Vol.
171, No.
September
3, 1990
28,
BIOCHEMICAL
AND
ACTIVATION
RESEARCH
COMMUNICATIONS Pages
OF LATENT
BUMAN NEUTROPHIL
OXYGEN SPECIES Herkko
Saari', Yrjij
'IVt"
BIOPHYSICAL
1990
Department
Kimmo
of
AND SWINE
1,3
Suomalainen
, Otso
and Timo
Sorsa2'3
of Medicine,
Helsinki
University
Medical
BY REACTIVE
PROTRASES
T. Konttinen'
Hospital, 'Department
COLLAGENASE
979-987
Helsinki,
Lindyl,
Central
Finland
Chemistry,
University
of
Helsinki,
Finland 3Department
Received
August
of
6,
Periodontology, Helsinki,
University Finland
of
Helsinki,
1990
: The ability of various reactive oxygen species and serine proteases to activate latent collagenase (matrix metalloproteinase-1) purified from human neutrophils was examined. Latent 70-75 kD human neutrophil collagenase (HNC) was efficiently activated by known non-proteolytic activators phenylmercuric chloride (an organomercurial compound) and gold thioglucose(Au(I)-salt). Corresponding degree of activation was achieved by reactive oxygen species including hypochlorous acid (H202) and hydroxyl radical generated WOCl) , hydrogen peroxide by hypoxanthine/xanthine oxidase (HX/XAO). The presence of trace amounts of iron and EDTA were necessary and even enhanced H,O, induced activation of latent HNC. This activation could be abolished by an iron chelator desferrioxamine and a hydroxyl radical scavenger mannitol. HOC1 induced activation of latent HNC was not affected by desferrioxamine and mannitol. Thus, these compounds do not inhibit the active/activated form of HNC. Latent HNC could also be activated by trypsin and chymotrypsin but not by plasmin and plasma kallikrein. The ability of mannitol and desferrioxamine to inhibit the H,O,induced activation of HNC suggests the transition metal dependent Fenton reaction to be responsible for localized and/or site-specific generation of hydroxyl radical/hydroxyl radical -like oxidants to act as the activating oxygen species. Our results support the ability of myeloperoxidase derived HOC1 to act as a direct oxidative activator of HNC and further suggest the existence of a new/alternative oxidative activation O1990 Academic Press,Inc. pathway of HNC involving hydroxyl radical. SUMMARY
Connective such
as
believed
tissue
destruction
rheumatoid
arthritis
to
from
result
in and
the
inflammatory
periodontal
action
of
several
diseases diseases
is
types
of
0006-291X/90 979
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
BIOCHEMICAL
Vol. 171, No. 3, 1990
proteolytic
enzymes.
capable
of
tissue Although
the
involved
in
are not
3.4.24.7)
(5) *
Concomitantly
in
species
human
exert
neutrophil (6-8). by
of a direct primary
subsequently
proteases extracts
neutrophils
generate
activates effects on latent
role
(HNC)
HNC (6-8).
been
reactive
(5).
oxygen
activity
species
have
especially reported
Furthermore,
of HOC1 on HNC, to
to
reactive
70-75
oxygen
kD HNC purified
both
activation instead
be mediated
cathepsin (8).
been those
(HOCl) has been presented,
HNC proteolytically
of
potential
oxidants,
have
milieu
regulation
of reactive
HOC1 on neutrophil
various
in the
cytotoxic
derived
action of
neutrophils
extracellular
myeloperoxidase,
action
Triggered
to be
the
effects
acid
are considered
to
and
latent
diseases
metalloproteinase-1,
collagenase
oxidative
metalloproteinases
(matrix
a pivotal
Neutrophil
importance.
inflammatory
(l-4).
form
of the
and inhibit
the
enzymes
connective
primary
neutrophils
triggered
of HNC by hypochlorous
here
seen in
a latent
results
generated
the
matrix
microbicidal
Inconsistent
activate
of
collagenase
that
neutrophil's
reported
sources
of the
secrete
E.C.
on
be of
clarified,
source
metalloproteinases
extracellular
to
destruction
completely
of
suggested
cellular
known to
oxygen
are
RESEARCH COMMUNICATIONS
the matrix
a wide range
tissue
a potential are
Among these
degrading
components
AND BIOPHYSICAL
by
G which
We have studied
species from
and
serine
neutral
salt
of human neutrophils. MATERIAL
AND
METHODS
Reasents Native type I collagen was purified from human skin as described (9). Xanthine oxidase (grade III, free of proteolytic impurities) chloride bean (2) I phenylmercuric (PMC) , soy trypsin inhibitor (SBTI), gold thioglucose (GTG), trypsin, chymotrypsin, human plasmin, plasma kallikrein and synthetic serine protease substrates (see below) were purchased from Sigma (St. Louis, Missouri, USA). HOC1 was obtained from the Pharmacy of the University of Helsinki. All other reagents were of the highest commercially available grade. 980
Vol.
171,
No.
