(Received for publication, August 27, 1996, and in revised form, October 11, 1996)
From the Joint Program in Neonatology, Department of
Pediatrics, Harvard Medical School, Children's Hospital, Boston,
Massachusetts 02115-5737, the § Department of Dermatology,
University of Pennsylvania School of Medicine, Philadelphia,
Pennsylvania 19104, and ¶ LXR Biotechnology,
Richmond, California 94804
The squamous cell carcinoma antigen (SCCA) serves
as a serological marker for more advanced squamous cell tumors.
Molecular cloning of the SCCA genomic region revealed the presence of
two tandemly arrayed genes, SCCA1 and SCCA2.
Analysis of the primary amino acid sequences shows that both genes are
members of the high molecular weight serpin superfamily of serine
proteinase inhibitors. Although SCCA1 and SCCA2 are nearly identical in
primary structure, the reactive site loop of each inhibitor suggests
that they may differ in their specificity for target proteinases. SCCA1 has been shown to be effective against papain-like cysteine
proteinases. The purpose of this study was to determine whether SCCA2
inhibited a different family of proteolytic enzymes. Using recombinant
DNA techniques, we prepared a fusion protein of glutathione
S-transferase and full-length SCCA2 . The recombinant SCCA2
was most effective against two chymotrypsin-like proteinases from
inflammatory cells, but was ineffective against papain-like cysteine
proteinases. Serpin-like inhibition was observed for both human
neutrophil cathepsin G and human mast cell chymase. The second order
rate constants for these associations were on the order of ~1 × 105 M1 s
1 and
~3 × 104 M
1
s
1 for cathepsin G and mast cell chymase, respectively.
Moreover, SCCA2 formed SDS-stable complexes with these proteinases at a stoichiometry of near 1:1. These data showed that SCCA2 is a novel inhibitor of two physiologically important chymotrypsin-like
serine proteinases.
The squamous cell carcinoma antigen
(SCCA)1 serves as a serological marker for
more advanced squamous cell tumors of the cervix, lung, and oropharynx
(reviewed in Refs. 1 and 2). SCCA is not specific for malignant
tissues, however, as the protein(s) is detected in the suprabasal
levels of normal stratified squamous epithelia of the skin and mucus
membranes (3, 4) and in the pseudostratified ciliated columnar
epithelia of the conducting airways.2 The
functional role of SCCA in both normal and malignant cells has not been
elucidated. Biochemical analysis of SCCA by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) reveals a single
band with a molecular mass of ~45 kDa (5). Chromatofocusing, however,
separates SCCA into neutral (pI 6.25) and acidic (pI
6.25) fractions (4, 5). The neutral isoform of SCCA is detected in both
malignant and normal epithelial cells (4, 6). In contrast, the acidic
isoform is present in tumor cells, especially those located at the
periphery of the tumor, and in the sera of cancer patients with well
differentiated SCC (4).
A cDNA for SCCA was cloned and sequenced by Suminami et
al. (7). The putative translation product agreed with the limited peptide sequences that were available for SCCA (7). Comparisons of the
primary amino acid sequence to those in the protein data bases revealed
that SCCA belongs to the high molecular weight family of
ine
roteinase
hibitor
(serpins) (7). The serpin superfamily has evolved over 500 million years with representatives found in viruses, plants, protozoa, insects, and higher vertebrates (8). In mammals, inhibitory-type serpins regulate serine proteinases involved in, for example, coagulation, fibrinolysis, inflammation, cell
migration, and extracellular matrix remodeling (reviewed in Refs.
9-12). However, some serpins have lost the ability to inhibit serine
proteinases but have evolved specialized functions such as hormone
transport and blood pressure regulation (9).
Molecular cloning of the SCCA genomic region revealed the presence of two nearly identical (92%), tandemly arrayed SCCA genes (13). These genes, SCCA1 and SCCA2, likely correspond to the neutral and acidic forms, respectively, of SCCA. In addition, SCCA1 and SCCA2 are flanked by two other closely related serpin genes, plasminogen activator inhibitor type-2 (PAI2) and maspin within a 300-kilobase pair region of 18q21.3 (13). The putative translation product of the more telomeric gene, SCCA1, agrees with that of the cDNA reported by Suminami et al. (7), whereas the more centromeric gene, SCCA2, represents a novel sequence. Alignments between the amino acid sequences of SCCA1 and SCCA2 with those of other serpins suggest that both SCCAs are inhibitory-type serpins (13, 14).
The reactive site loop (RSL) of serpins interacts with the active site
of the proteinase and thereby confers both functionality and
specificity to the serpin molecule (reviewed in Refs. 10, 12, and
15-17). Examination of the RSL may provide insight into the types of
proteinases inhibited by SCCA1 and SCCA2. Although SCCA1 and SCCA2 are
nearly identical, significant differences between their RSLs suggest
that SCCA1 and SCCA2 inhibit different types of proteinases (Fig. 1).
Residues flanking the putative scissile bonds within the RSLs (between
the P1 and P1 residues according to the numbering system of Schechter
and Berger (18)) of SCCA1 and SCCA2 are Ser-Ser and
Leu-Ser, respectively (13). Bovine
1-anti-chymotrypsin (
1ACT) is the only
other serpin known to contain a Ser-Ser at the reactive site
(19). As the target enzymes that interact with bovine
1ACT have not been identified, we cannot predict a
priori the types of proteinases that can be inhibited by SCCA1. In
contrast, both heparin-cofactor II (HCII) and human
1ACT
contain Leu-Ser residues at their reactive sites (14). Both
serpins inhibit chymotrypsin (CT)-like serine proteinases, and HCII is
a physiologic inhibitor of thrombin in the presence of heparans and
dermatan sulfate (reviewed in Ref. 20). Based on these findings, SCCA2
may share in the inhibitory profile defined by HCII or human
1ACT.
The purpose of our current investigations is to determine experimentally whether SCCA1 and SCCA2 are inhibitory-type serpins. In a separate study, we have shown that SCCA1 is a novel cross-class inhibitor of the papain-like cysteine proteinases; cathepsins L, S, and K.3 In this report, we demonstrate that SCCA2 is an inhibitor of the chymotrypsin-like serine proteinases, cathepsin G (catG) and human mast cell chymase (HMC).
A 1.2-kilobase pair DNA fragment containing the
complete coding sequence of SCCA2 was generated by polymerase
chain reaction from a plasmid containing the SCCA2 cDNA (13). The
forward (5-GCGCCCGGG
ATGAATTCACTCAGTGAAGCC-3
) and reverse (5
-GCGCCCGGGATTCGGTACCAGTGACAGACTAATTGCATCTA-3
) primers were designed to facilitate in-frame insertion into the pGEX-2T bacterial expression vector (Pharmacia, Uppsala, Sweden).
The SCCA2 coding sequence was amplified as described previously (21), digested with the restriction endonuclease BamHI (site underlined in forward primer) and ligated into the BamHI/SmaI site of the vector pGEX-2T. Recombinant clones were analyzed by DNA sequencing to verify that the coding sequence of SCCA2 was intact and in-frame with GST (22).
Purification of GST-SCCA2 Fusion ProteinThe GST-SCCA2
fusion protein was batch-purified using glutathione-Sepharose 4B beads
(Pharmacia). A 200-ml culture of 2 × YT (23), containing 2%
glucose and 100 µg/ml ampicillin, was incubated overnight in a
37 °C shaker. Eight hundred milliliters of 2 × YT, 2%
glucose, 100 µg/ml ampicillin was added to the overnight culture, and
the suspension was incubated at 37 °C until the
A600 = 0.5-1.0. Expression was induced by the
addition of 1 ml of 0.5 M
isopropyl-1-thio--D-galactopyranoside (Boehringer
Mannheim) and incubated at 37 °C for 4 h. Cells were harvested
by centrifugation and lysed by incubating for 30 min on ice in 60 ml of
prep buffer (100 mM NaCl, 100 mM Tris-HCl, pH
8.0, 50 mM EDTA, 2% Triton X-100) supplemented with 1.5 mg/ml lysozyme and 10 µg/ml phenylmethylsulfonyl fluoride
(Sigma). The lysate was cleared by centrifugation at 12,000 × g for 30 min. Two milliliters of 50%
glutathione-Sepharose 4B, equilibrated in prep buffer, was mixed with
60 ml of lysate. The slurry was gently shaken for 30 min at 4 °C.
