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Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1415-1422
On the Role of the Proform-Conformation for Processing and
Intracellular Sorting of Human Cathepsin G
By
Daniel Garwicz,
Anders Lindmark,
Ann-Maj Persson, and
Urban Gullberg
From the Department of Hematology, Lund University, Lund, Sweden.
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ABSTRACT |
The serine protease cathepsin G is synthesized during the
promyelomonocytic stage of neutrophil and monocyte differentiation. After processing, including removal of an amino-terminal propeptide from the catalytically inactive proform, the active protease acquires a
mature conformation and is stored in azurophil granules. To investigate
the importance of the proform-conformation for targeting to granules, a
cDNA encoding a double-mutant form of human preprocathepsin G lacking
functional catalytic site and amino-terminal prodipeptide (CatG/Gly201/ Gly19Glu20) was
constructed, because we were not able to stably express a mutant
lacking only the propeptide. Transfection of the cDNA to the rat
basophilic leukemia RBL-1 and the murine myeloblast-like 32D cl3 cell
lines resulted in stable, protein-expressing clones. In contrast to
wild-type proenzyme,
CatG/Gly201/Gly19Glu20 adopted a
mature conformation cotranslationally, as judged by the early
acquisition of affinity to the serine protease inhibitor aprotinin,
appearing before the carboxyl-terminal processing and also in the
presence of the Golgi-disrupting agent brefeldin A. The presence of a
mature amino-terminus was confirmed by amino-terminal radiosequencing.
As with wild-type proenzyme,
CatG/Gly201/Gly19Glu20 was
proteolytically processed carboxyl-terminally and glycosylated with
asparagine-linked carbohydrates that were converted into complex forms.
Furthermore, it was targeted to granules, as determined by subcellular
fractionation. Our results show that the initial proform-conformation
is not critical for intracellular sorting of human cathepsin G. Moreover, we demonstrate that double-mutant cathepsin G can achieve a
mature conformation before carboxyl-terminal processing of the proform.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
CATHEPSIN G IS A chymotrypsin-like
protease with antimicrobial activity1 and belongs to a
superfamily of serine proteases stored in cytoplasmic granules of
hematopoietic cells.2 Together with leukocyte elastase,
proteinase 3, and the catalytically inactive protease-homologue
azurocidin, whose genes are clustered on chromosome 19p13.3,3 cathepsin G (encoded on chromosome
14q11.2)4 is synthesized during the promyelocyte stage of
neutrophil differentiation.5,6 These 4 proteins,
collectively called serprocidins, are stored in the azurophil
(peroxidase-positive; primary) granules.1,2,6 Cathepsin G
or a cathepsin G-like enzyme has also been detected in monocytes, a
subset of mast cells, and basophils.7-9 In addition to its
capability of killing bacteria and fungi (a function that seems to be
independent of its catalytic activity), cathepsin G has several
possible catalytic functions in normal and pathological events,
indicated by in vitro studies; eg, leukocyte migration by acting as a
chemotactic agent for neutrophils and monocytes10 and
participation in an alternative pathway of leukocyte initiation of
coagulation by activating coagulation factor X11 and factor
V.12
The hematopoietic serine proteases are synthesized as catalytically
inactive proenzymes (zymogens) that, after removal of an amino-terminal
propeptide, often a dipeptide, are stored in granules as active
enzymes.13-17 Procathepsin G is activated simultaneously with, or directly after, transfer to granules,13,18-20 but
a fraction of the zymogen is constitutively secreted. The activation is
thought to be catalyzed by dipeptidyl peptidase I,21 a
thiol protease that removes the amino-terminal dipeptide of several
members of the hematopoietic serine protease
family.16,17,22 In addition to the amino-terminal
propeptide, procathepsin G has a carboxyl-terminal extension of 11 to
12 amino acids, which is removed during processing of the
proform.13,18,19,23,24 The enzyme mediating this removal
has not been identified and the role of the carboxyl-terminal extension
is unclear.
Several mechanisms have been proposed to be responsible for sorting of
proteins destined for storage in granules.2,25 However, the
mechanisms for sorting of the serprocidins are unknown. It could be
hypothesized that sorting of cathepsin G is restricted to protein in
proform-conformation, in analogy to recent reports on
pro-opiomelanocortin26 and prorenin,27 in which
binding of a processing enzyme is important for sorting to granules.
The main aim of the present study was to investigate whether the
proform-conformation of cathepsin G is important for intracellular sorting of the zymogen. To this end, we transfected cDNA encoding mutated forms of human preprocathepsin G lacking the amino-terminal propeptide (expected to adopt a mature conformation cotranslationally) to the rat basophilic/mast cell line RBL-1 and the murine myeloblast like 32D cl3 cell line, cellular models recently used for
investigations on the processing of neutrophil serine
proteases.19,20,28,29 We demonstrate that the
proform-conformation of procathepsin G is not critical for sorting to
granules or for constitutive secretion, but rather may serve to protect
the cell from the catalytic activity of cathepsin G. In addition, we
show that conversion into a mature conformation can occur independently
of carboxyl-terminal processing.
