Role of the Occluding Loop in Cathepsin B Activity*

(Received for publication, September 19, 1996, and in revised form, October 17, 1996)

Chantal Illy Dagger §, Omar Quraishi Dagger §, Jing Wang Dagger , Enrico Purisima Dagger , Thierry Vernet §par and John S. Mort §**Dagger Dagger

From the Dagger  Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada, the  Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada, the par  Institut de Biologie Structurale, 41 avenue des Martyrs, 38027 Cedex 1, France, the ** Joint Diseases Laboratory, Shriners Hospital for Children, and Department of Surgery, McGill University, Montreal, Quebec H3G 1A6, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Within the lysosomal cysteine protease family, cathepsin B is unique due to its ability to act both as an endopeptidase and a peptidyldipeptidase. This latter capacity to remove C-terminal dipeptides has been attributed to the presence of a 20-residue insertion, termed the occluding loop, that blocks the primed terminus of the active site cleft. Variants of human procathepsin B, where all or part of this element was deleted, were expressed in the yeast Pichia pastoris. A mutant, where the 12 central residues of the occluding loop were deleted, autoprocessed, albeit more slowly than the wild type proenzyme, to yield a mature form of the enzyme with endopeptidase activity comparable with the wild-type cathepsin B, but totally lacking exopeptidase activity. This deletion mutant showed a 40-fold higher affinity for the inhibitor cystatin C, suggesting that the occluding loop normally restricts access of this inhibitor to the active site. In addition, the binding affinity of the cathepsin B propeptide, which is a potent inhibitor of this enzyme, was 50-fold increased, consistent with the finding that the loop reorients on activation of the proenzyme. These results suggest that the endopeptidase activity of cathepsin B is an evolutionary remnant since, as a consequence of its membership in the papain family, the propeptide must be able to bind unobstructed through the full length of the active site cleft.


INTRODUCTION

The lysosomal cysteine protease cathepsin B is unique within the papain superfamily in that it acts both as an endopeptidase and an exopeptidase. Thus in addition to making internal cleavages (1), it also removes C-terminal dipeptide units from the substrate (peptidyldipeptidase activity) (2). The x-ray crystal structure of cathepsin B suggests the molecular basis for this dual character. Relative to other papain-like proteases, cathepsin B contains an extra structural element termed the "occluding loop" (3), which blocks off one end of the substrate binding cleft (see Fig. 1a). As proposed by Schechter and Berger (4), the protease active site cleft is considered to consist of a series of subsites, each accommodating an amino acid residue of the peptide substrate. Those subsites binding the residues C-terminal to the peptide bond undergoing cleavage are termed the primed sites (S1' to S3') and accept the P1' to P3' substrate residues. The subsites accepting the N-terminal side of the substrate, the P1 to P3 residues, are termed the unprimed sites (S1 to S3, with the numbering in each case starting with the residues bordering the cleavage site). The structure of rat cathepsin B containing the covalently bound inhibitor Z-Arg-Ser(OBz)-chloromethylketone (where Z represents benzyloxycarbonyl and Bz represents benzoyl) (5) maps out the unprimed substrate binding subsites and indicates that the occluding loop plays a defining role in primed site specificity. The strategic position of two histidine residues in this loop (His110 and His111)1 suggests that they can act as acceptors for the negatively charged carboxylate of the P2' residue C terminus. The structure of a human cathepsin B complex with the E-642 analogue CA030 (the ethyl ester of epoxysuccinyl-Ile-Pro-OH) (6) shows this can indeed be the case. The free carboxyl group on the proline residue in the dipeptide moiety of this inhibitor interacts with these two histidines suggesting that Ile and Pro occupy the P1' and P2' subsites, respectively, as would be expected for substrate binding in the exopeptidase mode.


Fig. 1. Occluding loop mutants of cathepsin B. a, stereo view of the alpha -carbon trace of wild-type human cathepsin B (green) superimposed on a modelled structure of the mutant M1 (blue). Areas of complete overlap are also shown in blue. The disulfide bond between Cys108 and Cys119 is shown in pink. The side chains of residues involved in substrate binding and catalysis (Glu245, His110, His111, Cys29, and His199) are shown in full. The positions of residues delimiting the occluding loop mutants M1 (Pro107 and Thr120) and M2 (Ser104 and Asp124) are also indicated. b, schematic representation of the occluding loop of wild-type cathepsin B. The filled square on Asn113 represents the site of N-linked oligosaccharide substitution that was deleted in the wild-type enzyme used in this study due to the substitution S115A. c, schematic representation of mutant M1 indicating the junction between Pro107 and Thr120. d, schematic representation of mutant M2 showing the points of deletion and position of the tetrapeptide Glu-Gly-Val-Gln (gray background), corresponding to residues 89-92 of papain, inserted in the place of residues 123-126.
[View Larger Version of this Image (34K GIF file)]


While the occluding loop appears to play an important role in the binding of substrates that are cleaved by the exopeptidase activity of cathepsin B, it would be expected to interfere with the binding of extended peptides. It would also be expected to obstruct the binding of protein protease inhibitors such as the cystatins, which in the case of the papain/stefin B complex have been shown to form extensive interactions with the protease primed sites (7). Since the occluding loop represents a separate and relatively poorly ordered region of cathepsin B (5), we reasoned that it could be deleted without compromising the structure of the remaining enzyme.

We have previously demonstrated the efficient recombinant expression of rat cathepsin B as an alpha -factor fusion construct in the yeast Saccharomyces cerevisiae (8). This system proved much less effective for the expression of the human enzyme (9); however, we have recently shown that high levels of human cathepsin B can be produced using similar constructs in the methanotropic yeast Pichia pastoris (10). We have used this system to produce forms of human cathepsin B lacking the occluding loop and thus allowing us to evaluate its role in cathepsin B function.


EXPERIMENTAL PROCEDURES

Computer Modelling

The x-ray structures of cathepsin B (Protein Data Bank code 1cst (5)) and papain (Protein Data Band code 9pap (11)) formed the basis for structural modelling. The Kollman all atom force field and Powell minimizer were used with the program SYBYL 6.1 (Tripos Assoc. Inc.). The non-bonded cutoff was set at 8 Å and the dielectric constant to 80. The segment Cys108 to Cys119 in the cathepsin B occluding loop was deleted, and the remaining ends were joined through a peptide linkage (mutant M1, see Fig. 1, a and c). Energy minimizations were performed allowing Ser104 to Asp124 and residues within 8 Å to move, while the rest of the molecule remained fixed. The minimizations were judged to be complete when the root mean square of the gradients was smaller than 0.1 kcal/mol Å.

Construction of Human Cathepsin B Deletion Mutants

A cDNA construct, consisting of wild-type human procathepsin B as a fusion with the preproregion of yeast alpha -factor, had been prepared previously in pVT105 (a derivative of the multiple purpose yeast shuttle vector pVT100U (12), where the ADH promoter had been deleted). The consensus sequence for oligosaccharide substitution in the mature protein had been removed by the substitution S115A (9). XhoI and NotI restriction sites were then introduced into the C-terminal region of the alpha -factor proregion and the 3'-untranslated region of procathepsin B, respectively, to allow subcloning into the P. pastoris expression vector pPIC9 (Invitrogen). Using single-stranded DNA from the construct in pVT105, two deletion mutants of human procathepsin B were constructed by loop-out mutagenesis. The oligonucleotide 5'-CCG TAC TCC ATC CCT CCC ACG GGG GAG GGA GAT ACC-3' was used to remove Cys108 to Cys119, yielding the mutant M1 (see Fig. 1c). The oligonucleotide 5'-GTA GGG TGC AGA CCG TAC GAA GGT GTT CAA AAG TGT AGC AAA ATC TGT G-3' was used to delete residues Ser104 to Pro126 and add four amino acids from the papain sequence (Glu89-Gly90-Val91-Gln92) as a linker, giving the mutant M2 (Fig. 1d). Sequencing of the complete procathepsin B cDNA inserts following mutagenesis confirmed the expected sequences. Both constructs were digested with XhoI and NotI, and the procathepsin B fragment was subcloned into pPIC9.