3, 1990
Purification qelatinase
BIOCHEMICAL
of
AND
latent HNC serine nrotease
and
BIOPHYSICAL
RESEARCH
and measurement activities
COMMUNICATIONS
of
collaaenase,
Latent 70-75 kD HNC was purified from neutral salt extracts of human neutrophils as described (9,lO). Collagenase activity was measured using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for separation of the characteristic cleavage products resulting from the action of collagenase on intact native type I collagen. Collagen degradation was quantitated by densitometric scanning of Coomassie brilliant blue stained gels as described (10). Activation of latent collagenase by the organomercurial compound PMC, Au(I)-salt GTG,reactive oxygen species and serine proteases was carried out as described (g-12,21,22). Gelatinase activity was measured using heat denatured type I collagen as substrate at 37OC and the reaction products were analyzed by SDS-PAGE (14). Elastasecathepsin G- and trypsin -like activities were measured using ; mM synthetic substrates, succinyl-alanyl-alanyl-valineparanitroanilide, succinyl-alanyl-alanyl-propyl-phenylalaninebenzoyl-arginine-paranitroanilide, paranitroanilide and respectively, by spectrofotometric recordings at 405 nm as described previously (15).
RESULTS Molecular in
forms
Figures
was
1 and
fractioned molecular
(obtained
after of
and
HNC.
analyzed
fractions with A and
Half
of
activity
HNC
lyophilized
in
From
which
be extracted Hasty
and
the
coeluted (Fig.
from et
described
gelatinolytic
of
latent
activity
to
5)
active
SDS-PAGE.
detected
according used
for
were
collagenase
preparation
free
was
4 and
endogenously
by
shown
corresponding
fractions
could
shown)
lanes
assayed
and
band latent
3 A,
kD were
the
HNC are
fractions
composition
completely
(not
purified
(Fig.
and
70-75
kD protein
band
was
of
protein
Collagenase
purified
studies
HNC S-200
volumes
for
75 kD protein
the
1 mM PMC treatment)
almost
B).
of
Isolated
weight
a 70-75 the
The
3 A.
latency
on Sephacryl
apparent
forms
and
AND DISCUSSION
the al.
1 70-
(16).
activation
serine
protease
activities. Figure
2 demonstrates
HOC1 on latent were
found
10).
Addition
to
the
70-75
kD HNC.
activate
the
of
trace
effects Both
these
latent
amounts
of
1.0
reactive
HNC (Fig. (f-c.
981
mM H,O, and
5pM)
oxygen 2 A,
of
iron
lanes
1.0
mM
species l-4,
7-
and EDTA to
Vol.
171, No. 3, 1990
BIOCHEMICAL
B
AND BIOPHYSICAL
FRACTION
NUMBER
FRACTION
NUMBER
RESEARCH COMMUNICATIONS
Fiqure 1. Fractionation of latent human neutrophil collagenase (LHNC) by Sephacryl S-200. A. Fractions corresponding to apparent Mr of 70-75 kD were assayed for total (PMC+) and endogenous (PMC-) collagenase activity. Dll-12 and al and a2 indicate intact type I collagen dimers and monomers, respectively. Dll-12A and o1A and a2A indicate characteristic 3/4-cleavage products produced by HNC. B. SDS-PAGE analysis of corresponding lyophilizated fractions for protein contents. Molecular weight markers are indicated.
the
incubations
(Fig.
2 A,
both
lanes
(Fig.
inhibit
2 A, lane
Figure the
the 8).
compounds
This
do not
latent
changes
were in detected
The activation
2 A, lanes
activation
70-75
5 and 6),
of
indicates inhibit
latent that
the
apparent after
(Fig.
the
of
HNC induced
compounds did by HOC1 (Fig.
at concentrations
used these form
of H,O,, H,O,-EDTA-FeCl,
3 B,
molecular exposure
982
in
lanes
weight to
HNC by
by desferrioxamine
but these
changes
activation
latent
active/activated
kD HNC; no
observed
HNC enhanced
was inhibited
3 B shows the effects
weight
were
2 and 10).
H,O, and H20,-EDTA-FeCl,
and mannitol not
of H,O, and latent
of
70-75
known
HNC.
and HOC1 on
apparent
8-10).
of
molecular
Similarly,
no
kD latent
HNC
non-proteolytic
Vol.
171,
No.
3, 1990
activators serine be
BIOCHEMICAL
PMC proteases
kallikrein
(Fig.
molecular
weight
2,3,6,7).
The
(13,17,18,22)
being found
was
3,
lanes
kD
latent
(Fig. human
support
by
HNC
the
of
fibroblast-type
operative
on
latent
HNC.
in
ability
5
678
P (1
Among found 3-6),
and
plasma
trypsin in
(Fig.
recent
and
apparent
3 B,
involving
procollagenase Okada of
serine
lanes
documentation
cascade
Also,
to
lanes
plasmin
activation
the
2 B,
changes
human
activator
234
were
Activation
proteolytic
differences
chymotrypsin
incomplete
results
the
(12,14).
with
7-10).
COMMUNICATIONS
4,5)
HNC
with
70-75
present
and
noted
RESEARCH
lanes
latent
associated of
of as
of
2 B,
was
BIOPHYSICAL
trypsin
activation
chymotrypsin
have
(Fig.
