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INTRODUCTION |
Heparan sulfate is an ubiquitous glycosaminoglycan of animal cells
(1). These classes of compounds are heteropolysaccharides made up of
repeating units of disaccharides, an uronic acid residue, either
D-glucuronic acid or L-iduronic acid, and
D-glucosamine with N- and
6-O-sulfates and N-acetyl substitutions (2).
Heparan sulfate occurs at the cell surface and in extracellular matrix as proteoglycans. Most of cellular heparan sulfate derives from the
syndecans and glypicans proteoglycans. The syndecan family are
associated with the cell membranes via transmembrane core proteins (3,
4), and the glypican family is anchored by glycosilyl
phosphatidylinositol-anchor core proteins (5). Also, heparan sulfate
proteoglycans are present in basement membranes performing the
perlecan family (6).
Heparan sulfate and heparin are particular among glycosaminoglycans in
their ability to bind a large number of different proteins. Heparin-like glycosaminoglycans play a complex role in the
extracellular matrix, regulating a wide variety of biological process,
including hemostasis, inflammation, angiogenesis, growth factors, cell
adhesion, and others (7). Proteolytic enzymes control many of these
biological process. Several reports in the literature have demonstrated
that heparin-like glycosaminoglycans can modulate the activity of some serine proteinases and their natural inhibitors (8-11). On the other
hand, the interaction of cysteine proteinases with glycosaminoglycans has not been completely explored. In mammalians, lysosomal cysteine proteinases have been implicated in a variety of diseases involving tissue remodeling states, such as inflammation (12), parasite infection
(13), and tumor metastasis (14). Cathepsin B shows close structural
homology to the other cysteine proteases of the papain family (15). The
main feature that distinguishes cathepsin B is the presence of a large
insertion loop structure, termed occluding loop, which covers the
active site, occupying the S2'- S3' subsites of the enzyme (16).
It has been shown that lysosomal cysteine proteinases, specially
cathepsin B, can participate in tumor invasion by degradation of
extracellular matrix components (17). This can take place either
intracellularly by heterophagosomal activity of tumors cell (18) or
extracellularly by cell surface associated cathepsin B (14). It has
been demonstrated that the presence of cathepsin B at plasma membrane
results in focal dissolution of extracellular matrix proteins and
enables the tumor cell to invade (19, 20). Trafficking and targeting of
lysosomal enzymes is mostly mediated by mannose-6-phosphate receptor
pathways (21). However, several reports show that this class of
receptors is not sufficient for targeting of lysosomal enzymes along
intracellular routes, either by an alteration in these receptors (22)
or by changes in glycosylation pattern of lysosomal enzymes as observed
for cathepsin B in carcinoma cells (23). Indeed,
mannose-6-phosphate-independent targeting has been proposed for
cathepsin B in normal cell (24) and human colon carcinoma cell lines
(25). A high level of cathepsin B and qualitative changes in cathepsin
B protein expression, including abnormal pattern of glycosylation, may
be important in maintaining the malignant phenotype in carcinoma cell
(23). Alterations in cathepsin B expression, processing, and cellular
localization have been observed in several human tumor tissue; clinical
investigations have shown that cathepsin B are highly predictive
indicator for prognosis and diagnosis in cancer (26).
The mechanism of secretion and insertion of cathepsin B at the plasma
membrane are not fully understood (27). Cathepsin B is secreted by
normal and by tumor cells as the precursor forms (28), whereas many
types of tumors cells may also release mature, active form of cathepsin
B (29). However, it is not know whether the precursors are activated at
the plasma membrane or extracellularly. It has been shown that the
activation of cathepsins B and L occur autocatalytically triggered by
acidic pH and also by anionic polysaccharides such as dextran sulfate
and heparin (30, 31). The mature form of cathepsin B and L have been
shown to be rapidly inactivated at neutral or alkaline pH end by its
endogenous proteins inhibitors, mainly from the cystatin family (15).
On the other hand, it has been shown that membrane-bound forms of
cathepsin B are very resistant to inactivation at neutral pH (32).
Recently, we have shown that heparin and heparan sulfate bind papain
specifically; this interaction induces an increase of
-helix content
of papain, which stabilizes the enzyme structure even at alkaline pH
(33). These results strongly suggest that heparan sulfate may be an
important binding site of cysteine proteinases at cell surface and
basement membrane. Therefore, the study of the interaction of cathepsin
B with glycosaminoglycans is of significant interest for the
understanding about the biological role of this enzyme. In this work, a
combination of circular dichroism analysis, affinity chromatography,
cathepsin B mutants, and fluorogenic substrate assays were used to
characterize the interaction of cathepsin B with glycosaminoglycans.
