|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 6 (March 15), 1999:
pp. 2089-2097
The Intracellular Serpin Proteinase Inhibitor 6 Is Expressed in
Monocytes and Granulocytes and Is a Potent Inhibitor of the
Azurophilic Granule Protease, Cathepsin G
By
Fiona L. Scott,
Claire E. Hirst,
Jiuru Sun,
Catherina H. Bird,
Stephen P. Bottomley, and
Phillip I. Bird
From the Department of Medicine, Monash Medical School, Box Hill
Hospital, Box Hill, Australia; and the Department of Biochemistry and
Molecular Biology, Monash University, Clayton, Australia.
 |
ABSTRACT |
The monocyte and granulocyte azurophilic granule proteinases
elastase, proteinase 3, and cathepsin G are implicated in acute and
chronic diseases thought to result from an imbalance between the
secreted proteinase(s) and circulating serpins such as  1-proteinase inhibitor and 1-antichymotrypsin. We show here that the
intracellular serpin, proteinase inhibitor 6 (PI-6), is present in
monocytes, granulocytes, and myelomonocytic cell lines. In extracts
from these cells, PI-6 bound an endogenous membrane-associated serine proteinase to form an sodium dodecyl sulfate (SDS)-stable
complex. Using antibodies to urokinase, elastase, proteinase 3, or
cathepsin G, we demonstrated that the complex contains cathepsin G. Native cathepsin G and recombinant PI-6 formed an SDS-stable complex in
vitro similar in size to that observed in the extracts. Further kinetic
analysis demonstrated that cathepsin G and PI-6 rapidly form a tight
1:1 complex (ka = 6.8 ± 0.2 × 106
mol/L 1s1 at 17°C;
Ki = 9.2 ± 0.04 × 1010 mol/L).
We propose that PI-6 complements 1-proteinase inhibitor and
1-antichymotrypsin (which control extracellular proteolysis) by
neutralizing cathepsin G that leaks into the cytoplasm of monocytes or
granulocytes during biosynthesis or phagocytosis. Control of intracellular cathepsin G may be particularly important, because it has
recently been shown to activate the proapoptotic proteinase, caspase-7.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
IMPORTANT PHYSIOLOGICAL processes such as
blood coagulation, fibrinolysis, complement activation, embryo
implantation, extracellular matrix remodeling, and cell differentiation
involve proteolysis mediated by serine proteinases.1 The
activity of many of these proteinases can be controlled by one or more
members of the serine proteinase
inhibitor (serpin) protein superfamily.1 The
physiological significance of serpins is illustrated by the consequences of defects in their production or activity. Loss of the
thrombin inhibitor, antithrombin III, results in a greatly increased
risk of thrombosis; loss of the serpin C1 inhibitor, which is a
regulator of the classical complement activation pathway, leads to
angioedema; and loss of  1-proteinase inhibitor activity underlies
emphysema and liver cirrhosis.2
Inhibitory serpins typically form 1:1 complexes with their target
proteinases by acting as pseudosubstrates.1 The serpin undergoes a conformational change leading to stabilization of the
serpin-proteinase complex, which becomes resistant to a variety of
denaturants, including sodium dodecyl sulfate (SDS). The
serpin pseudosubstrate region is known as the reactive center and
consists of approximately 20 amino acids near the carboxy terminus.
Cleavage of the serpin by the proteinase may occur in this region
between two residues designated P1 and
P1'. The identity of the P1 residue largely dictates the specificity of the serpin-proteinase interaction, whereas those surrounding the cleavage site contribute to the affinity
of the interaction.
Although the vast majority of serpins are secreted glycoproteins
involved in regulating extracellular proteinases, it has recently
become clear that some function within cells. One example is the cowpox
virus serpin CrmA, which inhibits both the cytotoxic lymphocyte granule
proteinase, granzyme B, and the intracellular caspases involved in
cytokine maturation and apoptosis.3,4 Apart from CrmA,
several proteins belonging to the mammalian ovalbumin (ov) serpin group
also appear to function intracellularly. At present, this group
includes plasminogen activator inhibitor 2,5 the squamous
cell carcinoma antigens-1 and -2,6,7 monocyte/neutrophil elastase inhibitor,8 maspin,9 proteinase
inhibitor 6,10 proteinase inhibitor 8,11
proteinase inhibitor 9,11,12 bomapin,13 and
megakaryocyte maturation factor.14 The ov-serpins are
unusual because they lack an amino terminal signal peptide normally
required to initiate extracellular secretion of the protein, yet
several appear to exist in both intracellular and extracellular
forms.15 The physiological roles of these inhibitors are
not fully understood, but some may function in processes associated
with tumorigenesis and inflammation. For example, loss of maspin
correlates with increased malignancy and metastasis of breast
carcinomas9; increases in the levels and release of
squamous cell carcinoma antigen occur in squamous cell
carcinomas6; and increased expression and release of
plasminogen activator inhibitor 2 occurs from monocytes during
inflammation.16 Recent evidence suggests that proteinase inhibitor 9 is a regulator of granzyme B that may protect immune cells
against the effects of this cytotoxin.12
We originally described the ov-serpin proteinase inhibitor 6 (PI-6)
after a screen for novel thrombin inhibitors.17 Unlike the
majority of ov-serpins, PI-6 is apparently restricted to the cytoplasm
of cells and cannot be released via the conventional secretory
pathway.18 It is present in most tissues in capilliary endothelial cells, platelets, and epithelial cells.10,18
Sequence comparisons suggest that the P1 residue of PI-6 is
Arg,10 but although this explains the interaction of PI-6
with thrombin, subsequent analyses have shown that PI-6 is only a
moderately effective thrombin inhibitor that inhibits trypsin far more
efficiently.19,20 Of additional interest is the finding
that PI-6 is also a very good chymotrypsin inhibitor,21 due
to the presence of Met at the P2 position in the reactive center.
The broad distribution and dual inhibitory capacity of PI-6 suggests
that it fulfils an important regulatory or protective role associated
with proteolysis within cells. Yet, despite extensive study in vitro,
the identity of the proteinase(s) targeted by PI-6 in vivo remains
obscure. We show here that PI-6 is also produced in monocytes and
granulocytes and that it interacts with a membrane-associated proteinase expressed in these cells. We have identified this proteinase as the azurophilic granule component cathepsin G and have shown that
PI-6 inhibits it very efficiently. We propose that PI-6 functions to
protect the interior of cells against ectopic or inadvertant exposure
to cathepsin G.
| |
MATERIALS AND METHODS |
Reagents and antibodies.
Preparation of recombinant PI-6 has previously been
described.19 Recombinant cathepsin G was purchased from
Calbiochem (La Jolla, CA), and the cathepsin G substrate
N-succinyl-Ala-Ala-Pro-Phe-pNA was from Sigma (St Louis,
MO). Anti-urokinase plasminogen activator antiserum was
purchased from America Diagnostica (Sydney, Australia). Polyclonal anti-PI-6 antisera is described elsewhere.18
Monoclonal anti-PI-6 antibody (3A) was produced to recombinant PI-6 by
standard procedures.22 Hybridomas producing immunoreactive
antibody were identified using enzyme-linked immunosorbent assay
(ELISA) on immobilized recombinant PI-6 and indirect
immunofluorescence on PI-6 transfected COS cells.23 Rabbit
polyclonal antibodies24 recognizing cathepsin G, proteinase
3, and human leukocyte elastase were a kind gift from Dr J. Hoidal
(University of Utah, Salt Lake City, UT). Monoclonal 3C10 anti-CD14
antibody was a kind gift from Dr R. Steinman (Rockefeller Institute,
New York, NY).
