(Received for publication, July 5, 1995; and in revised form, August 30, 1995 )
From the
Lysosomal protective protein/cathepsin A is a serine
carboxypeptidase that forms a complex with -galactosidase and
neuraminidase. The enzyme is synthesized as a 54-kDa precursor/zymogen
and processed into a catalytically active 32- and 20-kDa two-chain
form. We have expressed in baculovirus-infected insect cells the human
one-chain precursor as well as the two separate subunits in order to
establish the mode of catalytic activation of the zymogen and the
assembly and activation of the two subunits. Infected insect cells
synthesize large quantities of the exogenous proteins, which are
glycosylated and secreted but not processed. Co-expression of the two
subunits results in their assembly into a two-chain form of 34- and
20-kDa with negligible enzymatic activity. Limited proteolysis with
trypsin of the 54-kDa precursor and the reconstituted 34- and 20-kDa
form gives rise to a fully active 32- and 20-kDa product. These results
enabled us to map the sites of proteolytic cleavage needed for full
activation of the cathepsin A zymogen. They further indicate that the
34- and 20-kDa form is a transient processing intermediate that is
converted into a mature and active enzyme by removal of a 2-kDa
``linker'' peptide from the COOH terminus of the 34-kDa
subunit.
A primary defect of lysosomal protective protein/cathepsin A
(PPCA) ()in humans causes the metabolic storage disorder
galactosialidosis ((1) ; reviewed in (2) ). This
disease is characterized by severely reduced activities of the enzymes
-D-galactosidase and N-acetyl-
-neuraminidase, secondary to absent or abnormal
PPCA. The reason for the additional combined deficiency relates to one
of the functions of PPCA protein, which is to associate with and
protect the two glycosidases, modulating their activity and stability
in lysosomes(1, 3, 4, 5) . The
primary structures of human, mouse, and chicken PPCAs are highly
conserved (6, 7, 8) and bear homology to
yeast and plant serine carboxypeptidases ((6) ; for reviews see
Refs. 9 and 10). Mammalian PPCAs have cathepsin A activity at lysosomal
pH but maintain a deamidase/esterase activity at neutral
pH(8, 11) . Furthermore, the human enzyme, purified
from platelets and lymphocytes, has been shown to function both in in vitro and in vivo assays on the inactivation of
selected neuropeptides, like substance P, oxytocin, and endothelin
I(11, 12, 13) .
PPCA is synthesized as a 54-kDa precursor that is glycosylated on two Asn residues and is enzymatically inactive(1, 6, 8) . The precursor dimerizes at neutral pH shortly after synthesis and is likely to be transported as a dimer to the lysosomes(14) . Once in the acidic lysosomal environment, the zymogen is cleaved into an enzymatically active 32- and 20-kDa two-chain form. However, the events involved in proteolytic activation have not been established until now. Here we have used baculovirus (BV)-expressed human precursor and two separate subunits to map the sites of cleavage and processing of human PPCA zymogen. We have identified a ``linker'' domain in the precursor molecule, located at the COOH terminus of the large subunit, which needs to be removed for full catalytic activation of the enzyme.
SF21 insect cells were infected with recombinant
baculoviruses AcHPP54, AcHPP32, and AcHPP20 separately or co-infected
with AcHPP32 and AcHPP20. Metabolic labeling and immunoprecipitation
analysis showed that PPCA precursor was synthesized in large quantities
and efficiently secreted but was not or poorly processed to the mature
two-chain product (Fig. 1, lanes 1 and 5). Its
estimated molecular mass of 54 kDa was similar to wild-type
precursor from human cultured fibroblasts(1) . Trace amounts of
low molecular weight polypeptides, visible both intra- and
extracellularly, were products of aspecific proteolysis, because
pulse-chase experiments showed no time-dependent conversion of the
precursor into mature protein (not shown). Single infections with
AcHPP32 and AcHPP20 resulted in the production of truncated
polypeptides with molecular masses of 34, 20, and 18 kDa, respectively (Fig. 1, lanes 2 and 3). Only the former,
which was 2 kDa larger than the corresponding wild-type subunit, was
secreted to some extent (Fig. 1, lane 6). The two
chains of 20 and 18 kDa (Fig. 1, lane 3) were different
glycosylation forms of the small subunit. Under nonreducing conditions
the secreted large chain migrated as a doublet, suggesting that within
this polypeptide partial and/or different intrachain disulfide bridges
may have formed (Fig. 1, lane 10). Co-infected cells
synthesized a large subunit of 34 kDa and three small subunits of 20,
19.5, and 18 kDa, respectively. The intermediate 19.5-kDa species was
unique for co-infected cells (compare lanes 3 and 4)
and was the only one of the small subunits found in the medium (Fig. 1, lanes 8 and 12). Co-expressed
polypeptides were immunoprecipitated in larger quantities both intra-
and extracellularly, compared with single infections and under
nonreducing conditions were resolved as one product of
54 kDa,
similar in size to the wild-type precursor (Fig. 1, lanes 9 and 12). These results suggest an early intracellular
association of the two separately synthesized polypeptides, which are
secreted in an associated state. Although they assemble, the 34-kDa
subunit remains 2 kDa bigger (Fig. 1, lane 8) and is
not further processed to the mature size.
Figure 1:
Metabolic labeling of PPCA precursor
and separate subunits in insect cells. Sf21 cells were either infected
with baculovirus construct AcHpp54 (54), AcHpp32 (32), or AcHpp20 (20),
or co-infected with AcHpp32 and AcHpp20 (32/20). 2 days after infection
newly synthesized proteins were labeled with
[S]methionine. Labeled proteins were
immunoprecipitated from cells and media using anti-54 antibodies.
Proteins were separated by SDS-polyacrylamide gel electrophoresis under
reducing (lanes 1-8) or nonreducing (lanes
9-12) conditions and visualized by fluorography. Exposure
time was 24 h. Molecular sizes are indicated. DTT,
dithiothreitol.
To determine whether the subcellular localization of over-expressed proteins could account for the lack of maturation, singly infected or co-infected cells were analyzed with electron microscopy. Immunostaining of ultrathin sections demonstrated an intracellular distribution that was similar for the different over-expressed proteins. Extensive gold labeling was restricted to structures in the cytoplasm corresponding to swollen cisternae of the endoplasmic reticulum (Fig. 2A) and the Golgi complex (Fig. 2B). Other subcellular organelles, including multivesicular bodies and fibrous structures, usually observed in insect cells infected with either wild-type or recombinant viruses(23, 24) , were totally devoid of gold particles. It appears therefore that none of the BV-expressed proteins reach a lysosome-like compartment, where proteolytic processing should occur.
Figure 2:
Subcellular localization of BV-expressed
proteins. Sf21 cells were infected with AcHPP54, AcHPP32, or AcHPP54
+ AcHPP32 and prepared for immunoelectron microscopy 48 h after
infection. Cryosections were incubated with anti-32 antibodies followed
by goat anti-rabbit IgG gold labeling. A, in all three cases
the over-expressed proteins were localized in swollen cisternae of the
endoplasmic reticulum (R), which have the appearance of large
vacuoles. B, gold particles were also clearly present in the
Golgi complex (G). n, nucleus; F, fibrous structures; MVB, multivesicular bodies. The magnifications were:
38,000 for A and 79,000
for B.
The amino terminus of the 20-kDa chain, isolated from human placenta and human platelets(6, 11) , starts with Met-299, which is preceded in the amino acid sequence of the precursor by a conserved arginine. This residue likely represents the site of initial cleavage. Circumstantial evidence has, however, indicated that complete maturation of the precursor may require more proteolytic steps that could occur at Arg-284, Arg-292, or Lys-296. Here we have monitored the processing and catalytic activation of human PPCA precursor and reconstituted subunits by digesting BV-derived secreted proteins with trypsin. To ascertain the occurrence of sequential processing steps, we have used a polyvalent rabbit antibody (anti-pep) raised against a peptide of 15-amino acids, 14 of which are derived from the COOH terminus of the large subunit. Concentrated medium samples containing either the precursor or the 34- and 20-kDa associated protein were incubated with a fixed amount of trypsin for increasing periods of time, and each reaction was stopped by the addition of trypsin inhibitor. Aliquots of each sample were tested on Western blots immunostained with anti-54 and anti-pep antibodies and were assayed for cathepsin A activity. As seen in Fig. 3(upper left panel), after 0.5 min of incubation with trypsin, part of the one-chain precursor was cleaved into a two-chain product of 34 and 20 kDa. Between 2 and 5 min, the 34-kDa form was gradually converted into a 32-kDa derivative, and complete maturation was achieved after 10 min. In contrast, the size of the 20-kDa chain did not vary. Prolonged digestion periods (15 and 30 min) led to aspecific degradation and lower yield of both subunits. Using anti-pep antibodies, only the 54- and 34-kDa polypeptides were detected, indicating that the COOH-terminal peptide of the 34-kDa species was lost upon conversion to the 32-kDa form (Fig. 3, middle left panel). Step-wise maturation of the precursor into a fully processed product was paralleled by a clear increase in cathepsin A activity, which was maximal after 15 min of digestion (Fig. 3, lower left panel). Similar maturation steps were observed for the reconstituted 34- and 20-kDa protein. Upon trypsin cleavage, the large chain was again converted from a 34- to a 32-kDa product, whereas the size of the small subunit did not change (Fig. 3, upper and middle right panels). However, the amount of both polypeptides, as detected on immunoblots, was significantly less than for wild-type precursor, resulting in an overall reduced cathepsin A activity. This was probably due to both a lower secretion of the reconstituted two-chain protein as well as a reduced stability. However, also in this case a clear increase in enzymatic activity was measured after digestion (Fig. 3, lower right panel).
