From the Department of Biochemistry and Molecular
Biology, The Pennsylvania State University College of Medicine,
Hershey, Pennsylvania 17033, the § Department of Medicine,
Division of Signal Transduction, Beth Israel Deaconess Medical Center,
Boston, Massachusetts 02215, the ¶ Department of Cell Biology,
Harvard Medical School, Boston, Massachusetts 02215, and the
Department of Biomolecular Sciences, University of Manchester
Institute of Science and Technology, Manchester M60 1QD, United
Kingdom
Received for publication, December 19, 2000, and in revised form, January 18, 2001
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ABSTRACT |
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Meprin A and B are highly regulated, secreted,
and cell-surface metalloendopeptidases that are abundantly expressed in
the kidney and intestine. Meprin oligomers consist of evolutionarily related Meprin A and B are zinc metalloendopeptidases composed of
evolutionarily related Mouse kidney meprin A (EC 3.4.24.18) is a homooligomer of Identification of substrates for proteases is a valuable step
toward elucidation of physiological function and provides a knowledge-based approach to inhibitor design. Previous studies have
shown that a variety of biologically active peptides and proteins are
hydrolyzed by meprins in vitro. For example, meprins cleave
blood pressure regulators such as bradykinin, metabolism mediators such
as parathyroid hormone, signaling molecules such as protein kinase A,
and basement membrane proteins such as entactin (1, 2, 16, 17). There
has been no systematic study, however, of the enzymatic differences
between meprin A and B from any species or of the contributions of the
individual subunits to activity. Enzymological data for meprin B are
particularly lacking even though meprin B appears to be a more
essential protease than meprin A as indicated by expression patterns in
mammalian tissues. The work herein describes previously unidentified
meprin substrates and provides the first detailed comparison of meprin Isolation of Meprin--
Mouse meprin A and B were isolated from
kidney brush border membranes of ICR and C3H/He male mice,
respectively. For kinetic analyses, meprin B was activated using
trypsin as previously described (18). Concentrations of meprins are
based on a subunit molecular mass of 90 kDa. Meprin A contains active
Materials--
Peptides, proteins, inhibitors, and
reagents were purchased from Sigma Chemical Co. with the following
exceptions. The nonsulfated cholecystokinin
(CCK)1 derivatives
Boc-CCK8NH2, CCK8NH2, CCK8,
CCK7NH2, CCK6NH2, and CCK4NH2 were
from Bachem. Somatostatin, gastrin-releasing peptide fragment 14-27
(GRP-(14-27)), sulfated-CCK (sCCK), sCCK8NH2,
cerulein, and human secretin were from American Peptide Co.
Neurotensin, kassinin, and CCK5NH2 were from Novabiochem.
Recombinant mouse osteopontin and goat anti-mouse osteopontin antibody
were obtained from R & D Systems. SuperSignal West Dura Extended
Duration Substrate was from Pierce. The dodecamer peptide library was
synthesized at the Tufts University Core Facility (Boston, MA).
Peptide Library Screen--
An amino-terminally acetylated
dodecamer peptide mixture (1 mM) consisting of a roughly
equimolar mixture of the 19 naturally occurring L-amino
acids, excluding cysteine, was incubated with either meprin A (33 nM) or B (10 nM) in 25 mM HEPES, pH
7.4, 100 mM NaCl, 5 mM CaCl2 at
37 °C until 5-10% of the peptides were digested. The mixture
contained the inhibitors pepstatin, leupeptin, L-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane,
3,4-dichloroisocoumarin, and bestatin to prevent hydrolysis by trace
amounts of protease impurities. Hydrolysis was terminated by heating to
100 °C for 2 min, and 10 µl of the mixture was subjected to Edman
degradation-based amino-terminal peptide sequencing. The data in each
sequencing cycle was normalized to the total molar amount of amino
acids in that cycle so that a value of 1 indicates the average value. Undigested peptides and the amino-terminal fragments of digested peptides are amino-terminally blocked and therefore do not contribute to the sequenced pool. The relative amount of a given amino acid residue present in a particular sequencing cycle indicates the preference for that residue at a particular position relative to the
cleavage site. Values for a given amino acid were corrected by
renormalizing to the average relative amount of that residue present
across the first nine sequencing cycles to correct for the distribution
of amino acids in the starting mixture.
