From the
When the 44-amino acid propeptide of human procathepsin D was
deleted by mutagenesis in vitro, the mature protein was stably
expressed and secreted from transfected mammalian cells. The secreted
protein was correctly folded as judged by its binding to
pepstatinyl-agarose. We were unable to detect lysosomal targeting of
the propeptide-deleted protein, and targeting was not restored by the
substitution of the propeptides from pepsin or renin. We conclude that
its propeptide is not essential for the folding of nascent cathepsin D.
Efficient lysosomal targeting in mammalian cells appears to require the
precursor form of the molecule.
Cathepsin D, an abundant, soluble hydrolase, is a member of the
aspartic proteinase gene family, which includes the secretory proteins
pepsin, chymosin, and renin (Tang and Wong, 1987), all of which share
substantial amino acid sequence identity. The mature enzymes show a
bilobed structure flanking a large active site cleft (Davies, 1990)
which, in the precursor form, is occluded by an approximately 45-amino
acid propeptide. Although not as highly conserved as the mature
sequences of the enzymes, the propeptides also show structural
similarities and several invariant residues (Foltmann, 1988; Koelsch
et al., 1994).
Unlike the other members of the aspartic
proteinase family, which are mostly secretory proteins, procathepsin D
is sorted to the lysosome. Before reaching the Golgi apparatus,
procathepsin D is modified so that the oligosaccharides linked to
asparagines 70 and 199 bear mannose 6-phosphates. Upon exiting the
Golgi, procathepsin D is sorted to a prelysosomal/late endosomal
compartment by binding to mannose 6-phosphate receptors. As the pH in
this compartment drops, the receptors dissociate and recycle, whereas
the ligand is delivered to the primary lysosome (Kornfeld, 1990;
Erickson, 1989). Procathepsin D can also follow a mannose
6-phosphate-independent pathway to the lysosome in some cell types
(Glickman and Kornfeld, 1993).
Once committed to the lysosome, human
procathepsin D undergoes three proteolytic maturation steps: cleavage
of its 44-amino acid propeptide, removal of a COOH-terminal dipeptide,
and digestion of a six-amino acid loop around residue 100 (Erickson,
1989; Hasilik, 1992; Delbrück et al., 1994). A 46-kDa
form, lacking the propeptide, occurs in both late endosomes and the
primary lysosome as a biosynthetic intermediate. When procathepsin D is
overexpressed, some of the 52-kDa form of the protein exits the cell
via the constitutive secretory pathway (Hasilik, 1992).
High
resolution structures are known for many mature aspartic proteinases
(Davies, 1990), including cathepsin D (Metcalf and Fusek, 1993; Baldwin
et al., 1993), but pepsinogen is the only gene family member
for which a precursor structure has been published (James and Sielecki,
1986; Hartsuck et al., 1992). In pepsinogen, relative to
pepsin, two differences are apparent: 1) the active site cleft is
occupied by the propeptide, and 2) the amino terminus moves about 40
Å from its position in the precursor form, upon activation,
replacing one of the strands in the connecting
All of the characterized aspartic proteinases are
synthesized via the endoplasmic reticulum/Golgi pathway and are exposed
to similar folding conditions and chaperones (Nilsson and Anderson,
1991). It is thus likely that all members of the family follow a
similar pathway of protein folding in vivo. Active renin has
been successfully expressed from transfected mammalian cells, after
deletion of its propeptide sequence by mutagenesis in vitro,
by a number of groups (Chidgey and Harrison, 1990; Chu et al.,
1990; Norman et al., 1992; Rothwell et al., 1993).
