pH-induced Conformational Transitions of the Propeptide of
Human Cathepsin L
A ROLE FOR A MOLTEN GLOBULE STATE IN ZYMOGEN
ACTIVATION*
Roman
Jerala
§,
Eva
erovnik¶,
Jurka
Kidri
, and
Vito
Turk¶
From the
Laboratory for Molecular Modeling and NMR
Spectroscopy, National Institute of Chemistry, Hajdrihova 19, and the
¶ Department of Biochemistry and Molecular Biology, Jozef Stefan
Institute, Jamova 39, 1000 Ljubljana, Slovenia
 |
ABSTRACT |
Synthesis of proteases as inactive zymogens
is a very important mechanism for the regulation of their activity. For
lysosomal proteases proteolytic cleavage of the propeptide is triggered by the acidic pH. By using fluorescence, circular dichroism, and NMR
spectroscopy, we show that upon decreasing the pH from 6.5 to 3 the
propeptide of cathepsin L loses most of the tertiary structure, but
almost none of the secondary structure is lost. Another partially
structured intermediate, prone to aggregation, was identified between
pH 6.5 and 4. The conformation, populated below pH 4, where the
activation of cathepsin L occurs, is not completely unfolded and has
the properties of molten globule, including characteristic binding of
the 1-anilinonaphthalene-8-sulfonic acid. This pH unfolding of the
propeptide parallels a decrease of its affinity for cathepsin L and
suggests the mechanism for the acidic zymogen activation.
Addition of anionic polysaccharides that activate cathepsin L
already at pH 5.5 unfolds the tertiary structure of the propeptide at
this pH. Propeptide of human cathepsin L which is able to fold
independently represents an evolutionary intermediate in the emergence
of novel inhibitors originating from the enzyme proregions.
 |
INTRODUCTION |
All lysosomal and most other proteases are synthesized in the form
of inactive precursors (1, 2). Propeptides are generally located
N-terminal to the mature enzyme, and activation of the enzyme is
accomplished by cis- or trans-cleavage of the
propeptide. Propeptides vary from a few (e.g. trypsin) to
more than 200 residues (e.g. cathepsin C). Longer
propeptides are generally strong and specific inhibitors of their
mature enzymes (3-7). In most cases propeptides are also indispensable
for correct folding of the enzymes (8). In some enzymes folding of the
mature form is extremely slow, and the propeptide assists in overcoming
the kinetic barrier (9), which may also be overcome by physicochemical factors such as high ionic strength in subtilisin, for example (10).
Proenzymes are quite often more stable than mature enzymes (11, 12) and
can represent a pool of latent enzyme until the activation occurs in
the proper conditions. Propeptides are also involved in targeting to
specific organelles (13, 14); they can affect posttranslational
modification such as glycosylation (15) and mediate interactions with
other molecules (16, 17). Propeptides can be cleaved either by other
proteases or by intra- or intermolecular autocatalysis. pH change is
one of the most common environmental parameters responsible for
triggering the activation of proteases, occurring in cysteine, aspartic
acid, and metalloproteases (1, 18, 19). Low pH is thought either to
increase the susceptibility of the propeptide as a substrate due to the
protonation of groups close to the cleavage site or to cause a
conformational change in the propeptide or enzyme.
Cathepsin L is one of the most active cysteine proteases and accounts
for most of the lysosomal cysteine protease activity (20). It has been
implicated in a range of processes including turnover of proteins
involved in growth regulation, tumor invasion and metastasis (21), and
bone resorption (22). The mouse analogue of procathepsin is secreted
from transformed mouse fibroblasts and has been at first called major
excreted protein (11). Procathepsin L is stable under neutral and
slightly alkaline pH conditions where mature cathepsin L is rapidly
inactivated (11). The proregion of cathepsin L is compulsory for its
correct folding either in vivo (23) or in vitro
(24). Activation of cathepsin L occurs autocatalytically below pH 4 (11, 25), and also at higher pH (around pH 5.5) in the presence of
anionic oligosaccharides such as dextran sulfate or heparin (26, 27).