Expression of Mutants in P. pastoris

For integration into the Pichia genome, the pPIC9 based constructs were linearized by cleavage with BglII and purified. The P. pastoris host strain GS115 (Invitrogen) was then transformed with the linearized constructs by electroporation. Positive transformants were grown for 2 days in medium containing glycerol as the carbon source followed by growth in the presence of methanol for a further 2 days to induce expression of recombinant protein. Protein secreted into the culture supernatants was analyzed by SDS-PAGE and Western blotting using sheep anti-human cathepsin B (13).

Purification of M1 and M2 Mutant Proteins

After concentration, supernatants were dialyzed against 50 mM sodium acetate, 1 mM EDTA (pH 5.0) to allow autoprocessing of proenzyme mutants. The mature mutant M1 was reversibly inactivated by addition of methylmethane-thiosulfonate (1 mM) and purified on an FPLC system (Pharmacia) using an SP-Sepharose fast flow column. The enzyme eluted with 150 mM NaCl. Mutant M2, which was not able to autoprocess, was purified in its proenzyme form using an SP-Sepharose fast flow column under the conditions described above. The proenzyme form of mutant M1 was purified by gel filtration chromatography using a Sephacryl 200HR column equilibrated in 25 mM Tris, 150 mM NaCl (pH 7.5).

Active Site Titration

The concentration of active wild-type enzyme was determined by titration using the cysteine protease inhibitor E-64 (Sigma) (14). The mutant M1 proved to be poorly inhibited by E-64, so enzyme concentrations were estimated by titration with recombinant human cystatin C prepared as described previously (15).

Enzyme Assays

Endopeptidase activity was determined spectrofluorometrically using the fluorogenic amidomethylcoumaryl substrate Z-Phe-Arg-MCA. Methylcoumarinyl-7-amide release was monitored at 25 °C on a SPEX Fluorolog-2 spectrofluorometer at 440 nm using an excitation wavelength of 380 nm. Two different buffers were used: 50 mM sodium acetate (pH 5.2), containing 200 mM NaCl, 1 mM EDTA, 2 mM DTT, and 1% acetonitrile, and 50 mM sodium phosphate (pH 6.0), containing 1 mM EDTA, 200 mM NaCl, 2 mM DTT, and 3% DMSO. The values of kcat and Km were determined using non-linear regression analysis (16).

The exopeptidase activity was measured using the synthetic substrate dansyl-Phe-Arg-Phe(NO2)-Leu, which was prepared as described by Pohl et al. (17) except that Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry was used. Assay conditions were 10 µM substrate, in 25 mM sodium citrate (pH 5.0) containing 0.25 M NaCl, 1 mM EDTA, 3% DMSO, 2 mM DTT at 25 °C. Dansyl release was monitored by excitation at 350 nm and emission at 535 nm.

The proteolytic activity of cathepsin B mutants against protein substrates was performed using azocasein (18) in 50 mM sodium acetate (pH 5.2), containing 200 mM NaCl, 1 mM EDTA, 2 mM DTT. After incubation at 30 °C for 30 min with 0.5% azocasein, proteins were trichloroacetic acid-precipitated, and A366 of the supernatants containing liberated peptides was measured.

pH Activity Profiles

kcat/Km values were measured at 0.1 pH intervals over the range of pH 3.0-8.4 based on the relationship v = [E][S] kcat/Km at [S] <<  Km. The reaction buffers were 50 mM sodium citrate (pH 3.0-6.0), 50 mM sodium phosphate (pH 5.8-7.8), and 25 mM sodium borate (pH 7.7-8.4). All buffers contained 200 mM NaCl, 1 mM EDTA, and 2 mM DTT. The pH for each reaction was checked immediately after measurement of the initial rate.

Thermostability Measurements

Cathepsin B (0.7 nM) in 50 mM sodium acetate buffer (pH 5.2) containing 200 mM NaCl, 1 mM EDTA was incubated at 20 or 30 °C for various times. The remaining enzymatic activity was measured using the same buffer system containing 2 mM DTT, 1% acetonitrile, and 5 µM Z-Phe-Arg-MCA.