Pepsinogen can be reversibly refolded following denaturation, whereas
conditions for successful folding of mature pepsin activity in the
absence of its propeptide have not been found, despite extensive
efforts (Ahmad and McPhie, 1978; Pain et al., 1985; Lin et
al., 1993). These results suggest that some, but probably not all,
aspartic proteinases can fold efficiently in the absence of their
propeptides. Thus renin is unlike bacterial
Conner (1992) reported that an imperfect deletion of
the propeptide sequence of human procathepsin D abrogated stable
expression of the transfected protein in mammalian cells. He also
demonstrated that attachment of the propeptide to the NH
We
therefore constructed and expressed in rodent and human cell lines a
mutant of human procathepsin D with a precise deletion of the
propeptide. Overexpression resulted in secretion of a correctly folded
protein capable of binding to the active site reagent pepstatin. We
were unable to detect delivery to the lysosome of the
propeptide-deleted cathepsin D. Targeting was not restored by
substitution of the propeptide from human pepsin or renin.
Enzymes for molecular biology were from New England Biolabs
(Beverly, MA) and used according to the manufacturer's
instructions. Radiochemicals were from DuPont NEN, tissue culture
reagents from Life Technologies, Inc., and biochemicals from Sigma.
Oligodeoxynucleotides were synthesized by the Biopolymer Sequencing and
Synthesis Facility, Department of Biochemistry and used without further
purification, following deblocking and lyophilization.
Expressed
aspartic proteinase polypeptides were immunoprecipitated by standard
procedures (Harlow and Lane, 1988; Faust et al., 1987),
followed by electrophoresis on denaturing, reducing 12.5%
polyacrylamide gels (Laemmli, 1970), and fluorography with ENHANCE
(DuPont NEN) according to the manufacturer's instructions.
Autoradiographic data were quantified by digitized densitometric
analysis using a Macintosh 840AV computer with video camera and
National Institutes of Health Image 1.55 software (Wayne Rasband,
National Institutes of Health, Bethesda, MD).
Cathepsin D was
immunoprecipitated with a rabbit polyclonal antibody prepared against
the mature human enzyme purified from placentas.
All of the protein
constructs tested here carry a widely used 13 amino acid epitopic
extension (Kolodziej and Young, 1991) derived from the human c- myc gene and recognized by an available monoclonal antibody (Evan
et al., 1985), which permits affinity purification of labeled
mutant protein from transfected cells (Sachdev et al., 1991).
It can also be used to monitor delivery of cathepsin D to the lysosome,
since the myc epitope is proteolytically removed there
(Pelham, 1988). We have shown (Sachdev et al., 1991)
Mutagenesis in
vitro followed the procedure of Kunkel et al. (1987).
Single-stranded template DNAs were purified from phagemids grown in
Escherichia coli CJ236 supplemented with uridine. Phagemids
were precipitated twice with polyethylene glycol before
deproteinization. Mutagenic oligonucleotides consisted of complementary
anchors flanking the mutated region (Sambrook et al., 1989).
The double-stranded DNA products were transformed into E. coli strain XL1-B, and the resultant colonies screened by filter
hybridization with
The
control version of procathepsin D, whose expression is driven by a
cytomegalovirus promoter, is referred to as procathepsin D-myc (CDM), a
52-kDa bis- N-glycosylated, full-length procathepsin D, with
its carboxyl terminus extended by a 13-amino acid myc epitope
(Sachdev et al., 1991). The expressed fusion protein was
originally constructed by Pelham (1988). A parallel version of human
prorenin with the myc extension has been described
elsewhere.
Tissue culture of human 293 and Chinese hamster CHO-L76
cells, transfection with DNAs, labeling with
[
Binding to
pepstatinyl-agarose was carried out by standard means (Conner, 1989).
When cathepsin D with its 44-amino acid propeptide deleted
(CDM
When human preprorenin cDNA is transfected in mammalian cells
with its propeptide sequence deleted, mature active protein is
expressed at levels only moderately below those of the wild-type
proenzyme (Chidgey and Harrison, 1989; Chu et al., 1991;
Norman et al., 1992; Rothwell et al., 1993). We thus
anticipated that similarly deleted procathepsin D could be expressed
successfully in mammalian cells. This prediction was confirmed by the
results shown in Fig. 1. The reduction in the level of expression
of procathepsin D relative to cathepsin D was similar to that seen in
the prorenin:renin experiments. It is possible that this reduction
reflects a decrease in the efficiency of folding of nascent
polypeptides in the endoplasmic reticulum in the absence of their
propeptides.
