©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Fluorescent Peptidyl Substrates as an Aid in Studying the Substrate Specificity of Human Prohormone Convertase PC1 and Human Furin and Designing a Potent Irreversible Inhibitor (*)

(Received for publication, May 4, 1995; and in revised form, June 9, 1995)

François Jean (§) Alain Boudreault Ajoy Basak Nabil G. Seidah (1) Claude Lazure (¶)

From the Neuropeptides Structure and Metabolism Laboratory and the J. A. De Sève Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montréal (affiliated with the University of Montréal), Montréal, Québec, Canada H2W 1R7

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The substrate specificities of two human prohormone convertases, furin and PC1, were examined with a series of 7-amino-4-methylcoumarinamide (MCA) containing peptidyl substrates. Using acetyl-Arg-Ser-Lys-Arg-MCA as model, P4 Arg substitution by Lys or Orn resulted for furin in a 538- and a 280-fold lower k/K value, but only in a 14- and 18-fold decrease for PC1. Substitution of P3 Ser by either Pro, Glu, or Lys does not modify significantly the k/K value for PC1, whereas furin activity is seriously impaired by the Glu substitution. Elongating the peptidyl sequence up to the P8 position decreases the k/K value for furin but not for PC1. In both the P3 or P5 Glu substitution, the decrease of k/K was due primarily to lower k rather than higher K, possibly because of the presence of a negatively charged side chain. Finally, an octapeptidyl chloromethane derivative proved to be a potent irreversible inhibitor of either PC1 and furin. The 811-fold difference in the apparent K/[I] (1.63 10^6 sM), and k/K determined with the corresponding peptidyl MCA substrate (2.01 10^3 sM), supports the proposal that cleavage of the acylenzyme represents the rate-limiting step for PC1 and furin.


INTRODUCTION

Recently, a number of convertases were discovered which share a common role in carrying out processing of protein and prohormone precursors at distinct single or pairs of basic residues. So far, six mammalian processing enzymes are known which exhibit significant functional and structural similarities to both yeast kexin and bacterial subtilisins. These subtilisin/kexin-like convertases (reviewed in (1, 2, 3, 4) ) are called furin/PACE, (^1)PC1/PC3, PC2, PACE4, PC4 and PC5/PC6. Apart from their structural relatedness to kexin, further evidence in favor of their roles in precursor processing in either the constitutive or the regulated pathway of secretion was deduced from cellular co-expression and localization studies. Thus, for example, overexpression of PC1 or PC2 together with proopiomelanocortin in mammalian cells led to specific processing of this precursor at the COOH-terminal side of pairs of basic residues(5) . Similarly, furin when expressed in both endocrine and non-endocrine cell types can also process various precursors at sites characterized by the presence of an Arg-Xaa-Lys/Arg-Arg sequence(6, 7) . In fact, the number of potential substrates recognized by furin is expanding rapidly to include peptide growth factors such as beta-nerve growth factor (8) and viral envelope glycoproteins such as hemagglutinin of influenza virus(9) . Analysis of the poly(A) mRNAs of the convertases in rat tissues and various cell lines by Northern blot analysis demonstrated a unique pattern for each enzyme(4) . Thus, although furin, PACE4, and PC5 mRNAs exhibit a widespread tissue distribution, only furin is ubiquitously expressed. In contrast, Northern and in situ hybridization analysis of PC1 and PC2 indicate that they are primarily expressed in tissues and cells containing secretory granules. Finally, PC4 mRNA is only expressed in germ cells of the testis and ovary.

The cellular co-expression of enzyme and substrate was extremely revealing in terms of intracellular activities of the convertases and their cleavage specificities. However, the limiting level of some intracellular convertases, such as furin, suggests that overexpression systems might not be the physiologically relevant way to analyze the functioning of a given convertase(10) . Therefore, in order to better understand the physiological role as well as the molecular characteristics of these enzymes, an in vitro analysis is mandatory. For this purpose, the use of heterologous expression systems offered a way to produce these enzymes at levels above that of endogenous contaminating enzymes. Until now, only few reports are available regarding the substrate specificities of recombinant furin or recombinant PC1 using in vitro experiments. Those include substrate precursors such as hproinsulin(11) , anthrax-protective antigen(12) , and human immunodeficiency virus envelope glycoprotein gp160 (13) and/or smaller peptidyl substrates(14, 15, 16) . Interestingly, as we and others have demonstrated previously, both recombinant furin and PC1 prefer substrates with an Arg residue occupying the P4 position relative to the cleavage site characterized by the presence of a pair of basic residues such as in Arg-Xaa-Lys/Arg-Arg or of a single basic residue such as in Arg-Xaa-Xaa-Arg.

The present comparative study of both human furin and PC1 using fluorogenic substrates aims at describing more precisely the biochemical basis behind their apparent overlapping substrate specificities. The preparation as well as the comparison of the cleavage efficiency as determined by measuring k/K of several 7-amino-4-methylcoumarinamide derivatives are reported. Finally, based upon this study, a peptidyl chloromethane derivative was prepared and shown to be a very potent irreversible inhibitor for both PC1 and furin, useful for specific labeling and titration purposes.