This mode of activation is very similar to that of other cysteine
proteases of the papain family (28, 29).
The crystal structure of the procathepsin L (30) shows that the
proregion consists of a compact domain composed of three
-helices
and a short stretch of
-strand up to the first 75 residues followed
by a less ordered, extended chain of the remaining 20 residues. This
domain lies above the active site cleft and inhibits the activity by
sterically preventing the access to the active site. The structure of
the proregion in procaricain is very similar (31), whereas in
procathepsin B it lacks the first helix (32, 33). The helical domain of
the proregion has a hydrophobic core containing three of the four
tryptophans, which provide a convenient spectroscopic probe for
investigating its conformation. The complete propeptide as well as its
fragments, which comprise the compact domain, are potent inhibitors of
cathepsin L (34). This inhibition is strongly pH-dependent
and drops sharply at the acidic pH. The main contribution of this
decrease is due to a decrease in the rate of formation of the
propeptide-enzyme complex (34). However the mechanism of
pH-dependent decrease in the Ki and resulting cathepsin L activation is unknown.
In the present study we were interested in the connection between the
conformation of the propeptide and the process of acid-induced zymogen
activation. Recombinant propeptide of cathepsin L in Escherichia coli has been prepared. We have applied fluorescence, circular dichroism, and nuclear magnetic resonance to investigate the structure of the free propeptide in solution. We show that the propeptide folds
into a defined tertiary structure at neutral pH, which has not yet been
observed for any propeptide of the lysosomal proteases. Moreover we
have found a distinct conformational change upon decreasing the pH.
Below pH 3.5, the pH of activation of the procathepsin L, the
propeptide adopts a molten globule conformation, which can explain a
decrease in the inhibitory constant and particularly the association
rate and provide a mechanism for the activation.
 |
EXPERIMENTAL PROCEDURES |
Materials
Restriction enzymes and Vent DNA polymerase were purchased from
New England Biolabs, and the DNA sequencing kit was from Amersham Pharmacia Biotech. Oligonucleotides were custom-synthesized by The
Great American Gene Co. (Ramona, CA).
Methods
Production of Recombinant Propeptides in E. coli--
The region
coding for the propeptide for human cathepsin L
(PRL)1 was amplified by
polymerase chain reaction using oligonucleotides 5'-GCGCATATGACTCTAACATTTGATCAC-3' and 5'-CGCGGATCCTACTCATAAA
ACAGAGGTTC-3'. Gene coding for the human preprocathepsin L was used as
a template. Additional methionine was added at the N terminus. For the
shortened propeptide PRL78, comprising 78 residues N-terminal of the
propeptide and including additional methionine, oligonucleotide 5'-
ATCGGGATCCTAGCCATTCATCACCTGCCTGA-3' was used instead of the 3' primer.
Polymerase chain reaction products were subcloned into the pET3a
expression vector (Novagen). Recombinant proteins were expressed in
E. coli using intracellular expression under T7 RNA
polymerase promoter inducible by 0.4 mM
isopropyl-1-thio-
-D-galactopyranoside. 15N-Labeled proteins were prepared by growing bacteria in
the M9 minimal medium supplemented with 1 g/liter 15N
NH4Cl (Isotec). Full-length propeptide and
propeptide-(1-78) of cathepsin L were produced at yields of
approximately 20 and 7 mg/liter bacterial culture. Both proteins were
produced in form of inclusion bodies, which were isolated by sonication
of the bacterial paste, suspended in 20 mM Tris, pH 8.0, 0.1 M NaCl, 0.1% Triton X-100, and centrifuged. The
resulting compact pellet was extensively washed in the same buffer and
finally in the buffer containing 1 M urea. Recombinant
proteins were purified on a gel filtration column under denaturing
conditions (8 M urea, 50 mM acetate, pH 3.3).
