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(Received for publication, May 11, 1995; and in revised form, July
24, 1995) From the
Tumor necrosis factor (TNF)-
Initially described for its anti-tumor activity(1) ,
tumor necrosis factor (TNF) Several studies in human and murine models have suggested that
TNF- In this report, we
describe the processing of in vitro translated 26-kDa
TNF-
Synthetic substrates MeOSuc-Ala-Ala-Pro-Val-pNA and
MeOSuc-Ala-Ala-Pro-Met-pNA were from Sigma.
Human
monocytes were obtained from healthy donors' leukophoresis bags.
Briefly, peripheral blood mononuclear cells were separated by standard
Ficoll-Hypaque density gradient centrifugation. The enriched population
of monocytes and lymphocytes were plated into dishes containing RPMI
supplemented with fetal calf serum and incubated for 30 min at 37
°C. The dishes were extensively washed with RPMI, leaving only
adherent monocytes.
Two overlapping fragments were generated in a initial
reaction using as template a wild-type TNF- TNF-delVal1 mutant, where the amino acid valine at position +1
is deleted, was obtained with a 33-mer oligonucleotide (upstream) 5`-
TCGAGAAGATGATCTTGCCTGGGCCAGAGGGCT-3` and a 28-mer oligonucleotide
(downstream) 5`-GGCCCAGGCAAGATCATCTTCTCGAACC-3`. TNF-Gly1 mutant,
where the amino acid valine at position +1 is substituted by a
glycine, was obtained with a 24-mer oligonucleotide (upstream) 5`-
TCGAGAAGATGATCTGCCTGCCTG-3` and a 24-mer oligonucleotide (downstream)
5`-CAGGCAGGCAGATCATCTTCTCGA-3`. TNF-Ala1 mutant, where the amino
acid valine at position +1 is substituted by an alanine, was
obtained with a 24-mer oligonucleotide (upstream)
5`-TCGAGAAGATGATCTGGCTGCCTG-3` and a 24-mer oligonucleotide
(downstream) 5`-CAGGCAGCCAGATCATCTTCTCGA-3`. The cDNAs encoding for
the mutated TNF-
Figure 1:
In vitro processing of human
TNF-
Figure 2:
Effects of protease inhibitors in the in vitro TNF-
In order to further confirm previous results, we studied the in
vitro processing activity of three purified serine proteinases:
human leukocyte elastase, cathepsin G, and PR-3. As shown in Fig. 3, whereas cathepsin G did not efficiently process the
TNF-
Figure 3:
In vitro processing activity of
human TNF-
Figure 4:
Effects of SLPI on TNF-
Figure 5:
Effect
of a ANCA positive serum-derived IgG on the in vitro TNF-
Figure 6:
In vitro processing activity of
human wild type or mutant TNF-
Figure 7:
Sequence analysis of 17-kDa cleavage
product. [
Using an in vitro TNF- Different biological
properties of PR-3 have been reported. PR-3 degrades a variety of
extracellular matrix proteins including elastin (18) ,
fibronectin, type IV collagen, and laminin(12) . In addition
PR-3 has a potent antimicrobial activity against both bacteria and
fungi (19, 20) . It cleaves and inactivates the human
C1 inhibitor leading to activation of the classical complement
pathway(21) , and it has been recently demonstrated that PR-3
has a potentiating effect of platelet activation (22) and may
play an important role in neutrophil-mediated endothelial
damage(23) . Finally, PR-3 has been shown to process in
vitro interleukin-8 (24) and the nuclear factor- The present study demonstrates that PR-3 is
capable of cleaving in vitro synthesized TNF- Recently, the existence of
a Zn Altogether our results show that PR-3 can play a role in cleaving
the TNF-
Volume 270,
Number 40,
Issue of October 06, pp. 23688-23692, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
(*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
is initially synthesized as a
membrane-bound, cell-associated 26-kDa protein that is further cleaved
to yield the soluble 17-kDa form. By using a radiolabeled in vitro translated TNF- precursor we detected a serine proteinase
processing activity present in crude membrane preparations of monocytic
cells able to generate a 17-kDa active protein. A similar processing
pattern was obtained using purified neutral serine proteinase
proteinase-3 (PR-3). Moreover, while a secretory leukocyte proteinase
inhibitor (a natural serine anti-proteinase) did not affect the in
vitro TNF- processing, IgG preparations containing high
titers of anti-PR-3 autoantibodies completely blocked this activity.
