From the
The hematopoietic neutral serine proteases leukocyte elastase
and cathepsin G are synthesized as inactive precursors, but become
activated by removal of an amino-terminal dipeptide and are stored in
granules. Moreover, the pro forms of elastase and cathepsin G show
carboxyl-terminal prodomains of 20 and 11 amino acids, respectively,
which are not present in the mature enzymes. To investigate mechanisms
for processing, activation, and granular targeting, we have utilized
transgenic expression of myeloid serine proteases in the rat
basophilic/mast cell line RBL-1 (Gullberg, U., Lindmark, A., Nilsson,
E., Persson, A.-M., and Olsson, I.(1994) J. Biol. Chem. 269,
25219-25225). Leukocyte elastase was stably expressed in RBL-1
cells, and the translation products were characterized by biosynthetic
labeling followed by immunoprecipitation, SDS-polyacrylamide gel
electrophoresis, and fluorography. Processing of a main pro form of 34
kDa into mature 31- and 29-kDa forms was demonstrated. Translocation of
mature forms to granule-containing fractions was shown by subcellular
fractionation experiments. The processed forms were enzymatically
active, judging by the occurrence of amino-terminal processing
demonstrated by radiosequence analysis, the acquisition of affinity for
the protease inhibitor aprotinin, and the appearance of elastase
activity in transfected RBL cells. To investigate the function of the
carboxyl-terminal prodomains, deletion mutants of leukocyte elastase
and cathepsin G lacking the carboxyl-terminal extension were
constructed and transfected into RBL cells. Our results show that as
full-length proteins, the deletion mutants were converted to active
enzymes and transferred to granules with kinetics similar to that of
wild-type enzymes. We conclude that human leukocyte elastase and
cathepsin G are converted into enzymatically active forms when
expressed in RBL cells and targeted for storage in granules; the
carboxyl-terminal prodomains are necessary neither for enzymatic
activation nor for targeting to granules in RBL cells.
A regulated process of proliferation and differentiation of
hematopoietic cells along distinct lineages of maturation results in
production of functionally competent blood cells. The neutrophil
granulocyte is the dominant cell of the myeloid lineage and is of major
importance in host defense. Characteristically, the neutrophil contains
particular cytoplasmic granules formed in a sequential manner during
its maturation and whose content is released during phagocytosis or
regulated secretion. Peroxidase-positive azurophil granules are
produced during the promyelocyte stage of differentiation, while the
subsequent myelocyte stage gives rise to specific peroxidase-negative
granules
(1, 2) .
The azurophil granule contains
typical lysosomal enzymes and can therefore be regarded as a
specialized form of lysosome. Besides lysosomal hydrolases, the
azurophil granules store bactericidal proteins that are unique for the
myeloid lineage
(3, 4, 5) . Among them are the
neutral serine proteases leukocyte elastase and cathepsin G, both
belonging to a superfamily of hematopoietic serine proteases including
granzymes of cytotoxic T-lymphocytes and certain mast cell proteases
(6, 7). These serine proteases are characteristically stored in
granules in a processed catalytically active form, but are transiently
present as inactive zymogens; activation is likely to follow the
post-translational removal of an amino-terminal dipeptide in a
pregranular
compartment
(6, 8, 9, 10, 11, 12) .
Lysosomal enzymes are often phosphorylated on mannose residues of
carbohydrate side chains in order to provide binding to mannose
6-phosphate receptors, which mediate the transfer to
lysosomes
(13) . However, accumulating evidence indicates that
alternative mechanisms, independent of mannose 6-phosphate receptors,
exist for traffic to lysosomes
(14, 15) . In particular,
the azurophil granule proteins seem to be independent of
mannose-6-phosphate for subcellular sorting, but the mechanisms by
which these proteins are retrieved from the secretory pathway and
targeted for storage in granules are unknown
(16) . A
heterogeneous pattern of oligosaccharide side chains among the
different types of azurophil granule proteins of neutrophils suggests
that carbohydrates are not of general importance for sorting.
Promyeloperoxidase shows ``high mannose'' oligosaccharide
side chains (17); procathepsin G and proelastase have complex
oligosaccharide side chains
(18) , while the antibacterial
defensins lack carbohydrates
(19) .