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MATERIALS AND METHODS |
Materials.
The eukaryotic expression vectors pCEP4 and pcDNA3 were from Invitrogen
(Leek, The Netherlands). The vectors provide a cytomegalovirus promoter-driven expression of introduced cDNA. The plasmids also confer
resistance to hygromycin B and geneticin, respectively, allowing
selection of recombinant cells.
35S-methionine/35S-cysteine (cell labeling
grade) and 3H-isoleucine were from Amersham (Amersham,
UK). Before use, 3H-isoleucine was
concentrated 10-fold in a vacuum centrifuge. Percoll and Protein
A-Sepharose CL4-B were from Pharmacia (Uppsala, Sweden).
Aprotinin-agarose was from Sigma (St Louis, MO).
N-glycosidase F, endoglycosidase H, and geneticin were from Boehringer
Mannheim (Mannheim, Germany). Hygromycin B was from Calbiochem (La
Jolla, CA). A polyclonal rabbit antiserum to cathepsin G was obtained by immunization of rabbits.30 Brefeldin A (BFA) was a gift
from Sandoz (Basel, Switzerland). The polyvinylidene
difluoride (PVDF) membranes were from Millipore (Bedford, MA). The
 -galactosidase (-gal) staining kit and the eukaryotic expression
vector pCMV, carrying the reporter gene -gal, were from
Invitrogen.
cDNA, mutagenesis, and construction of expression vectors.
Full-length cDNA for human preprocathepsin G (a gift from Dr Guy
Salvesen, Duke University, Durham, NC) was cloned into pCEP4, thus
creating the expression vector pCEP4/CatG.19 For
site-directed mutagenesis, preprocathepsin G cDNA was used as template
in a two-step spliced overlap extension polymerase chain reaction (SOE PCR)31 in the following way. In the first reaction, 2 separate amplifications with 100 ng of DNA template in 10-cycle PCRs
produced 2 fragments of preprocathepsin G cDNA overlapping each other
on the sequence around the amino-terminal dipeptide, Gly19
and Glu20 (numeration from the initial ATG translational
initiation site). By design of the primers, Gly19 and
Glu20 were deleted. The 5 - and 3-primers for
the cDNA sequence used were identical to those earlier used for
producing pCEP4/CatG, thus introducing the Kozak consensus leader
sequence for maximum translational efficiency32 and the
flanking restriction enzyme sites Kpn I and Not I for subsequent cloning into pCEP4 or pcDNA3 plasmids. The PCR primers in
the 2 amplifications were upstream 5 GA CTT CAG GGT
ACC GCC GCC ACC ATG CAG CCA CTC CTG CTT CTG CTG G
3 (no. 1), plus downstream 5 CCG GCC TCC GAT GAT  TGC
CTC AGC CCC AGT GG 3 (no. 2), and upstream 5 CC ACT GGG
GCT GAG GCA ATC ATC GGA GGC CGG GAG AGC 3 (no. 3), plus
downstream 5 G ACT TCA GGC GGC CGC TCA CAG
GGG GGT CTC CAT CTG ATC CAG C 3 (no. 4), respectively (start and
stop codons in bold, restriction enzyme sites underlined, deleted
Gly19 and Glu20 indicated with triangle). The
PCR products were isolated on agarose gel, mixed, and subjected to a
20-cycle splicing PCR amplification with primers no. 1 and 4, thus
creating full-length preprocathepsin G cDNA with deleted
Gly19 and Glu20 (Gly19Glu20). The PCR product was digested
by Kpn I and Not I, followed by isolation on agarose
gel and cloning into plasmid. Individual clones were isolated and
sequenced to verify the mutation and the integrity of the reading
frame. cDNA with correct nucleotide sequence was cloned to create an
expression vector for mutated preprocathepsin G. All PCR reactions were
performed in a Perkin Elmer 480 Thermal Cycler using
Pfu-polymerase (Stratagene, La Jolla, CA)
according to the manufacturer's instructions. Analogously, a cDNA
coding for a mutant form of preprocathepsin G lacking enzymatic activity (CatG/Gly201) was made by substituting
Ser201 (numeration from the initial ATG translational
initiation site) with Gly, using the mutant-primers 5 GGG GCC
TCC GCC ATC CCC CTT 3 (no. 2) and 5 AAG GGG GAT
GGC GGA GGC CCC 3 (no. 3) (Gly201
underlined) with wild-type preprocathepsin G as template. Identical
mutagenesis, but with preprocathepsin
G/Gly19Glu20 as template, resulted in a
double mutant
(CatG/Gly201/Gly19Glu20).
Cell culture.
The rat basophilic/mast cell line RBL-1 and murine myeloblast-like 32D
cl3 (clone 3) cells were grown as described.28 Cell cultures were kept in 5% CO2 at 37°C in a fully
humidified atmosphere. Exponentially growing cells were used in all
experiments. COS-7, an African green monkey kidney cell line, was
maintained in Dulbecco's modified Eagle's medium (DMEM;
GIBCO Ltd, Paisley, UK) supplemented with 10% heat-inactivated fetal
calf serum.
