(Received for publication, July 27, 1995; and in revised form, November 2, 1995)
From the
Cathepsin O2, a human cysteine protease predominantly present in
osteoclasts, has been functionally expressed in Spodoptera
frugiperda Sf9 cells using the Autographa californica nuclear polyhedrosis virus. Following in vitro activation
at pH 4.0 with pepsin, active enzyme with an apparent molecular weight
of 29,000 was obtained. N-terminal sequencing revealed the typical
processing site for cysteine proteases of the papain family with a
proline in the position adjacent to the N-terminal alanine residue. The
SP
subsite specificity of human cathepsin O2 is
similar to cathepsin S but distinguished from cathepsins L and B.
Similar to cathepsin S, cathepsin O2 is characterized by a bell-shaped
pH activity profile and is stable at pH 6.5 for 30 min at 37 °C.
Cathepsin O2 is further distinguished by its potent collagenolytic
activity against Type I collagen between pH 5 and 6, and elastinolytic
activity against insoluble elastin at pH 7.0.
Its capacity to efficiently degrade Type I collagen and its high expression in osteoclasts suggest that cathepsin O2 may play a major role in human osteoclastic bone resorption.
Bone tissue is constantly undergoing a process of formation and resorption. Osteoclasts are cells responsible for the bone resorbing process. Bone resorption includes demineralization and degradation of extracellular matrix proteins (Delaissé and Vaes, 1992). Type I collagen constitutes 95% of the organic matrix (Krane and Simon, 1994). In addition to the interstitial collagenase, the lysosomal cysteine proteases cathepsins B and L are thought to be involved in osteoclastic bone resorption (Delaissé and Vaes, 1992). Both enzymes are present in the lysosomes as well as in the acidified extracellular resorption lacuna of the osteoclast (Goto et al., 1993), and both proteases display the in vitro ability to degrade collagen Type I at acidic pH (Maciewicz et al., 1987; Delaisséet al., 1991). Cysteine protease inhibitors, such as E-64 and leupeptin, have been shown to prevent osteoclastic bone resorption (Delaisse et al., 1987; Everts et al., 1988). Cathepsin L is considered to be one of the main proteases involved in collagen degradation in bone (Maciewicz and Etherington 1988; Kakegawa et al., 1993). It is thought that the collagenolytic action of cysteine proteases is exerted preferentially in the most acidic part of the bone resorption lacuna close to the ruffled border at a pH around 3.5 or 4.5, whereas the zinc-containing collagenases are more active in the neutral environment at the interface between the demineralized and mineralized matrix (Delaisse and Vaes, 1992). Besides cathepsins L and B, a variety of cathepsin L- and B-like activities may participate in collagenolytic bone degradation. Page et al.(1992) isolated multiple forms of cathepsin B from osteoclastomas. These have an acidic pH optimum and the ability to degrade soluble and insoluble Type I collagen. Delaisse et al.(1991) identified a 70-kDa thiol-dependent protease in bone tissue, which is also capable of degrading Type I collagen.
More recently, a cDNA encoding a novel human cysteine protease was cloned independently by several groups (Shi et al., 1995; Inaoka et al., 1995; Brömme and Okamoto, 1995) and named cathepsin O, cathepsin K, and cathepsin O2. All three sequences are identical and represent the human equivalent of the OC2 gene cloned from rabbit osteoclasts (Tezuka et al., 1994). This novel human cathepsin is highly expressed in osteoclasts and displays an approximate 10-fold higher expression at the message level when compared with cathepsin L (Brömme and Okamoto, 1995). Saneshige et al.(1995) could show that cathepsin O2(K) is regulated at the transcriptional level by retinoic acid in osteoclasts. Retinoic acid is a potent regulator and enhances the proliferation and differentiation of many types of cells including osteoclasts. Shi et al.(1995) demonstrated an endopeptidase activity of this enzyme against fibrinogen at acidic pH when overexpressed in COS-7 cells. However, no data have been published describing the enzymatic properties of this protease and demonstrating its activity toward extracellular matrix proteins such as collagen and elastin. In this publication, we describe for the first time the functional expression of recombinant cathepsin O2, its purification, enzymology, and inhibitor profile as well as its ability to efficiently degrade matrix proteins.
Recombinant human cathepsin S was expressed using the baculovirus
expression sytem and purified as described elsewhere. ()Recombinant human cathepsin L was kindly provided by Dr.
J. S. Mort (Shriners Hospital for Crippled Children, Montreal, Canada).
All cathepsins used were electrophoretically homogeneous, and their
molarities were determined by active site titration with E-64 as
described by Barrett and Kirschke (1981).
