©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
An Extracellular Proteasome-like Structure from C6 Astrocytoma Cells with Serine Collagenase IV Activity and Metallo-dependent Activity on -Casein and -Insulin (*)

(Received for publication, November 1, 1994; and in revised form, December 6, 1994)

Indrasen S. Vaithilingam (1)(§) Warren McDonald (1) David W. Malott (2) Rolando F. Del Maestro (1)(¶)

From the  (1)Brain Research Laboratories, Experimental Research Unit, Department of Clinical Neurological Sciences, Division of Neurosurgery, University of Western Ontario, Victoria Hospital and the (2)Electron Microscopy Suite, Department of Pathology, Victoria Hospital, 375 South Street, London, Ontario N6A 4G5, Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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, alpha-casein, beta-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.


INTRODUCTION

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), (^1)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.


EXPERIMENTAL PROCEDURES

Materials

alpha-[^3H]Casein and I-beta-insulin were kindly provided by R. A. Cook. Human collagen IV, 3,4-dichloroisocoumarin, EDTA, and dimethyl sulfoxide were obtained from Sigma. Tissue inhibitor of metalloproteases (TIMP-2) was obtained from Amgen. [S]Methionine was obtained from ICN Biochemicals.

Origin of the Extracellular Proteasome

Non-conditioned 10% fetal calf serum and non-conditioned serum-free medium were examined for collagenase IV and general protease activities utilizing the method described in the section ``Proteolytic Assays.'' The extent of cell lysis was determined by assaying for the intracellular marker lactate dehydrogenase in homogenized cells and in serum-free conditioned medium according to the method of Wroblewski et al. (14). [S]Methionine studies were utilized to confirm that the proteasome-like enzyme arose from inside the C6 astrocytoma cells in culture. C6 cells were initially grown in 10% fetal calf serum for 24 h. The medium was removed and the cells were washed twice with serum-free medium, the cells were then placed in serum-free medium containing 10 µCi/ml [S]methionine and incubated for 4 h. The unconcentrated medium was then collected and run through the purification protocol listed below. The radioactive peak on the Superose 6 column, corresponding to the molecular weight of the proteasome, was characterized through inhibitor studies to identify this radioactive peak as the extracellular proteasome.

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 times 10^4 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 [^3H]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.

Preparation of Proteolytic Activities

C6 cells were initially grown to subconfluency in three Nunclon tissue culture flasks (surface area 175 cm^2), containing minimal essential medium, supplemented with 10% fetal calf serum (Myo-clone), penicillin, and streptomycin (Life Technologies, Inc., Missisauga, Ontario, Canada). Seven flasks (175 cm^2) each containing 50 ml of serum-free medium were inoculated with 3 times 10^6 cells. The cells were grown four days, at which point the conditioned medium was collected and concentrated. Serum-free conditioned medium was concentrated to 3 mg/ml on a YM-10 Amicon ultrafiltration membrane (Amicon, Oakville, Ontario, Canada). 200 µl of this sample was loaded onto a FPLC Mono Q HR 5/5 column (5 times 50 mm) resulting in two peaks of collagenase IV activity (data not shown).

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 times 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.

Proteolytic Assays

Proteolytic activity was assayed by measuring the degradation of alpha-[^3H]casein and [^3H]collagen type IV to products soluble in 100% (w/v) trichloroacetic acid. The alpha-casein and type IV collagen were labeled as described previously(3) . To maintain linearity of the proteolytic activity, 1-3 µg of protein was utilized in each assay. One unit of activity represents the amount of enzyme required to solubilize 1 µg of [^3H]collagen IV or alpha-[^3H]casein or beta-[^3H]insulin per h at 37 °C. Specific activity on each of the substrates is expressed as units per mg of protein.

Electrophoresis

5% discontinous (15) or continuous at pH 8.3 (16) nondenaturing gels were utilized. For the discontinuous gel, 10 µl of 1 mg/ml samples were loaded in buffer containing, 0.5 mM Tris-HCl, pH 6.8, 10% glycerol, and 0.05% (w/v) bromphenol blue. Similar sample conditions were followed for the continuous gel except that the sample buffer had a pH of 8.3. Two-dimensional electrophoresis was carried out according to the method of O'Farrell(17) . Standard low molecular weight markers obtained from Pharmacia were used to calibrate the 12% SDS gel of the second dimension. Two types of samples containing the extracellular proteasome were subjected to 5% native gel electrophoresis. One represented unconcentrated, unfractionated, or total conditioned free media, while the second was a proteasome sample purified through column chromatography. Nine lanes for each of these samples were run on this native gel. One lane was stained with Coomassie Blue to determine the number of bands (purity) and location of bands, while the remaining eight lanes were not stained. The unstained lanes were also sliced. The first slices (top of each lane) of each of the seven lanes were pooled and suspended in 70 µl of assay buffer. This was carried out for the subsequent slices. Elution was accomplished by storing the gel slices in buffer at 4 °C overnight. A Pasteur pipette was then used to draw the liquid and subsequently to wash the gel slices. 30 µl of the band arising from the ``unfractionated'' media while 10 µl of enzyme from the column purified sample was utilized per assay.

Electron Microscopy

An aliquot from the Superose 6 gel filtration column containing the 1000-kDa fraction was negatively stained with 1% uranyl acetate. The proteasome structure was observed using a Philips EM 300 electron microscope at a magnification of 928,000 times.

Inhibition Assay

Inhibitors were included in the collagenase IV assay, the alpha-casein, and beta-insulin assay. The total assay mixture in each tube for each of the three different assays was 500 µl. Inhibitors were preincubated with an aliquot of enzyme for 10 min at 22 °C before addition of the respective substrates and assay at 37 °C. Dichloroisocoumarin, diisopropyl fluorophosphate, and L-1-tosylamido-2-phenylethyl chloromethyl ketone were dissolved in 50% dimethyl sulfoxide (final concentration per assay was 5%). Phenylmethylsulfonyl fluoride, 1-chloro-3-tosylamido-7-amino-2-heptanone, and p-chloromercuribenzoate, L-cysteine, and N-ethylmaleimide, 1,10-phenanthroline were dissolved in 50% ethanol (final concentration per assay was 1.0%). EDTA, TIMP-1, TIMP-2, and E64 were dissolved in distilled water.

Peptidase Assays

Peptidase activity against the following synthetic substrates were measured: N-CBZ-Ala-Arg-Arg-4-methoxy-beta-naphthylamide, N-CBZ-Leu-Leu-Glu-beta-naphthylamide, and N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin. Each of the substrates were separately incubated at a concentration of 0.05 mM with 4 µg of the proteasome in buffer containing 50 mM Tris-Cl, 200 mM NaCl, at pH 7.5, including 5% Me(2)SO. The reaction mixture was incubated at 37 °C for 60 min. The reaction was stopped by the addition of 60 µl of 100% trichloroacetic acid. Enzyme activity was assessed by fluorometry according to the method of Ozaki et al.(18) . N-CBZ-Gly-Gly-Arg-beta-naphthylamide was detected by the method of Kramer et al.(19) . N-(3-[2-Furyl]acryolyl)-Leu-Gly-Pro-Ala was detected by the method of Van Wart et al.(20) . Hydrolysis of N-(3-[2-furyl]acryolyl)-Phe-Gly-Gly was detected by the method of Holmquist et al.(21) . N-(3-[2-Furyl]Acryolyl)-Gly-Leu-amide degradation was detected by the method of Feder et al.(22) . Finally, breakdown of N-CBZ-Gly-Gly-Leu-p-nitroanilide was detected by the method of Lyublinskaya et al.(23) .

Western Blot

Serum-free conditioned medium from C6 astrocytoma cells grown to day 3 was fractionated using filtration membranes: YM-10, YM-30, and XM-300. The 30-300-kDa fraction contained the active 68-kDa collagenase IV while the >300-kDa fraction contained the proteasome. Each of these two samples were electrophesed on a 4-15% SDS gradient gel (mini-protein ready gel, Bio-Rad). Proteins were transferred onto Immobilon-nitrocellulose (Millipore) for 4 h at 100 volts; transfer buffer included, 4.5 g of Tris, 21.6 g of glycine, 300 ml of MeOH, and 1200 ml of H(2)O. The blot was developed according to the Bio-Rad procedures (Amplified Alkaline Phosphatase Immuno-blot Assay Procedure). A primary rabbit anti-human antibody to recombinant 72-kDa collagenase IV was utilized (kindly provided by W. G. Stetler-Stevenson). Two modifications to this procedure: the primary antibody was incubated for 2 h and the low molecular weight blocker, peptide 175 (provided by W. G. Stetler-Stevenson), was also included in the blocking solution.


RESULTS

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 [^3H]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 circle. Collagenase IV activity of the eluted proteasome-like structure is represented by bullet, 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 (bullet). 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 times 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 times 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 times 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; alpha-casein and beta-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(2)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 times, respectively. This microenvironment did not affect the general protease activity of the extracellular proteasome. The activity of this complex on the general protein substrates alpha-casein and the beta-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 alpha-casein nor beta-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.




DISCUSSION

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 alpha-casein and beta-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, (^2)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 ^14C-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 alpha subunit of 27 kDa and beta 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 alpha-casein and beta-insulin.

EP degrades large complex substrates such as collagenase IV and relatively smaller polypeptides such as alpha-casein and beta-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.


FOOTNOTES

*
This work was supported in part by the Brain Tumor Foundation of Canada, the Victoria Hospital Research Development Fund, and the Royal Arch Masons of Canada, ``Release a Miracle Donation to Medical Research.'' The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Brain Tumor Foundation of Canada Fellowship.

To whom correspondence should be addressed: Brain Research Laboratories, Department of Clinical Neurological Sciences, Victoria Hospital, 375 South St., London, Ontario N6A 4G5, Canada. Tel.: 519-667-6603; Fax: 519-667-6766.

(^1)
The abbreviations used are: TIMP, tissue inhibitors of metalloproteases; EP, extracellular proteasome; CBZ, carbobenzoxy; FPLC, fast protein liquid chromatography.

(^2)
I. S. Vaithilingam, W. McDonald, and R. F. Del Maestro, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. R. A. Cook and Dr. V. J. Lang for use of the FPLC columns; Dr. S. Koval and D. Moyles, for initial electron microscopy work; and Jo-Ann Dunn for her secretarial assistance. We also thank W. G. Stetler-Stevenson for providing the anti-human antibody to the 72-kDa collagenase IV and Dr. K. E. Langley (Amgen, Thousand Oaks, CA) for providing the human recombinant TIMP-2.


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