Biotinylation of monoclonal anti-PI-6 antibody (3A).
One milligram of purified antibody was biotinylated by exposure to
sulfo-NHS-Biotin according to the manufacturer's instructions (EZ-Link; Pierce, Rockford, IL). Unbound biotin was
removed by extensive dialysis against phosphate-buffered saline (PBS).
Cell culture.
HL60, THP-1, and U937 cells were cultured in RPMI-1640 media
supplemented with 10% heat-inactivated fetal calf serum, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 50 µg/mL streptomycin. Cultures were maintained in a 5% CO2, 95% air mixture at 37°C
and passaged every 3 days.
Isolation of peripheral blood monocytes, neutrophils, and
lymphocytes.
Human monocytes were purified from whole blood using Nycoprep 1.068 according to the manufacturer's instructions. To isolate lymphocytes
and neutrophils, erythrocytes were removed from whole blood containing
0.15% (wt/vol) EDTA by precipitation using 1.2% Dextran 500 (wt/vol)
for 1 hour at room temperature. Neutrophils were pelleted from the
leukocyte-rich plasma by centrifuging over Ficoll Hypaque (Pharmacia,
Sydney, Australia) at 2,500g. The supernatant containing the lymphocytes was also retained. All cell preparations were washed repeatedly with PBS and lysed with 10 mmol/L Tris-HCl, 0.15 mmol/L NaCl, pH 7.6, containing 1% Triton X-100 and 1 µg/mL aprotinin, 150 µg/mL phenylmethyl sulfonyl fluoride
(PMSF), 0.5 µmol/L leupeptin, and 1 µmol/L
pepstatin. Total protein concentration was determined (Bio-Rad,
Hercules, CA), and equal amounts of total protein were
immunoblotted with rabbit polyclonal anti-PI-6 as described below.
Fluorescence-activated cell sorting (FACS) analysis.
All steps were performed at room temperature and during staining
samples were kept in the dark. One hundred microliters of whole blood
from a healthy volunteer was stained with a monoclonal antibody
recognizing CD14 (3C10). After 30 minutes, cells were washed twice in
PBS containing 1% neonatal calf serum and 0.02% sodium azide (FACS
buffer; used in all subsequent washes). Cells were incubated with sheep
antimouse Ig-PE (Silenus, Melbourne, Australia) for 30 minutes, washed twice, and then incubated in FACS Lysing Solution
(Becton Dickinson, Mountain View, CA) for 10 minutes.
Cells were collected and resuspended in FACS Permeabilizing Solution
(Becton Dickinson) for 10 minutes, washed, and then blocked in 10%
normal mouse serum for 30 minutes. Biotinylated monoclonal 3A
anti-PI-6 antibody was then added for 30 minutes. Cells were washed
twice and incubated with streptavidin-fluorescein isothiocyanate (FITC) (Amersham, Sydney, Australia) for 30 minutes. After two washes, cells were analyzed using a Becton Dickinson
FACScan and CellQuest software.
Immunoblotting.
Cells were washed with ice-cold PBS and subsequently lysed with 10 mmol/L Tris-HCl, 0.15 mmol/L NaCl, pH 7.6, containing 1% Triton X-100.
In some experiments, 1 µg/mL aprotinin, 150 µg/mL PMSF, 0.5 µmol/L leupeptin, 1 µmol/L pepstatin, 100 µmol/L EDTA, or a
cocktail of all the proteinase inhibitors were included in the lysis
buffer. To allow the PI-6 and cathepsin G interaction to occur, samples
were incubated at 37°C for 10 minutes after the addition of lysis
buffer. Cell lysates were centrifuged to remove cell debris, boiled in
Laemmli sample buffer containing 0.1 mol/L dithiothreitol
(DTT) for 5 minutes, and resolved on 10%
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions.25 After electrophoresis, proteins were
transferred to nitrocellulose membranes and incubated for 1 hour in
blocking buffer (5% skim milk powder in 20 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.6). Membranes were incubated for 1 hour with rabbit polyclonal anti-PI-6 antiserum or monoclonal 3A anti-PI-6 and washed
in 20 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.6. Horseradish peroxidase
(HRP)-conjugated sheep antirabbit Ig (Silenus) or
HRP-conjugated sheep antimouse Ig (Bio-Rad) were used as secondary
antibodies and were detected using enhanced chemiluminescence (DuPont,
Sydney, Australia).
Cell fractionation.
U937 cells were washed in PBS and resuspended in hypotonic buffer (50 mmol/L PIPES, 50 mmol/L KCl, 5 mmol/L EGTA, 2 mmol/L MgCl2,
5 mmol/L DTT, pH 7.6). Cells were sonicated on ice at 100 W five times
for 30 seconds. Samples were centrifuged at 1,000g to remove
nuclei and whole cells and then at 100,000g for 1 hour. The
supernatant was kept (cytosol) and the pellet was extracted with 1%
Triton X-100 in 10 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.6, for 1 hour
at 4°C (membrane). Protein concentrations were determined and equal
amounts of cytosol and membrane were mixed and incubated at 37°C
for 10 minutes before SDS-PAGE and immunoblotting with polyclonal
anti-PI-6 antisera as described above.
35S-Methionine labeling and immunoprecipitation.
U937 cells (2 × 106) were
[35S]methionine labeled for 3 hours, as previously
described.18 Labeled cells were lysed and
immunoprecipitated overnight at 4°C with 100 µL of 10% (wt/vol)
protein A-sepharose (Pharmacia Biotech Inc, Sydney,
Australia) and 1 µL of the indicated rabbit polyclonal
antiserum, and immune complexes were analyzed by 10% SDS-PAGE
according to Laemmli.25 Gels were enhanced in Amplify
(Amersham Corp) and visualized by fluorography.
Immunoprecipitation of the PI-6/cathepsin G complex from U937 cells.
U937 cells (2 × 106) were washed with ice-cold PBS,
lysed in 1 mL 50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 5 mmol/L EDTA,
0.25% (wt/vol) gelatin (NETGEL) containing 1% Nonidet P-40, and
incubated at 37°C for 10 minutes. A further 1 mL of NETGEL was
added and extracts were immunoprecipitated with 1 µL of the
appropriate antibody. Immune complexes were analyzed by 10% SDS-PAGE,
and separated proteins were transferred to a nitrocellulose membrane, blocked for 1 hour as described previously, and immunoblotted with
monoclonal 3A anti-PI-6 antibody. HRP-conjugated sheep antimouse Ig
(Bio-Rad) was used as the secondary antibody and was detected using
enhanced chemiluminescence.
Analysis of the PI-6/cathepsin G complex in vitro.
Recombinant PI-6 (2 ng) was mixed with 1 to 200 ng of cathepsin G in 20 mmol/L Tris, 0.15 mol/L NaCl and incubated at 37°C for 10 minutes.
Samples were separated by SDS-PAGE under reducing conditions and
immunoblotted with polyclonal anti-PI-6 antisera as described above.
Determination of kinetic parameters.
