Chymases are a group of mast cell serine proteinases that have
distinct substrate specificities (1, 2, 3) and are involved in diverse
functions, including inflammation, submucosal gland secretion, and
peptide hormone
processing(1, 2, 3, 4, 5, 6, 7, 8) .
Chymases are synthesized as inactive precursors but are stored in
secretory granules as active enzymes (9) with other
proteinases(10, 11) . The secretory granule of the
connective tissue mast cell also contains the highly anionic heparin
that is known to interact with the cationic chymase(12) . This
interaction is thought to prevent chymase autolysis and degradation by
chymase of other secretory granule proteins. Chymase activation in mast
cell secretory granules occurs by the removal of an acidic 2-residue
propeptide(13) . Studies with an inhibitor of DPPI (
)directly implicate a role for this proteinase in the in situ activation of prochymase in mast cells(14) .
By analogy with other serine proteinases(15) , the final step
in h-prochymase activation is likely to result from a conformational
change in the enzyme that occurs with the formation of an ion pair
between the NH
-terminal
-amino group of Ile
and the carboxylate side chain of Asp
(13) . It is critical that DPPI removes only the
2-residue propeptide, since further cleavage of the h-chymase NH
terminus would lead to enzyme inactivation. Because DPPI is a
nonspecific aminopeptidase (16) and is expected to sequentially
degrade the NH
terminus of h-chymase, we considered the
following questions. Does DPPI stably activate h-prochymase? If DPPI is
not specific for Glu-Xaa bonds, why is Glu
invariant in all
chymases? And, does heparin-binding to h-prochymase influence
DPPI-mediated activation of h-prochymase? Here we describe the effect
of heparin on h-chymase and present a mechanism for the
heparin-dependent activation of its zymogen by DPPI that address these
questions.
EXPERIMENTAL PROCEDURES
Materials
Spodoptera frugiperda (Sf9)
cells were obtained from the American Type Culture Collection (ATCC
DRL1711). pVL1393 and pBlueBacII were purchased from Invitrogen (San
Diego, CA). Baculovirus transfection module, including cationic
liposome, and linear Autographa californica nuclear
polyhedrosis virus (AcMNPV) was purchased from Invitrogen. Bovine DPPI
was purchased from Boehringer Mannheim. Porcine heparin sodium salt was
purchased from Sigma. All other chemicals and reagents were obtained
from Sigma.
Construction of a New Transfer Vector
(pBlueBacM)
The multiple cloning site of pBlueBacII was replaced
with one of pVL1393 to increase the multiple cloning sites. pVL1393 was
treated with SnaBI and BamHI to make a cassette
including multiple cloning site, which was ligated into pBlueBacII
using the same restriction enzyme sites. The new transfer vector,
containing the multiple cloning site of pVL1393 and the
-galactosidase gene, will subsequently be referred to as
pBlueBacM.
Site-directed Mutagenesis
Human chymase cDNA in
pBSIIKS was used as a template. 5`-End of the cDNA next to the
translation initiation codon has GAATTCCACC, which contains the
consensus Kozak sequence, CCACC(17) , and the EcoRI
site(13, 18) . We made two mutants of Glu
to Ala
(E2A) and Glu
to Lys
(E2K) using site-directed mutagenesis of unique site elimination,
as earlier described(19) . Briefly, three mutagenic primers
were synthesized. CAGGAAAGAAGATCTGAGCAAAAG was used to change the
unique site AflIII to BglII in pBSIIKS.
TGTGCCCCCGATGATGGCCCCAGCTTCAGCTCTGGA and
TGTGCCCCCGATGATCTTCCCAGCTTCAGCTCTGGA were mutagenic primers for the E2A
and E2K mutants, respectively. A 20-µl solution containing
phosphorylated primers of unique site and mutation (25 pmol), wild
chymase in pBSIIKS (0.025 pmol), Tris/HCl (20 mM, pH 7.5),
MgCl
(10 mM), and NaCl (50 mM) was heated
at 100 °C for 3 min. Primers were annealed quickly in an ice bath.
Primer-directed DNA synthesis was performed by adding 3 µl of
solution containing Tris/HCl (100 mM, pH 7.5), dNTPs (5 mM each), ATP (10 mM), and dithiothreitol (20 mM),
5 µl of dH
O, 1 µl of T4 DNA polymerase (3 units, U.