For the PRL refolding was performed by gel filtration thereby
transferring the protein from the denaturing buffer into 1 M urea, 20 mM Tris, pH 8.0. The protein was
further purified on the Q-Sepharose column in the presence of 1 M urea. Refolding of the PRL78 was performed by dilution
into 20 mM phosphate buffer, pH 8.0, dialyzed, and further
purified using Q-Sepharose column in the same buffer. For spectroscopic
experiments propeptides were dialyzed against the water and adjusted to
pH 8. Any precipitate was removed by filtration, and corresponding
buffer was added for each pH value.
Fluorescence--
A Perkin-Elmer model LS-50 luminescence
spectrometer was used for fluorescence measurements. Tryptophan
emission of PRL was measured using an excitation wavelength of 290 nm.
Three scans from 300 to 410 nm were recorded at a speed rate of 180 nm
per min. PRL concentration was 0.02 mg/ml. ANS fluorescence was excited at 370 nm, and the emission spectra were measured from 400 to 610 nm.
Two scans at scan rate of 220 nm per min were averaged. 10 µl of ANS
(4 × 10
3 M) was added to 620 µl of
the same PRL solutions as for the tryptophan emission, giving a molar
ratio of ANS to protein of 35. Buffers for pH measurements were 10 mM Tris-HCl, pH 8.0, 10 mM potassium phosphate,
pH 7.6 to 6, 15 mM sodium acetate, pH 5.7 to 3.8, and 15 mM glycine buffer, pH 3.5 to 2.2. pH 2 and lower was
achieved by the addition of HCl to the protein in water.
Circular Dichroism--
CD spectra were measured on an Aviv
model 62A DS CD spectrometer, equipped with a thermostated cell holder.
All measurements were performed at 18 °C to avoid thermal
denaturation and to make less mobile any structure present. Near and
far UV CD spectra were measured in cells with a path length of 10 and 1 mm, respectively. Base-line spectra were subtracted from each spectrum.
Concentration of the proteins was determined from the absorbance at 280 nm taking into account the extinction coefficients calculated from the
amino acid composition (2.27 for 1
PRL and 2.4 for PRL78). Typical concentrations of the propeptides were 20 and 5 µM for
the near and far UV CD spectra, respectively. Results were converted
into mean residue ellipticities in the far UV region (250-190 nm) and into molar ellipticities in the near UV region (340-250 nm).
Stopped-flow Fluorescence--
Stopped-flow experiments were
performed on an SX-17MV Applied Photophysics stopped-flow
spectrofluorimeter. A protein sample at a concentration of 200 µg/ml
at pH 7.2 was mixed in a stopped-flow chamber with a 50 mM
glycine HCl buffer at pH 3.5. As a control the propeptide was mixed
with 50 mM potassium phosphate buffer at pH 7.2. Experiments were performed at 18 °C.
Nuclear Magnetic Resonance--
NMR spectra were measured on an
INOVA 600 Varian NMR spectrometer equipped with a triple resonance
probe and z gradients. Concentration of PRL78 was around 0.5 mM in 20 mM buffers. Two-dimensional nuclear
Overhauser effect spectroscopy spectra were measured on proteins
dissolved in D2O at pH 7.0, without the pH adjustment due
to the solvent. Spectral width was 12 ppm with 256 increments in
indirect dimension and 2048 points in direct dimension. Free induction
decays were apodized with a sine square function.
1H-15N HSQC spectra were measured with spectral
widths of 1700 and 3500 Hz for the 15N and 1H
dimensions, respectively. Protein samples were dissolved in 90%
H2O, 10% D2O. Quadrature detection was
achieved with States-time proportional phase increment method. Pulsed
field gradients were used to suppress water signal, which was further
decreased by time domain spectral deconvolution. Data were transformed
using FELIX software (MSI) on a Silicon Graphics workstation.