The activity of rPI-6 was measured by inhibition of active
site-titrated thrombin using procedures described in Duranton et al.26 The active site concentration of cathepsin G was
determined using 1-proteinase inhibitor as previously
described26 using the substrate
suc-Ala-Ala-Pro-Phe-pNA in 50 mmol/L Tris, 150 mmol/L NaCl,
0.1% (wt/vol) polyethylene glycol (PEG), pH 7.8, at
20°C. Determination of the stoichiometry of inhibition was
performed as previously described27 using the substrate
suc-Ala-Ala-Pro-Phe-pNA in 50 mmol/L Tris, pH 7.8, 150 mmol/L
NaCl, 0.1% (wt/vol) PEG at 20°C. The inhibition
(Ki) and second order association rate (ka) constants were determined as previously
described.28 Briefly, the second order rate constant was
measured by mixing cathepsin G and PI-6 at equimolar concentrations (10 nmol/L) for variable periods of time before the addition of substrate
(0.2 mmol/L); the residual enzyme activity was then measured (Molecular
Dynamics plate reader; Molecular Dynamics, Sunnyvale, CA)
and the temperature of the incubation was maintained at 17°C. All
data presented represent the mean of five determinations.
| |
RESULTS |
Expression of PI-6 in peripheral blood leukocytes.
PI-6 was originally detected in cytosolic extracts from K562 cells,
which are human myeloleukemia cells that maintain the potential to
differentiate along the monocytic, granulocytic, or erythroid
lineages.17 It is also present in the megakaryocytic cell
line MEG-01 and in platelets.10 These observations
suggested that PI-6 is normally expressed in myeloid cell lineages. To
examine the distribution of PI-6 in human leukocytes, we used FACS to analyze permeabilized peripheral blood leukocytes stained with a
monoclonal antibody specific for PI-6. This antibody recognizes PI-6 by
indirect immunofluorescence and immunoblotting23 but does
not recognize the ov-serpins most closely related to PI-6, namely
proteinase inhibitor 8, proteinase inhibitor 9, monocyte/neutrophil elastase inhibitor, and plasminogen activator inhibitor 2 (data not shown).
As shown in Fig 1, the monoclonal antibody
marked the granulocytic (R2) population of leukocytes, but not the
small lymphocyte (R1) population (T and B cells). Double-staining with
an anti-CD14 monoclonal antibody confirmed that PI-6 is in the
monocyte/granulocyte population. PI-6 was present in both CD14-high and
CD14-low R2 cells, which correspond to monocytes and granulocytes,
repectively.
View larger version (16K):
[in this window]
[in a new window]
| Fig 1.
PI-6 is expressed in peripheral blood monocytes and
granulocytes. Leukocytes from fresh, normal peripheral blood were
analyzed by FACS after staining for PI-6 and CD14. The left-hand panel
shows a scatter plot of the distribution of leukocytes into the small
lymphocyte (R1) and monocyte/granulocyte (R2) populations. The central
and right hand panels show the results of two-color FACS analysis of
the R1 and R2 populations simultaneously stained with the anti-PI-6
monoclonal antibody 3A (x-axis) and an antibody recognizing the
monocyte/granulocyte differentiation antigen CD14 (y-axis). The R1
population is not significantly marked by either antibody, whereas the
R2 population is stained by both.
|
|
Detection of a PI-6/proteinase complex in monocyte/macrophage cell
lines.
To analyze the expression of PI-6 in cells of monocyte/macrophage
lineage in more detail, we used the well-established and characterized
human lines HL60, THP-1, and U937. These resemble myeloid cells
arrested at different stages of differentiation. For example, HL60
cells resemble less mature precursor cells and can be induced to
differentiate either along the monocyte/macrophage pathways by exposure
to phorbol esters or tumor necrosis factor or along the granulocyte
pathway by exposure to retinoic acid.29 By contrast, THP-1
and U937 cells resemble more mature cells that have acquired monocytic
characteristics.30
As shown in Fig 2A, PI-6 was detected in
extracts of U937 and THP-1 cells, but surprisingly most of the material
detected by both polyclonal and monoclonal antibodies to PI-6 was in
the form of a higher molecular weight species. The size of this species (56 kD) is too small to represent a PI-6 dimer, but it is reminiscent of an SDS-resistant complex that may form when a serpin interacts with
a serine proteinase.1 To determine whether the 56-kD
species is present in normal monocytes or granulocytes, PBLs were
fractionated into monocyte, neutrophil, and lymphocyte populations, and
cytosolic extracts were prepared. Overall, the distribution of PI-6
immunoreactive material in the purified leukocytes was consistent with
the FACS analysis (Fig 1). The 56-kD species was present in monocytes
but not in neutrophils or lymphocytes (Fig 2B).
View larger version (115K):
[in this window]
[in a new window]
| Fig 2.
PI-6 and a 56-kD PI-6/proteinase complex are found in
myelomonocytic cells. (A) HL60, THP-1, and U937 cells were lysed and
equal amounts of protein were separated by 10% SDS-PAGE under reducing
conditions and immunoblotted using polyclonal anti-PI-6 antisera or
monoclonal anti-PI-6 antibody. (B) Monocytes, neutrophils, and
lymphocytes were isolated from peripheral human blood as described in
Materials and Methods. Cells were lysed in the presence of 1 µg/mL
aprotinin, 150 µg/mL PMSF, 0.5 µmol/L leupeptin, and 1 µmol/L
pepstatin to minimize degradation by plasma proteinases. Equal amounts
of protein were separated by 12.5% SDS-PAGE under reducing conditions
and immunoblotted using polyclonal anti-PI-6 antisera. Both gels
contain 2 ng of recombinant PI-6 (rPI-6) loaded as a positive control.
The lower band in the rPI-6 sample represents serpin cleaved in the
reactive loop by nonspecific proteolysis during purification and
storage.
|
|
Establishment of a stable serpin-proteinase complex requires that the
proteinase is active, and complex formation may be perturbed or
abolished in the presence of compounds that bind the active site of the
proteinase.1 To test whether a proteinase is present in the
56-kD species, extracts of U937 cells were prepared in the presence of
a variety of protease inhibitors including EDTA, PMSF, aprotinin,
leupeptin, and pepstatin. As shown in Fig
3, the 56-kD species disappeared in the presence of the serine
proteinase inhibitors PMSF and aprotinin, but not in the presence of
inhibitors of other classes of proteases such as EDTA, leupeptin, and
pepstatin. From these results we concluded that the 56-kD species
represents PI-6 complexed with a serine proteinase produced by myeloid
cells.
View larger version (32K):
[in this window]
[in a new window]
| Fig 3.
The 56-kD PI-6/proteinase complex contains a serine
proteinase and is formed after lysis. U937 cells were lysed in the
presence of 1 µg/mL aprotinin, 150 µg/mL PMSF, 0.5 µmol/L
leupeptin, 1 µmol/L pepstatin, 100 µmol/L EDTA, or a mixture of all
these inhibitors. Samples were incubated at 37°C for 10 minutes,
separated by 10% SDS-PAGE, and immunoblotted using polyclonal
anti-PI-6 antisera.
|
|
PI-6 was essentially undetectable by immunoblotting of cytosolic
extracts from undifferentiated HL60 cells (Fig 2A), suggesting that
PI-6 expression is induced as monocytes mature. Addition of recombinant
PI-6 to HL60 cell extracts did not result in the appearance of the
56-kD PI-6-proteinase complex, indicating that the proteinase is also
not present in these precursor type cells (data not shown). Treatment
of HL60 cells with phorbol myristate acetate
(PMA)-induced monocyte/macrophage differentiation as
assessed by morphological alterations and acquisition of adhesiveness. Under these conditions, high levels of PI-6 were produced (data not
shown). By contrast, exposure of HL60 cells to retinoic acid induced
granulocytic differentiation as assessed by NBT assay,29 and lower levels of PI-6 were detected under these conditions (data not
shown). These results are consistent with the FACS analysis showing the
presence of PI-6 in peripheral blood monocytes and granulocytes and
suggest that PI-6 expression is induced at specific stages of
differentiation of these cells.
