Characterization of rat cathepsin E and mutants with
changed active-site residues and lacking propeptides and
N-glycosylation, expressed in human embryonic kidney
293T cells
Takayuki Tsukuba
1
, Shinobu Ikeda
2
, Kuniaki Okamoto
2
, Yoshiyuki Yasuda
1
, Eiko Sakai
2
,
Tomoko Kadowaki
1
, Hideaki Sakai
2
and Kenji Yamamoto
1
1 Department of Pharmacology, Graduate School of Dental Science, Kyushu University, Fukuoka, Japan
2 Department of Oral Molecular Pharmacology, