Journal
A Cytosolic Raymond Department
M. Thomas,
of Medicine, R.A.C.),
Inhibitor of Human and Cathepsin
VA Medical
and Department
of Leukocyte
Neutrophil G
William M. Nauseef, Shankar S. lyer, Phillip J. Stone, and Robert A. Clark Center
and the College
of Biochemistry,
Boston
of Medicine, University
Biology
University School
of Iowa,
of Medicine,
W. Peterson,
City (R.M.T.,
Boston,
(1991)
Elastase
Michael Iowa
50:568-579
W.M.N.,
Massachusetts
5.5.1.,
M.W.P.,
(P.J.S.)
The neutrophil serine proteinases elastase and cathepsin G produce connective tissue injury, the extent of which depends on the balance between these enzymes and their inhibitors. The most important of these inhibitors is a1-proteinase inhibitor, a member of
a superfamily
of homologous
proteins
known as serpins.
Neutrophil
cytosol
inhibited
the
activities of human neutrophil elastase and cathepsin G in a dose-dependent fashion. To demonstrate formation of an enzyme-inhibitor complex, we combined 1251-elastase or 1251-cathepsin G with neutrophil cytosol or a1-proteinase inhibitor and analyzed the products by polyacrylamide gel electrophoresis. Unbound elastase and cathepsin G each migrated to an apparent molecular weight of 25 kDa. In the presence of cytosol from neutrophils both radiolabeled enzymes migrated with a relative size of 68 kDa, whereas in the presence of a1-proteinase inhibitor the relative size was 85 kDa. Enzyme-inhibitor complexes were stable in sodium dodecyl sulfate at 100#{176}Cbut were dissociated by hydrolysis in ammonium hydroxide (1 .5 mol/L) at 37#{176}C. Formation of each complex was prevented by pretreatment of elastase or cathepsin G with diisopropylfluorophosphate, indicating that the inhibitor binds to the active site of the enzyme. Exposure of either a1-proteinase inhibitor or neutrophil cytosol to the myeloperoxidase-H2O2-halide system prevented complex formation, suggesting the presence of an oxidizable amino acid at the binding site of the inhibitor. By electrophoretic analysis, the molecular weight of the cytosolic inhibitor was 43 kDa and neutrophils contained approximately 1 attomol of inhibitor per cell. The isoelectric points of the elastase and cathepsin G inhibitor were 5.5-5.9 and inhibitors of the two proteinases coeluted using size exclusion chromatography. These data demonstrate that human neutrophil cytosol contains a single serpinlike protein that inhibits elastase and cathepsin G. The inhibitor may be important in protecting the intracellular environment from proteolytic injury during degranulation.
Key words:
serine cytes
proteinase,
serine
proteinase
INTRODUCTION Human neutrophil elastase (HNE) and cathepsin G (CG) are serine proteinases contained in the azurophilic granules of neutrophils (PMNs) [12,32]. Substrates for both enzymes include a variety of connective tissue components vessels, and
of various organs joints [15,16,41].
of these enzymes which an imbalance
is most develops
ases and their inhibitors and activity results in localized
including The clinical
evident under between the
lung, blood significance conditions in serine protein-
the net increase in proteolytic connective tissue injury. Of
particular clinical interest is hereditary -proteinase inhibitor (a1PI) deficiency, a disorder characterized by reduced levels of P1 in plasma and lung fluids thereby leading to unopposed proteinase activity and culminating in pulmonary emphysema [23]. In addition, PMN serine proteinases have been implicated in the pathogenesis of the
more
common
type
of
emphysema
associated
cigarette smoking and of rheumatoid arthritis a1PI is the prototype of a family of homologous proteins called serine proteinase © 1991
Wiley-Liss,
Inc.