EXPERIMENTAL PROCEDURES

Materials

All amino acid derivatives (L-form) were purchased from Calbiochem. The reagents and solvents for solid-phase peptide synthesis were from Applied Biosystems (Mississauga, Ontario, Canada), whereas other reagents and solvents for liquid-phase peptide synthesis were bought from the Armand Frappier Institute (Laval, Québec, Canada). The fluorogenic substrate pGlu-Arg-Thr-Lys-Arg-MCA was obtained from Peptide International (Louisville, KY), whereas all other MCA derivatives were prepared in our laboratory. The solid-phase peptide synthesis was carried out using an Applied Biosystems 430A automatic peptide synthesizer model using Fmoc chemistry. Amino acid analyses were performed following 18-h hydrolysis in 5.7 N HCl at 110 °C in vacuo, and the hydrolysates were analyzed using a modified Beckman 120C autoanalyser equipped with a Varian DS 604 integrator/plotter and using post-column ninhydrin detection.

The crude peptidyl derivatives were purified by RP-HPLC as described previously(17) . TLC was performed on silica gel precoated (0.2 mm thickness) aluminium sheets (Kiesel gel, Merck Co.). The following solvent systems were used for ascending TLC (v/v): A, CHCl(3)/MeOH (24:1); B, CHCl(3)/MeOH (2:1); C, CHCl(3)/MeOH (12:1); D, n-BuOH/HOAc/H(2)O/pyridine (15:3:12:10); and E, CHCl(3)/MeOH (10:7). The spots were revealed by UV illumination or by spraying with molybdate/sulfuric acid or dilute ninhydrin.

The two human endoproteases, prohormone convertase 1 (hPC1) and hfurin, were obtained from the medium of somatomammotroph GH(4)C(1) cells following infection with the respective recombinant vaccinia virus as described(15) . The two proteases were partially purified through anion exchange chromatography prior to use and their enzymatic activities determined using a fluorometric assay as described previously(15) . One unit of enzymatic activity is defined as the amount of enzyme necessary to release 1 pmol of AMC from the fluorogenic substrate acetyl-Arg-Ser-Lys-Arg-MCA per minute.

Methods

Preparation of Peptidyl 4-Methyl-7-coumarylamide

All peptidyl-MCA derivatives were synthesized according to the scheme described in Fig. 1. The method will be described in detail using the fluorogenic peptide acetyl-Arg-Ser-Lys-Arg-MCA (II), whereas only the final chemical characterization data will be reported for the other derivatives.


Figure 1: Synthetic scheme for the preparation of acetyl-Arg-Ser-Lys-Arg-MCA (II). A, synthesis of the fluorogenic amide (I). Part B: Synthesis of the fully protected tripeptide (IIa). Part C: Condensation of peptide segments and final deprotection of the peptidyl-substrate (IIb).



Preparation of Fmoc-Arg(Pmc)-MCA (I)

Fmoc-Arg(Pmc)-OH (2.4 g; 3.2 mmol) was dissolved in dimethylformamide (4 ml) and cooled to -20 °C. N-Methylmorpholine (0.39 g; 3.8 mmol) and isobutyl chloroformate (0.5 g; 3.8 mmol) were added. The mixture was stirred for 30 min at -20 °C under nitrogen atmosphere prior to addition of AMC (0.5 g; 2.9 mmol). The reaction was stirred at room temperature and compound I was recovered by preparative TLC, R(A): 0.27. The overall yield of the purified fluorogenic amide was 80-85% (based on weight); FAB-MS: m/z 820 (M + H); ^1H NMR: data are in agreement with the proposed structure.

Preparation of Acetyl-Arg-Ser-Lys-Arg-MCA (II)

The synthesis of acetyl-Arg(Pmc)-Ser(tBu)-Lys(Boc)-OH (IIa) was accomplished on an automated peptide synthesizer using the Fmoc chemistry in combination with the preloaded acid labile Rink resin (Fmoc-Lys(Boc)-Rink resin; 0.5 mmol/g resin)(18) . The following side chain protections were used: Boc for Lys, tBu for Ser, and Pmc for Arg. After the removal of the last Fmoc-protecting group, the resin was treated (2 h) with an excess of acetic anhydride (1 mmol). The acetyl-Arg(Pmc)-Ser(tBu)-Lys(Boc)-Rink resin was suspended in a solution of 1% trifluoroacetic acid, dichloromethane (v/v) for 2 min and then the resin was washed with TFE. These steps were repeated two to three times. Evaporation of the solvent gave a white solid residue. TLC revealed the presence of a single component (R(B): 0.58) identified as IIa by its ^1H NMR spectra and its FAB-MS spectra (m/z 854 (M + H)). The overall yield of the protected tripeptide was 85-90%, based on powder weight.