As for typical lysosomal
enzymes, the processing of azurophil serine proteases involves late
proteolytic cleavage in a pregranular or granular
compartment
(16) . As a consequence, prodomains are eliminated by
proteolysis during the processing of the precursor into the mature
enzyme. In addition to an amino-terminal dipeptide, the pro forms of
leukocyte elastase and cathepsin G contain carboxyl-terminal prodomains
not found in the mature enzymes
(8) . Judging by comparison of
the predicted sequence from cDNA
(20, 21) with the amino
acid sequence of the mature protein
(22) , the carboxyl-terminal
prodomain of leukocyte elastase comprises 20 amino acids. Cathepsin G
is also subject to carboxyl-terminal processing, and although not
exactly defined, cleavage may take place after Ser
The aim of this work
was to investigate the role of the carboxyl-terminal prodomains of
leukocyte elastase and cathepsin G for enzymatic activation and
granular targeting. We have recently utilized a transgenic expression
model for myeloid serine proteases in a rat basophilic/mast cell line
and showed that human cathepsin G is adequately processed,
enzymatically activated, and targeted to granules for storage in these
cells
(23) . In the present work, we demonstrate that transgenic
human leukocyte elastase is similarly processed and sorted to granules
in this cell line. To investigate a possible role of the
carboxyl-terminal prodomains in intracellular sorting or enzymatic
activation, cDNA deletion mutants that lacked the prodomains were
utilized. We found that the carboxyl-terminal prodomains of elastase
and cathepsin G are dispensable for granular targeting in RBL cells and
that deletion of these domains does not seem to interfere with folding
and enzymatic activation of the native protein.
The aim of this work was to elucidate processing and sorting
mechanisms for the granule serine protease family of myeloid
origin
(3, 6) . Certain mast cell proteases belong to
this family of enzymes, and therefore, the rat basophilic/mast cell
line RBL should also be equipped with processing and sorting mechanisms
for the hematopoietic serine proteases. Indeed, granzyme A and
cathepsin G cDNAs transfected into this cell line both produced
functional enzymes
(23, 32) . For this reason, RBL cells
were employed in both our previous
(23) and present studies. Our
data show that transgenic human leukocyte elastase is converted into
active enzyme and targeted to granules in RBL cells. Moreover, this
study demonstrates that both leukocyte elastase and cathepsin G lacking
the carboxyl-terminal prodomain are still targeted to dense
granule-containing fractions and converted into catalytically active
forms. Thus, the carboxyl-terminal extension of these enzymes is
obviously unnecessary for sorting and enzymatic activation in RBL
cells.
Generally speaking, lysosomal enzymes are synthesized as
larger precursors that undergo limited proteolysis during and after
intracellular sorting, involving cleavage of both amino-terminal and
carboxyl-terminal prodomains
(16) . In some cases, either amino-
or carboxyl-terminal prodomains have proved important for folding,
intracellular targeting, and enzymatic activation of the
protein
(33) . However, the biological role of prodomain
processing remains obscure for most lysosomal enzymes. Our results show
that processing and sorting of human leukocyte elastase transfected
into RBL cells follow a scheme that corresponds to previous findings
concerning the processing of endogenous neutrophil
elastase
(6, 8, 18, 34) and are similar
to results from studies on endogenous and transgenic cathepsin
G
(23) . The mature form of leukocyte elastase had an apparent
mass of 31 kDa and was found in dense granule fractions isolated by
Percoll density gradient separation. This form appeared in granule
fractions after 90 min of chase, suggesting that transport into
granules requires <2 h. The 31-kDa product was stable for at least 5
h, although some slow processing into a 29-kDa form did occur. Several
lines of evidence strongly indicate that the protein was converted into
enzymatically active forms; the acquisition of affinity for the serine
protease inhibitor aprotinin was demonstrated, and it was confirmed by
radiosequencing for both elastase and cathepsin G that amino-terminal
processing occurred with removal of the activation dipeptide. Moreover,
we also demonstrated the presence of elastase activity in extracts of
transfected RBL cells. A fraction of newly synthesized leukocyte
elastase failed to be retained in the cells and was secreted as
precursor forms, which is a common feature in lysosomal enzyme
processing
(16) .
In the promyelocyte, leukocyte elastase and
cathepsin G are stored in azurophil granules, which can be regarded as
specialized forms of lysosomes, and they seem to be targeted to
granules in a mannose 6-phosphate-independent manner
(16) . In
yeast, some enzymes contain prodomains that are necessary and
sufficient for mannose 6-phosphate-independent targeting into the
vacuole, the yeast analogue of the mammalian
lysosome
(35, 36) . Also in mammalian cells, mechanisms
for lysosomal delivery that are independent of mannose 6-phosphate
exist
(14, 15) . A transient mannose
6-phosphate-independent membrane association has been demonstrated for
procathepsins D, C, and L (37-40). Among these, the membrane
association of cathepsin D involves interaction with prosaposin that
binds to cathepsin D immediately after synthesis in the endoplasmic
reticulum
(41) . The recent demonstration that the prodomain of
cathepsin L, which shows a close resemblance to vacuolar sorting
sequences of enzymes in yeast, binds to an intracellular transmembrane
proenzyme receptor
(42, 43) makes a functional role of
prodomains in lysosomal mannose 6-phosphate-independent targeting
likely. And thus, the carboxyl-terminal prodomains of leukocyte
elastase and cathepsin G may be necessary for granular
sorting
(8) . Consequently, carboxyl-terminal deletion mutants
were constructed to investigate this hypothesis. Our results show that
deletion of the prodomain does not interfere with sorting, as the
deletion mutants were transferred to granules with the same kinetics as
full-length proteins. Targeting mechanisms in RBL cells may, however,
be different from those operating in the promyelocytes, as the granules
of RBL cells are not identical to azurophil granules of promyelocytes.
The timing for cleavage of the carboxyl-terminal prodomains of
elastase and cathepsin G was reported to parallel the removal of an
amino-terminal activation dipeptide
(8) . However, other data
suggesting that the carboxyl-terminal processing may precede cleavage
of the amino-terminal dipeptide
(23) are further supported by
our results from radiosequencing indicating that following 2 h of
chase, when the carboxyl-terminal processing was accomplished judging
by reduction in apparent mass, the removal of the amino-terminal
dipeptide was still incomplete. These data suggest that amino-terminal
processing and activation of the protein need to take place on protein
lacking the carboxyl-terminal prodomain. The present demonstration that
the carboxyl-terminal deletion mutants of elastase and cathepsin G were
transformed into active enzymes, as judged by the acquisition of
affinity for aprotinin and the occurrence of elastase activity in
transfected cells, strongly argues against an important function of the
carboxyl-terminal prodomains in enzyme folding and prevention of
premature activation of the zymogen forms. However, the question of
whether or not previous removal of the carboxyl-terminal prodomain is a
prerequisite for the cleavage of the amino-terminal dipeptide to occur
is still not resolved. Among the myeloid serine proteases, azurocidin
has been reported to have a carboxyl-terminal prodomain, comprising
three amino acids, that is removed during processing
(44) . For
other members of the hematopoietic serine protease superfamily, the
information is incomplete, and it is not known whether all members are
carboxyl-terminally processed. Whatever the case, our present data
indicate that the carboxyl-terminal prodomain seems to be dispensable
for folding, activation, and granular targeting of the enzymes. Besides
neutral serine proteases, the azurophil granules contain several
lysosomal hydrolases and thus expose the content to a highly
proteolytic environment. Similarly, the granules of RBL cells, used in
this experimental model, store both serine proteases and lysosomal
enzymes
(45, 46) . Thus, the carboxyl-terminal processing
of leukocyte elastase and cathepsin G might merely be the result of
exposure of protease-sensitive regions, but the enzyme(s) responsible
for this action are not identified.
In conclusion, this
investigation has demonstrated that human leukocyte elastase, following
transfection into rat RBL cells, is targeted for storage in granules
and converted into enzymatically active forms. Furthermore, the
carboxyl-terminal prodomains of both leukocyte elastase and cathepsin G
were found to be dispensable for enzymatic activation as well as
granular targeting of the enzymes in RBL cells.
We thank Dr. Björn Hultberg for help with
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
to
remove 11 amino acids
(6, 8) .
Materials
The eukaryotic expression vectors
pRC/CMV and pCEP4 were from Invitrogen (Leek, The Netherlands). Both
vectors provide cytomegalovirus promoter-driven expression of
introduced cDNA and confer resistance to Geneticin and hygromycin B,
respectively, which allows selection of recombinant cells.