 |
RESULTS |
Conformation of the Free Propeptide--
The fluorescence emission
spectrum of the recombinant propeptide of human cathepsin L (PRL) at
neutral pH has a maximum at 335 nm, showing that tryptophan residues
are predominantly in a hydrophobic environment shielded from the
solvent. The near UV CD spectrum exhibits fine structure characteristic
of a folded protein (Fig. 1) with high
positive ellipticity centered at 280 nm with a shoulder at 288 nm and a
trough at 294 nm. This indicates rigid asymmetric environments of the
aromatic residues, of which four are tryptophans. The near UV CD
spectrum does not change significantly between pH 8 and 6.5. Far UV CD
spectra indicate that the free propeptide contains a high content of
-helix (44%, predicted from the neural network program k2d (35)),
consistent with the crystal structure of the propeptide in procathepsin
L. By using ellipticity at 220 nm as a probe the propeptide undergoes a
cooperative and reversible transition upon heating with a midpoint at
45 °C (not shown), as expected for a protein with a defined tertiary
structure. Full-length propeptide aggregates at concentrations above
0.5 mg/ml, so we have also produced fragment 1-78 of the PRL (PRL78).
This fragment comprises the compact domain of the proregion and
contains all four tryptophan residues. It has a lower tendency to
aggregate compared with the full-length propeptide, being particularly
suited for the NMR experiments. Fluorescence emission maximum of the
PRL78 is at 336 nm, and near and far UV CD spectra are closely similar
to the spectra of PRL (not shown), implying that the region with
defined tertiary structure (or at least core with aromatic residues) is
limited to this region. Two-dimensional nuclear Overhauser effect
spectroscopy spectrum of the PRL78 at pH 7.0 displays many nuclear
Overhauser effects between the aromatic and aliphatic side chain
protons (Fig. 2), as additional evidence
for the tertiary structure interactions.

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Fig. 1.
Near UV CD spectra of PRL at different pH
values. Buffer concentration was 10 mM and protein
concentration was 20 µM. From top to
bottom pH values were 7.8, 6.5, 5.0, 3.6, and 2.27. Results
were converted into molar ellipticities.
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Fig. 2.
Two-dimensional nuclear Overhauser effect
spectroscopy spectrum of PRL78 in the native state, pH 7.0. Concentration of the protein was 0.2 mM. The number of
transients was 128; temperature was 18 °C; and mixing time was 150 ms.
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|
pH-dependent Conformational Changes of the
Propeptide--
Ellipticity in the near UV CD spectra decreases with
decreasing pH (Fig. 1) indicating unfolding of tertiary structure upon acidification. Fluorescence emission intensity decreases likewise with
a midpoint of transition around pH 5.5 (Fig.
3). Wavelength maximum of the
fluorescence decreases from 335 nm at pH 8 (state N) to 342 nm at pH
5.4 and persists at 346 nm in the pH range 4.8 to 3.6 (state X).
Further lowering the pH causes an additional increase in the wavelength
maximum, reaching the
max of 353 nm below pH 3, characteristic for the solvent-exposed tryptophan residues. Far UV CD
spectra remain quite similar upon decrease of pH with only a small
difference in shape at pH below 4 (Fig. 4). This indicates that the secondary
structure content of the propeptide is conserved. The acidic state
(state A) is thus characterized by a high content of the secondary
structure and the absence of the aromatic core and persistent tertiary
structure. Presence of the secondary and absence of persistent tertiary
structure are two of the characteristics of molten globule type
conformations (36). The ANS binding to propeptide, as monitored by
fluorescence emission at 478 nm, is low at neutral pH values and
increases in the pH range from 3.5 to 1.5 (Fig.
5), where there is almost no tertiary
structure left but still high amounts of secondary structure, again
confirming the presence of molten globule type conformation. At even
lower pH the propeptide transforms into an unfolded state which does
not bind ANS, as shown also for other proteins (37). Higher ionic
strength shifts the transition toward higher pH values and increases
the aggregation of the X state (not shown). The ANS molecules were
efficiently excited by the process of energy transfer from tryptophans,
when we used excitation wavelength of 293 nm. This shows a close
proximity of the hydrophobic, ANS binding surfaces to tryptophans.
Unfolding of the truncated propeptide PRL78, monitored by near and far
UV CD, followed very closely unfolding of the full-length propeptide.