activators no
plasmin
GTG
tested,
efficient
whereas
not
and
AND
(19,20) and
Nakanishi
proteases
to
91oH12 PA CZA
BI
234567891o
Figure 2. The effects of H,O,, HzOz-EDTA-FeClz and HOC1 on neutrophil collagenase (LHNC). LHNC (5pg enzyme protein) + 1mM HzO,+type Lane 1. Lane 2. LHNC+lmM HZ02+5pM EDTA+5fiM FeCl,+collagen LHNC+1.5mM H,O,+collagen Lane 3. Lane 4. LHNC+1.5mM H,0,+5pM EDTA+5pM FeCl,+collagen As lane 1+2OmM mannitol+lmM desferrioxamine Lane 5. As lane 4+2OmM mannitol+lmM desferrioxamine Lane 6. LHNC+lmM HOCl+collagen Lane 7. As lane 7+2OmM mannitol+lmM desferrioxamine Lane 8. Lane 9. As lane 1 Lane 10. As lane 2 Lane 11. LHNC+collagen Lane 12. LHNC+lmM PMC+collagen The effects of trypsin, chymotrypsin, plasmin B. kallikrein on LHNC. Tvne I collagen Lane 1. LHNC (5pg enzyme protein)+collagen Lane 2. LHNC (5~4 enzyme protein) treated with Lane 3. molar ratio I:i for 10 min+collagen LHNC treated with trypsin in molar ratio Lane 4. min+collagen 3 and 4, but chymotrypsin Lanes 5 and 6. As lanes as Lanes 7 and 8. As lanes 3 and 4, but plasmin As lanes 3 and 4, but plasma Lanes 9 and 10. activator 011-12 and al and a2 indicate intact type I collagen Rll-12A and alA and respectively. monomers, characteristic 3/4-cleavage products produced by A.
983
latent
human
I collagen
and
plasma
trypsin 1:l
in for
as activator _activator __,_ , KalliKreln
30
as
dimers and a2A indicate HNC.
Vol.
BIOCHEMICAL
171, No. 3, 1990
A 12
kD
kD 5
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
345
7
? 3 4 5 6
8 9 10
9266-
43-
Figure 3. SDS-PAGE A. fractions
analysis during
of
the
protein of
purification
Contents
latent
of
collagenase (LHNC). of human neutrophils Lane 1. Neutral salt extract Lane 2. Sepharose 6-B fraction Lane 3. 0.3 M NaCl-peak/QAE-Sephadex A-50 Lane 4. 0.5 M NaCl-peak/Cibacron Blue Sepharose kD-peakfsephacryl S-200, see also Fig. Lane 5. 70-75 Purification was carried out according to refs. Molecular weight markers are indicated. B. The effects of serine proteases and reactive oxygen on
VariOuS
human neutrophil
1 (9110). species
LHNC.
Lane 1. Lane 2.
LHNC (1Opg enzyme protein) LHNC treated with trypsin in molar ratio 1:l for min Lane 3. As lane 2, but chymotrypsin treatment Lane 4. LHNC treated with 1mMPMC for 30 min Lane 5. LHNC treated with 1.5 mMGTG for 30 min Lane 6. As lane 2, but trypsin treatment for 30 min Lane 7. As lane 3, but chymotrypsin treatment for 30 min Lane 8. LHNC treated with 1 mMH,O, for 30 min Lane 9. LHNC treated with 1mMH,O,-5pM EDTA-5fiM FeCl, for min Lane 10. LHNC treated with 1mMHOC1 for 30 min Molecular weight markers are indicated. act
as matrix
metalloproteinase
metalloproteinases activation
seem
potent
and that
type-specific
the concept
and direct their
activators
activation
proteolytic
activators,
Au(I)-salts
(11,12,20,23).
exclusive
have
(21).
Thus,
30
matrix
proteolytic
pathways.
Our resultssupport are
to
activators
10
inhibition
that of
potential i.e.
reactive
latent
organomercurials
of HNC exposed 984
species
human HNC (5,6,22)
is comparable
Vissers
oxygen
and Winterbourn to reactive
to other and
certain
described oxygen
non-
an
species
Vol.
171, No. 3, 1990
BIOCHEMICAL
However,
(7).
activated
they
by
activator
of
(7).
It
HNC by
species
enzyme
molecule
ability
to
which
then
have
previously
is
noteworthy
involves
the
(20,22).
initially by time
endogenously
activate
the
to
its
that
that
hydroxyl
present
radical
reaction
(24)
peroxide
may react
resulting with
bound
by
of trace
added to can
act
activation amount
enzyme
as
have
the
enzyme, (23).
latent
of
iron,
We
generated
by
HNC to the
same
The present of
iron
incubations
or
catalysts
of
localized
of
itself,
the
amounts
the
HzOZ through
of
the
potential
and GTG (22).
trace
the
of
acetate
from to
form
activates
vivo,
formation
seem to
-system
either
in
group
radical
the presence
metals,
activation and reactive
reactants
catalytic
by
denaturation
thiol
latent
HNC-
activated
Au(I)-salts
these
oxidase
hydroxyl
following
suspectible
All
found
transition
or
HNC completely
once
same critical
may loose
demonstrate
medium
that
rather
as aminophenylmercuric
or other
crude
an organomercurial
organomercurials,
hypoxanthinelxanthine
results
study
the mechanism of the non-proteolytic
oxygen
extent
their acetate,
HNC is
Evidently latent
in
phenylmercuric
organomercurials (23).
used
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fenton
HNC. Hydrogen present
in
according
to
the the
reaction: Fe3+ + H,O, + Fe'+ + O,- + 2H+
Ferrous
iron
thus
formed
may then
catalyze
the
Fenton
reaction
as follows: H,O, + Fe 2+ + Fe 3+ + OH- + OH' The hydroxyl collagenase
radical
by cleaving
coordination to cysteic
site acid
(or other
the
of active that
The formation iron
may then
bond between site
zinc
which
radical is able 985
Cysn
activation and the
atom and oxidize
can not be liganded
of hydroxyl
metal)
cause the oxidative
by the requires
to undergo
zinc the redox
of fourth
cysteine atom (20). presence reactions.
of
Vol.