Transfection procedure.
RBL-1 and 32D cl3 cells were transfected using the BioRad Gene Pulser
(Bio Rad, Hercules, CA) with electrical settings 960 µF and 300 V, as
previously described.19,28 After electroporation, geneticin
or hygromycin B was added to select for recombinant clones expressing
the geneticin-resistance gene of pcDNA3 or the hygromycin B-resistance
gene of pCEP4. Individual clones growing in the presence of antibiotic
were isolated, expanded into mass cultures, and screened for expression
of cathepsin G by biosynthetic labeling. Clones with the most
pronounced expression were chosen for further experiments. COS-7 cells
were transiently transfected in 10-cm plastic dishes with 4 µg of
plasmid DNA per 106 cells, using the diethyl aminoethyl
(DEAE)-dextran method.33 Twenty-four to 48 hours after transfection, the expression of cathepsin G was detected by
biosynthetic labeling of cells. In experiments with cotransfection of
pCMV, COS-7 cells were transiently cotransfected in
2.3-cm wells with 0.2 to 0.3 µg of pCMV and equal or
double amounts of plasmid DNA, encoding wild-type or mutated forms of
preprocathepsin G in the pcDNA3 vector, per 50 × 103
cells.
Biosynthetic labeling.
Biosynthetic radiolabeling of newly synthesized proteins was performed
essentially as described elsewhere.19 Unless otherwise indicated, cells were starved for 30 minutes in
methionine/cysteine-free medium followed by pulse-labeling with
35S-methionine/35S-cysteine for 30 minutes. In
chase experiments after pulse-labeling, cells were resuspended in
complete medium. At timed intervals, cells were withdrawn and subjected
to extraction of whole cells or homogenization and subsequent
subcellular fractionation.
Radio sequence analysis.
For determination of the amino-terminal processing of procathepsin G,
biosynthetic labeling with 3H-isoleucine, followed by amino
acid sequencing, was performed essentially as described.20
Briefly, cells were incubated in medium supplemented with
3H-isoleucine (200 µCi/mL) to achieve metabolic labeling
of synthesized proteins. After pulse-labeling for 30 minutes, cell
lysates were prepared and immunoprecipitation was performed. After
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), the proteins were transferred to a PVDF
membrane, which was subjected to autoradiography. Radioactive bands
were excised and subjected to amino acid degradation according to Edman
(performed by the Biomedical Service Unit, Lund University, Lund,
Sweden). The initial 9 to 10 degradation cycles were assayed for
radioactivity by use of a scintillation counter.
Subcellular fractionation.
Subcellular fractionation was performed essentially as previously
described.19 Briefly, the cell homogenate was fractionated in a Percoll density gradient after which 9 fractions were collected with all cytosol in fraction no. 9. The distribution of lysosomes and
Golgi elements in the density gradient was determined by assaying -hexosaminidase and galactosyl transferase. Peak activities of -hexosaminidase and galactosyl transferase in subcellular fractions from RBL wild-type cells were localized in fractions no. 1 and 2 and
no. 5 through 8, respectively, and from 32D wild-type cells in
fractions no. 2 and 6.19,28
Immunoprecipitation.
For immunoprecipitation, whole cells or Percoll-containing subcellular
fractions were solubilized essentially as previously described.19 Biosynthetically labeled cathepsin G was
immunoprecipitated by addition of a polyclonal rabbit antiserum to
cathepsin G and protein A-sepharose and was subjected to SDS-PAGE on a
5% to 20% (or a 10% to 20% precast Tris-Glycine gel; Novex, San
Diego) gradient gel followed by fluorography essentially as
described.19
Adsorption to aprotinin-agarose.
Adsorption to aprotinin-agarose was performed essentially as described
by Salvesen and Enghild13 and modified.19
Briefly, cells were lysed and the lysate was allowed to react with a
suspension of aprotinin-agarose. The aprotinin with bound material was
washed, followed by incubation with elution buffer to obtain release of bound material. Cathepsin G remaining in the lysate after adsorption to
aprotinin-agarose (lacking affinity to aprotinin) or eluted (with
affinity to aprotinin) was immunoprecipitated and subjected to SDS-PAGE
and fluorography.
Digestion with endoglycosidase H and N-glycosidase F.
The susceptibility of procathepsin G to digestion with endoglycosidase
H (Endo H) was determined essentially as described.23 Digestion with N-glycosidase F was performed according to the manufacturer's instructions. Briefly, immunoprecipitates, collected as
described above, were dissolved in buffer and boiled for 5 minutes.
After centrifugation, the supernatant was collected and Endo H or
N-glycosidase F was added. Control incubations were treated
identically, except that no enzyme was added. After incubation for 16 to 24 hours, the samples were subjected to SDS-PAGE and fluorography.
-gal staining and statistical analysis.