Interaction of Cystatin C with M1 Mutant and Wild-type Cathepsin B

Enzymes and human cystatin C were incubated either in 50 mM sodium acetate buffer (pH 5.2), containing 200 mM NaCl, 1 mM EDTA, 2 mM DTT, and 1% acetonitrile, or in 50 mM sodium phosphate (pH 6.0), containing 1 mM EDTA, 200 mM NaCl, 2 mM DTT, and 3% DMSO, for 5 min at room temperature. The residual enzyme activity was measured using 5 µM Z-Phe-Arg-MCA. Values of Ki were calculated by non-linear regression analysis using the general equation describing tight binding inhibition. Precise cystatin C concentrations were determined by titration with papain (19).

Inhibition by the Cathepsin B Propeptide

The rat cathepsin B propeptide (PCB1, residues 1-56) was prepared as described previously (20). Inhibition experiments were performed either in 50 mM sodium acetate (pH 5.2), 200 mM NaCl, 1 mM EDTA, 2 mM DTT, 1% acetonitrile or in 50 mM sodium phosphate (pH 6.0), 100 mM NaCl, 0.2 mM EDTA, 2 mM DTT, 0.025% DMSO using 5 µM Z-Phe-Arg-MCA as substrate. Under these conditions, progress curves for the inhibition of the mutant M1 and wild-type cathepsin B by PCB1 at pH 5.2 and 6.0 followed typical slow-binding kinetics as defined by the equation,
[P]=v<SUB><UP>s</UP></SUB>t+[(v<SUB><UP>i</UP></SUB>−v<SUB><UP>s</UP></SUB>) (1−<UP>exp</UP>(<UP>−</UP>k<SUB><UP>obs</UP></SUB>t))]/k<SUB><UP>obs</UP></SUB>
where P is the product formed, vi and vs are the initial and steady-state velocities, respectively, t is the reaction time, and kobs is the apparent second order rate constant for inhibition. Nonlinear regression analysis using the program Enzfitter (Elsevier-Biosoft, Cambridge, U. K.) gave the individual parameters (vi, vs and kobs) for each progress curve. For each data set, the enzyme-inhibitor dissociation constant (Ki) values were obtained from the relationship vi/vs - 1 = [I]/Ki (21). The plot of kobs versus [I] remained linear over the range of inhibitor concentrations studied (2-20 nM), giving an estimate of kon and confirming that inhibition by PCB1 occurred by a one-step process (20). The dissociation rate constant koff was calculated from the relationship Ki = koff/kon.

SDS-PAGE Analysis

Proteins were treated with 10 µM of E-64. When necessary, they were deglycosylated with endoglycosidase H. After precipitation with methanol (4 volumes), deglycosylation was performed in 25 mM sodium citrate buffer (pH 5.0) by incubation with 1 unit of endoglycosidase H (Boehringer Mannheim) for 1 h at 37 °C. Proteins were analyzed by gel electrophoresis (12%) in the presence of SDS under reducing conditions.


RESULTS

Modelling of Cathepsin B Occluding Loop Mutants

The possibility that the cathepsin B occluding loop could be deleted without causing significant perturbation of the remaining protein was investigated by molecular modelling. Inspection of the x-ray crystal structure of cathepsin B suggested that the disulfide-bonded segment, Cys108 to Cys119, could be deleted and a peptide bond formed between Pro107 and Thr120 (Fig. 1c). Energy minimization of the resulting structure, the mutant M1 (Fig. 1a), showed that, on the formation of this bond, only a few residues on the surface of the molecule would be displaced, suggesting that deletion of this segment would not seriously affect the overall structure of the molecule.

Relative to papain, which lacks the occluding loop, the mutant M1 still included eight residues of this structural element. For the complete deletion, the segment 104-126 was removed, and the resulting gap closed using a connecting tetrapeptide derived from the homologous region of papain (Fig. 1d). Superimposition of the modified cathepsin B structure (the mutant M2) with that of papain suggested that these changes could be accommodated without radical restructuring of the protein. Energy minimization of this more drastically modified form was not attempted.

Expression and Purification of the Procathepsin B Mutants

Deletion of part or all of the occluding loop was accomplished by loop-out mutagenesis. Both mutant proteins were expressed and secreted at levels comparable with the wild-type human enzyme (approximately 20 mg/liter of culture medium) and as previously observed for rat procathepsin B (10). The mutant M1 precursor was purified by gel filtration, and the proenzyme form of the mutant M2 was purified by ion exchange chromatography. About 10 mg of pure proenzymes were obtained in each case (Fig. 2). The recombinant cathepsin B proenzymes expressed in P. pastoris were heterogeneous due to modification of the N-linked oligosaccharide moiety on the proregion and migrated with apparent molecular masses ranging from 40 to 52 kDa with a dominant band at the leading edge of the smear (Fig. 2, lanes 1 and 5). Following enzymatic deglycosylation with endoglycosidase H, each cathepsin B form resolved as a single band of the expected size of 36.5 kDa (Fig. 2, lanes 2 and 6), migrating slightly faster than the deglycosylated wild-type proenzyme. The far UV circular dichroism spectra of the mutant proenzymes were similar to that of the wild-type proenzyme, suggesting correct folding (data not shown).


Fig. 2. SDS-PAGE of purified mutants M1 and M2 (Coomassie Blue staining). Lane 1, proenzyme M1; lane 2, deglycosylated proenzyme M1; lane 3, mature M1 after processing (still containing a small amount of proenzyme); lane 4, mature wild-type human cathepsin B; lane 5, proenzyme M2; and lane 6, deglycosylated proenzyme M2. Positions of molecular mass standards are indicated on the left.
[View Larger Version of this Image (54K GIF file)]


In Vitro Processing of the M1 and M2 Mutants

Wild-type procathepsin B is readily processed upon activation at acid pH (9), leading to the generation of the mature, active, single chain form of the enzyme (Fig. 2, lane 4). Processing of the M1 precursor to yield a proteolytically active enzyme proceeded at a much slower rate than that observed for the wild-type enzyme (Fig. 2, lane 3). Five days at pH 5.0 were required to totally activate the mutant M1, whereas the wild type enzyme was completely processed following overnight dialysis at 4 °C. Processing was accelerated somewhat under lower pH conditions. Inclusion of the cysteine protease inhibitor, E-64, during dialysis prevented maturation of the precursor, suggesting that activation occurred as the result of autoprocessing. N-terminal sequencing demonstrated a three residue extension relative to the fully processed lysosomal form. Attempts to process the mutant M1 using extrinsically added proteases (pepsin, papain, and proteinase K) were unsuccessful as the protein was either totally resistant or rapidly degraded (data not shown).

The mature mutant M1 was purified by ion-exchange chromatography on an SP-Sepharose column. From 1 liter of culture medium, 4 mg of pure mature M1 could be obtained, which migrated as a single band with a slightly higher mobility than wild-type cathepsin B (Fig. 2, lane 3). The mutant M2 did not autoprocess at acidic pH, even following lengthy incubation at 37 °C. Processing could be attained in the presence of exogenous wild-type cathepsin B but not with other extrinsically added proteases such as pepsin, papain, or proteinase K. This conversion was shown to be both time- and dose-dependent as in the case of rat procathepsin B (C29S) (8). Further characterization of the mature mutant M2 was not carried out due to the inability to separate it from the wild-type cathepsin B used for processing.

Thermal Inactivation of the Mature M1 Mutant

The mutant M1 was found to be much more sensitive to thermal inactivation than the wild-type enzyme (Fig. 3). Loss of activity followed simple first order kinetics. At 30 °C, inactivation of the mutant M1 occurred with a t1/2 of 11 min compared with 110 min for the wild-type enzyme. A similar differential effect was seen at 20 °C (t1/2 of 28 and 280 min, respectively).


Fig. 3. Thermal inactivation of mutant M1 and wild-type cathepsin B. Enzymes were preincubated at pH 5.2 for the times indicated at 30 °C (a) or at 20 °C (b) and then assayed for endopeptidase activity using Z-Phe-Arg-MCA. Mutant M1 (black-square); wild-type cathepsin B (bullet ).
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Endo- and Exopeptidase Activities of the M1 Mutant

Kinetic analysis was carried out with synthetic and protein substrates. Kinetic parameters, determined using the amidomethylcoumaryl substrate, Z-Phe-Arg-AMC, showed that removal of the external part of the loop had no significant effect on the kcat/Km value at pH 5.2 (Table I) although both Km and kcat showed a 5-fold decrease for the mutant M1 compared with the wild-type enzyme. At pH 6.0, kcat/Km was about 1.6-fold lower for the mutant M1 (Table I), consistent with a decrease in kcat at this pH. Little change in Km was observed between pH 5.2 and 6.0. 

