(Received for publication, August 15, 1995)
From the
The neutral protease cathepsin G belongs to a family of hematopoietic serine proteases stored in the azurophil granules of the neutrophil granulocyte. To investigate the function of asparagine-linked carbohydrates in neutrophil serine proteases, we constructed a mutant cDNA, coding for human cathepsin G deficient of a functional glycosylation site, for use in a transgenic cellular model. Wild type and mutant cDNA were stably expressed in the rat basophilic/mast cell line RBL and in the murine myeloblast-like cell line 32D. Biosynthetic labeling, followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography, showed that carbohydrate-deficient cathepsin G was synthesized as a 29-kDa proform in both cell lines. The proform was proteolytically processed into a stable form with an apparent molecular mass of 27.5 kDa, indicating removal of the carboxyl-terminal prodomain. The mutant cathepsin G was enzymatically activated as determined by acquisition of affinity to aprotinin, a serine protease inhibitor. As for wild type cathepsin G, small amounts of the unprocessed form of the mutated enzyme were released from the cells, while the major part was transferred to a granular compartment as demonstrated by subcellular fractionation. Thus, neither processing leading to enzymatic activation nor granular sorting was obviously affected by the lack of oligosaccharides on the mutant cathepsin G. Our results therefore indicate that glycosylation is not essential for these processes. In addition to the previously utilized cell line RBL, we propose the 32D cell line as a suitable cellular model for transgenic expression of human neutrophil serine proteases.
The azurophil granules of neutrophil granulocytes contain lysosomal hydrolases and can therefore be regarded as specialized forms of lysosomes(1, 2) . The azurophil granules also store bactericidal proteins which are unique for the myeloid lineage(3, 4, 5) . Among them are the neutral serine proteases cathepsin G and leukocyte elastase, which belong to a superfamily of hematopoietic serine proteases also including granzymes of cytotoxic T lymphocytes and certain mast cell proteases(6, 7, 8) . Hematopoietic serine proteases are stored in cytosolic granules as active enzymes 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(9, 10, 11, 12, 13) .
Early processing of lysosomal enzymes involves modifications of
carbohydrate side chains with phosphorylation of mannose residues
followed by translocation to lysosomes mediated by receptors for
mannose 6-phosphate(14, 15, 16) . In cells
from patients with I cell disease phosphotransferase activity is
defective, and consequently mannose 6-phosphate cannot be added during
processing. Therefore, lysosomal enzymes are constitutively secreted
from the cells with resulting intracellular deficiency of most
lysosomal enzymes(17) . The packaging of serine proteases into
azurophil granules is different from that of typical lysosomal enzymes
and seems to be consistently independent of the mannose 6-phosphate
receptors(18) . Specific sorting mechanisms involved in
transfer of proteins for storage in azurophil granules have not been
identified. Human cathepsin G contains one consensus site for
asparagine-linked glycosylation (Asn, numeration according
to (19) ). Accordingly, biochemical characterization of
cathepsin G has demonstrated asparagine-linked carbohydrates which are
processed into complex forms(20) . Similarly, the other members
of the neutrophil serine protease family, i.e. leukocyte
elastase, proteinase 3, and azurocidin, also contain asparagine-linked
carbohydrates (20, 21, 22, 23) . In
general, functions of asparagine-linked oligosaccharides include
effects on folding and conformation as well as a role in recognition
events(24, 25) . For example, glycosylation of the
lysosomal enzyme
-glucosidase is required for catalytic activity (26) , and carbohydrates of the cysteine protease propapain A
are important for subcellular transport and secretion of the
enzyme(27) . However, a functional role for the carbohydrates
of the serine proteases of the azurophil granules has not been
demonstrated.
The aim of this work was to determine whether the
asparagine-linked oligosaccharide has a critical role for folding,
processing, or granular targeting of cathepsin G. We have recently used
the rat basophilic leukemia cell line RBL-1 as a transgenic model for
studying the processing of transfected human cathepsin G and leukocyte
elastase (28, 29) . Therefore, we have utilized
site-directed mutagenesis for elimination of the glycosylation site by
substitution of Asn with Gln followed by transfection of
the mutated cDNA to RBL cells in order to investigate the consequences
of absent glycosylation. To further strengthen our data, we have
extended the studies by transfecting wild type and mutated cathepsin G
to murine myeloid 32D cells. Our results demonstrate that, after
transfection to RBL or 32D cells, cathepsin G lacking asparagine-linked
carbohydrates is expressed as a proform which is processed into active
enzyme. The oligosaccharide-deficient protein is also targeted for
granular storage, thus questioning the importance of asparagine-linked
carbohydrates of cathepsin G.
Figure 1:
Processing of
transgenic cathepsin G/Gln in RBL cells. RBL/cathepsin
G/Gln
cells were pulse-labeled with
[
S]methionine/[
S]cysteine
for 30 min followed by chase for up to 4 h. At indicated points, 20
10
cells were withdrawn and subjected to
solubilization and immunoprecipitation with anti-cathepsin G. In
addition, cathepsin G was immunoprecipitated from the incubation medium
after each period of chase. The immunoprecipitates were run in SDS-PAGE
in a 5-20% gradient gel, and subsequent fluorography was
performed. The fluorogram was exposed for 7 days. The positions of
newly synthesized 29-kDa procathepsin G/Gln
and the
27.5-kDa processed form are indicated with arrows to the right. Numbers to the left in this and subsequent
figures are the values of molecular mass
standards.
Following
translation, the proform of cathepsin G/Gln was converted
into a 27.5-kDa form which was stable during 4 h of chase (Fig. 1). This reduction of molecular mass most likely
represents carboxyl-terminal proteolytic processing, as demonstrated
for endogenous and transgenic cathepsin G and leukocyte
elastase(9, 28, 29) . No further processing
was evident during the time of the experiment. Thus the absence of
carbohydrates on cathepsin G apparently did not affect proteolytic
processing or the stability of the protein. Small amounts of the
unprocessed proforms of both mutant, hardly visible in Fig. 1,
and of wild type cathepsin G (data not shown) were released into the
medium, as commonly seen with lysosomal enzymes (17) . No
obvious difference between the extracellular release of cathepsin
G/Gln
and wild type cathepsin G was evident.
Figure 2:
Adsorption of cathepsin G/Gln to aprotinin-agarose. RBL/cathepsin G/Gln
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. 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. The fluorogram was exposed for 7 days. Arrows to the right indicate newly synthesized
procathepsin G/Gln
(29 kDa) and the processing form of
27.5 kDa.
Figure 3:
Targeting of transgenic cathepsin
G/Gln to granules in RBL cells. RBL/cathepsin G/Gln
cells were pulse-labeled for 30 min followed by chase for 90 min
and 5 h. At times indicated, 100
10
cells were
homogenized after which subcellular fractionation was performed, with
subsequent collection of nine subcellular fractions (fraction no. 9
containing all cytosol). Fractions were solubilized and subjected to
immunoprecipitation with anti-cathepsin G. Analyses of
immunoprecipitates were as described in the legend to Fig. 1.
The fluorograms were exposed for 7 days. The positions of 29-kDa
procathepsin G/Gln
and the 27.5-kDa processing form are
indicated with arrows to the right.
Figure 4:
Processing of transgenic cathepsin G and
cathepsin G/Gln in 32D cells. A, 32D/cathepsin G
cells and B, 32D/cathepsin G/Gln
cells were
pulse-labeled with
[
S]methionine/[
S]cysteine
for 30 min followed by chase for up to 4 h. At indicated points, 20
10
cells were withdrawn and subjected to
solubilization, immunoprecipitation, and analyses as described in the
legend to Fig. 1. The fluorograms were exposed for 7 days. The
positions of the proforms of 32.5 and 29 kDa, respectively, and the
processing forms are indicated with arrows to the right.
Figure 5:
Granular targeting of transgenic cathepsin
G and cathepsin G/Gln in 32D cells. A,
32D/cathepsin G cells and B, 32D/cathepsin G/Gln
cells were pulse-labeled for 30 min, followed by chase for 90 min
and 5 h. At times indicated, 100
10
cells were
homogenized after which subcellular fractionation, immunoprecipitation,
and subsequent analyses were performed as described in the legends to Fig. 1and Fig. 3. The fluorograms were exposed for 7
days. The positions of the proforms of 32.5 and 29 kDa, respectively,
and the processing forms are indicated with arrows to the right.
The aim of the present work was to investigate the role of asparagine-linked carbohydrates in the cellular processing of cathepsin G. For this purpose we utilized site-directed mutagenesis to create a mutant form of cathepsin G, lacking a functional glycosylation site. Mutant or wild type cDNA was transfected into target cells and the processing of wild type and mutant protein was compared. Our results demonstrate that the mutant, carbohydrate-deficient, form of cathepsin G was synthesized in RBL and 32D cells as a proform with apparent molecular mass of 29 kDa.This is close to the calculated molecular mass of the protein core (26.9 kDa) and consistent with the previous characterization of wild type cathepsin G in RBL cells, which showed an apparent proform of 32.5 kDa, containing asparagine-linked oligosaccharides of approximately 3.5 kDa(28) . The proform of the carbohydrate-deficient mutant was processed into a 27.5-kDa form, indicating proteolytic removal of a 1.5-kDa prodomain, again similar to the processing of the wild type protein. The transfer of mutant cathepsin G to a granular compartment in RBL and 32D cells, as demonstrated by subcellular fractionation experiments, was not obviously affected by the lack of oligosaccharides. We also demonstrated that mutant cathepsin G was enzymatically activated in RBL cells, as judged by the acquisition of affinity to aprotinin, which indicates that carbohydrates are dispensable for efficient processing of cathepsin G.
