From the Biotechnology Research Institute, National
Research Council of Canada, Montreal, Quebec H4P 2R2, Canada and the
** Joint Diseases Laboratory, Shriners Hospital for Children, and
Department of Surgery, McGill University, Montreal,
Quebec H3G 1A6, Canada
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ABSTRACT |
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The autocatalytic processing of procathepsin L was investigated in vitro using purified recombinant proenzyme expressed in Pichia pastoris. Pure intermolecular processing was studied by incubating the mutant procathepsin L (C25S), which cannot autoactivate with a small amount of mature active cathepsin L. The results clearly establish that, contrary to recent reports, intermolecular processing of procathepsin L is possible. The main cleavage sites are located at or near the N terminus of the mature enzyme, in an accessible portion of the proregion, which contains sequences corresponding to the known substrate specificity of cathepsin L. Contrary to procathepsins B, K, and S, autocatalytic processing of procathepsin L can generate the natural mature form of the enzyme. A continuous assay using the substrate benzyloxycarbonyl-Phe-Arg 4-methylcoumarinyl-7-amide hydrochloride has also been used to obtain information on the nature of the steps involved in the autocatalytic processing of wild-type procathepsin L. Processing is initiated by decreasing the pH from 8.0 to 5.3. The influence of proenzyme concentration on the rate of processing indicates the existence of both unimolecular and bimolecular steps in the mechanism of processing. The nature of the unimolecular event that triggers processing remains elusive. Circular dichroism and fluorescence measurements indicate the absence of large scale conformational change in the structure of procathepsin L on reduction of pH. However, the bimolecular reaction can be attributed to intermolecular processing of the zymogen.
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INTRODUCTION |
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Cathepsin L is a lysosomal cysteine protease that plays a major role in intracellular protein degradation (1, 2). Like many mammalian proteases, cathepsin L is synthesized as an inactive preproenzyme, which is subsequently processed to the mature form (3, 4). Cleavage of the 96-residue proregion, which is located between the signal sequence and the N terminus of the mature enzyme, is necessary to generate the fully active 221-residue mature enzyme (5); therefore, the proregion serves as a regulator of catalytic activity. Accordingly, the propeptide of cathepsin L has been shown to be a potent inhibitor of the mature protease (6). The proregion is also required for the proper folding of the protein (7). Procathepsin L is stable at high pH, and the proregion protects the protein from the denaturing effect of neutral to alkaline pH (8). The proregion also mediates the pH-dependent membrane association of procathepsin L, which may play a role in transport to the lysosome or activation of the proenzyme (9). In addition to its role in protein degradation, evidence has accumulated for the participation of cathepsin L in various physiological and pathological processes, e.g. tumor invasion and metastasis (10, 11), bone resorption (12), spermatogenesis (13), and arthritis (14, 15). In some cases, extracellular events involve the zymogen form (e.g. Refs. 16 and 17), and the role for the proregion in regulation of the catalytic activity and stabilization of procathepsin L could be very important.
The recently determined three-dimensional structure of human procathepsin L has demonstrated the molecular basis for the inhibition of this cysteine protease by its propeptide (18). However, details of the mechanism for conversion of the zymogen to the mature enzyme remain obscure. Cleavage of the proregion to yield active mature cathepsin L can occur autocatalytically under acidic conditions (16), but it is not yet clear whether processing occurs by intramolecular and/or intermolecular events. Procathepsin L was recently reported to process exclusively by an intramolecular reaction mechanism (8, 19). However, this is difficult to visualize, considering the position of the N terminus of mature cathepsin L relative to the active site (18). In the case of procathepsin B, it has been reported that, similar to procathepsin L, processing can be activated in vitro under acidic conditions by an autocatalytic mechanism (20, 21). Evidence has been presented for the contribution of a unimolecular process (i.e. independent of proenzyme concentration) for the conversion of procathepsin B to mature cathepsin B, and this was considered to reflect an intramolecular processing step (22). However, it has been demonstrated that intermolecular processing of procathepsin B is also possible (20). In addition, the observation that proenzyme activation seems to proceed faster than proteolytic processing suggested that the mechanism involves two steps: proenzyme activation followed by cleavage to yield the mature enzyme (22).