with
[16,17]. structurally inhibitors
inhibitor,
polymorphonuclear
or serpins [8] . Members identified in plants, insects, kingdom [13,20,33]. Most lular
fluids,
although
horse
PMNs
has
recently
leuko-
of this family and throughout serpins are found
a cytosolic been
have been the animal in extracel-
inhibitor
determined
of
HNE
by amino
in acid
Abbreviations: a1PI. a1-proteinase inhibitor: CG. cathepsin G: DFP, diisopropylfluorophosphate: HPLC. high performance liquid chromatoraphy: HNE. human neutrophil elastase; MPO. myeloperoxidase: PBS, phosphate buffered saline: PMNs, polymorphonuclear leukocytes: SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel ehectrophoresis: ATP, adenosine 5’ triphosphate: EGTA, ethyleneglycoh-bis-(3-aminoethyl ether) N.N,N’.N’-tetraacetic acid: MCA. succinyl(O-methyh)-ahanyl-alanyl-prolyl-valyl-4-methyl-cOulllaryl-amide AMC. aminomethylcoumarin Received
February
12. 1991 : accepted
March
26.
1991.
Portions of this work were presented at the Annual National of the American Federation for Clinical Research held April 1 . 1989 in Washington. D.C. Reprint requests: William icine, College of Medicine.
M. Nauseef. University
Meeting 28-May
Department of Internal Medof Iowa. Iowa City. IA 52242.
A Cytosolic sequence
analysis
serpin-like
porcine. lysates
ovine, human
of
More
to
cytosolic
recently
be
a
serpin
inhibitors
[33].
In
been
identified
have
I 19,21
bovine PMNs monocyte-derived Remold-O’Donnell
et al.
their studies to characterization lysates of freshly isolated
of monocytes
Our studies confirm and extend cytosolic inhibitor of HNE and monocytes. and cultured myeloid
this
hypotonic
addition, in
,27], and macrophages have
in
in the [35].
characterization CG in human cell lines.
lysis
nuclear
layer
of a PMNs,
PMNs cells
were
were
(ATP)
( 3-aminoethyl
( DFP) ovalbumin
,
ether)
N
‘
L-alanyl-L-alanyl-L-alanyl
washed
,
, adenosine
5
tn-
‘
ethyleneglycol-bis-
,N ‘-tetraacetic methyl
ester
acid
( EGTA),
[(ala)3
methyl
adherent [6].
mono-
dishes
at 37#{176}C
monocytes
were
once
KCI,
pH
and
7.0,
and
cultured
, N ‘-bis-(2-ethanesulfonic
and
1 mmol/L
unbroken
centrifuged granules.
at
and
nuclei.
treated
with
10 mm represented
cells in [5].
at 5#{176}C
Supernatant
for 20 mm supernatant
at 4#{176}C for
supernatant
acid)
and
as previously described at I ,000g for 10 mm
cells
12.400g granule-free
ATP)
20 mm. Subsequently by nitrogen cavitation
with ATP centrifuged
to sediment
The
monocytes
times in relaxation buffer with ATP 3 mmol/L NaCl, 3.5 mmol/L MgCl,,
(2 mmol/L) on ice for washed and disrupted
final
Petni
569
The
[5].
in sterile
piperazine-N
230,000g
G
3
relaxation buffer The cavitate was
Diisopropylfiuorophosphate
erythrocytes
placed
washed
( 100 mmol/L
DFP were
Cathepsin
of Cytosols
I 0 mmol/L
AND METHODS
phosphate
and
of residual was
Preparation
(PIPES),
MATERIALS Reagents
of Elastase
overnight. After washing, gently scraped into suspension
the [34].
extended
inhibitor and PMNs
Inhibitor
was
at 4#{176}Cto pellet was centrifuged at
to remove the cytosol
membranes. fraction and
The was
ester], and N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine p-nitnoanilide were obtained Chemical Co. (St. Louis, MO). Human
from Sigma a1Pl was ob-
free of HNE and CG activity at a detection limit of 95% purity were blood using sequential
1-lypaque-Ficoll
density
isolated dextran
centnifugation
from sedi, and
Elmer,
the cleav-
Oak
buffer, X
added
0.6 7.4),
at
and
which
CG activity
was
(2.7 1.0
MCA,
1
quantities
of
volume
of 2
a total point
All
10 mmol/L
p.mol/L
varied
fluorescence was recorded as a function range of quantities of HNE added (0-2 cence response was linear.