Identification of Novel Meprin Substrates--
Naturally
occurring peptides (100 µM) were incubated with meprin A
or B (2 nM) for 4 h. The incubation was carried out at 37 °C in 20 mM Tris-HCl, 150 mM NaCl, pH
7.5, containing the same inhibitors as above. The reaction was
terminated by the addition of EDTA to 10 mM and
trifluoroacetic acid (TFA) to 0.05% v/v. Samples were then subjected
to high performance liquid chromatography (HPLC) analysis using a POROS
R2H (4.6 × 100 mm) column and a Brownlee Newguard 7-µm
RP-18 (3.2 × 15 mm) guard column. Peptides were eluted with a
linear gradient of acetonitrile in 0.1% TFA at a constant flow rate of
3.2 ml min Kinetic Constant Determination--
Kinetics for peptide
hydrolysis by meprins were determined by quantitative HPLC analysis of
reaction mixtures using the same buffer conditions as the peptide
screen. Meprin concentrations in reactions were between 1 and 4 nM depending on efficiency of proteolysis. Hydrolysis was
limited to 20% and was calculated by monitoring loss of substrate peak
area. Velocity (µM min Identification of Cleavage Sites--
Peptides (50 µM) were incubated with meprin A or B (2 nM)
at 37 °C in 20 mM Tris-HCl, 150 mM NaCl, pH
7.5, in a final volume of 100 µl for 5 min to 6 h depending on
the efficiency of hydrolysis. Total peptide was subjected to HPLC using
a Spheri-5 ODS 5 micron (4.6 × 250 mm) column and a Brownlee
Newguard 7-µm RP-18 (3.2 × 15 mm) guard column. Peptides were
eluted with a linear gradient of acetonitrile in 0.1% TFA at a
constant flow rate of 1.2 ml min Meprin Activity against Extracellular Matrix Proteins--
The
extracellular matrix proteins gelatin, collagen I and IV, laminin, and
fibronectin (20 µg) were incubated with meprin A or B (20 nM) for 18 h at 25 °C in 20 mM
Tris-HCl, 150 mM NaCl, pH 7.5, with a final volume of 40 µl. Proteolysis was terminated by the addition of EDTA to 10 mM. The sample was boiled in SDS-PAGE sample buffer
containing Degradation of Osteopontin by Meprin--
Recombinant mouse
osteopontin (50 µg ml Inhibition Profiles of Meprins--
The inhibition profiles of
meprin A and B were compared using actinonin and
Pro-Leu-Gly-hydroxamate (PLG-(NHOH)). Meprin A or B (2 nM)
was preincubated with various concentrations of inhibitor for 20 min
before addition of substrate. Reactions were performed in 20 mM Tris-HCl, 150 mM NaCl, pH 7.5, and
sCCK8NH2 (50 µM) was used as
substrate. Substrate hydrolysis was limited to 25% and was
determined by quantitative HPLC analyses. The concentrations of
inhibitors ranged from 12.5 nM to 50 µM for
actinonin and from 250 nM to 500 µM for
PLG-(NHOH). The level of inhibition was determined by comparing the
decrease in substrate concentration in the presence and absence of inhibitor.
Homology Models of Meprin Meprin A and B Have Distinct Specificities for Preferred Amino
Acids at or Near the Cleavage Site of Substrates as Determined by
Peptide Libraries--
Peptide libraries were used to identify
preferred amino acids of substrates at or near the cleavage site and to
map the primed subsite2
binding region of meprins (25). A completely random dodecapeptide mixture acetylated at the amino terminus was partially digested with
meprin A or B, and the digested peptides were subjected to amino-terminal peptide sequencing. For meprin B the strongest specificity was seen at the P1' site of the substrate where acidic residues were preferred (Fig. 1).
Aspartic acid at the amino terminus of peptide products was
particularly prevalent with a signal 2.5- to 41-fold greater than other
residues. Glutamic acid was also selected but to a lesser extent. The
selectivity decreased further away from the scissile bond; with 41-, 3.4-, 2.1-, and 1.5-fold differences between the highest and lowest
relative molar amounts for the P1', P2', P3', and P4' subsites,
respectively. There was no clear preference for a single amino acid at
the P2' through P4' sites, aside from a slight preference for proline
in P3'. However, there was a selection against basic residues at P2'
and P3' as well as a slight selection against aspartic acid and
arginine side chains in P4'.
Meprin A had a completely different profile to meprin B for amino acid
preference at the P1' subsite (Fig. 2).
There was a selection for small and aromatic residues (S > F > A > T > M > Y > G) at the P1' position. The
only similarity between the enzymes at the P1' site was a strong
selection against proline. The profiles at the P2' subsite were also
dissimilar for the two enzymes. Proline was the most preferred amino
acid at the P2' position for meprin A, with at least a 2-fold larger
signal than other residues. There was also a slight selection against
leucine, asparagine, and basic amino acids in the P2' subsite. Proline
was also the preferred residue at the P3' site, although to a lesser
extent than the P2' subsite. Charged residues and tyrosine were
slightly disfavored. The specificity signature at P3' was strikingly
similar to that of meprin B, and there was little or no specificity at
P4'. The data indicated that, for both enzymes, at least three primed
subsites contribute to substrate binding specificity (Figs. 1 and 2).
In addition, meprin A and B have very different specificities in the
P1' and P2' subsites.
Comparison of Naturally Occurring Peptides as Substrates of
Meprins--
Specific peptides were tested as meprin substrates to
further define substrate preferences (Table
I). The peptides were chosen based on the
knowledge of meprin peptide bond specificity and the peptide
localization in vivo. In an initial screen of potential substrates it was noted that peptides of the gastrointestinal tract
(e.g. GRP-(14-27), sCCK8NH2, and gastrin 17)
were relatively good substrates of meprins. The screen employed 25 bioactive peptides that fell into four groups. The first group,
GRP-(14-27), sCCK8NH2, secretin, glucagon, neuropeptide Y,
and cerulein were susceptible to hydrolysis by both meprin A and B. GRP-(14-27) was efficiently hydrolyzed by both meprins under the
conditions used; the intact peptide was almost completely hydrolyzed
after 4 h. The second group, bombesin, neurotensin, luteinizing
hormone releasing hormone (LHRH), bradykinin, Kinetic Parameters for Meprin Cleavage of Bioactive
Peptides--
A kinetic study was conducted to better characterize the
substrates of meprins identified in the initial screen. The kinetic constants, kcat and Km as
well as the specificity constant kcat/Km (Table
II) were determined by directly fitting data to the Michaelis-Menten equation by nonlinear regression analysis
as described under "Experimental Procedures." All peptides tested
exhibited typical Michaelis-Menten kinetics (data not shown). Velocity
was determined by monitoring the loss of substrate peak area rather
than the appearance of substrate. This approach was taken due to the
presence of multiple cleavage sites as evident by more than two product
peaks for most substrates (data not shown). The Km
values for meprin A ranged from 116 for GRP-(14-27) to 425 µM for bradykinin; for meprin B the range was from 7.1 for gastrin and 211 µM for sCCK8NH2. Meprin A
had kcat values between 11.8 for secretin and
88.3 s Meprins Have Distinct Differences in Peptide Bond
Specificity--
Peptides that were relatively good substrates of
meprins were further characterized to determine peptide bonds cleaved.
The peptides were digested with either meprin B (Table
III) or meprin A (Table
IV), and products were identified by
MALDI-TOF. Cleavage was evident at more than one site in all instances
except meprin B hydrolysis of orcokinin, sCCK8NH2, peptide
YY, and kinetensin and meprin A hydrolysis of bradykinin (Tables III
and IV). In general meprin B appeared to be a more specific protease
than meprin A. Bonds with an acidic residue in the P1' position were
cleaved by meprin B in most instances (Table III). Out of 20 scissile
bonds determined, 13 sites were amino-terminal to an acidic residue. Residues seen multiple times at P1' were aspartic acid (9 times), glutamic acid (4 times), and glycine (twice), consistent with the
peptide library data (Fig. 1). Meprin B cleaved gastrin 17, orcokinin,
glucagon, sCCK8NH2, secretin, peptide YY, and neuropeptide Y with an acidic residue at P1' (Table III). However, GRP-(14-27) and
kinetensin, which lack acidic residues, were also hydrolyzed. This
demonstrated a distinct preference, but not an absolute requirement, for acidic residues in the P1' site of substrates by meprin B. The data
also indicated a lack of substrates with a basic residue in the P2'
position, which is consistent with peptide library data in which both
lysine and arginine are strongly selected against (Fig. 1). Meprin B is
able to accommodate all types of amino acids in P1 showing a lower
stringency for specific residues in this subsite. Glutamic acid is seen
the most often (6 out of 20 times). Proline was frequently seen in the
unprimed region (8 times). Interestingly, meprin B was able to act as
both an amino- and carboxypeptidase (e.g. orcokinin
and sCCK8NH2, respectively). In general, meprin A tended to
cleave bonds that have small uncharged or hydrophobic residues in their
P1 and P1' positions (Table IV). The presence of a proline residue in
meprin A substrates was frequent. Proline was seen at P4 (4 times), P3
(twice), P2' (5 times), and P3' (twice). Proline was never seen at P1
or P1'.
The Hydrolytic Efficiency of Meprin B Is Dependent on Peptide
Length--
Previous work indicated that meprin A has a preference for
substrates with a minimum of eight amino acids (28). To test the
peptide length requirement for efficient hydrolysis by meprin B,
derivatives of sCCK8NH2 were used as substrates (Table
V). Meprin B cleaved sCCK8NH2
at a single site toward the carboxyl terminus (Table III). The
sCCK8NH2 derivatives allowed the determination of the
unprimed subsites contribution, peripheral to the scissile bond as well
as the contribution of the carboxyl terminus toward substrate
recognition. The data indicated that meprin B had a preference for a
minimum of six amino acids. CCK4NH2 and CCK5NH2 were relatively poor substrates (17 and 15% hydrolyzed, respectively, compared with 56% for sCCK8NH2), whereas
CCK6NH2 and larger peptides were relatively good
substrates. The effects of modifications within sCCK8NH2
were also examined. The absence of sulfation at the tyrosine residue
resulted in a poorer substrate compared with the parent peptide (35 and
56% hydrolysis, respectively). Blockage at the amino termini
(Boc-CCK8NH2) increased the susceptibility to hydrolysis
(59% hydrolyzed compared with 35% for CCK8NH2). The
presence or absence of amidation at the carboxyl terminus resulted in larger differences toward hydrolysis. The free acid (CCK8)
was 76% hydrolyzed compared with 35% hydrolysis for
CCK8NH2. Cerulein, a CCK analog, was hydrolyzed 60% in
4 h. This peptide is identical to sCCK8NH2 except for
a methionine in place of a threonine residue, carboxyl-terminal to the
sulfated tyrosine, and an amino-terminal extension of pyroglutamic acid
and glutamine. The free acid was hydrolyzed to the greatest extent of
all CCK derivatives tested.
Meprin Degradation of Extracellular Matrix Proteins--
To
determine the ability of meprins to degrade extracellular matrix
components, gelatin, fibronectin, collagens I and IV, and laminin were
incubated with meprins, and the products were subjected to SDS-PAGE
(Fig. 3, upper panel). Gelatin
proved to be the best substrate under the conditions used. After an
18-h incubation with meprin A or B, intact gelatin was extensively hydrolyzed by both enzymes. Fibronectin was sensitive to both enzymes,
yielding similar patterns of hydrolysis. The major protein bands above
200 kDa seen in the control were hydrolyzed by both meprin A and B,
yielding bands that migrated slightly faster and reproducibly in both
cases. Collagens I and IV and laminin were resistant to hydrolysis at
25 °C (data not shown). Collagen I was sensitive to hydrolysis by
meprin B at 37 °C, possibly due to some local unfolding of the
triple helix (data not shown).
Meprin B Hydrolysis of Osteopontin--
To test the ability of
meprin B to hydrolyze proximal to acidic residues in the context of a
protein rather than a short peptide, osteopontin was used as a
substrate (Fig. 3, lower panel). This 294-amino acid protein
has a high percentage of acidic residues (23%). Meprin B effectively
degraded the protein in a time-dependent fashion. Over a
30-min incubation the intact protein decreased markedly as detected by
Western blot analysis. Meprin A showed little if any ability to degrade
osteopontin under the conditions used; the 60-min time point shows
slightly less protein than the control.
Inhibitor Profiles of Meprins--
The active sites of meprins
were further mapped using the inhibitors PLG-(NHOH) and actinonin. The
crystal structure of astacin complexed with PLG-(NHOH) has shown that
the inhibitor binds the unprimed subsite region (accession number 1QJJ
(29)). The hydroxamate moiety ligates the zinc, whereas the peptide
moiety binds the unprimed subsites and proline binds P3, leucine P2,
and the glycine P1. The IC50 values toward meprin A
and B were similar to one another with values of 30 and 50 µM, respectively (Fig. 4).
This was in the same range as the reported Ki value
toward astacin, which has a Ki of 16 µM (29). Unexpectedly, actinonin was a much more potent
inhibitor of both meprin A and B compared with astacin, even though it
has a very similar structure to PLG-(NHOH). The IC50 values
were over 300- and 125-fold higher against meprin A and B,
respectively, at 100 and 400 nM, respectively. In contrast,
the Ki toward astacin is 8-fold lower at 130 µM (30).
The work herein clearly demonstrates marked differences in the
preferences of meprin A and B for the P1' and P2' subsites of
substrates. Meprin B is predominantly an Asp/Glu-N peptidase as shown
by the peptide library studies and individual substrate data, selecting
acidic residues in the P1' site (Fig. 1 and Table III). By contrast,
meprin A selects a variety of small and hydrophobic amino acids in the
P1' site and clearly prefers proline residues in the P2' sites (Fig. 2
and Table IV). Both proteases have extended binding sites and prefer
substrates of at least 6 amino acids (Ref. 28 and Table V). The
different specificities of the two subunits implicate diverse functions.
Amino acid sequence analyses and homology models of the and/or
subunits. The work herein was carried out to identify bioactive peptides and proteins that are susceptible to
hydrolysis by mouse meprins and kinetically characterize the hydrolysis. Gastrin-releasing peptide fragment 14-27 and gastrin 17, regulatory molecules of the gastrointestinal tract, were found to be
the best peptide substrates for meprin A and B, respectively. Peptide
libraries and a variety of naturally occurring peptides revealed that
the meprin
subunit has a clear preference for acidic amino acids in
the P1 and P1' sites of substrates. The meprin
subunit selected for
small (e.g. serine, alanine) or hydrophobic
(e.g. phenylalanine) residues in the P1 and P1' sites, and
proline was the most preferred amino acid at the P2' position. Thus,
although the meprin
and
subunits share 55% amino acid identity
within the protease domain and are normally localized at the same
tissue cell surfaces, they have very different substrate and peptide
bond specificities indicating different functions. Homology models of
the mouse meprin
and
protease domains, based on the astacin
crystal structure, revealed active site differences that can account
for the marked differences in substrate specificity of the two subunits.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and/or
subunits. They are members of the
astacin family and are highly expressed in brush border membranes of
the intestine and renal proximal tubules (1, 2). Meprins are
particularly abundant in mouse juxtamedullary nephrons and constitute
~5% of total protein in renal brush border membranes (3). The meprin
and
subunits are expressed early in embryonic development of
mouse kidney and intestine, by day 11, and have different patterns of
expression in the suckling phase and after weaning (4). Homologous
enzymes are found in rat and human kidney and intestine (1, 5, 6).