PRL78 was used for the two-dimensional 1H-15N
HSQC NMR experiments due to its higher solubility. At pH 3.3, when the
protein is in the A state, dispersion of the amide cross-peaks in the
H-N plane is typical for a partially structured conformation, whereas
in the unfolded state, in the presence of 8 M urea (U state), dispersion is much decreased (Fig.
6), proving that the A state is not
completely unfolded. Stopped-flow fluorescence experiments were
performed by transferring the PRL78 from pH 7.2 to 3.5 in order to
evaluate the rate of acidic unfolding. Fluorescence decrease occurred
within the mixing time of the device (Fig.
7), indicating a rate of unfolding faster
than 100 s
1.

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Fig. 3.
Intrinsic fluorescence of the PRL as a
function of pH. Top, fluorescence intensity as a function of
pH; bottom, maximum of fluorescence emission wavelength of
PRL as a function of pH.
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Fig. 4.
Far UV CD spectra of PRL at different pH
values. Secondary structure content remains conserved. Spectra
were measured at 18 °C in 20 mM buffers and 5 µM protein concentration. Mean residue
ellipticities were calculated assuming mean residue molecular
weight of 115.
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Fig. 5.
ANS fluorescence of the PRL as a function of
pH. From top to bottom pH values were pH
2.2, 3.1, 4.3, 5.5, and 6.4. Ratio of ANS: protein = 35, exc = 370 nm, em = 478 nm (see
inset).
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Fig. 6.
Two-dimensional HSQC spectra of the A and D
states of PRL78 in H2O at pH 3.3. Top, D state
at pH 3.3 and 8 M urea; bottom, A state at pH
3.3. Spectra were recorded at 25 °C, and protein concentration was
0.2 mM; spectral widths were 1700 and 3500 Hz for the
15N and 1H dimensions; number of
transients = 16.
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Fig. 7.
Stopped-flow fluorescence of the acid
unfolding of the propeptide of cathepsin L. Propeptide in the 5 mM phosphate buffer, pH 7.2, was rapidly mixed with the 50 mM buffer at the pH 3.5 ( ) and 7.2 ( ) in the mixing
chamber of the Applied Photophysics stopped-flow
spectrofluorimeter.
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Unfolding of the Propeptide in the Presence of Anionic
Polysaccharides--
Addition of dextran sulfate to PRL78 at pH 5.5 causes a complete elimination of the near UV CD ellipticity, showing
that the tertiary structure is destroyed (Fig.
8). Ellipticity in the far UV is also
much decreased at this pH, different from the propeptide in the absence
of dextran sulfate. At lower pH the negative far UV ellipticity
increases again. In the presence of dextran sulfate at pH 5.5 aggregation at micromolar concentrations was also observed from the UV
absorption spectra. This demonstrates that dextran sulfate unfolds the
propeptide already at pH values below ~5.5.

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Fig. 8.
Unfolding of the propeptide of cathepsin L in
the presence of dextran sulfate. Near UV CD spectra of the
propeptide at the pH 5.5 in the absence ( ) and presence ( ... )
of dextran sulfate (20 µg/ml).
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 |
DISCUSSION |
Structure of the Free Propeptide--
Propeptides of proteases
characterized in the literature are either completely unfolded or
exhibit a limited content of secondary structure (38, 39), with the
exception of the 166-residue propeptide of
-lytic protease and
activation domain of carboxypeptidase B (9, 40). Secondary structure
was in some cases induced by co-solvents such as polyethylene glycol or
methanol (38, 41). We have shown that the free propeptide of cathepsin
L and its fragment (PRL78) are at neutral pH folded into a compact
structure with defined secondary and tertiary structure. The structure
has, like the proregion in zymogen, a high content of
-helix. The near UV CD spectrum is very similar to the difference between the
spectra for procathepsin and cathepsin L (compare Fig. 2 and Fig.
3A in Ref. 34), suggesting that the conformations of the free propeptide and proregion in zymogen are probably similar. The
proregion is essential for folding of cathepsin L (23), and
particularly based on its ability to fold independently, as demonstrated in this work, it is a good candidate for a folding cofactor of the mature enzyme, similar to the propeptide
-lytic protease (9) or subtilisin (42), which, however, does fold per
se. Previously it has been reported that the propeptide of cathepsin L is devoid of persistent tertiary structure (34); however,
those measurements were performed in the presence of 10% acetonitrile
and at pH 5.5, where most of the tertiary structure is already
destroyed. Propeptides of papain and papaya protease IV have also been
expressed and shown to contain some secondary structure (39).