S. Biochemical Corp.) and 1 µl of T4 DNA ligase (4 units, U. S.
Biochemical Corp.). The reaction mixture was incubated at 37 °C for
90 min, then transformed into Escherichia coli strain
BMH71-81 mut S. After overnight incubation at 37 °C, plasmid
DNA was purified, and 0.5 µg was digested with AflIII (10
units, U. S. Biochemical Corp.) in 20-µl volume. The digested DNA
was transformed into E. coli (DH5
) and plated on LB
plates containing ampicillin. Each clone was screened by the treatment
with BglII. The clone with BglII was then subjected
to nucleotide sequencing(20) .
Construction of Recombinant Baculovirus
Clones
Wild-type, E2A, and E2K h-chymase cDNA in pBSIIKS were
treated with EcoRI and NotI and subcloned into
pBlueBacM. Using the approach of Summers and Smith(21) ,
recombinant baculoviruses of wild-type h-chymase and its E2A and E2K
mutants were produced by homologous recombination following
cotransfection of 2
10
Sf9 cells with 5 µg of
plasmid and 1 µg of linear virus (wild-type ACMNPV) DNAs using
cationic liposome and then purified by repeated plaque assay.
Sf9 Growth and Infection
Sf9 cells were maintained
at 27 °C in serum-free media (Excel 401; JRH Bioscience). Viral
stocks were produced by infection at low multiplicity and were stored
at 4 °C. Viral titers were usually greater than 10
plaque-forming units/ml. For protein production, cells were grown
in a spinner flask (500 ml or 1 liter) in SF900IISFM (Life
Technologies, Inc.) or Excel 401 media. Cells were infected at 1
10
cells/ml with a multiplicity of infection of 10
for each virus, using the procedures described by Summers and
Smith(21) .
Purification of rh-prochymase, Its Mutants, and
rh-chymase
rh-prochymase and its mutants were purified using a
heparin affinity HPLC column, as described by Urata et al.(13) . To produce rh-chymase, rh-prochymase in Sf9 culture
media was incubated with DPPI at 27 °C for 24 h. rh-chymase was
separated from unreacted rh-prochymase and purified to homogeneity
using a heparin affinity HPLC column as described by us
previously(13) . Amino-terminal sequencing of pure
rh-prochymase, E2A rh-prochymase, E2K rh-prochymase, and rh-chymase
indicated that the NH
terminus primary structure of these
proteins was GEIIGGTEXKPHSRP, GAIIGGTEXKPHSRP,
GKIIGGTEXKP, and IIGGTEXKPHSRP, respectively. Protein
sequencing was performed on an Applied Biosystems model 470A gas phase
Sequencer (Foster City, CA) by Dr. K. S. Misono, Cleveland Clinic
Foundation.In Tris/HCl buffer, pH 8.0, containing 0.3 M KCl, the kinetic constants for the hydrolysis of Ang I by
rh-chymase and chymase isolated from the human heart are similar (Table 1). Concentrations of KCl much lower than 0.3 M cause the highly cationic chymase to aggregate and
precipitate(2) .
Enzyme Kinetics
K
and k
values for the conversion of Ang I to Ang II
by rh-chymase, rh-prochymase, and its mutants were determined, as
described previously by Kinoshita et al.(3) .
h-prochymase Activation by DPPI
Purified
rh-prochymase and its E2A and E2K mutants (100 fmol each) were
incubated with 0.1 unit of bovine DPPI in 20 mM phosphate
buffer, pH 6.0, containing KCl (0.3-1.7 M) in the
presence or absence of 10 units of heparin at 37 °C in total volume
of 20 µl. Reactions were terminated by the addition of 3.0 mMN-ethylmaleimide. To circumvent problems associated with
the effects of heparin on rh-chymase activity assay, the final KCl
concentration was adjusted to 1.7 M, and 10 units of heparin
was added to samples where rh-prochymase activation was performed
without heparin. To assay for rh-chymase activity in the sample
following the activation with DPPI, 0.75 mM Ang I in 40 µl
of 500 mM Tris/HCl buffer, pH 8.0, containing 0.015% Triton
X-100, was added to the incubation mixture and incubated at 37 °C
for 20 min. The reaction was terminated by the addition of ice-cold
ethanol. Conversion of Ang I to Ang II was quantitated by reverse phase
HPLC, as described previously by us(3) .
h-prochymase Activation by Synthetic Peptides Mimicking
the NH
Terminus of h-prochymase and h-chymase
The
following peptides were synthesized by Dr. K. S. Misono, Cleveland
Clinic Foundation: h-prochymase-(1-16), GEIIGGTECKPHSRPY;
h-chymase-(3-16), IIGGTECKPHSRPY; h-chymase-(3-10),
IIGGTECK; and h-chymase-(3-4), II. These peptides were purified
on a C
reverse-phase HPLC column (Vydac), as described
previously by Kinoshita et al.(3) . Peptides were
purified to a peptide purity exceeding 99%. rh-prochymase was
preincubated with synthetic peptides mimicking the NH
terminus of h-prochymase or h-chymase for 5 min at 37 °C in
30 mM Tris/HCl buffer, pH 8.0, containing KCl (0.3 to 1.7 M), and 0.01% Triton X-100 in total volume 50 µl. To assay
for chymase activity, 2.5 µl of 10 mM Ang I was then added
to this incubation mixture, which was then further incubated at 37
°C for 20 min. The reaction was terminated by the addition of
ice-cold ethanol. Conversion of Ang I to Ang II was quantitated by
reverse-phase HPLC, as described previously by us(3) .
DPPI Assay Using Gly-Phe-
-naphthylamide as
Substrate
DPPI activity was determined, as described by McGuire et al.(16) , using Gly-Phe-
-naphthylamide (100
µM) as substrate in 20 mM phosphate buffer, pH
6.0.
Digestion by DPPI of Peptides Mimicking the NH
Terminus of h-prochymase
10 nmol of
h-prochymase-(1-16) was incubated with 0.05 unit of bovine DPPI
in 20 mM sodium phosphate buffer, pH 6.0, containing 0.3 M KCl at 37 °C for 30 min. The reaction was terminated by the
addition of ice-cold ethanol. The samples were evaporated to dryness,
dissolved in 125 µl of distilled water, and the reactant and
product peptides separated by reverse-phase HPLC on a C
column (Vydac) pre-equilibrated in 0.1% trifluoroacetic acid
containing 7% acetonitrile. The column was developed with a 7-min
linear acetonitrile gradient (7-40%) at a flow rate of 2 ml/min.
RESULTS AND DISCUSSION
Heparin Both Binds to and Inhibits
rh-chymase
Recombinant rh-chymase, in a Tris/HCl buffer, pH 8.0,
containing 0.3 M KCl, efficiently hydrolyzes the Phe-His bond
in the decapeptide Ang I to yield Ang II and His-Leu (Table 1).
Heparin (500 units/ml) produces an
15-fold reduction in Ang I
hydrolysis (i.e.k
) by rh-chymase,
without affecting K
(Table 1). Increasing
the KCl concentration to 1.7 M leads to a partial reversal of
the heparin inhibitory effect (
60% of the control incubation
lacking heparin). It is unlikely that the heparin inhibitory effect is
due to a sequestration of the substrate. Such an effect would decrease
the concentration of free Ang I in the assay and result in an increase
in apparent K
. These observations are, however,
consistent with a masking of the substrate-binding site of rh-chymase
by heparin. The partial reversal by KCl is likely due to the disruption
of the electrostatic interactions between rh-chymase and heparin.
Supporting this contention are the findings that rh-chymase binds to a
heparin affinity column in buffers containing 0.3 M KCl and
can be eluted at a KCl concentration of 1.7 M(13) .
Thus, the high concentration of heparin in the mast cell granule not
only immobilizes the enzyme, but also keeps the mature chymase in a
largely inactive state. The IC
for the inhibition of
rh-chymase by heparin is
10 units/ml (data not shown), suggesting
that upon chymase secretion, the resulting decrease in heparin
concentration may lead to a restoration of enzyme activity.
Heparin Is Required for the DPPI-dependent Activation of
rh-prochymase
DPPI is a thiol proteinase with a pH optimum of
6.0 (16) . The pH optimum of DPPI is consistent with its
proposed function as a prochymase-activating enzyme(14) , since
the pH within the mast cell secretory granule has been estimated to be
about 5.5(22) . In a sodium phosphate buffer, pH 6.0,
containing 0.3 M KCl, heparin (500 units/ml) produces a
10-fold increase in the maximal activation of rh-prochymase by DPPI (Fig. 1). The EC
for this heparin effect is
0.005 unit of heparin/ml (or
400 pM) (Fig. 1D). Proteoglycans related to heparin, such as
chondroitin sulfate and keratan sulfate that are not present in chymase
containing connective tissue mast cells, could not mimic this heparin
effect (Fig. 1D). Thus the facilitatory effect of
heparin on DPPI-dependent rh-prochymase activation is of high affinity
and specificity. Because high levels of heparin are found in mast cell
granules, in the studies described below, a heparin concentration of
500 units/ml was used.
Figure 1:
Effect of heparin on the activation by
DPPI of rh-prochymase and its E2A and E2K mutants. A,
effect of heparin on the time-dependent activation of rh-prochymase by
DPPI. To control for the effects of KCl and heparin on the chymase
activity assay, the final KCl concentration was adjusted to 1.7 M, and 10 units of heparin was added to samples where
rh-prochymase activation was performed without heparin. The horizontal dotted line indicates the activity of rh-chymase
assayed in buffer containing 1.7 M KCl and 10 units of
heparin. B and C, effect of heparin on the
time-dependent activation of E2A and E2K mutants of h-prochymase by
DPPI. D, effect of heparin sulfate, chondroitin sulfate, and
keratan sulfate on the maximal activation of rh-prochymase by DPPI.
Assuming an average heparin molecular weight of 50 kDa (heparin is sold
as a mixture of molecular masses, ranging between 10 and 100 kDa), the
EC
for the heparin effect on rh-prochymase activation is
400 pM. E, rh-prochymase (100 fmol) was incubated
with 0.1 unit of bovine DPPI in 20 mM phosphate buffer, pH
6.0, containing 1.7 M KCl in the presence (dotted lines and closed circle) or absence (solid lines and open circle) of 10 units of heparin at 37 °C in total
volume of 20 µl. Filled triangles show the results from
incubations where 10 units of heparin were added after initial 2-h
incubations without heparin. One unit of chymase activity is defined
here as 1 pmol of Ang II generated/s. All values are the mean ±
S.E., n = 3.
The 2-residue rh-prochymase propeptide,
Gly-Glu, is acidic. Thus, we initially anticipated that the propeptide
may interact with one of the several basic residues present on the
surface of rh-prochymase and that this interaction could prevent an
appropriate presentation of the zymogen NH
terminus for
DPPI, an effect that could be prevented by the highly anionic heparin.
If this event is simply due to electrostatic shielding, the effect of
heparin should be mimicked by high salt. However, increasing the KCl
concentration from 0.3 to 1.7 M did not improve the activation
of rh-prochymase by DPPI (Fig. 1A). It is unlikely that
the effect of heparin on the DPPI-dependent activation of rh-prochymase
is due to an activation of DPPI, because DPPI does not bind to heparin
affinity supports even in the presence of a low KCl concentration (0.1 M), and heparin does not facilitate the hydrolysis by DPPI of
the small synthetic DPPI substrate Gly-Phe-
-naphthylamide (data
not shown). Collectively, these findings suggest that a high affinity
binding of heparin to the zymogen exposes the propeptide so that it can
be cleaved by DPPI. Detachment of the acidic propeptide from a basic
site on the enzyme surface by direct competition with the acidic
heparin is not a plausible explanation of the heparin effect, however,
because high salt cannot mimic the effect of heparin on the zymogen
activation process. We suggest that the binding of heparin to the
zymogen (through a heparin-binding site) produces a local allosteric
effect which weakens the interaction that immobilizes the propeptide
(through a heparin-sensitive propeptide attachment site) on the zymogen
surface.
Replacing Glu
with Ala or Lys Prevents
Heparin-dependent Activation of rh-prochymase
The propeptide
sequences in mammalian prochymases are conserved in two respects: the
propeptide is 2 residues in length, and Glu
is invariant.
Nevertheless, studies with dipeptide-
-naphthylamide substrates
suggest that DPPI has no particular preference for cleavage at Glu-Xaa
bonds(16) . To examine if Glu
plays a role in the
appropriate presentation of the rh-prochymase NH
terminus
for cleavage by DPPI, we studied the effect of heparin on
DPPI-dependent activation of E2A and E2K mutants of rh-prochymase. In
these mutants, where Glu
was replaced with a hydrophobic
Ala or a basic Lys, two effects were observed. First, the E2A and E2K
mutants were not completely inactive like the wild-type proenzyme (Table 1), suggesting that Glu
plays an important
role in keeping the zymogen inactive. Second, although the basal (i.e. heparin-independent) activation by DPPI of the E2A and
the E2K mutants was similar to the level of activation seen with the
wild-type rh-prochymase, the facilitatory effects of heparin on this
activation process was abolished in these mutants (Fig. 1, B and C). This latter finding may be interpreted as
follows. Glu
may help direct the rh-prochymase NH
terminus to one of two states: one which is readily susceptible
to activation by DPPI and one which is resistant to such activation.
The relative ratio of DPPI activation-sensitive state:DPPI
activation-resistant state is approximately 1:15 (specific activity of
the zymogen after a 3-h incubation with DPPI:specific activity of
rh-chymase; Fig. 1). Importantly, heparin can change the
rh-prochymase NH
terminus from a DPPI activation-resistant
state to a DPPI activation-sensitive state. It is unlikely that there
is a rapid equilibrium of the zymogen NH
terminus between
the DPPI activation-resistant and DPPI activation-sensitive states,
because activation by DPPI is not progressive after
10% of the
rh-prochymase is activated (Fig. 1E). Furthermore, in
the DPPI activation-resistant state the NH
terminus is not
subject to sequential degradation by DPPI (which would lead to
permanent inactivation), since the addition of heparin at the end of a
2-h incubation of rh-prochymase with DPPI leads to full zymogen
activation (Fig. 1E).In the E2A and the E2K
rh-prochymase mutants the NH
terminus again adopts two
states, DPPI activation-sensitive and DPPI activation-resistant, their
ratio being about 1:8 (specific activity of the mutant zymogen after a
3-h incubation with DPPI:specific activity of rh-chymase; Fig. 1). However, in these rh-prochymase mutants, the DPPI
activation-resistant state cannot be converted to a DPPI
activation-sensitive state by heparin. These observations suggest that
Glu
plays an important role in allowing the zymogen to
adopt a state that is initially resistant to the actions of DPPI, but
is susceptible to a DPPI-mediated activation in the presence of
heparin, an electrostatic interaction between Glu
and a
buried (i.e. high salt-resistant) basic residue may help
direct the zymogen NH
terminus to such a heparin-sensitive
site. When h-prochymase is copackaged with heparin in the Golgi
apparatus and/or secretory granules of the mast cell, an interaction
with the heparin proteoglycan would allow the NH
terminus
to adopt a DPPI activation-sensitive state. We speculate that in the
highly anionic matrix of the mast cell secretory granule, the anionic
property of Glu
could also prevent an association of
NH
terminus with the matrix. The
``free-floating'' NH
terminus of an otherwise
immobilized h-prochymase would then be easily susceptible to cleavage
by DPPI, which does not bind to heparin. These proposed events are
summarized in Fig. 2, A-C.
Figure 2:
A model of h-prochymase activation. A, following its synthesis in the rough endoplasmic reticulum
of the mast cell, the NH
terminus of h-prochymase is likely
to be firmly attached to a region of the proenzyme which is a
heparin-sensitive site. This attachment renders the majority of the
h-prochymase NH
termini resistant to proteolytic cleavage
by DPPI and thus may prevent premature activation of the enzyme. Based
on molecular modeling studies, Sali et al.(35) have
reported that the substrate-binding site of mouse chymase 5 is located
in between two positively charged domains. Molecular modeling studies
on h-chymase, also, indicate the presence of two clusters of positively
charged residues on the surface of this enzyme,
which may
be the putative heparin-binding sites. Because a disruption of
electrostatic interactions by heparin, but not by high salt, can
facilitate the presentation of the zymogen NH
terminus for
cleavage by DPPI, we make a distinction here between the
heparin-sensitive site where the zymogen NH
terminus is
attached and the heparin-binding site. We propose that the binding of
heparin to its binding sites induces a conformational change in the
zymogen structure which disrupts the electrostatic interaction that
keeps the zymogen NH
terminus attached to the enzyme
surface. B, when h-prochymase is copackaged with heparin the
NH
terminus likely adopts a free-floating position, whereas
the rest of the proenzyme is attached to the heparin matrix by
electrostatic interactions. C, in the free-floating position,
the h-prochymase NH
terminus is efficiently cleaved by
DPPI. The newly formed NH
terminus of h-chymase is rapidly
captured by the activation groove. D, the capture of the
h-chymase NH
terminus both prevents further degradation of
the NH
terminus by DPPI and causes enzyme
activation.
Following Propeptide Cleavage, NH
Terminus
Capture by the Activation Groove Activates Chymase
In several
proenzymes, for example, renin(23) , the propeptide blocks the
substrate binding site and keeps the enzyme in an inactive state. In
the trypsin family of serine proteinases, a buried ion pair between the
-amino group of the NH
-terminal Ile and the
carboxylate side chain of Asp that has a position penultimate to the
active site serine is necessary for enzyme activation(15) . An
analogous buried ion pair (i.e. between Ile
and
Asp
) has also been identified in the crystal structure of
rat chymase 2(24) . In the activation of rh-prochymase by DPPI,
high concentrations of KCl (1.7 M) do not inhibit the
activation process, either in the presence or absence of heparin (Fig. 1). The lack of inhibitory effect of KCl would indicate
that this ``activation'' ion pair is stabilized by forces
besides a simple electrostatic interaction. Molecular modeling of
h-chymase, based on the crystal structure of rat chymase 2, indicates
that the hydrophobic side chains of Ile
and Ile
are not solvent accessible. Also, several backbone hydrogen bonds
exist between the h-chymase NH
terminus and the rest of the
protein (for example, between Ile
HN and Ser
O, Thr
HN and Glu
O, Thr
O
and Glu
HN, and Cys
HN and Leu
O). (
)These interactions could serve to facilitate the
formation of the activation ion pair.To obtain an efficient
presentation of the propeptide for cleavage by DPPI, we considered that
in the zymogen-heparin complex, the NH
terminus would not
be closely associated with the zymogen surface. A close association
could hinder the binding of the propeptide to DPPI and hence its
cleavage. However, once the propeptide is enzymatically removed, the
new NH
terminus could then be captured by an unoccupied
activation groove and lead to the formation of the activation ion pair.
To determine if a specific unoccupied activation groove is present on
the surface of rh-prochymase, we determined if a synthetic peptide
based on the structure of the h-chymase NH
terminus could
activate rh-prochymase.
Human chymase-(3-16), a synthetic
peptide corresponding to the first 14 residues of h-chymase, was able
to activate rh-prochymase in a buffer containing 1.7 M KCl (Fig. 3). The activation of rh-prochymase by
h-chymase-(3-16) was sequence-specific, since
h-prochymase-(1-16) was completely ineffective at activating
rh-prochymase. The activation by h-chymase-(3-16) was rapid (Fig. 3B), and the effect was concentration-dependent (Fig. 3A); at a concentration of 200 µM,
the activation of rh-prochymase by h-chymase-(3-16) was
4% of
the theoretical specific activity of rh-chymase. This low degree of
zymogen activation by 200 µM h-chymase-(3-16) as
compared with the degree of activation seen after zymogen processing by
DPPI is probably due to the fact that the synthetic h-chymase
NH
-terminal peptide is not tethered to the rest of the
enzyme as is the natural NH
terminus. The effective
concentration of a ligand tethered adjacent to its binding site may be
as high as 1 M.
Figure 3:
Effect of peptides mimicking the NH
terminus h-chymase on rh-prochymase activity. A, h-chymase-(3-10) and h-chymase-(3-16), peptides
mimicking the first 8 and 14 residues of the h-chymase NH
terminus, respectively, activated rh-prochymase.
h-chymase-(3-4), a dipeptide mimicking the first 2 residues of
the h-chymase NH
terminus, and h-prochymase-(1-16), a
peptide mimicking the first 16 residues of the h-prochymase NH
terminus, were ineffective at activating rh-prochymase. B, effect of preincubation time on the activation of
rh-prochymase by 0.2 mM h-chymase-(3-16). C,
effect of KCl concentration on the activation of rh-prochymase by
h-chymase-(3-16). All values are the mean ± S.E., n = 3.
Decreasing the concentration of KCl from
1.7 to 0.3 M did not enhance the
h-chymase-(3-16)-dependent activation of rh-prochymase (Fig. 3C), suggesting that the buried activation ion-pair must
form once the newly formed NH
terminus is captured by the
activation groove on the enzyme surface. Thus, this initial capture
must not only involve electrostatic interactions, but may involve
hydrogen bonding and hydrophobic interactions. Indeed, the finding that
an increase in the KCl concentration from 0.3 to 1.7 M, which
is expected to enhance the hydrophobic effect, led to a 6-fold increase
in rh-prochymase activation by h-chymase-(3-16) supports the
latter contention (Fig. 3C). Similarly, in the presence
of 1.7 M KCl, heparin-facilitated activation of rh-prochymase
by DPPI was 60% greater than in the presence of 0.3 M KCl (Fig. 1A). In the natural activation of h-prochymase,
hydrogen bonding and side chain hydrophobic interactions may bring the
h-chymase NH
terminus close to the enzyme surface, like the
closing of a zipper, ultimately forcing the formation of the activation
ion pair. To characterize the putative activation groove, we examined
the effectiveness of synthetic peptides of varying lengths, mimicking
the NH
terminus of h-chymase, in their ability to activate
rh-prochymase. h-chymase-(3-10) and h-chymase-(3-16) were
equally effective, but h-chymase-(3-4) was ineffective in
activating rh-prochymase (Fig. 3A). These findings
indicate that the unoccupied part of the activation groove serves to
capture, at most, the first 8 residues of the rh-chymase NH
terminus.
Capture of the rh-chymase NH
Terminus by the
Activation Groove Likely Protects the Newly Formed NH
Terminus from Further Degradation by DPPI
Previous studies
have indicated that DPPI has a broad specificity with regard to amino
acid preferences in the P
and P
positions(
); however, P
Pro and P
Arg and Lys are not tolerated(16) . Since the first four
dipeptides in the h-prochymase NH
terminus sequence
represent appropriate substrate cleavage sites for DPPI, we were
intrigued as to why rh-prochymase was not inactivated during a
prolonged incubation with DPPI (Fig. 1E). To examine
the possibility that cleavage of a single dipeptide from the
rh-prochymase NH
terminus results in a local change in the
secondary structure which prevents DPPI from cleaving the next
dipeptide, DPPI was incubated with h-prochymase-(1-16), a
synthetic 16-residue peptide mimicking the NH
terminus of
h-prochymase, and h-chymase-(3-16), a synthetic 14-residue
peptide mimicking the NH
terminus of h-chymase. Analyzing
by HPLC the time-dependent degradation by DPPI of
h-prochymase-(1-16) and h-chymase-(3-16) we observed that
both these peptides are rapidly degraded by the sequential removal of
NH
terminal dipeptides (data not shown). This finding
argues against the possibility that a DPPI-resistant local change in
the secondary structure occurs at the NH
terminus of
rh-chymase once the propeptide is cleaved. We speculate that in the
secretory granule of the mast cell, the capture of the newly formed
h-chymase NH
terminus by the activation groove protects it
from further degradation by DPPI. Moreover, an important consequence of
this capture is enzyme activation. These proposed events are summarized
in Fig. 2, C and D.
Conclusions
Presentation and packaging are
important parameters in most biological processes. In mast cell
secretory granules that contain an anionic proteoglycan matrix,
activation of the matrix-immobilized cationic h-prochymase has required
specialization. The key feature of this specialization appears to be an
acidic 2-residue propeptide that allows efficient presentation of the
h-prochymase NH
terminus for cleavage by DPPI. This
specialization appears not to be unique for h-prochymase. Many highly
cationic leukocyte serine proteinases, including all
chymases(18, 25, 26, 27) , human
cathepsin G(28) , and granzymes B-F and G(29) ,
contain a 2-residue propeptide with a highly conserved Glu
.
DPPI has been implicated in the activation of several of these
proteinases (14, 30) . It is interesting that
neutrophil elastase which is packaged with cathepsin G, has a 2-residue
propeptide, containing a Glu
(31) . Pancreatic
elastases, on the other hand, have large propeptides, 10-14
residues in length, with an Arg at the activation
site(32, 33) . Our studies suggest that the occurrence
of Glu
in the 2-residue propeptide of cationic leukocyte
serine proteinases is important for NH
terminus positioning
and not because of a processing enzyme preference for a P
acidic residue.