In this study, we have obtained information on processing events by identifying the propeptide fragments generated during autocatalytic processing of procathepsin L in vitro. Pure intermolecular processing was studied by using a mutant of procathepsin L where the critical catalytic residue Cys25 is replaced by a serine, therefore preventing intramolecular cleavage of the proenzyme. The intermolecular processing was carried out by exogenously added active cathepsin L. The recombinant activable procathepsin L as well as the C25S mutant have been expressed in Pichia pastoris and purified to homogeneity. The two variants of recombinant procathepsin L have also been used to set up a continuous assay for kinetic studies and to investigate possible conformational changes that could be linked to the triggering of processing. Based on the results of these studies, structural aspects of the autocatalytic processing of procathepsin L are discussed.
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EXPERIMENTAL PROCEDURES |
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Materials-- Recombinant procathepsin L (C25S) was expressed and purified as described previously (6). In addition to the active site mutation, the proenzyme also lacks the glycosylation site (Thr110 in Asn108-Asp109-Thr110 replaced by Ala). The vector (pPic9) and P. pastoris strain GS115 were purchased from Invitrogen Corp. (San Diego, CA). The substrate benzyloxycarbonyl-L-phenylalanyl-L-arginine 4-methylcoumarinyl-7-amide hydrochloride (Cbz-Phe-Arg-MCA)1 and the irreversible inhibitor E-64 (1-[[(L-trans-epoxysuccinyl)-L-leucyl]amino]-4-guanidino)butane) were purchased from IAF Biochem International Inc. (Laval, Canada). The cathepsin L propeptide (4p-90p)2 used for fluorescence and circular dichroism measurements was obtained as described previously (6).
Expression and Purification of Recombinant Human Procathepsin L
and Cathepsin L--
Human procathepsin L was expressed in P. pastoris as an -factor fusion construct as described previously
for procathepsin L (C25S) (6). The culture medium (pH 6.5-7.0) was
centrifuged (4000 × g, 15 min), and the supernatant
was concentrated using an Amicon Spiral Concentrator with a YM10
membrane followed by a stirred cell with an Amicon PM10 membrane. The
sample was then dialyzed overnight at 4 °C against 25 mM
Tris-HCl buffer, pH 8.0, containing 2 mM EDTA. The solution
was applied to a DEAE-cellulose column (2.5 × 17.5 cm)
equilibrated with the previous buffer (pH 8.0) at a flow rate of 2 ml/min. Elution was carried out using a 500-ml linear gradient to 0.5 M NaCl. The presence of procathepsin L in the fractions was
detected by SDS-PAGE and measurement of activity under conditions for
processing to the mature enzyme. Since, contrary to mature cathepsin L,
the proenzyme is stable at high pH (16), purification of the proenzyme
at pH 8 ensures that no mature enzyme is present in the procathepsin L
preparations. The yield of procathepsin L was approximately 4 mg/liter
of buffered minimal glycerol complex culture medium.
Identification of Processing Products-- The propeptide fragments generated during processing of procathepsin L were separated and purified by reverse-phase HPLC on an analytical Vydac C4 column (4.6 × 250 mm) using a flow rate of 1 ml/min and a two-step gradient (0-30% CH3CN in 10 min followed by 30-50% CH3CN in 35 min, all in 0.1% trifluoroacetic acid). The identity of the propeptide fragments was assigned by N-terminal sequencing using an Applied Biosystems 473A pulse liquid sequencer and mass spectrometric analysis on a triple quadrupole mass spectrometer (API III LC/MS/MS system; Sciex, Thornhill, Canada). Processing was also followed by SDS-PAGE followed by electrophoretic transfer onto a Pro-Blott membrane (ABI), and the N-terminal sequence of cathepsin L obtained after maturation of the proenzyme was determined.