Formalin-fixed and paraffin-embedded osteoclastoma sections were deparaffinized in xylene and rehydrated with a 100, 95, 70, 50% alcohol series. The sections were washed with phosphate-buffered saline, pH 7.4, boiled in Citra antigen retrieval buffer as described (Biogenex, San Ramon, CA), and allowed to cool to room temperature. The sections were blocked with 5% normal goat serum, 0.1% (v/v) Triton X-100, phosphate-buffered saline, and 3% hydrogen peroxide, and incubated with control rabbit IgG (2.5 µg/ml) or affinity-purified anti-cathepsin O2 antibodies (250 ng/ml). Specific immunoreactivity was detected with a biotin/streptavidin-horseradish peroxidase-based detection system (Biogenex). After development of the chromogen (3,3`-diaminobenzidine), the sections were washed with water and treated with a 50, 70, 95% alcohol series. The sections were counterstained with 0.025% (w/v) fast green FCF, 95% alcohol, further treated with 100% alcohol and xylene, and permanently mounted for microscopy.
The kinetic constants V and K
were obtained by
nonlinear regression analysis using the program Enzfitter
(Leatherbarrow, 1987).
Inhibition of cathepsin O2 was assayed at a constant substrate (5 µM Z-FR-MCA) and enzyme concentration (1 nM) in the presence of different inhibitor concentrations in the substrate assay buffer. Cathepsin O2 was preincubated with the inhibitors for 10 min, and the reaction was started with substrate. Residual activity was monitored, and percent inhibition was calculated from the uninhibited rate.
The pH stability of cathepsins O2, S, and L was studied at three different pH values. Recombinant human cathepsins O2, S, and L were incubated at 37 °C in 100 mM sodium acetate buffer, pH 5.5, in 100 mM potassium phosphate buffer, pH 6.5, and in 100 mM Tris-HCl, pH 7.5, containing 5 mM dithiothreitol and 2.5 mM EDTA. Incubating for 0.5, 1, 2 and 4 h, the activity remaining was determined using 5 µM Z-FR-MCA for cathepsin O2 (100 mM potassium phosphate buffer, pH 6.5) and cathepsin L (100 mM sodium acetate buffer, pH 5.5) and 5 µM Z-VVR-MCA for cathepsin S (100 mM potassium phosphate buffer, pH 6.5).
Soluble calf skin Type I collagen was diluted to 0.4 mg/ml into 100 mM sodium acetate buffer, pH 4.0, 5.0, and 5.5, in 100 mM potassium phosphate buffer, pH 6.0 and 6.5, and 100 mM Tris-HCl, pH 7.0, containing 2 mM dithiotreitol, 2 mM EDTA. Human cathepsins O2, S, and L and bovine trypsin (Sigma) were incubated at concentrations of 100 nM enzyme concentration for 10 h at 28 °C. To measure the gelatinase activity of cathepsins O2 and S, Type I collagen was heated for 10 min at 70 °C prior to incubation with the proteases. In the presence of 0.1 nM cathepsin O2, 0.2 nM cathepsin L and 2 nM cathepsin S, respectively, the reaction mix was incubated for 30 min at 28 °C. The samples were subjected to SDS-polyacrylamide electrophoresis using 4-20% Tris/glycine gels (Novex, San Diego, CA).
The precursor of cathepsin O2 can be efficiently transformed into mature active enzyme in presence of pepsin at pH 4.0. The digest of crude cellular extract or of concentrated culture media supernatant results in a time-dependent disappearance of precursor and generation of mature enzyme (29 kDa) via an intermediate of 36 kDa (Fig. 1). In parallel with this process an increase of E-64 inhibitable activity measured at pH 7.5 is observed.
Figure 1: Maturation of procathepsin O2 with pepsin. Aliquots of the culture supernatant containing procathepsin O2 were incubated with pepsin (0.4 mg/ml) at 40 °C in 100 mM sodium acetate buffer, pH 4.0. The incubation was stopped by adding sample buffer. The times of digestion are as indicated. Molecular mass standards (kDa) are indicated in the right margin.
Figure 2: SDS-PAGE of purified recombinant human cathepsin O2 (Coomassie Blue staining). Lane 1, crude Sf9 fraction; lane 2, after passage through n-butyl fast Flow; lane 3, after passage through Mono S. Molecular mass standards are indicated in the right lane.
The purified enzyme is a single-chain enzyme and exhibits an apparent molecular mass of 29 kDa in a 4-20% Tris/glycine SDS gel under reducing conditions. Treatment with endoglycosidases H and F as well as N-glycosidase F did not result in a shift in the molecular weight, which implies that the protease is not glycosylated (data not shown). Human cathepsin O2 has two potential glycosylation sites in its mature sequence (Brömme and Okamoto, 1995). However, both sites have either a proline residue adjacent to the asparagine or to the threonine, so that their use is unlikely (Gavel and von Heijne, 1990). Additionally, cathepsin O2 contains one putative glycosylation site in the propart close to the processing site between the mature enzyme and the propart. Again, no shift in molecular weight of the proenzyme was observed after overnight treatment with endoglycosidases H and F as well as N-glycosidase F.
