(Received for publication, August 26, 1996, and in revised form, December 13, 1996)
From the Lombardi Cancer Center and Department of Cell Biology, Georgetown University Medical Center, Washington, D. C. 20007
The 170-kDa membrane-bound gelatinase, seprase,
is a cell surface protease, the expression of which correlates with the
invasive phenotype of human melanoma and carcinoma cells. We have
isolated seprase from cell membranes and shed vesicles of LOX human
melanoma cells. The active enzyme is a dimer of
N-glycosylated 97-kDa subunits. Sequence analysis of three
internal proteolytic fragments of the 97-kDa polypeptide revealed up to
87.5% identity to the 95-kDa fibroblast activation protein (FAP
), the function of which is unknown. Thus, we used reverse
transcription-polymerase chain reaction to generate a 2.4-kilobase
cDNA from LOX mRNA with FAP
primers. COS-7 cells transfected
with this cDNA expressed a 170-kDa gelatinase that is recognized by
monoclonal antibodies directed against seprase. Sequence analysis also
showed similarities to the 110-kDa subunit of dipeptidyl peptidase IV
(DPPIV). Like DPPIV, the gelatinase activity of seprase was completely
blocked by serine-protease inhibitors, including diisopropyl
fluorophosphate. Seprase could be affinity-labeled by
[3H]diisopropyl fluorophosphate, but the proteolytically
inactive 97-kDa subunit could not, confirming the existence of a serine protease active site on the dimeric form. Proteolytic activity is lost
upon dissociation into its 97-kDa subunit following treatment with
acid, heat, or cysteine and histidine-modifying agents. We conclude
that seprase, FAP
, and DPPIV are related serine integral membrane
proteases and that seprase is similar to DPPIV, the proteolytic activities of which are dependent upon subunit association.
Seprase was originally identified from a human malignant melanoma cell line LOX, which exhibited aggressive behavior in experimental metastasis (1, 2). It is a membrane glycoprotein with gelatinase activity that is expressed and localized at the invasion front during invasion into the ECM1 by human melanoma, breast carcinoma cells, and chicken embryo fibroblasts transformed by Rous sarcoma virus (3-6). The protease is a hydrophobic glycoprotein soluble in Triton X-100 and in SDS (3). Seprase shows gelatinolytic activity as demonstrated by gelatin zymography (3, 4). The enzyme maintains maximal activity at neutral pH, which can be further enhanced by SDS buffer, EDTA, the cysteine protease inhibitor trans-epoxysuccinyl-L-leucylamido(4-guanidino)-butane and dithiothreitol. However, seprase has a peculiar protease inhibitor profile; it is inhibited by the protease inhibitors PMSF and NEM (3).
A major problem in purifying seprase was the apparent low level of this
protein in cultured cell lines. Because LOX cells produced a large
quantity of membrane vesicles that were enriched in seprase, we
isolated seprase from the cell membranes and shed vesicles in LOX
conditioned medium and characterized its subunit composition and its
enzymatic activity. We found that the protease had an apparent
molecular mass of 170 kDa composed of proteolytically inactive 97-kDa
subunits. Amino acid analysis of internal proteolytic fragments of the
97-kDa polypeptide revealed similarity to the 95-kDa FAP (7) and the
110-kDa subunit of human DPPIV (8-10). RT-PCR analysis suggests an
identical size for the mRNA encoding the 97-kDa seprase subunit
from LOX cells as well as the 95-kDa FAP
from WI-38 fibroblasts.
When a 2.4-kilobase cDNA amplicon generated by RT-PCR from LOX
mRNA using FAP
primers was expressed in COS-7 cells, it produced
a 170-kDa gelatinase that was recognized by mAbs directed against
seprase. In addition, this report demonstrates that like DPPIV (11,
12), seprase requires dimerization for its gelatinase activity. This
gelatinase activity was completely blocked by the serine-protease
inhibitors, DFP, PMSF, AEBSF, and APSF. Dimeric seprase could be
affinity-labeled by [3H]DFP, but the proteolytically
inactive 97-kDa subunit could not. These data demonstrate structural
regulation of protease activity by the formation of a serine protease
active site upon association of its subunits. Based on its protease
characteristics and on the deduced amino acid sequence (GenBank
accession number U76833[GenBank]) from its cDNA, we suggest that seprase and
DPPIV represent novel serine integral membrane proteases, the
proteolytic activity of which is regulated by subunit association.