The beads were collected by centrifugation at 500 × g,
and washed three times in 10 ml of PBS (0.01 M phosphate
buffer, 27 mM KCl, 137 mM NaCl, pH 7.4).
Attempts to cleave SCCA2 from GST using thrombin resulted in
inactivation of the inhibitor. Therefore, the entire fusion protein was
eluted in three 1-ml washes of Glutathione Elution Buffer (10 mM reduced glutathione, 50 mM Tris-HCl, pH
8.0). The eluate containing the fusion protein was aliquoted and
frozen.
Human neutrophil
elastase (HNE), CT, plasmin, proteinase 3 (PR3), catL, catG,
1ACT, catB, and
1-proteinase inhibitor
(
1PI) were purchased from Athens Research & Technology,
Inc. (Athens, GA). Prostate specific antigen (PSA) was purchased from
Scripps Laboratories (San Diego, CA). Cathepsins K and S were
generously provided by Dr. Harold Chapman (Brigham and Women's
Hospital, Boston, MA). HMC was purified from human skin as described
(24, 25). Human trypsin, and urokinase-type plasminogen activator (u-PA) were purchased from Sigma. Thrombin was
purchased from Calbiochem. Granzyme B was kindly provided by Dr. Chris
Froelich of Evanston Hospital of Evanston, IL. Enzyme substrates
were purchased from Sigma
(succinyl-Ala-Ala-Pro-Phe-para-nitroanilide (Succ-AAPF-pNA), methoxy-Succ-Ala-Ala-Pro-Val-pNA (MeO-Succ-AAPV-pNA), and
Val-Leu-Lys-pNA (VLK-pNA)), Bachem Bioscience, Inc. (King of
Prussia, PA) (benzyloxycarbonyl-Arg-Arg-pNA (Z-RR-pNA), Glu-Gly-Arg-pNA
(EGR-pNA), and butyloxycarbonyl-Ala-Ala-Asp-pNA (Boc-AAD-pNA)), and
Molecular Probes, Inc. (Eugene, OR)
((Z-Pro-Arg)2-R110 ((Z-PR)2-R110) and
(Z-Phe-Arg)2-R110
((Z-FR)2-R110)).
PBS reaction buffer was used with the enzymes catG, HMC, HNE, CT, plasmin, thrombin, trypsin, and u-PA. Cathepsin reaction buffer (50 mM sodium acetate, pH 5.5, 4 mM dithiothreitol, 1 mM EDTA) was used with catL, catB, catK, and catS. Unique reaction buffers were used with PR3 (200 mM NaCl, 50 mM Tris-HCl, pH 6.7), granzyme B (PBS, 4 mM dithiothreitol) and PSA (PBS, 0.1% Tween 20). The reaction buffer for HMC was either PBS or 1 M NaCl buffer (1 M NaCl, 0.2 M Tris, pH 8.0, 0.1% Tween 20).
Determination of Enzyme ConcentrationsTrypsin was
calibrated by the method of Chase and Shaw (26) using
p-nitrophenyl-p-guanidinobenzoate, except that
Tris-HCl was used in place of sodium barbiturate. The concentration of
1PI was standardized against calibrated trypsin.
Cathepsin G was calibrated against the standardized
1PI.
1ACT was calibrated against the standardized catG. The
concentration of HMC was determined by activity of the enzyme in 1.8 M NaCl, 0.45 M Tris-HCl, pH 8.0, 10%
Me2SO, using 1.0 mM Succ-AAPF-pNA (25). The
specific activity of HMC in this buffer was 0.025
A410/min/pmol. The concentrations of SCCA2
were determined by Bradford analysis (Bio-Rad Protein Assay Kit II) and
amino acid composition analysis by post-column ninhydrin detection on a
Beckman 6300 amino acid analyzer (Beckman Instruments, Fullerton,
CA).