[S]Methionine/[
S]cysteine
(cell labeling grade) and [
H]isoleucine were from
Amersham International (Buckinghamshire, UK). Prior to use,
[
H]isoleucine was concentrated 10-fold in a
vacuum centrifuge. Percoll and protein A-Sepharose CL-4B were from
Pharmacia (Uppsala). Hygromycin B, protein G-agarose,
aprotinin-agarose, and
N-succinyl-Ala-Ala-Ala-p-nitroanilide were from
Sigma. Geneticin was from Boehringer Mannheim (Mannheim, Federal
Republic of Germany). Rabbit polyclonal antisera to leukocyte elastase
and to cathepsin G were obtained by immunization of
rabbits
(24, 25) .
cDNA, Mutagenesis, and Construction of Expression
Vectors
Human cDNA for leukocyte elastase was produced from
total RNA from the HL-60 leukemic cell line using reverse transcription
and PCR(
)
amplification of cDNA with primers
based on a published nucleotide sequence
(21) using the Gene
Amp® RNA PCR kit (Perkin-Elmer) according to the
manufacturer's instructions. By designing the PCR primers, the
Kozak consensus leader sequence for maximum translational efficiency
(26) was introduced, and the flanking restriction enzyme sites
HindIII and NotI were included for subsequent cloning
into plasmid. PCR primers were as follows: upstream,
5`-GACTTCAGAAGCTTGCCACCATGACCCTCGGCCGCCGACTCG-3`; and
downstream, 5`-GACTTCAGGCGGCCGCTCAGTGGGTCCTGCTGGCCGGGTCCGG-3`
(start and stop codons in boldface). The resulting PCR product was
cloned into pRC/CMV to create the expression vector pRC/CMV-elastase.
Similarly, cDNA for cathepsin G (generously provided by Dr. G.
Salvesen, Duke University, Durham, NC) was cloned into the expression
vector pCEP4-CatG as described
(23) . A carboxyl-terminal
deletion mutant of leukocyte elastase was created by PCR amplification
of leukocyte elastase cDNA with the downstream primer
5`-GACTTCAGGCGGCCGCTCATTGGATGATAGAGTCGATCCAG-3`, thereby
introducing a stop codon (boldface) after Gln
.
Analogously, a deletion mutant of cathepsin G was constructed using the
downstream primer
5`-GACTTCAGGCGGCCGCTCAGCTTCTCATTGTTGTCCTTATCC-3`, introducing a
stop codon (boldface) after Ser
. The deletion mutants of
leukocyte elastase and cathepsin G were sequenced and cloned into
pRC/CMV and pCEP4 to create the expression vectors
pRC/CMV-elastase
248-267 and pCEP4-CatG
245-255,
respectively. All cDNA constructs were sequenced to verify the
integrity of the reading frame and the desired mutations. The deletion
mutants are depicted schematically in Fig. 1.
Figure 1:
Schematic view of
the structures of leukocyte elastase and cathepsin G and the
carboxyl-terminal deletion mutants. Amino acids are indicated by
one-letter code.
Transfection Procedure
RBL-1 cells were
transfected using the Bio-Rad electroporation apparatus. Transfection
of RBL-1 cells by electroporation was performed with electrical
settings of 960 microfarads and 300 V as described
elsewhere
(23) . After electroporation, Geneticin (2 mg/ml) or
hygromycin B (1200 units/ml) was added to select for recombinant clones
expressing the Geneticin resistance gene of pRC/CMV or the hygromycin B
resistance gene of pCEP4, respectively. Individual clones growing in
the presence of antibiotic were isolated, expanded into mass cultures,
and screened for expression of elastase or cathepsin G by biosynthetic
labeling. Clones with the most pronounced expression were chosen for
further experiments.
Cell Culture
The rat basophilic/mast cell line
RBL-1 was a gift from Dr. L. Hellman. Cells were grown in RPMI 1640
medium supplemented with 10% heat-inactivated fetal calf serum
(complete medium) (GIBCO Ltd., Paisley, UK) in 5% CO at 37
°C in a fully humidified atmosphere. Exponentially growing cells
were used in all experiments.
Biosynthetic Labeling
Biosynthetic labeling of
newly synthesized proteins was performed as described
elsewhere
(23) . Unless otherwise indicated, cells were starved
for 30 min, followed by pulse labeling with
[S]methionine/[
S]cysteine
for 30 min. In chase experiments following pulse labeling, cells were
resuspended in complete medium. At timed intervals, cells were
withdrawn and subjected to extraction or homogenization and subsequent
subcellular fractionation.