The human amelanotic melanoma cell line LOX was obtained from Professor Oystein Fodstad, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo, Norway. The human embryonic lung fibroblast line WI-38 and African Green monkey kidney fibroblast line COS-7 were obtained from the American Type Culture Collection (Rockville, MD). Cell culture materials and most protease inhibitors were obtained as described (3). [3H]DFP was purchased from DuPont NEN (Boston, MA). 35S-labeled Tran label, DFP, and AEBSF were obtained from ICN Biomedical, and APSF was obtained from Dr. Jörg Sturzebecher (Erfurt, Germany). Three hybridoma cell lines, D8, D28, and D43, which secreted monoclonal IgG (all class 2a) antibodies, were derived from the fusion of rat myoloma Y3 cells and the spleen cells of Sprague-Dawley rats that were immunized for four 2-week intervals with 50 µg of partially purified seprase derived from human placenta.
RT-PCR, DNA Cloning, and Expression in COS-7 CellsTotal
RNA was isolated from LOX and WI-38 cells using the RNA Stat-60 kit
(Tel-Test "B", Inc.). Reverse transcription was carried out with 6 µg of total RNA using oligo(dT)12-18 as the primer. The
reaction was catalyzed by Superscript II RNase H reverse
transcriptase, as directed by the manufacturer (Life Technologies,
Inc.). Two oligonucleotide primers were synthesized which correspond to
a sense sequence within the 5
untranslated region (FAP 1) and an
antisense sequence within the 3
untranslated region (FAP 6) of the
published FAP
cDNA sequence (7). A ~2.4-kilobase amplicon
generated with either LOX RNA or WI-38 RNA and the FAP 1 (5
-CCACGCTCTGAAGACAGAATT-3
(no. 161-181)) and FAP 6 (5
-TCAGATTCTGATACAGGCT-3
(no. 2523-2505)) primers using the Expand
Long Template PCR System (Boehringer Mannheim) and a Perkin Elmer
GeneAmp 9600 cycler was isolated from a 1% agarose gel using a Qiagen
gel extraction kit. Purified cDNA (40 ng) was ligated to 30 ng of
the pCR3.1 mammalian expression vector. Ligation, transformation, and
selection of recombinant clones were carried out using the Eukaryotic
TA cloning kit (Invitrogen). A recombinant plasmid, clone pA15, was
purified using the Qiagen Plasmid Maxi kit. Transfection of COS-7 cells was carried out by electroporation using a Bio-Rad Gene Pulser II
system (Conditions: 0.3 kV and 950 µF). Each electro-transformation was carried out on ~5 × 106 cells in a volume of
0.5 ml using 20 µg of plasmid. Transfection efficiency was ~20%,
as determined by immunofluorescence. To enrich for cells that express
seprase, transfected cells were immunoselected using a panning
procedure (13) with the mAbs D8 and D28.
LOX and transfected COS-7 cells were fixed with 3% paraformaldehyde in PBS for 15 min and stained with mAbs D8 or D28 for confocal microscopic analysis as described previously (4).