The propeptide-deleted (
We were initially
concerned that the propeptide-deleted cathepsin D might display some
proteolytic activity during its sorting within acidic intracellular
compartments. Such activity could be deleterious for the expressing
cells. When we combined the
Conner (1992) expressed a
mutant of procathepsin D with most of the propeptide sequence removed.
The encoded protein retains its 20-amino acid signal peptide, followed
by 8 amino acids which replace the first 2 residues of the mature
protein. This 8-amino acid replacement consists of LVRI (the first 4
residues of the propeptide) plus RNSG. The construct which we have
reported here fuses the signal peptide directly to residue +1 of
the mature protein. Conner's mutant protein was unstable when
expressed in mammalian cells. In pepsinogen the first 6 residues of the
propeptide sequence form the first strand of a six-stranded
antiparallel
Sagherian et al. (1994) and Tao et al. (1994) have
expressed propeptide deletions of the lysosomal enzymes
The observation by van den Hazel et al. (1993) that
formation of active proteinase A in yeast requires co-expression of the
propeptide in cis or in trans may reflect requirements for this
sequence in vacuolar targeting (Klionsky et al.,
1988) and not directly in protein folding. It is also possible that the
propeptide region of the endogenous cathepsin D in our mammalian cells
transfected with the CDM
Baranski et al. (1990, 1992) have concluded that the major
lysosomal targeting determinant (as assayed in Xenopus oocytes) of cathepsin D lies in the carboxyl-terminal lobe, in
particular Lys-203 and residues 265-292, although lesser
contributions are made by the amino-terminal portion, including the
propeptide, as well (Cantor et al., 1992). Glickman and
Kornfeld (1993) tested many of the mutants used by Baranski et al. (1990, 1992) and Cantor et al. (1992) in Epstein-Barr
virus-transformed B lymphocytes from a patient with I-cell disease
(phosphotransferase deficiency). They concluded that the propeptide
sequence did not contribute to lysosomal targeting in this system. From
these results propeptide-deleted cathepsin D would be expected to
target to the lysosome.
We were thus surprised by the results in
Fig. 3
that showed deletion of the propeptide blocked targeting of
the protein to the lysosome in transfected Chinese hamster cells. To
confirm this observation, we combined the
Conner (1992) demonstrated that attachment of the cathepsin D
propeptide to secretory
In an attempt to restore the zymogen conformation to
cathepsin D deleted of its propeptide, we replaced the cathepsin D
propeptide by the similar propeptides from human pepsinogen A and human
prorenin. These peptides show less sequence identity than the mature
proteins, although they conserve a number of critical residues and the
capability to form several
Our results
suggest that the targeting to the lysosome of human procathepsin D in
mammalian fibroblasts requires determinants in the propeptide sequence.
Such targeting determination occurs in the routing of proteinase
precursors to the vacuole in yeast (Klionsky et al., 1988;
Valls et al., 1990). The modification of certain blood
clotting factors depends on propeptide sequence determinants which
signal
We thank Drs. P. M. Hobart, R. T. Taggart, and I. M.
Samloff for generously supplying antibodies and cDNA clones, Deepali
Sachdev for helpful discussions, and Richard Sunvison for assistance
with the pepstatinyl agarose binding experiments.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-sheet, which was
previously contributed by the NH
terminus of the
propeptide. The major folded domains and the active site cleft itself
show no gross conformational changes. Crystallographic data are
unavailable for the precursor forms of renin or cathepsin D, but a
striking feature of all the aspartic proteinases is their high degree
of tertiary structural similarity (Davies, 1990). The conservation of
the protein folding pattern in the gene family has made it practical to
create useful computer graphics models of aspartyl proteases for which
sequence but not crystallographic data are available (Hutchins and
Greer, 1991). Thus Hsueh and Baxter (1991) have suggested for prorenin
that the precursor folds into a three-dimensional structure very
similar to that of pepsinogen. Procathepsin D also probably shares
substantial structural similarity to pepsinogen.
(
)
-lytic protease, whose
large propeptide sequence functions as a specific template for the
productive folding of the mature polypeptide chain (Baker et
al., 1992). A number of other secreted subtilisin-related
proteases also require their propeptides to fold correctly (Shinde and
Inouye, 1994).
terminus of
-lactalbumin did not direct the fusion protein
to lysosomes. We wondered if an exact deletion of its propeptide would
permit mature cathepsin D to fold directly, when expressed in the
endoplasmic reticulum of mammalian cells, and if this deletion would
impair targeting to the lysosome, since Baranski et al. (1992)
have shown that the propeptide sequence may contribute determinants for
mannose 6-phosphate modification in Xenopus oocytes.
(
)
Rabbit polyclonal antibodies against human pepsinogen A and
recombinant human prorenin were generously supplied by Dr. I. M.
Samloff (Sepulveda Veterans Administration Hospital, Los Angeles, CA)
and Dr. P. M. Hobart (Pfizer Central Research, Groton, CT). Protein
molecular weight markers for gel electrophoresis were labeled with
[
C]formaldehyde (ICN, Irvine, CA) by reductive
methylation (Jentoft and Dearborn, 1979).
that both human
(293) and rodent (CHO)
(
)
cells efficiently express myc-extended
procathepsin D (CDM) and prorenin in transient transfection assays.
Expressed proteins were stable for 24 h, as shown by pulse-chase
experiments.
Fra et al. (1993) have demonstrated
that COOH-terminally extended versions of CDM can be retained in the ER
lumen. The COOH-terminal epitopic extension offers a probe of the
proteolytic environment to which nascent aspartic proteinase precursors
are exposed, without altering intracellular sorting. Horst et al. (1993) fused lysozyme to the COOH terminus of procathepsin D to
monitor delivery to the lysosomes of CHO cells.
P-end-labeled mutagenic
oligonucleotides. Filters were repetitively washed at increasing
temperatures and autoradiographed to identify clones with no mismatches
to the oligonucleotides (Sambrook et al., 1989). Mutants were
verified by restriction mapping and partial DNA sequencing (Trevino
et al., 1993). DNAs for transfection into mammalian cells were
purified by the polyethylene glycol precipitation protocol described in
Sambrook et al. (1989) and used without CsCl banding.
The 44-amino acid propeptide sequence (residues
44 through
1) of human procathepsin D was deleted from
CDM by mutagenesis in vitro (Kunkel et al., 1987)
with a 30-mer oligonucleotide complementary to the last 5 codons of the
prepeptide and the first 5 codons of the mature sequence (Faust et
al., 1985). Cathepsin D mutants lacking the glycosylation site at
Asn
have been described.
(
)
These
were combined with the
44/
1 mutation by ligating
fragments of the single mutants via a unique FspI restriction
site at base pair 775 of the cDNA. A series of six mutants with the
propeptides interchanged with the bodies of the mature proteins of CDM,
pepsinogen, and RNM were constructed with 30-mer oligonucleotides and
an overlap extension PCR mutagenesis procedure (Ho et al.,
1989). We use the cathepsin D numbering convention of Faust et al. (1985), in which the propeptide is numbered from
44 to
1.
S]methionine, and immunoprecipitations have
been described.
