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Protein Science (2005), 14:303-315. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 The Protein Society
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Stability of mutant serpin/furin complexes: Dependence on pH and regulation at the deacylation step

Erick K. Dufour, Antoine Désilets, Jean-Michel Longpré and Richard Leduc

Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4

Reprint requests to: Richard Leduc, Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4; e-mail: Richard.Leduc{at}USherbrooke.ca; fax: (819) 564-5400.

(RECEIVED April 28, 2004; FINAL REVISION October 6, 2004; ACCEPTED October 6, 2004)


   Abstract
TOP
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Furin proteolytically cleaves a wide variety of proprotein substrates mainly within the trans-Golgi network (TGN) but also at the cell membrane and in endosomal compartments where pH is more acidic. Incorporation of furin recognition sequences within the reactive site loop (RSL) of {alpha}1-antitrypsin (AT) leads to the production of furin inhibitors. In an attempt to design more stable, potent, and specific serpin-based inhibitors, we constructed a series of AT and {alpha}1-antichymotrypsin (ACT) mutants by modifying the P7–P1 region of their RSLs. The biochemical properties of these variants were assessed by evaluating their propensity to establish SDS-resistant complexes with furin in a variety of conditions (pH 6.0–9.0) and by measuring their association rate constants. The effect of pH during the initial steps of complex formation was minimal, suggesting that the acylation step is not rate-limiting. The decrease in stoichiometry of inhibition (SI) values observed in AT variants at high pHs was a result of the reduced pH-dependent deacylation rate, which is rate-limiting in this mechanism and which suggests increased complex stability. Conversely, the SI values for ACT mutants had a tendency to be lower at acidic pH. Transiently transfecting HEK293 cells with these mutants abolished processing of the pro-von Willebrand factor precursor but, interestingly, only the ACT variants were secreted in the media as uncleaved forms. Our results suggest that reengineering the reactive site loops of serpins to accommodate and target furin or other serine proteases must take into account the intrinsic physicochemical properties of the serpin.

Keywords: furin; serpin mechanism; convertases; serine proteases; {alpha}1-antitrypsin; {alpha}1-antichymotrypsin; protease inhibitors

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04843305.


   Introduction
TOP
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Proteolytic processing is a ubiquitous mechanism for the production of biologically active polypeptides. A wide variety of precursors or proproteins of serum proteins, hormones, growth factors, cell surface receptors, extracellular matrix proteins, bacterial toxins, and viral coat proteins must undergo proteolytic cleavage in the exocytic and endocytic pathways to liberate their active moieties. Evidence accumulated over the past 15 years suggests that this event involves a family of calcium-dependent serine proteases called subtilisin-like proprotein convertases (Steiner 1998; Bergeron et al. 2000). Among the seven members of this family, furin has been the most thoroughly studied (Thomas 2002). Furin is a ubiquitously expressed enzyme that processes precursor molecules within the trans-Golgi network/ endosomal system. The wide variety of proproteins cleaved by this protease highlight its crucial role in numerous cellular and pathological events.

The initial characterization of the enzymatic signature of furin spearheaded efforts to design compounds aimed at inhibiting this convertase. One of the particularities of the specificity of furin is the requirement for arginine both at the P1 and P4 sites of the scissile bond, Arg-Xaa-Xaa-Arg{downarrow}, which forms the minimal recognition site of the enzyme (Molloy et al. 1992; Watanabe et al. 1992). With the recent elucidation of the crystal structure of mouse furin (Henrich et al. 2003), it has become clear how specific acidic residues forming the S1 and S4 subsites within the catalytic pocket interact with the basic residues at the P1 and P4 positions of substrates, thus influencing their affinity for the enzyme. Taken together, these data form the starting point for the design of furin inhibitor compounds. Many reports have demonstrated how a variety of templates ranging from simple polyarginine compounds to engineered eglin C proteins can inhibit furin (Angliker 1995; Boudreault et al. 1998; Lazure et al. 1998; Cameron et al. 2000; Komiyama and Fuller 2000; Komiyama et al. 2003).

Another class of inhibitors, serpins (serine protease inhibitors), are naturally occurring proteins that play a vital role in the regulation of serine protease activity (Gettins 2002). Serpins function by initially binding to the catalytic pocket of the target enzyme through their reactive site loop (RSL). The recognition of the serpin by the protease is largely attributed to the P1–P1 bond of the RSL, but other determinants are also involved. Subsequent to the binding of the serpin to its protease, it is proposed that a refolding step occurs during which the RSL is inserted into the center of {beta}-sheet A of the serpin to form a highly stable and kinetically trapped covalent serpin–protease complex. The solved crystallographic structure of the {alpha}1-antitrypsin–trypsin complex illustrates, at the atomic level, how this refolding step occurs (Huntington et al. 2000).

Furin is the prototype for mammalian proprotein processing convertases (Siezen et al. 1994; Henrich et al. 2003), which follows the serine protease mechanism of utilizing the archetypal catalytic triad of serine, histidine, and aspartate. These proteases also have a fourth conserved catalytic residue, the so-called oxyanion hole asparagine, which acts as a hydrogen bond donor to stabilize the buildup of negative charges on the scissile carbonyl during the transition state (Siezen and Leunissen 1997). Specific proteolytic autoactivation of furin is pH-dependent (Thomas 2002). However, the importance of the pH effect on the proteolytic maturation of furin substrates, activity catalysis, and inhibition is an unknown factor with regard to the overall enzymatic signature of furin. In the present study, we focused on the properties of various AT and ACT mutants in furin inhibition. The results revealed the dynamics and mechanisms of serpin/furin complex formation at different pHs.


   Materials and methods
TOP
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials
DNA modification enzymes were from New England Bio-lab. Culture reagents were from Invitrogen Corp. Escherichia coli strains BL21 and BL21(DE3)pLysS and the pET-32a expression vector were from Novagen. Human AT and ACT cDNAs were from the American Type Culture Collection (ATCC). The pQE-31 expression vector, Ni2+-nitriloacetic acid affinity resin, and anti-His5 antibodies were from Qiagen. Fluorogenic substrates, boc-Arg-Val-Arg-Arg-4-methyl-7-amidocoumarin (boc-RVRR-MAC), and decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-CMK) were from Bachem Bioscience Inc. Monoclonal anti-hfurin antibodies (MON-152) were from Alexis Biochemicals. AT-PDX and AT-Pittsburgh cDNAs were a generous gift from Dr. Gary Thomas (Vollum Institute) and Hedral Corp. Pro-vWF cDNA was from the Sanquin Blood Supply Foundation. Anti-vWF and anti-{alpha}1-antichymotrypsin polyclonal antibodies were from Dako Diagnostics Canada Inc. Anti-{alpha}1-antitrypsin polyclonal antibody was from Novocastra Laboratories Ltd.

Cell culture
Human embryonic kidney HEK293 and HEK293-C4 cells, a stable cell line overexpressing human furin (Denault 2000; Denault et al. 2002), were cultured in high glucose Dulbec-co’s modified Eagle’s medium containing 10% FBS, 2 mM L-glutamine, 50 IU/mL penicillin, and 50 µg/mL streptomycin.

Site-directed mutagenesis
The construction of AT mutants has been described previously (Dufour et al. 2001). ACT mutants with their signal peptide removed were produced by PCR and inserted into the BamHI/HindIII site of pCR2.1 vector (Invitrogen) before being subcloned into the prokaryotic expression pET-32a vector. ACT variants were produced as thioredoxin fusion proteins.

Construction, expression, and purification of the AT and ACT variants
The expression of AT variants in pQE-31 has been described previously (Dufour et al. 1998, 2001). For the ACT variants, purification of the native proteins was essentially the same as for the AT variants, with slight modifications. All purification steps were carried out at 4°C. The E. coli BL21 (AT variants) or BL21(DE3)pLysS (ACT variants) cells were grown at 30°C in TB medium containing 50 µg/mL carbenicillin to an A600 = 0.8 for AT variants and in TB containing 50 µg/mL carbenicillin and 34 µg/mL chloramphenicol to an A600 = 0.4 for ACT variants. Isopropyl-{beta}-D-thiolgalactopyranoside was then added to a final concentration of 1 mM and 200 µM for the induction of AT and ACT variants, respectively, followed by a 4.5- to 5-h incubation. Cells (0.5–1.0 liters) were harvested by centrifugation and resuspended in 10% of the initial volume in S buffer (50 mM sodium phosphate [pH 8.0], 300 mM NaCl, 10% glycerol, 15 mM {beta}-mercaptoethanol, 1 mM phenyl-methylsulfonyl fluoride, 500 µL of protease inhibitor cocktail solution, and 1 mM EDTA) containing 1 mg/mL of lysozyme for 30 min. Lysate was then sonicated (4 x 20-sec pulses) on ice. Cell debris were removed by centrifugation at 11,000g for 25 min. Ni2+-nitriloacetic acid resin equilibrated with S buffer was added to the supernatant. The slurry was stirred overnight, washed twice with W buffer (50 mM sodium phosphate [pH 6.5], 1.5 M NaCl, 5% glycerol, 5% ethanol, 20 mM imidazole, 15 mM {beta}-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride), and bound proteins were eluted with E buffer (50 mM sodium phosphate [pH 6.5], 300 mM KCl, 5% glycerol, 250 mM imidazole, and 500 µL of a protease inhibitor cocktail). Fractions containing recombinant proteins were desalted using PD-10 columns (Amersham Bioscience) or Amicon Ultra-15 concentrators (Millipore) in enterokinase buffer (20 mM Tris-HCl [pH 7.5], 50 mM NaCl, and 2 mM CaCl2). The thioredoxin portion of the ACT variants was removed by digestion with 5 U enterokinase (New England Biolab) for 16 h at room temperature. Further purification of serpin variants was achieved by Mono-Q HR anion exchange. Protein concentrations were determined by Bradford assay using bovine serum albumin as standard. All preparations were quantified by densitometry using SYPRO ruby (Bio-Rad) stained SDS-PAGE gels.

For expression in HEK293 cells, wild-type ACT cDNA (obtained from ATCC) was first subcloned from pBR322 into pST-Blue1 AccepTor (Novagen) before being introduced into the BamHI/EcoRI site of pcDNA3 (Invitrogen). ACT variants were then produced by site-directed PCR mutagenesis.

General kinetic methods
Human furin (hfur/714, a soluble form of the enzyme) was expressed and purified as described previously (Denault et al. 2000). The concentration of catalytically active hfur/714 was determined by titration with the active site-directed irreversible inhibitor dec-RVKR-cmk as described (Jean et al. 1998). Enzymatic activity was determined by the cleavage of the fluorogenic substrate boc-RVRR-MAC (excitation, 370 nm; emission, 441 nm). All enzymatic assays were carried out in 100 mM HEPES (pH 7.5), containing 1 mM CaCl2, 1 mM {beta}-mercaptoethanol, and 500 µg/mL BSA. To measure the pH dependence of the SI and kass parameters, the enzymatic assays were performed with the same buffer composition which was adjusted over a pH range of 6.0–9.0. The data obtained at pH 6.0–9.0 were fitted to the hyperbolic Michaelis-Menten rate equation to determine values of Km, kcat, and Vmax for the substrate boc-RVRR-MAC.

Determination of the stoichiometry of inhibition
hfur/714 was used to determine the molar ratio of the AT and ACT variants needed for complete inhibition of the enzyme. The stoichiometry of inhibition (SI) values for the inhibition of hfur/714 were determined by incubating AT and ACT variants at different concentrations and pH in a total volume of 300 µL at 37°C with a fixed concentration of hfur/714 for 1 h. The residual amidolytic activity was determined by the addition of 100 µM boc-RVRR-MAC and the reactions were stopped by the addition of 5 mM EDTA with a 1-h incubation. The residual activity fraction of substrate hydrolysis was plotted as a function of inhibitor concentration. The amount of inhibitor required to completely inhibit the enzyme was calculated by nonlinear regression fitting with a tight-binding titration equation:


(1)

The SI was determined by regression analysis of the dependence of residual furin activity on [I]o/[E]o or by SI = [I]o/ [E]o.

Slow-tight-binding inhibition kinetics
Slow-tight-binding inhibition experiments were conducted under pseudofirst-order conditions. Each assay consisted of four to 11 reactions with varying amounts of serpin and a control reaction without inhibitor. The assays were started by the addition of a constant amount of enzyme (800 pM) to a solution containing the fluorogenic substrate (200 µM) and an appropriate inhibitor concentration in kinetic buffer with a pH ranging from 6.0 to 9.0. Progress curves were obtained with the following final concentrations of reactants: 800 pM hfur/714; 200 µM Boc-RVRR-MAC; 3–30 nM AT-PDX, AT-EK1, AT-EK5, and ACT-EK2 at pH 6.0–9.0; 40–280 nM AT-EK2 at pH 6.0–7.0; 20–180 nM AT-EK2 at pH 8.0; 5–60 nM AT-EK2 at pH 9.0; 30–150 nM AT-EK3 at pH 6.0–9.0; 10–100 nM AT-EK4 at pH 6.0–9.0; 20–200 nM ACT-EK3 at pH 6.0–7.0; 50–260 nM ACT-EK3 at pH 8.0–9.0; and 250–600 nM ACT-EK5 at pH 6.0–9.0. The duration of the inhibition reactions by progress curve kinetics was 100 min. Spontaneous substrate hydrolysis was measured in separate experiments. The background rate of substrate hydrolysis in the absence of enzyme was subtracted from the data prior to estimating the rate constants. Considering an irreversible reaction ({nu}s = 0, kdiss = 0) for each serpin variant, the collected data were determined by fitting to the integrated rate equation for slow tight binding inhibition (Bieth 1995).


(2)

The data were fitted by nonlinear regression using Enzfitter (Biosoft) to obtain values for the initial velocity ({nu}o), the steady-state velocity ({nu}s), and the apparent first-order rate constant (k') for the establishment of the steady-state equilibrium of the protease–inhibitor complex. A second order rate constant (k*) was determined by plotting a series of k' versus the respective inhibitor concentration and measuring the slope of the line (k* = {Delta}k'/{Delta}[I]). Since the inhibitor was competing with the substrate, the second-order rate constant kass was corrected for the substrate concentration and the KM of hfur/714 for the substrate at different pH (pH 6.0 = 86 µM; pH 7.0 = 44 µM; pH 8.0 = 20 µM; pH 9.0 = 19 µM) in order to calculate the kass (M –1 sec–1). This relationship is kass = k* x (1 + [S]/KM).

Analysis of complex formation by SDS-PAGE
The ability of AT and ACT variants to form SDS-stable complexes with hfur/714 at different pH values was assessed by incubating 30–150 nM AT-PDX, AT-EK1, AT-EK5, and ACT-EK2, 500 nM–2 µM AT-EK2 and AT-EK3, 150–200 nM AT-EK4, and 350 nM–1 µM ACT-EK3 and ACT-EK5 with hfur/714 (30 nM) in 180 µL of modified kinetic buffer containing 20 µg/mL BSA for 20 min at 37°C. The reactions were stopped with 5 mM EDTA, and the reaction mixtures were lyophilized and then boiled in Laemmli buffer under reducing conditions. Proteins were separated on 8% or 10% SDS-PAGE gels and transferred to nitrocellulose membranes, which were then blocked with 5% nonfat dry milk in TBS. Western blotting was performed using anti-hfur/714 (MON-152), anti-{alpha}1-antichymo-trypsin, or anti-pentaHis antibodies to detect the complexes, hfur/714, ACT, and His-tagged proteins, respectively. The membranes were developed using the Lightning protocol (Perkin Elmer Life Sciences).