Compartmentalization of PI-6 and the proteinase in U937 cells.
Our previous studies have demonstrated that PI-6 is a cytosolic
serpin,17,18 which raised the possibility that the
interacting proteinase is also cytosolic. An alternative scenario is
that the proteinase is in a specific compartment within the cell, such as a membrane-bound granule, and that the interaction observed with
PI-6 occurs primarily after lysis of the cells. To distinguish these
possibilities, we fractionated U937 cells into membrane (100,000g pellet) and cytosolic components (after
100,000g supernatant) and analyzed the distribution of PI-6 and
the complex. As shown in Fig 4A, PI-6 was
present in the cytosolic fraction, but not in the membrane fraction.
The complex was not detected in either the cytosolic or membrane
fraction, but became apparent after the two fractions were mixed. This
indicated that the proteinase is in a separate, membranous compartment
that is normally inaccessible to PI-6. When recombinant PI-6 was added
to the whole-cell extracts, the amount of complex formed was much
larger than normally observed, indicating that the amount of endogenous
PI-6 is limiting in the extracts (Fig 4B). Taken together, these
results suggest that the U937 proteinase interacting with PI-6 is in a
separate, membranous compartment, that it is in excess to the amount of
PI-6 contained within the cytoplasm, and that the complex observed in
U937 cell extracts is probably formed after lysis.
View larger version (91K):
[in this window]
[in a new window]
| Fig 4.
The protease is in a membrane bound subcellular
compartment and is in excess to PI-6. (A) U937 cells were fractionated
into cytosol and Triton X-100 soluble membrane fractions as described
in Materials and Methods. Equal amounts of cytosolic protein, membrane
protein, and a 1:1 mixture of both were incubated at 37°C for 10 minutes. Samples were separated by 10% SDS-PAGE under reducing
conditions and immunoblotted using polyclonal anti-PI-6 antisera to
visualize the 56-kD complex. The 63-kD band present in the first two
lanes is nonspecific and was recognized by preimmune sera (not shown).
(B) U937 cells were lysed and incubated at 37°C for 10 minutes with
or without 2 ng recombinant PI-6 (rPI-6). Samples were separated by
10% SDS-PAGE under reducing conditions and immunoblotted using
polyclonal anti-PI-6 antisera. Two nanograms of recombinant PI-6 was
included in a separate lane as a positive control.
|
|
The PI-6/proteinase complex contains cathepsin G.
It is well established that myelomonocytic cells produce the serine
proteinases urokinase plasminogen activator, elastase, proteinase 3, and cathepsin G (reviewed in Borregaard and Cowland31). Numerous studies have shown that these proteinases are contained within
membrane-bound granules in resting cells and are released into
phagocytic vesicles and secreted during inflammation. It is also known
that expression of the elastase, cathepsin G, and proteinase 3 is
differentiation stage-specific and is downregulated on terminal
differentiation into macrophages.32
Because PI-6 can inhibit urokinase in vitro,19 we used
urokinase-specific antiserum to determine by immunoblotting whether this proteinase is present in the 56-kD complex. Although urokinase was
present in the U937 cells, we were unable to demonstrate any PI-6-urokinase interaction in the cell extracts, and experiments using
purified proteinase and inhibitor indicated that a PI-6-urokinase SDS-stable complex would be much larger than 56 kD (data not shown). We
therefore concluded that urokinase and PI-6 do not interact detectably
in U937 cell extracts.
To determine whether the 56-kD complex contains elastase, proteinase 3, or cathepsin G, we obtained rabbit antisera specific for each of these
proteinases24 (a kind gift of Dr J. Hoidal, Department of
Internal Medicine, University of Utah Health Sciences Center, Salt Lake
City, UT). As shown in Fig 5A, when U937
cells were metabolically labeled using 35S-methionine,
elastase, proteinase 3, or cathepsin G, which are all approximately 30 kD in size, could be immuoprecipitated from cell extracts using the
appropriate antiserum. The higher molecular (70 kD) species observed in
the elastase immunoprecipitate probably represents an
elastase-monocyte/neutrophil inhibitor complex (see Discussion). The
45-kD species seen in all the samples probably represents actin, which
commonly contaminates immunoprecipitates.
View larger version (113K):
[in this window]
[in a new window]
| Fig 5.
PI-6 coimmunoprecipitates with cathepsin G from U937
cells as a 56-kD complex. (A) U937 cells were
[35S]methionine labeled, lysed, and immunoprecipitated
with rabbit polyclonal antibodies recognizing cathepsin G, proteinase
3, or human leukocyte elastase. Immune complexes were separated by
12.5% SDS-PAGE under reducing conditions. (B) U937 cells were lysed
and incubated at 37°C for 10 minutes. Extracts were
immunoprecipitated with rabbit polyclonal antibodies recognizing
cathepsin G, proteinase 3, or human leukocyte elastase. Immune
complexes were separated by 12.5% SDS-PAGE and separated proteins were
immunoblotted using monoclonal 3A anti-PI-6 antibody.
|
|
Because these antisera do not recognize denatured antigen, they could
not be used directly in immunoblotting experiments to detect the PI-6
complex in U937 cell extracts. Instead, we used the antisera to
immunoprecipitate the target proteinases from cell extracts and then
used the anti-PI-6 monoclonal antibody to determine by immunoblotting
whether any PI-6 or complex had been coprecipitated. As a control, we
also used rabbit anti-PI-6 serum to immunoprecipitate the complex from
U937 extracts. Figure 5B shows that, of the four rabbit antisera used
in the experiment, only the cathepsin G and PI-6 sera
immunoprecipitated proteins that were detected using the PI-6
monoclonal antibody. The major species in each case was 56 kD in size,
indicating that the PI-6 proteinase complex in U937 cells almost
certainly contains cathepsin G.
Kinetics of the interaction of PI-6 and cathepsin G.
To confirm the interaction of PI-6 and cathepsin G, native human
cathepsin G was incubated with purified recombinant PI-6 in varying
ratios. This resulted in the formation of a complex identical in size
to the complex formed in U937 cell extracts (Fig 6). As expected for a typical
serpin-serine proteinase interaction, the amount of complex formed
varied with the ratio of proteinase to inhibitor. As the amount of
proteinase in the reaction increased, the amount of complex decreased
and was accompanied by the appearance of lower molecular weight forms
that probably represent the serpin cleaved in the reactive center by
cathepsin G.
View larger version (36K):
[in this window]
[in a new window]
| Fig 6.
Recombinant PI-6 and native cathepsin G form an
SDS-stable complex in vitro. Recombinant PI-6 (2 ng) was mixed with 1 to 200 ng of native cathepsin G and incubated at 37°C for 10 minutes. Samples were resolved by 10% SDS-PAGE under reducing
conditions and then immunoblotted with polyclonal anti-PI-6
antisera.
|
|
We then used standard procedures to analyze the kinetics of the PI-6
and cathepsin G interaction.26-28 The stoichiometry of inhibition of cathepsin G by PI-6 was found to be 1.0, and the inhibitory constant (Ki) was 9.2 ± 0.04 × 1010 mol/L, indicating very tight 1:1 binding. The
second order rate constant (ka) for the inhibition
of cathepsin G by PI-6 was measured at 17°C as 6.8 ± 0.2 × 106 mol/L1s1.
All attempts to accurately measure the ka at higher
temperatures failed because the reaction was too fast. Under
physiological conditions we estimate the lower limit for the
ka is 107
mol/L1s1.
| |
DISCUSSION |
Mononuclear phagocytes and neutrophils play key roles in the
elimination of bacterial and fungal pathogens via the generation of
reactive oxygen species and secretion of azurophilic granule cytotoxins
into phagocytic vesicles.31,33,34 Among the azurophilic granule cytotoxins are the neutral serine proteinases cathepsin G,
proteinase 3, and elastase. Apart from contributing to the destruction
of phagocytosed targets, these proteinases are sometimes secreted and
have pronounced effects on the extracellular matrix, cytokine
processing,35 chemotaxis,36 and platelet
activation.37 In addition, it has been shown recently that
cathepsin G also has the ability to proteolytically activate the
intracellular cysteine proteinase, caspase-7, suggesting that in some
circumstances cathepsin G may participate in apoptosis.38
Examples of extracellular targets of the granule proteinases include
connective tissue components such as fibronectin, elastin, proteoglycans, and collagen types III and IV,39-42 and it
has long been recognized that one or more of these proteinases are
implicated in cell and tissue injury in several acute and chronic
diseases, including emphysema, cystic fibrosis, asthma, arthritis, and
psoriasis.43-46 The pathological effects of the proteinases
are thought to arise through a localized imbalance between their levels
and the levels of their major serum inhibitors, 1-proteinase
inhibitor (1-antitrypsin) and 1-antichymotrypsin. This hypothesis
is supported by the observation that 1-proteinase inhibitor
deficiency is associated with the development of
emphysema.47
Clearly, control of these powerful proteinases is crucial for normal
tissue homeostasis. In this study we have shown that monocytes and
granulocytes produce PI-6 and that PI-6 is a potent inhibitor of
cathepsin G. We believe that the PI-6-cathepsin G interaction is
physiologically relevant because (1) the kinetics of the
PI-6-cathepsin G interaction show that complex formation is rapid and
tight, with the deduced association rate constant at the upper end of
the scale of known serpin-proteinase interactions1; and (2)
PI-6 and cathepsin G are produced in the same physiological compartment, suggesting that an interaction occurs in vivo. In contrast, the ov-serpin, squamous cell carcinoma antigen-2 (SCCA-2), has also recently been demonstrated to have cathepsin G inhibitory capacity.48 However, the interaction between SCCA-2 and
cathepsin G is 100-fold less efficient than the PI-6-cathepsin G
interaction, and there is no evidence to suggest that SCCA-2 and
cathepsin G colocalize in a physiologically relevant compartment.
Indeed, it is more likely that SCCA-2 is an inhibitor of mast cell
chymase, because it is expressed in the epithelium where
chymase-producing mast cells reside.48
The identity of the PI-6 P1 residue in the context of an
interaction with cathepsin G is presently unresolved. PI-6 was
initially described as an inhibitor of trypsin-like serine proteinases
that by sequence comparisons with other serpins has a P1
Arg.10 However, a subsequent study showed that it also
inhibits chymotrypsin.21 Peptide analysis in this study
demonstrated that PI-6 uses a Met upstream and adjacent to the Arg as
the P1 residue for interactions with chymotrypsin and
confirmed that it uses the P1 Arg for interactions with
trypsin. Thus, PI-6 is a dual function inhibitor with overlapping reactive sites.
Because cathepsin G is considered to resemble chymotrypsin in overall
structure and substrate preference,49 these results might
imply that Met rather than Arg in the reactive center of PI-6 is
crucial for cathepsin G binding. However, cathepsin G possesses an
unusual active site pocket that may accomodate a variety of bulky
P1 residues,50 and a very recent study has shown that it can efficiently cleave substrates after basic amino acids.51 According to this study, cathepsin G exhibits dual and equal trypsin and chymotrypsin specificities, hence it is conceivable that the PI-6-cathepsin G interaction can accomodate Arg
or Met as the P1 residue. Distinguishing these
possibilities will require peptide analysis of PI-6 cleaved by
cathepsin G; however, the absence of Met in the corresponding position
in mouse PI-652 suggests that Arg is the likely
P1 residue.
Studies by Remold-O'Donnell et al8,53,54 have elegantly
demonstrated that monocytes and neutrophils produce another ov-serpin, monocyte/neutrophil elastase inhibitor (M/NEI), that rapidly
inactivates leukocyte elastase and, to a lesser extent, proteinase 3 and cathepsin G. M/NEI is present in the cytoplasm of monocytes and
neutrophils and is thought to be released from cells during
inflammation primarily to control elastase. Although cathepsin G has a
different substrate preference to both elastase and proteinase 3 (which
prefer small hydrophobic P1 residues), the hydrophobic
nature of the PI-6 reactive center raises the possibility the latter
proteinases also interact with PI-6. At present, we cannot formally
exclude this, but the failure to coimmunoprecipitate elastase or
proteinase 3 with PI-6 from U937 extracts suggests either that
productive interactions do not occur or that cathepsin G competes very
efficiently with elastase and proteinase 3 for a limited amount of
PI-6.
Interestingly, M/NEI is highly homologous to PI-6 and the genes
encoding these inhibitors are part of a cluster on human chromosome 6,55 suggesting a close evolutionary relationship and
similarities or redundancy in function. Besides PI-6 and M/NEI,
monocytes and neutrophils produce a third ov-serpin, plasminogen
activator inhibitor 2 (PAI-2).5 This serpin is thought to
be the major regulator of urokinase, which is present in the specific
granules and is secreted during inflammation.31 PAI-2
exists predominantly within cells but can also be secreted on cell
stimulation.16 The secreted form of PAI-2 is thought to
regulate the extracellular activity of urokinase, but the function of
the intracellular form is unknown.
Between them, PI-6 and M/NEI have the potential to efficiently regulate
three major monocyte/granulocyte granule proteinases and to complement
the serum serpins 1-proteinase inhibitor and 1-antichymotrypsin
in vivo. It is interesting to consider why both extracellular and
intracellular inhibitors of granule proteinases exist. We believe that
the answer may lie in the dual functions of these proteinases as
enzymes acting within phagocytic vesicles to destroy microbes or other
ingested material and as extracellular modulators of the inflammatory
response. We postulate that, whereas the serum serpins act to contain
extracellular collateral damage mediated by the granule proteinases,
PI-6 and M/NEI act to contain intracellular damage occurring as
microbes or tissue debris are ingested and destroyed. If the vacuole
integrity is breached during phagocytosis, any proteinases released
into the interior of the cell would be rapidly inactivated by PI-6 and
M/NEI. This may be particularly important in light of the recent
finding that cathepsin G can activate a pro-apoptotic proteinase
(caspase-7)38 and thus has the potential to induce
apoptosis if released into the cytoplasm of the host cell.
In support of this model, it is known that oxidative stress such as
exposure to hydrogen peroxide can induce lysosomal rupturing in
cultured fibroblasts, leading to the release of lysosomal contents, including proteases, into the cell (discussed in Brunk et
al56). Although cells can apparently tolerate and repair
lysosomal ruptures to a certain degree, as the amount of damage
increases cells may undergo apoptosis (moderate damage) or necrosis
(substantial damage). Given that myeloperoxidase is a key component of
azurophilic granules and that phagocytic vesicles and lysosomes have
many structural similarities, we argue that some leakage of phagocytic
vesicle contents due to oxidative stress is likely to occur in
monocytes, macrophages, and neutrophils. To avoid death and remain
functional, these cells would require vesicle repair mechanisms similar
to those described in fibroblasts56 as well as the means of
countering the potentially cytotoxic components of leaking vesicles,
such as cathepsin G and elastase.