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EXPERIMENTAL PROCEDURES |
Materials--
Cathepsin B from human liver was purchased from
Calbiochem Co.; nonglycosylated cathepsin B S115A, denominated
wild-type cathepsin, and its punctual mutants H110A and H111A were
subcloned, as proenzymes, into the vector pPIC9 and expressed in yeast
Pichia pastoris as described previously (34). The
concentration of the active enzyme was determined by titration using
the cysteine proteinase inhibitor E-641 (35). Active forms of
cathepsin B were stored at 4 °C in 50 mM sodium acetate
buffer (pH 5.0) containing 10 µM methyl
methane-thiosulfonate. The irreversible inhibitor E-64, azocasein, the
fluorogenic amidomethylcoumaryl substrate Cbz-FR-MCA, and dextran
sulfate (5,000 Da) were purchased from Sigma. The intramolecularly
quenched fluorogenic substrate Abz-FRA(
-Dnp)K was synthesized using
solid phase chemistry as described previously (36). In the experiments
we have used a size-defined (10 kDa) bovine lung heparin (The
Upjohn Co.), prepared by using size exclusion column approach (2, 7);
heparan sulfate (16,000 Da) from bovine lung were a generous gift from Dr. P. Bianchini (Opocrin Research Laboratories, Modena, Italy) (2);
dermatan sulfate (12,000 Da) and chondroitin sulfate (25,000 Da) were
purchased from Seikagaku Kogyo Co. (Tokyo, Japan). Heparin-Sepharose resin was purchased from Amersham Pharmacia Biotech.
Kinetic Measurements--
Cathepsin B activities were monitored
spectrofluorometrically using the fluorogenic substrates Cbz-FR-MCA and
Abz-FRA(
-Dnp)K on a thermostatic Hitachi F-2000 spectrofluorometer.
For Cbz-FR-MCA assays, the excitation and emission wavelengths were set
at 380 and 460 nm, respectively. The assay with Abz-FRA(
-Dnp)K was
monitored at 420 nm using an excitation wavelength of 320 nm (37).
Prior to the assay, the methyl methane-thiosulfonate-inhibited enzymes were activated by incubation for 5 min at 25 °C in 50 mM
sodium phosphate (pH 6.0), 200 mM NaCl, 1 mM
EDTA, and 2 mM dithiothreitol. For the determination of pH
activity profiles, the kinetics of Cbz-FR-MCA and Abz-FRA(
-Dnp)K
hydrolysis were performed in absence or in presence of different
glycosaminoglycans concentrations at 37 °C in 50 mM
sodium phosphate (pH 5.8-8.0), 50 mM citrate (pH 3.0-5.9)
containing 200 mM NaCl, 1 mM EDTA, and 2 mM DTT. The substrate concentrations were kept 20-fold
below the Km values. The progress of the reaction
was continuously monitored by the fluorescence of the released product.
The initial rates were determined, and the
kcat/KM values were obtained by dividing the initial rates by enzyme and substrate concentrations. The pH activity profiles for cathepsin B are extremely complex, which
precludes the use of well defined models to curve fit the data.
Therefore, the empirical equations were included solely for clarity of
the graphical display (34). The kinetic model depicted in Equation 1
can describe the effect of heparin on the hydrolysis of
Abz-FRA(
-Dnp)K by cathepsin B.
|
(Eq. 1)
|
where S is Abz-FRA(
-Dnp)K, I is heparin,
KS is the substrate dissociation constant, and
KI is heparin dissociation constant. The influence
of heparin upon proteolytic activity of cathepsin B was also assayed
against protein substrate using azocasein as described previously (38).
Effect of Heparin on E-64 Induced Inactivation of Cathepsin
B--
The kinetics of cathepsin B inactivation by E-64 was done under
pseudo first-order conditions at various heparin concentration as
described previously (33).
The Alkaline pH-induced Inactivation--
The kinetics of
alkaline pH-induced inactivation of cathepsins B and its mutants were
done at different glycosaminoglycan concentrations in 50 mM
Tris-HCl buffer (pH 8.0) containing 200 mM NaCl, 1 mM EDTA, and 2 mM dithiothreitol. The
inactivation of the enzymes was performed in the presence of 10 µM Cbz-FR-MCA. Progress of the reaction was monitored
continuously by the fluorescence of the released product. The obtained
exponential decay curves could be best fitted according to the
first-order relationship shown in Equation 2.
|
(Eq. 2)
|
where P and P
are the
product concentrations at a given time and at infinite time,
respectively, and kobs is the observed first-order rate of alkaline pH-induced enzyme inactivation (39). The
influence of heparin and heparan sulfate upon the observed first-order
rate cathepsins B inactivation can be described by Equation 3.
|
(Eq. 3)
|
where keh and ke
are the observed first-order rates in presence and in absence of
heparin, respectively; KH is the apparent
heparin-cathepsin B dissociation constant; Hep is heparin; and
is
the parameter of limit for kobs in presence of heparin.
Identification of Cleavage Site for Substrates--
The
determination of the cleaved bonds for the peptide Abz-FRA(
-Dnp)K by
cathepsin B was done by HPLC and mass spectrometry analysis as
described previously (33).
Circular Dichroism Spectrometry--
Circular dichroism spectra
were recorded in a JASCO J-700 spectropolarimeter equipped with a
stopped flow chamber and thermostated cell holder. Far ultraviolet
measurements (260-200 nm) were performed at 37 °C scanning at rate
of 10 nm/min on wild-type cathepsin B solution of 0.05 mg/ml in 0.05-cm
cells. Circular dichroism spectra of cathepsin B-glycosaminoglycans
interactions were done in 50 mM sodium phosphate buffer (pH
6.0) containing 200 mM NaCl. The observed ellipticity was
normalized to units of degrees cm2/dmol. Base-line
recordings in the presence or in absence of glycosaminoglycans were
routinely made and used to correct cathepsin B spectra. Cathepsin B
spectra were analyzed for the percentage of secondary structural elements as described previously (40).
Effect of Glycosaminoglycans on Alkaline pH-induced Unfolding of
Cathepsin B--
The amount of
-helix structure disruption of
cathepsin B induced by alkaline pH was continuously monitored by CD
spectra measuring the mean residue ellipticity at 222 nm. Alkaline
pH-induced denaturation of cathepsin B was performed in absence or in
presence of different heparin concentrations at 37 °C in 50 mM Tris-HCl buffer (pH 8.0) containing 200 mM
NaCl. The influence of alkaline pH on
-helix structure of wild-type
cathepsin B is a first-order relationship described by Equation 4
(39).
|
(Eq. 4)
|
where Ehelix is the amount of
-helix
structures of wild-type cathepsin B at a given time of pH 8.0 exposition; E0 is cathepsin B
-helix content,
dependent on alkaline pH, at zero time of inactivation; Kunfolding is the observed first-order rate of
pH 8.0-induced unfolding of cathepsin B; and R is the
residual
-helix content, independent of pH 8.0.
Heparin-Sepharose Affinity Chromatography--
Wild-type
cathepsin B (1 µM) dissolved in 50 mM sodium
phosphate buffer (pH 6.0) was applied on a heparin-Sepharose column (3 ml) previously equilibrated at 4 °C in the same buffer. A linear NaCl gradient (0-1 M) was used to elute the bonded
material. The collected fractions were monitored by cathepsin B
enzymatic activity upon substrate Cbz-FR-MCA.
 |
RESULTS |
Cathepsin B Binds Heparin-Sepharose--
The interaction of
cathepsin B with heparin was observed on heparin-Sepharose affinity
chromatography. Cathepsin B was eluted from heparin-Sepharose column at
0.42 M of ionic strength (Fig. 1). This interaction could be disrupted
specifically by the previous addition of 100 µM of
heparin or heparan sulfate to cathepsin B solution. Other sulfated
glycosaminoglycans, such as dermatan sulfate and chondroitin sulfate
were not capable of dislodge this binding. These results show that the
binding of cathepsin B to heparin is specific and seems to be governed
mainly by electrostatic interactions. These data led us to investigate
the possible influence of glycosaminoglycans upon cathepsin B
activity.

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Fig. 1.
Cathepsin B binds heparin-Sepharose
column. Cathepsin B (1 µM) dissolved in 50 mM sodium phosphate buffer (pH 6.0) was chromatographed on
a heparin-Sepharose column ( ). Cathepsin B was preincubated with 100 µM heparin and then submitted to heparin-Sepharose column
( ). A linear NaCl gradient (0-1 M) was used to elute
the bonded material.
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The Influence of Heparin upon Cathepsin B pH Activity
Profiles--
Cathepsin B is generally considered to possess both
endo- and exopeptidase activity (41-43). To study the influence of
heparin upon endopeptidase activity of cathepsin B, we used the
substrates Cbz-FR-MCA to analyze the subsites S2 to
S1 of cathepsin B. To monitor the influence of heparin upon
dipeptidyl carboxypeptidase activity of cathepsin B, we used the
substrate Abz-FRA(
-Dnp)K, covering the cathepsin B subsites from
S2 to S'2. The HPLC and mass spectrometry
analysis showed that Arg-Ala is the only peptide bond cleaved by
cathepsin B on substrate Abz-FRA(
-Dnp)K. The presence of heparin did
not change the pattern of cleavage of these peptides by cathepsin B.
The pH activity profile for the dipeptidyl carboxypeptidase activity of
cathepsin B is very different from that observed for its endopeptidase
activity; the maximum activity observed for the hydrolysis of substrate
Abz-FRA(
-Dnp)K was at pH 5.0 (Fig. 2B), whereas the pH dependence
for hydrolysis of Z-FR-MCA is characterized by a gradual increase in
activity when the pH is raised from 3 to 8 (Fig. 2A). The
effect of heparin on pH activity profiles of wild-type human cathepsin
B was analyzed by monitoring the enzyme-catalyzed hydrolysis of the
fluorogenic substrates. As showed in Fig. 2, when cathepsin B was
assayed with substrate Z-FR-MCA in the presence of 100 µM
heparin, no significant effect on the endopeptidase pH activity
profiles was observed (Fig. 2A). On the other hand, when
heparin was assayed with the substrate Abz-FRA(
-Dnp)K and used to
monitor the dipeptidyl carboxypeptidase activity of cathepsin B (Fig.
2B), a dramatic effect of heparin was observed upon the pH
activity profile. Basically, heparin promoted a general decrease in
observed Abz-FRA(
-Dnp)K hydrolysis second-order
kcat/Km rates and shifted the
cathepsin B pH activity profile about 0.5 unit to the right. This
inhibition promoted by heparin is strongly dependent on pH, because no
inhibition was observed when cathepsin B was assayed above pH 7. Also,
the inhibition promoted by heparin at pH 5.0 is very lower than that promoted at pH 6.0. More importantly, heparin did not show effect upon
E-64 cathepsin B inactivation activity (data not shown), and E-64 binds
the subsites Sn of cathepsin B (44). These data suggest that the occluding loop of cathepsin B is controlling the
heparin efficiency, and that the subsites S'n
are involved in the interaction with heparin.

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Fig. 2.
The pH activity profiles of cathepsin B. The kinetics of Cbz-FR-MCA (A) and Abz-FRA( -Dnp)K
(B) hydrolysis were performed in absence ( ) or in the
presence 100 µM heparin concentration ( ) at 37 °C
in 50 mM sodium phosphate (pH 5.9-7.9), 50 mM
citrate (pH 3.0-5.8) containing 200 mM NaCl, 1 mM EDTA, and 2 mM dithiothreitol.
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|
Inhibition of the Exopeptidase Activity of Cathepsin B by
Heparin--
The effect of heparin upon cathepsin B exopeptidase
activity, at pH 6.0, can be described by a linear competitive type
inhibition depicted in Equation 1. The influence of heparin upon
substrate dissociation constant is observed in Fig.
3. Basically, heparin-cathepsin B
interaction did not affect Abz-FRA(
-Dnp)K catalytic constant; the
presence of the substrate Abz-FRA(
-Dnp)K at active site of cathepsin
B excludes the binding of heparin. The results show that heparin only
binds free cathepsin B with a dissociation constant of
KI = 41 ± 3 µM.

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Fig. 3.
Effect of heparin on affinity of the
cathepsin B for Abz-FRA( -Dnp)K. The
influence of heparin concentration on cathepsin B dipeptidyl
carboxypeptidase activity was determined spectrofluorometrically as
described under "Experimental Procedures."
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The exopeptidase activity of cathepsin B is related to the presence of
two histidine residues, His110 and His111,
located at occluding loop performing the
S'2 subsite. His110 and
His111 residues can interact with the C terminus
carboxylate of a substrate (16, 34, 45). Site-directed mutagenesis has
been used to investigate the importance of the His110 and
His111 for the effect promoted by heparin upon dipeptidyl
carboxypeptidase activity of cathepsin B. The effect of the
substitution of the His110 or His111 residues
by alanine can be observed in Table I.
Kinetic parameters, determined using the C terminus free carboxylate
substrate, Abz-FRA(
-Dnp)K, showed that the substitution of
His110 or His111 residues by alanine promoted a
significant decrease in the peptidyldipeptidase activity of cathepsin
B, as expected (45). The
kcat/KM value observed for
the wild-type enzyme was 1.31 × 106
M
1·s
1, and the
kcat/KM value was about
6.5-fold lower for the mutant H111A and about 3.5-fold for the mutant
H110A. These effects are related to the decrease in
kcat; little effect in KM was
observed. Surprisingly, the substitution of His111 residue
by alanine was able to abolish the effect promoted by heparin upon
substrate dissociation constant; the KM value was
the same in the presence of 100 µM heparin (13.9 ± 0.9 µM) as in the absence of heparin (16.3 ± 0.8 µM). On the other hand, heparin showed similar effect
upon mutant H110A heparin and wild-type enzyme; for both enzymes, the
substrate dissociation constant was increased 3.5-fold by 100 µM heparin. These data indicate that heparin changes the
dissociation constant of the cathepsin B-substrate interaction for the
substrates able to reach the S'2
interaction site. The interaction of heparin with wild-type cathepsin B
and H110A and H111A mutants did not affect the
kcat values.
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Table I
Kinetic parameters for hydrolysis of Abz-FRA ( -Dnp)K by wild-type
and mutants of human cathepsin B and in the presence of heparin
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Effects of Glycosaminoglycans on pH-induced Inactivation of
Cathepsin B--
The deprotonation of the catalytic residue
His199, catalyzed by OH
ions, is considered a
crucial event for the alkaline irreversible inactivation of cathepsin
B. The alkaline pH-induced inactivation, as well as the unfolding of
cathepsin B, have been shown to be a first-order process (39). Fig.
4 shows the influence of heparin upon the
first-order inactivation rate of cathepsin B at pH 8.0. Clearly, the
data show that presence of heparin is decreasing the first-order rate
of cathepsin B inactivation at pH 8.0. The kinetic parameters were
determined by fitting the data to Equation 3 using nonlinear
regression. The results show that heparin binds wild-type cathepsin B
with a dissociation constant of KH = 18 ± 2 µM. Also, heparin induced a 5.1-fold decrease in the first-order inactivation rate of cathepsin B; the
kobs value was decrease from 8.2 ± 0.6 ms
1 to 1.6 ± 0.1 ms
1 (Table
II).

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Fig. 4.
Effects of heparin on pH-induced inactivation
of cathepsin B. The influence of heparin concentration on kinetics
of alkaline pH-induced inactivation of cathepsins B (A) were
done in 50 mM Tris-HCl buffer (pH 8.0) containing 200 mM NaCl, 1 mM EDTA, and 2 mM
dithiothreitol, in the presence of 10 µM Cbz-FR-MCA at
various heparin concentration: control (+) and 5 (*), 10 ( ), 20 ( ), 40 ( ), 80 ( ), 120 ( ), and 200 ( ) µM
heparin. Progress of the reaction was monitored continuously by the
fluorescence of the released product. B, cathepsin B
first-order inactivation rate in function of heparin
concentration.
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Table II
Effect of heparin on the rate of inactivation of wild-type and mutants
of human cathepsin B at pH 8.0
t1/2 = ln2/kobs in the absence of
heparin and teh1/2 in the presence of
heparin. kobs is the observed first-order rate of
alkaline pH-induced enzyme inactivation. keh and
ke are observed first-order rates in presence and in
absence of heparin, respectively; KH is the apparent
heparin-cathepsin B dissociation constant. is the ratio of mutant
and wild type cathepsin B forms dissociation constants, and is the
parameter of limit for kobs in presence of heparin.
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|
The mutants H110A and H111A were also used to investigate the
importance of histidine residues in the alkaline pH-induced inactivation of cathepsin B. As shown previously, the deletion of the
occluding loop from cathepsin B resulted in a decrease of the pH and
thermal stability of enzyme (45). Also, it has been shown that
His110-Asp22 salt bridge is an important
contact between the occluding loop and the central
-helix, and this
interaction can contribute to the stability of these structural
elements. On the other hand, His111 residue is not involved
in electrostatic interactions with elements of the enzyme (16, 34). The
effect of heparin and the substitution of the His110 or
His111 residues by alanine on the alkaline pH-induced
inactivation of enzymes can be observed in Table II. The data showed in
Table II are consistent with these propositions. The change of
His110 by alanine promoted a 4-fold decrease in the pH
stability as the inactivation of the mutant H110A occurred with a
t1/2 of 22 s compared with 84 s for the
wild-type enzyme. On the other hand, the change of His111
by alanine had no significant effect on the enzyme stability; the
inactivation of the mutant H111A occurred with a
t1/2 of 75 s compared with 84 s for the
wild-type enzyme. Nonetheless, the substitution of His111
residue by alanine reduced 5-fold the efficiency of heparin to protect
the enzyme against alkaline pH-induced inactivation, the ratio
/
observed to the wild-type enzyme was 5.1 compared with 1.0 for the
mutant H111A. This effect is related to decrease the parameter
,
from 5.1 to 2.6, and increase of heparin dissociation constant
(parameter
), from 18 to 48 µM (Table II). However, heparin showed a similar effect upon mutant H110A and wild-type enzyme
to protect these enzymes against alkaline pH-induced inactivation. In
general, the effect of heparin on the first-order inactivation rate
promoted by alkaline pH is comparable with the effect promoted by
heparin upon dipeptidyl carboxypeptidase activity of cathepsin B (Table
I), showing that the residue His111 is related to the
interaction of cathepsin B with heparin.
To probe the specificity for cathepsin B interaction, other
glycosaminoglycans were tested. Table III
shows that, besides heparin, only heparan sulfate was able to increase
the stability of cathepsin B at alkaline pH and, simultaneously,
inhibited the dipeptidyl carboxypeptidase activity of cathepsin B. Other sulfated glycosaminoglycans tested, namely dermatan sulfate and
chondroitin sulfate, were not able to decrease the cathepsin B affinity
for the substrate or protect the enzyme stability at alkaline pH.
Regarding the sulfonation of dextran sulfate, we have determined that
this compound has a stronger effect than that observed for heparin on
cathepsin B activity. It is possible to conclude that the electrostatic effect is important to the bind more than the structural features of
the glycosaminoglycans.
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Table III
The second-order constant kcat/KM for the hydrolysis of
Abz-FRA( -Dnp)K and the rate of inactivation kobs at pH 8.0 of wild-type human cathepsin B in the presence of different
glycosaminoglycans
kobs is the observed first-order rate of alkaline
pH-induced enzyme inactivation.
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In addition, we have proceeded initial experiments related to the
influence of smaller fragments of heparin. The minimum size that
exhibited the same effect showed by heparin was a low molecular mass
heparin (4,000 Da). Heparin tetrasaccharide
(
Idu,2S
GlcNS,6S
Idu, 2S
GlcNS,6S) and disaccharide
(
Idu,2S
GlcNS,6S) were not able to protect the enzymatic activity
against the alkaline pH inactivation. On the other hand, the
oligosaccharides containing between 3 and 8 disaccharide units showed a
partial effect in the protection against alkaline pH denaturation
experiments (data not shown).
Effects of Heparin on Cathepsin B Circular Dichroism
Spectra--
The effect of heparin on cathepsin B conformation was
analyzed by CD spectroscopy. Fig.
5A show that the presence of
100 µM heparin causes a significant change in the
spectral envelope of the cathepsin B, leading a decrease of the
ellipticity value at [
]222 nm. These data show that
heparin increases the helicity of cathepsin B, suggesting that this
change is due to cathepsin B heparin interaction. As expected, the
interaction between cathepsin B and heparin was disrupted by the
addition of 0.5 M NaCl (Fig. 5B). The spectrum
obtained in the presence of 0.5 M NaCl is very similar to
the spectrum obtained for the cathepsin B alone in the presence of 0.5 M NaCl.

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Fig. 5.
Effects of heparin on cathepsin B circular
dichroism spectra. A, cathepsin B CD spectra were
determined at pH 6.0 as described under "Experimental Procedures."
The CD spectra were performed in absence of heparin ( ) or in the
presence of 100 µM heparin ( ). B, the
cathepsin B circular dichroism spectra were obtained at 0.5 M NaCl in the absence ( ) or in the presence ( ) of 100 µM heparin.
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The Influence of Heparin upon Unfolding of Cathepsin B Induced by
Alkaline pH--
Inactivation of human cathepsin B is accompanied by
large structural changes and highly dependent on pH, temperature, and ionic strength of the medium (39). The break of the
thiolate-imidazolium ion pair, catalyzed by alkaline pH, as well as the
disruption of other important salt bridges, such as
Asp22-His110 and
Arg116-Asp224, located at the occluding loop
(16, 34), and two interdomain bonds,
Asp40-Arg202 and
Arg41-Glu163, (16, 39) are responsible for the
destabilization of the cathepsin B central
-helix, which contains
the active site Cys29 residue. Fig.
6A shows the measurements of
circular dichroism of cathepsin B at pH 5.5 and 8.0. The results show
that a drastic increase in the ellipticity values was observed when
cathepsin B was exposed at pH 8.0, indicating a large loss of the
native structure. Table IV summarizes the
secondary structure content of cathepsin B in presence or in absence of
heparin at different pH levels. A large decrease in the
-helix
content was observed at pH 8.0, whereas the
-sheet contents
increases. At pH 5.5, the
-helix and
-sheet contents were 34.2 and 21%, respectively, and at pH 8.0, the
-helix and
-sheet
contents were 14.6 and 35.5%, respectively. However, when cathepsin B
was preincubated with 100 µM heparin, the amount of
structure unfolding, induced by alkaline pH, was largely decreased.

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Fig. 6.
Heparin prevents the loss of cathepsin B
-helix structures induced by alkaline pH.
A, the CD analysis of cathepsin B were proceeded at pH 5.5 ( ) and 8.0 ( ), and pH 8.0 in the presence ( ) of 100 µM heparin. Other experimental details were as described
under "Experimental Procedures." B, the rate of loss of
cathepsin B -helix structures at pH 8.0 in absence ( ) or in
presence ( ) of 100 µM heparin.
