenesulfonate, which is known to bind to hydrophobic sites in proteins, inhibits the cleavage of the C-subunit by KSMP (45).
(iii) While a copolymer composed of Glu and Tyr is cleaved by KSMP, a
polymer of Glu amino acid residues alone is not cleaved by it and acts
as a competitive inhibitor of this proteinase (7, 45). (iv) The
hydrophobic proteinase inhibitor chymostatin inhibits the cleavage of
the C-subunit by KSMP though at relatively high concentrations (
10
3 M) (3). (v) In general, members of the
astacin family of peptidases have been shown to cleave peptides
containing hydrophobic amino acid residues and to possess an
arylamidase activity (26).
It is, therefore, possible that the degradation of gastrin by meprin
may draw its affinity from the interaction of meprin
with the
cluster of acidics (Km = 1.5 µM) while
the fast cleavage at the Met-Asp bond (which will inactivate gastrin) may originate from the high Kcat value (63.4 s
1) for this cleavage. It is also possible that the
cleavage at the cluster of acidics facilitates the subsequent
evacuation of the active site.
Meprins are
synthesized as inactive proenzymes whose prosequences are removed when
they are called upon to act. One of the plausible mechanisms for an
auto-inhibition of enzymes is the blocking of the active site by a
substrate-like prosequence, which can then be removed for the purpose
of activation by the enzyme itself or by another enzyme, in response to
an appropriate regulatory stimulus. In view of the specificity profile
described above for meprin , we looked into its prosequence,
searching for features related to the specificity ensuing from this
study and for a difference between meprin
and meprin
that might
reflect the known difference in specificity between these two subunits
(24). At the same time, we attempted to find out whether, in view of
the specificity implicated in this study, there may be evidence
supporting or disproving an autoinhibition mechanism of the type
described above. Scheme 2 illustrates such a comparison for the rat,
mouse, and human prosequences of meprins
and
. From this
comparison, it is evident that (i) the prosequence of meprin
possesses a stretch of acidic and hydrophobic amino acids (in line with
the specificity profile described above). This is especially prominent
in the stretch D55-I74, say in the rat, in
which out of 20 amino acids, seven are acidic (mostly Asp), seven are
hydrophobic (mostly Ile and Leu), and not one is a basic amino acid.
(ii) The parallel stretch in promeprin
has six hydrophobics but
only three acidics and one basic amino acid. (iii) Five of the acidic
and hydrophobic residues in the
prosequence come in pairs of DI or
DL, raising the possibility that this repeated motif may have a
distinct inhibitory significance. These suggestions regarding the
difference in specificity between the
and the
subunits of
meprin, and regarding the molecular basis of their autoinhibition, will
hopefully shed light from a new angle on the biorecognition of these
metalloendopeptidases. However, their ultimate proof will have to rely
on molecular modeling studies (based on the astacin structure (46)) and
possibly to await the determination of the three-dimensional structures
of these precursors by x-ray crystallography.
This paper provides evidence to show that the cleavage of KSMP in
the C-subunit occurs at E332-E333, within the
cluster of acidic amino acids (D328-E334) of
this kinase. The Km values of KSMP and of meprin (which we recently showed to reproduce KSMP activity) for the C-subunit
are below 1 µM. The Km for peptides
containing a stretch of four Glu residues are in the micromolar range,
suggesting a significant contribution of this cluster of acidics to the
biorecognition of meprin
. This conclusion is supported by
experiments with a series of C-subunit mutants with deletions and
mutations in the cluster of acidics. In addition, hydrophobic amino
acids vicinal to the cleavage site seem to play an important role in
the function of this proteinase, increasing its
Kcat.
These studies unveil a new specificity profile for meprin ,
suggesting new candidate substrates that are 1-2 orders of magnitude better (lower in their Km and higher in their
Kcat) than substrates commonly used for meprin
. Specifically, the search for substrates that have such a cluster
of acidics and hydrophobics and are accessible to meprin
under
physiological conditions, pointed at the hormone gastrin as a potential
target. Indeed, we show here that, at least in vitro, meprin
cleaves gastrin at its cluster of five glutamic acid residues and
also at the Met-Asp bond within its WMDF-NH2 sequence whose
unmodified structure has been claimed to be indispensable for all the
known biological activities of gastrins (33, 34). The latter meprin
cleavage will lead to the inactivation of gastrin and thus to the
control of its activity. In view of the fact that meprins have been
implicated in key biological processes such as growth, development, and
tissue remodeling, the search for such target substrates, and the
demonstration that they occur in vivo, is quite an important
task.