The NH
-terminal sequencing of the reaction products
obtained with either membrane preparations or PR-3 showed that cleavage
occurs in both cases between Val and Arg
.
These results together with cellular expression and localization of
PR-3 suggest a potential role for this enzyme as an accessory TNF-
processing enzyme.
- is actually a pleiotropic
cytokine that plays a key role as mediator of inflammation and cellular
immune response(2) . This cytokine has been shown to be
involved in the pathology of diseases such as septic shock, cancer,
AIDS, rheumatoid arthritis, or malaria(3, 4) . The
gene for TNF- encodes for a surface transmembrane biologically
active 26-kDa precursor, that is subsequently cleaved to release the
17-kDa soluble protein(5) . It has been suggested that the
membrane-bound form of TNF- can be implicated in the paracrine
activities of TNF- in tissues while systemic activities of
TNF- may be associated with the secreted form(5) . release may be dependent on the activity of one or more
serine proteases. For example, N-p-tosyl-L-arginine methyl
ester, a specific serine proteinase inhibitor, has been reported to
suppress the secretion of TNF-
without affecting the level of
TNF- mRNA or the expression of its cell surface form(6) .
Serine proteinase inhibitors were also shown to suppress the secretion
of TNF- from murine activated macrophages(7) . Moreover,
mice pretreated with the serine proteinase inhibitor
-antitrypsin (-AT) were not able to
secrete TNF- in response to D-galactosamine/lipopolysaccharide thus becoming fully
protected against D-galactosamine/lipopolysaccharide-induced
hepatitis(8) . Recent reports suggest, however, the implication
of a metalloprotease in the processing of TNF-(9) .
Indeed, a metalloproteinase activity capable of generating the 17-kDa
moiety from recombinant TNF- precursor was partially purified from
the monocytic cell line THP-1 membranes. A series of hydroxamate
inhibitors of matrix metalloproteases have been shown to inhibit the
release of TNF- without reducing the cell-associated activity and
to protect mice challenged with lethal doses of
endotoxin(9, 10, 11) . using cellular fractions derived from human monocytes or
monocytic cell lines. Such processing generated active 17-kDa TNF-
and could be blocked with serine proteinase inhibitors. Experimental
evidences suggest that proteinase-3 (PR-3) is the enzyme responsible
for this in vitro observed activity. The potential
physiological relevance of these findings are discussed.
Reagents
Human leukocyte neutrophil elastase and
cathepsin G are from Calbiochem Biochemicals. PR-3 and sera containing
anti-neutrophil cytoplasmic autoantibodies (ANCA) were obtained from
Wieslab AB (Lund, Sweden).-AT,
3,4-dichloroisocoumarin (DCIC), E-64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane),
leupeptin, and pepstatin were purchased from Sigma.
Methoxysuccinyl-Ala-Ala-Pro-Val chloromethylketone
(MeOSuc-Ala-Ala-Pro-Val-CMK) was from Bachem, Inc. (Torrance, CA).
