(Received for publication, November 1, 1994; and in revised form, December 6, 1994)
From the
An extracellular proteasome-like (EP) structure has been
isolated from serum-free media conditioned by C6 astrocytoma cells. EP
has a native molecular mass of 1000 kDa and is composed of three
subunits, two isoelectric variants at 70 kDa and one at 65 kDa. The
extracellular proteasome degraded collagen IV, -casein,
-insulin, and certain synthetic peptide substrates. A 68-kDa type
IV collagenase, identified as the activated form of gelatinase A, was
also isolated from this medium. The type IV collagenase activity of the
proteasome was sensitive to serine protease inhibitors, while the
68-kDa collagenase IV represented the matrix metalloprotease gelatinase
A. The general protease activity of the proteasome was sensitive to
metalloprotease inhibitors. Western blot analysis indicates a sequence
relationship between the 68-kDa type IV collagenase and either one or
both of the 70-kDa isoelectric variants of the proteasome; however, the
two enzymes appear to be distinct functionally. Comparison with known
proteasomes indicates that EP represents a novel proteasome. The
complexity of degradative enzymes in the extracellular microenvironment
implies that complete inhibition of tumor growth requires at least a
combination of serine and metalloprotease inhibitors.
Tumor cells release a variety of degradative enzymes(1, 2, 3, 4, 5) . Enhanced proteolytic activity has been correlated with tumor growth, tumor-induced angiogenesis, and tumor invasiveness(6, 7) . The involvement of C6 astrocytoma cells in angiogenesis may be due to their enhanced levels of both collagenase IV activity and general protease activity. In vivo, these activities are maximal prior to the rapid phase of C6 astrocytoma growth(3, 4, 5) . Proteolytic enzymes are involved in providing an extensive vascular environment, permitting further tumor growth(4) . The initiation of angiogenesis requires the physical breakdown of the basement membrane. Degradation of collagen IV represents a significant step in this process, since it composes between 60 and 90% of the basement membrane(8) .
Type IV collagenases belong to the matrix
metalloprotease family of enzymes. This group of collagenases are
sensitive to the metalloprotease inhibitors: tissue inhibitors of
metalloproteases (TIMP-1 or TIMP-2), ()EDTA, and
1,10-o-phenanthroline. Type IV collagenases are secreted into
the extracellular microenvironment in a proenzyme form which is
subsequently activated. This extracellular location of type IV
collagenases allows contact with, and degradation of the basement
membrane(5) .
The multiple degradative activities of proteasomes may be involved in the degradation of the vascular basement membrane during tumor-associated angiogenesis. At least four different proteasomes have been identified. Two represent intracellular proteasomes, the most extensively studied being the mammalian 20 S multicatalytic proteasome (9) and the archaebacterial proteasome isolated from Thermoplasma acidophilum(10) . The remaining two proteasomes appear to have migrated past the cell membrane of Escherichia coli, subsequently located in the periplasmic space of E. coli(11) . Proteasomes represent a unique group of degradative enzymes, characterized by multiple activities. Their importance in cell physiology and pathophysiological events is implied by their wide distribution in bacterial and mammalian cells(9, 10, 11) , where they are found in the nucleus, cytoplasm, and endoplasmic reticulum (12, 13) and the periplasmic space of E. coli(11) .
In working toward a goal of inhibiting tumor-associated angiogenesis, the extracellular enzymes secreted from C6 astrocytoma cells, which degrade collagen IV and nonfibrous proteins, were isolated and characterized in this study.
Previous
studies (3, 4, 5) indicated that C6
astrocytoma cells released collagenase IV continuously throughout
growth; also the magnitude of this activity fluctuated during this
period. In this study, serum-free medium conditioned by C6 astrocytoma
cells was obtained for each of the five days of growth. Ten plates of
serum-free medium were seeded with 3 10
cells on
day 0. Conditioned medium was collected from two plates each day and
concentrated 50-fold. This medium was not changed on a daily basis, to
allow for continuous growth. The extracellular proteasome activity and
gelatinase A activity were determined utilizing
[
H]collagen IV as the substrate. The levels of
extracellular proteasome activity throughout growth were assessed by
adding 2.5 mM EDTA to the assay to block gelatinase activity.