The 293 (human embryonal kidney) cell line
was obtained from the ATCC. CHO-L76 cells (Cockett et al.,
1991) were from Celltech Ltd. (Berkshire, United Kingdom). Both 293 and
CHO-L76 cells express an adenoviral E1a gene product to enhance
transcription from the cytomegalovirus promoter (Gorman et
al., 1990). Cells were transiently transfected by CaPO
precipitation (Gorman et al., 1990). Transfection
efficiencies were reproducibly greater than 50% as monitored by the
inclusion of 10% pSV2-
-gal DNA and assaying aliquots of cell
lysates for
-galactosidase activity (Gorman, 1985). Two days after
transfection, the cells were changed to Dulbecco's modified
Eagle's medium minus methionine for 2 h, then supplemented with
100 µCi/ml [
S]methionine overnight, or as
indicated in the figure legends. Medium was removed and cell lysates
prepared by three freeze-thaw cycles in the presence of 0.2% Triton
X-100. All samples were treated with a protease inhibitor mix (Faust
et al., 1987) and immunoprecipitated.
44/
1) was expressed in human 293 cells, it was
abundantly secreted, at a level about one-third that of the undeleted
protein, CDM (Fig. 1). This same moderate reduction of expression
is seen when human preprorenin cDNA is transfected in mammalian cells
with its propeptide deleted (Chidgey and Harrison, 1989; Chu et
al., 1991; Norman et al., 1992; Rothwell et al.,
1993).
Figure 1:
Cathepsin D with its propeptide deleted
is secreted from mammalian cells. Human kidney 293 cells transfected
with DNAs expressing procathepsin-myc, CDM ( lanes 1) or
CDM44/
1 ( lanes 2) were labeled overnight
and immunoprecipitated proteins from cell lysates and conditioned media
analyzed.
Both secreted intact procathepsin D-myc and
propeptide-deleted proteins bound efficiently to pepstatinyl-agarose
(Fig. 2), indicating correct folding of the active site.
Immunoprecipitation of the fractions unbound to pepstatinyl agarose
showed that the majority of secreted cathepsin D polypeptides, with or
without the propeptide attached, had bound to the column (data not
shown).
Figure 2:
Secreted CDM and 44/
1
both bind efficiently to pepstatinyl agarose. Conditioned media
containing CDM ( lanes 1 and 2) or
CDM
44/
1 (lanes 3-5) were prepared as in Fig.
1. Lanes 1 and 3 show total labeled proteins in the
media. Lanes 2 and 4 show material specifically bound
to pepstatinyl agarose at pH 3.6 and eluted at pH 8.5. Lane 5 shows the pepstatinyl agarose unbound material. Samples were not
immunoprecipitated.
Human cells cannot readily be used to assay lysosomal
targeting of human procathepsin D, because of the inability to
distinguish between endogenous and transfected proteins. However,
rodent cells express a single chain (46 kDa) enzyme, whereas
lysosomally processing the transfected human protein to a two-chain (31
+ 14 kDa) form (Conner et al., 1989; Horst and Hasilik,
1991). Xenopus oocytes do not process human procathepsin D to
this two-chain form (Faust et al., 1987). When we transfected
CDM44/
1 into rodent CHO-L76 cells, we were unable to
detect any lysosomal delivery of the human protein in multiple
experiments ( Fig. 3and data not shown). Isidoro et al. (1991) have shown that transfected human procathepsin D does not
compete efficiently for targeting to the lysosome with the endogenous
precursor in hamster BHK cells. Therefore we considered it important
also to test the lysosomal targeting of human propeptide-deleted
cathepsin D in human cells.
Figure 3:
Propeptide-deleted cathepsin D does not
target to the lysosome. The experiment was the same as in Fig. 1,
except CHO-L76 cells were used.
We combined the 44/
1
mutation with ones at the second N-linked glycosylation site
Asn
. These latter mutants are not blocked in lysosomal
targeting (since the oligosaccharide at Asn
still supports
the mannose 6-phosphate-mediated pathway) but can be distinguished from
the endogenous human protein.