N-terminal sequencing of the mature protease processed in presence
of pepsin revealed the typical processing site described for cysteine
proteases of the papain family with a proline adjacent to the
N-terminal alanine (NH-APDSVDYRKKGYVTPVKN) (Rowan et
al., 1992). Since a cleavage by pepsin after arginine (position
-1) is very unlikely, it is possible that this site was generated
after an initial cleavage by pepsin in the proregion followed by an
aminopeptidase activity present in the Sf9 cell extract or by an active
intermediate of cathpsin O2 generated by pepsin. Most aminopeptidases
will stop at an alanine residue (position 1 of the mature enzyme
sequence) due to the subsequent proline residue. This mechanism of
processing involving aminopeptidases is also discussed for procathepsin
B (Rowan et al., 1992).
In contrast, autocatalytically activated cysteine proteases frequently have at their processing site an N-terminal extension of 3-6 amino acids from the propart (Brömme et al., 1993). No activation of the precursor was observed by addition of purified active cathepsin O2 at pH 4.5 (data not shown), indicating that neither an initial cis nor trans autoactivation of cathepsin O2 within the lysosomes is likely. This contrasts related cysteine proteases such as papain and cathepsin S, which exhibit a potential autocatalytic activation pathway (Vernet et al., 1990; Brömme et al., 1993).
The activating enzyme of cathepsin O2 within the osteoclast could be the aspartyl protease cathepsin D, which is present in osteoclastic lysosomes but secreted at low levels into the resorption lacuna (Goto et al., 1993).
Figure 3:
k/K
values for the hydrolysis of Z-XR-MCA by cathepsins O2,
S, L, and B (normalized to the best substrate = 1). Cathepsin O2 (Z-LR-MCA) 257,900 M
s
; Cathepsin S (Z-LR-MCA) 243,000 M
s
; Cathepsin L (Z-FR-MCA) 5,111,000 M
s
); Cathepsin B (Z-FR-MCA) 460,000 M
s
(data for cathepsins
S, L, and B were from Brömme et
al.(1994)).
The catalytic efficiency (k/K
) of cathepsin O2
toward dipeptide substrates is comparable with that of cathepsins S and
B but is approximately 1 order of magnitude lower than that of
cathepsin L. Interestingly, the K
values for
cathepsin O2 are comparable with those determined for cathepsin L. The K
value reflects to some extent the affinity of
the substrates for the protease. This trend is even more obvious for
the tripeptide substrate, Z-LLR-MCA, which displays a K
value as low as 4
10
M (Table 2). However, in contrast to cathepsins S and L, the k
values are almost 2 orders of magnitude lower
for cathepsin O2.
Figure 4:
pH activity profile for recombinant human
cathepsin O2. The k/K
values were obtained by measuring the initial rates of
Z-FR-MCA hydrolysis and by dividing by enzyme and substrate
concentration.
Human cathepsin O2 is more stable than cathepsin L at slightly acidic to neutral pH values but less stable than cathepsin S (Table 4). Approximately 50% of the cathepsin O2 activity remains after 1 h at 37 °C and pH 6.5, whereas essentially no cathepsin L activity could be observed under these conditions.
However, it must
be considered that the pH stability was determined without substrate
protection, which usually increases the pH stability. In the
[H]elastin degradation assay with cathepsin O2,
an increase of solubilized
H fragments was still observed
after 2 h at pH 7.0.
Figure 5:
Elastinolytic activity of recombinant
human cathepsin O2 at pH 4.5, 5.5, and 7.0 in comparison to cathepsins
S and L and pancreatic elastase (substrate, H-labeled
insoluble elastin).
The
collagenolytic activity against Type I collagen was determined between
pH 4.0 and 7.0. Cathepsin O2 extensively degrades Type I collagen
between pH 5.0 and 6.0 at 28 °C, whereas the degradation at pH 4.5
and 7.0 is much less pronounced (Fig. 6A). The primary
cleavage seems to occur in the telopeptide region since the monomers released from the
and
components are slightly
smaller. Additionally cleavage may also occur within the
monomers. It is yet unclear whether the cleavage occurs in the intact
helical region or in unraveled
monomers. Major fragments of Type
I collagen observed after cathepsin O2 action have the size of
70-80 kDa. Cathepsin L also cleaves in the telopeptide region,
but essentially no small molecular weight fragments were detected under
the conditions used. The effective pH range for the collagenolytic
activity of cathepsin L is more acidic when compared with that observed
for cathepsin O2 (between pH 4.0 and 5.5). Cathepsin S seems to reveal
only a very weak collagenolytic activity. In contrast, tissue
collagenases cleave the monomers into 3/4 and 1/4 fragments (Gross and
Nagai, 1965). No degradation of Type I collagen was observed with
trypsin at equal enzyme concentration compared with cathepsin O2,
showing that the integrity of the triple helix of the collagen used was
not impaired (data not shown).