In contrast, when gelatinase A activity was assessed, 1 mM 3,4-dichloroisocoumarin was added to block EP activity.
Each peak of
collagenase IV activity from the Mono Q column was pooled and
concentrated to 2 mg/ml using a YM-10 Amicon ultrafiltration membrane,
simultaneously exchanged with gel filtration buffer (50 mM Tris-Cl, 200 mM NaCl at pH 7.5). 200 µl of each peak
was loaded onto a FPLC Superose 12 HR 10/30 gel filtration column (10
300 mm). Each of the gel filtration columns were calibrated
using the molecular weight standards denoted on the column
chromatographs. The gel filtration standards were separately dissolved
in column buffer to 2 mg/ml, 200 µl of this sample was run on the
column.
Each peak from the Mono Q column eluted at significantly different times when run on the the Superose 12 column, indicating both a high molecular weight and a low molecular weight collagenase IV. For a more accurate determination of the size of the high molecular weight collagenase IV peak, a Superose 6 column was utilized with the gel filtration standards.
Serum-free medium conditioned by C6 cells was utilized to
isolate and characterize the extracellular proteolytic activities.
Control studies were carried out to ensure that the extracellular
activities were not the result of proteolytic activity already present
in non-conditioned medium, nor due to cell lysis. Collagenase IV
activity was neither present in 10% fetal calf serum nor serum-free
media. General protease activity present in non-conditioned media
containing serum contributed less than 3% of total activity to the
fetal calf serum-conditioned medium. General protease activity was not
present in serum-free medium. In conjunction with these control medium
studies, [S]methionine studies confirm that the
extracellular proteasome with collagenase IV activity was released from
C6 cells and was not initially present in the media (Fig. 2B).
Figure 2:
A, two collagenase IV activities from C6
conditioned medium separated on a FPLC Superose 12 gel filtration
column. One-min fractions were collected during elution at a flow rate
of 0.3 ml/min. Day 4 serum-free medium conditioned by C6 astrocytoma
cells was concentrated to 3 mg/ml and resolved into two peaks of
collagenase IV activity using an elution buffer containing: 50 mM Tris-Cl, 200 mM NaCl at pH 7.5. Collagenase IV activity
was determined using [H]collagen IV as the
substrate. Protein levels were measured in optical density at 280 nm.
The collagenase activity is reported in disintegrations per min (dpm). The elution times for the respective high molecular
weight markers are denoted by
. B, elution profile of
the high molecular weight collagenase IV peak on a FPLC Superose 6 gel
filtration column. The first peak off the Superose 12 column was pooled
and chromatographed on a Superose 6 column for an accurate
determination of the molecular weight. Two and one-half min fractions
were collected during elution at a flow rate of 0.2 ml/min using an
elution buffer containing: 50 mM Tris-Cl, 200 mM NaCl
at pH 7.5. Collagenase IV activity and extracellular
S-labeled activity is represented as dpm (see
``Experimental Procedures''). Elution times for the molecular
weight markers are represented by
. The elution time for the
proteasome is denoted by
. Collagenase IV activity of the eluted
proteasome-like structure is represented by
, while the eluted
S-labeled proteasome is indicated by
.
To ensure that the extracellular location of the collagenase IV enzymes and general proteases in the serum-free conditioned medium were not due to cell lysis, activity of the intracellular enzyme marker lactate dehydrogenase was examined. The ratio of extracellular to intracellular lactate dehydrogenase activity was 0.0025/0.17, thereby only 1.5% lactate dehydrogenase activity was present in the extracellular media. Cell lysis did not appear to be significant in these studies.
Day 0, represents the time when the C6 cells are placed in serum-free medium after growing in fetal calf serum (Fig. 1). Collagenase IV activity was absent at this time point, subsequently, total collagenase IV activity increased to peak on day 2 of growth, followed by a decline. These data confirms that the extracellular proteolytic activities found were not present in the non-conditioned medium, but were released at varying levels from growing C6 astrocytoma cells in culture.
Figure 1:
Levels of
collagenase IV activity during growth of C6 astrocytoma cells. The
collagenase IV activities of the proteasome and gelatinase A were
obtained for each of the five days of C6 growth in serum-free medium.
50 µl of conditioned medium from each of the five days were assayed
for collagenase IV activity. Total collagenase IV activity (absence of
inhibitors) is represented by (-). Proteasome activity was
determined by adding EDTA to the collagenase IV assay ().