Enzyme inhibition was determined by
mixing enzyme and inhibitor in the appropriate buffer and incubating
for 30 min at 25 °C. Residual enzyme activity was determined by
adding the appropriate substrate and measuring its hydrolysis over time
(velocity) using the UVmax microplate reader (Molecular Devices,
Sunnyvale, CA) or the FluorImager 575 (Molecular Dynamics, Sunnyvale,
CA). Due to its enzymatic activity, PSA was incubated with inhibitors
at 37 °C for 2 h. Initial assays were performed at a ratio of
inhibitor/enzyme (I/E) of approximately 10/1. When no
inhibition was detected, I/E ratios were increased up to
~100/1. The concentrations of enzyme, inhibitor, and substrate are
listed in Table I, and the buffers are listed above. Percent
inhibition = 100 × (1 (velocity of inhibited enzyme
reaction/velocity of uninhibited enzyme reaction)).
|
Assays were performed in a volume of 100 µl in low-binding microtiter plates (Costar 9017, Costar, Cambridge, MA). Varying amounts of inhibitor were incubated with enzyme for 15-30 min at 25 °C. Ten microliters of substrate was added to terminate the reaction. The velocity of substrate hydrolysis (i.e. the release of pNA over time) was determined by measuring the A405 using the UVmax microplate reader. The partitioning ratio of the inhibitor-enzyme association was determined by plotting the fractional activity (velocity of the inhibited enzyme reaction/velocity of the uninhibited enzyme reaction) versus the ratio of the inhibitor to enzyme ([I]0/[E]0) (27). Linear regression analysis was used to determine the x intercept (i.e. the stoichiometry of inhibition (SI)).
Second Order ReactionsThe association rate constant for the interaction of catG and HMC with SCCA2 was determined under second order conditions (28). Equimolar amounts of protein and inhibitor were incubated at 25 °C for varying periods of time. The reaction was stopped by the addition of substrate (final volume 110 µl), and the velocity of the free enzyme activity was measured using the UVmax plate reader. Velocity was converted to free enzyme concentration using an enzyme concentration standard curve. The rate of change in the amount of free enzyme over time is described as shown below, where the slope of the plot of reciprocal remaining free enzyme (1/Ef) over time (t) yields a second order rate constant (ka).
![]() |
(Eq. 1) |
The interaction of SCCA2 with HMC was also determined by the progress curve method (29). Under pseudo-first order conditions, a constant amount of enzyme was mixed with different concentrations of inhibitor and excess substrate. The rate of product formation was measured on the UVmax plate reader. Since the inhibition of HMC is assumed to be irreversible over the course of the reaction, product formation is described as shown below, where the amount of product formation (P) proceeds at an initial velocity (vz) and is inhibited over time (t) at a rate (kobs).
![]() |
(Eq. 2) |
![]() |
(Eq. 3) |
Proteins were mixed with 2 × loading buffer (4% SDS, 20% glycerol, 120 mM Tris-HCl, pH
6.8, 0.01% bromphenol blue, 28 mM -mercaptoethanol), heated to 95 °C for 5 min, and separated by SDS-PAGE (10%
acrylamide, %T:%C = 19:1) according to the method of Laemmli
(30). The running buffer (pH 8.3) was 25 mM Tris base, 250 mM glycine, 0.1% SDS. Protein bands were visualized after
staining in a solution containing 0.25% Coomassie Brilliant Blue
R-250, 45% methanol, 10% acetic acid. For amino acid sequence
analysis, the ~4-kDa cleavage fragment formed after mixing SCCA2 with
catG was isolated by electrophoresis through a 5-20% gradient gel and
by electroblotting to a polyvinylidene difluoride membrane
(Immobilon-PSQ, Millipore, Bedford, MA) (31). N-terminal peptide
sequence analysis was performed using a peptide sequencer (Applied
Biosystems, Foster City, CA).
A rabbit polyclonal antisera was raised against SCCA by immunization of a New Zealand White rabbit with the GST-SCCA2 fusion protein. The antisera was passed over a glutathione-Sepharose 4B-GST column, and its specificity was tested on immunoblots containing GST-SCCA1, GST-SCCA2, ovalbumin, and GST. As expected, the polyclonal anti-SCCA reagent detected both SCCA2 and SCCA1, but not the closely related serpin, ovalbumin. The preimmune sera did not detect any of these proteins. Other tests of antisera specificity included: (i) detection of the appropriately sized 45-kDa band on immunoblots of cell lysates prepared from SCCA1 or SCCA2 transfected cell lines but not from mock transfected, non-SCCA-expressing controls; and (ii) immunoprecipitation of in vitro translated, 35S-labeled SCCA1 or SCCA2 proteins but not the control luciferase protein. Translation products were generated using a coupled transcription-translation in vitro expression system (Promega, Madison, WI) with plasmids containing the cDNA of SCCA2, SCCA1, or luciferase under the control of a T7 promoter.
Proteins separated by SDS-PAGE were electroblotted at 100 V for 1 h at 4 °C, onto reinforced nitrocellulose (NitroPlus, Micron Separations, Inc., Westborough, MA) as described (32). The transfer buffer was 25 mM Tris base, pH 8.0, 190 mM
glycine (pH 8.3). Membrane-bound protein was detected using the Western
Light chemiluminescence kit from Tropix (Bedford, MA), per the
manufacturer's instructions. In brief, the blot was washed three times
in wash buffer (PBS, pH 7.4, 0.1% Tween 20) and incubated for 1 h
in I-Block (Tropix) blocking buffer (PBS, pH 7.4, 0.1% Tween 20, 0.2%
I-Block). The blot was incubated for 1 h with primary antisera.
The primary detection antibodies were the rabbit polyclonal antisera
raised against SCCA (for the purpose of detecting the purified
GST-SCCA2 fusion protein unadsorbed antiserum at 1/100,000 dilution in
blocking buffer was used), catG (1/5,000 dilution, Athens Research
Biotechnologies), and HMC (1/1,000 dilution, Ref. 33). After three
rinses in wash buffer, the blot was incubated with a secondary antibody
(1/10,000 dilution of an alkaline phosphatase-conjugated goat
anti-rabbit antibody provided in the Western Light kit) and then washed
two times in wash buffer and three times in chemiluminescent assay buffer (0.1 M diethanolamine-HCl, pH 9.8, 1.0 mM MgCl2, 5% NitroBlock). The blot was
incubated for 5 min with detection substrate (either CSPD or CDP-Star
(Tropix) diluted to 0.25 mM in chemiluminescent assay
buffer). Chemiluminescence was detected by photography. To remove bound
antibodies prior to re-probing, the blot was incubated at 65 °C for
20 min in a solution containing 62.5 mM Tris-HCl, pH 8.0, 2.0% SDS, 0.1 M -mercaptoethanol.
A
Leu-Ser at the putative reactive center (P1-P1),
suggested that SCCA2 could inhibit chymotrypsin-like proteinases such as CT, HMC, catG, or PSA (Fig. 1). However, predictions
of inhibitory specificity based only on reactive site residues are
unreliable. Therefore, we screened for SCCA2 inhibitory activity by
incubating the serpin with a panel of serine and cysteine proteinases
prior to the addition of the appropriate enzyme substrate (Table
I). SCCA2 inhibited completely the enzymatic activity of
the chymotrypsin-like proteinases HMC and catG, but showed no
inhibitory activity against CT or PSA. SCCA2 also showed a modest
inhibition of PR3, but not HNE. SCCA2 showed no activity against
trypsin-like proteinases, granzyme B, or several papain-like cysteine
proteinases (cathepsins B, L, and K). SCCA2 in the presence or absence
of heparin did not inhibit thrombin.
Upon
binding with their target proteinases, most serpins form complexes that
are stable to denaturation in SDS, even in the presence of reducing
agents (34). This stability indicates the presence of a covalent bond
between the enzyme and inhibitor, which arises through stabilization of
a covalent intermediate analogous to that formed during peptide bond
hydrolysis (35-38). To determine whether SCCA2 forms such complexes,
the serpin was mixed with different proteinases and then incubated at
95 °C for 5 min in the presence of 2% SDS and 14 mM
-mercaptoethanol. Samples were analyzed by SDS-PAGE (Fig.
2A). The complex should appear as a high
molecular weight band reflective of the combined weights of SCCA2
(GST-SCCA2, Mr ~71,000) plus the proteinase.
SCCA2 formed a 90-100-kDa SDS-stable complex with both HMC
(~Mr 30,000) and catG
(~Mr 23,500), but not with CT, HNE, trypsin,
catS (very similar to catL and catK) or catB (Fig. 2A). The
presence of SCCA2-HMC and SCCA2-catG complexes were confirmed by
immunoblotting. The antisera specific for SCCA (Fig. 2B) and
either HMC (Fig. 2C) or catG (Fig. 2D) bound to
the same high molecular weight band.
SCCA2 served as a substrate for CT, HNE, trypsin, and catS, as these enzymes cleaved SCCA2 into several smaller fragments. A small amount of SCCA2 appeared to form a complex with PR3 (Fig. 2B, lane 4). However, the majority of the serpin was cleaved into several smaller fragments. These results suggested that SCCA2 inhibited catG and HMC via formation of a tight complex, whereas SCCA2 inhibited PR3 via simple competition with the substrate, or by a serpin-like mechanism with a high partitioning ratio (see below).
The Reactive Site of SCCA2The formation of an SDS-stable
complex suggested that proteinase inhibition occurred via an
interaction between the enzyme's active site and the serpin's RSL. If
this were the case, then transition from a tetrahedral intermediate to
a stable acyl-enzyme intermediate would result in cleavage of the
putative P1-P1 bond and release of an ~4-kDa, C-terminal fragment
from the serpin. The N terminus of the released fragment should
correspond to the P1
residue. To test this hypothesis, SCCA2-catG
complexes were separated by SDS-PAGE and electroblotted to a
polyvinylidene difluoride membrane. A 4-kDa fragment was identified and
subjected to N-terminal amino acid sequencing. Six cycles yielded a
sequence of SSPSTN. This sequence matched the P1
-P6
residues of
SCCA2 (Fig. 1) and confirmed that the Leu-Ser residues
served as the reactive site in SCCA2's interaction with catG.
The interaction between a
classical inhibitory-type serpin and its target proteinase usually
results in the formation of a tight complex at a stoichiometry of 1:1
(10). Under certain conditions, however, parallel substrate-like
reactions are present, which result in proteinase-mediated hydrolysis
and inactivation of the serpin. The degree to which the
serpin-proteinase complex preferentially partitions toward the
substrate rather than the inhibitor pathway is reflected by SI values
greater than 1 (39). The SI for a serpin-proteinase reaction is
determined by titration of the proteinase with the inhibitor and
extrapolating the data to the
[I]0/[E]0 ratio required for
complete enzymatic inhibition. When varying amounts of SCCA2 were
incubated with a fixed amount of either catG or HMC in PBS (~140
mM NaCl), the SI was ~1 and 1.5, respectively (Fig.
3). In a previous report, the SI of 1ACT with HMC decreased from ~6.0 to ~4.5 as the ionic strength of the
buffer increased from ~150 mM NaCl to 1 M
NaCl (24). Therefore, we repeated the SCCA2 and HMC titration in 1 M NaCl buffer. The SI decreased to ~1 (Fig. 3).
Collectively, these data showed that SCCA2 formed stable complexes with
either catG or HMC at near 1:1 stoichiometry, and that little SCCA2 was
partitioning into the substrate pathway.
Kinetics of the Interaction between SCCA2 and Chymotrypsin-like Proteinases
To determine the rate of complex formation between
SCCA2 and either catG or HMC, second order rate constants
(ka) were measured. Since the interaction of SCCA2
with HMC and catG was near 1:1, the ka was
determined under second order conditions (28). Equimolar amounts of
SCCA2 and proteinase were incubated in the absence of substrate. At
various time points, the reaction was stopped by the addition of
substrate and the remaining free enzyme activity was measured. The
ka for the interaction was calculated by using a
simple linear regression formula (Equation 1). The
ka of SCCA2 and catG under second order conditions
was 1.0 × 105 M1
s
1 (Fig. 4, Table II). The
interaction of SCCA2 and HMC was measured under second order conditions
using the 1 M NaCl buffer previously described in the
kinetic analysis of
1ACT and HMC (24, 25). The
ka for the interaction of SCCA2 and HMC was 2.8 × 104 M
1 s
1 (Fig.