The acetyl-Arg(Pmc)-Ser(tBu)-Lys(Boc)-Arg(Pmc)-MCA (IIb) was obtained by condensing Ia and IIa using the activating reagent TBTU(19) . Compound I (74.1 mg; 90.5 mmol) was added to a solution of 20% piperidine, dimethylacetamide. The reaction mixture was concentrated under reduced pressure and the residue washed with dimethylacetamide several times. The free amine Ia was then added to a solution of compound IIa (75 mg; 90.4 mmol), triethylamine (91 mg; 87.8 mmol) and TBTU (29 mg; 90.4 mmol) in 3 ml of dimethylacetamide, and the resulting mixture was stirred at ambient temperature. After 30 min, TLC analysis showed a major compound; R(C): 0.42. The reaction mixture was precipitated using an ice-cold acid solution (pH 3; HCl). The resulting precipitate was filtered and dried in vacuo. Purification through preparative TLC plates using solvent system C afforded compound IIb (60-65% yield based on weight). FAB-MS: m/z 1434 (M + H). Its ^1H NMR spectrum was fully consistent with its structure.

The final deprotection of the peptidyl-MCA derivative IIb was performed by reacting with reagent K (20) for 4 h at room temperature. After appropriate dilution with water and lyophilization, the residue was repetitively washed with ether to remove the excess of scavengers. The material was finally purified by RP-HPLC and eluted at 31.7 min (32% CH(3)CN). FAB-MS: m/z 745 (M + H). Amino acid analysis: Ser(1), Lys(1), Arg(2). The ^1H NMR spectrum was fully consistent with its structure.

For acetyl-Ser-Lys-Arg-MCA (III): FAB-MS, m/z 589 (M+H); RP-HPLC, R = 29.05 min (29% CH(3)CN). Amino acid analysis: Ser(1), Lys(1), Arg(1).

For acetyl-Arg-Glu-Lys-Arg-MCA (IV): FAB-MS, m/z 787 (M + H); RP-HPLC, R = 29.05 min (29% CH(3)CN). Amino acid analysis: Glu(1), Lys(1), Arg(2).

For acetyl-Arg-Lys-Lys-Arg-MCA (V): FAB-MS: m/z 786 (M + H). RP-HPLC: R(t) = 30.95 min (31% CH(3)CN). Amino acid analysis: Lys(2), Arg(2).

For acetyl-Arg-Pro-Lys-Arg-MCA (VI): FAB-MS, m/z 755 (M + H); RP-HPLC, R = 30.95 min (31% CH(3)CN). Amino acid analysis: Pro(1), Lys(1), Arg(2).

For acetyl-Arg-Phe-Ala-Arg-MCA (VII): FAB-MS, m/z 748 (M + H); RP-HPLC, R = 38.95 min (39% CH(3)CN). Amino acid analysis: Ala(1), Phe(1), Arg(2).

For acetyl-Lys-Ser-Lys-Arg-MCA (VIII): FAB-MS, m/z 717 (M + H). RP-HPLC, R = 29.75 min (30% CH(3)CN). Amino acid analysis: Ser(1), Lys(2), Arg(1).

For acetyl-Orn-Ser-Lys-Arg-MCA (IX): FAB-MS, m/z 703 (M + H); RP-HPLC, R = 31.75 min (32% CH(3)CN). Amino acid analysis: Ser(1), (Orn + Lys)(1), Arg(1).

For acetyl-Tyr-Glu-Lys-Glu-Arg-Ser-Lys-Arg-MCA (X): FAB-MS, m/z 1294 (M + H). RP-HPLC, R = 31.70 min (32% CH(3)CN). Amino acid analysis: Ser(1), Glu(2), Tyr(1), Lys(2), Arg(2).

Preparation of Peptidyl Chloromethane Acetyl-Tyr-Glu-Lys-Glu-Arg-Ser-Lys-Arg-CH(2)Cl (XI)

This compound was prepared by condensing acetyl-Tyr(tBu)-Glu(tBu)-Lys(Boc)-Glu(tBu)-Arg(Pmc)-Ser(tBu)-Lys(Boc)-OH and NH(2)-Arg(Z(2))-CH(2)Cl, which was obtained by treating Boc-Arg(Z(2))CH(2)Cl (FAB-MS: m/z 575 (M + H)) with 25% trifluoroacetic acid, dichloromethane for 30 min. The condensation was accomplished using the activating reagent TBTU as above described. After 50 min, TLC analysis revealed one major product with a R(E): 0.44. Following precipitation with an ice-cold HCl aqueous solution (pH 3.0), the resulting precipitate was filtered and dried in vacuo. Purification through preparative TLC using solvent system E afforded compound XIa: acetyl-Tyr(tBu)-Glu(tBu)-Lys(Boc)-Glu(tBu)-Arg(Pmc)-Ser(tBu)-Lys(Boc)-Arg(Z(2))-CH(2)Cl (FAB-MS: m/z 2129 (M + H). Following complete deprotection of compound XIa by HF, the desired product XI was finally purified by RP-HPLC, whereupon it eluted as a single peak with a R of 31.00 min and was characterized by amino acid analysis, Ser(1), Glu(2), Tyr(1), Lys(2), Arg(2), and by ion-spray mass spectrometry, M+: 1169.74.