Gelatinase A activity was determined by adding 3,4-dichloroisocoumarin
to another aliquot of conditioned media (
). Each of these
activities are plotted as units per mg of extracellular protein
released by C6 astrocytoma cells. The data noted for each time point
represent the mean ± S.E of three
experiments.
To identify and
characterize the protease(s) present in the day 2 peak, a purification
protocol was established, which utilized a FPLC Mono Q anion-exchange
column followed by a FPLC Superose 12 HR 10/30 (Fig. 2A) and Superose 6 HR 10/30 gel filtration
columns (Fig. 2B). Day 2 medium was resolved into two
peaks of collagenase IV activities (Fig. 2A). The first
peak represented a 1000-kDa collagenase IV activity while the second
peak contained the low molecular weight collagenase IV activity at 68
kDa. A more accurate determination of the molecular weight for the
first peak was obtained utilizing a Superose 6 column, which eluted at
57 min, indicating a molecular mass of 1000 kDa (Fig. 2B). All column buffers utilized in this
purification protocol did not contain calcium to eliminate the
possibility of aggregation of the 68-kDa collagenase IV into the higher
molecular weight collagenase IV. Purified aliquots of the 68-kDa
collagenase IV were subjected to 5, 10, and 15 mM Ca. The latter two concentrations of calcium
resulted in aggregation of the 68-kDa enzyme into dimers and trimers,
higher aggregation units, however, were not obtained.
Peak activity column fractions were analyzed on a 5% native gel (Fig. 3A) illustrating a single protein band. This native band was also observed in unconcentrated and nonfractionated conditioned medium. Gel purified enzyme and buffer elution of the band from both the fractionated and nonfractionated samples were similar in biochemical characteristics, attributed to that of the extracellular proteasome. The denatured form of this protein was subjected to two-dimensional electrophoresis, indicating three subunit bands, two isoelectric variants at 70-kDa (Fig. 3, B2 and B3), and a single band at 65 kDa (Fig. 3, B4). The activated form of the matrix metalloprotease (68 kDa) is illustrated in Fig. 3, C2. Electron microscopy demonstrated a circular structure 11 nm in diameter (Fig. 4), which appears to represent an end-on view of an elongated cylinder(10, 13) . These purification and structural studies identified the high molecular weight collagenase IV as a proteasome as defined by Rivett (9) and Tanaka et al.(12) .
Figure 3:
A
native molecular size of the EP and the relationship of the subunit
structure of EP and gelatinase A. A, serum-free medium was
concentrated to 3 mg/ml on a YM-10 Amicon ultrafiltration membrane. 200
µl of this sample was loaded onto a FPLC Mono Q HR 5/5 column (5
50 mm), the proteasome peak of collagenase IV activity was
pooled and chromatographed on a FPLC Superose 6 HR 10/30 gel filtration
column (10
300 mm) eluting as a major collagenase IV peak at a
molecular size of 1000-kDa. Several tubes of this peak were
individually analyzed by native electrophoresis for percentage
purification on a 5% native gel. The proteasome fraction migrated as a
single band under native electrophoresis. B, the subunit
structure of the single proteasome band was determined by analyzing an
aliquot on two-dimensional electrophoresis. Lane 1 represents
low molecular weight standards. Lanes 2-4 indicate the
denatured proteasome resulting in two subunit, pI variants at 70 kDa
and one at 65 kDa. C, lane 1 shows the molecular
standards. Lane 2 indicates that the second peak of the
Superose 12 gel filtration column was concentrated and run on a
SDS-polyacrylamide gel electrophoresis to yield the active 68-kDa
collagenase IV band.
Figure 4:
Structure of the extracellular proteasome.
An aliquot from the Superose 12 gel filtration column containing the
1000-kDa purified fraction was negatively stained with 1% uranyl
acetate. The structure of the proteasome was then examined utilizing a
Philips EM 300 electron microscope at a magnification 928,000.
Each of the three proteasomes shown contain an average inside diameter
of 11 nm.
For characterization and comparison with
known proteasomes, the extracellular proteasome and the 68-kDa enzyme
were tested against a variety of natural substrates (Table 1).