A minor portion of the mature forms of wild type
cathepsin G and elastase in RBL cells is slowly processed with further
reduction in molecular mass of approximately 1
kDa(28, 29) . This late processing, occurring at a
point of time when transfer to granules is accomplished, was also
visible when wild type cathepsin G was expressed in 32D cells, seen as
a tight double band after four hours of chase (Fig. 4A)
and could involve further proteolytic processing or late trimming of
carbohydrates. However, contrary to what was seen with wild type
cathepsin G, the mutant form of cathepsin G (cathepsin
G/Gln) did not show any late reduction in molecular mass,
whether expressed in RBL or 32D cells ( Fig. 1and 4B).
Our results therefore indicate that the late processing of wild type
cathepsin G is the result of trimming of carbohydrates in granular
structures rather than further proteolytic processing. Different
structures of the sugar chains of cathepsin G and leukocyte elastase in
neutrophil granulocytes have been reported(37) ; typical
biantennary chains, commonly found on secreted
glycoproteins(38) , were present in a small fraction of the
enzymes, while the major forms had truncated oligosaccharide chains.
Since similar short structures of sugar chains are found on several
lysosomal enzymes, it was hypothesized that the biantennary
chain-containing cathepsin G and leukocyte elastase constitute a minor
fraction of the enzymes, processed in a separate pathway, with specific
functions involving extracellular secretion(37) . However, our
results which indicate that late trimming of the carbohydrates is
possible in the milieu of the maturing granules, rather support the
alternative interpretation that the truncated forms arise by
degradation of the complex chains by glycosidases present in the
granules.
A major concern when studying transgenically expressed
proteins, is the relevance of the target cell chosen for transfection.
The granules of the basophilic/mast cell like RBL cell line contain
lysosomal enzymes and mast cell serine proteases (32, 39, 40) and transfected cathepsin G and
leukocyte elastase are adequately processed in this cell
line(28, 29) . However, mechanisms for granular
targeting in RBL cells could be different from those of
promyelocyte-like cells, in which cathepsin G and leukocyte elastase
are normally formed and stored. Therefore, we extended our study of
processing and granular targeting of carbohydrate-deficient cathepsin G
(cathepsin G/Gln) to include myeloblastic 32D cells. The
32D cell line has successfully been employed for transfection of
defensin, another component of azurophil granules(41) . Similar
to the results from RBL cells, a stable expression of cathepsin G and
cathepsin G/Gln
was seen in 32D cells. Furthermore,
processing of the proforms and targeting of processed forms to granules
were also indistinguishable from what was seen in RBL cells. Thus also
in myeloblast-like cells, the carbohydrates of cathepsin G do not seem
to be important for stability or subcellular transport.
Most lysosomal hydrolases carry phosphorylated mannose residues enabling a mannose 6-phosphate receptor-mediated transfer to a prelysosomal compartment(14, 15, 16) . The sorting of cathepsin G and leukocyte elastase, on the other hand, seems to be independent of the mannose 6-phosphate receptors(17) . Alternative targeting mechanisms to azurophil granules have not been demonstrated. A transient mannose 6-phosphate-independent membrane association can possibly be involved in the subcellular trafficking of some lysosomal enzymes(42, 43, 44, 45, 46, 47, 48) . This binding to membranes in the endoplasmic reticulum or the Golgi apparatus in some cases involves prodomains of the proteins. Similar mechanisms could play a role in the sorting of cathepsin G and leukocyte elastase as both proteins exhibit C-terminal extensions which are proteolytically removed after transfer to granules(7, 9, 28, 29) . However, previous results from experiments with deletion mutants indicate that transfer to granules occurs independently of the C-terminal prodomains of cathepsin G and leukocyte elastase(29) . Thus data argue against an important function of the carboxyl-terminal prodomains in sorting of these enzymes. Similarly, the present results indicate that the oligosaccharide structures are also dispensable for granular targeting of cathepsin G. The sorting mechanisms for these hematopoietic serine proteases therefore remain obscure.
In conclusion, the present investigation has demonstrated that cathepsin G lacking a functional glycosylation site can be stably expressed in both rat basophilic RBL cells and murine myeloid 32D cells. The absence of carbohydrates did not have any obvious consequence for stability, processing, activation, or granular targeting of the protein, thus strongly arguing against an important role for carbohydrates of cathepsin G in these processes.