Figure 6: SDS-PAGE of type I collagen (soluble calf skin collagen) after digestion with recombinant human cathepsins O2, L, and S. A, collagenase activity. Digestion of soluble calf skin collagen at 28 °C and at pH 4.0, 5.0, 5.5, 6.0, 6.5, and 7.0 by human cathepsins O2, S, and L (each 50 nM) for 12 h is shown. The reaction was stopped by the addition of 10 µM E-64. Untreated soluble collagen was used as standard (S). B, gelatinase activity. Digestion of denatured soluble calf skin collagen (10 min heated at 70 °C) at 28 °C and at pH 4.0, 5.0, 5.5, 6.0, 6.5, and 7.0 by human cathepsin O2 (0.1 nM), human cathepsin L (0.2 nM) and human cathepsin S (2 nM) is shown. Molecular mass standards are indicated in the right lane.
In addition to its collagenase activity, cathepsin O2 displays a powerful gelatinase activity. At 0.1 nM concentration of the enzyme, denatured collagen is totally degraded within 30 min within a pH range of 5.0-7.0. In contrast, cathepsin L displays its gelatinase activity only in the pH range between 4.5 and 5.5 (Fig. 6B). Cathepsin S is active between pH 4.0 and 7.0, but it displays a significantly weaker activity than that of cathepsins O2 and L.
Figure 7:
Immunohistochemical staining of human
cathepsin O2 in a human osteoclastoma. A, osteoclastoma,
stained with affinity-purified anti-cathepsin O2 antiserum,
magnification 400. B, osteoclastoma, stained with
control rabbit IgG, magnification
400
.
The pH value in the subosteoclastic resorption zone underneath the ruffled border is approximately between pH 3.5 and 4.5 (Fallon, 1984; Silver et al., 1988). However, it can be assumed that in the lower part of the resorption lacuna, at the interface between the demineralized and mineralized matrix, a pH gradient toward a neutral pH value exists due to the buffering capacity of dissolved bone salts. This area of the resorption lacuna is the proposed compartment for the action of interstitial collagenase, which is only active at slightly acidic to slightly alkaline pH (Delaisse and Vaes, 1992). The results presented for the pH stability, pH activity profile as well as the in vitro collagenase activity of cathepsin O2, suggest that this novel cysteine protease is active at the site where interstitial collagenase is active and that both activities may act in a concerted manner on Type I collagen. Besides cathepsin S (Brömme et al., 1993), cathepsin O2 is the second known human papain-like cysteine protease that displays a bell-shaped pH profile with a pH optimum at 6.0. Cathepsin L, due to its high instability at neutral pH (Turk et al., 1993) is excluded from this site and may be involved in a later degradation stage of collagen in the more acidified microenvironment of the resorption lacuna. Demonstration of cathepsin O2 secretion into the bone resorption lacuna will need to be achieved in order to verify this hypothesis.
In addition, cathepsin O2 is distinguished by a very potent gelatinase activity in the pH range between 5 and 7, whereas the gelatinase activity of cathepsin L is limited to the pH range between 4 and 5.5. Consequently, a rapid degradation of the collagen cleavage products released by the interstitial collagenase is possible in the pH micro environment of the tissue collagenase.
Studies with cysteine protease inhibitors, E-64, leupeptin, and diazo methanes, clearly demonstrate that cysteine protease inhibitors are capable of inhibiting bone resorption (Delaisse et al., 1980, 1987; Debari et al., 1995). Since all three types of inhibitors are very effective on cathepsin O2, it is likely that these inhibition studies reflect to a large extent the inhibition of cathepsin O2.
In conclusion, cathepsin O2, is highly expressed in osteoclasts. Cathepsin O2 is distinguished from classical human cysteine proteases like cathepsins L and B by its increased stability at neutral pH as well as its ability to efficiently hydrolyze Type I collagen and elastin at pH values above 6.0. The expression of cathepsin O2 protein in osteoclasts as well as its enzymatic properties imply a central role in normal bone remodelling as well as in pathological processes, such as osteoarthritis, osteoporosis, and multiple myeloma osteoclastomas. The design of potent cathepsin O2 inhibitors could be an important contribution in the efforts to arrest these pathological processes.