Figure 3: Limited proteolysis with trypsin of 54-kDa precursor and 34- and 20-kDa reconstituted two-chain protein. Aliquots of medium concentrates containing the 54-kDa precursor and 34- and 20-kDa associated protein were incubated at 37 °C with 1 µg of trypsin in the presence of bovine serum albumin (1 mg/ml) for the indicated periods of time. Reactions were stopped with 3 µg of trypsin inhibitor. Samples in lanes 1 and 9 were untreated. At time 0 (lanes 2 and 10), the samples were treated with trypsin inhibitor prior to the addition of trypsin. A portion of each sample was separated by SDS-polyacrylamide gel electrophoresis, followed by electroblotting and immunostaining with anti-54 and anti-pep antibodies. Cathepsin A activity toward the acylated dipeptide benzyloxycarbonyl-phenylalanyl-alanine was measured in each aliquot. One milliunit (mU) of activity is defined as the enzyme activity that releases one nanomole of alanine/minute.
Catalytic activation of precursor and reconstituted products was confirmed by the ability of trypsin-processed proteins to bind the serine protease inhibitor DFP. As shown in Fig. 4(lanes 1-6), only the mature 32-kDa subunit, generated after trypsin digestion of the precursor, was recognized by the radiolabeled inhibitor. The 34-kDa form was apparently unable to bind DFP, although it was present at 0.5-5 min digestion time points (see Fig. 3). The inability to bind the inhibitor was particularly evident in the case of the undigested 34- and 20-kDa protein (lane 7), which showed no radioactive signal, confirming that it has only marginal enzymatic activity. However, this associated form was particularly susceptible to proteolytic cleavage, because immediately after addition of trypsin (time 0, lane 8), a substantial conversion to the 32-kDa product took place. For both wild-type precursor and reconstituted protein, the highest levels of cathepsin A activity were measured at digestion time points in which maximal binding was observed (Fig. 4, lanes 3-5 and 9-10). As seen in the previous experiment, the overall amount of reconstituted and digested two-chain product was again considerably lower than trypsin-cleaved precursor. All together these data point to the 34- and 20-kDa product as being a processing intermediate, transiently occurring during proteolytic maturation of PPCA precursor. This process is probably mediated by a trypsin-like protease. Removal of a COOH-terminal peptide from the 34-kDa subunit is essential for catalytic activation.
Figure 4:
Binding of serine carboxypeptidase
inhibitor DFP to trypsin digested 54 kDa precursor and reconstituted
two-chain protein. Trypsin digestions of precursor and 34- and 20-kDa
two-chain product were performed as described in the legend to Fig. 3. 1 µCi of [H]DFP was added to
40 µl of trypsin digest, followed by immunoprecipitation with
anti-54 antibodies. Immunoprecipitated proteins were separated on
SDS-polyacrylamide gels under reducing conditions and visualized by
fluorography. Exposure times were 2 weeks for lanes 1-6 and 3 months for lanes 7-12. Molecular
sizes are indicated. Cathepsin A activity was measured as described in
the legend to Fig. 3.