In addition to countering proteinases leaking from phagocytic vesicles,
PI-6 and M/NEI could also serve to inactivate any proteinase molecules
that are misdirected into the cytoplasm during biosynthesis or
degranulation. This model can also be extended to explain the function
of intracellular PAI-2 by postulating it is present to control any
urokinase (which is not directed into the phagocytic vesicle) that
escapes into the cell during biosynthesis or degranulation.
Alternatively, it could be argued that PI-6 and M/NEI (perhaps like
PAI-2) form an intracellular pool of inhibitors that can be rapidly
released under specific conditions to operate at high local
concentrations in the extracellular milieu. However, release of M/NEI
from monocytes or neutrophils has not been convincingly demonstrated,
whereas we have shown that PI-6 cannot be detected in media conditioned
by U937 and other cells and that it cannot be released from cells
through the classical secretory pathway.18 In addition,
both M/NEI and PI-6 are sensitive to oxidation that indicates that they
would be short-lived once outside the cell.8,10
Finally, although PI-6 is a very efficient cathepsin G inhibitor and
our evidence points to a physiological role in monocytes and
granulocytes, the fact remains that it is expressed in endothelial cells and many types of epithelial cells that do not produce cathepsin G and it is also a good inhibitor of trypsin-like serine
proteinases.18 Thus, it is possible that the role of PI-6
extends beyond the control of cathepsin G and that it regulates one or
more proteinases that have yet to be identified.
| |
ACKNOWLEDGMENT |
The authors are grateful to Dr J. Hoidal for the generous gift of
antisera to cathepsin G, proteinase 3, and elastase and to Dr R. Steinman for the anti-CD14 antibody. We also thank C. O'Malley for
advice and assistance with hematological procedures and Dr E. Randle-Barrett and Dr R. Pike for discussions.
| |
FOOTNOTES |
Submitted May 11, 1998; accepted November 11, 1998.
Supported by a grant from the National Health and Medical Research
Council, Australia.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Phillip I. Bird, PhD, Department of
Medicine, Clive Ward Centre, Box Hill Hospital, Box Hill 3128, Australia; e-mail: Phil.Bird{at}med.monash.edu.au.
| |
REFERENCES |
1.
Potempa J, Korzus E, Travis J:
The serpin superfamily of proteinase inhibitors: Structure, function and regulation.
J Biol Chem
269:15957, 1994[Free Full Text]
2.
Salem H:
The natural anticoagulants.
Clin Haematol
15:371, 1986[Medline]
[Order article via Infotrieve]
3.
Gagliardini V, Fernandez P-A, Lee RK, Drexler HCA, Rotello RJ, Fishman MC, Yuan J:
Prevention of vertebrate neuronal cell death by the crmA gene.
Science
263:826, 1994[Abstract/Free Full Text]
4.
Miura M, Zhu H, Rotello R, Hartwieg EA, Yuan J:
Induction of apoptosis in fibroblasts by IL-1 beta-converting enzyme, a mammalian homologue of the C. elegans cell death gene ced-3.
Cell
75:653, 1993[Medline]
[Order article via Infotrieve]
5.
Ye RD, Ahern SM, Le Beau MM, Lebo RV, Sadler JE:
Structure of the gene for human plasminogen activator inhibitor-2. The nearest mammalian homologue of chicken ovalbumin.
J Biol Chem
264:5495, 1989[Abstract/Free Full Text]
6.
Suminami Y, Kishi F, Sekiguchi K, Kato H:
Squamous cell carcinoma antigen is a new member of the serine protease inhibitors.
Biochem Biophys Res Commun
181:51, 1991[Medline]
[Order article via Infotrieve]
7.
Schneider SS, Schick C, Fish KE, Miller E, Pena JC, Treter SD, Hui SM, Silverman GA:
A serine proteinase inhibitor locus at 18q21.3 contains a tandem duplication of the human squamous cell carcinoma antigen gene.
Proc Natl Acad Sci USA
92:3147, 1995[Abstract/Free Full Text]
8.
Remold-O'Donnell E, Chin J, Alberts M:
Sequence and molecular characterisation of human monocyte/neutrophil elastase inhibitor.
Proc Natl Acad Sci USA
89:5635, 1992[Abstract/Free Full Text]
9.
Zou Z, Anisowicz A, Hendrix MJC, Thor A, Neveu M, Sheng S, Rafidi K, Seftor E, Sager R:
Maspin, a serpin with tumor-supressing activity in human mammary epithelial cells.
Science
263:526, 1994[Abstract/Free Full Text]
10.
Coughlin P, Sun J, Cerruti L, Salem HH, Bird P:
Cloning and molecular characterization of a human intracellular proteinase inhibitor.
Proc Natl Acad Sci USA
90:9417, 1993[Abstract/Free Full Text]
11.
Sprecher CA, Morgenstern KA, Mathewes S, Dahlen JR, Schrader SK, Foster DC, Kisiel W:
Molecular cloning, expression, and partial characterization of two novel members of the ovalbumin family of serine proteinase inhibitors.
J Biol Chem
270:29854, 1995[Abstract/Free Full Text]
12.
Sun J, Bird CH, Sutton V, McDonald L, Coughlin PB, De Jong TA, Trapani JA, Bird PI:
A cytosolic granzyme B inhibitor related to the viral apoptotic regulator cytokine response modifier A is present in cytotoxic lymphocytes.
J Biol Chem
271:27802, 1996[Abstract/Free Full Text]
13.
Riewald M, Schleef RR:
Molecular cloning of bomapin (protease inhibitor 10), a novel human serpin that is expressed specifically in the bone marrow.
J Biol Chem
270:26754, 1995[Abstract/Free Full Text]
14.
Tsujimoto M, Tsuruoka N, Ishida N, Kurihara T, Iwasa F, Yamashiro K, Rogi T, Kodama S, Katsuragi N, Adachi M, Katayama T, Nakao M, Yamaichi K, Hashino J, Haruyama M, Miura K, Nakanishi T, Nakazato H, Teramura M, Mizoguchi H, Yamaguchi N:
Purification, cDNA cloning, and characterization of a new serpin with megakaryocyte maturation activity.
J Biol Chem
272:15373, 1997[Abstract/Free Full Text]
15.
Remold-O'Donnell E:
The ovalbumin family of serpin proteins.
FEBS Lett
315:105, 1993[Medline]
[Order article via Infotrieve]
16.
Belin D:
Biology and facultative secretion of plasminogen activator inhibitor-2.
Thromb Haemost
70:144, 1993[Medline]
[Order article via Infotrieve]
17.
Coughlin PB, Tetaz T, Salem HH:
Identification and purification of a novel serine proteinase inhibitor.
J Biol Chem
268:9541, 1993[Abstract/Free Full Text]
18.
Scott FL, Coughlin PB, Bird C, Cerruti L, Hayman JA, Bird P:
Proteinase inhibitor 6 cannot be secreted, which suggests it is a new type of cellular serpin.
J Biol Chem
271:1605, 1996[Abstract/Free Full Text]
19.
Sun J, Coughlin P, Salem H, Bird P:
Production and characterization of recombinant human proteinase inhibitor 6 expressed in Pichia pastoris.
Biochim Biophys Acta
1252:28, 1995[Medline]
[Order article via Infotrieve]
20.
Morgenstern KA, Sprecher C, Holth L, Foster D, Grant FJ, Ching A, Kisiel W:
Complementary DNA cloning and kinetic characterization of a novel intracellular serine proteinase inhibitor: Mechanism of action with trypsin and factor Xa as model proteinases.