Existence of tertiary structure in the free propeptide is particularly
interesting in light of the fact that the homologues of the proregion
of cathepsin L-cytotoxic T-cell lymphocyte antigen-2 (CTLA-2
and
CTLA-2
) are expressed in murine T-cells and mastocytes as
independent proteins (43) and are functional as inhibitors of cysteine
proteases (44). This represents an interesting demonstration of a
co-evolution of the enzyme and inhibitor as one protein (proenzyme), until the proregion is able to fold independently, which is a necessary
condition for the independent existence of the propeptide as an
autonomous protein. Of all the proregions of cysteine proteases, CTLA-2
are most similar to the proregion of cathepsin L. A phylogenetic tree
of the proregions of cathepsins L, S, K, B, and H (not shown) has been
constructed which suggests that divergence of CTLA-2 and a proregion of
cathepsin L occurred by gene duplication after divergence of cathepsin
L and other cysteine proteases. Similarity between the CTLA-2 and
proregion of cathepsin L is, as in proregions of other cysteine
proteases, concentrated in the first 80 residues, corresponding to the
compact domain. Gene structures of the CTLA-2s are not yet known.
Proregions of human and murine cathepsin L are coded by the exons 2-4
(45, 46). The second exon codes for the signal sequence and the first
helix of the proregion, whereas exon 3 starts precisely at the
beginning of the longest helix 2 and includes the hairpin with the
extended strand. Exon 4 starts at the beginning of the third helix and
includes the linker between the compact proregion domain and the
enzyme. The end of the fourth exon does not correspond to the border
between the proregion and the mature enzyme but also includes 19 residues of the mature enzyme. CTLA-2
and
, on the other hand,
are 13 residues longer than the proregion of cathepsin L, so that they could have arisen by a duplication of the first five exons of the
procathepsin L. Another plausible explanation is a point mutation in a
copy of a procathepsin L gene giving rise to a premature stop codon.
Several pseudogenes of procathepsin L have been found in human (47),
estimated to have arisen by a duplication event 40-50 million years
ago. Three of them were sequenced, and stop codons were found at amino
acid positions 35 and 59 of the proregion. Intrinsic stability of the
propeptide per se thus suggests that pseudogenes of
cathepsin L, where the termination occurs after the coding region for
the compact domain of the proregion (>78 residues of the proregion),
might in fact be functional proteins.
Acidic Unfolding of the Propeptide and Activation of the
Zymogen--
Activation of the protease zymogen must occur under
conditions where the propeptide no longer inhibits the enzyme. This can be accomplished either by cleavage of the proregion into noninhibitory fragments, generally by another protease as for example in the coagulation cascade (48), or by changes in the conformation of the
proregion in response to changes in the medium (e.g. acidic pH, binding of anionic oligosaccharides or membranes) with subsequent cleavage either intra- or intermolecularly (49). We have shown that the
free propeptide of cathepsin L undergoes a conformational transition
upon decreasing the pH. Two different, partially structured conformations were observed, one in the pH range from 6 to 4 which has
a low amount of tertiary structure and is more prone to aggregation, and the second, the acidic state, which has almost no tertiary structure but a high amount of secondary structure and is populated in
the pH range below 4, where the activation of cathepsin L occurs in vitro. This A state also binds to ANS and so has the
attributes of a typical molten globule state. It has been shown
previously that the inhibitory activity of the propeptide of cathepsin
L decreases with decreasing pH (34). This decrease is mainly due to the
decrease in the association rate constant, showing that in the acidic
pH conformational rearrangements have to be made in order to bind
propeptide to cathepsin L. The kinetic rate of dissociation, on the
contrary, is almost independent of pH. In the tertiary structure of the
proenzyme there are few electrostatic interactions between the
proregion and enzyme (30) compared with those within the proregion.