Human secretory protease inhibitor (hSLPI) was from R& systems
(Abingdon, UK).Cells
HL-60, U937, Raji, and Jurkat human cell
lines were obtained from American Type Culture Collection, Rockville,
MD. Cells were grown in suspension (at 37 °C, 5% CO) in
RPMI 1640 (Life Technologies, Inc.) supplemented with 10% fetal calf
serum, 10 mM Hepes, 1 mM sodium pyruvate, 2 mML-glutamine, 1% penicillin and streptomycin.Preparation of Membrane/Particulate
Fractions
Cells were washed twice with ice-cold Dulbecco's
phosphate-buffered saline (PBS). The cells were collected by
centrifugation at 1200 rpm, resuspended at a density of
10
/ml in lysis buffer (Tris, 10 mM, pH 7.5, EDTA,
1 mM), and homogenized at 200 rpm with a motor-driven
Potter-Elvehjem (Teflon/glass) homogenizer. The homogenate was
centrifuged at 400 
g for 5 min. Pellets were
discarded, and the supernatant was ultracentrifuged at 160,000 g for 2 h. The cytosol fractions (supernatants) were stored at
-80 °C, and pellets containing membrane/particulate fraction
were resuspended at a density of 7 10 cells/ml in
membrane buffer (10 mM Tris, 250 mM sucrose),
homogenized with 15 passes of the Teflon/glass homogenizer at 200 rpm,
and stored at -80 °C. The protein concentrations of the
fractions were determined by a colorimetric BCA assay (Pierce).Preparation of Radiolabeled Precursor TNF-
A
0.8-kilobase cDNA, obtained from HL-60 RNA and containing the entire
coding sequence of TNF- precursor, was inserted into KpnI/SacI-digested pBS-SK+ plasmid DNA and
propagated in Escherichia coli. Purified plasmid was
transcribed in vitro by using a T7 RNA polymerase and
translated in vitro in a rabbit reticulocyte lysate system
(TNT T7-coupled reticulocyte lysate system, Promega
Biotech Inc.) in the presence of 40 µCi of
[
S]Cys (1 mCi = 37 MBq; Amersham Corp.)
for 1 h at 30 °C to produce [
S]Cys-labeled
26-kDa TNF-
.Assay for in Vitro TNF-
Cleavage of TNF- Precursor
Cleavage precursor was performed by
incubating 200 µl of in vitro translated TNF-
precursor with different amounts of crude membrane cellular extract in
a final volume of 1.5 ml in 20 mM Hepes, pH 7.5, 2 mM
dithiothreitol, 10% (v/v) glycerol. Each reaction was incubated for 1 h
at 30 °C and immunoprecipitated with 10 µl of rabbit anti-human
TNF- polyclonal antisera (PS30, Monosan) and 60 µl of protein
A-Sepharose (Pharmacia). Sepharose pellets were washed four times,
resuspended, and boiled for 3 min in 80 µl of 0.25 M Tris-Cl, pH 6.8, 10% SDS, 0.5% bromphenol blue, 0.5 M dithiothreitol, and 50% glycerol. Samples were migrated on a 13.5%
SDS-polyacrylamide gel and autoradiographied.Preparation of Human TNF-
Mutants were generated by polymerase chain reaction with
oligonucleotides encoding an Ala/Val site where the valine residue at
position +1 is either deleted or substituted by glycine or
alanine. Precursor
Mutants precursor cDNA
obtained from HL-60 RNA and a complementary set of oligonucleotides,
both of which include the point mutation. In a subsequent reaction the
two fragments were joined using flanking oligonucleotides as primers. proteins were cloned into the KpnI/SacI-digested pBS-SK+ plasmid DNA. The
mutations were verified by sequence analysis.Sequencing of Cleavage Product
The TNF-
precursor was translated in vitro as described above except
that 80 µCi of L-[3,4-
H]valine (1
mCi = 37 MBq; Amersham) was used as radiolabeled amino acid.
Cleavage reactions were performed using 100 µg of HL-60 membrane
proteins or 1 µg of purified PR-3. Radiolabeled TNF- product
was purified from the processing reaction by migration on SDS-PAGE gel
followed by transfer to poly(vinylidene difluoride) membrane.
NH-terminal sequencing of radiolabeled cleavage product was
performed by automated Edman degradation on a 470 A gas phase
microsequencer (Applied Biosystems, Inc.). Each degradation cycle
product was counted in a liquid scintillation counter (Beckman
Instruments).Determination of Enzymatic Activity
Proteolysis of
the synthetic substrates for elastase and PR-3
(MeO-Suc-Ala-Ala-Pro-Val-pNA) or cathepsin G
(MeO-Suc-Ala-Ala-Pro-Met-pNA) was assayed in 200 µl, total volume,
consisting of 2 mM of the corresponding substrate in PBS with
0.1% (v/v) Tween-80, 1.25%(v/v) dimethyl sulfoxide, and 25 nM enzyme. pNA release was followed by continuously measuring the
change of absorbance at 405 nm at 30 °C during a 1-h period using a
Microplate reader ThermoMax (Molecular Devices). Specific activities
determined under these conditions for elastase, PR-3, and cathepsin G
were 20, 0.2, and 6 units/mg, respectively. One unit corresponds to 1
µmol/min released pNA.