PPCA is a lysosomal serine protease with pleiotropic
biological properties. It binds in its normal state to the enzymes
-galactosidase and neuraminidase, rendering them stable and active
in lysosomes; it also hydrolyzes as carboxypeptidase and/or
deamidase/esterase a variety of bioactive peptides, depending upon the
pH conditions used in the assay. When deficient or defective in humans,
it causes the lysosomal storage disease galactosialidosis. Because its
protective and catalytic functions are distinct(8) , we and
others have postulated a role for PPCA in the local inactivation of
selected neuropeptides, with or without the aid of the two
glycosidases(8, 11, 12, 13) . The
protein is synthesized in mammalian tissues and cultured cells as a
one-chain precursor that is enzymatically inactive. Proteolytic
conversion to a disulfide-linked two chain product triggers catalytic
activation. To investigate the process of zymogen activation and to
assess the capacity of the separately synthesized subunits to assemble
into an active enzyme, we have used the baculovirus system (for review
see (25) and references therein) to express human PPCA in
insect cells, either as one-chain precursor or as two separate
subunits.
BV-encoded proteins are synthesized in large amounts and
transported from the endoplasmic reticulum to the Golgi complex but do
not seem to be targeted to a lysosome-like organelle. It is conceivable
that this transport step either requires a recognition marker on
lysosomal proteins specific for insect cells or the process is
saturated by the high concentration of newly synthesized proteins. The
subcellular distribution of both PPCA precursor and reconstituted
subunits could however explain their inadequate intracellular
maturation, which may depend on a lysosome-associated protease.
Although it is known that the degree of proteolytic processing varies
among heterologous proteins expressed with the baculovirus
system(25) , identical results were obtained by us with mouse
PPCA and human -galactosidase expressed in insect cells (
)and by others with human
-galactosidase,
-glucosidase, and
-hexosaminidase (26, 27, 28, 29) . Of the three
separately synthesized proteins, mainly the wild-type precursor is
secreted in a large quantity. Interestingly, however, the co-expressed
subunits are able to form interchain disulfide bridges and are secreted
only in the associated state. They evidently require conformational
changes for their secretion, acquired only when both domains are
present. Using baculovirus co-expression vectors, other investigators
have demonstrated appropriate formation of functional immunoglobulin
heterodimers, which are efficiently secreted, and of disulfide-bridged
interleukin 5 homodimers(30, 31) .
The focus of the
experimental work presented here is the process of enzymatic activation
of human PPCA zymogen, which enabled us to map the linker domain
between the two chains, whose removal is required for activation. Both
wild-type precursor and reconstituted two-chain form can be subjected
to partial proteolysis with trypsin, and the cleaved products have
maximal cathepsin A activity. Our results suggest that the in vitro activation of BV-produced precursor mimics its in vivo processing taking place in at least two steps: an endoproteolytic
cleavage resulting in a transient intermediate of 34- and 20-kDa,
followed by trimming of the last 14 amino acids at the COOH terminus of
the large chain. Furthermore, we provide evidence that the 34- and
20-kDa reconstituted product is probably identical to the partially
processed intermediate, which is a naturally occurring catalytically
inactive form. A similar processing pattern was observed earlier for
PPCA precursor over-expressed in COS-1 cells(8, 14) .
In this case the 34- and 20-kDa intermediate is seen only transiently,
indicating that its half-life may be too short and its quantity too low
to be detected in normally expressing cultured fibroblasts. There is an
interesting analogy between the processing and activation of PPCA and
that of other serine carboxypeptidases. This family of proteases
comprises single- and two-chain enzymes that are present in different
species, ranging from yeast to fungi, plants, and
humans(9, 10) . Some of the plant peptidases have been
purified in an active form composed of two chains that have been
sequenced at their NH termini(32) . In the case of
barley carboxypeptidase I, the two mature subunits of the enzyme
originate from a single-chain precursor that in addition contains a
stretch of amino acids separating the two chains(33) . This
closely resembles the processing of PPCA zymogen and provides another
verified example in a homologous carboxypeptidase of COOH-terminal
trimming after the initial endoproteolytic step. The analogy in
maturation events might indicate a similar function for the linker
domain in the plant carboxypeptidase and PPCA precursors, namely to
keep these forms in an inactive state. In yeast carboxypeptidase Y such
an inactivation function resides within its propeptide segment, which
must obstruct the access of a substrate to the preformed active
site(34) . Limited proteolysis with trypsin removes this
inactivating segment and exposes the active site. Removal of the linker
domain during maturation of the PPCA zymogen may induce major
conformational changes on the protein necessary to uncover its active
site. Alternatively, this peptide may only mask the active site on an
already correctly folded polypeptide. These hypotheses are currently
verified on the basis of the three-dimensional structure of the
uncleaved precursor molecule.