Meprins are also expressed in leukocytes of intestinal lamina propria
and in cancer cells and are consequently implicated in inflammation and
cancer growth and metastasis (7, 8).
subunits, or a heterooligomer of
and
subunits (2, 9). Mouse
kidney meprin B (EC 3.4.24.63) is a homooligomer of
subunits (10).
The multidomain
and
meprin subunits are highly glycosylated and
form disulfide-linked dimers and higher order oligomers by noncovalent
interactions (11, 12). Meprins containing at least one
subunit
remain membrane-bound by virtue of a transmembrane domain located near
the carboxyl terminus of
subunits (1). Mature meprin
homooligomers contain no transmembrane domain and are found in mouse
urine (13). The expression of the meprin
subunits in mice is
strain-dependent (1). Random-bred mice (such as ICR) and
many inbred strains of mice (e.g. C57BL/6) express both
meprin
and
in the adult kidney, and these strains possess heterooligomeric forms of meprin A. Some inbred mouse strains (such as
C3H/He) only express meprin
in the adult kidney and therefore have
only meprin B. Furthermore, meprin
subunits in mouse kidney exist
primarily in the proenzyme form, thus these subunits are latent (14).
This is in contrast to the rat kidney enzyme, as well as meprins from
mouse, rat, and human intestine where the propeptide is removed and the
enzymes are fully active (1, 15). Therefore, meprin A and B isolated
from adult mouse kidney are novel in that they can be used to determine
the activities of the
and
subunits, respectively.
and
substrate and peptide bond specificity.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits and inactive
subunits. Trypsin-treated meprin B has
only
subunit activity.
1. Peptides were monitored at
A220. Percent hydrolysis was then calculated by
monitoring the decrease in the substrate peak area, compared with time zero.
1) was plotted
against the average substrate concentration for the reaction
([S]avg (µM)) and fitted directly to the
Michaelis-Menten equation by nonlinear regression analysis.
[S]avg was calculated using the equation,
[S]avg = ([S]0 + [S]f)/2,
where [S]0 and [S]f equal initial and final
substrate concentrations, respectively, as described previously (19).
The Km values of meprin A hydrolysis of neuropeptide
Y and meprin B hydrolysis of neuropeptide Y, secretin, peptide YY, and
kinetensin were too high for accurate measurement. Therefore, the
kcat and Km constants were not individually determined. Instead the specificity constant kcat/Km was determined using
the method of Fersht (20) where
kcat/Km = V0/([E]·[S]), a simplification of the Michaelis-Menten equation that applies when Km
[S].
1. Appropriate peptide
peaks were extensively dried and then dissolved in a matrix solution of
-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.3% TFA.
This was then spotted on a PerSeptive Biosystems stainless steel
plate. Products and substrates were identified using a Perceptive
Biosystems Linear Voyager matrix-assisted laser desorption ionization
time of flight (MALDI-TOF) machine with continuous extraction. The
accelerating voltage was set at 18,750, and the laser intensity was
between 175 and 300 in positive mode except for sCCK8NH2
detection, which was in the negative mode. Between 60 and 240 scans
were averaged. The machine was calibrated using the PerSeptive
Biosystems Sequazyme Peptide Mass Standards kit. Data were evaluated
using Grams/386 software version 3.04 (Galactic Industrial Corp.).
Where required, amino-terminal sequence analysis was performed to
identify peptides. Neuropeptide Y and peptide YY and their products
were not separated by HPLC due to poor resolution. Instead, total
peptide was bound to a ZipTip (Millipore) in 1% TFA, washed
extensively to remove nonpeptide impurities, and eluted in 70%
acetonitrile. Total peptide was then dissolved in
-cyano-4-hydroxycinnamic acid solution and treated as above.
-mercaptoethanol, and proteins were resolved on a 4-15%
PAGE gradient gel. Protein was visualized with Coomassie Brilliant Blue.