171,
The
No.
3, 1990
BIOCHEMICAL
production
of
endogenous
iron
activation
hydroxyl
present
radicals
Generally, activate
the
suggest specific,
the
well
way
as
other
generation and/or
of be the
neutrophils
simplest to
degranulation enzymes proteolytic cathepsin (5,6,8,9)
of shown
activation
cascades
G;both would
being not
formation
components
necessarily
degrade
may
may,
site very
indeed,
be an
neutrophils
as
activate
their increased radical oxidative
NADPH-oxidase for
triggered
collagenase
contents,
of
to be
locating
mechanism their
than
appropriate
neutrophil
HNC
enzyme.
of hydroxyl
to of
the
The described
direct
granule
participitate
of
involving
activate
to
attack
of
This
activated
specific
random
metal)
oxidants.
endogenously
the
more
means
and most
Thus
endogenously
compensatory
HNC involving
(24).
deficient
to
-like
the
radical
enzyme.
of HzOz and subsequent
activation
be
other
myeloperoxidase
by
rather
hydroxyl
(or
(25)
of
would
of
of the
radical
result
COMMUNICATIONS
catalyzed
solution
would
iron
site
by
hydroxyl
would
by
cells
collagenase
It
be
moiety
reactions
(2).
for
bulk
protein
production
active
alternative
the
such
catalyzed to the
the
RESEARCH
may
may be the
enzyme
that
close
in
on
however,
BIOPHYSICAL
radical
of collagenase
hydroxyl
AND
upon
because
other
non-proteolytic
and
(f.ex.myeloperoxidase, azurophilic
granules)
be required.
Acknowledgments: This work has been financially supported by the Finnish Medical Association Duodecim, the Niilo Helander Foundation, the Finnish Cancer Foundation, the Finnish Cultural Foundation, the Arthritis Foundation in Finland, the Wellcome Foundation and the Oskar ijflunds Foundation. REFERENCES 1. 2. 3.
Werb, Z. (1989) Textbook of Rheumatology, 3rd ed. (Kelley, W.N. et al. eds.), Saunders, Philadelphia pp. 300-309. Sorsa, T., Saari, H., Konttinen, Y. T., Suomalainen, K., Lindy; S., and Uitto, V-J. (1989) Int. J. Tiss. React. 11, 153-159. Sorsa, T., Suomalainen, K., Helenius, J., Lindy, S., Saari, H., Konttinen, Y. T., and Uitto, V-J. (1990) N. Engl. J. Med. 323, 133-134. 986
Vol.
171, No. 3. 1990
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Birkedal-Hansen, H. (1988) J. Oral. Pathol. 17, 445-451. Weiss, S. J. (1989) N. Engl. J. Med. 320, 365-376. Weiss, S. J., Peppin, G. J., Ortix, X., Ragsdale, C., and Test, S. T. (1985) Science (Wash. D.C.) 227, 747-749. Vissers, M. C., and Winterbourn, C. C. (1987) Biochem. J. 245, 277-280. Capodici, C. C., and Berg, R. A. (1989) Agents and Action 27, 481-484. Sorsa, T. A. (1987) Stand. J. Rheumatol. 16, 167-175. Sorsa, T., Suomalainen, K., Turto, H., and Lindy, S. (1985) Med. Biol. 63, 66-72. Lindy, S., Sorsa, T., Suomalainen, K., and Turto, H. (1986) FEBS Lett. 208, 23-25 Sorsa, T., Suomalainen, K., Turto, H., and Lindy, S. (1987) Biosci. Rep. 7, 965-968. Uitto, V-J., Suomalainen, K., and Sorsa, T. (1990) J. Periodont. Res. 225, 135-142. Lindy, S., Sorsa, T., Suomalainen, K., and Turto, H. (1988) Stand. J. Rheumatol. suppl. 67, 5-9. Bieth, J., Spiess, B., and Wermuth, C-G-(1974) Biochem. Med. 11, 350-357. Hasty, K. A., Hibbs, M. S., Kang, A. H., and Mainardi, C. L. (1986) J. Biol. Chem. 261, 5645-5650. Ph.D. thesis, University of Helsinki, Sorsa, T. (1989) Finland, ISBN 952-90102-8-l. pp. l-62. Knauper, V., KrZmer, S., Reinke, H., and Tschesche, H. (1990) Eur. J. Biochem. 189, 295-300. Berg, R. A., Capodici, C. C., and D'Armiento, J. (1989) In: Therapeutic approaches to inflammatory diseases (Lewis, M. J. et al. eds.) pp. 84-90. Springman, E. B., Angleton, E-L., Birkedal-Hansen, H., and Van Wart, H. (1990) Proc. Acad. Sci., USA, 87, 364-368. I. (1989) FEBS L&t. 249, 353Okada, Y., and Nakanishi, 356. Sorsa, T., Saari, H., Konttinen, Y. T., Uitto, V-J., and Lindy, S. (1989) N. Engl. J. Med. 321, 327-328. Uitto, V-J., Turto, H., Huttunen, A., Lindy, S., and Uitto, J. (1980) Biochem. Biophys. acta 613, 168-177. Biochem. J. Halliwell, B., and Gutteridge, J. M. (1984) 219, l-14. Klebanoff, S. J. (1988) In: Inflammation: basic principles and clinical correlates (Gallin, J. I. et al. eds.) Raven Press, New York pp. 391-344.