Table I.

Kinetic constants for the hydrolysis of Z-Phe-Arg-MCA by mutant M1 and wild-type cathepsin B


Enzyme kcat (s-1) Km (mM) kcat/Km (M-1 s-1)

pH 5.2 
  Wild-type 41.3  ± 6.1 0.110  ± 0.010 375,000  ± 31,000
  Mutant M1 6.89  ± 1.4 0.0212  ± 0.002 327,000  ± 26,000
pH 6.0 
  Wild-type 42.2  ± 7.0 0.105  ± 0.009 422,000  ± 29,000
  Mutant M1 4.44  ± 1.1 0.0179  ± 0.003 261,000  ± 22,000

Activity of the mutant M1 was also tested against the protein substrate, azocasein (Table II). The deletion mutant was able to cleave this substrate with a specific activity similar to that of the wild-type enzyme. This activity was completely abolished in the presence of the cysteine protease inhibitor E-64. These estimates of the proteolytic activity of the mutant M1 are probably underestimates since, as shown in Fig. 3, there were substantial activity losses during the incubation times used in these assays.

Table II.

Activity of mutant M1 and wild-type cathepsin B towards azocasein

Enzymes were incubated with azocasein in the buffer 50 mM sodium acetate (pH 5.2), 200 mM NaCl, 1 mM EDTA for 1 h at 20 °C or 30 minutes at 30 °C. For reaction in the presence of E-64, enzymes were preincubated with the inhibitor (10 µM) for 5 min before assay.
Enzyme Specific activity

µg of azocasein degraded/mg of enzyme/min
20 °C
  Wild-type 262  ± 15
  Mutant M1 150  ± 16
30 °C
  Wild-type 480  ± 8
  Mutant M1 422  ± 9
  Wild-type + E-64 15  ± 0.2
  Mutant M1 + E-64 18  ± 0.2

The peptidyldipeptidase activity of the mutant M1 was measured using the quenched fluorescence substrate dansyl-Phe-Arg-Phe(NO2)-Leu. Under the experimental conditions used, no exopeptidase activity was detectable for the mutant M1 in acetate or citrate buffers at pH 5.0, while native cathepsin B showed activity (kcat/Km of 432,000 M-1 s-1) comparable with that determined previously by Pohl et al. (17).

pH Activity Profiles Using the Substrate Z-Phe-Arg-MCA

As shown previously, wild-type cathepsin B exhibits a complex pH-activity profile that can best be fitted to a model involving five dissociation events, three in the ascending limb and two in the descending limb (22). Deletion of the occluding loop had a dramatic effect on the pH-activity profile (Fig. 4). Maximal activity was observed at pH 5, while the high levels of activity found for wild-type cathepsin B at pH 7-8 were not found with the mutant M1. Below pH 4.5, the activity dropped very rapidly. This loss of activity was in part the result of the reduced stability of the mutant M1 at acidic pH.


Fig. 4. pH activity profiles of mutant M1 and wild-type human cathepsin B. Activity was measured using Z-Phe-Arg-MCA. Wild-type cathepsin B (bullet ); mutant M1 (open circle ).
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Inhibition of the M1 Mutant by Human Cystatin C

Since it had been proposed that the occluding loop of cathepsin B would obstruct the binding of cystatins to the active site (7), inhibition of the mutant M1 by human cystatin C was studied. At pH 6.0, a Ki value of 0.17 nM for the wild-type cathepsin B/cystatin C interaction was found, consistent with values obtained by others (23, 24) (Table III). However, the mutant M1 exhibited a 40-fold higher affinity for the inhibitor. The same differential effect was observed at pH 5.2. However, the affinity of both forms of the enzyme for the inhibitor was lower than at the higher pH.