Loss of glycosylation lowers
the apparent M
of the human heavy chain from about
31,000 to 29,000 (Horst and Hasilik, 1991). When the mutants N199A,
N199D, and N199S were combined with
44/
1, no
targeting to the lysosome was detectable in transfected human 293 cells
(Fig. 4). The data in Fig. 4and in similar experiments
(not shown) were analyzed densitometrically. In those lanes, such as
lanes 4 and 5, where the mutant did not target to the
lysosome, the background in the region where the targeted band should
have been seen was always less than 5% of the density seen for a
successfully targeted mutant. Thus, in these experiments targeting was
reduced at least 20-fold, which was the limit of detection in the
experiment with the highest background. The mutant N199T also gave the
same result (not shown). The secreted, nontargeting mutants appear to
be of somewhat higher M
than expected. We are
presently testing whether this is the result of modification of the
oligosaccharide to the complex type in the Golgi apparatus.
Figure 4:
Lysosomal targeting of monoglycosylated
forms of procathepsin D is abrogated by deletion of the propeptide.
Transfected 293 cells were analyzed as in Fig. 1. A shows cell
lysates and B the corresponding conditioned media. Lanes 1 and 2 are mock-transfected and CDM positive controls.
Lane 3 is CDM N199D, which removed the second glycosylation
site. Lanes 4-6 combine the 44/
1
propeptide deletion with mutations at Asn-199; lane 4 is
N199S, lane 5 is N199A, and lane 6 is N199D.
Successful delivery to the lysosome of an N199X mutant protein results
in the appearance of a 29-kDa cathepsin D heavy chain
band.
To
ascertain if the cathepsin D propeptide carried lysosomal targeting
determinants, cathepsin D, with or without an N199T mutation, was
expressed with its propeptide sequence replaced by that from human
pepsin or human renin. No lysosomal targeting in the absence of the
cathepsin D propeptide sequence was detected (Fig. 5 A).
The mutant proteins were expressed productively as judged by the stable
secretion of precursors into the conditioned media
(Fig. 5 B). The decreased mobility of the secreted
protein in lane 8 (cathepsin D with the propeptide of renin)
may be the consequence of altered oligosaccharide processing at
Asn.
Figure 5:
Cathepsin D targeting to the lysosome
requires its own propeptide sequence. Transfected 293 cells were
analyzed as in Fig. 1. A shows cell lysates, and B shows the corresponding conditioned media. Lane 1 is CDM
positive control. Lanes 2 and 3 are N199D and N199T
mutants, which target to the lysosome. Lanes 4 and 5 are CDM44/
1 and
CDM
44/
1,N199A, which do not target to the lysosome.
Lanes 6 and 7 are CDM N199T with the cathepsin D
propeptide replaced by the propeptide of human renin or human
pepsinogen, respectively. (The sample for lysate ( lane 6) was
lost during handling.) Lanes 8 and 9 correspond to
lanes 6 and 7, but without the mutation at Asn-199.
Lane 10 is the mock-transfected negative
control.
44/
1)
protein bound efficiently to pepstatinyl agarose (Fig. 2). Such
binding has been used as a criterion of correct folding for the
expressed protein, both precursor and mature forms (Conner and Udey,
1990), as well as for chimeras between procathepsin D and pepsinogen
(Glickman and Kornfeld, 1993). Our results (not shown) indicated that
the majority of the secreted, propeptide-deleted cathepsin D was
productively folded. The data in Figs. 1 and 2 suggest that the
propeptide of cathepsin D facilitates protein folding in vivo but is not essential for the process.
44/
1 deletion with the
conversion of the essential active-site Asp-33 to Ser, there was no
change in the expression pattern seen in Fig. 1(data not shown).
A parallel active site mutation in pepsinogen abolished catalytic
activity, without perturbing folding of the active site cleft, as
judged by successful binding of the mutant pepsinogen to pepstatinyl
agarose (Lin et al., 1989).