Subcellular Fractionation
Subcellular
fractionation was performed as described elsewhere
(23) .
Briefly, the cell homogenate was fractionated in a Percoll density
gradient, after which nine fractions were collected, with fraction 9
containing all cytosol. The distribution of lysosomes and Golgi
elements in the density gradient was determined, by assaying
-hexosaminidase and galactosyltransferase as described
elsewhere
(27, 28) , to fractions 1-3 and
5-8, respectively (data not shown).
Immunoprecipitation
For immunoprecipitation, whole
cells or Percoll-containing subcellular fractions were solubilized, and
biosynthetically labeled elastase or cathepsin G was immunoprecipitated
and subjected to electrophoretic analysis (SDS-PAGE) followed by
fluorography as described previously
(18, 23) .
Adsorption to Aprotinin-Agarose
Adsorption to
aprotinin-agarose was performed essentially as described by Salvesen
and Enghild
(8) and as previously described in
detail
(23) . Briefly, cells were lysed, and the lysate was
allowed to react for 60 min at room temperature with 200 µl of a
50% suspension of aprotinin-agarose/10 cells. The aprotinin
with bound material was washed, followed by incubation for 45 min at 4
°C in 5 ml of elution buffer (50 mM sodium acetate, pH
4.5, 300 mM NaCl) to obtain the release of bound material.
Elastase or cathepsin G in the eluate or remaining in the lysate after
adsorption to aprotinin-agarose (lacking affinity for aprotinin) was
immunoprecipitated as described
(23) .
Radiosequencing
For determination of the
amino-terminal sequence of processed elastase and cathepsin G,
biosynthetic labeling with [H]isoleucine followed
by amino acid sequencing was performed in the following way. Cells (1
10
) were starved for 30 min in 50 ml of
isoleucine-free RPMI 1640 medium (Select-Amine kit, GIBCO Ltd.) in
order to reduce intracellular isoleucine supplies, after which the
cells were incubated in 25 ml of identical medium supplemented with
[
H]isoleucine (200 µCi/ml) to achieve
metabolic labeling of synthesized proteins. During the subsequent
chase, cells were incubated in 50 ml of complete medium. Following
pulse labeling for 30 min and chase for 2 h, respectively, cell lysates
were prepared, and immunoprecipitation was performed as described
above. Following SDS-PAGE, the proteins were transferred to a
polyvinylidene difluoride membrane by semidry electroblotting, and the
membrane was subjected to autoradiography for 1 week. By guidance of
the autoradiogram, radioactive bands were excised and subjected to
Edman amino acid degradation, performed by the Biomedical Service Unit
at University of Lund. The initial 10 degradation cycles were assayed
for radioactivity using a scintillation counter.
Enzymatic Assay
The enzymatic activity of elastase
was assayed using
N-succinyl-Ala-Ala-Ala-p-nitroanilide as
substrate
(29) . A cell extract was prepared and assayed as
described elsewhere
(30) with minor modifications. Briefly, 1
10
cells were washed once in phosphate-buffered
saline, and the cell pellet was frozen and thawed twice, after which it
was resuspended in 0.9 ml of 100 mM Tris-Cl, pH 8.5,
containing 1 M MgCl
and 0.1% Triton X-100. After
30 s of sonication with an ultrasonicator (Kistner Lab, Stockholm,
Sweden) to obtain complete homogenization of the cells, 2 ml of 5
mM Tris-Cl, pH 8.5, containing 1 M NaCl and 0.1%
Triton X-100 was added, and the lysate was centrifuged at 32,000
g at 4 °C for 2 h to remove DNA. Of the remaining
lysate, 500 µl was mixed with 250 µl of 200 mM
Tris-Cl, pH 8.5, containing 1 M NaCl, and the mixture was
heated to 40 °C, after which 250 µl of 2 mMN-succinyl-Ala-Ala-Ala-p-nitroanilide (dissolved in
dimethyl fluoride) was added. The reaction mixture was incubated for 30
min, after which the reaction was stopped by the addition of 500 µl
of soybean trypsin inhibitor (200 µg/ml), and the absorbance at 410
nm was read. A standard curve was obtained by replacing the cells with
various amounts of porcine pancreatic elastase (Sigma). Measurements
were performed within a linear range of 0.15 to 1.5 A (83-830 units/ml elastase).