Table III.

Dissociation constants for the inhibition of mutant M1 or wild-type human cathepsin B by wild-type human cystatin C


Enzyme Ki (nM) (pH 5.2) Ki (nM) (pH 6.0)

Wild-type 5.2  ± 0.7 0.17  ± 0.05
Mutant M1 0.18  ± 0.02 0.004  ± 0.0008

Inhibition of M1 Mutant by the Synthetic Propeptide of Cathepsin B

The propeptide of cathepsin B is a potent inhibitor of the mature enzyme (20). The recently determined procathepsin B structure (25, 26) indicates the occluding loop adopts a different conformation in the proenzyme so that the propeptide is able to bind through the entire active site cleft. Such a reorientation of the occluding loop must occur for the free propeptide to bind to the mature enzyme. In agreement with this proposal, the affinity of the free cathepsin B propeptide for the mutant M1 was found to be 54-fold higher than for the wild-type enzyme at pH 6.0 (Table IV). A similar differential effect was seen at pH 5.2. In agreement with the proposal that the occluding loop must be reoriented during propeptide binding, the on rate is increased in the case of the mutant M1, whereas the off rate is essentially unchanged.

Table IV.

Equilibrium and kinetic data for the interactions between human wild-type cathepsin B or mutant M1 and the cathepsin B propeptide


Enzyme Ki (nM) kon (M-1 s-1) koff (s-1)

pH 5.2 
  Wild-type 7.0  ± 0.4 0.3  × 105 2.1  × 10-4
  Mutant M1 0.2  ± 0.023 7.2  × 105 1.4  × 10-4
pH 6.0 
  Wild-type 5.4  ± 0.6 0.5  × 105 2.7  × 10-4
  Mutant M1 0.1  ± 0.03 20.2  × 105 2.0  × 10-4


DISCUSSION

The occluding loop of cathepsin B represents a unique structural element relative to the other members of the papain superfamily. The three-dimensional structures of cathepsin B (3) and of a cathepsin B complex with an inhibitor occupying the S1' and S2' subsites (6) provide clues with respect to the exopeptidase activity. However, the relationship of the loop to endopeptidase activity is not clear. In order to study the role of the occluding loop, we have expressed a mutant of cathepsin B (M2) engineered to mimic the local structure of the other members of the papain superfamily. Although the folding of the precursor of this cathepsin B mutant did not appear to be altered significantly, as shown by circular dichroism spectroscopy (data not shown), this mutant did not autoprocess. In contrast, a more subtle deletion (M1) did allow processing to occur, albeit at a reduced rate relative to the wild-type precursor. Since the activation of wild type and mutant M1 procathepsin B proceeds by an autoactivation mechanism, the inability of the mutant M2 to undergo processing may be related to a stronger binding of the propeptide to the active site region. The slower autoprocessing of the mutant M1 and its higher affinity for the free propeptide support this proposal.

Studies of the mature mutant M1 provide insight into the functional role of the loop. Its partial truncation reduced the stability of the molecule indicating that this structural element contributes to the stability of the protein. In fact, the three dimensional structure of cathepsin B shows that, while the loop is attached to the left domain of the enzyme, residues 108-119 interact with the right domain, suggesting that this region of the protein may be stabilized by interdomain interactions. In this regard, it was shown previously that interdomain perturbation in papain affects enzyme stability (27).

The three-dimensional structure also suggested an explanation of the unique ability of cathepsin B to act as an exopeptidase (3, 6). Indeed, while the mutant M1 was proteolytically active, it showed no detectable exopeptidase activity, validating the previously proposed role of the loop residues His110 and His111 as "acceptors" for the negative charge on the substrate P terminus. On the other hand, the Km for the endopeptidase substrate Z-Phe-Arg-MCA decreased five-fold, showing that the peptide is able to bind to the active site in a more efficient manner. Similar endoprotease activity against a protein substrate for the wild type and M1 was demonstrated although the value for the mutant M1 is likely to have been an underestimate due to its reduced stability.