The expression of -gal was detected by -gal staining performed
with the -gal staining kit, according to the manufacturer's instructions. The numbers of positive cells in each well were counted
in a light microscope. The statistical analysis was performed with a
two-sided Wilcoxon signed rank test.
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RESULTS |
Establishment of stable cell clones expressing mutant forms of
cathepsin G.
By use of site-directed mutagenesis as described in the Materials and
Methods, a mutant form of preprocathepsin G lacking the amino-terminal
propeptide (CatG/Gly19Glu20) was cloned into pCEP4 (pCEP4/CatG/Gly19Glu20) and into the
pcDNA3 vector (pcDNA3/CatG/Gly19Glu20). However, despite 5 separate transfections of RBL-1 cells using these
constructs, it was not possible to establish stable cell clones
expressing detectable amounts of cathepsin G. Likewise, transfections
of RBL cells using a muristerone-inducible expression system based on
the expression plasmids pVgRXR and pInd (Invitrogen) did not result in
any detectable synthesis when
CatG/Gly19Glu20 was cloned into the
pInd-vector (15 clones were screened), whereas 3 clones expressing
cathepsin G were found when wild-type preprocathepsin G was cloned into
vector (data not shown). Furthermore, it was not possible to detect any
synthesis of cathepsin G in simian COS-7 cells transiently transfected
with CatG/Gly19Glu20, although the protein
was strongly expressed after transfection of wild-type preprocathepsin
G to these cells (data not shown).
In contrast, transfection of mutant preprocathepsin G lacking
functional catalytic site (CatG/Gly201) resulted in stable
clones of both RBL and 32D cells expressing the protein. Because the deletion of the amino-terminal propeptide might result in a
cotranslational activation of the protease, it was hypothesized that
the difficulties in obtaining stable expression of
CatG/Gly19Glu20 resulted from a premature
emergence of catalytic activity. To test this hypothesis further, we
transiently cotransfected wild-type preprocathepsin G or the 2 mutant
forms of preprocathepsin G cloned into the pcDNA3 expression vector
together with the expression vector pCMV to COS-7 cells. The total
numbers of -gal-positive cells in each well were counted 1 to 5 days after transfection and mean values were calculated for each day.
Three days after transfection, there was a significant difference
(P < .05) between the number of cells expressing -gal
after cotransfection with CatG/Gly19Glu20
compared with the cells cotransfected with wild-type or double-mutant
preprocathepsin G (Fig 1). On day 5 after
transfection, the number of -gal-positive cells decreased in all
wells, probably due to an unspecific decrease in the expression of
pCMV (not shown in Fig 1). To circumvent the possible obstacle in
the form of a premature catalytic activation, a double-mutant form of
preprocathepsin G
(CatG/Gly201/Gly19Glu20), lacking both Ser201, essential for catalytic activity, and
the amino-terminal propeptide, was transfected to RBL or 32D cells. These transfections resulted in stable clones expressing the
double-mutant form of procathepsin G.
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| Fig 1.
-gal positivity after cotransfection of pCMV and
wild-type or mutated preprocathepsin G forms to COS-7 cells. COS-7
cells (50 × 103) were transiently cotransfected with 0.2 to 0.3 µg of pCMV and ( ) pcDNA3/CatG, ( )
pcDNA3/CatG/Gly201/Gly19Glu20,
or ( ) pcDNA3/CatG/Gly19Glu20 in equal or
double amounts in six 12-well plates using the DEAE-dextrane method.
Cells were stained using the -gal kit and the absolute numbers of
-gal-positive cells were determined in a light microscope. The
diagram shows the mean values of -gal-positive cells 1 to 4 days
after transfection. Values are from 4 separate experiments (with 8 wells counted on day 3).
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The processing of the mutant forms of procathepsin G is similar to
that of wild-type protein.
The processing of the mutant forms of procathepsin G was investigated
by biosynthetic labeling and pulse-chase experiments as described
in the Materials and Methods. Figure 2A
demonstrates the biosynthesis of wild-type cathepsin G in RBL/CatG
cells. A specifically immunoprecipitated protein with an apparent
molecular mass of 32.5 kD, representing the proform of cathepsin G, was detected after 30 minutes of radio-labeling. During chase of the label,
processing of the 32.5-kD proform to a 31-kD form was observed within
30 minutes, and after 2 hours of chase the conversion to the 31-kD form
seemed complete. This reduction of molecular mass most probably
represents the removal of the carboxyl-terminal peptide extension. In
addition, a minor fraction was further processed to a 30-kD form (not
indicated in Fig 2). Upon analysis of the incubation medium, 32.5-kD
procathepsin G was observed. These results are consistent with those
earlier published.13,19,20,28
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| Fig 2.
Processing of wild-type and double-mutant procathepsin G
in RBL cells. (A) RBL/CatG and (B)
RBL/CatG/Gly201/Gly19Glu20 cells were pulse-labeled with
35S-methionine/35S-cysteine for 30 minutes
followed by chase of the label for up to 4 hours. At indicated points,
20 × 106 cells were withdrawn and subjected to
solubilization and immunoprecipitation with polyclonal anti-cathepsin G
antiserum. In addition, cathepsin G was immunoprecipitated from the
incubation medium after each period of chase. The immunoprecipitates
were run in SDS-PAGE in a 10% to 20% and a 5% to 20% gradient gel,
respectively, whereupon fluorography was performed. The fluorograms
were exposed for 6 days and 2 weeks, respectively. The different
processing forms of cathepsin G are indicated with arrows to the right.
Numbers to the left in this and subsequent figures are the molecular
weight values of molecular weight standards.
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Upon analysis of the processing of mutant procathepsin G lacking
functional catalytic site (CatG/Gly201) (data not shown) and of the double mutant
(CatG/Gly201/Gly19Glu20) (Fig
2B) transfected to RBL cells, similar results were obtained. Thus, both
mutant forms of procathepsin G showed a processing pattern matching
that of wild-type enzyme after transfection to RBL cells. Also, the amount of proform released into the medium, as compared with retained protein, was similar for the different forms of procathepsin G.
Results from transfection of 32D cells further substantiated that lack
of both the amino-terminal propeptide and a functional catalytic site
does not interfere with processing of the protein. Thus, both
wild-type28 and the double-mutant form of procathepsin G
was processed from a 32.5-kD proform into a 31-kD form and to a minor
extent to a 30-kD form (data not shown). However, processing of both
wild-type and mutated procathepsin G was not completed after 4 hours of
chase, indicated by the presence of both the 32.5-kD proform and the
31-kD form, consistent with earlier results suggesting a retarded
processing in 32D cells as compared with RBL cells.28,29
Release of the proform into the medium was observed also in 32D cells.
The double-mutant procathepsin G acquires a mature amino-terminal
cotranslationally.
By deletion of the two-residue amino-terminal propeptide, which
normally is removed in pregranule or granule structures 60 to 90 minutes after translation,13,19 we assumed that the signal peptidase site34 would be preserved, leading to the
cotranslational appearance of a mature amino-terminus lacking the
propeptide. CatG/Gly201/Gly19Glu20 in RBL
cells was subjected to radiosequencing after biosynthetic labeling with
3H-isoleucine. Figure 3 shows
that, after 30 minutes of radio-labeling of
RBL/CatG/Gly201/Gly19Glu20
cells, peak radioactivity appeared in the initial fractions of the
amino acid-sequencing reaction, indicating the presence of a mature
amino-terminus with 2 isoleucines. However, the expected amino-terminus
starting with 2 isoleucines should result in the appearance of peak
radioactivity only in the first 2 fractions. The fact that 3, and not
only 2, of the initial fractions contained substantial amounts of
radioactivity could indicate that more than one signal peptidase
cleavage site is present, resulting in a mixture of newly synthesized
mutant procathepsin G, with a portion of the protein having a remaining amino acid from the signal peptide before the 2 isoleucines of the
normal, mature amino-terminus of cathepsin G. Alternatively, the
appearance of radioactivity in fractions beyond the initial 2 fractions
may be due to inefficient peptide degradation during the
radiosequencing reaction. Similar results were obtained with 32D/CatG/Gly201/Gly19Glu20
cells, demonstrating the presence of amino-terminal isoleucines after
radio-labeling (data not shown), indicating the cotranslational
appearance of a mature amino-terminal peptide sequence.
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| Fig 3.
Amino-terminal radiosequencing of double-mutant
procathepsin G in RBL cells.
RBL/CatG/Gly201/Gly19Glu20 cells
were pulse-labeled with 3H-isoleucine for 30 minutes as
described in the Materials and Methods. After pulse-labeling, 100 × 106 cells were subjected to solubilization,
immunoprecipitation, SDS-PAGE, and transfer to a PVDF membrane by
Western blotting. Radioactive bands containing pulse-labeled 32.5-kD
CatG/Gly201/Gly19Glu20 were
excised and subjected to amino acid degradation. The amount of
radioactivity in the initial 9 cycles of each sequence analysis is
shown (dpm, disintegrations per minute).
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The double-mutant procathepsin G acquires a mature conformation
cotranslationally.
Removal of the amino-terminal propeptide of procathepsin G is a
prerequisite for the conformational change leading to catalytic activation of the zymogen.13 The activation is reflected by the simultaneous acquisition of affinity to the serine protease inhibitor aprotinin.13,19 However, it should be noted that affinity for aprotinin is not necessarily equivalent to proteolytic capacity, as demonstrated by the strong affinity to aprotinin of
azurocidin, a catalytically inactive serine protease
homologue.35 Rather, the affinity to aprotinin parallels
the conformational change due to removal of the
propeptide,13 including formation of an internal salt
bridge between the amino-terminal isoleucine and an aspartic acid close
to the active catalytic site.36 To investigate the
conformational status of the double-mutant procathepsin G, adsorption
to aprotinin was performed. Wild-type cathepsin G, pulse-labeled for 40 minutes, did not show any affinity to aprotinin, indicating persistent
proform-conformation (Fig 4A). However,
after chase of the label for 1 hour, a substantial amount of the
protein was bound to aprotinin, indicating conversion into mature
conformation. The acquisition of affinity to aprotinin occurred
concurrently with a reduction in the apparent molecular mass,
representing the carboxyl-terminal processing. The expected cotranslational appearance of a mature conformation of
CatG/Gly201/Gly19Glu20 was
confirmed, as shown in Fig 4B. Already after 40 minutes of pulse-labeling, a considerable amount of the double-mutant procathepsin G was bound to aprotinin and the fraction of protein, with affinity for
the protease inhibitor, increased further during chase of the label.
This increase occurred concomitantly with the carboxyl-terminal processing. Normally, the removal of the amino-terminal dipeptide and the carboxyl-terminal peptide extension of procathepsin G occurs
in post-Golgi structures, as demonstrated by the inhibition of
processing by brefeldin A.18 To further substantiate that CatG/Gly201/Gly19Glu20 achieved
a mature conformation without further proteolytic processing, we
investigated the acquisition of affinity to aprotinin after
biosynthetic radio-labeling in the presence of brefeldin A. Brefeldin A
induces the disassembly of the Golgi complex, thus blocking the
transport of proteins from the endoplasmic reticulum (ER) to the Golgi
apparatus.37 Figure 5A demonstrates that
brefeldin A completely abrogates the processing of wild-type
procathepsin G, as judged by the lack of affinity to aprotinin and the
absence of carboxyl-terminal processing, thus confirming previous
data.18
CatG/Gly201/Gly19Glu20, on the
other hand, showed affinity to aprotinin despite the presence of
brefeldin A with inhibition of proteolytic processing (Fig 5B). Thus,
it can be concluded that
CatG/Gly201/Gly19Glu20 obtains a
mature conformation also in the absence of transfer to post-Golgi structures in which proteolytic processing normally occurs.
Furthermore, removal of the carboxyl-terminal peptide extension is
probably not necessary for enzymatic activation, because the
double-mutant procathepsin G did bind to aprotinin in the absence of
proteolytic processing.
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| Fig 4.
Adsorption of wild-type and double-mutant cathepsin G to
aprotinin-agarose. (A) RBL/CatG and (B)
RBL/CatG/Gly201/Gly19Glu20 cells
were labeled with 35S-methionine/35S-cysteine
for 40 minutes and chased for 1 and 3 hours. At timed intervals,
aliquots of labeled cells (20 × 106) were withdrawn for
analyses. Cell lysis and adsorption to aprotinin-agarose were performed
as described in the Materials and Methods. No affinity to aprotinin
represents labeled protein that did not bind to aprotinin (non-active
conformation), whereas material with affinity to aprotinin was eluted
after binding (active conformation). After immunoprecipitation and
SDS-PAGE, fluorography was performed. The fluorograms were exposed for
7 days.
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| Fig 5.
Adsorption of wild-type and double-mutant cathepsin G to
aprotinin-agarose in the presence of brefeldin A. (A) RBL/CatG and (B)
RBL/CatG/Gly201/Gly19Glu20 cells
were preincubated with brefeldin A (5 µg/mL) for 60 minutes,
whereupon pulse-labeling and chase of the label were performed in the
continued presence of brefeldin A. Lysis and adsorption to aprotinin
was performed as described in the legend to Fig 4. The fluorograms were
exposed for 7 days.
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The double-mutant procathepsin G acquires complex carbohydrates and
is proteolytically processed.
To characterize the processing of the present mutant forms of
procathepsin G, digestion with N-glycosidase F was performed. As
earlier demonstrated,19 pulse-labeled procathepsin G showed a molecular mass of 29 kD after digestion with N-glycosidase F, indicating the presence of asparagine-linked carbohydrates of approximately 3.5 kD (data not shown). After 3 hours of chase, the
molecular mass of the processed protein was, upon digestion with
N-glycosidase F, reduced from 31 to 27.5 kD, indicating that proteolytic processing of approximately 1.5 kD took place. When the
double mutant was examined, similar results were found
(Fig 6), demonstrating that
CatG/Gly201/Gly19Glu20 is
proteolytically processed to the same extent as the wild-type
proenzyme. These results indicate that the proteolytic processing of
procathepsin G is not dependent on autocatalysis, because both mutant
forms, lacking enzymatic activity, were proteolytically processed
similar to wild-type proenzyme.
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| Fig 6.
Digestion of double-mutant cathepsin G with N-glycosidase
F. RBL/CatG/Gly201/Gly19Glu20
cells were pulse-labeled with
35S-methionine/35S-cysteine for 30 minutes,
whereupon the label was chased for 3 hours. At each time point, 50 × 106 cells were lysed and cathepsin G was
immunoprecipitated. Half of the material was subjected to digestion
with N-glycosidase F (indicated with "+"). Material without
added N-glycosidase F, but otherwise treated identically, is shown as
controls. The fluorogram was exposed for 5 days.
|
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Early after synthesis, procathepsin G acquires resistance to digestion
with Endo H, demonstrating processing of its oligosaccharides into
complex forms.19,23 Because this processing is known to take place in the Golgi, acquisition of resistance to Endo H
demonstrates the translocation of the protein from the ER to the Golgi.