Biosynthesis, Processing, and Granular Sorting of
Transgenic Human Leukocyte Elastase in RBL Cells
Stable clones
of RBL cells expressing human leukocyte elastase (RBL/elastase cells)
were established by transfection with pRC/CMV-elastase. The
biosynthesis of transgenic elastase was characterized by biosynthetic
labeling and immunoprecipitation as described under ``Experimental
Procedures.'' No labeled material was specifically
immunoprecipitated from wild-type RBL cells (data not shown).
Fig. 2
shows pulse labeling of RBL/elastase cells, followed by
chase for the time periods indicated. After 30 min of pulse labeling, a
main translation product of 34 kDa was found. In addition, two minor
products with apparent masses of 37 and 32 kDa, respectively, were
precipitated. The 32-kDa form was found to be an initial translation
product, as it was the dominating form after 10 min of pulse labeling
and was converted into the 34-kDa form following chase of the label for
20 min (data not shown); this processing from 32 to 34 kDa most
probably represents glycosylation events. The nature of the 37-kDa form
is not clear. After 2 h of chase, almost all of the labeled material
was reduced to 31 kDa, most likely corresponding to proteolytic removal
of the carboxyl-terminal prodomain
(8) , and prolonged chase
resulted in the appearance of an additional 29-kDa form,
distinguishable after 4 h of chase. A substantial amount of the
proteolytically unprocessed forms of transgenic elastase was released
into the culture medium (Fig. 2), which is commonly seen with
lysosomal enzymes
(16) . The intracellular processed forms were
stable during the 4 h of the experiment, without obvious signs of
degradation. These data suggest the initial synthesis of a 34-kDa pro
form of elastase that with time is carboxyl-terminally processed into a
31-kDa form and, to some extent, further into a 29-kDa form.
Figure 2:
Processing of transgenic leukocyte
elastase. RBL/elastase cells were pulse-labeled with
[S]methionine/[
S]cysteine
for 30 min, followed by chase for up to 4 h. At the indicated time
points, 2
10
cells were withdrawn and subjected to
solubilization and immunoprecipitation with anti-elastase as described
under ``Experimental Procedures.'' In addition, elastase was
immunoprecipitated from the incubation medium after each chase period.
The immunoprecipitates were subjected to SDS-PAGE on a 5-20%
gradient gel, and subsequent fluorography was performed as described
under ``Experimental Procedures.'' The fluorogram was exposed
for 7 days. The positions of newly synthesized 34-kDa proelastase and
the 31- and 29-kDa processing forms are indicated with arrows to the right. Numbers to the left in this figure and in Figs.
3-5 are the M
values of molecular weight
standards.
To
correlate the processing with subcellular translocation, pulse-chase
experiments were performed, followed by subcellular fractionation.
After 30 min of labeling, most of the newly synthesized elastase was
present in fractions of intermediate density, corresponding to the
endoplasmic reticulum and Golgi elements (Fig. 3), with the
34-kDa form as the dominant one. Following 90 min of chase, most of the
34-kDa pro form was processed into a 31-kDa form and transferred to
dense fractions corresponding to granules. Labeled transgenic elastase
still remained, after 5 h of chase, in dense fractions, and a minor
portion was converted into a 29-kDa form. These data are analogous to
those shown for transgenic cathepsin G in RBL cells
(23) and
indicate that proteolytic processing of proelastase occurs during or
after transfer to a granular compartment, where the mature enzyme is
stored.
Figure 3:
Targeting of leukocyte elastase to
granules. RBL/elastase cells were pulse-labeled for 30 min
(A), followed by chase for 90 min (B) and 5 h
(C). At the times indicated, 1 10
cells
were homogenized, after which subcellular fractionation was performed,
with subsequent collection of eight 0.8-ml subcellular fractions
(fraction 9 contained all cytosol) as described under
``Experimental Procedures.'' Fractions were solubilized and
subjected to immunoprecipitation with anti-elastase. Analyses of
immunoprecipitates were as described in the legend to Fig. 2. The
fluorograms were exposed for 7 days. The positions of 34-kDa
proelastase and the 31- and 29-kDa processing forms are indicated with
arrows to the right.
Processing and Granular Sorting of Carboxyl-terminal
Deletion Mutants of Leukocyte Elastase and Cathepsin G in RBL
Cells
By transfection of RBL cells with carboxyl-terminally
deleted cDNA for leukocyte elastase or cathepsin G, cell clones were
established expressing elastase (RBL/elastase248-267) and
cathepsin G (RBL/CatG
245-255), respectively, lacking the
carboxyl-terminal prodomains. The deletion mutants are depicted
schematically in Fig. 1. Fig. 4shows the initial synthesis
of a 31-kDa form in RBL/elastase
248-267 cells. Thus, the
initially synthesized form of the carboxyl-terminal deletion mutant (31
kDa) corresponds to that of the processed full-length enzyme (31 kDa),
supporting the notion that the carboxyl-terminal prodomain is removed
during processing of the full-length proenzyme from 34 to 31 kDa
(Fig. 2). Similar to RBL/elastase cells, the
RBL/elastase
248-267 cells showed, after 30 min of labeling,
a small amount of elastase of lesser size (30 kDa), which disappeared
while chasing the label. As for the full-length protein, this smaller
(30 kDa) form may represent an early translation product that is, as
the result of early glycosylation events, converted to the 31-kDa form.
After 4 h of chase, a minor part was further processed into a 29-kDa
form. A considerable amount of the labeled material was secreted into
the medium, but after
1 h of chase, the intracellular labeled
protein remained stable during the 4 h of the experiment. Most of the
extracellular release occurred during the 1st h of chase, after which
the secreted forms disappeared to some extent (Fig. 4). As for
the full-length protein, the stability of the intracellular enzyme
suggests transfer to a storage compartment, and translocation of newly
synthesized elastase lacking the carboxyl-terminal prodomain was indeed
demonstrated by subcellular fractionation. Fig. 5shows that
elastase
248-267 is targeted to dense fractions corresponding
to granules, analogous to what was shown for full-length elastase in
Fig. 3
. Fig. 5also demonstrates that 30 min of pulse
labeling of RBL/elastase
248-267 cells results in two visible
translation products of 31 and 30 kDa. As seen in experiments with
full-length elastase, the smaller product disappeared by chasing the
label, which probably reflects glycosylation of the protein. Following
90 min of chase, the main initial translation product of 31 kDa was
transferred to dense fractions, where a minor part was further
processed into a 29-kDa form, distinguishable after 5 h of chase.
Likewise, it was found that cathepsin G lacking the carboxyl-terminal
prodomain (CatG
245-255) was targeted to granule-containing
fractions (data not shown). These results demonstrate that the
carboxyl-terminal deletion mutants of leukocyte elastase and cathepsin
G are, like full-length proenzymes, efficiently sorted for storage in a
granular compartment in RBL cells.
Figure 4:
Processing of transgenic leukocyte
elastase248-267. RBL/elastase
248-267 cells were
pulse-labeled with
[
S]methionine/[
S]cysteine
for 30 min, followed by chase for up to 4 h. At the indicated time
points, 2
10
cells were withdrawn and subjected to
solubilization, immunoprecipitation, and subsequent analyses as
described in the legend to Fig. 2. The fluorogram was exposed for 7
days. The positions of 31-kDa elastase
248-267 and the 29-kDa
processing form are indicated with arrows to the
right.
Figure 5:
Targeting of leukocyte
elastase248-267 to granules. RBL/elastase
248-267
cells were pulse-labeled for 30 min (A), followed by chase for
90 min (B) and 5 h (C). At the times indicated, 1
10
cells were homogenized, after which subcellular
fractionation, immunoprecipitation, and subsequent analyses were
performed as described in the legend to Fig. 3. The fluorograms were
exposed for 7 days. The positions of 31-kDa elastase
248-267
and the 29-kDa processing form are indicated with arrows to
the right.
Enzymatic Activation and Amino-terminal Processing of
Transgenic Leukocyte Elastase and Cathepsin G
Leukocyte elastase
and cathepsin G are synthesized as inactive zymogens, but are
enzymatically activated in a pregranular or granular compartment. The
removal of an amino-terminal dipeptide, catalyzed by the thiol protease
dipeptidyl peptidase I, is a prerequisite for enzymatic
activation
(8, 11) . To investigate the activation of the
transfected serine proteases in RBL cells, their affinity for the
serine protease inhibitor aprotinin was investigated. By pulse labeling
and then chasing the label, followed by adsorption to
aprotinin-agarose, we demonstrated the acquisition of affinity to
aprotinin for transgenic elastase, elastase248-267, and
CatG
245-255. Fig. 6shows similar results for all
three enzyme variants, with binding of most of the labeled material to
aprotinin after 2-5 h of chase. These results are consistent with
results from experiments with endogenous elastase and cathepsin G
(8) as well as with transgenic cathepsin G
(23) , thus
suggesting amino-terminal removal of the dipeptide, leading to
enzymatic activation. Indeed, using a radiosequencing technique,
amino-terminal processing with removal of a dipeptide was indicated for
both transgenic elastase (Fig. 7A) and cathepsin G
(Fig. 7B). The amino acid sequences of mature elastase
and cathepsin G begin with one and two isoleucines, respectively
(Fig. 1). This is in contrast to the immature forms, which
contain the activation dipeptide (Ser-Glu and Gly-Glu, respectively)
prior to the isoleucines. Cells were labeled with
[
H]isoleucine for 30 min, followed by chase for 2
h, and radiosequencing was performed as described under
``Experimental Procedures.'' After 30 min of labeling, 34-kDa
proelastase and 32.5-kDa procathepsin G showed, upon sequencing, the
presence of radioactivity in the third fraction, corresponding to amino
acid 3 in the amino-terminal sequence, indicating the presence of the
activation dipeptide followed by labeled isoleucine (Fig. 7).
Following 2 h of chase, however, the processed forms of elastase and
cathepsin G with masses of 31 kDa showed considerable radioactivity in
fraction 1 of the sequencing chromatogram, thus indicating that
cleavage of the dipeptide had occurred. The persisting radioactivity in
fraction 3 following chase indicates that activation was incomplete at
this point, which is concordant to results above showing incomplete
acquisition of affinity for aprotinin after 2 h of chase
(Fig. 6).
Figure 6:
Adsorption to aprotinin-agarose.
RBL/elastase, RBL/elastase248-267, and
RBL/CatG
245-255 cells were labeled with
[
S]methionine/[
S]cysteine
for 30 min and chased for up to 5 h. At timed intervals, aliquots of
labeled cells (15
10
) were withdrawn for analysis.
Cell lysis and adsorption to aprotinin-agarose were performed as
described under ``Experimental Procedures.'' A demonstrates the immunoprecipitates of labeled protein that did
not bind to aprotinin (enzymatically nonactive). In B, the
aprotinin-bound material was eluted, and immunoprecipitation with
specific antiserum, SDS-PAGE, and fluorography were performed as
described under ``Experimental Procedures.'' The fluorograms
were exposed for 7 days. Arrows to the right indicate newly
synthesized proelastase (34 kDa), elastase
248-267 (31 kDa),
CatG
245-255 (31 kDa), and their processing
forms.
Figure 7:
Radiosequencing of transgenic elastase and
cathepsin G. RBL/elastase (A) and RBL/CatG (B) cells
were pulse-labeled with [H]isoleucine for 30 min
(upper panels), followed by chase for 2 h (lower
panels) as described under ``Experimental Procedures.''
Following pulse labeling and chase, 1
10
cells were
withdrawn and subjected to solubiliza-tion, immunoprecipitation,
SDS-PAGE, and transfer to polyvinylidene difluoride membrane as
described under ``Experimental Procedures.'' Radioactive
bands containing pulse-labeled 34-kDa proelastase and 32.5-kDa
procathepsin G and the 31-kDa processing form of elastase and cathepsin
G following 2 h of chase were excised and subjected to amino acid
degradation. The amount of radioactivity in the initial 10 cycles of
each sequence analysis is shown.
To estimate the relative enzymatic activity in RBL
cells expressing transgenic elastase, an enzymatic assay was employed
as described under ``Experimental Procedures.'' As might be
predicted from the results of experiments with adsorption to aprotinin
(Fig. 6), RBL/elastase248-267 cells contained elastase
activity comparable to that of RBL/elastase cells. The elastase
activity in RBL/elastase
248-267 cells was 561 ± 95
units, and the corresponding value for RBL/elastase cells was 490
± 108 units, while wild-type RBL cells contained 75 ± 27
units (per 5
10
cells, ± S.E., n = 3). These results demonstrate that transgenic elastase is
enzymatically activated and that a deletion of the carboxyl-terminal
prodomain obviously does not interfere with the conversion of the
zymogen form into catalytically competent enzyme. A similar enzymatic
assay for cathepsin G
(31) could not be employed due to strong
interference of endogenous proteases from the RBL cells.
-hexosaminidase assays.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.