Further characterization of substrate specificity by EP utilized
synthetic peptide substrates. Although the extracellular proteasome
complex was isolated and identified by collagenolytic IV activity;
-casein and
-chain of insulin also represent adequate
substrates. To determine if the catalytic sites were distinct for each
of the different substrates, inhibitor studies were
performed(10) . Collagenase IV activity was inhibited by the
serine protease inhibitors, 3,4-dichloroisocoumarin and
phenylmethylsulfonyl fluoride. The hydrophobic microenvironment
provided by Me
SO and ethanol, the solvents in which these
inhibitors were dissolved, enhanced the collagenase IV activity of the
extracellular proteasome by 4 and 2.2
, respectively. This
microenvironment did not affect the general protease activity of the
extracellular proteasome. The activity of this complex on the general
protein substrates
-casein and the
-chain of insulin were
inhibited by the metalloprotease inhibitors EDTA,
1,10-o-phenanthroline, and TIMP-2.
The specific activity of
EP on collagen IV is at least two times greater than that of gelatinase
A. Unlike gelatinase A, the collagenase IV activity of EP is inhibited
by serine protease inhibitors rather than metalloprotease inhibitors.
Also gelatinase A does not degrade -casein nor
-insulin. At
least two independent catalytic sites appear to be present on the
proteasome.
For the synthetic peptide substrates tested, EP does not contain peptidylglutamyl-peptide hydrolyzing activity nor chymotrypsin-like activity (Table 2). EP degrades only one of the two trypsin-like substrates utilized. Collagenase activity and angiotensin converting enzyme activity was observed using the respective synthetic peptide substrates. EP did not hydrolyze the thermolysin-like nor the subtilisin-like substrates.
Western blot analysis indicated that both the 70-kDa subunit band and the 68-kDa bond bound the antibody to human 72-kDa enzyme (Fig. 5). Ongoing studies will determine if one or both of the 70-kDa pI variants of the protease binds to the antibody. The 65-kDa subunit band, however, did not react with the antibody. This suggests that at least one of the 70-kDa variants is related to the 68-kDa matrix metalloprotease through protein sequence homology, although differing in sensitivity to protease inhibitors.
Figure 5: Relationship of the proteasome subunits and the activated form of gelatinase A. Purified samples of the proteasome and gelatinase A were blotted. Anti-human antibody to 72-kDa collagenase IV was utilized to determine the relationship of the 68-kDa collagenase IV and the subunit of the proteasome (EP). A shows binding of the antibody only to the 70-kDa form(s). C shows binding of the antibody to the 68-kDa gelatinase A.
Growing C6 astrocytoma cells in monolayer culture release at least two types of collagenase IV activities into serum-free conditioned medium, in addition to general protease activities. The serine-sensitive type IV collagenase activity is associated with a novel EP of 1000 kDa, while the 68-kDa activity represents the activated form of the 72-kDa matrix metalloprotease gelatinase A.
These studies were conducted in the absence of calcium to eliminate
the possibility of aggregation, even though the presence of 10 or 15
mM Ca does not induce aggregation of the
68-kDa collagenase IV to form the 1000-kDa collagenase IV. Also these
two enzymes represent two different types of collagenase IV activities.
Unconcentrated, dilute serum-free conditioned medium still contained
the 1000-kDa collagenase IV activity, excluding the possibility that
concentration of the 68-kDa unit resulted in the higher molecular
weight collagenase IV. The lower molecular weight collagenase IV does
not degrade noncollagenase IV substrates, whereas the proteasome also
degrades
-casein and
-insulin. The varying levels of each of
these collagenases during C6 growth in culture, further distinguishes
the 68-kDa collagenase IV from the 1000-kDa collagenase IV.
Although
the two type IV collagenase activities are distinguishable by inhibitor
studies, antibody studies suggested a relationship between the 70-kDa
subunits (ongoing studies will determine if the antibody binds to one
or both of the 70-kDa pI variants) of the proteasome and the 68-kDa
enzyme. According to Western blot analysis, the 65-kDa subunit of the
proteasome does not appear to be related to either of these collagenase
IV activities. These findings in addition to the inhibitor studies
carried out on gelatin zymography, ()indicate that the type
IV collagenase activity of the proteasome is associated with one or
both the 70-kDa subunits. The remaining 65-kDa subunit of the
proteasome may then be responsible for the general protease activity.
Each characteristic of EP, if considered separately does not constitute a unique finding. However, a combination of these particular characteristics on a single extracellular proteasome has not been reported. Immunocytochemical studies indicate that proteasomes are distributed intracellularly in the nucleus and cytoplasm of cells from a variety of tissues(24, 25, 26) . Proteolytic activity has been detected in human serum from tumor-bearing patients(1, 27, 28) . The proteasome included in this group appears to be located extracellularly, possibly through lysis of tumor cells and hepatocytes associated with liver injury(28) . In contrast, EP is released by intact growing C6 astrocytoma cells.
A high molecular weight
metallo-collagenase IV (>2000 kDa) has been isolated from human
carcinoma tissue(29) . Another proteasome sensitive to
3,4-dichloroisocoumarin has been isolated in the archaebacterium T.
acidophilum(10) . This bacterial proteasome hydrolyses
peptide substrates and C-methylated casein, however, type
IV collagenase activity has not been examined. While the 20 S mammalian
multicatalytic proteasome and the archaebacterial proteasome
demonstrate trypsin, chymotrypsin, and peptidylglutamyl peptide
hydrolyzing activities, the EP differs in that it contains trypsin-like
activity, collagenase activity, and angiotensin converting enzyme
activity on synthetic peptide substrates. Studies utilizing synthetic
substrates may be informative in the classification of protease
activity, regarding the amino acids adjacent to the cleavage site
(either, basic, aromatic, or glutamic), however, little is understood
about the degradation of natural protein substrates. Also studies
utilizing synthetic peptide substrates for classification of proteases
may be misleading as suggested by Baumeister et
al.(30) . Information on the EP activity of the synthetic
substrates studied should only be utilized for initial comparison,
utilizing similar studies conducted on other known proteasomes. The
bacterial proteasome isolated from Thermoplasma represents the
simplest subunit structure for a proteasome to date, being composed of
two types of subunits: an
subunit of 27 kDa and
subunit of
25 kDa(31) . EP has three subunits, two pI variants at 70 kDa
and a single band at 65 kDa. EP is not composed of the 22-35-kDa
range of multiple subunits characteristic of other mammalian
proteasomes(1, 4) . Comparison of the subunit
structures thereby further differentiates the EP from the
archaebacterial proteasome and the mammalian 20 S proteasome.
Proteasomes contain more than one independent active site on the
same protein complex(9) . Each site is sensitive to particular
inhibitors, and accommodates certain substrates. EP contains at least
two active sites. A serine active site which appears to be involved in
the degradation of collagenase IV and a site sensitive to
metalloprotease inhibitors which degrades -casein and
-insulin.
EP degrades large complex substrates such as
collagenase IV and relatively smaller polypeptides such as -casein
and
-insulin. The digestion of multiple substrates and the
extracellular location of EP may account for the breakdown of many
protein components of the basement membrane structure and extracellular
space(4, 5) . The levels and type of collagenase IV
activity seen is dependent on the growth phase of C6 astrocytoma cells.
After initial cell plating (day 2: early log), EP represents the major
collagenase IV activity observed, whereas with continued growth (day 4:
stationary), gelatinase A is the major contributor to collagen IV
degradation. At the present time it is not clear what role the varying
levels of the two different types of collagenase IV play in the
initiation of angiogenesis, however, this finding does indicate that
effective inhibition of collagenase IV activity may require a
combination of serine protease inhibitors and metalloprotease
inhibitors.
The release of collagenase IV activities from C6 astrocytoma cells, which are sensitive to two different types of inhibitors, may result in a more controlled degradation of the basement membrane. If either a serine or a metalloprotease inhibitor was available at different times in vivo, it would appear that only one of the two activities of the EP would be expressed at this time. This study indicates that the inclusion of both types of protease inhibitors may result in effective inhibition of the extracellular collagenase IV activities released from C6 cells.
To our knowledge, EP represents the first extracellular proteasome not released through cell lysis. The combined characteristics for EP have not been observed for proteasomes known to date. The multicatalytic ability together with the substrate specificity of this extracellular proteasome suggests it may play a significant role in the initiation of angiogenesis. The development of strategies for the inhibition of tumor-associated angiogenesis should take into account the activities of this proteasome-like structure.
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