Biochemistry
33:3432, 1994[Medline]
[Order article via Infotrieve]
21.
Riewald M, Schleef RR:
Human cytoplasmic antiproteinase neutralizes rapidly and efficiently chymotrypsin and trypsin-like proteinases utilizing distinct reactive site residues.
J Biol Chem
271:14526, 1996[Abstract/Free Full Text]
22.
Harlow E, Lane D:
Antibodies. A Laboratory Manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1988.
23.
Scott F, Paddle-Ledinek JE, Cerruti L, Coughlin PB, Salem HH, Bird PI:
Proteinase inhibitor 6 (PI-6) expression in human skin: Induction of PI-6 and a PI-6/proteinase complex during keratinocyte differentiation.
Exp Cell Res
245:263, 1998[Medline]
[Order article via Infotrieve]
24.
Rao NV, Rao GV, Marshall BC, Hoidal JR:
Biosynthesis and processing of proteinase 3 in U937 cells.
J Biol Chem
271:2972, 1996[Abstract/Free Full Text]
25.
Laemmli UK:
Cleavage of structural proteins during assembly of the head of bacteriophage T4.
Nature
227:680, 1970[Medline]
[Order article via Infotrieve]
26.
Duranton J, Adam C, Bieth JG:
Kinetic mechanism of the inhibition of cathepsin G by 1-antichymotrypsin and 1-proteinase inhibitor.
Biochemistry
37:11239, 1998[Medline]
[Order article via Infotrieve]
27.
Hopkins PCR, Stone SR:
The contribution of the conserved hinge region residues of 1-antitrypsin to its reaction with elastase.
Biochemistry
3:15872, 1995
28.
Salvesen G, Nagase H:
Inhibition of proteolytic enzymes, in
Beynon RJ,
Bond JS
(eds):
Proteolytic Enzymes: A Practical Approach. Oxford, UK, IRL, 1989, p 83.
29.
Collins SJ:
The HL-60 promyelocytic leukemia cell line: Proliferation, differentiation, and cellular oncogene expression.
Blood
70:1233, 1987[Abstract/Free Full Text]
30.
Dickinson JL, Antalis TM:
Tissue factor and plasminogen activator inhibitor expression in the differentiation of myeloid leukemic cells.
Leukemia
7:864, 1993[Medline]
[Order article via Infotrieve]
31.
Borregaard N, Cowland JB:
Granules of the human neutrophilic polymorphonuclear leukocyte.
Blood
89:3503, 1997[Free Full Text]
32.
Welgus HG, Senior RM, Parks WC, Kahn AJ, Ley TJ, Shapiro SD, Campbell EJ:
Neutral proteinase expression by human mononuclear phagocytes: A prominent role of cellular differentiation.
MATRIX Suppl
1:363, 1992[Medline]
[Order article via Infotrieve]
33.
Spitznagel JK:
Antibiotic proteins of human neutrophils.
J Clin Invest
86:1381, 1990
34.
Lehrer RI, Ganz T:
Antimicrobial polypeptides of human neutrophils.
Blood
76:2169, 1990[Free Full Text]
35.
Padrines M, Wolf M, Walz A, Baggiolini M:
Interleukin-8 processing by neutrophil elastase, cathepsin G and proteinase 3.
FEBS Lett
352:231, 1994[Medline]
[Order article via Infotrieve]
36.
Chertov O, Ueda H, Xu LL, Tani K, Murphy WJ, Wang JM, Howard OMZ, Sayers TJ, Oppenheim JJ:
Identification of human neutrophil-derived cathepsin G and azurocidin/CAP37 as chemoattractants for mononuclear cells and neutrophils.
J Exp Med
186:739, 1997[Abstract/Free Full Text]
37.
Evangelista V, Rajtar G, de Gaetano G, White JG, Cerletti C:
Platelet activation by fMLP-stimulated polymorphonuclear leukocytes: The activity of cathepsin G is not prevented by antiproteinases.
Blood
77:2379, 1991[Abstract/Free Full Text]
38.
Zhou Q, Salvesen GS:
Activation of pro-caspase-7 by serine proteases includes a non-canonical specificity.
Biochem J
324:361, 1997
39.
Janoff A, Feinstein G, Malemud CJ, Elias JM:
Degradation of cartilage proteoglycan by human leukocyte granule neutral proteinases: A model of joint injury. I. Penetration of enzyme into rabbit articular cartilage and release of 35SO4-labeled material from the tissue.
J Clin Invest
57:615, 1976
40.
McDonald JA, Kelley DG:
Degradation of fibronectin by human leukocyte elastase.
J Biol Chem
255:8848, 1980[Abstract/Free Full Text]
41.
Gadek JE, Fells GA, Wright DG, Crystal RG:
Human neutrophil elastase functions as a type III collagen "collagenase".
Biochem Biophys Res Commun
95:1815, 1980[Medline]
[Order article via Infotrieve]
42.
Mainardi CL, Dixit SN, Kang AH:
Degradation of type IV (basement membrane) collagen by a proteinase isolated from human polymorphonuclear leukocyte granules.
J Biol Chem
255:5435, 1980[Abstract/Free Full Text]
43.
Janoff A:
Elastase in tissue injury.
Annu Rev Med
36:2207, 1985
44.
Lee CT, Fein AM, Lippman M, Holtzman H, Kimbel P, Weinbaum G:
Elastolytic activity in pulmonary lavage fluid from patients with adult respiratory-distress syndrome.
N Engl J Med
304:192, 1981[Abstract]
45.
Bruce MC, Poncz L, Klinger JD, Stern RC, Tomashefski JF, Dearborn DG:
Biochemical and pathologic evidence for proteolytic destruction of lung connective tissue in cystic fibrosis.
Am Rev Respir Dis
132:529, 1985[Medline]
[Order article via Infotrieve]
46.
Weiss SJ:
Tissue destruction by neutrophils.
N Engl J Med
320:365, 1989[Medline]
[Order article via Infotrieve]
47.
Laurell C-B, Eriksson S:
The electrophoretic alpha1-globulin pattern of serum in alpha1-antitrypsin deficiency.
Scand J Clin Lab Invest
15:132, 1963
48.
Schick C, Kamachi Y, Bartuski AJ, Cataltepe S, Schechter NM, Pemberton PA, Silverman GA:
Squamous cell carcinoma antigen 2 is a novel serpin that inhibits the chymotrypsin-like proteinases cathepsin G and mast cell chymase.
J Biol Chem
272:1849, 1997[Abstract/Free Full Text]
49.
Caughey GH:
Serine proteinases of mast cell and leukocyte granules. A league of their own.
Am J Respir Crit Care Med
150:S138, 1994
50.
Hof P, Mayr R, Huber R, Korzus E, Potempa J, Travis J, Powers J, Bode W:
The 1.8 A crystal structure of human cathepsin G in complex with Suc-Val-Pro-PheP-(oPh)2 a Janus-faced proteinase with two opposite specificities.
EMBO J
15:5481, 1996[Medline]
[Order article via Infotrieve]
51.
Polanowska J, Krokosynska I, Czapinska H, Watorek W, Dadlez M, Otlewski J:
Specificity of human cathepsin G.
Biochim Biophys Acta
1386:189, 1998[Medline]
[Order article via Infotrieve]
52.
Sun J, Rose JB, Bird P:
Gene structure, chromosomal localization and expression of the murine homologue of human proteinase inhibitor 6 (PI6) suggests divergence of PI6 from the ovalbumin serpins.