of CG Enzymatic
IL).
excitation of 480 buffer CaCl,,
and 20
with
106 CE), last
PiCMG mmol/L
glucose, pH
or combined
(0-15 was
Brook,
at 37#{176}Cwith an emission wavelength
10 mmol/L
alone
cytosol
Assay
i.e. , the
by measuring
cuvettes contained 138 mmol/L NaCl,
MgCl.,.
HNE
determined
(Perkin
phosphate
ml.
(CE),
volume of cytosol. was determined by
Activity
were performed of 360 nm and
mmol/L
PMN
KS).
was
sodium of
equivalents
nonfluorescent coumanin peptide, MCA to the AMC [36]. Fluorescence was measured using a Perkin Elmer LS5 fluorescence
spectrofiuorometer
ig
Products,
in cell
represented in a given concentration of cytosol of Lowry [24].
the
change
in
of time. Over the rig), the fluores-
Activity
determined
by measuring
the cleavage
of the peptide Suc-Ala-Ala-Pro-Phe-p-nitroanilide to the photo-absorbant product, p-nitroaniline [ 1 1 ,3 I ] . All reactions were measured using a Perkin Elmer 320 dual beam spectrophotometer with an absorbance wavelength of4l0 nm at 25#{176}C. Sample cuvettes contained buffer (0. 1 mole/L Tris, pH 8.3), substrate (30 jimole/L), CG (4 rig) alone
or combined
with
varied
quantities
of PMN
cytosol
570
Thomas
(0-40
x
et al.
106 CE),
and
a total
ence cuvettes were identical CG was added last to the absorbance
was
HNE isolated
except sample
measured
and
added
(0-4
of time. fig),
Refer-
Over
the
the absorbance
of HNE
extracted sputum
il.
that CG was omitted. cuvette at which point
Radiolabeling
and CG were from purulent
of 600
as a function
range ofquantities ofCG response was linear. Purification
volume
and
1 h. The
gel
was
destained
acid and dried by vacuum. formed using an intensifying film (Rochester, NY) and weights of bands identified
and purified from PMNs by the method of Mar-
log
Mr,
DFP Treatment
of 125I-HNE or 125l-CG
The DFP pmol
irreversible
active
and
placed
on
ice
for
samples
were
method described by McFarlane [30]. (Ala)3 methyl ester (2.5 mg), a substrate of HNE, was added prior to iodination in order to prevent labeling of the active site [14]. The (Ala)3 methyl ester and unbound 251_ were
by SDS-PAGE
NH4OH
Hydrolysis
was
determined
by
yielding a labeled The concentration of
measuring
the
A,8()
(ex-
tinction coefficient 2.9 x l0 M cm ‘) [2] and labeled enzyme retained 90% of its enzymatic activity when compared with equivalent quantities of unlabeled HNE. The specific activity of the ‘251-HNE was 3.5 x l0 ‘
Ci/mol. Purified CG using the lactoperoxidase
(500
rig) was iodinated method described
(0.5 mCi) by Marchal-
onis [26]. The concentration of ‘25I-CG was determined by measuring the A280 (extinction coefficient 1 .9 X l0 M cm ‘) [2]. In contrast to HNE, CG lost the majority of its enzymatic activity to the synthetic peptide, SucAla-Ala-Pro-Phe-p-nitroanilide after iodination . How‘
ever,
this
V’s
I-CG activity --
was
not due
.