Interactions between the proregion and the enzyme are achieved mainly
through the short
-strand, the conformation of which is secured by
the second, longest, helix. The first helix of the proregion does not
form any contacts with cathepsin L, but its deletion decreases the
inhibition by 2 orders of magnitude (34), showing the importance of
tertiary structure of the propeptide for the interactions with the
enzyme. In the case of propapain, optimum pH of activation was
increased by one pH unit through the introduction of an additional
charge into the proregion by an F38H mutation (50), consistent with the role of electrostatic interactions in disruption of the tertiary structure of the proregion.
It is likely that the propeptide could be stabilized by the cathepsin L
as it has been demonstrated for subtilisin (51), where propeptide
per se does not possess persistent tertiary structure. However, procathepsin L has approximately one-tenth of the enzymatic activity of the mature cathepsin L (11), and active site inhibitors can
be bound to the proenzyme (11), indicating either an exchange between
the active and inactive forms of the proenzyme or that in solution the
complex between the enzyme and proregion is not as tight as in the
crystal. At acidic pH, once the proregion dissociates from the enzyme
it would rapidly be unfolded into the molten globule state with a low
rate of association with the enzyme and could be cleaved off either
intra- or intermolecularly. We have demonstrated with stopped-flow
experiments that at pH 3.5 unfolding from the N into the A state is a
fast process which may be able to compete with formation of the
propeptide-enzyme complex.
It has been observed for other proteases that the conformational
change accompanies the activation, as in the case of the matrix
metalloproteases (52, 53). Formation of molten globule conformations
under acidic conditions is a rather common phenomenon (54-60) and
probably occurs due to the nonspecific long range electrostatic interactions caused by protonation at low pH (61). Unfolding of a
domain of a protein could represent a more general mechanism of
regulation employing a pH switch. For this mechanism two domains of a
protein with different pH stability should be discernible and connected
by a linker as between the propeptides and cysteine proteases.
Activation in the presence of anionic oligosaccharides appears to
unfold the propeptide by unfolding its conformation and thereby
increasing its susceptibility to proteolysis. Histidine residues are
probably responsible for binding to dextran sulfate below their
pK which coincides with the pH around 5.5 where this type of
activation occurs. The binding motif of the proregion of mouse
cathepsin L which is responsible for its binding to the microsomal
membranes has been identified (62, 63). This motif is restricted to a
nonapeptide which includes several basic residues and histidines. A
similar effect to that of the anionic oligosaccharides might be
exhibited by the anionic phospholipid containing membranes, which have
been shown to shift induction of molten globule formation to higher pH
(64, 65). Molten globule conformation has been implicated in
physiological processes such as membrane insertion and pore formation
(36, 66, 67) or chaperone binding (68) and acid activation of the
proteases may be another role of this type of protein conformation.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Marko
Dolinar (Jozef Stefan Institute) for the donation of the clone
for human procathepsin L and Andreja Majerle (National Institute of
Chemistry) for help in preparation of the PRL78 expression construct.
We thank Dr. R. H. Pain for critical reading of the manuscript and
useful suggestions.
 |
Note Added in Proof |
Since the submission of the
manuscript, two papers connected with the acidic activation of cysteine
proteases have appeared. Maubach et al. (69) have described
conformational changes in the propeptide of cathepsin S, but with no
defined tertiary structure. Menard et al. (70) have shown
that the structure of the complete procathepsin L does not experience
large conformational changes upon decreasing the pH.
 |
FOOTNOTES |
*
This work was supported by the Slovenian Ministry of Science
and Technology.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: Laboratory for
Molecular Modeling and NMR Spectroscopy, National Institute of Chemistry, POB 3034, Hajdrihova 19, 1000 Ljubljana, Slovenia. Fax: 386 61 125 9244; E-mail: roman.jerala{at}ki.si.
1
The abbreviations used are: PRL, propeptide of
cathepsin L; ANS, 1-anilinonaphthalene-8-sulfonic acid.
 |
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