©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Human Prochymase Activation
A NOVEL ROLE FOR HEPARIN IN ZYMOGEN PROCESSING (*)

(Received for publication, October 13, 1994; and in revised form, November 3, 1994)

Marohito Murakami Sadashiva S. Karnik Ahsan Husain (§)

From the Department of Molecular Cardiology, Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Human prochymase is packaged with heparin in mast cell granules and appears to be activated by dipeptidylpeptidase I. We show that a high affinity interaction between heparin and prochymase allows the 2-residue propeptide to be cleaved by dipeptidylpeptidase I. A conserved Glu in the propeptide is necessary for this heparin effect. Following propeptide cleavage, capture of the newly generated NH(2) terminus by an ``activation groove'' on the enzyme activates the enzyme and concurrently prevents a progressive degradation of the NH(2) terminus by dipeptidylpeptidase I. Surrogate peptide studies show that the activation groove is unoccupied in prochymase and is specific for the chymase NH(2) terminus. These observations indicate that heparin is an important cofactor in the prochymase activation process and explain how dipeptidylpeptidase I, a nonspecific processing enzyme, can effect a specific cleavage of the zymogen propeptide.


INTRODUCTION

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 (^1)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(2)-terminal alpha-amino group of Ile^3 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(2) terminus would lead to enzyme inactivation. Because DPPI is a nonspecific aminopeptidase (16) and is expected to sequentially degrade the NH(2) 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^2 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 beta-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^2 to Ala^2 (E2A) and Glu^2 to Lys^2 (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(2) (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(2)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 (DH5alpha) 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 times 10^6 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 10^8 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 times 10^6 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(2) 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(m) 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(2) 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(18) 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(2) 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-beta-naphthylamide as Substrate

DPPI activity was determined, as described by McGuire et al.(16) , using Gly-Phe-beta-naphthylamide (100 µM) as substrate in 20 mM phosphate buffer, pH 6.0.

Digestion by DPPI of Peptides Mimicking the NH(2) 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(18) 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 approx15-fold reduction in Ang I hydrolysis (i.e.k) by rh-chymase, without affecting K(m) (Table 1). Increasing the KCl concentration to 1.7 M leads to a partial reversal of the heparin inhibitory effect (approx60% 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(m). 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 approx10 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 approx0.005 unit of heparin/ml (or approx400 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 approx400 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(2) 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-beta-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^2 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^2 is invariant. Nevertheless, studies with dipeptide-beta-naphthylamide substrates suggest that DPPI has no particular preference for cleavage at Glu-Xaa bonds(16) . To examine if Glu^2 plays a role in the appropriate presentation of the rh-prochymase NH(2) 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^2 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^2 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^2 may help direct the rh-prochymase NH(2) 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(2) 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(2) terminus between the DPPI activation-resistant and DPPI activation-sensitive states, because activation by DPPI is not progressive after approx10% of the rh-prochymase is activated (Fig. 1E). Furthermore, in the DPPI activation-resistant state the NH(2) 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(2) 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^2 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^2 and a buried (i.e. high salt-resistant) basic residue may help direct the zymogen NH(2) 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(2) 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^2 could also prevent an association of NH(2) terminus with the matrix. The ``free-floating'' NH(2) 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(2) 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(2) 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,^2 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(2) terminus for cleavage by DPPI, we make a distinction here between the heparin-sensitive site where the zymogen NH(2) 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(2) terminus attached to the enzyme surface. B, when h-prochymase is copackaged with heparin the NH(2) 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(2) terminus is efficiently cleaved by DPPI. The newly formed NH(2) terminus of h-chymase is rapidly captured by the activation groove. D, the capture of the h-chymase NH(2) terminus both prevents further degradation of the NH(2) terminus by DPPI and causes enzyme activation.



Following Propeptide Cleavage, NH(2) 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 alpha-amino group of the NH(2)-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^3 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^3 and Ile^4 are not solvent accessible. Also, several backbone hydrogen bonds exist between the h-chymase NH(2) terminus and the rest of the protein (for example, between Ile^4 HN and Ser O, Thr^7 HN and Glu O, Thr^7 O and Glu HN, and Cys^9 HN and Leu O). (^2)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(2) 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(2) 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(2) 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 approx4% 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(2)-terminal peptide is not tethered to the rest of the enzyme as is the natural NH(2) 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(2) 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(2) terminus, respectively, activated rh-prochymase. h-chymase-(3-4), a dipeptide mimicking the first 2 residues of the h-chymase NH(2) terminus, and h-prochymase-(1-16), a peptide mimicking the first 16 residues of the h-prochymase NH(2) 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(2) 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(2) 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(2) 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(2) terminus.

Capture of the rh-chymase NH(2) Terminus by the Activation Groove Likely Protects the Newly Formed NH(2) 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(1) and P(2) positions(^3); however, P(1) Pro and P(2) Arg and Lys are not tolerated(16) . Since the first four dipeptides in the h-prochymase NH(2) 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(2) 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(2) terminus of h-prochymase, and h-chymase-(3-16), a synthetic 14-residue peptide mimicking the NH(2) 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(2) 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(2) 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(2) 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(2) 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^2. 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^2(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^2 in the 2-residue propeptide of cationic leukocyte serine proteinases is important for NH(2) terminus positioning and not because of a processing enzyme preference for a P(1) acidic residue.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL33713, HL44201, and RR04871 and by a grant from the Japan Health Sciences Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 Molecular Cardiology, Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195-5069. Tel.: 216-444-2057; Fax: 216-444-9410.

(^1)
The abbreviations used are: DPPI, dipeptidylpeptidase I; rh, recombinant human; Sf9 cells, Spodoptera frugiperda cells; Ang I, angiotensin I; Ang II, angiotensin II; HPLC, high performance liquid chromatography.

(^2)
S. S. Sung and A. Husain, unpublished data.

(^3)
The nomenclature used for the individual amino acids (P(1), P(1)`, etc.) of a substrate and the subsites (S(1), S(1)`, etc.) of the enzyme is that of Schechter and Berger(34) .


ACKNOWLEDGEMENTS

We thank Dr. Shen-Shu Sung for the molecular modeling studies and Dr. Kunio S. Misono for protein sequence analysis; Manuel J. Glynias, and Drs. Larry Stern, William E. Serafin, and Robert M. Graham for discussion and comments on this manuscript; and Dennis Wilk for technical assistance.


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