Immunoprecipitation of 170-kDa Gelatinase and Its 97-kDa SubunitProtein A-Sepharose beads (15 µl; Pierce) were coated with rabbit anti-rat IgG (24 µg; Amersham Corp.), and rat mAbs D43 or D28 (25 µg each) prior to incubation with cell detergent extract. Alternatively, mAbs D8, D28, and D43 were coupled to CNBr-Sepharose 4B (Pharmacia Biotech Inc.) according to the manufacturer's instructions. Protease preparations were incubated with the antibody-coated beads for 2 h at 4 °C. The antigen-antibody complex-coated beads were collected by centrifugation at low speed (1300 × g), supernatants were removed, and the beads were washed four times with 1% Triton X-100 and 1 mM EDTA in TBS, pH 8.0, at 4 °C (buffer A). The beads were then washed with 1.0% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS in TBS, pH 8.0 (RIPA buffer). Bound proteins were eluted with 2 × SDS sample buffer containing 0.1 M dithiothreitol at 37 °C or 80 °C for 10 min, and the SDS-solubilized samples were analyzed by gelatin zymography, SDS-PAGE autoradiography, or immunoblotting. For immunoprecipitations requiring metabolic labeling, cells were incubated overnight with 0.025 mCi/ml 35S-labeled Tran label (ICN Biomedical) in methionine-free Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 1% complete Dulbecco's modified Eagle's medium, 2 mM glutamine, and 1% penicillin/streptomycin. For assaying the gelatin-degrading activity of seprase, heat-denatured rat tail type I collagen (gelatin) was incubated at 37 °C for 45 h in TBS, in the presence of seprase immobilized on mAbs D8, D28, and D43 coupled to Sepharose beads. Following digestion, the supernatants were collected, and the digested gelatin was resolved by SDS-PAGE (7.5% acrylamide gel). The gels were stained by Coomassie Brilliant Blue.
Immunoaffinity PurificationWe isolated seprase from 100 liters of LOX cell conditioned media and 15 ml of LOX cell pellet using WGA and organomercurial chromatography as described (3). Alternatively, seprase was enriched by a 40% ammonium sulfate cut of LOX cell RIPA lysates. Protein suspensions were spun at 10,000 × g for 30 min, and the pellet was resuspended in 1/5 volumes with TBS containing 1% octylglucoside. Resuspended proteins were dialyzed against 2 × 500 ml of 1% octylglucoside/TBS. The ammonium sulfate concentrated cell lysate was cleared by centrifugation at 10,000 × g for 30 min at 4 °C. The cleared lysate was loaded onto a BSA precolumn and then onto a mAb D28 affinity column. Column chromatography was carried out at 4 °C. The D28 mAb column was washed with five bed volumes of buffer A and five bed volumes of RIPA buffer. Seprase was eluted with 50 mM glycine buffer, pH 2.4, containing 1% octylglucoside, and fractions (1.5 ml) were immediately neutralized with 1.5 M Tris buffer, pH 8.8. Fractions were analyzed by SDS-PAGE, and protein bands were visualized using silver staining.
Partial Amino Acid Sequence DeterminationAmino acid sequence analysis was performed as described by Matsudaira (14). The immunoaffinity purified 97-kDa subunit was reduced by 20 mM dithiothreitol and then alkylated by 200 mM NEM. About 10 µg of the alkylated subunit was resolved on SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Boehringer Mannheim; sequencing grade) in the presence of 1% hydrogenated Triton X-100 (Sigma)/10% acetonitrile/100 mM Tris-HCl, pH 8.0, for 24 h at 37 °C. Protein bands migrating at 97 kDa were stained with Ponceau S and excised from the membrane, and Lys-C digestion was performed as described (15). Following digestion, samples were centrifuged, and the supernatant was transferred to an HPLC injection vial (Applied Bio Systems). Peptide isolation (200 µl) was performed by HPLC (Applied Bio Systems model 130) using a Vydac C18 column (2.1 × 250 mm) with a flow rate of 150 µl/min at room temperature. HPLC buffers were: buffer A, 0.1% trifluoroacetic acid/Milli-Q water; and buffer B, 0.08% trifluoroacetic acid/acetonitrile. The gradient was 1.6-29.5% B (0-63 min), 29.6-60% B (63-95 min), and 60-80% B (95-105 min). The column was then washed with 80% buffer B for 12 min at 150 µl/min and re-equilibrated with 1.6% buffer B for 50 min at 300 µl/min. Peptide elution was monitored at 220 nm. Individual polypeptide peaks on HPLC were collected and subjected to Edman degradation using the Hewlett Packard Sequencer (model G1000A) with an on-line PTH analyzer (Hewlett-Packard model 1090).
Isoelectric Focusing and Superose 12 Gel Filtration Liquid ChromatographySeprase, isolated through phase partitioning and
WGA-affinity chromatography from LOX cell Triton X-114 extracts, was
further purified by isoelectric focusing (Rotofor cell; Bio-Rad).
Ampholite solution (2%) in PBS containing 10% glycerol and 1% Triton
X-100 was prefocused for 1 h. WGA-purified proteins were applied
at the neutral pH range, and the Rotofor was run for another 4 h. Samples were harvested and analyzed by gelatin zymography and immunoblotting. Fractions, at pI 5, containing seprase were pooled and
stored at 80 °C. An aliquot was applied to a gel filtration column
(Superose 12; Pharmacia Biotech Inc.) previously equilibrated with 10%
glycerol and 1% Triton X-100 in PBS. The sample was loaded and eluted
at a flow rate of 0.05 ml/min and 0.1 ml/min, respectively. The void
volume was collected in 0.5-ml fractions, and the remaining samples
were collected in 0.2-ml fractions.
The 170-kDa
form of seprase isolated by isoelectric focusing, the 97-kDa subunit
obtained from mAb D28-affinity chromatography, or seprase from LOX cell
lysate immobilized on mAbs D8, D28, and D43 beads was incubated in the
presence of 33 µM [3H]DFP (10 µCi, 6 Ci/mmol; DuPont NEN) in 0.1% Tween 20/TBS (TBS-T), pH 7.5, for 1 h at room temperature. The free [3H]DFP was washed out
with TBS by consecutive rounds of dilution/concentration using a
Centricon concentrator (Amicon Inc.), and the
[3H]DFP-labeled seprase was resolved by SDS-PAGE under
nonboiling/nonreducing conditions. [3H]Seprase
immobilized on antibody beads was washed with TBS-T and eluted with SDS
by reducing and boiling. The eluate was then subjected to SDS-PAGE.
Proteins were fixed with 40% methanol/10% acetic acid for 30 min. The
gel was incubated with Amplify solution (Amersham Corp.) for 20 min at
room temperature and dried. Autoradiograms were exposed for 3, 10, or
20 days at 80 °C using Hyperfilm (Amersham Corp.).
Proteases were electrophoresed on SDS gels and analyzed on a substrate overlay membrane (Enzyme Systems Products) coupled with the fluorescent substrate Ala-Pro-7-amino-4-trifluoromethyl coumarin (16). The membrane was moistened in 0.5 M Tris-HCl, pH 7.8, placed against the gel, and incubated at 37 °C in a humidified chamber. The membrane was then removed from the gel and air dried. The DPPIV activity of individual proteases was monitored by detecting 7-amino-4-trifluoromethyl coumarin released from the substrate using a long wave length ultraviolet lamp.
Gelatin Zymography and Immunoblotting of Seprase in the Presence of Protease Inhibitors, Buffers at Different pH, and Different TemperaturesSeprase isolated from LOX lysates was treated with inhibitors for various classes of proteases, exposed to different pH buffers, or incubated at temperatures of 40 °C, 50 °C, or 60 °C. To prepare the protease for exposure to different buffers, seprase in 1% Triton X-114 was partitioned three times using saline to replace the Tris buffer. The resulting detergent phase was incubated with buffers at pH 4 (0.06 M citric acid/0.08 M sodium phosphate), pH 5 (0.05 M citric acid/0.1 M sodium phosphate), and pH 6 (0.04 M citric acid/0.12 M sodium phosphate) on ice for 10 min. Treated protease preparations were subjected to gelatin zymography and immunoblotting using mAbs D8 and D43.
Other MethodsProtein determinations were made using the bicinchoninic acid assay (Pierce) with bovine serum albumin as the standard protein. The absorbance was measured at 562 nm using an LKB Ultrospec 4050 (Pharmacia Biotech Inc.) or at dual absorbance of 540 and 490 nm using a Bio-Rad microtiter plate reader. For immunoblotting, proteins were separated by SDS-PAGE with 7.5 or 10% polyacrylamide gels, transferred to nitrocellulose sheets (Schleicher & Schuell) by electroblotting, and subsequently probed with mAbs. Blots were incubated with hybridoma supernatants diluted 1:30 in TBS-T for 1 h at room temperature. Immunoreactive polypeptides were visualized using the enhanced chemiluminescence detection system (Amersham Corp.) as described previously (17).
Seprase was detectable by immunofluorescence with mAbs
D8, D28, and D43 on cell surface extensions and membrane vesicles of LOX cells (Fig. 1, A-E). Confocal
microscopic analysis of cells cultured on glass coverslips and stained
with mAb D28 shows intense seprase localization on membrane extensions
at the leading edge, lamellipodia, of the cell (Fig. 1,
A-C). When cells were cultured on fibronectin-coated
cross-linked gelatin films and stained with mAb D28, seprase became
concentrated at invadopodia, specialized protrusions of the ventral
membrane, that contacted the film (Fig. 1D). In addition,
seprase could be observed on shed vesicles and on surface extensions in
the cell-gelatin film interface (Fig. 1E). These results
support the observation that seprase can be localized to the cell
surface lamellipodia, invadopodia, and on shed vesicles.
Immunoblotting analysis showed that mAbs D8 and D28 recognized seprase
(170 kDa) and its 97-kDa subunit, whereas mAb D43 labeled only dimeric
seprase (Fig. 1F). Immunoprecipitation of metabolically labeled cells resulted in elution of a major 97-kDa protein band following washing with both 1% Triton X-100/TBS and RIPA buffer (Fig.
2A, lane 1), but multiple components were
eluted when only 1% Triton X-100/TBS was used for washing (Fig.
2A, lane 2). Monoclonal antibody D28 was used for affinity
purification of seprase from LOX cell membranes and shed vesicles.
Fractions (1-3) were analyzed by silver staining (Fig. 2B, lanes
1-3) and immunoblotting with mAb D8 (Fig. 2B, lanes
4-6). The conditions used to elute seprase from the mAb column
(glycine buffer, pH 2.4) resulted in elution of the proteolytically
inactive 97-kDa subunit (Fig. 2B). Analysis of seprase to
determine the extent of its glycosylation indicated that the 97-kDa
subunit undergoes a reduction of apparent molecular mass of
approximately 20 kDa after exposure to N-glycosidase F (Fig.
2C, lanes 1 and 2). D8 (data not shown) and D28
mAbs recognized the deglycosylated 97-kDa subunit (Fig. 2C, lanes
3 and 4), indicating that their epitopes did not
include the N-linked side chains. These results show that
seprase is composed of monomeric, N-glycosylated 97-kDa
subunits.
Partial Amino Acid Sequence Analysis of the 97-kDa Protease Subunit
Initial attempts to obtain protein sequence data from the
97-kDa subunit were not successful, suggesting that its N terminus may
be blocked. Three internal proteolytic fragments were generated from
the 97-kDa subunit by Lys-C digestion, purified by HPLC, and subjected
to microsequencing. Comparison of the HPLC patterns from the
proteolytic digest of the 110-kDa subunit of human placental DPPIV and
the 97-kDa subunit of seprase revealed differences (data not shown),
suggesting that seprase was distinct from DPPIV. Sequence analysis of
the three peptides, which were 10, 12, and 8 amino acids in length,
exhibited 80, 66.7, and 87.5% identities, respectively, with the
corresponding deduced sequences of FAP. In addition, the 10- and
12-mers exhibited 70 and 33.3% identities with that of DPPIV,
respectively (Fig. 3). These results suggest that the 97-kDa subunit of seprase may be highly homologous to the 95-kDa FAP
and related to the 110-kDa subunit of DPPIV.
Cloning and Expression of Seprase
We carried out RT-PCR of
LOX total RNA using oligonucleotide primers that corresponded to the 5
untranslated region (FAP 1) and the 3
untranslated region (FAP 6) of
the FAP
cDNA (7). The resultant ~2.4-kilobase amplicon was
subcloned into the pCR3.1 mammalian expression vector (clone pA15). To
confirm that this amplicon encoded the 97-kDa subunit of seprase, we
transfected COS-7 cells with pA15 or the vector alone (pA11). Two mAbs,
D8 and D28, which had been shown previously (4) to recognize epitopes on both seprase and its monomeric 97-kDa subunit, were used for the
detection of surface expression, Western blot analysis, and gelatinolytic activity upshift. As can be seen in Fig.
4, mAb D8 specifically stained cells that had been
transfected with pA15 (Fig. 4A), whereas only background
staining was observed for those cells transfected with vector alone
(Fig. 4B). In a functional assay, detergent extracts of
immunoselected pA15- and pA11 (vector alone)-transfected COS-7 cells
and LOX cells were assayed for proteolytic activity by gelatin
zymography. The pA15-transfected cells gave rise to a gelatinolytic
band at ~170 kDa (Fig. 4C, lane 3) that corresponds with
the region of lysis produced by the LOX cell detergent extract (Fig.
4C, lane 4) in contrast to the vector-transfected COS-7 cell
extracts, which show no specific band of lysis (Fig. 4C, lanes
1 and 2). In addition, the identity of the
gelatinolytic activity was confirmed by the formation of seprase-D8
(Fig. 4C, lanes 6 and 8) and seprase-D28 (Fig.
4C, lanes 7 and 9) complexes that upshifted the
proteolytic activity. The immunoblot shows that pA15-transfected cells
(Fig. 4D, lane 3) express the 97-kDa monomeric subunit of
seprase that is also present in the control LOX cell extract (Fig.
4D, lane 2) but not in the mock-transfected cells (Fig.
4D, lane 1). Furthermore, RT-PCR with the FAP
primers,
FAP 1 and FAP 6, generated identical amplicons from LOX and WI-38
fibroblast mRNA (data not shown). These results support our initial
observation that the 97-kDa subunit of seprase is related to the 95-kDa
FAP
.
Size and ECM Substrate Specificity of Seprase
Previously, we
found that the 170-kDa gelatinase derived from LOX cells was present as
a >400-kDa complex in nonionic detergents, including Triton X-100,
Triton X-114, and octyl glucoside, and in WGA-agarose affinity-purified
material (3). The >400-kDa complex eluted in the void volume fractions
on Sephacryl S-200 gel filtration chromatography (data not shown).
Isolation of WGA-purified seprase by isoelectric focusing (at pI 5),
followed by Superose 12 gel filtration liquid chromatography, exhibited
major forms of about 200 and 230-490 kDa (Fig.
5A). Seprase, 170 kDa (Fig. 5A, insert,
lane 1), when subjected to gelatin zymography under nonboiled
conditions showed a diffuse clear zone between 150 and 200 kDa (Fig.
5A, insert, lane 3). Gelatin zymography of the 97-kDa subunit (Fig. 5A, insert, lane 2) did not show any activity,
indicating that it is proteolytically inactive (Fig. 5A, insert,
lane 4).
Substrate specificity studies using zymography have shown that seprase degrades gelatin but not laminin, fibronectin, fibrin, or casein (3). Thus, we examined possible ECM substrates, including type I collagen, type IV collagen, laminin, and fibronectin, using a soluble proteolytic assay. Seprase was immobilized on mAbs D8-, D28-, and D43-Sepharose beads. Native type I and type IV collagens, when incubated with the immobilized protease, were apparently not digested (data not shown), but thermally denatured type I (Fig. 5B, lanes 2-4) and type IV collagens (data not shown) incubated at 37 °C were digested into smaller polypeptides. Seprase did not degrade fibronectin and laminin under these conditions (data not shown). Also, based on its protein sequence similarity with DPPIV, we investigated whether seprase possessed DPPIV proteolytic activity using DPPIV derived from placental tissue as a positive control (Fig. 5C, lane 2). We did not detect any DPPIV activity for seprase (Fig. 5C, lane 1).
Identification of Seprase as a Serine ProteaseInitial
classification studies suggested that seprase is a serine protease
sensitive to PMSF and the sulfhydryl-modifying agent NEM (3). To
further classify the enzyme, we examined the effects of various
inhibitors specific for serine proteases by gelatin zymography (Fig.
6A). In addition to inhibition of the 170-kDa
gelatinase by PMSF, other serine-protease inhibitors, AEBSF (5 mM), APSF (0.05 mM), and DFP (0.005 mM), were found to completely inhibit the 170-kDa
gelatinase, whereas the cysteine protease inhibitor
trans-epoxysuccinyl-L-leucylamido(4-guanidino)-butane enhanced it (Fig. 6A). These inhibitors did not dissociate
seprase into its 97-kDa subunit at the concentrations used to inhibit its enzymatic activity (data not shown). Other inhibitors of aspartate, serine, or metalloproteases, including pepstatin (5 mM),
benzamidine (10 mM), EDTA (5 mM), and
1,10-phenanthroline (2 mM), and cysteine protease
inhibitors, including leupeptin (0.1 mM), iodoacetic acid
(1 mM), iodoacetamide (1 mM), and
trans-epoxysuccinyl-L-leucylamido(4-guanidino)-butane (5 mM), had no inhibitory effect on the activity of the
170-kDa gelatinase. The inhibition study suggests that seprase contains a catalytically active serine residue(s).
[3H]DFP affinity labeling experiments showed that the label was associated with both the 170-kDa form (Fig. 6B, lanes 1 and 3) and the larger forms (Fig. 6B, lanes 4-7) but not with the 97-kDa monomer (Fig. 6B, lane 2). When labeling was performed on seprase immunoabsorbed to mAbs D8 (Fig. 6B, lanes 3, 4, and 7), D28 (Fig. 6B, lane 5), and D43 (Fig. 6B, lane 6), the radioactive label was detected on the 97-kDa subunit because seprase was dissociated upon elution by boiling. The active site affinity labeling is specific because [3H]DFP labeling was abolished when seprase was preincubated with cold DFP (Fig. 6B, lanes 7 and 8). [3H]DFP labeling of seprase appeared to alter mobility of the enzyme on SDS gels (Fig. 6B, lane 1). In addition, the isolated 97-kDa subunit could not be labeled with [3H]DFP (Fig. 6B, lane 2) and failed to degrade gelatin (Fig. 5A, insert, lane 4). Furthermore, seprase could not be affinity labeled by the cysteine protease inhibitors biotin-Phe-Ala-CHN2 or biotin-Phe-CH2Cl (data not shown). We have, therefore, demonstrated that seprase, like DPPIV, is a serine protease and that its 97-kDa subunit is not active.
Dimerization Is an Essential Structural Requirement for Generation of the Seprase Serine Protease Active Site(s)The effects of
acid, heat, and amino acid-modifying agents, such as NEM and DEPC, in
inhibiting seprase gelatinase activity have been investigated. The
sensitivity of the seprase dimeric structure and its gelatinase
activity to buffers at different pHs (Fig.
7A), incubation at different temperatures
(Fig. 7B), and treatment with the histidine-modifying agent
DEPC (Fig. 7C) was investigated using gelatin zymography and
immunoblotting. Gelatinase activity was measured by zymography, and
changes in subunit association were detected by immunoblotting with mAb
D8 that recognizes both seprase and its 97-kDa subunit and with mAb D43
that recognizes only seprase. At pH lower than 5 (Fig. 7A), incubation at temperatures above 50 °C (Fig. 7B), or DEPC
treatment at concentrations over 5 mM (Fig. 7C), loss of
gelatinase activity was always accompanied by the disappearance of the
170-kDa form and the appearance of the 97-kDa subunit. A similar effect
to DEPC was observed for the cysteine-modifying agent NEM but not for
the serine protease inhibitors PMSF, AEBSF, APSF, and DFP at the
concentrations that inhibited the gelatinase activity (data not shown).
Thus, acidic pH, heat, NEM, and DEPC treatment inhibit the seprase
gelatinase by altering its subunit association, which is necessary for
its catalytic activity.
This study focused on the characterization of melanoma seprase in terms of its amino acid sequence, expression of its cDNA, classification of its proteolytic activity, and structure/activity relationships. Seprase is localized at cell surface invadopodia, lamellipodia, and vesicles. These results, together with the previous observation that LOX cells with higher levels of seprase display a more invasive phenotype than those with lower levels (4), make it important to identify and characterize this protease.
Previously, seprase was defined by its gelatinolytic activity rather
than by its isolation (3, 4). Here we have used nondenaturing
chromatographic techniques followed by D28 immunoaffinity chromatography to purify a Triton X-100-soluble gelatinase from LOX
cell membranes. We have also isolated seprase using isoelectric focusing and have determined that it has an isoelectric point of 5. We
show here that heat-denatured type I collagen is a substrate for
seprase (Fig. 5B) but not native type I collagen, supporting the notion that it is a gelatinolytic endopeptidase. Also, we showed
that seprase is a serine protease by virtue of its inhibition by the
serine protease inhibitors DFP, PMSF, APSF, and AEBSF and by
affinity-labeling with [3H]DFP. Consistent with these
results, the deduced amino acid sequence of a cDNA clone that
encodes the 97-kDa subunit of seprase (GenBank accession number U76833[GenBank])
is highly homologous to those of the nonclassical serine protease DPPIV
(8-10) and the putative nonclassical serine protease FAP (7) in
their catalytic regions.
The affinity-purified material gave a single band at 97-kDa by silver staining and was recognized by mAbs D8 and D28. This 97-kDa subunit was devoid of proteolytic activity, as determined by gelatin zymography, a soluble gelatin degradation assay, and DFP affinity labeling. Lack of [3H]DFP labeling of the 97-kDa form could be due to the fact that serine proteases need to be in an active conformation to accept phosphorylation of the OH group of their catalytic site serine (18). However, we do know that each 97-kDa subunit of seprase contains the consensus motif of serine proteases, GXSXG, as well as the other residues that make up the catalytic triad (GenBank accession number U76833[GenBank]).
Most secreted ECM-degrading enzymes are proteolytically activated from their zymogen precursors; however, formation of a dimeric structure appears to be necessary for expression of the high molecular mass dipeptidase activity of DPPIV (11, 12, 19). We have shown that like DPPIV (20, 21), seprase required dimerization to exhibit its gelatinase activity, which was completely blocked by the serine-protease inhibitors DFP, PMSF, AEBSF, and APSF. In addition, seprase could be affinity-labeled by [3H]DFP, but the proteolytically inactive 97-kDa subunit could not. These results are further confirmed by our COS-7 cell expression experiments in which the expressed 97-kDa subunit dimerized to produce the active form of seprase. These data demonstrate that there is structural regulation of the proteolytic activity of seprase; the formation of the serine protease active site(s) is dependent on the association of two subunits. However, the mechanisms by which seprase subunit association is regulated are not yet understood.
We speculate that this structural switch for proteolytic activity may be a common mechanism for the regulation of serine integral membrane proteases, including seprase and DPPIV. The requirement for a dimeric structure for seprase gelatinase activity is supported by our studies showing a correlation between structure stability and gelatinase activity at various pH, temperatures, and in the presence of the amino acid-modifying agents NEM and DEPC. We have shown that seprase was not active when samples were exposed to acidic conditions (pH <5), to high temperatures (>50 °C), or to 5 mM NEM or DEPC. In all cases, protease inactivation was accompanied by protease dissociation into the 97-kDa subunits. Similarly, the dimeric 150-220-kDa DPPIV has been reported to be active and accessible to DFP labeling, but the 110-kDa monomeric DPPIV was not (11, 22). These properties of membrane-bound enzymes point to the possibility that subunits may assemble to form active enzymes at sites of protease action, i.e. the cell surface. Furthermore, the fact that seprase is sensitive to an acidic environment suggests that seprase may be inactivated in an endocytic compartment. Thus, increasing or decreasing the stability of an oligomeric complex may be a means for cells to regulate the proteolytic activity of membrane proteases.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U76833[GenBank].
We are most grateful to Susette C. Mueller for critical reviews of this work, to Steve Akiyama and Scott W. Argraves for help with the initial stage of this study, to Jörg Sturzebecher for providing APSF and several newly synthesized serine protease inhibitors, and to Tom Winters for help with the gel filtration experiment.