1) was incubated with meprin A or
B (10 nM) at 37 °C in 20 mM Tris-HCl, 150 mM NaCl, pH 7.5, in a final volume of 40 µl. A 6-µl
sample was removed at various times and immediately mixed with an equal
volume of 20 mM EDTA to terminate hydrolysis. The sample
was boiled in SDS-PAGE sample buffer containing
-mercaptoethanol and
subjected to electrophoresis on a 4-15% gradient gel. Osteopontin was
detected by Western blot analysis using goat anti-mouse osteopontin as
a primary antibody (1:1000), rabbit anti-goat IgG peroxidase conjugate
as a secondary antibody (1:5000), with SuperSignal West Dura Extended
Duration Substrate used as a horseradish peroxidase substrate for
detection purposes.
and
Protease
Domains--
The protease domain structures of the mouse
and
subunits were determined by knowledge-based homology modeling using the program Modeller (21). The input consisted of a sequence alignment of
the catalytic domains of meprin and astacin, and the coordinates obtained from the Rutgers Protein Data Bank for the crystal structure of astacin (accession number 1AST (22)). The catalytic zinc was modeled
by coordinating it to the equivalent residues that were determined for
astacin. The model quality was assessed using the program PROCHECK (23)
and was found to be comparable in stereochemical quality to a low
resolution crystal, typical of homology models. Surface representations
were prepared using WebLab Viewer 3.7 (Molecular Simulations Inc., San
Diego, CA).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Preferred amino acids in substrate subsites
P1' to P4' for meprin B. An acetylated dodecamer peptide library
mixture (1 mM) containing roughly equimolar amounts of all
amino acids at each position, except cysteine, was incubated with
meprin B (10 nM) until the library was between 5 and 10%
digested. The incubation was in 25 mM HEPES, 100 mM NaCl, 5 mM CaCl2, pH 7.4, at
37 °C. Amino-terminal sequencing of the resulting products allowed
for the elucidation of amino acid preference for meprin B at each
primed site. The data in each sequencing cycle was normalized to the
total molar amount of amino acids in that cycle so that a value of 1 indicates the average value.
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Fig. 2.
Preferred amino acids in substrate subsites
P1' to P4' for meprin A. The same acetylated dodecamer peptide
library mixture (1 mM) described in Fig. 1 was incubated
with meprin A (33 nM) until the library was between 5 and
10% digested. Amino-terminal sequencing of the resulting products
allowed for the characterization of amino acid preference for meprin A
at each primed subsite.
-melanocyte
stimulating hormone (
-MSH), substance P, valosin, parathyroid
hormone fragment 13-34, vasoactive intestinal peptide, and angiotensin
I were only susceptible to meprin A. The third group of peptides was
susceptible to meprin B only; orcokinin and gastrin 17 were
particularly well hydrolyzed together with peptide YY and kinetensin.
The final group of peptides was resistant to both enzymes; these
peptides were [Lys8]-vasopressin, somatostatin, kassinin,
oxytocin, and
-neurokinin. The screen identified 11 peptides not
previously known to be cleaved by meprins. Novel meprin A activity was
seen against GRP-(14-27), sCCK8NH2, secretin,
glucagon, cerulein, bombesin, and vasoactive intestinal peptide. In
addition, novel substrates of meprin B identified were GRP-(14-27),
sCCK8NH2, secretin, glucagon, neuropeptide Y, cerulein,
orcokinin, peptide YY, and kinetensin.
Percent hydrolysis of bioactive peptides by meprin A and B
1 for GRP-(14-27). The kcat
values of meprin B lay between 12.4 for gastrin and 26.8 s
1 for glucagon. Of all the peptides tested in this
study, meprin B hydrolysis of gastrin 17 was found to give the highest
specificity constant (17.5 × 105
M
1 s
1). This was predominantly
due to the low Km value of 7.1 µM.
This value is over 6-fold lower than any other Km determined for either enzyme. The fluorogenic bradykinin analog substrate, 2-aminobenzoyl-RPPGFSPFRK-(dinitrophenyl)-G is commonly used
to study meprin A activity (27). However, bradykinin is a relatively
poor substrate due to a high Km (425 µM (26)). This high Km leads to the
low specificity constant of 5.1 × 104
M
1 s
1. Rates of hydrolysis for
neuropeptide Y by meprin A and neuropeptide Y, secretin, peptide YY,
and kinetensin by meprin B were linear with respect to substrate
concentration at high peptide concentrations, therefore, the individual
kcat and Km values were not directly determined. Instead the specificity constant
kcat/Km was determined
directly. As expected, these values were the lowest seen for all
substrates, the lowest value being 0.133 × 105
M
1 s
1 for meprin B hydrolysis
of secretin.
Kinetic constants for meprin A and B against bioactive peptides
Cleavage sites in peptides by meprin B
).
In instances of multiple cleavage sites double arrows (
)
represent the major site(s) of cleavage. The subscript "NH2"
represents amidation at the carboxy terminus; the asterisk represents
sulfation at the tyrosine; p represents pyro.
Cleavage sites in peptides by meprin A
).
In instances of multiple cleavage sites double arrows (
)
represent the major site(s) of cleavage. The subscript "NH2"
represents amidation at the carboxy terminus; the asterisk represents
sulfation at the tyrosine; p represents pyro; Ac- represents
N-acetylated at the amino terminus.
Percent hydrolysis of cholecystokinin derivatives by meprin B
View larger version (47K):
[in a new window]
Fig. 3.
Degradation of proteins by meprins.
Upper panel, the extracellular matrix proteins gelatin and
fibronectin (20 µg) were incubated with meprin A or B (20 nM) or no enzyme ( ) in a final volume of 40 µl. The
reactions were conducted in 20 mM Tris-HCl, 150 mM NaCl, pH 7.5, at 25 °C for 18 h. The reaction
was terminated by addition of EDTA (10 mM), and samples
subjected to electrophoresis on a 4-15% reducing denaturing gradient
SDS-PAGE gel. Proteins were visualized with Coomassie Brilliant Blue.
For control lanes (
) substrates were incubated without meprins:
lanes A, meprin A-treated; lanes B, meprin
B-treated. Lower panel, mouse osteopontin (50 µg
ml
1) was incubated with meprin A or B at 37 °C in 20 mM Tris-HCl, 150 mM NaCl, pH 7.5 for various
times as indicated. Meprins were at 10 nM. Remaining
osteopontin was detected by Western blot analysis after SDS-PAGE on a
4-15% gradient gel. Representative data from at least three
independent experiments are shown.
View larger version (13K):
[in a new window]
Fig. 4.
Inhibition of meprins by hydroxamate
inhibitors. Meprin A or B (2 nM) was preincubated with
inhibitor before addition of sCCK8NH2 (50 µM)
as substrate. Reactions were performed in 20 mM Tris-HCl,
150 mM NaCl, pH 7.5. Substrate hydrolysis was determined by
quantitative HPLC analyses. The concentrations of hydroxamate
inhibitors ranged from 12.5 nM to 50 µM for
actinonin and from 250 nM to 500 µM for
PLG-(NHOH). The level of inhibition was determined by comparing the
decrease in substrate concentration in the presence and absence of
inhibitor. IC50 values of inhibition toward meprins as well
as structures of actinonin and PLG-NHOH are shown.
Ki values for astacin are shown for comparison (29,
30).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
protease domains yield insights into the substrate specificity differences and activities of meprins compared with other astacin family members, such as crayfish astacin and mouse bone morphogenic protein-1 (BMP-1; Fig. 5 and Ref. 1).
There are nine basic residues in the meprin
sequence that are not
basic in the equivalent positions in the meprin
protease domain.
Three of the basic residues are within the active site cleft of mouse
meprin
(Arg-147, Lys-185, and Lys-214; Fig. 5) and have the
potential to form a salt bridge with acidic residues of substrates. For
example, Phe-161 in meprin
, six amino acids carboxyl-terminal to
the zinc HEXXH binding site, is equivalent to the Arg-147
position in meprin
. Astacin and meprin
have aromatic residues
at this position, whereas meprin
and BMP-1 have basic residues.
Meprin
and BMP-1 have a preference for acidic residues in P1' of
substrates (Fig. 1, Table III, and Ref. 31). BMP-1 hydrolyzes several
proteins with a P1' aspartic acid. Thus, it appears that meprin
and
BMP-1 are similar proteins with respect to activity. Residues Tyr-199 and Lys-185 of mouse meprin
and
, respectively, form the floor of the proposed S1' subsites (Fig. 5). Thus, this is a potential site
of interaction between meprin
and acidic side chains of substrates
such as gastrin. The CCK free acid, CCK8, is a better substrate for
meprin B than CCK8NH2, which is consistent with the
preference of meprin
for an acidic moiety in or around the P2'
position of substrates.
View larger version (110K):
[in a new window]
Fig. 5.
Active site clefts of the protease domains of
mouse meprin and
. Homology models of the protease
domains of mouse meprin
and
were produced using Modeller as
described under "Experimental Procedures." Upper panels,
left and right, surface representations of the
front of the protease domains of mouse meprin
and
protease
domains, respectively. The unprimed (S) and primed
(S') binding regions are shown. Lower panels,
left and right, enlarged views of the mouse
meprin
and
active site clefts, respectively. This view is from
the perspective of the primed subsite region through the active site
clefts toward the unprimed subsite region of the domains.
Yellow indicates the catalytic center. Blue and
red indicates basic and acidic residues respectively, and
all other residues are colored gray. Key residues are shown
and are numbered according to the full-length sequence starting from
the initiator methionine.
The Tyr-199 residue of mouse meprin protrudes into the active site
cleft. This may explain why prolines are preferred at proximal sites to
the scissile bonds in substrates. Prolines impart rigidity and
stability to peptide backbones and have the ability to form cis peptide
bonds within the backbone of substrates. This may allow the peptide to
kink around the protruding tyrosine, thus allowing the peptide to fit
in the cleft. Consequently, the cis conformation of a peptide bond may
be preferred by meprins.
The inhibition of meprins by PLG-(NHOH) was expected, because both
enzymes select for prolines in the unprimed substrate sites (e.g. bradykinin and gastrin 17 for meprin A and B,
respectively). The crystal structure of astacin complexed with
PLG-(NHOH) shows that the inhibitor binds along a -edge strand,
which consists of Trp-114 through Tyr-116 (accession number 1QJJ (29)).
The tyrosine is not conserved in the meprins; Met-143 and Ser-128 are
in the equivalent positions in meprin
and
, respectively. Therefore the pocket in astacin is likely smaller than in both meprins.