4, Table II).
|
The ka for the interaction between SCCA2 and HMC was
determined also under pseudo-first order conditions using the progress
curve method (29). This technique also permitted a direct comparison of
the ka for SCCA2-HMC versus
1ACT-HMC associations. HMC was incubated with an excess
of SCCA2 or
1ACT in the presence of substrate. The
progress of enzyme inactivation was followed and represented as a
simple decay with a rate, kobs. The
kobs obtained at different concentrations
(Equation 2) were plotted against the inhibitor concentrations. The
slope of this line (k
) and the Km of the
substrate were used to calculate the overall second order rate constant
as described by Equation 3. The ka for SCCA2-HMC and
1ACT-HMC in PBS were 3.7 × 104
M
1 s
1 and 3.7 × 103 M
1 s
1,
respectively (Fig. 5). Although the
ka for the
1ACT-HMC interaction is
lower than that of SCCA2-HMC, the former value is also less than that
reported (ka = 2.1 × 104
M
1 s
1) for the
1ACT-HMC interaction analyzed under slightly different assay conditions (24). The ka for the interaction
with SCCA2-HMC (2.6 × 104
M
1 s
1) did not change
appreciably by performing the assays in 1 M NaCl buffer
(data not shown). The ka for the SCCA2-HMC
interaction, regardless of the buffer used, was similar to that
obtained under second order conditions (Table II).
A comparison of the primary structure of SCCA2 to those of other
serpins led us to predict that this molecule could serve as a
proteinase inhibitor. However, this prediction was tentative due to the
presence of a rare hydrophobic residue at the P14 position and a unique
glutamic acid residue at the P2 position of the RSL (Fig. 1). These
deviations from the RSL consensus sequence prompted us to determine
experimentally whether SCCA2 was a proteinase inhibitor. The results of
this study indicated unequivocally that SCCA2 is an inhibitory-type
serpin. Based on SDS-PAGE and in vitro kinetic analyses,
SCCA2 was shown to bind stably and inhibit two chymotrypsin-like serine
proteinases, HMC and catG. SCCA2 was unable to inhibit two other
enzymes with similar substrate specificities, CT and PSA. SCCA2 showed
no serpin-like inhibitory activity against neutrophil elastase or
trypsin-like serine proteinases. SCCA2 demonstrated modest inhibitory
activity against the neutrophil-derived, elastolytic enzyme, PR3.
However, SDS-PAGE showed minimal complex formation and extensive
cleavage of SCCA2. This suggested that SCCA2 inhibited PR3 by
simple competition with the substrate or by a serpin-like mechanism
with a high partitioning ratio. Although the inhibition of PR3 by SCCA2
may prove to be of little physiologic significance, this interaction
may signify the ability of SCCA2 to interact with other
non-chymotrypsin-like serine proteinases. If this proves to be the
case, it will be interesting to determine whether the Leu-Ser
residues serve as the reactive site for these interactions or if
other residues in the RSL are involved. 2-Antiplasmin, for example, uses overlapping residues in the RSL to inhibit plasmin and CT (40).
The ability of SCCA2 to form SDS-stable complexes with its target
proteinases confirmed that the inhibitory mechanism conforms with that
described for most inhibitory-type serpins (reviewed in Refs. 9, 10,
16, 17, and 39). Most likely, this mechanism involves the formation of
a stable tetrahedral or acyl-enzyme intermediate formed by a linkage
between the -O of the enzymes' active site serine and the
-carboxyl of SCCA2's P1 leucine. Subsequent cleavage of the P1-P1
bond should yield a ~4-kDa C-terminal fragment with an N terminus
corresponding to the P1
serine residue of SCCA2. Indeed, such a
fragment was detected after the binding of SCCA2 with catG. This
observation supports the notion that SCCA2 interacts with its target
serine proteinases via the RSL and that like most serpins, functions as
a suicide-substrate inhibitor (11, 39, 41-43).
The inhibition of catG and HMC by SCCA2 provides the first insight into the types of serine proteinases inhibited by this serpin. Cathepsin G is a cationic neutral serine proteinase that is synthesized by cells in the myelomonocytic series, especially neutrophils and mast cells (44, 45). The active form of catG is stored, along with other proteinases, in secretory granules (46). Upon release of the granules, catG preferentially cleaves peptide bonds on the C-terminal side of aromatic or leucine residues. Known catG substrates include laminin, type IV collagen, fibronectin, elastin, proteoglycans, immunoglobulins, complement components, clotting factors, and cytokines (47-52). Cathepsin G also cleaves pro-forms of interleukin-8 (53) and several matrix metalloproteinases (54, 55), converts either angiotensinogen or angiotensin I to the vasoactive polypeptide angiotensin II (56, 57), and activates platelets, lymphocytes, and macrophages (58-60). Cathepsin G is cytotoxic to some mammalian cells (61), and, similar to other serine proteinases in the azurophilic granule, catG has a bactericidal effect (62, 63). However, the bactericidal activity is not mediated by the catalytic domain.
Considering the diversity of catG substrates, it will be important to
determine whether SCCA2 is a physiologic inhibitor of this proteinase.
1ACT and
1PI are two well studied serpins
that inhibit catG at 1:1 stoichiometry. Using second order rate
constants (ka) as a measure of potency,
1ACT (ka ~5 × 107
M
1 s
1) and
1PI
(ka ~4 × 105
M
1 s
1) appear to be better
inhibitors of catG than SCCA2 (ka ~1 × 105 M
1 s
1) (28).
However, a ranking based on ka cannot in itself be
used to predict the physiologic potency of these serpins in vivo. For example, factors such as local concentration of the inhibitor, the susceptibility of the proteinase to inhibition (e.g. some proteinases bound to cell surface receptors or
heparans are relatively resistant to inhibition; Refs. 64 and 65), the
presence of serpin inactivators (e.g. oxidants, pH, and
other proteinases; Ref. 28), and the availability of serpin-proteinase complex clearance mechanisms (66) will affect the overall ability of a
serpin to modulate activity of its target proteinase. Thus, a better
understanding of the pattern of SCCA2 expression, secretion, and its
local concentration should help determine whether SCCA2 is a
physiologic inhibitor of catG.
HMC is a cationic, neutral serine proteinase that also cleaves peptide
bonds C-terminal of aromatic or leucine residues (reviewed in Ref. 67).
HMC is stored exclusively in the secretory granules of a subset of mast
cells. Chymase-containing mast cells are located in the dermis, the
submucosa of the large and small intestine, and the subepithelium of
the nasal mucosa, bronchi, and bronchioles (68). HMC converts
angiotensin I to angiotensin II (69) and pro-interleukin-1 (70) to
their active forms. Other chymase substrates include substance P, VIP,
type IV collagen, fibronectin, and proteoglycans (67). Chymase is a
potent secretagogue of bronchial serous glands (71) and enhances
histamine-induced vascular permeability (72).
Considering the diversity of HMC activities, it also will be important
to determine whether SCCA2 is a physiologic inhibitor of this
proteinase. SCCA2's inhibition of HMC proteolytic activity may be the
most significant yet described for a member of the serpin family
against this mast cell proteinase. Previously, 1ACT and,
to a lesser extent,
1PI were shown to inhibit HMC (24). In a direct comparison, the rate of association for SCCA2-HMC was 1 order of magnitude greater than that for
1ACT-HMC.
Furthermore, SCCA2 formed inhibitory complexes at near 1:1
stoichiometry, whereas the SI for
1ACT (~4.5) and
1PI (~5.0) is markedly greater (24). The SI results
suggested that most of the SCCA2-HMC complexes partition into the
inhibitory pathway, whereas the majority of
1ACT- and
1PI-HMC complexes partition into the substrate pathway (25). Thus, the predominantly plasma-derived serpins,
1ACT and
1PI, may not be the first line
of defense against a proteinase that achieves its highest concentration
and activity in extravascular compartments. In this regard, preliminary
immunohistochemical studies show that local SCCA2 expression occurs in
the epithelium adjacent to the submucosal sites known to harbor
chymase-positive mast cells (67). These data support the notion that
SCCA2 possesses the physiochemical traits and is in the appropriate
location to protect the epithelial barrier from the potentially
damaging effects of mast cell chymase.
The data presented in this report suggest a role for SCCA2 in regulating catG- and HMC-induced inflammation and tissue degradation within the epithelia of the skin and lung. However, it is unclear what role, if any, this inhibitory activity plays in the development or progression of squamous cell tumors. The generation of reagents (e.g. monoclonal antibodies) that discriminate between SCCA2 and SCCA1 will be critical in determining whether SCCA2 can interact with other epidermally derived proteinases that, for example, are involved in tumor invasion, cellular differentiation, inflammation, and wound repair.