Preparation of I-Labeled Acetyl-Tyr-Glu-Lys-Glu-Arg-Ser-Lys-Arg-CHCl

Compound XI (0.88 µg) was iodinated using the lactoperoxidase method as reported previously(21) . The reaction was stopped after 60 s with 1 ml of aqueous 0.1% trifluoroacetic acid (v/v) and the mixture applied to a Sep-Pak C(18) cartridge that was washed sequentially with 20 ml of 1.0% (v/v) acetonitrile and then eluted with five fractions of 1 ml of 60% (v/v) acetonitrile containing 0.1% trifluoroacetic acid. The recovered material was purified by RP-HPLC as above mentioned. Only one iodinated form was observed, which eluted at 33% CH(3)CN and was stored in solution at -20 °C until used. The specific activity was estimated at 2.69 fmol/200 cpm.

Fluorometric Assays and Determination of the Kinetic Constants K(m)and V

Each fluorogenic substrate was kept as a 10 mM stock solution in dimethyl sulfoxide at -20 °C. For each assay, the substrate was incubated with proteinase sample in a total volume of 100 µl containing 50 mM sodium acetate (pH 6.0) and 5.0 mM CaCl(2) for hPC1 (3.4 units) and 50 mM sodium acetate (pH 7.0) and 1.0 mM CaCl(2) for hfurin (2.3 units). Incubations for the determination of kinetic constants were typically carried out in the presence of various concentrations (corrected for peptide content) of fluorogenic peptidyl substrates for 6 h at 25 °C before stopping the reaction by addition of 50 µl of acetic acid. All assays were performed in duplicate, and the average value is given. The values of K and V(max) were determined using a computer-assisted algorithm (Enzfitter; Elsevier Science Publishers B. V., Amsterdam). All fluorescence measurements were made with a Perkin-Elmer MPF-3L spectrofluorimeter using an excitation wavelength set at 370 nm and an emission wavelength set at 460 nm.

Inhibitor Profile and Active Site Titration of hPC1 and hfurin

In duplicate, hPC1 (1.3 units) was mixed with various concentrations (corrected for peptide content) of compound XI in a total volume of 100 µl of sodium acetate (pH 5.5) containing 5 mM CaCl(2) and incubated for 1 h at room temperature. The activity remaining was assayed by adding acetyl-Arg-Ser-Lys-Arg-MCA to a final concentration of 80 µM. Following incubation for 4 h at 25 °C, the reaction was stopped by adding 50 µl of acetic acid, and the AMC released was determined fluorimetrically. Similarly, a set of experiments was accomplished for hfurin (3.3 units) using 50 mM sodium acetate buffer (pH 7.0) and containing 1 mM CaCl(2).

Progressive Development of Inhibition Produced by the Peptidyl Chloromethane (XI) against hPC1

Inactivation reactions of hPC1 were conducted at 25 °C in 1.2 ml of 50 mM sodium acetate buffer (pH 6.0), containing 5.0 mM in CaCl(2). Inactivation reactions were initiated by adding hPC1 (30.4 units) to a solution of compound XI (30 nM). At various time points, from 2 to 90 min, 20 µl of the reaction mixture was removed and added to 80 µl of assay mixture containing 40 µM acetyl-Arg-Ser-Lys-Arg-MCA as substrate. The residual activity was determined after a 22-h incubation period. In order to monitor for time points of less than 2 min, inactivation reactions were made with 0.82 unit of hPC1 in a solution containing 1.5 nM compound XI, and the residual activity was determined after a 4 h incubation period. The same experiment was done in parallel without addition of compound XI to monitor the intrinsic stability of the enzyme in the conditions used.


RESULTS

Design and Synthesis of Peptidyl-MCA Derivatives

In order to investigate and define the primary structure requirements necessary for an efficient cleavage of the scissile peptide bond by the convertase PC1 and furin, we synthesized a number of peptidyl-MCA substrates and assayed their efficiency of cleavage with respect to the bimolecular rate constant k/K. In doing so, we developed a new synthetic strategy for rapid and efficient preparation of peptidyl-MCAs (Fig. 1). The method used previously relied on the Zimmerman's procedure(22) , which involved exclusively liquid-phase chemistry. That procedure was improved by including an automated solid-phase peptide synthesis step followed by direct coupling to a previously prepared amino acid-MCA moiety. This procedure allowed the preparation of the 10 peptidyl derivatives in a very short period of time and in very good yield.

Effect of Elongating the Peptidyl Sequence on the Overall Efficiency of Cleavage

As shown in Tables I and II, both hPC1 and hfurin exhibited similar reactivity toward compounds II and XII, both of which contain the tetrapeptide motif found at the junction between the pro- and mature enzyme in the zymogen molecule, namely Arg-Ser-Lys-Arg for hPC1 and Arg-Thr-Lys-Arg for hfurin. However, when compared with compound II, the removal in compound III of the Arg residue in P4 resulted in a 101-fold decrease in k/K for hPC1 and in an almost complete absence of cleavage by hfurin under the same conditions. Unexpectedly, comparison of compounds II and X revealed that k/K decreased by a factor of 43 for hfurin as compared with only 2 for hPC1. Moreover, for hfurin, this significant reduction is mainly due to an effect on the k that decreased by a factor of 30 in contrast to the corresponding K, which remains almost constant. Clearly, whereas a tetrapeptide containing P4 (Arg) is important for the efficient cleavage of a peptidyl substrate by either hPC1 and hfurin, elongation up to the P8 residue appears to modify significantly the overall efficiency of cleavage by furin but less so by PC1. The presence of a negatively charged glutamic acid in the P5 position could possibly be responsible for such an effect in the case of hfurin as discussed in the next paragraph.

Effect of the P3 Residue on the Overall Catalytic Efficiency of Cleavage

Comparison of compounds II and IV demonstrated that the introduction of a negative residue at the P3 position also results in a significant decrease in k when assayed in the presence of hfurin with an overall 138-fold decrease in k/K. In contrast, a similar comparison for hPC1 revealed only a small decrease in k/K. In general, our data on the effect of substitutions at the P3 position on the overall efficiency of cleavage indicate a greater sensitivity of hfurin as compared with hPC1 with k/K ranging from 5.8 10^1 to 2.5 10^3 sM for compounds IV, V, and VI compared with 1.6 10^3 to 1.9 10^3 sM, respectively.

Effect of the P4 Residue on the Overall Catalytic Efficiency of Cleavage

The importance of the P4 Arg residue is further substantiated by using a peptidyl substrate in which this position is occupied by a Lys (VIII) or an ornithine (Orn, IX) residue. Upon comparison with compound II, the net effect, as observed for hfurin, resulted, respectively, in a 538-fold and a 280-fold decrease in the relative ratio k/K, whereas a comparatively much lower 14- and 18-fold decrease was observed for hPC1. Although poorly cleaved, the peptidyl substrate IX exhibiting a K equal to 28 µM represents a better recognition motif for hfurin than the peptidyl substrate VIII, which exhibits a K equal to 106 µM.

The Efficiency of Cleavage of the Arg-Xaa-Xaa-Arg Peptidyl Motif

Finally, we investigated the ability of hPC1 and hfurin to cleave at a single basic amino acid of the type Arg-Xaa-Xaa-Arg. Compound VII, of which structure is identical to the last four residues of the chicken proalbumin sequence, is 5.8 times more efficiently cleaved by hPC1 than by hfurin. However, as indicated in Table 1and Table 2, this single basic amino acid site is much less efficiently cleaved than, for example, compound II (55-fold less for hfurin and 4.8-fold less for hPC1). These results indicate that the decrease in the k/K in relation to II is mainly through substrate discrimination in the ground state (K) rather than in the transition state (k). Nevertheless, those results clearly demonstrate the capability of either furin and especially PC1 in recognizing and cleaving at a single basic amino acid in a peptidyl sequence contributing a P4 Arg residue.





Peptidyl Chloromethane as Highly Potent Irreversible Inhibitor of PC1 and Furin

In order to develop an irreversible inhibitor of hPC1, we decided to synthesize a chloromethane-containing derivative based on the results obtained using the peptidyl-MCA derivatives. The compound X appeared to be a good candidate as it is cleaved quite efficiently (second best k/K for hPC1, Table 1) and as it contains a tyrosine that can be used for labeling purpose. As shown in Fig. 2, the determination of the parameter K/[I], indicative of the overall potency of an irreversible inhibitor, yields a value of 1.63 10^6 sM which represents so far the most potent chloromethane derivative reported for hPC1. Used as an active site titrant as shown in Fig. 3A, the amount of enzymatically active hPC1 (648 pmol) thus estimated is in good agreement with the value obtained (529 pmol) using a radioimmunoassay with an NH(2)-terminal directed antibody against mPC1. Similarly, the titration of hfurin yielded an estimated value of enzymatically active enzyme in good agreement with the one calculated using the k value reported by Hatsuzawa et al.(23) using the pGlu-Arg-Thr-Lys-Arg-MCA fluorogenic substrate. Indeed, as shown in Table 2, a value of 5.7 ± 0.4 µM (K) and 4.8 10 s (k) are obtained for hfurin compared with the reported values of 5.8 µM and 1.15 10 s for mfurin. The difference in the k value can either be explained by the species difference and/or by the different temperature at which the assays were performed. In fact, we observed a significant increase in the k value for both hPC1 and hfurin, when the assays are performed at 37 °C instead of 25 °C as reported herein (data not shown). Thus, even though the k of compound X, when compared with the k obtained with compound II, is lower for either hPC1 and for hfurin, an identical peptidyl sequence coupled to a chloromethane moiety results in the formation of a covalent enzyme-inhibitor complex. In fact, assuming that in certain experimental conditions the K/[I] approaches k(2)/K(24) , the bimolecular rate constant of the overall inactivation reaction of hPC1 (k(2)/K) is 811-fold higher than the bimolecular reaction of the cleavage efficiency of the scissile peptide bond (k/K). Since the mechanism for inactivation by peptidyl chloromethane involves formation and stabilization of a tetrahedral hemiketal (Michaelis complex), this step is essentially identical to what occurs during stabilization of the tetrahedral transition state upon substrate hydrolysis. However, as suggested by Powers (25) and further documented by Ringe et al.(26) , the hemiketal oxyanion displaces chloride to give an epoxy intermediate, which is then attacked by the catalytic histidine at the less hindered carbon to regenerate the hemiketal. Therefore, the main difference between substrate hydrolysis and inactivation by a peptidyl chloromethane is related to the second step of the overall mechanism, namely (i) the deacylation step for the hemiketal complex or (ii) the His alkylation of the Ser-hemiketal adduct for the chloromethane derivative. Thus, the discrepancy observed for the bimolecular rate constant for compound X (k/K: 2.01 10^3 sM) and XI (K/[I]: 1.63 10^6 sM) as well as the burst kinetics observed for furin (27) strongly suggest that the deacylation of the acylenzyme is a rate-limiting step for either hPC1 or hfurin.


Figure 2: Time-dependence of inhibition of hPC1 enzymatic activity toward acetyl-Arg-Ser-Lys-Arg-MCA by the octapeptidyl chloromethane inhibitor. The progressive development of inhibition produced by the reaction of hPC1 with the octapeptidyl chloromethane inhibitor is plotted as a semilogarithmic curve in accordance with Kitz and Wilson's representation(24) . The enclosed graph represents the graphical determination of the pseudo-first-order inactivation rate constant (K) derived from the plot of ln v/v(0)versus time.




Figure 3: Active site-titration and affinity labeling of hPC1 and hfurin with the octapeptidyl chloromethane inhibitor. Panel A: active site titration of hPC1 using the procedure as described in Materials and Methods. Panel B: RP-HPLC chromatogram of the radioiodinated octapeptidyl chloromethane inhibitor used as labeling agent for both hPC1 and hfurin. Elution conditions are described under Materials and Methods. The arrow indicates the elution position of the Tyr-containing unlabeled peptide as identified by amino acid analysis. Panel C: affinity labeling of hPC1 (10.2U) and hfurin (6.9U) obtained from the medium of recombinant vaccinia virus infected GH(4)C(1) cells. Aliquots of each medium were incubated in the presence of 10 mM EDTA (lane A) or 1 mM CaCl(2) (lane B) at pH 7.0 for hfurin or in the presence of 10 mM EDTA (lane D) or 5 mM CaCl(2) (lane C) at pH 6.0 for hPC1 with 2.4 10^6 cpm of the radiolabeled octapeptidyl chloromethylketone at 37 °C for 90 min. Each sample was then boiled in Laemli sample buffer and 1/3 was loaded on a 6% SDS-polyacrylamide gel and electrophoresed. The gel was then dried and the radioactive bands revealed by radiography.



The use of the radioiodinated inhibitor (Fig. 3B) resulted in the labeling of three molecular weight forms of hPC1, as shown in Fig. 3C, corresponding to 85, 74, and 66 kDa. The labeling of these three forms is found to be calcium-dependent as it is blocked by addition of 10 mM EDTA and represents, so far, the first example of an active site labeling agent for hPC1. The same set of experiments, done in parallel with the secreted shed form of hfurin, led to a calcium-dependent labeling of a molecular form corresponding to the predicted molecular mass form of 80 kDa (Fig. 3C).


DISCUSSION

The data of Table 1and Table 2reveal substantially reduced values of k, when measured with pGlu-Arg-Thr-Lys-Arg-MCA, for both hPC1 and hfurin as compared with those obtained with the yeast-related enzyme kexin (21-45 s) with similar peptidyl-MCA substrates(28) . One possible explanation could be related to the bulkiness of the MCA moiety rendering it a poor leaving group in the case of the mammalian prohormone convertases. In agreement, we recently demonstrated that substitution at the P`1 position by a sterically hindered group can lead to a competitive inhibitor(16) . Alternatively, these mammalian enzymes may require more extensive interactions with the substrate brought about by (i) stabilization of the substrate by elongation in the COOH-terminal direction or (ii) the presence of an yet undefined specificity-determining residues in the COOH-terminal region of the substrate. In support of hypothesis (i), the cleavage with hPC1 of an intramolecularly quenched fluorogenic peptidyl substrate containing the peptidyl sequence corresponding to the native hproPC1 sequence (positions 77-90) results in a 6-fold increase in the V(max) when compared with X without any substantial change in the K(29) . These data are in full agreement with those recently published (30) and suggest that k values are affected in a positive way by the sequence of the peptidyl substrate COOH-terminal to the cleavage site. Clearly more work is needed to clarify this aspect and define what are the determinants responsible for this substantial modulation of the k value.

As indicated in Table 1and Table 2, a discrepancy is observed for PC1 and furin in the overall efficiency of cleavage (k/K) of synthetic peptidyl substrates incorporating a negatively charged residue in the P3 and P5 positions. Comparing compound II with compound X, the effect observed for furin is mostly related to a decrease in the k parameter, since the K is more or less identical. Interestingly, among the many sequences sensitive to cleavage by furin identified by substrate phage display(31) , only one incorporated a negatively charged residue at P5 and none at P3. This observation can be rationalized in light of the recently proposed three-dimensional model of the active site and substrate-binding region of the convertases(32) . Thus, contrary to furin, of which subsites S3 and S5 are proposed to contain a negatively charged residue (Glu), PC1 appears to contain a neutral beta-branched residue (Ile). Therefore, with furin, the substrates IV and X in which a Glu is occupying the P3 and P5 positions, respectively, will experience an electrostatic repulsion upon entering the subsite cavities of the enzyme, resulting in a substantially decreased value of k. In fact, the relatively higher efficiency of cleavage of compound XII by furin with respect to X also indicates that, to observe the decrease in k value, the free carboxylate moiety of the glutamic acid side chain is needed as the presence of a pyroGlu moiety in P5 resulted in an efficient cleavage by the enzyme. This last aspect was described earlier by substitution of the trypsin active site S1 Asp-189 residue by a Ser residue which led to a loss of complementary with the P1 Arg or Lys residue and to a decrease in binding energy of 6.6 Kcal mol(33) . Similarly, these observations could also be extended to the prohormone convertases PACE4 and PC5, since an aspartic acid residue is predicted to occupy the S3/S5 subsites in contrast to PC2 and PC1, which contain a Phe and Ile residue, respectively. Furthermore, recent studies by Walker et al. (10) , using hemagglutinin mutants of a virulent avian influenza virus as a model, indicated that replacement, within the cleavage site of the P5 Lys residue by a Glu, completely abolished cleavage by purified furin in vitro. Upon co-expression with the vaccinia virus expression system, the same mutant hemagglutinin was, however, cleaved, hinting that either other cellular convertases can perform such processing or that the achieved cellular furin:substrate ratio is high enough to allow such a cleavage. Similarly, cellular proteolytic maturation of the HIV-1 envelope glycoprotein gp160 into gp120 and gp41 is accomplished at a highly conserved tetrapeptide sequence (Arg-Glu-Lys-Arg) for which furin has been proposed as a candidate processing enzyme(9, 13) . In both studies, only a small portion of the gp160 was actually cleaved. Although it was reported to be due to the slow exit of gp160 from the endoplasmic reticulum, (^2)we further propose that the Glu at P3 may also contribute to this phenomenon. Other structural features, including the three-dimensional structure of gp160(34) , may also be responsible. Noteworthy is the fact that when one compares compounds IV and V, substitution of the P3 Glu for a P3 Lys leads to the recovery of catalytic efficiency; a similar effect of a P3 Lys had been reported previously, since it had an enhancing effect on the inhibitory activity of certain synthetic inhibitors(13) .

The substitution of Ser at P3 by a conformationally restricted amino acid such as Pro results in an 11-fold decrease in k/K for furin as compared with only 2-fold for PC1. This effect of Pro substitution may suggest that the active site of furin is more sterically restricted than PC1 with respect to the topology in the S1 to S4 subsites of the substrate binding region. Interestingly, Wasley et al.(35) showed that the endogenous chinese hamster ovary cell processing enzymes are inefficient at processing overexpressed profactor IX at the Arg-Pro-Lys-Arg site. This inefficiency can be circumvented by increasing the amount of enzyme through co-expression of furin, but not of PACE4. In addition, the three-domain vasopressin precursor in guinea pig neurohypophysis is incompletely processed in contrast to complete processing as observed in rat and other mammals(36) . Interestingly, the interdomain sequence comprising the guinea pig COOH-terminal sequence of MSEL-neurophysin and NH(2)-terminal sequence of the copeptin contains a P3 Pro residue as compared with, for example, an Arg in the bovine and ovine sequences.

Another important aspect was the demonstration of the highly selective nature of the S4 subsite of furin, when compared with PC1. The significant decrease observed in the k/K rate constant for compounds VIII and IX relative to II indicates that both the guanidinyl moiety and the spatial geometry of the side chain are important components for an efficient cleavage. In fact, the results obtained with a genetically engineered proinsulin (PI) molecule, whereupon insertion of furin-like recognition sites at both the B/C and/or C/A junctions led to correct and efficient cleavage by the endogenous enzymes in the constitutive pathway(37) , are confirmed by the data presented herein. On the other hand, in the case of PC1, it can be seen that Lys (VIII) and even Orn (IX) can substitute for the Arg in P4 without major effect on the k. Nevertheless, it can be said that the presence of a positively charged side chain containing residue at P4 appears advantageous as illustrated by studies centered on the processing of proinsulin(38, 39) . These demonstrated that mouse PC1 activity is dependent upon the presence of a Lys at P4 position as it cleaves very well human PI-I, but processes more slowly the rat or murine PI-II (P4, Met) at the B/C junction and not at all at the hPI-I C/A junction (P4, Leu).

Concerning the peptidyl sequence Arg-Xaa-Xaa-Arg as a recognition motif for furin, we demonstrate with the compound VII that PC1 not only cleaves this sequence but does it in a more efficient way than furin (6 times more efficient (k/K). Actually, it was shown recently that PC1 can cleave the prodynorphin precursor at a single Arg residue preceded by a P8 Arg, releasing the C-peptide(40) . Therefore, one should be cautious in reporting (41, 42) the neuroendocrine PC1 as a processing enzyme cleaving exclusively at pairs of basic sites. Furthermore, the peptidyl substrate VII is identical to the last four residues of the chicken proalbumin precursor sequence which was shown to be efficiently and specifically cleaved in vitro by recombinant mouse PC1(43) . The extremely low k/K observed for furin (55 times lower than observed with II) can be best explained by the high K value (345 µM), suggesting that chicken proalbumin precursor will at best be only moderately cleaved by furin in vivo.

Finally, the usefulness of the chloromethane derivative as a tool for labeling and titration purpose for both PC1 and furin was demonstrated in the present paper. In the case of PC1, three molecular forms were labeled by the I-chloromethane octapeptidyl inhibitor and corresponded to molecular forms observed previously(15, 44) , namely, the 80-85 kDa form and the 74- as well as the 66-kDa forms (two carboxyl-terminally truncated PC1). Moreover, the high value observed for the kinetic parameter (K/[I]: 1.63 10^6 sM) underlines its potential usefulness as a complementary tool to Western blotting techniques in order to investigate the different forms of convertases found in various neuroendocrine cells and tissues. In the case of furin, one molecular form of 80 kDa was labeled using the iodinated chloromethane derivative. This shed form was observed previously using Western blot analysis and corresponds to the carboxyl-terminally truncated form secreted in the medium using the full-length recombinant vaccinia cDNA of furin.

Clearly more work is needed to define all of the structural subsites refinement of PC1 and furin in order to get a better understanding of the mechanism of recognition and cleavage of endogenous substrate by those two convertases. The principal advantage of using synthetic peptidyl substrate as a probe for mapping the substrate specificity of enzyme is clearly highlighted by allowing access to kinetic constants such K, k, and k/K as well as to relative transition state binding energies, Delta(DeltaG), which measure the selectivity of catalysis(45) . As shown in Fig. 4, in the particular case of hPC1 and hfurin, the high degree of selectivity of furin as compared with PC1 is well illustrated by, for example, Delta(DeltaG) values of up to 3.7 Kcal/mol for single amino acid substitutions at P4 and of 3 Kcal/mol for discrimination between Glu and Ser residue in P3. Furthermore, the good agreement between the results described herein with those using (i) co-expression studies of proprotein and convertases, (ii) in vitro studies with native proproteins, as well as (iii) the three-dimensional model proposed for furin, demonstrated that fluorescent peptidyl substrates can help in elucidating some important elements of substrate recognition in proprotein processing and can lead to the design of potent irreversible inhibitors.


Figure 4: The importance of the substrate side chains at P(n) position for the transition-state stabilization. The difference in transition-state stabilization between the preferred (acetyl-Arg-Ser-Lys-Arg-MCA denoted as A) and the other substrates denoted as B is given by Delta(DeltaG) = RT ln [(k/K)(A)/(k/K)(B)] as described previously in(45) . The solid triangles represent values obtained with hfurin whereas the solid circles represent data obtained with hPC1.




FOOTNOTES

*
This study was made possible by Program Grant PG-11474 from the Medical Research Council of Canada and by a Protein Engineering Network Centres of Excellence network grant. 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.

§
Recipient of a studentship from the Fonds pour la Formation des Chercheurs et l'Aide à la Recherche (F.C.A.R.) and from the Fonds des Etudes Supérieures of the University of Montréal.

Research fellow of the Fonds de la Recherche en Santé du Québec (F.R.S.Q.). To whom correspondence should be addressed: Neuropeptides Structure and Metabolism Laboratory, Clinical Research Institute of Montréal, 110 Pine Ave. West, Montréal, Québec, Canada H2W 1R7. Tel.: 514-987-5593; Fax: 514-987-5542.

(^1)
The abbreviations used are: PACE, pair of basic amino acids cleaving enzyme; AMC, 7-amino-4-methylcoumarin; Boc, t-butyloxycarbonyl; tBu, t-butyl; FAB-MS, fast atom bombardment mass spectrometry; Fmoc, 9-fluorenylmethoxycarbonyl; MCA, 4-methylcoumaryl-1-amide; PC, prohormone convertase (the prefix h, r, or m denotes the human, rat, or murine prohormone convertase respectively); Pmc, 2,2,5,7,8-pentamethylchroman-6-sulfonyl; RP-HPLC, reverse phase high performance liquid chromatography; TBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium tetrafluoroborate; TFE, trifluoroethanol; TLC, thin layer chromatography; Z, benzyloxycarbonyl; PI, proinsulin. The substrate positions are denoted P(i) . . . P(2), P(1), P`(1), P`(2), . . . P`(J) in accordance with the site of cleavage.

(^2)
F. Vollenweider, S. Benjannet, M. Chrétien, C. Lazure, G. Thomas, and N. G. Seidah, submitted for publication.


ACKNOWLEDGEMENTS

We express our sincere thanks to Dr. M. Evans (Chemistry Department, University of Montréal), Dr. Brian Marsden (Clinical Research Institute of Montréal), and Dr. S. A. St-Pierre (INRS-Santé, Université du Québec) for performing the FAB-MS, ^1H NMR and the HF cleavage, respectively. We also thank Dr. Y. Konishi (Biotechnology Research Institute of Montréal) for providing the Boc-Arg(Z(2))-CH(2)Cl derivative. Thanks are due to Dany Gauthier and Diane Savaria for technical assistance. We also express our sincere thanks to Dr. Didier Vieau for critical reading of the manuscript.


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