-sheet connecting the two major lobes. Upon
activation to pepsin, this strand is replaced by the mature amino
terminus (James and Sielecki, 1986; Hartsuck et al., 1992).
The positions of the first 12 residues of the mature sequence differ
greatly in the crystal structures of pepsinogen and pepsin. The first
10 or so residues of both pro- and mature forms of other aspartic
proteinases are probably also constrained by the requirement to fold
into the six-stranded
-sheet. We think it likely that
Conner's mutant, whose sequence was dictated by available
restriction enzyme sites in the DNA sequence, violated the constraints
on this region of the cathepsin D protein sequence. Richo and Conner
(1994) have recently shown that mutation of residues
Leu
, Ile
, or Val
in the human cathepsin D propeptide does not block expression or
targeting to the lysosome in mouse Ltk
cells.
-hexosaminidase B and cathepsin L, respectively, in mammalian
cells. In neither case was the deleted protein able to complete folding
or exit the endoplasmic reticulum. However, both mutants were
constructed using restriction sites in the cDNA sequences, so that the
proteins retained some residues from their precursor amino termini.
44/
1 vector is similarly
able to catalyze folding in trans. This, however, leaves unexplained
the abrogation of lysosomal targeting by deletion of the propeptide.
44/
1
deletion with a series of mutations
which eliminate heavy
chain glycosylation at Asn-199. Deletion of the propeptide from the
monoglycosylated mutants confirmed that the propeptide was necessary
for efficient targeting to the lysosome. Failure to reach the lysosome
was not the result of protein instability, since these double mutants
expressed well and were secreted into the medium of the transfected
cells. Lysosomal targeting was depressed to the limit of detection, at
least 20-fold, by the deletion of the propeptide. A series of lysine to
glutamic acid mutations in the carboxyl-terminal lobe of procathepsin D
was without effect on lysosomal targeting in transfected
fibroblasts,
suggesting that the failure of the propeptide
deletion to target in not an artifact of the mammalian cell system.
-lactalbumin did not convert the fusion to
a lysosomal protein, although the folding of the propeptide in this
construct is unknown. Horst et al. (1993) fused lysozyme to
the carboxyl terminus of wild-type human procathepsin D. When expressed
in CHO cells, the fusion protein was transported to the lysosome and
cleaved. The lysozyme partner had acquired mannose 6-phosphate
modification on an introduced N-linked glycosylation site. The
results of Conner (1992) and Horst et al. (1993) are
compatible with a model in which the major determinants of lysosomal
targeting are carried on the surface of mature cathepsin D rather than
primarily on the propeptide. We were therefore puzzled why the presence
of the propeptide was necessary for lysosomal targeting in the
experiments presented here. In view of the conformational differences
between the precursor and mature forms of aspartic proteinases,
discussed above, it is possible that the propeptide does not itself
carry significant targeting information, but rather the targeting
information resides primarily in the mature protein sequence but is
only correctly configured when the protein is in the proenzyme
conformation.
-helical segments (Foltmann, 1988;
Koelsch et al., 1994). Fusek et al. (1991) have shown
that the human cathepsin D propeptide is a strong inhibitor of bovine
and chicken pepsins, whereas the chicken pepsin propeptide showed no
binding to bovine cathepsin D. These data suggested that interchanging
the propeptides of aspartic proteinases by mutagenesis in vitro should be practical for some combinations. When we carried out
this experiment (Fig. 5), we observed that the renin propeptide
permitted folding and secretion of the mutant protein but lysosomal
delivery was not restored. The propeptide from pepsinogen was less
effective in promoting expression of the fusion protein, consistent
with the results of Fusek et al. (1991).
-carboxylation in a pre-Golgi compartment (Furie and Furie,
1991). Mannose 6-phosphate addition could proceed by a similar
mechanism in mammalian fibroblasts, whereas Xenopus oocytes
(Baranski et al., 1990, 1992) may recognize different features
of human procathepsin D than those which are important in the
homologous targeting assay we have used here.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.