Fluorogenic Assay for Monitoring Proenzyme Processing-- Processing of procathepsin L was followed in a continuous manner by carrying out the reaction in a 3-ml quartz fluorimeter cuvette in the presence of the substrate Cbz-Phe-Arg-MCA and measuring fluorescence as a function of time. The conversion of inactive procathepsin L to active enzyme leads to hydrolysis of the substrate, and fluorescence of the MCA product was monitored using excitation and emission wavelengths of 380 and 440 nm, respectively. Processing was initiated by lowering the pH from 8.0 (pH of the stock solution of procathepsin L) to 5.3 (pH of the assay). Reactions were carried out at 25 °C in presence of 50 mM sodium citrate buffer, 0.2 M NaCl, 2 mM EDTA, 2 mM dithiothreitol, and 10% CH3CN. The reaction mixture was stirred continuously in the cuvette during the reaction. The product versus time curves were fitted to the equation,
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(Eq. 1) |
Circular Dichroism and Fluorescence Measurements--
Circular
dichroism measurements were performed on a Jobin Yvon CD6 dichrograph
as described previously (6). Spectra were recorded at 25 °C in 20 mM buffer (sodium acetate, pH 4.0, sodium phosphate, pH 6.0 and 8.0) and in the presence of 10% CH3CN. Samples were
preincubated for several hours in each buffer before recording the
spectra. The concentration of procathepsin L (C25S) was 28 µM. The results are expressed in terms of molar
ellipticity []. Fluorescence spectra were recorded at 25 °C on
an Aminco SLM 4800C spectrofluorimeter in the ratio mode using an
excitation wavelength of 287 nm. The concentrations of procathepsin L
(C25S) and cathepsin L propeptide (4p-90p) were 0.7 and 0.4 µM, respectively. For fluorescence polarization
experiments, the excitation light (287 nm; bandwidth, 4 nm) was
polarized with a Glan-Thompson polarizer, and the emission wavelength
was set at 348 nm (bandwidth, 16 nm) for the cathepsin L propeptide
(4p-90p) and 332 nm (bandwidth, 16 nm) for procathepsin L (C25S).
Tryptophan polarization was measured with the spectrofluorimeter in the
L format. Emission and excitation polarizers were alternatively changed
in vertical and horizontal orientations to measure
I
and I
. The instrument
averaging parameter was set at 20. The steady-state anisotropy
(r) was calculated using the relation r = (I
GI
)/(I
+ 2GI
), where G = I
/I
with the excitation
polarizer oriented for horizontal polarization. The r values
were averages of 15 measurements on the same sample.
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RESULTS AND DISCUSSION |
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Identification of Cleavage Sites in the Autocatalytic Intermolecular Processing of Procathepsin L-- Recombinant procathepsin L (C25S) has been expressed in P. pastoris at a yield, after purification, of approximately 10 mg/liter of buffered minimal glycerol complex culture medium. Autocatalytic intermolecular processing was investigated by incubating procathepsin L (C25S) with a small amount of mature active cathepsin L at pH 5.1. Purified procathepsin L (C25S) was detected as a single band that migrates at an apparent molecular mass of 39 kDa on SDS-PAGE (Fig. 1). Upon incubation with cathepsin L, two new bands with apparent molecular masses of 30 and 10 kDa were observed. These bands result from the conversion of the proenzyme to the single chain mature form of cathepsin L and propeptide fragment(s). It must be noted that the molecular weights of the precursor (proenzyme) and mature forms of cathepsin L are 35.8 and 24.2 kDa, respectively, but that these proteins migrate with higher apparent molecular masses on SDS-PAGE (23). The mature enzyme resulting from intermolecular processing of procathepsin L (C25S) was purified by ion exchange chromatography (SP-Sepharose), while the propeptide fragments were isolated by HPLC. The protein and peptides were subjected to N-terminal amino acid sequence analysis. In addition, the propeptide fragments were also characterized by ion spray mass spectrometry. The results are given in Table I.
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Continuous Monitoring of Wild-type Procathepsin L Autocatalytic
Processing--
To obtain information on the nature of the steps
involved in the autocatalytic processing of wild-type procathepsin L,
we have used a continuous assay based on the hydrolysis of the
substrate Cbz-Phe-Arg-MCA by the active enzyme generated in the
process. Typical progress curves obtained at varying concentrations of the proenzyme are shown in Fig.