Actinonin is bulkier than PLG-(NHOH) in the region that is predicted to
bind along the
-edge strand. Actinonin is a much better inhibitor of
meprins than it is of astacin, perhaps due to steric hindrance with
astacin that would not occur with the meprins. The improved inhibition
of meprins by actinonin over PLG-(NHOH) could reflect the presence of
an additional methylene group within the carboxyl-terminal residue of
the peptide backbone in actinonin. This may provide more optimal
spacing between the metal chelating hydroxamate moiety and the peptide
backbone and side chains of the inhibitor.
Meprins are the only known endopeptidases in brush border membranes that degrade proteins; other proteases in these specialized cell surface membranes either degrade only small polypeptides (e.g. neprilysin) or are exopeptidases such as angiotensin converting enzyme or leucine aminopeptidase (32). Thus, meprins may initiate degradation of proteins in the lumen of the kidney proximal tubule, act as sheddases in this location, or activate/inactivate polypeptide hormones in the glomerular filtrate. In the intestine, meprins are most active in the ileum where there is an active immune system (33). The location at this site and proteolytic capacity of meprins implicates them in the hydrolysis of proteins and formation of peptides that are presented to antigen-producing cells. The presence of meprins in leukocytes and cancer cells also implies functions for meprins in cytokine activation or degradation and in hydrolysis of basement membrane components.
Peptides of the gastrointestinal tract appear to be among the best substrates identified for meprins and are of particular interest because of the expression of meprins in the intestine. Gastrin 17, cerulein, and sCCK8NH2 have an identical stretch of five amino acids at their carboxyl termini, the four terminal amino acids confer the total biological activity of these peptides (34). Therefore meprins have the ability to inactivate these important regulatory molecules. The gastrointestinal peptides regulate the movement, secretory activity, and growth of the intestinal tract and pancreas, and thus the concentration of these peptides must be highly regulated.
The kidney is known to play a major role in the clearance of many
plasma polypeptides such as glucagon and sCCK8NH2
(e.g. Ref. 35). Patients with chronic renal failure also
have elevated levels of peptides in the blood that are involved in gut
motility, hunger, and satiety. Neurotensin, peptide YY, substance P,
vasoactive intestinal peptide, GRP-(14-27), and gastrin are all
elevated during chronic renal failure probably due to a decrease in
metabolism of circulating peptide (36, 37). This points toward a role of renal brush border proteases, including meprins, in the catabolism of circulating peptides and thus the recapture of amino acids and/or
the alteration of urinary peptides. The finding that osteopontin is a
substrate for meprin B is intriguing. Experimental hydronephrosis results in an accumulation of osteopontin protein within the lumen of
the proximal tubule at a time that meprin protein is markedly decreased
(38, 39). The absence of meprins may account for some of the
accumulation of peptides and proteins that contribute to a cascade of
events that lead to fibrosis and end-stage renal disease.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Bruce Stanley for assistance with the MALDI-TOF analyses, Anne Stanley for amino-terminal sequence analyses of individual peptides, Elizabeth Piro for peptide sequencing of the peptide libraries, and Michael Berne for the synthesis of the peptide library.
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FOOTNOTES |
---|
* This work was supported by the American Heart Association Predoctoral Fellowship 9910075U (to G. P. B.), by Department of Defense Grant DAMD17-98-1-8143 (to G. L. M.), and by National Institutes of Health Grants DK19691 and DK54625 (to J. S. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, H171, The Pennsylvania State University College of Medicine, Hershey, PA 17033-0850. Tel.: 717-531-8586; Fax: 717-531-7072; E-mail: jbond@psu.edu.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M011414200
2 The nomenclature for the interaction of proteases with their substrates is from Schecter and Berger (24). The substrate amino acid residues are called P (for peptide), the subsites on the protease that interact with the substrate are called S (for subsite). The residues on the amino-terminal side (also known as the unprimed residues) of the scissile bond (bond that is cleaved during hydrolysis) are numbered P1 through P6 counting outward. The residues on the carboxyl-terminal side (also known as primed residues) of the scissile bond are numbered P1' through P6'. Thus, hydrolysis occurs between the P1 and P1' residues. The subsites on the protease are termed S1 through S6 and S1' through S6' to complement the substrate residues that interact with the enzyme.
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ABBREVIATIONS |
---|
The abbreviations used are:
CCK, nonsulfated cholecystokinin;
GRP-(14-27), gastrin releasing peptide
fragment 14-27;
sCCK, sulfated-cholecystokinin;
TFA, trifluoroacetic
acid;
HPLC, high performance liquid chromatography;
MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight;
BMP-1, bone
morphogenic protein-1;
-MSH,
-melanocyte stimulating hormone;
LHRH, luteinizing hormone releasing hormone;
PAGE, polyacrylamide gel
electrophoresis;
PLG-(NHOH), Pro-Leu-Gly-hydroxamate.
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