In Vitro TNF-
Crude membrane and cytosol fractions were prepared
from the myelomonocytic cell lines HL-60 and U937 as well as from human
monocytes and tested for their ability to cleave in vitro translated and [ Processing by Crude Cell Membrane
PreparationsS]Cys radiolabeled 26-kDa
TNF-
. Reaction products were analyzed by SDS-PAGE and
autoradiography after immunoprecipitation with an anti-TNF-
polyclonal antibody. While cytosol fractions did not exhibit any
processing activity, incubation of TNF- precursor with crude
membrane fractions prepared from these cells generated a 17-kDa band
that comigrated with a recombinant I-labeled TNF-
(Fig. 1A). A 24-kDa band could be systematically
detected under these conditions. In the in vitro assay, the
crude membrane extract derived from HL-60 was significantly more active
than those from monocytes and U937. No processing could be observed
when TNF- precursor was incubated with crude membranes obtained
from Jurkat (human T cell line) or Raji (human B cell line). Titration
of HL-60 membrane fractions showed that the processing activity was
dose-dependent (Fig. 1B). Thus 100 µg of total
protein derived from the HL-60 membrane fraction almost completely
processed the 26-kDa precursor in 60 min. Interestingly, when 100
µg of crude membrane extracts were used in the assay, the 24-kDa
band disappeared, suggesting that this band could result of the use of
an intermediate cleavage site located in the 14-kDa precursor portion
of TNF-. When assayed using the L929 assay, the cleavage product
was as active as the precursor form, indicating that the 17-kDa protein
resulting from the in vitro processing was biologically active
(data not shown).
precursor by crude membrane fractions prepared from different
cell sources. A, the 26-kDa in vitro translated
TNF- precursor (lane 1) was incubated for 1 h at 30
°C with 100 µg of membrane/particulate fraction proteins
prepared from HL-60 (lane 3), U937 (lane 4), human
monocytes (lane 5), Jurkat (lane 6), or Raji (lane 7). The reaction products were visualized by
autoradiography after immunoprecipitation and SDS-PAGE. I-Labeled 17-kDa TNF-
is shown in lane 2.
B, the 26-kDa in vitro translated TNF- precursor (lane 4) was incubated with 1, 10, and 100 µg (lanes
1, 2, an 3, respectively) of HL-60 membrane
fraction proteins, and reaction products were analyzed as described
above.
Effect of Protease Inhibitors on the in Vitro Processing
of TNF-
In
order to characterize the enzymatic nature of the proteolytic activity
found in the crude membrane extracts of HL-60, we tested the effect of
a series of protease inhibitors in the TNF- and Activity of Purified Serine Proteinases cleavage assay. The
results shown in Fig. 2indicate that the serine proteinase
inhibitors DCIC, -AT, and MeO-Suc-Ala-Ala-Pro-Val-CMK
efficiently inhibited the generation of 17-kDa TNF- by HL-60
membrane proteins. In contrast, E-64, pepstatin, EDTA, and leupeptin,
which are specific inhibitors of cysteine, aspartate, metallo, and
serine/cysteine proteinases, respectively, failed to inhibit the in
vitro processing of TNF-. These results strongly suggest that
the in vitro TNF- processing activity detected in HL-60
membrane proteins is dependent on one or several serine proteinases.
processing activity. The 26-kDa in
vitro translated TNF- precursor was incubated with 10 µg
of HL-60 crude membrane fraction proteins in the absence or presence of
250 µM DCIC, 1 mg/ml -AT, 250
µM MeO-Suc-Ala-Ala-Pro-Val-CMK, 5 mM EDTA, 200
µM E-64, 500 µM leupeptin, or 50 µM pepstatin A, before immunoprecipitation, SDS-PAGE, and
autoradiography. Results were analyzed by scanning and are expressed as
percentage of the activity found in controls performed in the presence
of the solvents used for each inhibitor.