When radio-labeled protein from
RBL/CatG/Gly201/Gly19Glu20 cells
was subjected to digestion with Endo H, it was demonstrated that almost all protein was Endo H-resistant after 15 minutes of chase (data not
shown), thus indicating that the early processing of the double-mutant form was identical to that of wild-type procathepsin G.
The mutant forms of procathepsin G are translocated to granules in
RBL and 32D cells.
Transfected procathepsin G is translocated to granules, as indicated by
the transfer with time to dense subcellular fractions containing
granules.19,20,28 The intracellular distribution of
CatG/Gly201/Gly19Glu20 in
transfected RBL cells or 32D cells was investigated by use of
subcellular fractionation.
CatG/Gly201/Gly19Glu20 was
translocated to dense fractions in RBL cells
(Fig 7) and in 32D cells (data not shown).
Similar results were obtained for CatG/Gly201 (data not
shown). No obvious differences between the subcellular transfer of
wild-type procathepsin G and that of the mutant forms of procathepsin G
were evident. In contrast to what is seen in whole cell lysates (Fig
2B), a minor amount of the 32.5-kD form is still visible after 4 hours
of chase. The reason for this is not clear, but it could be the higher
resolution in subcellular fractionation, compared with whole cells. The
results demonstrate that procathepsin G lacking the amino-terminal
dipeptide and thus cotranslationally adopting a mature conformation is
efficiently sorted to granules.
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| Fig 7.
Targeting of double-mutant procathepsin G to granules in
RBL cells.
RBL/CatG/Gly201/Gly19Glu20 cells
were pulse-labeled for 30 minutes followed by chase for 90 minutes and
4 hours. At the times indicated, 100 × 106 cells were
homogenized, after which subcellular fractionation was performed, with
subsequent collection of eight 0.8 mL subcellular fractions with
decreasing density and with fraction no. 9 containing all cytosol.
Fractions were solubilized and subjected to immunoprecipitation with
polyclonal anti-cathepsin G antiserum. Analyses of immunoprecipitates were as described in the legend to Fig 2. The different processing forms of cathepsin G are indicated with arrows to the right. The fluorograms were exposed for 3 weeks.
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| |
DISCUSSION |
In the present work, we have transfected mutant forms of human
preprocathepsin G to 2 rodent hematopoietic cell lines (RBL-1 and 32D
cl3) to investigate the importance of the proform-conformation of
cathepsin G for targeting to granules. Our results suggest that
premature absence of the amino-terminal propeptide impairs cell
survival. This conclusion is based on the fact that we were not able to
establish stable cell clones expressing a mutant form of procathepsin G
lacking only the amino-terminal propeptide
(CatG/Gly19Glu20). Moreover, transient
transfection of this mutant to the simian kidney cell line COS-7 failed
to result in detectable amounts of protein, in contrast to a comparable
transfection of wild-type preprocathepsin G to COS-7 cells. Further,
cotransfection of the mutant form of preprocathepsin G with a plasmid
expressing -gal resulted in a statistically significant lower number
of -gal-positive cells compared with controls 3 days after
transfection. These results are partially in contrast to those from
transient transfections of COS-7 cells with mutant granzyme B lacking
the amino-terminal propeptide, showing synthesis of propeptide-deleted
granzyme B.14,16 However, in these experiments, the levels
of granzyme B were in fact lower in cells transfected with the
propeptide-deleted form of this protease than in cells transfected with
wild-type enzyme.16 It should be emphasized that our
present results should be interpreted cautiously, because they only
provide indirect lines of evidence for the adverse effect of
prematurely activated procathepsin G. However, recent reports have
shown that leukocyte elastase38 as well as cathepsin
G39 can in fact induce apoptosis, in the latter case by
activating pro-caspase-7.
In any case, our results led us to perform extended site-directed
mutagenesis, also inactivating the functional catalytic site, and
thereby stable clones expressing procathepsin G lacking the
amino-terminal propeptide were easily obtained. This enabled us to
investigate the role of the conformations of the protein for sorting to
granules. Furthermore, the importance of the carboxyl-terminal processing for acquisition of a mature conformation could be studied.
Both azurophil granule and lysosomal proteins are often synthesized as
larger precursors, which are subjected to late proteolytic processing
in granule structures. Because the three-dimensional structures of the
proforms probably differ from those of the mature proteins, recognition
for sorting to granules could be limited to unprocessed forms that,
upon arrival in granules, are converted to mature proteins. The
proform-conformation would then be a prerequisite for sorting to
granules. Some reports are compatible with this notion; in experiments
with a mutant form of human prochymase lacking the amino-terminal
dipeptide, it was suggested that the absence of the dipeptide rendered
the nascent protein susceptible to intracellular proteolysis, possibly
due to aberrant cellular trafficking, implying an important role of the
dipeptide for subcellular sorting.15 Recent reports on
neuroendocrine cells also indicate an important role of the
proform-conformation for sorting; some neuroendocrine prohormones (eg,
proinsulin and proenkephalin) bind specifically to a membrane-bound
form of the processing enzyme carboxypeptidase E, which functions as a
sorting receptor to regulated secretion.26 Also,
recognition of the protease cleavage site in prorenin is sufficient to
direct proteins to regulated secretion.27 Therefore, it
could be that processing enzymes of neutrophil serine proteases, by
binding to the proform of the enzyme, are important also for sorting of
this family of proteins. However, the present data argue against this
concept. It was shown that double-mutant procathepsin G, lacking the
amino-terminal propeptide and functional catalytic site, was
cotranslationally converted to a mature conformation, as judged by the
mature amino-terminal sequence and early acquisition of affinity to the
protease inhibitor aprotinin. Furthermore, this activation occurred
also in the presence of brefeldin A, indicating that it took place in
pre-Golgi or Golgi structures. Thus, the double-mutant procathepsin G
did not adopt the proform-conformation, but was instead
cotranslationally, or shortly after, adopting the mature conformation
and should therefore not be a substrate for conformation-dependent
processing enzymes. Nevertheless, the amount of protein released into
the medium versus that transferred to granules was not increased, which
let us conclude that sorting to granules is not restricted to the
proform of cathepsin G. Neither was the amount of double-mutant
procathepsin G retained intracellularly increased, arguing against the
alternative hypothesis that conversion of proform into a mature
conformation causes retention, by that constituting a mechanism for
cellular retention and granule formation. Thus, the
proform-conformation of procathepsin G seems not to have a role in
sorting, but rather acts to keep the zymogen catalytically inactive
during intracellular transfer of the protein.
Despite the cotranslational emergence of a mature amino-terminal
sequence, the fraction of mutant protein with affinity to aprotinin,
indicating conformational maturation, increased with time. This is
unlikely to be due to further proteolytic processing, because this
fraction also increased in the presence of brefeldin A, which inhibits
transfer to compartments where proteolytic processing normally occurs.
Processing of carbohydrates is also a far-fetched explanation, because
the asparagine-linked oligosaccharides on cathepsin G are dispensable
for conformational maturation.28 Furthermore, it was
recently demonstrated by x-ray crystallography that the single
carbohydrate chain on cathepsin G is distant from the active site and
in fact points away from it. Possibly, the increased affinity to
aprotinin could reflect the stabilization of the mature conformation by
formation of internal salt bridges found in the mature
protein.36
The crystal structure of cathepsin G confirms that mature cathepsin G
is carboxyl-terminally truncated, ending with Ser or Phe, which could
be a potential autocatalytic site.36 The carboxyl-terminal processing of cathepsin G might follow activation and result from intramolecular or intermolecular autocatalysis in analogy to the proteolytic processing of the lysosomal cysteine protease cathepsin B40,41 and mast cell tryptase,42 which involves
the action of the hydrolase itself. However, the finding of similar
processing of mutant procathepsin G deficient of a functional catalytic
site and wild-type proenzyme in both RBL and 32D cells makes it
unlikely that the carboxyl-terminal processing is mediated by cathepsin G itself. But, it cannot be entirely excluded that endogenous cathepsin
G-like enzymes in RBL or 32D cells are responsible for an
intermolecular carboxyl-terminal processing. A functional role for the
carboxyl-terminal extension has not been proven. The presence of the
carboxyl-terminal peptide extension of procathepsin G does not seem to
be necessary for initial folding, activation, or sorting.20 Because the double-mutant procathepsin G adopted a mature conformation (as judged by the affinity to aprotinin) in the absence of
carboxyl-terminal processing, the present results demonstrate that
enzymatic activation can occur without this processing.
| |
FOOTNOTES |
Submitted February 12, 1998;
accepted April 14, 1998.
Supported by the Swedish Medical Research Council (Project No. 11546),
the Greta and Johan Kock Foundation, the Alfred Österlund Foundation, the Crafoord Foundation, the Anna-Greta Crafoord
Foundation, Funds of Reumatikerförbundet, the John Persson
Foundation, the Swedish Society for Medical Research, and Funds of
Lunds sjukvårdsdistrikt.
Presented in part at the European Congress for Molecular Cell Biology
(ECBO), Brighton, UK, March 22-25, 1997 (abstract no. H-5041) and at
the European Molecular Biology Organization (EMBO)-Workshop "Protein
sorting and processing in the secretory pathway," Annaberg/St Martin, Austria, January 13-18, 1998.
Address reprint requests to Daniel Garwicz, MD, Research
Dept. 2, E-blocket, University Hospital, SE-221 85 Lund, Sweden; e-mail: Daniel.Garwicz{at}hematologi.lu.se.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors are most grateful for the expert technical assistance by
Eva Nilsson and Karin Svensson.
| |
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Abstract
Introduction
Abstract