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Table IV
The influence of heparin upon far UV (200-260 nm) CD spectra of
wild-type human cathepsin B at pH 5.5 and pH 8.0
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In an attempt to compare rates of the inactivation and of unfolding of
cathepsin B, the conformational change of cathepsin B, induced by
alkaline pH, were monitored continuously by circular dichroism at 222 nm (Fig. 6B). The alkaline pH-induced unfolding of cathepsin
B is shown to be a first-order process (39). The experimental curves
were fitted to Equation 4, and the first-order constants were
determined; in absence of heparin Kunfolding = 7.8 ± 0.4 ms
1, and in the presence of 100 µM heparin the observed constant was decreased 3.1-fold
(Kunfolding = 2.5 ± 0.2 ms
1). The rate constants of conformational change of
cathepsin B were very similar to results show in Fig. 4; in absence of
heparin kobs = 8.2 ± 0.6 ms
1, and in presence 100 µM heparin
kobs = 2.6 ± 0.2 ms
1,
suggesting that both heparin effects are linked.
Activity of Cathepsin B toward Azocasein Substrate--
The
experiments with azocasein showed that heparin does not exert influence
in the endopeptidase activity of cathepsin B cleaving protein
substrates. In addition, it was also possible to observe that heparin
protects cathepsin B from alkaline pH denaturation in the cleavage of
azocasein, used as a model for protein substrates (Fig.
7).

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Fig. 7.
Protection against pH alkaline inactivation
of cathepsin B promoted by heparin in the cleavage of azocasein.
Preactivated cathepsin B was preincubated at pH 8.0 for the times
indicated at 37 °C and then assayed for endopeptidase activity using
azocasein, in the absence ( ) or in the presence ( ) of heparin (20 µM).
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DISCUSSION |
We observed that cathepsin B interacted with heparin-Sepharose
resin (Fig. 1). Cathepsin B is generally considered to possess both
endo- and exopeptidase activity (41-43, 45). The binding of heparin to
cathepsin B did not perturb its endopeptidase activity upon the
fluorogenic substrate Z-FR-MCA (Fig. 2A) or upon azocasein used as a model for a protein substrate (Fig. 7). Also, heparin binding
was not capable of counteracting the inhibitory activity of E-64 (data
not shown). Because Z-FR-MCA and E-64 interact with cathepsin B at
S1 and S2 subsites (16,
34, 45), we can conclude that heparin binding to cathepsin B does not
affect Sn subsites of the enzyme. On the other
hand, it was observed that heparin inhibits cathepsin B exopeptidase
activity (Fig. 2B). The inhibition promoted by heparin upon
dipeptidyl carboxypeptidase activity of cathepsin B strongly suggests
that heparin interacts with cathepsin B at the occluding loop region
and contains residues His110 and His111 at
subsites S'2 and
S'3 that can interact with the lysin
C-terminal carboxylate of the substrate Abz-FRA(
-Dnp)K (16, 41,
45).
The same above effect was then assessed by site-directed mutagenesis
studies. Indeed, as shown in Table I, the mutant H111A was not
inhibited by heparin, but the mutant H110A was inhibited by heparin at
the same extension as the wild-type enzyme was. These data clearly show
the main role of His111 on the interaction between heparin
and cathepsin B.
It has been shown that the deletion of the occluding loop from
cathepsin B results in a decrease of the pH and thermal stability of
enzyme (45). It was also observed that
His110-Asp22 salt bridge is an important
contact between the occluding loop and the central
-helix, and this
interaction can contribute to the stability of these structural
elements (16, 34, 46). On the other hand, His111 residue is
not involved in electrostatic interactions with elements of the enzyme
(16, 34). The mutants H110A and H111A were also used to investigate the
importance of histidine residues in the alkaline pH-induced
inactivation of cathepsin B. Table II shows that the efficiency of
heparin to protect cathepsin B against alkaline pH-induced inactivation
is very dependent on His111. These results show that the
effect of heparin on the first-order inactivation rate promoted by
alkaline pH is comparable with the effect promoted by heparin upon
dipeptidyl carboxypeptidase activity of cathepsin B (Table I). The poor
inhibition of membrane-bound forms of cathepsin B by compounds like
CA-030, which requires interactions with His110 and
His111 residues, is probably related to the interactions of
the enzyme with heparan sulfate from the cell surface (34).
The interaction between cathepsin B and heparin or heparan sulfate is
specific, as other sulfated glycosaminoglycans, namely chondroitin
sulfate and dermatan sulfate, were not able to inhibit the dipeptidyl
carboxypeptidase activity of cathepsin B or induce protection against
alkaline pH inactivation (Table III). On the other hand, dextran
sulfate, a more sulfonated polymer, has a stronger effect than heparin
on cathepsin B activity. It is possible to conclude that the
electrostatic effect is more important to the binding than the
structural features of glycosaminoglycans.