precursor, elastase and PR-3 generated a 17-kDa protein in a
dose-dependent manner. It should be noted that PR3 was more efficient
than elastase to generate the 17-kDa TNF-. Moreover PR-3
reproduced the same pattern of proteolysis (17- and 24-kDa bands)
previously found with HL-60 membrane fractions. Among the natural
serine proteinase inhibitors, the secretory leukoproteinase inhibitor
(SLPI) has been shown to inhibit both elastase and cathepsin G but not
PR-3(12, 13) . The proteolytic activity of elastase
and PR-3 on the synthetic substrate MeO-Suc-Ala-Ala-Pro-Val-pNA was
studied in the presence of different concentrations of recombinant
SLPI. This molecule completely inhibited the elastase activity at a 5:1
molar ratio without affecting the PR-3 activity (data not shown). In
the in vitro TNF- cleavage assay, SLPI (1 µg)
inhibited the weak processing activity of elastase without affecting
the PR-3 one (Fig. 4). In addition, the same concentration of
SLPI did not inhibit the processing activity of HL-60 membrane
fraction, suggesting that elastase was not implicated in this reaction.
precursor by purified serine proteinases. The 26-kDa in vitro translated TNF- precursor (lane 10) was
incubated for 1 h at 30 °C in the presence of 8, 80, or 800 ng of
neutrophil elastase (lanes 2, 3, and 4) or
PR-3 (lanes 5, 6, and 7) or 1 µg of
cathepsin G (lane 8), and reaction products were analyzed by
SDS-PAGE and autoradiography after immunoprecipitation. In lane 1 is shown the in vitro processing obtained with 100 µg
of HL-60 crude membrane fraction proteins. I-labeled
17-kDa TNF-
is shown in lane
9.
in vitro processing. Aliquots of 800 ng of elastase, 80 ng of PR-3, or 100
µg of HL-60 crude membrane preparation proteins were incubated with
1 µg of SLPI for 10 min at room temperature before cleavage assay
on the in vitro translated TNF- precursor,
immunoprecipitation, SDS-PAGE, and autoradiography. Results were
analyzed by scanning and are expressed as percentage of the activity
found in controls performed in the absence of
SLPI.
Effect of Purified IgG from Wegener's
Granulomatosis (WG) Patients on the in Vitro Processing of
TNF-
Classic ANCA are specific markers for active WG. These
antibodies are specifically directed against
PR-3(14, 15, 16) . Recently, IgG from
patients with active WG were shown to significantly inhibit PR-3
proteolytic activity(17) . We therefore evaluated the
inhibitory capacity of IgGs purified from five WG patients' sera
on the PR-3 enzymatic activity measured on the synthetic substrate
MeO-Suc-Ala-Ala-Pro-Val-pNA. IgG derived from the serum IO5
PR
inhibited the PR-3 activity at a 50-fold molar excess without affecting
the activity of elastase. The incubation of purified PR-3 or elastase
with 100 µg of IO5 IgGs inhibited the PR-3 but not the
elastase-mediated TNF- in vitro processing activity (data
not shown). At the same concentration, IO5 IgGs completely inhibited
the processing activity of the monocytes and HL-60-derived membrane
fractions (Fig. 5). These results support that PR-3 is most
probably the enzyme responsible for the in vitro cleavage of
26-kDa TNF- by HL-60 or monocyte-derived crude membrane
preparations.
cleavage activity. Aliquots of 10 µg of HL-60 membrane fraction
proteins (lanes 2, 3, and 4) or 100 µg
of human monocyte membrane fraction proteins (lanes 5, 6, and 7) were incubated for 30 min at 37 °C with
buffer (lanes 2 and 5), 100 µg of
PRIO5-derived IgGs (lanes 3 and 6) or 100
µg of control human IgG (lanes 4 and 7) before
the cleavage assay on the in vitro translated TNF-
precursor (lane 1).
Identification of the Cleavage Site of 26-kDa TNF-
We have constructed three mutants in
which the residue Val
by HL-60 Membrane Proteins (the NH
-terminal amino
acid in the secreted TNF-) has been either deleted or substituted
by Gly or Ala. HL-60-derived membrane preparations were unable to
generate any 17-kDa protein from either the deleted (Fig. 6) or
glycine-substituted (data not shown) mutants. Interestingly, the
generation of the 24-kDa band was not modified in these two mutants. In
contrast, any major changes could be detected in the mutant in which
Val was substituted by an Ala (data not shown). These
results indicate that Val
is crucial for the in vitro detected TNF-
processing activity. To identify the amino
terminus of the 17-kDa cleavage product,
[H]Val-labeled 26-kDa TNF- was cleaved by
HL-60-derived membrane fraction, immunoprecipitated, electrophoresed,
transferred to poly(vinylidene difluoride) membrane, and subjected to
automated sequencing. Peaks of radiolabeled Val were detected at cycles
12, 15, and 16 (Fig. 7). The same radioactivity pattern was
observed when purified PR-3 was used in the in vitro cleavage
assay (data not shown). Our results show that cleavage of the TNF-
precursor by both PR-3 and HL-60 membrane preparations occurs between
Val and Arg
.
precursors by HL-60 membrane
preparations. In vitro translated wild type TNF-
precursor (lane 1) or a valine deletion mutant precursor (lane 7) were incubated for 1 h at 30 °C with 80 ng of
PR-3 (lanes 2 and 5, respectively) or 10 µg of
HL-60 membrane fraction proteins (lanes 3 and 6,
respectively). The reaction products were visualized by autoradiography
after immunoprecipitation and SDS-PAGE. I-labeled 17-kDa
TNF-
is shown in lane 4.
H]Val-labeled TNF- precursor was
cleaved with 100 µg of HL-60 crude membrane preparation proteins
and the cleavage product was sequenced after SDS-PAGE and transferred
to poly(vinylidene difluoride) membrane. Fractions from the sequence
run were counted for associated radioactivity. Peaks of radioactivity
were found in cycles 12, 15, and 16 corresponding to the Val amino
acids of mature TNF-, which are plotted on the ordinate axis.
Amino acids are depicted by the single-letter
code.
precursor cleavage
assay, we have identified a serine proteinase activity in the crude
membrane fractions from monocytic cells, which is capable of generating
a bioactive 17-kDa TNF- form. Experiments carried out to
characterize the enzymatic nature of this activity suggest that the
neutral serine proteinase PR-3 or a related enzyme is responsible for
this effect. First, purified PR-3 processed the TNF- precursor
with a pattern identical to the one obtained with the crude membrane
preparations. In addition to the 17-kDa protein, a 24-kDa band was
observed when the TNF- precursor was incubated with active
membrane fractions or PR-3. Second, SLPI, a natural serine
anti-proteinase secreted by cells of mucosal surfaces that interacts
with both cathepsin G and elastase but is devoid of inhibitory activity
against PR-3(12, 13) , did not affect the proteolytic
activity of the membrane preparations. Third, purified IgG prepared
from an ANCA-positive serum (previously shown to specifically interfere
with the PR-3 in vitro proteolytic activity) completely
inhibited the in vitro processing activity of the membrane
fractions. Finally, the NH-terminal sequence of the 17-kDa
product derived from the proteolysis with both crude membrane extracts
or purified PR-3 were shown to be identical.
B
subunit p65(25) . At the cellular level, PR-3 is not only
localized in the azurophil granules of granulocytes, but is also
present in small granules of monocytes(26) , in human
endothelial cells(27) , and in mastocytes(28) . Several
stimuli such as TNF- or IL-8 can even induce translocation of PR-3
from the intragranular loci to the cell surface of polymorphonuclear
leukocytes(29) . precursor
in a site-specific manner between Val and
Arg
, thus generating a 17-kDa TNF-
with an Arg at its
NH terminus. The importance of this site was confirmed by
using TNF- mutants in which Val was either deleted or
changed by Ala or Gly (Fig. 6). Accordingly, studies previously
conducted to map the active site of PR-3 showed that the preferred P1
residue is a small aliphatic amino acid such as valine or
alanine(30) . As described above, an additional 24-kDa band was
generated in the in vitro cleavage assay by PR-3, thus
indicating the existence of a second proteolytic site in the TNF-
precursor. This second proteolytic site is more probably located in the
14-kDa prosequence because (i) it disappeared with high amounts of
membrane preparations (Fig. 1B) or when longer
incubation times were performed (data not shown), and (ii) membrane
preparations did not cleave the recombinant soluble 17-kDa TNF-
(data not shown). A potential site theoretically susceptible to
generate a 24-kDa protein is located between alanine 15 and leucine 16.
This is in agreement with the studies on the primary specificity of
PR-3 against the insulin-B chain showing that a major site of cleavage
was an alanine/leucine bond(12) .-containing endopeptidase capable of cleaving the
26-kDa TNF-
to a 17-kDa form beginning at Val was
reported(9, 11) . Val
was previously
shown to correspond to the NH
terminus of the TNF-
secreted by cultured cell lines(31, 32) . This,
together with the in vivo efficacy of metalloprotease
inhibitors to block TNF-
secretion(9, 10, 11) , strongly suggests that
the enzyme primarily responsible for TNF- processing is a
metalloprotease. Our results suggest, however, that accessory sites and
perhaps accessory enzymes could exist to generate active TNF-.
Indeed, we demonstrated that Val-Arg
is a
possible alternative cleavage site. Additional sites could exist since
it was shown that deletion of residues between Val
and
Pro
did not affect the generation of active
TNF-
(33) . Furthermore, pulse-chase studies suggest that
the processing of TNF- primarily takes place at the cell
surface(34, 35) , raising the possibility of an
extracellular cleavage of TNF- by serum proteinases.
Interestingly, PR-3 is present in large amounts in the serum of normal
subjects and its levels are significantly high in patients with
connective tissue disease(36) . Along with this line, the
serine proteinase inhibitor -AT was shown to block
TNF- release in vitro(37) and in
vivo(8) . More recently, the TNF- concentration in
synovial fluid of rheumatoid arthritis patients was shown to be
inversely correlated with -AT activity (38) . precursor to generate a bioactive form. The relevance of
PR-3-mediated TNF- processing under normal and pathological
situations remains to be elucidated.
)-AT, - antitrypsin; ANCA,
anti-neutrophil cytoplasmic autoantibodies; CMK, chloromethylketone;
DCIC, 3,4-dichloroisocoumarin; E-64, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane;
MeO, methoxy; Suc, succinyl; pNA, p-nitroanilide; PBS,
phosphate-bufffered saline; PR-3, proteinase-3; SLPI, secretory
leukoproteinase inhibitor; WG, Wegener's granulomatosis; PAGE,
polyacrylamide gel electrophoresis.
We thank Drs. A. Diu, H. Fridman, and R. Westwood for
stimulating discussions and critical reading of the manuscript. We are
also grateful to Dr. F. Fassy for help with some experiments.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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C. R. D. Carter, K. M. Whitmore, and R. Thorpe The significance of carbohydrates on G-CSF: differential sensitivity of G-CSFs to human neutrophil elastase degradation J. Leukoc. Biol., March 1, 2004; 75(3): 515 - 522. [Abstract] [Full Text] [PDF]
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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]
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M. Kaup, K. Dassler, C. Weise, and H. Fuchs Shedding of the Transferrin Receptor Is Mediated Constitutively by an Integral Membrane Metalloprotease Sensitive to Tumor Necrosis Factor alpha Protease Inhibitor-2 J. Biol. Chem., October 4, 2002; 277(41): 38494 - 38502. [Abstract] [Full Text] [PDF]
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Y. M. van der Geld, P. C. Limburg, and C. G. M. Kallenberg Proteinase 3, Wegener's autoantigen: from gene to antigen J. Leukoc. Biol., February 1, 2001; 69(2): 177 - 190. [Abstract] [Full Text]
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U. Bank and S. Ansorge More than destructive: neutrophil-derived serine proteases in cytokine bioactivity control J. Leukoc. Biol., February 1, 2001; 69(2): 197 - 206. [Abstract] [Full Text]
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C. Coeshott, C. Ohnemus, A. Pilyavskaya, S. Ross, M. Wieczorek, H. Kroona, A. H. Leimer, and J. Cheronis Converting enzyme-independent release of tumor necrosis factor alpha and IL-1beta from a stimulated human monocytic cell line in the presence of activated neutrophils or purified proteinase 3 PNAS, May 25, 1999; 96(11): 6261 - 6266. [Abstract] [Full Text] [PDF]
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C. Herman and Y. Chernajovsky Mutation of Proline 211 Reduces Shedding of the Human p75 TNF Receptor J. Immunol., March 1, 1998; 160(5): 2478 - 2487. [Abstract] [Full Text] [PDF]
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