.
to disruption
.
still exhibited of the ‘25I-CG
.
of the active
.
binding was 5.0
to x
DFP. The l0 Ci/mol.
site
since .
specific
--
I-HNE
I-’S
or
‘--
I-CG
and the products analyzed the formation of enzyme
was
combined.
with
PMN
cytosol
I’S
to varied P1 (5- 15
iig) and placed on ice for 15 mm. Samples were denatured by adding an equivalent volume of SDS sample buffer (2.3% SDS, 5% 3 mercaptoethanol, 5% glycerol, and 62 mmol/L Tris HC1, pH 6.8) and heated to 100#{176}C for 5 mm. Bromophenol blue tracking dye in 50% glycerol phoresed stacking Laemmli voltage Coomassie
was into
added to samples 10% acrylamide gels
which were electrowith 3.5% acrylamide
gel according to the method described by [22]. Electrophoresis was performed at constant overnight and gels stained for protein with R-250 blue
in 50%
methanol/lO%
acetic
molecular
acid
site
serine
proteinase
inhibitor,
20
mm.
In parallel,
untreated
or
enzyme added
was
( 187.5 ng I-HNE to PMN cytosol or
incubated
on and
ice
for
15
or to
250 ng a1PI and
mm
prior
to
autoradiography.
of HNE-lnhibitor
Complex
without
added
samples before
were analysis
NH4OH
HPLC Fractionation PMN by
were
analyzed
incubated in a shaker by SDS-PAGE and
cytosol
(300
in parallel.
All
bath at 37#{176}C for autoradiography.
1 h
of PMN Cytosol il or 70
x
106 CE)
was
analyzed
at 25#{176}C using a Beckman Spherogel TSK-3000 molecular sizing column (Beckman Instruments, San
HPLC
SW
Ramon, CA). Relaxation buffer was buffer at a flow rate of 0.5 mI/mm. standards included thyroglobin (670 kDa),
ovalbumin
vitamin
B-12
(I .3
(44 kDa), myoglobin kDa). Absorbance
and 0.50 ml fractions of each fraction were
used as a running Molecular weight kDa), IgG (158 at
(17 kDa), 280 nm
were collected. combined with
and was
Aliquots ‘25I-HNE
(375 ng) or ‘25I-CG (250 ng) and analyzed by SDSPAGE and autoradiography. The molecular weight of the inhibitor was determined by comparing its elution time to
by SDS-PAGE to demonstrate inhibitor complexes. ‘251-HNE
( 1 87.5-375 ng) or I-CG (250 ng) was added amounts of PMN cytosol (0- 1 5 x 10 CE) or
high
PMN cytosol or a1PI was combined with 187.5 ng of ‘251-HNE and incubated on ice for 15 mm to allow complex formation. NH4OH ( 1 .5 mol/L, final concentration) was added to HNE alone or to HNE complexed with either PMN cytosol or a1PI. Duplicate samples
recorded (75 pi)
SDS-PAGE V’s
known
i15
analysis
125!
‘251-HNE
using
( 1 1 .5 nmol) was combined with 25I-HNE (86.2 in 50 p.1 H,O) or ‘25I-CG ( 140 pmol in 50 .tl H,O)
DFP-treated ‘251-CG)
by dialysis, active site.
acetic
Autoradiography was perscreen and Kodak X-Omatic stored at -80#{176}C. Molecular in gels were calculated from
plots of Rf vs. weight standards.
HNE (500 p.g) in I ml of citrate buffer (200 mmol/L glycine, 200 mmol/L NaCI, 20 mmol/L sodium citrate, pH 9.3) was combined with 0.5 mCi ofNa using the
removed a preserved
methanol/lO%
CG
todam [25,28]. Purified CG contained 0.5% HNE contamination as assessed by enzymatic activity and purified HNE contained no detectable CG enzymatic activity.
subsequently enzyme with
in 15%
for
plots of standards
the relative elution vs. log Mr.