3A. The rate of substrate
hydrolysis increases with time due to time-dependent
release of cathepsin L from procathepsin L until a constant rate is
obtained that corresponds to the activity of fully processed cathepsin
L. The curves can be fitted to a model considering a first order
increase in rate from an initial value vPE, which
should correspond to the activity of procathepsin L, to a final value
vE, which corresponds to the activity of mature
cathepsin L (Fig. 3B). From the results of nonlinear
regression analysis, no significant activity of the proenzyme against
the substrate Cbz-Phe-Arg-MCA could be detected within experimental
error. The first-order rate constant for autocatalytic processing,
kobs, can be replotted as a function of the
proenzyme concentration in each assay. As shown in Fig. 3C,
the rate of processing, kobs, increases linearly with proenzyme concentration, which indicates the presence of a
bimolecular reaction most probably corresponding to intermolecular processing of procathepsin L by active cathepsin L. In addition, as the
concentration of proenzyme tends toward 0, the extrapolated rate
constant is equal to 0.9 × 103 s
1,
indicating also the presence of a unimolecular event, independent of
the concentration of the proenzyme, in the processing of procathepsin L.
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Circular Dichroism and Fluorescence Measurements--
Processing
of procathepsin L occurs by an autocatalytic mechanism at low pH while
the proenzyme is stable at pH 8.0. To probe for possible conformational
changes at low pH required for triggering of processing, we have used
circular dichroism and fluorescence measurements of procathepsin L
(C25S). The near-UV CD spectrum is sensitive to a asymmetry of aromatic
side chains, particularly for tryptophan residues, and can give a
qualitative evaluation of changes in tertiary structure. A strong
increase in circular dichroism was observed previously when the
cathepsin L propeptide bound to cathepsin L (6). This was attributed to
the presence of hydrophobic interactions involving tryptophan residues
in the cathepsin L-propeptide complex, which are absent in the
propeptide or cathepsin L alone. From examination of the procathepsin L
crystal structure, it can be seen that three tryptophan residues
(Trp12p, Trp15p, and Trp35p) form
part of the hydrophobic core of the N-terminal globular domain of the
proregion (18). In addition, tryptophan residues are also found at the
binding site of the proregion on the enzyme (Trp189,
Trp193). If conformational changes occur involving these
portions of the proregion, we would expect the near-UV CD spectrum to
be affected. As shown in Fig.
4A, there is virtually no
difference in the near-UV circular dichroism signals for pH 4.0, 6.0, and 8.0. The far-UV CD spectrum gives a measure of the protein
secondary structure. The signal observed for procathepsin L (C25S) with
a maximum at 190 nm and two minima at 210 and 220 nm is characteristic
of an /
protein. Again very little difference is observed between the CD spectra obtained at various pH values (Fig. 4B).
These results indicate that there is no large scale conformational
change in the structure of procathepsin L when the pH is lowered from 8.0 to 4.0. If conformational changes do occur, they must be very localized and minimal or involve only a small fraction of the molecules
in solution at any one time and thus go undetected by CD.
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Nature of the Steps Involved in the Autocatalytic Processing of Procathepsin L-- The results of the present study clearly establish that intermolecular processing of procathepsin L occurs and can generate the correct mature form of the enzyme. Cleavage takes place mainly in a portion of the proregion located close to or at the N terminus of the mature enzyme. The intermolecular cleavage sites are accessible and contain sequences corresponding to the known substrate specificity of cathepsin L. By comparison, procathepsins B, K, and S yield mature enzyme forms containing N-terminal extensions of several amino acids (20, 27, 28). In a recent study published while this manuscript was in preparation, evidence was obtained that also supports the involvement of an intermolecular process in the activation of procathepsin K (27). Cleavages were observed close to the C-terminal portion of the proregion, as observed in the present study. The preferred cleavage sites in procathepsin K occurred one or two amino acids after each proline residue in the proregion. These sites are located in regions that are predicted to be fairly accessible for proteolysis based on the structure of procathepsin L.
Evidence supporting the involvement of a unimolecular step in the processing of procathepsin L was also obtained in the present study using a continuous assay. The existence of a unimolecular event in the processing mechanism of cysteine protease zymogens has been suggested previously based on similar experiments with procathepsin B and propapain (22, 31). Although that process has frequently been ascribed to intramolecular processing of the proenzyme, direct evidence for intramolecular cleavage has been difficult to obtain, and the nature of this event remains elusive. Whether or not the unimolecular step corresponds to an intramolecular process cannot be ruled out or directly supported by the present work. By comparing the three-dimensional structures of cathepsin L (32) and procathepsin L (18), no rearrangement of the N terminus of the mature enzyme is observed following processing; therefore, intramolecular cleavage cannot occur directly at the N-terminal position of cathepsin L unless major conformational changes are involved. The initial intramolecular proteolytic cleavage must occur elsewhere in the proregion, generating a catalytically active processing intermediate that could be further processed to yield mature cathepsin L. No processing intermediates other than those resulting from cleavage near the C terminus of the proregion have been observed in this study. However, such intermediates might be unstable (i.e. rapidly degraded) or present in such small amounts as to be undetectable. The existence of a processing intermediate has been suggested for the processing of recombinant propapain, but the intermediate could not be identified (31). Possible insight into the nature of such an intramolecular cleavage was provided recently by a very interesting study using a mutant of propapaya proteinase IV (24). Propapaya proteinase IV cannot autoprocess, presumably due to its own restricted specificity for glycine in P1. A mutant of propapaya proteinase IV containing a glycine residue at the proregion-mature enzyme boundary was shown to process autocatalytically. Interestingly, a partially processed proenzyme was observed, resulting from cleavage after a glycine residue located at a position corresponding to Gly77p in procathepsin L, and it was proposed that this cleavage could correspond to the initial proteolytic event. In the crystal structure of procathepsin L, it can be seen that Gly77p is located at the active site, in proximity to the catalytic cysteine residue. From a sequence alignment of 39 cysteine protease precursors (18), this residue is usually found to be a small amino acid, allowing the proregion segment to pack in close proximity to the active site cysteine residue of the proenzyme. It must be noted, however, that the proregion binds in the opposite direction to that of a normal substrate and cannot be cleaved by the normal catalytic mechanism (33). Alternatively, the unimolecular step could represent a process other than an intramolecular cleavage reaction. For example, a rate-limiting conformational change that exposes the active site and renders the proenzyme active (e.g. dissociation of the proregion from the active site) would correspond to a unimolecular process. An "activated" proenzyme could then cleave another procathepsin L molecule by an intermolecular mechanism. In that regard, it is interesting to note that the rate constant for the unimolecular process obtained from the intercept in Fig. 3B (9 × 10 ![]() |
ACKNOWLEDGEMENT |
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We thank Dr. John D. Doran for helping with the procathepsin L cloning.
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FOOTNOTES |
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* This work was supported by grants from the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazil (to E. C.), NATO, and the Ministère de la recherche et de l'espace, France (to E. D.). This is NRCC Publication 41400. The Biotechnology Research Institute and McGill University are members of the Protein Engineering Network of Centres of Excellence, sponsored by the government 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.
§ To whom correspondence should be addressed: Biotechnology Research Institute, 6100 Royalmount Ave., Montreal, Quebec H4P 2R2, Canada. Fax: 514-496-5143; E-mail: robert.menard{at}nrc.ca.
¶ Visiting scientist from Instituto Butantan, C.P. 65, 05504, São Paulo, Brazil.
Visiting scientist from Institut National de la Recherche
Agronomique, BP 1627, 44316 Nantes Cedex 03, France.
1 The abbreviations used are: Cbz-Phe-Arg-MCA, benzyloxycarbonyl-L-phenylalanyl-L-arginine 4-methylcoumarinyl-7-amide hydrochloride; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography.
2 Residues in the propeptide are identified with the suffix p. Numbering is based on the sequence of human procathepsin L starting at the N-terminal amino acid of the proregion (residue 1p). The C-terminal amino acid of the proregion corresponds to position 96p and is followed by the N-terminal residue (position 1) of the mature enzyme.
3 R. Ménard, E. Carmona, S. Takebe, and R. Coulombe, unpublished data.
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REFERENCES |
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