987
171, No.
September
3, 1990
28,
BIOCHEMICAL
AND
ACTIVATION
RESEARCH
COMMUNICATIONS Pages
OF LATENT
BUMAN NEUTROPHIL
OXYGEN SPECIES Herkko
Saari', Yrjij
'IVt"
BIOPHYSICAL
1990
Department
Kimmo
of
AND SWINE
1,3
Suomalainen
, Otso
and Timo
Sorsa2'3
of Medicine,
Helsinki
University
Medical
BY REACTIVE
PROTRASES
T. Konttinen'
Hospital, 'Department
COLLAGENASE
979-987
Helsinki,
Lindyl,
Central
Finland
Chemistry,
University
of
Helsinki,
Finland 3Department
Received
August
of
6,
Periodontology, Helsinki,
University Finland
of
Helsinki,
1990
: The ability of various reactive oxygen species and serine proteases to activate latent collagenase (matrix metalloproteinase-1) purified from human neutrophils was examined. Latent 70-75 kD human neutrophil collagenase (HNC) was efficiently activated by known non-proteolytic activators phenylmercuric chloride (an organomercurial compound) and gold thioglucose(Au(I)-salt). Corresponding degree of activation was achieved by reactive oxygen species including hypochlorous acid (H202) and hydroxyl radical generated WOCl) , hydrogen peroxide by hypoxanthine/xanthine oxidase (HX/XAO). The presence of trace amounts of iron and EDTA were necessary and even enhanced H,O, induced activation of latent HNC. This activation could be abolished by an iron chelator desferrioxamine and a hydroxyl radical scavenger mannitol. HOC1 induced activation of latent HNC was not affected by desferrioxamine and mannitol. Thus, these compounds do not inhibit the active/activated form of HNC. Latent HNC could also be activated by trypsin and chymotrypsin but not by plasmin and plasma kallikrein. The ability of mannitol and desferrioxamine to inhibit the H,O,induced activation of HNC suggests the transition metal dependent Fenton reaction to be responsible for localized and/or site-specific generation of hydroxyl radical/hydroxyl radical -like oxidants to act as the activating oxygen species. Our results support the ability of myeloperoxidase derived HOC1 to act as a direct oxidative activator of HNC and further suggest the existence of a new/alternative oxidative activation O1990 Academic Press,Inc. pathway of HNC involving hydroxyl radical. SUMMARY
Connective such
as
believed
tissue
destruction
rheumatoid
arthritis
to
from
result
in and
the
inflammatory
periodontal
action
of
several
diseases diseases
is
types
of
0006-291X/90 979
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
BIOCHEMICAL
Vol. 171, No. 3, 1990
proteolytic
enzymes.
capable
of
tissue Although
the
involved
in
are not
3.4.24.7)
(5) *
Concomitantly
in
species
human
exert
neutrophil (6-8). by
of a direct primary
subsequently
proteases extracts
neutrophils
generate
activates effects on latent
role
(HNC)
HNC (6-8).
been
reactive
(5).
oxygen
activity
species
have
especially reported
Furthermore,
of HOC1 on HNC, to
to
reactive
70-75
oxygen
kD HNC purified
both
activation instead
be mediated
cathepsin (8).
been those
(HOCl) has been presented,
HNC proteolytically
of
potential
oxidants,
have
milieu
regulation
of reactive
HOC1 on neutrophil
various
in the
cytotoxic
derived
action of
neutrophils
extracellular
myeloperoxidase,
action
Triggered
to be
the
effects
acid
are considered
to
and
latent
diseases
metalloproteinase-1,
collagenase
oxidative
metalloproteinases
(matrix
a pivotal
Neutrophil
importance.
inflammatory
(l-4).
form
of the
and inhibit
the
enzymes
connective
primary
neutrophils
triggered
of HNC by hypochlorous
here
seen in
a latent
results
generated
the
matrix
microbicidal
Inconsistent
activate
of
collagenase
that
neutrophil's
reported
sources
of the
secrete
E.C.
on
be of
clarified,
source
metalloproteinases
extracellular
to
destruction
completely
of
suggested
cellular
known to
oxygen
are
RESEARCH COMMUNICATIONS
the matrix
a wide range
tissue
a potential are
Among these
degrading
components
AND BIOPHYSICAL
by
G which
We have studied
species from
and
serine
neutral
salt
of human neutrophils. MATERIAL
AND
METHODS
Reasents Native type I collagen was purified from human skin as described (9). Xanthine oxidase (grade III, free of proteolytic impurities) chloride bean (2) I phenylmercuric (PMC) , soy trypsin inhibitor (SBTI), gold thioglucose (GTG), trypsin, chymotrypsin, human plasmin, plasma kallikrein and synthetic serine protease substrates (see below) were purchased from Sigma (St. Louis, Missouri, USA). HOC1 was obtained from the Pharmacy of the University of Helsinki. All other reagents were of the highest commercially available grade. 980
Vol.
171,
No.
3, 1990
Purification qelatinase
BIOCHEMICAL
of
AND
latent HNC serine nrotease
and
BIOPHYSICAL
RESEARCH
and measurement activities
COMMUNICATIONS
of
collaaenase,
Latent 70-75 kD HNC was purified from neutral salt extracts of human neutrophils as described (9,lO). Collagenase activity was measured using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for separation of the characteristic cleavage products resulting from the action of collagenase on intact native type I collagen. Collagen degradation was quantitated by densitometric scanning of Coomassie brilliant blue stained gels as described (10). Activation of latent collagenase by the organomercurial compound PMC, Au(I)-salt GTG,reactive oxygen species and serine proteases was carried out as described (g-12,21,22). Gelatinase activity was measured using heat denatured type I collagen as substrate at 37OC and the reaction products were analyzed by SDS-PAGE (14). Elastasecathepsin G- and trypsin -like activities were measured using ; mM synthetic substrates, succinyl-alanyl-alanyl-valineparanitroanilide, succinyl-alanyl-alanyl-propyl-phenylalaninebenzoyl-arginine-paranitroanilide, paranitroanilide and respectively, by spectrofotometric recordings at 405 nm as described previously (15).
RESULTS Molecular in
forms
Figures
was
1 and
fractioned molecular
(obtained
after of
and
HNC.
analyzed
fractions with A and
Half
of
activity
HNC
lyophilized
in
From
which
be extracted Hasty
and
the
coeluted (Fig.
from et
described
gelatinolytic
of
latent
activity
to
5)
active
SDS-PAGE.
detected
according used
for
were
collagenase
preparation
free
was
4 and
endogenously
by
shown
corresponding
fractions
could
shown)
lanes
assayed
and
band latent
3 A,
kD were
the
HNC are
fractions
composition
completely
(not
purified
(Fig.
and
70-75
kD protein
band
was
of
protein
Collagenase
purified
studies
HNC S-200
volumes
for
75 kD protein
the
1 mM PMC treatment)
almost
B).
of
Isolated
weight
a 70-75 the
The
3 A.
latency
on Sephacryl
apparent
forms
and
AND DISCUSSION
the al.
1 70-
(16).
activation
serine
protease
activities. Figure
2 demonstrates
HOC1 on latent were
found
10).
Addition
to
the
70-75
kD HNC.
activate
the
of
trace
effects Both
these
latent
amounts
of
1.0
reactive
HNC (Fig. (f-c.
981
mM H,O, and
5pM)
oxygen 2 A,
of
iron
lanes
1.0
mM
species l-4,
7-
and EDTA to
Vol.
171, No. 3, 1990
BIOCHEMICAL
B
AND BIOPHYSICAL
FRACTION
NUMBER
FRACTION
NUMBER
RESEARCH COMMUNICATIONS
Fiqure 1. Fractionation of latent human neutrophil collagenase (LHNC) by Sephacryl S-200. A. Fractions corresponding to apparent Mr of 70-75 kD were assayed for total (PMC+) and endogenous (PMC-) collagenase activity. Dll-12 and al and a2 indicate intact type I collagen dimers and monomers, respectively. Dll-12A and o1A and a2A indicate characteristic 3/4-cleavage products produced by HNC. B. SDS-PAGE analysis of corresponding lyophilizated fractions for protein contents. Molecular weight markers are indicated.
the
incubations
(Fig.
2 A,
both
lanes
(Fig.
inhibit
2 A, lane
Figure the
the 8).
compounds
This
do not
latent
changes
were in detected
The activation
2 A, lanes
activation
70-75
5 and 6),
of
indicates inhibit
latent that
the
apparent after
(Fig.
the
of
HNC induced
compounds did by HOC1 (Fig.
at concentrations
used these form
of H,O,, H,O,-EDTA-FeCl,
3 B,
molecular exposure
982
in
lanes
weight to
HNC by
by desferrioxamine
but these
changes
activation
latent
active/activated
kD HNC; no
observed
HNC enhanced
was inhibited
3 B shows the effects
weight
were
2 and 10).
H,O, and H20,-EDTA-FeCl,
and mannitol not
of H,O, and latent
of
70-75
known
HNC.
and HOC1 on
apparent
8-10).
of
molecular
Similarly,
no
kD latent
HNC
non-proteolytic
Vol.
171,
No.
3, 1990
activators serine be
BIOCHEMICAL
PMC proteases
kallikrein
(Fig.
molecular
weight
2,3,6,7).
The
(13,17,18,22)
being found
was
3,
lanes
kD
latent
(Fig. human
support
by
HNC
the
of
fibroblast-type
operative
on
latent
HNC.
in
ability
5
678
P (1
Among found 3-6),
and
plasma
trypsin in
(Fig.
recent
and
apparent
3 B,
involving
procollagenase Okada of
serine
lanes
documentation
cascade
Also,
to
lanes
plasmin
activation
the
2 B,
changes
human
activator
234
were
Activation
proteolytic
differences
chymotrypsin
incomplete
results
the
(12,14).
with
7-10).
COMMUNICATIONS
4,5)
HNC
with
70-75
present
and
noted
RESEARCH
lanes
latent
associated of
of as
of
2 B,
was
BIOPHYSICAL
trypsin
activation
chymotrypsin
have
(Fig.
activators no
plasmin
GTG
tested,
efficient
whereas
not
and
AND
(19,20) and
Nakanishi
proteases
to
91oH12 PA CZA
BI
234567891o
Figure 2. The effects of H,O,, HzOz-EDTA-FeClz and HOC1 on neutrophil collagenase (LHNC). LHNC (5pg enzyme protein) + 1mM HzO,+type Lane 1. Lane 2. LHNC+lmM HZ02+5pM EDTA+5fiM FeCl,+collagen LHNC+1.5mM H,O,+collagen Lane 3. Lane 4. LHNC+1.5mM H,0,+5pM EDTA+5pM FeCl,+collagen As lane 1+2OmM mannitol+lmM desferrioxamine Lane 5. As lane 4+2OmM mannitol+lmM desferrioxamine Lane 6. LHNC+lmM HOCl+collagen Lane 7. As lane 7+2OmM mannitol+lmM desferrioxamine Lane 8. Lane 9. As lane 1 Lane 10. As lane 2 Lane 11. LHNC+collagen Lane 12. LHNC+lmM PMC+collagen The effects of trypsin, chymotrypsin, plasmin B. kallikrein on LHNC. Tvne I collagen Lane 1. LHNC (5pg enzyme protein)+collagen Lane 2. LHNC (5~4 enzyme protein) treated with Lane 3. molar ratio I:i for 10 min+collagen LHNC treated with trypsin in molar ratio Lane 4. min+collagen 3 and 4, but chymotrypsin Lanes 5 and 6. As lanes as Lanes 7 and 8. As lanes 3 and 4, but plasmin As lanes 3 and 4, but plasma Lanes 9 and 10. activator 011-12 and al and a2 indicate intact type I collagen Rll-12A and alA and respectively. monomers, characteristic 3/4-cleavage products produced by A.
983
latent
human
I collagen
and
plasma
trypsin 1:l
in for
as activator _activator __,_ , KalliKreln
30
as
dimers and a2A indicate HNC.
Vol.
BIOCHEMICAL
171, No. 3, 1990
A 12
kD
kD 5
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
345
7
? 3 4 5 6
8 9 10
9266-
43-
Figure 3. SDS-PAGE A. fractions
analysis during
of
the
protein of
purification
Contents
latent
of
collagenase (LHNC). of human neutrophils Lane 1. Neutral salt extract Lane 2. Sepharose 6-B fraction Lane 3. 0.3 M NaCl-peak/QAE-Sephadex A-50 Lane 4. 0.5 M NaCl-peak/Cibacron Blue Sepharose kD-peakfsephacryl S-200, see also Fig. Lane 5. 70-75 Purification was carried out according to refs. Molecular weight markers are indicated. B. The effects of serine proteases and reactive oxygen on
VariOuS
human neutrophil
1 (9110). species
LHNC.
Lane 1. Lane 2.
LHNC (1Opg enzyme protein) LHNC treated with trypsin in molar ratio 1:l for min Lane 3. As lane 2, but chymotrypsin treatment Lane 4. LHNC treated with 1mMPMC for 30 min Lane 5. LHNC treated with 1.5 mMGTG for 30 min Lane 6. As lane 2, but trypsin treatment for 30 min Lane 7. As lane 3, but chymotrypsin treatment for 30 min Lane 8. LHNC treated with 1 mMH,O, for 30 min Lane 9. LHNC treated with 1mMH,O,-5pM EDTA-5fiM FeCl, for min Lane 10. LHNC treated with 1mMHOC1 for 30 min Molecular weight markers are indicated. act
as matrix
metalloproteinase
metalloproteinases activation
seem
potent
and that
type-specific
the concept
and direct their
activators
activation
proteolytic
activators,
Au(I)-salts
(11,12,20,23).
exclusive
have
(21).
Thus,
30
matrix
proteolytic
pathways.
Our resultssupport are
to
activators
10
inhibition
that of
potential i.e.
reactive
latent
organomercurials
of HNC exposed 984
species
human HNC (5,6,22)
is comparable
Vissers
oxygen
and Winterbourn to reactive
to other and
certain
described oxygen
non-
an
species
Vol.
171, No. 3, 1990
BIOCHEMICAL
However,
(7).
activated
they
by
activator
of
(7).
It
HNC by
species
enzyme
molecule
ability
to
which
then
have
previously
is
noteworthy
involves
the
(20,22).
initially by time
endogenously
activate
the
to
its
that
that
hydroxyl
present
radical
reaction
(24)
peroxide
may react
resulting with
bound
by
of trace
added to can
act
activation amount
enzyme
as
have
the
enzyme, (23).
latent
of
iron,
We
generated
by
HNC to the
same
The present of
iron
incubations
or
catalysts
of
localized
of
itself,
the
amounts
the
HzOZ through
of
the
potential
and GTG (22).
trace
the
of
acetate
from to
form
activates
vivo,
formation
seem to
-system
either
in
group
radical
the presence
metals,
activation and reactive
reactants
catalytic
by
denaturation
thiol
latent
HNC-
activated
Au(I)-salts
these
oxidase
hydroxyl
following
suspectible
All
found
transition
or
HNC completely
once
same critical
may loose
demonstrate
medium
that
rather
as aminophenylmercuric
or other
crude
an organomercurial
organomercurials,
hypoxanthinelxanthine
results
study
the mechanism of the non-proteolytic
oxygen
extent
their acetate,
HNC is
Evidently latent
in
phenylmercuric
organomercurials (23).
used
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fenton
HNC. Hydrogen present
in
according
to
the the
reaction: Fe3+ + H,O, + Fe'+ + O,- + 2H+
Ferrous
iron
thus
formed
may then
catalyze
the
Fenton
reaction
as follows: H,O, + Fe 2+ + Fe 3+ + OH- + OH' The hydroxyl collagenase
radical
by cleaving
coordination to cysteic
site acid
(or other
the
of active that
The formation iron
may then
bond between site
zinc
which
radical is able 985
Cysn
activation and the
atom and oxidize
can not be liganded
of hydroxyl
metal)
cause the oxidative
by the requires
to undergo
zinc the redox
of fourth
cysteine atom (20). presence reactions.
of
Vol.
171,
The
No.
3, 1990
BIOCHEMICAL
production
of
endogenous
iron
activation
hydroxyl
present
radicals
Generally, activate
the
suggest specific,
the
well
way
as
other
generation and/or
of be the
neutrophils
simplest to
degranulation enzymes proteolytic cathepsin (5,6,8,9)
of shown
activation
cascades
G;both would
being not
formation
components
necessarily
degrade
may
may,
site very
indeed,
be an
neutrophils
as
activate
their increased radical oxidative
NADPH-oxidase for
triggered
collagenase
contents,
of
to be
locating
mechanism their
than
appropriate
neutrophil
HNC
enzyme.
of hydroxyl
to of
the
The described
direct
granule
participitate
of
involving
activate
to
attack
of
This
activated
specific
random
metal)
oxidants.
endogenously
the
more
means
and most
Thus
endogenously
compensatory
HNC involving
(24).
deficient
to
-like
the
radical
enzyme.
of HzOz and subsequent
activation
be
other
myeloperoxidase
by
rather
hydroxyl
(or
(25)
of
would
of
of the
radical
result
COMMUNICATIONS
catalyzed
solution
would
iron
site
by
hydroxyl
would
by
cells
collagenase
It
be
moiety
reactions
(2).
for
bulk
protein
production
active
alternative
the
such
catalyzed to the
the
RESEARCH
may
may be the
enzyme
that
close
in
on
however,
BIOPHYSICAL
radical
of collagenase
hydroxyl
AND
upon
because
other
non-proteolytic
and
(f.ex.myeloperoxidase, azurophilic
granules)
be required.
Acknowledgments: This work has been financially supported by the Finnish Medical Association Duodecim, the Niilo Helander Foundation, the Finnish Cancer Foundation, the Finnish Cultural Foundation, the Arthritis Foundation in Finland, the Wellcome Foundation and the Oskar ijflunds Foundation. REFERENCES 1. 2. 3.
Werb, Z. (1989) Textbook of Rheumatology, 3rd ed. (Kelley, W.N. et al. eds.), Saunders, Philadelphia pp. 300-309. Sorsa, T., Saari, H., Konttinen, Y. T., Suomalainen, K., Lindy; S., and Uitto, V-J. (1989) Int. J. Tiss. React. 11, 153-159. Sorsa, T., Suomalainen, K., Helenius, J., Lindy, S., Saari, H., Konttinen, Y. T., and Uitto, V-J. (1990) N. Engl. J. Med. 323, 133-134. 986
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171, No. 3. 1990
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Birkedal-Hansen, H. (1988) J. Oral. Pathol. 17, 445-451. Weiss, S. J. (1989) N. Engl. J. Med. 320, 365-376. Weiss, S. J., Peppin, G. J., Ortix, X., Ragsdale, C., and Test, S. T. (1985) Science (Wash. D.C.) 227, 747-749. Vissers, M. C., and Winterbourn, C. C. (1987) Biochem. J. 245, 277-280. Capodici, C. C., and Berg, R. A. (1989) Agents and Action 27, 481-484. Sorsa, T. A. (1987) Stand. J. Rheumatol. 16, 167-175. Sorsa, T., Suomalainen, K., Turto, H., and Lindy, S. (1985) Med. Biol. 63, 66-72. Lindy, S., Sorsa, T., Suomalainen, K., and Turto, H. (1986) FEBS Lett. 208, 23-25 Sorsa, T., Suomalainen, K., Turto, H., and Lindy, S. (1987) Biosci. Rep. 7, 965-968. Uitto, V-J., Suomalainen, K., and Sorsa, T. (1990) J. Periodont. Res. 225, 135-142. Lindy, S., Sorsa, T., Suomalainen, K., and Turto, H. (1988) Stand. J. Rheumatol. suppl. 67, 5-9. Bieth, J., Spiess, B., and Wermuth, C-G-(1974) Biochem. Med. 11, 350-357. Hasty, K. A., Hibbs, M. S., Kang, A. H., and Mainardi, C. L. (1986) J. Biol. Chem. 261, 5645-5650. Ph.D. thesis, University of Helsinki, Sorsa, T. (1989) Finland, ISBN 952-90102-8-l. pp. l-62. Knauper, V., KrZmer, S., Reinke, H., and Tschesche, H. (1990) Eur. J. Biochem. 189, 295-300. Berg, R. A., Capodici, C. C., and D'Armiento, J. (1989) In: Therapeutic approaches to inflammatory diseases (Lewis, M. J. et al. eds.) pp. 84-90. Springman, E. B., Angleton, E-L., Birkedal-Hansen, H., and Van Wart, H. (1990) Proc. Acad. Sci., USA, 87, 364-368. I. (1989) FEBS L&t. 249, 353Okada, Y., and Nakanishi, 356. Sorsa, T., Saari, H., Konttinen, Y. T., Uitto, V-J., and Lindy, S. (1989) N. Engl. J. Med. 321, 327-328. Uitto, V-J., Turto, H., Huttunen, A., Lindy, S., and Uitto, J. (1980) Biochem. Biophys. acta 613, 168-177. Biochem. J. Halliwell, B., and Gutteridge, J. M. (1984) 219, l-14. Klebanoff, S. J. (1988) In: Inflammation: basic principles and clinical correlates (Gallin, J. I. et al. eds.) Raven Press, New York pp. 391-344.
987
