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

-

A cytosolic inhibitor of human neutrophil elastase and cathepsin G.

The neutrophil serine proteinases elastase and cathepsin G produce connective tissue injury, the extent of which depends on the balance between these ...
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