High molecular weight inhibitors also provide a method to probe active site accessibility. The recently determined three-dimensional structures of rat (25) and human (26) procathepsin B reveal that the occluding loop is a mobile element. In the precursor molecule, the loop adopts an alternate conformation to that occupied in the mature enzyme, no longer impeding access to the primed side of the active site cleft. Indeed, the free cathepsin B propeptide must trigger a similar conformational transition on binding to the mature enzyme as it is a potent, specific inhibitor of cathepsin B (20) implying that it has the ability to displace the occluding loop. This finding also implies that shortening the loop should increase the binding affinity of the free propeptide to cathepsin B, a prediction confirmed on removal of 12 residues of the loop in the mutant M1 which enhanced the affinity of the free propeptide by more than 50-fold over that for the wild-type enzyme. As expected from the mechanism of the interaction, the on rate is increased while the off rate remains essentially unchanged.

It was pointed out previously that the conformation of the occluding loop in mature cathepsin B is incompatible with cystatin binding (3), as reflected by the high Ki value for formation of the cystatin C/cathepsin B complex relative to that of cysteine proteases lacking the occluding loop (28). This is a similar situation to that described above for the binding of the propeptide and suggests that a significant alteration of the loop position occurs when the inhibitor binds to the cathepsin B active site. Indeed, partial deletion of the loop, as in the mutant M1, increases the affinity of cathepsin B for cystatin C by over 40-fold relative to the wild-type enzyme, a value similar to that found for propeptide inhibition. The Ki value for the mutant M1 approaches that reported for cathepsin L (23), a cysteine protease lacking the occluding loop. All of these kinetic and structural observations are consistent with the predictions from molecular modelling of increased access to the active site on deletion of the loop.

Although the influence of the loop on the active site accessibility is clear, the occluding loop also has an influence on the hydrolytic activity of cathepsin B. For instance, the reduction of kcat by over 6-fold at pH 5.2 for the mutant M1 shows that the gain of binding efficiency (lower Km) is offset by a reduction in substrate turnover. Also, modification of the loop lowers the optimal pH of activity by over 2.5 pH units although the exact meaning of this effect remains obscure. The lack of symmetry of the pH-activity profile of wild-type cathepsin B has been attributed to the proximity of the loop and the protonation state of residues His110 and His111 (22). Shortening of the loop with the consequent removal of these two residues promotes a more symmetrical profile. Taken together, these data show the dramatic effect of the loop on the mechanism of activity.

Besides its demonstrated role in exopeptidase activity, the occluding loop of the cathepsin B clearly limits access of macromolecules to the active site. It is interesting to speculate on the evolution of cathepsin B from an ancestral papain-like cysteine protease (29). The addition of the occluding loop to facilitate peptidyldipeptidase activity had to remain compatible with the propeptide binding pattern and activation mechanism common to this family of enzymes, as demonstrated by the structures of procathepsin B and procathepsin L (25, 30). We propose, therefore, that the endoprotease activity retained by cathepsin B is an evolutionary remnant and that the main role of cathepsin B in its natural environment, the lysosome, is to act as an exopeptidase. The residual endopeptidase activity, while not essential intracellularly, still allows the enzyme to play an extracellular role as an endopeptidase that is poorly inhibited by cystatins (31). This may have important pathological consequences.


FOOTNOTES

*   This work was supported by the Protein Engineering Network of Centers of Excellence and the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Member of the Protein Engineering Network of Centers of Excellence.
Dagger Dagger    To whom correspondence should be addressed: Joint Diseases Laboratory, Shriners Hospital for Children, 1529 Cedar Ave., Montreal, Quebec, Canada H3G 1A6. Tel.: 514-849-6208; Fax: 514-842-5581; E-mail: mc60{at}musica.mcgill.ca.
1    Residue numbering relates to mature cathepsin B.
2    The abbreviations used are: E-64, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane; Z-Phe-Arg-MCA, benzyloxycarbonyl-L-phenylalanyl-L-arginine 4-methylcoumarinyl-7-amide; PAGE, polyacrylamide gel electrophoresis; DMSO, dimethyl sulfoxide; DTT, dithiothreitol.

Acknowledgments

We thank M.-C. Magny for preparing wild-type cathepsin B and Drs. A. C. Storer and P. Lindahl for valuable discussions.


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