The interaction of heparin with cathepsin B can be monitored by CD
spectroscopy analysis. Fig. 5A shows that heparin
significantly decreases the molar ellipticity of the cathepsin B CD
spectra at [
]222 nm, showing that the presence of
heparin increases the
-helix content of the enzyme. As expected,
this effect was dependent on ionic strength. Addition of 0.5 M NaCl to heparin-cathepsin B solution causes a spectral
change consistent with the disruption of the heparin-cathepsin B
complex (Fig. 5B).
As already shown in the literature (39), a dramatic increase in the
ellipticity molar value at [
]222 nm was detected at pH
8.0, suggesting a large decrease in the
-helix content of the enzyme
at alkaline pH (Fig. 6A). However, when cathepsin B was
preincubated with heparin, the amount of
-helix structure disruption
induced by alkaline pH was decreased (Table IV). The Fig. 6B
shows that the rate of unfolding of cathepsin B at pH 8.0 was decreased
by the presence of heparin at the same extension that first-order
inactivation rate of cathepsin B at alkaline pH was.
The effect of heparin on the rate of unfolding of cathepsin B at pH 8.0 (Fig. 6B) is comparable with the first-order inactivation rate promoted by alkaline pH (Table II) that by its turn is related to
the inhibition of heparin upon the dipeptidyl carboxypeptidase activity
of cathepsin B (Table I). Most of these effects are related to the
interaction of heparin with His111 residue of the cathepsin
B occluding loop. Taken together, these results show that, in all
cases, heparin binding is perturbing cathepsin B in a similar manner.
The presence of cathepsin B at the plasma membrane results in focal
dissolution of extracellular matrix proteins and enables the tumor cell
to invade the tissue (14, 17-20). Our results suggest that the cell
surface heparan sulfate can anchor the membrane forms of cathepsin B,
and such complexation affects the cathepsin B activities. The coupling
of cathepsin B with heparan sulfate increases its half-life 5-fold
(t1/2) at physiological pH and, quite probably,
potentiates the endopeptidase activity of the enzyme at the cell
surface. In addition, it was also possible to observe that cathepsin B
is protected by heparin from alkaline pH denaturation in the cleavage
of protein substrates (Fig. 7). The endopeptidase activity of cathepsin
B is related to the degradation of extracellular matrix proteins (19,
20). These results are in agreement with the observation that the
membrane-bound forms of cathepsin B are very resistant to inactivation
at neutral pH (32). As previously mentioned, the mechanism of secretion and insertion of cathepsin B on the plasma membrane are not fully understood (27, 29). Mannose-6-phosphate-independent targeting has been
proposed for cathepsin B. So, according to this scenario, the cell
surface heparan sulfate proteoglycans can be anchoring a pool of the
membrane forms of cathepsin B.
Recently it has been shown that cathepsin B colocalizes with annexin II
tetramer on the surface of tumor cells (47). In addition, annexin II
tetramer was also shown to bind heparin with high affinity dissociation
constant (Kd = 32 nM) (48). These
results and our present data strongly suggest that heparan sulfate and
annexin II tetramer together can act as an important binding site for
cathepsin B on the cell surface. Moreover, the cell surface heparan
sulfate proteoglycans are in a constant turnover, as a result of its
continuous secretion and endocytosis (49-51). It has been shown that
some proteins that are bound to heparan sulfate glycosaminoglycans
chains are endocytosed together with proteoglycans, e.g.
fibroblast growth factor (52), trombospodin (53), and lipoprotein
lipases (54). It is interesting to observe that in the lysosomal
vesicles there is a high concentration of cathepsin B (26) and that the
heparan sulfate is also present in this compartment during its
intracellular traffic (49-51). These observations suggest that the
mechanism of insertion of cathepsin B on the plasma membrane and its
cellular traffic can be dependent on heparan sulfate proteoglycans
present at cell surface. In addition, this hypothesis is also supported
by the perinuclear cathepsin B location in tumor cells (23, 26, 55), as
also observed for the cellular distribution of heparan sulfate
complexed to fibroblast growth factor (52). This intracellular location
of cathepsin B may play a role in nuclear functions, becoming a part of
the dramatic phenotypic transformation, known as "activation," observed in carcinogenic process (56).
Acknowledgments--
We thank Drs. Robert Ménard
(Biotechnology Research Institute, Montréal, Québec,
Canada) and John S. Mort (McGill University, Montréal,
Québec, Canada) for supplying the wild type and mutants of
cathepsin B and Dr. Michel Goldberg (Institut Pasteur, Paris, France)
for helping in CD analysis.