J Biol Chem
270:16089, 1995[Abstract/Free Full Text]
53.
Remold-O'Donnell E, Nixon JC, Rose RM:
Elastase inhibitor. Characterization of the human elastase inhibitor molecule associated with monocytes, macrophages, and neutrophils.
J Exp Med
169:1071, 1989[Abstract/Free Full Text]
54.
Sugimori T, Cooley J, Hoidal JR, Remold-O'Donnell E:
Inhibitory properties of recombinant monocyte/neutrophil elastase inhibitor.
Am J Respir Cell Mol Biol
13:314, 1995[Abstract]
55. Sun J, Stephens R, Mirza G, Kanai H, Ragoussis J, Bird PI: A
serpin gene cluster on human chromosome 6p25 contains PI6, PI9 and
ELANH2 which have a common structure almost identical to the 18q21
ovalbumin serpin genes. Cytogenet Cell Genet (in press)
56.
Brunk UT, Dalen H, Roberg K, Hellquist HB:
Photo-oxidative disruptiion of lysosomal membranes causes apoptosis of cultured human fibroblasts.
Free Radic Biol Med
23:616, 1997[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
M. Bots and J. P. Medema
Serpins in T cell immunity
J. Leukoc. Biol.,
November 1, 2008;
84(5):
1238 - 1247.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. L. Scott, J. Sun, J. C. Whisstock, K. Kato, and P. I. Bird
SerpinB6 is an Inhibitor of Kallikrein-8 in Keratinocytes
J. Biochem.,
October 1, 2007;
142(4):
435 - 442.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Zvonic, M. Lefevre, G. Kilroy, Z. E. Floyd, J. P. DeLany, I. Kheterpal, A. Gravois, R. Dow, A. White, X. Wu, et al.
Secretome of Primary Cultures of Human Adipose-derived Stem Cells: Modulation of Serpins by Adipogenesis
Mol. Cell. Proteomics,
January 1, 2007;
6(1):
18 - 28.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Suzuki, H. Kobayashi, Y. Tanaka, N. Kanayama, and T. Terao
Reproductive failure in mice lacking inter-alpha-trypsin inhibitor (ITI) - ITI target genes in mouse ovary identified by microarray analysis
J. Endocrinol.,
October 1, 2004;
183(1):
29 - 38.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. L. Scarff, K. S. Ung, H. Nandurkar, P. J. Crack, C. H. Bird, and P. I. Bird
Targeted Disruption of SPI3/Serpinb6 Does Not Result in Developmental or Growth Defects, Leukocyte Dysfunction, or Susceptibility to Stroke
Mol. Cell. Biol.,
May 1, 2004;
24(9):
4075 - 4082.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. C. M. Strik, A. Wolbink, D. Wouters, B. A. Bladergroen, A. R. Verlaan, I. S. van Houdt, S. Hijlkema, C. E. Hack, and J. A. Kummer
Intracellular serpin SERPINB6 (PI6) is abundantly expressed by human mast cells and forms complexes with {beta}-tryptase monomers
Blood,
April 1, 2004;
103(7):
2710 - 2717.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. H. Roberts, S. Marttila, S. K. Rasmussen, and J. Hejgaard
Differential gene expression for suicide-substrate serine proteinase inhibitors (serpins) in vegetative and grain tissues of barley
J. Exp. Bot.,
October 1, 2003;
54(391):
2251 - 2263.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. E. Hirst, M. S. Buzza, C. H. Bird, H. S. Warren, P. U. Cameron, M. Zhang, P. G. Ashton-Rickardt, and P. I. Bird
The Intracellular Granzyme B Inhibitor, Proteinase Inhibitor 9, Is Up-Regulated During Accessory Cell Maturation and Effector Cell Degranulation, and Its Overexpression Enhances CTL Potency
J. Immunol.,
January 15, 2003;
170(2):
805 - 815.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. C. Strik, B. A. Bladergroen, D. Wouters, W. Kisiel, J. H. Hooijberg, A. R. Verlaan, P. L. Hordijk, P. Schneider, C. E. Hack, and J. A. Kummer
Distribution of the Human Intracellular Serpin Protease Inhibitor 8 in Human Tissues
J. Histochem. Cytochem.,
November 1, 2002;
50(11):
1443 - 1454.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Benarafa, J. Cooley, W. Zeng, P. I. Bird, and E. Remold-O'Donnell
Characterization of Four Murine Homologs of the Human ov-serpin Monocyte Neutrophil Elastase Inhibitor MNEI (SERPINB1)
J. Biol. Chem.,
October 25, 2002;
277(44):
42028 - 42033.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Korkmaz, S. Attucci, E. Hazouard, M. Ferrandiere, M. L. Jourdan, M. Brillard-Bourdet, L. Juliano, and F. Gauthier
Discriminating between the Activities of Human Neutrophil Elastase and Proteinase 3 Using Serpin-derived Fluorogenic Substrates
J. Biol. Chem.,
October 11, 2002;
277(42):
39074 - 39081.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. A. Hamerman, F. Hayashi, L. A. Schroeder, S. P. Gygi, A. L. Haas, L. Hampson, P. Coughlin, R. Aebersold, and A. Aderem
Serpin 2a Is Induced in Activated Macrophages and Conjugates to a Ubiquitin Homolog
J. Immunol.,
March 1, 2002;
168(5):
2415 - 2423.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Takamiya, M. Takeda, A. Yoshida, and H. Kiyama
Expression of Serine Protease Inhibitor 3 in Ocular Tissues in Endotoxin-Induced Uveitis in Rat
Invest. Ophthalmol. Vis. Sci.,
October 1, 2001;
42(11):
2427 - 2433.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. H. Bird, E. J. Blink, C. E. Hirst, M. S. Buzza, P. M. Steele, J. Sun, D. A. Jans, and P. I. Bird
Nucleocytoplasmic Distribution of the Ovalbumin Serpin PI-9 Requires a Nonconventional Nuclear Import Pathway and the Export Factor Crm1
Mol. Cell. Biol.,
August 15, 2001;
21(16):
5396 - 5407.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. V. Terskikh, M. C. Easterday, L. Li, L. Hood, H. I. Kornblum, D. H. Geschwind, and I. L. Weissman
From the Cover: From hematopoiesis to neuropoiesis: Evidence of overlapping genetic programs
PNAS,
July 3, 2001;
98(14):
7934 - 7939.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. A. Bladergroen, M. C. M. Strik, N. Bovenschen, O. van Berkum, G. L. Scheffer, C. J. L. M. Meijer, C. E. Hack, and J. A. Kummer
The Granzyme B Inhibitor, Protease Inhibitor 9, Is Mainly Expressed by Dendritic Cells and at Immune-Privileged Sites
J. Immunol.,
March 1, 2001;
166(5):
3218 - 3225.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Kato, T. Kishi, T. Kamachi, M. Akisada, T. Oka, R. Midorikawa, K. Takio, N. Dohmae, P. I. Bird, J. Sun, et al.
Serine Proteinase Inhibitor 3 and Murinoglobulin I Are Potent Inhibitors of Neuropsin in Adult Mouse Brain
J. Biol. Chem.,
April 27, 2001;
276(18):
14562 - 14571.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. A. Irving, R. N. Pike, A. M. Lesk, and J. C. Whisstock
Phylogeny of the Serpin Superfamily: Implications of Patterns of Amino Acid Conservation for Structure and Function
Genome Res.,
December 1, 2000;
10(12):
1845 - 1864.
[Abstract]
[Full Text]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION