(Received for publication, September 6, 1995; and in revised form, November 16, 1995)
From the
Proteinase 3 is a human polymorphonuclear leukocyte serine
proteinase that degrades elastin in vitro and causes emphysema
when administered by intratracheal insufflation into hamsters.
Proteinase 3, stored in the azurophilic granules, is expressed in
progenitor cells of myeloid origin. In the present study, the
biosynthesis, processing, and intracellular transport of the enzyme was
investigated in the human myelomonocytic cell line U937. Proteinase 3
is initially identified as a 35-kDa precursor and converted into the
29-kDa mature form within 3 h. By using a combination of techniques
including amino-terminal sequencing, we identified the 35-kDa form as a
zymogen containing an activation dipeptide but lacking the
amino-terminal 25 residues, presumably the result of cleavage by a
signal peptidase. Tunicamycin treatment and alkalinization of acidic
cell compartments with NHCl did not prevent the processing
of the proteinase 3 zymogen into the mature form, suggesting that the
enzyme is targeted to the cytoplasmic granules by a mechanism other
than the mannose 6-phosphate receptor. Brefeldin A inhibited the
zymogen processing, suggesting that the dipeptide cleavage occurred in
a post-Golgi organelle. The enzyme responsible for the removal of the
dipeptide is a cysteine proteinase since E-64d, a class-specific
inhibitor, prevented processing. However, treatment of cells with a
dipeptidyl peptidase I inhibitor, Gly-Phe-diazomethyl ketone and with
the lysosomotropic agents, NH
Cl and chloroquine, did not
prevent dipeptide cleavage, indicating that the processing enzyme for
proteinase 3 is not dipeptidyl peptidase I. In contrast,
Gly-Phe-diazomethyl ketone inhibited cleavage of the dipeptide from
cathepsin G. This indicates that processing of proteinase 3 is distinct
from that of cathepsin G. Proteinase 3 is also processed at the
COOH-terminal extension. Cleavage takes place next to Arg-222,
suggesting that a trypsin-like proteinase is involved in the
COOH-terminal processing.
Proteinase 3 (PR-3, ()EC 3.4.21.76), is a third
neutral serine proteinase in azurophilic granules of human
polymorphonuclear leukocytes (PMNL)(1, 2) , distinct
from elastase (HLE, EC 3.4.21.37) and cathepsin G (Cat G, EC
3.4.21.20). PR-3 degrades several structural proteins in
vitro, including elastin, suggesting that it plays a role in
several PMNL-mediated physiologic and pathologic
events(1, 3) . Physiologically, the proteolytic
activity of PR-3 may facilitate the movement of neutrophils from the
vasculature through basement membranes at sites of inflammation (4) or may assist in the digestion of phagocytosed
microorganisms(5) . Pathologically, the elastolytic property of
PR-3 suggests a role in the development of emphysema. This possibility
is supported by the demonstration of emphysematous lesions in the lungs
of hamsters following intratracheal insufflation of PR-3(1) .
The prospect that PR-3 also is a mediator of airway injury is
strengthened by its resistance to inhibition by secretory leukocyte
protease inhibitor, the primary serine proteinase inhibitor in human
upper respiratory tract (3, 6) .
PR-3, in addition
to its proteolytic activity against extracellular matrix proteins, has
a variety of other potentially important actions. PR-3 is identical to
myeloblastin, which has been ascribed a central role in the control of
growth and differentiation of leukemic cells (7) . Recently,
PR-3 was found to degrade the 28-kDa mammalian heat shock
protein(8) , previously linked to differentiation of normal and
neoplastic cells and to cleave the nuclear factor B subunit
p65(9) . PR-3 also has microbicidal activity that is
independent of its serine proteinase activity(10) . Perhaps
most importantly, PR-3 is the antigen recognized by
cytoplasmic-staining anti-neutrophil cytoplasmic autoantibodies in
patients with Wegener's
granulomatosis(11, 12, 13) , a disease
characterized by a prominent neutrophilic vasculitis. PR-3 could
contribute to the pathogenesis of this disease either by inactivating
complement pathway inhibitor (C1) (14) or by its presence on
the surfaces of PMNL and human endothelial cells, turning these cells
into targets for activation by cytoplasm-staining anti-neutrophil
cytoplasmic autoantibodies(15, 16) . Because of these
diverse roles, an in-depth knowledge of factors that influence the
expression of PR-3 are of substantial importance. In the present
investigation, we report on the biosynthesis, processing, and
intracellular transport of this enzyme.
Figure 1: PMNL serine proteinases primary structure, location of peptide sequences used for peptide antisera production, and proposed COOH-terminal cleavage sites. The amino acid sequences are deduced from the cDNA and genomic studies reported for PR-3 (7, 34, 56, 57, 58) HLE(26, 59) , and Cat G(60) . All of the residues are numbered starting with the first amino acid residue of the active proteinase. The residues in the prepro region are assigned negative numbers relative to the first amino acid residue. The peptide sequences used for raising antisera are shown in boldface. Using conventional protein sequencing methodology, the sequence obtained for the COOH-terminal tryptic peptide of purified PR-3 (3) and that reported for purified HLE (61) are shown in italics. The sequence obtained for the peptide resulting from the cleavage of each proteinase by idosobenzoic acid is underlined. The arrow next to Arg indicates the probable cleavage site of the COOH-terminal extension peptide.
At the completion of the pulse or chase period, cells were
transferred to ice and recovered by centrifugation at 10,000 g for 15 s. Cell pellets lysed with 1 ml of chilled
radioimmunoprecipitation assay buffer, pH 8, containing 10 mM Tris/Tris-HCl, 140 mM NaCl, 1% (v/v) Nonidet P-40, 0.5%
(w/v) sodium deoxycholate, 0.1% (w/v) SDS, and a proteinase inhibitor
mixture (1 mM phenanthroline, 50 µM 3,4-dichloroisocoumarin, 10 µM E-64)(20) .
Cell lysates and conditioned media were each mixed with 50 µl of a 10% suspension of protein A-Sepharose CL-4B beads in radioimmunoprecipitation assay buffer and incubated for 1 h at 4 °C with gentle agitation. Supernatants were recovered by centrifugation, and then 50 µl of protein A beads, preincubated with 20 µl of the appropriate antiserum, was added and incubated overnight at 4 °C with gentle mixing. Protein A-bound immune complexes were then washed, suspended in 40 µl of SDS sample buffer, boiled for 5 min, and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography or radiosequencing.
Figure 2:
Pulse-chase experiment showing processing
of PR-3 in U937 cells. Approximately 2 10
/ml U937
cells were pulsed with 100 µCi/ml
[
S]methionine for 30 min at 37 °C following
a 30-min preincubation with methionine-deficient media. The labeled
cells were chased with nonradioactive media for the time periods
indicated ranging from 0 to 3 h. The labeled proteins were
immunoprecipitated from cell lysates with PR-3 antibody and analyzed by
SDS-PAGE and fluorography. Note that the 35-kDa form of PR-3 present
after the pulse period (0 h) was completely converted to a 29-kDa form
by 3 h of chase. The apparent molecular weights of the biosynthetic
forms were calculated from their electrophoretic mobilities relative to
standards.
To further investigate
the nature of the precursor processing, we used radiosequencing to
identify the amino terminus of the precursor forms of PR-3. For these
studies, cells were labeled with [H]isoleucine
since isoleucine is at the amino terminus of mature PR-3 (see Fig. 1). The results are shown in Fig. 3. The 35-kDa form
of PR-3 contained an isoleucine at position 3. Thus, the earliest
identifiable biosynthetic form (35 kDa) was pro-PR-3, which consists of
a dipeptide preceding the amino-terminal isoleucine of mature PR-3 (see Fig. 1). These results confirmed that the 25-amino acid signal
peptide was removed co-translationally. In the later biosynthetic form
of PR-3 (29 kDa), the prosegment
(Ala
-Glu
) was removed resulting
in isoleucine at position one.
Figure 3:
Protein sequencing of
[H]isoleucine-labeled biosynthetic forms of PR-3
in U937 cells. Biosynthetic forms of PR-3 were immunoprecipitated from
U937 cells using PR-3 antiserum following 1 h of pulse and 4 h of
chase. The immunoprecipitates were subjected to SDS-PAGE and blotted to
proBlott membrane. The protein bands of interest were cut out from the
membrane and sequenced using Edman degradation followed by
scintillation counting of radioactivity of the product released during
each cycle. The radioactivity (counts/min) released from each
degradation cycle was plotted against cycle number depicted on the abscissa. Single-letter amino acid code of the pro-PR-3 or
mature PR-3 sequence is shown below the cycle numbers to match the
position of the labeled amino acid.
Figure 4:
Biosynthesis of PR-3 in tunicamycin
treated U937 cells. Cells were preincubated for 30 min in the presence
of indicated concentrations of tunicamycin (prepared as a 10 mg/ml
stock in dimethylformamide) and then pulsed with
[S]methionine for 30 min in the continued
presence of inhibitor. Cell lysates were immunoprecipitated with PR-3
antiserum and analyzed by SDS-PAGE and fluorography. Lane 1,
immunoreactive form isolated after pulse in the absence of tunicamycin. Lanes 2-4, immunoreactive forms isolated after pulse in
the presence of 0.1, 0.5, and 1 µg/ml tunicamycin, respectively.
Note the difference in the size of the earliest biosynthetic form of
PR-3 in the lysates of tunicamycin treated cells (lanes
2-4) and untreated cells (lane
1).
Next we conducted chase experiments with
[H]isoleucine in the presence of tunicamycin to
explore the role of glycosylation in PR-3 trafficking and processing.
The radiosequence analysis demonstrated that inhibition of
glycosylation did not prevent the isoleucine shift from the third to
the first position during the chase period (data not shown). Thus,
glycosylation is not essential for cleavage of the pro dipeptide and by
inference is not critical for transport of the protein through
intracellular compartments.
Figure 5:
BFA
blocks the processing of zymogens of PR-3, HLE, and Cat G. To cells in
an isoleucine deficient medium, BFA (2.5 mg/ml in ethanol) was added to
a final concentration of 5 µg/ml and incubated for 1 h. Following
the preincubation, the cells were labeled with
[H]isoleucine for 1 h and chased for 4 h in the
presence of BFA. At the end of the chase period, cells were
immunoprecipitated with proteinase-specific antisera, and the purified
labeled proteins were analyzed as described in legend to Fig. 3.
Note that isoleucine remains in position 3, position 3, and positions 3
and 4 in PR-3, HLE, and Cat G, respectively, showing that BFA treatment
prevents excision of the dipeptides.
Figure 6:
Inhibition of cysteine proteinases with
E-64d in U937 cells prevents cleavage of activation dipeptide from PR-3
zymogen. Cells were preincubated for 1 h with 0.24 mM E-64d
prior to labeling with [H]isoleucine for 1 h and
chased for 4 h in the continued presence of the inhibitor. At the end
of chase period, cells were immunoprecipitated with proteinase-specific
antisera, and the purified labeled proteins were analyzed as described
in legend to Fig. 3. Note that the inhibitor blocks the cleavage
of the activation dipeptides from PR-3, HLE, and Cat
G.
Since E-64d is known to inhibit calcium-dependent cysteine proteinases (calpains) within cells(32) , we examined the effects of EGTA (1 and 5 mM), a general calcium-dependent proteinase inhibitor, and other metalloproteinase inhibitors, EDTA (1 and 5 mM) and o-phenanthroline (100 µM), on the processing of PR-3. These inhibitors did not block the processing ruling out the possibility that calpain-type proteinases are involved.
Figure 7:
Influence
of lysosomotropic agents on PR-3 processing. Cells were preincubated
for 1 h with 5 mM NHCl (A) or 25
µM chloroquine (B) prior to labeling with
[
H]isoleucine for 1 h and chased for 4 h in the
continued presence of the agent. At the end of chase period, cells were
immunoprecipitated with proteinase-specific antisera, and the purified
labeled proteins were analyzed as described in legend to Fig. 3.
Note that NH
Cl did not inhibit processing of any of the
proteinases. In contrast, chloroquine inhibited processing of Cat G,
and partially inhibited the processing of HLE but had no effect on
PR-3.
We have previously reported the identification(1) ,
biochemical characterization(3) , and gene structure (34, 35) of PR-3, an elastolytic PMNL serine
proteinase. The present investigation focused on the biosynthesis,
processing, and intracellular transport of the enzyme. The biosynthesis
studies showed that PR-3 is initially detectable as a 35-kDa form and
over 3 h is converted to a 29-kDa protein, a size identical to the
mature enzyme. The mass of the larger precursor was comparable with the
33-kDa size deduced from the cDNA, for PR-3 comprised 256 amino acids
plus the two carbohydrate chains at the Asn-linked glycosylation sites.
However, our data, which include 1) the failure to immunoprecipitate
the precursor with an antibody directed to the NH-terminal
prepro region and 2) the radiosequence analysis that identified Ile in
the third position in the 35-kDa precursor, demonstrate that removal of
the 25-amino acid signal peptide occurs early in the biosynthesis of
the enzyme probably in a co-translational fashion. The amino-terminal
cleavage occurs between Ala
and Ala
and fits the rules predicted for signal peptidases according to
von Heijne(36) . The signal peptide cleavage results in the
earliest identifiable precursor being a zymogen containing a dipeptide
(Ala
-Glu
) before the
NH
-terminal Ile of the active enzyme. This, however, leaves
a disparity between the observed molecular mass of the earliest
precursor (35 kDa) and the predicted molecular mass (30.6 kDa) of the
glycosylated protein minus the signal peptide. This difference may be
explained by the reduced mobility of the protein in SDS-PAGE due to its
cationic nature and by ``trimming'' of the Asn-linked
oligosaccharides(37) .
Following trimming of the
oligosaccharides, the maturation of pro-PR-3 to the biologically active
proteinase proceeds via excision of the dipeptide
(Ala-Glu
) at the acidic residue.
This dipeptide is homologous to other proteinases of hematopoietic
origin including HLE and Cat G(28, 29, 38) ,
human mast cell chymase(39) , mouse mast cell
chymase(40) , and mouse cytotoxic lymphocyte granzyme
B(41) . In the case of Cat G and suggested for the other
hematopoietic cell-derived serine proteinases, the cysteine
exopeptidase DPP-I has been reported to be the processing enzyme
involved in the excision of the dipeptide(29) . This prompted
us to investigate its role in the processing of PR-3 (and HLE). When
DPP-I was inhibited by GF-CHN
, the processing of pro-Cat G
was prevented, but pro-PR-3 and pro-HLE were processed at their acidic
residues. In addition, alkalinization of the secretory granules with
NH
Cl, which should prevent the dipeptide removal by DPP-I,
since the enzyme requires an acidic milieu for activity, had no effect
on the processing of pro-PR-3, pro-HLE, or pro-Cat G. In contrast to
the results of the present investigation, Lindmark et al.(42) reported that NH
Cl inhibited the
processing of both HLE and Cat G in U937 cells. However, the site of
inhibition in the processing pathway was not determined, and prevention
of the dipeptide removal was not demonstrated.
Chloroquine, which
has a similar effect on the pH of the granules as NHCl,
surprisingly showed differential effects on the processing of the PMNL
serine proteinases. The processing of pro-Cat G was inhibited, but that
of pro-PR-3 and pro-HLE was not. This raises the possibility that the
observed effect of both the DPP-I inhibitor and chloroquine on the
processing of pro-Cat G may be related to the blockage of the transport
of the enzyme through the subcellular organelles rather than inhibition
of DPP-I and that the transport of Cat G may be different from that of
PR-3 and HLE. While it is clear from our data that DPP-I is not
involved in the processing of PR-3 or HLE, inhibition of the dipeptide
excision by E-64d establishes that a cysteine proteinase, perhaps
related to DPP-I, is involved in this processing step. Ongoing work in
our laboratory is aimed at identifying the processing enzyme.
In the
present investigation, analysis of the COOH terminus established
arginyl residues as the final two amino acids of that mature PR-3
(Arg-Arg
). Thus PR-3 is shorter by seven
amino acid residues on the COOH-terminal side than would be predicted
from the cDNA. The COOH termini for HLE and Cat G are monoarginyl
residues (Arg
and Arg
, respectively, Fig. 1). This implicates a basic amino acid directed
endoproteinase in the processing of the COOH termini of these enzymes.
Recently several mammalian subtilisin-like convertases have been
identified that cleave at dibasic and monobasic sites of
proproteins(43, 44, 45, 46) . Among
these enzymes, furin would be a candidate enzyme for processing of PMNL
proteinases. However, furin is localized in the Golgi
compartment(47) . While studies by Salvesen and Enghild (28) on the processing of Cat G are consistent with removal of
its COOH-terminal extension peptide in the Golgi, our results suggest
that the COOH-terminal extension of PR-3 is likely removed
co-translationally, indicating that its processing enzyme is probably
localized to the endoplasmic reticulum. In addition, it has been shown
that furin requires a precursor protein comprising the consensus
cleavage site Arg-Xaa-(Lys/Arg)-Arg or Arg-Xaa-Xaa-Arg (47, 48, 49, 50) , indicating
importance for an Arg at P
(
)position. None of
the PMNL proteinases contain Arg at the P
position. Thus
basic amino acid directed endoproteinases other than furin likely are
involved in processing the PMNL proteinases. In preliminary studies, N
-p-tosyl-L-lysine
chloromethyl ketone and phenylmethanesulfonyl fluoride did not inhibit
the processing of the COOH terminus, suggesting that the responsible
enzyme, although trypsin-like, is likely not a serine proteinase.
The azurophilic granules of PMNL are considered analogous to lysosomes (51) of nonmyeloid cells. In the present investigation, we compared the targeting of PR-3 to granules with sorting of lysosomal enzymes. Following synthesis, lysosomal enzymes in nonmyeloid cells are transported from endoplasmic reticulum to Golgi, during which time high mannose side chains are modified and mannose 6-phosphate residues are added as a recognition marker for receptor targeting to the lysosome. In the acidic intralysosomal environment, the lysosomal enzyme dissociates from the receptor and the proenzyme is proteolytically processed. We found that PR-3 zymogen was not processed to the mature form when we treated cells with BFA, an agent that specifically blocks protein transport distal to the Golgi, suggesting that the PR-3 precursor is transported beyond the Golgi compartment before processing of the propetide. The cleavage of the propeptide from the PR-3 precursor in the presence of tunicamycin suggests that the transport of PR-3 to the proper cell compartment is not mannose 6-phosphate receptor-mediated. In addition, agents that increase the pH of acid compartments did not prevent the processing of PR-3 precursor into the mature form, further evidence that the mannose 6-phosphate receptor is not required for the transport to the storage granules and processing of PR-3. These findings are consistent with results from previous investigations of various azurophilic granule proteins such as myeloperoxidase (52) and defensins(53) , indicating that the sorting mechanisms are distinct from those of typical lysosomal enzymes (54) .
In summary, the processing of PR-3
in U937 cells requires three proteolytic cleavages, two on the
amino-terminal side and one on the carboxyl-terminal side of 256-amino
acid prepro-PR-3. The initial amino-terminal cleavage results in the
removal of a 25-amino acid endoplasmic reticulum-targeting signal
sequence. In additional cleavages, the dipeptide
(Ala-Glu
) propiece from the
amino-terminal side preceding Ile
and seven-amino acid-long
peptide COOH-terminal to Arg
are removed leaving the
222-amino acid PR-3 found in PMNL. Our results show that the processing
of PR-3 in U937 cells is distinct from that of Cat G.