Isoelectric In
order
time
of
molecular
weight
Focusing to
determine
the
isoelectric
point
of
the
cytosolic inhibitor, isoelectric focusing was performed using the Rotofor cylindrical focusing chamber (BioRad). About 5 ml of neutrophil cytosol (l0 CE) in relaxation buffer was diluted to 55 ml with water containing pH 3-10 ampholytes to give a final ampholyte concentration of 1%. This material was then loaded into the chamber, maintained at 5#{176}C, and electrofocused at 12 W constant power for 5-6 h. At the end of focusing 20
A Cytosolic fractions
were
determined. NaCl (0.5
collected
and
Fractions mol/L) and
temperature
to dissociate
quots of each ‘25I-CG and
the
pH of each
were then incubated protein
fraction evaluated
were by
fraction
supplemented for 30 mm from
was
at
with room
ampholytes.
combined SDS-PAGE
Ali-
with ‘25I-HNE and autoradiog-
or
Oxidation of Inhibitors by the MPO-H202-Halide System
for
(5-15 .ag) or HPLC fractions (50 purified PMN cytosolic inhibitor
to components mU/mI MPO,
of the 32 p.mol/L
.al) containing were exposed
MPO-H2O2-halide H2O,, 100 mmol/L
system NaCI,
(32 and
of Elastase
inhibitor activity were Proteins were electrotransferred
(25 nmol/L was washed
20 mm
and
dried
The
quantity
azide Each
then added combined
varied
ng)
and
Ligand In
analyzed
and
autoradiography.
Blotting
order
cytosolic
by SDS-PAGE
to
determine
inhibitor,
the
PMN
molecular
cytosol
weight
and
of
HPLC
the
fractions
separated (90
[8].
‘251-HNE
of
analyzed resulting
by SDS-PAGE autoradiogram
inhibitor
complex
inhibitor
fluorophore
AMC.
of elastase Purified
PMN cytosol were analyzed. cytosol.
added
(HNE)
measured
activity
the cleavage
HNE alone
Inhibition
by the addition
of neutrophil
ofthe nonfluorescent
(1 .0 g)
was
determined
bands
were
cut
from
the
Using the and HNEgel
and
1251
radioactivity quantitated (Beckman Gamma 5500B). Assuming the molecular weight of HNE to be 29 kDa, moles of complexed inhibitor were calculated based on
siconds Inhibition
with 140 pH to
of the
and autoradiography. as a guide, the HNE
units
Fig. 1 .
by SDSvolt-hours)
that HNE binds to inhibitor with as is the case for other serpins or 562.5 ng) was combined with cytosol (0. 125-15 X 106 CE) and
(375
quantities
Content
Fluor#{149}sc#{149}ncs
assay continuously
571
to autoradiography.
of the cytosolic
based on the assumption I : 1 molar stoichiometry
to inactivate the MPO. with ‘25I-HNE (187.5
prior
Determination of the Cellular Cytosolic Inhibitor
phosphate buffer, pH 7.0). In and/or H2O7 were omitted. All for 15 mm at 37#{176}C,and sodium
was then
G
or 88 pCi/L) in PBS for 2 h at 4#{176}C. 4 times in 0.05% Tween 20 in PBS
300 mmol/L sodium control samples, MPO samples were incubated ( 18 mmol/L) sample was
and Cathepsin
to nitrocellulose paper and the blot was blocked 0.05% Tween 20 in phosphate buffered saline (PBS, mmol/L NaCI, 10 mmol/L sodium phosphate buffer, 7.0) for 2 h. The nitrocellulose was then exposed ‘25I-HNE The blot
raphy.
a1PI partially
containing PAGE.
Inhibitor
or HNE combined
of HNE activity
cytosol.
coumarin with varied
was a linear function
An enzymatic
peptide,
MCA to the of CE of of the amount of
numbers
572
Thomas
et al.
CsIl
#{149}qulval.nts 0
97.4
of
5
1
PMN
a
cytosol
10
15
-
Is
-