Immunoprecipitations
HEK293 and HEK293-C4 cells were transfected with 4 µg of pro-vWF cDNA or cotransfected with 0.5 µg or 4 µg of serpin variant cDNAs using Fugene 6 (Roche Corp.) in 6-cm plates. Twenty-four hours posttransfection, cells were washed with PBS and metabolically labeled in Met–/Cys–minimum essential medium containing 10% dialyzed FBS and 75 µCi [35S]Met/Cys (Expre35S35S labeling mix; Per-kin-Elmer Life Sciences) for 4 h. Media were harvested and divided into three equal parts for incubation either with anti-vWF (1:500), anti-{alpha}1-antichymotrypsin (1:500), anti-{alpha}1-antitrypsin (1:500), or M2 anti-FLAG (for detection of furin at 1:500) followed by protein A/G-agarose. The immunoprecipitates were resolved on a 7% SDS-PAGE gel for vWF, an 8% SDS-PAGE gel for hfur/714, and a 10% SDS-PAGE gel for serpin variants.


   Results
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 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Production of AT and ACT variants
In order to examine the inhibitory properties of serpin variants toward human furin, we designed, constructed, expressed, and purified a series of AT and ACT mutants incorporating a variety of furin recognition sequences within their RSL (Fig. 1Go). The AT variants were produced as polyhistidine (N-terminal) fusion proteins (Dufour et al. 2001), while ACT variants were expressed as thioredoxin fusion proteins. For ACT, we found that these conditions reduced protein degradation and increased the solubility of the expressed proteins. All variants were produced as soluble proteins and were purified under native conditions from total cytoplasmic proteins by either a two-step procedure (AT variants) using nickel affinity column chromatography and MonoQ ion exchange chromatography or a three-step procedure (ACT variants) consisting of an additional enterokinase digestion step for the removal of the thioredoxin moiety. Figure 2AGo shows that the purified recombinant wild-type and AT variants migrated as 47-kDa proteins, whereas the purified recombinant wild-type and ACT variants migrated as 42-kDa proteins (Fig. 2BGo). All purified serpins were estimated to be >90% pure by densitometric analysis.



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  		Figure 1. Schematic reactive site loop sequences of recombinant wild-type AT and ACT and their variants. Single-letter amino acid sequences of the reactive site loop of wild-type and AT- and ACT-derived serpin variants. The amino acids at positions P7–P3' within the RSL are displayed according to the nomenclature proposed by Schechter and Berger (1967). The residues substituted in the AT and ACT reactive site loop variants are underlined. The recognition site for elastase (Johnson and Travis 1978) and chymotrypsin (Cooperman et al. 1993) and the cleavage site for furin (Jean et al. 1998) are marked by asterisks between the P1–P1' scissile bond.

 


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  		Figure 2. Purification of recombinant wild-type AT and ACT and their variants. Analysis of the purified His-tagged wild-type and variant serpins expressed in prokaryotic cells. Recombinant proteins were purified from isopropyl-{beta}-D-thiogalactopyranoside-induced bacterial cell extracts using Ni2+-nitrilotriacetic acid affinity resin and MonoQ column chromatography. The proteins were subjected to 10% SDS-PAGE and stained with Coomassie Blue. (A) Purification of recombinant wild-type AT and AT variants (1 µg of protein). (B) Purification of recombinant wild-type ACT and ACT variants (1 µg of protein).

 
Influence of pH on the stoichiometry of furin inhibition by AT and ACT variants
We evaluated the SI for each AT and ACT variants by titration experiments. Active-site titration of furin was determined using the active site-directed irreversible inhibitor dec-RVKR-cmk. Serpins are mechanism-based inhibitors and follow a branched reaction pathway (Scheme 1Go). According to this mechanism, serpin inhibitory activity is affected by the ratio of the rate constant for the substrate pathway (k3), which generates cleaved, uncomplexed serpin (I*), and the rate constant for the inhibitory pathway (k4), which generates the stable serpin–enzyme complex (EI*). This ratio is reflected in the stoichiometry of inhibition, SI [SI = 1 + k3/k4]. The SI value indicates the "efficiency" of a serpin for a given protease, which equals the number of moles of inhibitor required to completely inhibit 1 mole of a target protease. For cognate serpin–protease complexes such as AT-elastase, the reaction flows almost entirely toward stable complex formation, resulting in SI values close to 1 (Hopkins and Stone 1995). Table 1Go shows the pH effect on the SI values of AT and ACT variants toward furin. For all AT variants we observed a decrease in SI as a function of an increase in pH. Three variants (AT-EK2, AT-EK3, and AT-EK4) had very high SI values in all pH conditions, indicating that the reaction flow for these serpins partitioned to the pathway generating the cleaved form (k3). However, this behavior changed dramatically when more basic pH conditions were used, as can be seen when SI values of >200 for AT-EK2 at pH 6.0 were reduced to 46 at pH 9.0. Lower SI values were obtained with AT-PDX, AT-EK1, and AT-EK5, with the most efficient variant being AT-EK5 at basic pH (SI of 1.5).



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  		Scheme 1. Branched pathway for the suicide substrate inhibition mechanism of serpin action. This scheme shows the accepted minimal kinetic mechanism for the reaction of a serpin (I) with a proteinase (E) to form a stable, covalent inhibited complex (EI*). Initial formation of the noncovalent Michaelis complex (EI) is followed by acylation of the enzyme by the serpin to form the initial acyl-enzyme (EI') (first part of proteolytic process). The intermediate complex (EI') can partition (branch point in the mechanism) to undergo a conformational rearrangement, resulting, through the inhibitor pathway, in the formation of the final inhibited complex (EI*). EI* slowly decomposes through a single pathway, resulting in the release of cleaved serpin and active enzyme with rate constant k5. The proteinase component of the complex may also be cleaved at one or more sites by free proteinase (P) (either the same proteinase as E or a different one) to give E*I*, which cannot dissociate. This step has been well characterized for antichymotrypsin (Cooperman et al. 1993; Stavridi et al. 1996) and for heparin cofactor II (Han et al. 1997). According to the proposed kinetic scheme of Nash et al. (2000), this inhibition mechanism incorporates the possibility that the steps with rate constants k2 and k4 might be reversible, and that EI* can decompose either by direct hydrolysis (with rate constant k5) or by reversion to EI'. The equilibrium dissociation constant of the Michaelis complex (Ks) represents the affinity between the inhibitor and the enzyme.

 

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  		Table 1. Influence of pH on stoichiometry of inhibition between the reactive site loop of {alpha}1-antitrypsin and {alpha}1-antichymotrypsin in reactions with furin
 
In contrast, the SI of ACT variants decreased as the pH shifted to more acidic values. Indeed, the SI of ACT-EK2, which reconstitutes a typical furin recognition motif (RTRR), shifted from 4.7 at pH 9.0 to 2.7 at pH 6.0. Replacing Arg by Lys at the P2 position, which yielded ACT-EK3, significantly increased the SI (2.7–13 at pH 7.0), which is similar to the shift observed for the AT homolog. Interestingly, the EK5 homologs of AT and ACT behaved quite differently. ACT-EK5 had very high SI values (>200) while AT-EK5, as previously mentioned, had an SI of 1.5 at pH 9.0. This is likely the result of the effect of the P5 residue, which is a glutamate in AT and a lysine in ACT.

Influence of pH on kass
We next examined whether the AT and ACT variants possessed the time- and concentration-dependent characteristics of serpins, namely slow tight-binding inhibition kinetics at various pH. Reaction kinetic curves for all variants were performed under conditions where Io ≥ SI x Eo, such that the effect of the cleavage pathway on kass could be ignored (Waley 1985). All AT and ACT variants tested had slow tight-binding inhibition kinetic behaviors (data not shown) (Hopkins and Stone 1995; Dufour et al. 2001). Table 2Go shows the effect of pH on kass and k'ass of each AT and ACT variant in reaction with furin. In serpin–protease reactions with an SI > 1, consumption of the inhibitor by hydrolysis may influence the magnitude of kass (Djie et al. 1996). Therefore, a change in SI could thus confound the interpretation of the pH effect on the inhibition pathway. Considering the k'ass (kass x SI) therefore represents the true second-order rate constant of the formation of the covalent acyl–enzyme intermediate (EI') (Scheme 1Go), and is a way of normalizing inhibition rate constants for comparative purposes when the mechanism suggests such an adjustment is warranted. Indeed, we observed that the association rates for each AT or ACT variant were not greatly influenced by pH, with k'ass values between 105 and 106 M–1 sec–1. The lower values obtained (5.5 x 104 M–1 sec–1) were for ACT-EK3 at pH 9.0 with the highest association rates (1.1 x 107 M–1 sec–) for AT-EK2 at pH 9.0. These results demonstrate that, in general, the association rate with furin of the various mutants is primarily a function of the inhibitory pathway and is not significantly related to a change in flux through the hydrolytic pathway.


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  		Table 2. Kinetic association parameters determined for the reaction of {alpha}1-antitrypsin and {alpha}1-antichymotrypsin reactive-site loop variants with furina,b
 
We also show the influence of pH on Ki values for each AT and ACT variants (Table 3Go). In general, we observe that for each variant, Ki values diminish with increasing pH in accordance with the relationship which defines Ki as kdiss/kass. However, because of the high SI values of AT-EK2, AT-EK3, and ACT-EK5, high Ki values are obtained (nM) with corresponding elevated kdiss.


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  		Table 3. Ki values from titration curve for each serpin variant at different pH
 
Effect of pH on serpin/furin complex formation
A hallmark of the serpin–serine protease interaction is the formation of a complex that is not dissociated by heating and incubation in SDS. Quench-flow experiments revealed that these complexes were formed very rapidly (milliseconds) (see Fig. 7Go, below). We determined whether the pH influenced the ability to form SDS-stable, irreversible serpin/furin complexes. Figures 3Go (AT) and 4Go (ACT) show that immunoblotting with anti-furin (MON-152) or anti-serpin (anti-His for AT or anti-ACT) enabled us to determine that the variants formed characteristic SDS-stable complexes (C) that migrated with an apparent molecular mass of ~200 kDa. Cleaved inhibitor, Ic (43 kDa for AT, 41 kDa for ACT), uncleaved inhibitor (47 kDa for AT, 45 kDa for ACT), cleaved complex (C*) (72 kDa for AT furin, 102 kDa for ACT furin), and free enzyme (E) (83 kDa) are also shown. Of the six AT variants studied, we noticed that AT-EK2 and AT-EK3 did not readily associate with furin at pH 6.0, as shown by the weak band at 200 kDa. However, increasing the pH led to an enhanced rate of complex formation. Slight increases in basic pH-dependent complex formation were observed for other AT variants. Moreover, using the anti-His antibody, we showed that AT variants that do not possess the P6 Arg in the furin recognition motif (AT-PDX, AT-EK1, and AT-EK4 but not AT-EK5) produced a cleaved complex form (C*) at pH 6.0. This would represent the entire serpin associated with a proteolytically processed form of furin. Last, all AT variants were cleaved (Ic) thereby flowing to the hydrolytic pathway k3. Wild-type AT did not interact with furin under various pH conditions (Fig. 3GGo).



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  		Figure 7. Quenched-flow/immunoblot analysis of rate of complex formation of AT-EK4 and ACT-EK2 reactions with furin. Initial concentrations of 210 nM for the variant serpins (A,B) and 30 nM for furin ([I]o/[E]o ratio of 7:1) were mixed for various quench times and the reactions were stopped with 5 mM EDTA. Final concentrations were 70 nM (serpins) and 10 nM (furin). Noncomplexed and complexed furin immunoreactivity was revealed by MON-152 monoclonal antibody. C, enzyme–serpin complex; E, enzyme. The result is representative of two independent experiments.

 


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  		Figure 3. Immunoblot analysis of complex formation stability at different pH between AT reactive site loop variants and furin. Ninety (90) nM of AT-PDX (A) and AT-EK1 (B), 1.5 µM of AT-EK2 (C), 1 µM of AT-EK3 (D), 200 nM of AT-EK4 (E), 70 nM of AT-EK5 (F) and 1 µM of wt-AT (G) were incubated with 30 nM of furin at 37°C for 20 min in 180 µL of modified kinetic buffer containing 20 µg/mL BSA. The reactions were stopped by the addition of 5 mM EDTA. The samples were lyophilized then boiled in Laemmli buffer. (Upper panel) The noncomplexed and complexed furin were revealed by MON-152. (Bottom panel) The noncomplexed and complexed AT variants were detected by anti-His-tag. C, enzyme–serpin complex; C*, cleaved complex; E, enzyme; I, uncleaved serpin; Ic, cleaved serpin. The result is representative of three independent experiments.

 


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  		Figure 4. Immunoblot analysis of complex formation stability at different pH between ACT reactive site loop variants and furin. Ninety (90) nM of ACT-EK2 (A), 500 nM of ACT-EK3 (B), 700 nM of ACT-EK5 (C), and 1 µM of wt-ACT (D) were incubated with 30 nM of furin at 37°C for 20 min in 180 µL of modified kinetic buffer containing 20 µg/mL BSA. The reactions were stopped by the addition of 5 mM EDTA. The samples were lyophilized then boiled in Laemmli buffer. (Upper panel) The noncomplexed and complexed furin were revealed by MON-152. (Bottom panel) The noncomplexed and complexed ACT variants were detected using an antichymotrypsin polyclonal antibody. C, enzyme–serpin complex; C*, cleaved complex; E, enzyme; I, uncleaved serpin; Ic, cleaved serpin. The result is representative of three independent experiments.

 
When ACT variants were incubated with furin, we observed a different pattern (Fig. 4Go). The rate of irreversible inhibitory complex (C) formation for ACT-EK2 (Fig. 4AGo) in reactions with furin appeared greater at pH 6.0, as shown by the higher intensity of free enzyme (E) when compared to pH ranging from 7.0 to 9.0. For ACT-EK3 (Fig. 4BGo), the formation of SDS-stable complexes at different pH remained constant. Despite its very high SI value, ACT-EK5 (Fig. 4CGo) also formed an irreversible complex (C) with furin under different conditions, but a major species of 102 kDa corresponding to cleaved complex (C*) was detected at pH 7.0–9.0, but not at pH 6.0, using MON-152 antibodies. This could explain the high SI values obtained for ACT-EK5. All ACT variants, like AT variants, were cleaved, as shown by the presence of lower molecular weight forms (Ic). Wild-type ACT did not interact with furin under various pH conditions (Fig. 4DGo).

Inhibition of furin-dependent processing of ProvWF by AT and ACT variants
To examine the efficiency of each AT and ACT construct at inhibiting intracellular furin-dependent processing, HEK293 and HEK293-C4 (a cell line overexpressing furin) cells were cotransfected with a construct expressing pro-vWF, a known furin substrate (Van de Ven et al. 1991), together with constructs expressing various serpin variants. In the absence of inhibitors, only ~50% of vWF was secreted into the medium as the fully processed mature form (Fig. 5AGo). Overexpression of vWF by transfection of HEK293 cells does not allow endogenous furin to process all the 330-kDa precursor to the mature 220-kDa form of vWF (de Wit and van Mourik 2001), most likely because the proprotein processing system is saturated. The coexpression of vWF with each AT and ACT variant, but not the wild-type AT or ACT, inhibited pro-vWF proteolytic processing in HEK293 cells (Fig. 5AGo, upper panel). In general, AT variants seemed to be more efficient than ACT variants at abolishing processing, as indicated by the patterns generated when 0.5 µg of DNA was transfected. Interestingly, AT-EK2 and AT-EK3 were detected both as cleaved and uncleaved forms, whereas AT-PDX and AT-EK1 did not demonstrate this behavior in a cellular context. None of the ACT variants generated a cleaved form (Ic) in our conditions, unlike the AT variants. To more fully assess this result, we examined the biochemical behavior of each AT and ACT variant in conditions where furin is overexpressed. The results clearly demonstrated that, while serpin/furin complexes were detected (C), all AT variants (Fig. 5BGo, upper panel) were cleaved (Ic), whereas ACT variants remained intact.



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  		Figure 5. Inhibition of maturation of provWF by different AT and ACT reactive site loop variants in cellulo. (A) (Upper panel) The ability of each AT and ACT variant to inhibit processing of provWF in HEK293 cells. HEK293 cells were transfected either with 4 µg of provWF vector or cotransfected with 0.5 µg or 4 µg of constructs expressing the different AT and ACT reactive site loop variants, as indicated above the panel. At 24 h posttransfection, the cells were metabolically labeled with [35S]Met/Cys for 4 h, after which medium was harvested. VWF proteins were immunoprecipitated with anti-vWF. Inhibition of provWF maturation was analyzed on 8% SDS-PAGE. Lane mock, pcDNA3 control. ProvWF, immature form of vWF; vWF, mature form. (Lower panel) Differential rates of generation of cleaved serpin. The serpins remaining in the medium were immunoprecipitated with anti-AT or anti-ACT. The proteins were analyzed by 10% SDS-PAGE. I, uncleaved serpin; Ic, cleaved serpin. (B) (Upper panel) Differential rates of generation of cleaved serpin in stable HEK293-C4 cells. The serpins remaining in the medium were immunoprecipitated with anti-AT or anti-ACT antibodies. The proteins were analyzed by 10% SDS-PAGE. Lane mock, pcDNA3 control. C, Enzyme–serpin complex; I, uncleaved serpin; Ic, cleaved serpin. (Lower panel) The complex formation between various reactive site loop serpins in the medium. Furin was immunoprecipitated with anti-M2. Proteins were analyzed by 8% SDS-PAGE. C, enzyme–serpin complex; E, enzyme. The result is representative of two independent experiments.

 
Effect of pH on furin hydrolysis of a peptide substrate
The effect of pH on furin catalyzed hydrolysis of the substrate boc-RVRR-MAC is shown in Figure 6Go. Upon increase of the pH from 6.0 to 9.0, KM decreased linearly up to pH 7.5 and then remained constant. Over this pH interval, kcat occurred as a bell-shaped form with a maximum at pH 7.0. This led to an overall increase of kcat/KM over pH 6.0 to 8.0 indicating enhanced catalytic efficiency of furin over this pH range.



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  		Figure 6. Effect of pH on furin catalysis of the peptide substrate boc-RVRR-MAC. Parameters KM, kcat, and kcat/KM are reported in A, B, and C, respectively. Kinetic assays were performed in 100 mM HEPES (pH 6.0–9.0), 1 mM CaCl2, 1 mM {beta}-mercaptoethanol, and 500 µg/mL BSA. Values were determined by the initial-rate method and were obtained by nonlinear regression analysis to fit the data to the Michaelis-Menten rate equation.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Furin is a ubiquitously expressed transmembrane serine protease that participates in some of the most basic processes of cellular function by cleaving precursor proteins into their active entities (Thomas 2002). These cleavages occur in various cellular organelles where pH conditions vary. A recent study focusing on the autoproteolytic activation of the furin zymogen demonstrated that, following the first intramolecular cleavage (Leduc et al. 1992) and transport to acidified TGN/endosomal compartments, furin cleaves the bound propeptide at a second internal P1/P6 Arg site (-Arg70-Gly-Val72-Thr-Lys-Arg75-), resulting in propeptide dissociation and enzyme activation (Anderson et al. 2002). This suggests that noncanonical furin recognition motifs (where the P4 Arg is missing) may be pH sensitive.

In this study, we introduced furin recognition sequences into the RSLs of AT and ACT in an attempt to design more potent and stable serpin-based inhibitors. The RSLs of serpins defines their ability to recognize and inhibit a particular protease. RSL modifications reassign the targets and change the serpin–protease interaction kinetics. We and others have previously shown that modifying the RSL of AT does not affect the nature of its essential biochemical properties such as the formation of SDS-stable irreversible complexes with furin (Dufour et al. 1998, 2001; Jean et al. 1998; Anderson et al. 2002). In addition to further modifying the RSL of AT with other furin recognition motifs, we hypothesized that the various biochemical properties of ACT (Schechter et al. 1997) may yield more efficient inhibitors in certain cellular environments where pH tends to be acidic. The RSL (P7–P1 residues) of ACT was thus modified to introduce various furin recognition sequences (Watanabe et al. 1992), and the properties of the resulting variants were evaluated.

Initially, we expected that the true second association rate constant (k'ass) of all serpin variants would significantly change as a function of pH. This is because the chymase-AT reaction causes an increase in SI and a marked decrease in kass, whereas decreases in SI are observed with ACT (Schechter et al. 1997). In our conditions of varying pH, we did not observe any striking differences in k'ass for the AT and ACT furin mutants. We confirmed the rapid association with furin by quench-flow experiments, which are more an evaluation of the final reaction complex. Complex formation times <7.2 msec at different pH were reached by all AT variants (e.g., AT-EK4; Fig. 7AGo). However, slower complex formation rates were observed with the ACT-EK2/furin pair (~1000 msec) (Fig. 7BGo). It is probable that the reduced complex formation time of AT furin pairs is caused by different RSL conformational structures. Indeed, the ACT loop is helical (Wei et al. 1994), whereas the AT loop is an extended {beta}-strand (Elliott et al. 1996, 1998). We can presume that the extended {beta}-strand conformation of the AT loop is a better fit for the substrate-binding pocket of furin than the helical structure of the ACT loop at the time of association. Our results thus revealed that the canonical furin sequences (RXXR, RXRXXR, or RX(K/R)R) are pH-insensitive, i.e., that the substrate binding rate to the catalytic pocket of furin is independent of the different canonical motifs and is not a rate-limiting enzymatic step.

Serpins undergo the same first kinetic steps that normal substrates do in that proteolysis is not complete but rather stops at the acyl–enzyme intermediate stage (i.e., acylation step, k2). Like all serine proteases, furin uses this enzymatic mechanism to cleave the peptide bond following a minimum of four kinetically significant steps: (1) substrate binding, (2) acylation, (3) deacylation, and (4) release of the N-terminal product. While the presteady-state behavior of furin has yet to be established with certainty, studies indicate that furin, like Kex2, a well-characterized yeast homolog of furin (Rockwell et al. 2002), exhibits burst kinetics in the cleavage of substrates with P1 Arg. This suggests that the acylation step is not rate-limiting for cleavage by furin (Bravo et al. 1994; Krysan et al. 1999). With the k'ass values of the various furin recognition motifs in the serpin variants used in this study, we show with strong kinetic evidence that the initial association and acylation steps in the serpin mechanism are also not rate-limiting.

How can we thus explain the increased stability of irreversible inhibitory complexes at high pH for certain variants? We previously demonstrated that the complex formation between AT-EK2 and furin was very rapid at neutral pH (Dufour et al. 2001), which has been confirmed by quench-flow experiments. These complexes are then dissociated after a few minutes, generating active furin and cleaved inhibitor. We showed that this is because the RSL P6 Arg within the {beta}-sheet structure of the serpin acts as a repulsion center with regard to the hydrophobic environment of the serpin’s central body (Dufour et al. 2001). Since acylation rates remain constant as a function of pH, the increased stability observed for the AT-EK2–furin complex (i.e., pH 9.0; Fig. 3CGo) can be explained by a reduced deacylation rate of the acyl–enzyme complex. This could also explain the decreased SI values for AT-EK2 and all AT variants at high pH. Since furin may possess similar kinetic properties to Kex2, i.e., the deacylation step is rate-limiting and pH-dependent, this would explain the exceptionally high substrate specificity seen with Kex2 and mammalian convertases. This is in marked contrast to the behavior of the related subtilisin family of serine proteases, for which acylation is the rate-limiting step (Perona and Craik 1995) and which exhibit comparatively low substrate specificities (Gron et al. 1992).

Introduction of various arginine residues within the P1–P6 region of AT probably affect the stability of the complex with furin due either to new or absent interactions with the body of the cleaved serpin. Based on the crystal structure of AT it is clear that the charged side chain of Arg in the P4 and P6 position renders the complex less stable due to their contact with the hydrophobic regions of the serpin. Moreover, the added arginines may also contribute to increasing the stability of the complex at high pH, which is in contrast to most serpin–enzyme pairs (Calugaru et al. 2001; Plotnick et al. 2002), by creating local steric and charge impediments thereby influencing the accessibility of the hydroxyl ion to perform the deacylation step.

We examined the effect of the AT and ACT variants in wild-type HEK293 cells on furin-dependent processing of pro-vWF. This precursor is a substrate that is efficiently cleaved by furin (Wise et al. 1990). Expression of the AT and ACT variants in these cells abolished processing of pro-vWF, as demonstrated by the absence of the mature form (Fig. 5AGo). However, we noted that ACT variants seemed to be less efficient than AT variants at abolishing processing when similar serpin expression levels were compared (i.e., at 0.5 µg DNA). Interestingly, the biochemical behavior of the variants in vitro was reconstituted in cellulo, i.e., AT variants were readily cleaved by furin while ACT variants were not. Using cells that overexpressed furin (HEK293-C4 cells) (Fig. 5BGo), we again detected completely cleaved AT variants in the medium of HEK293-C4 cells while all the ACT variants were detected as intact, uncleaved forms. The higher stability of ACT in acidic environments (Schechter et al. 1997) compared to AT may contribute to maintaining the protein in an intact form.

Apart from processing precursors within the exocytic pathway, furin also participates in cleaving such precursors as Pseudomonas exotoxin A in endosomes (Chiron et al. 1994; Corboy and Draper 1997) and Bacillus anthracis protective antigen at the cell surface (Collier and Young 2003). It would thus be tempting to speculate that ACT variants may provide an interesting alternative to AT variants in the context of inhibiting furin-dependent processing of such precursors.


   Acknowledgments
 
We are grateful to Dr. Klaus Klarskov for mass spectroscopy analysis of the cleaved serpins. We especially thank Dr. Peter Gettins for helpful discussions and comments on the manuscript. This work was supported by grants from the Canadian Institutes of Health Research (CIHR). R.L. holds a Fonds de la Recherche en Santé du Québec (FRSQ) Senior Scholarship.


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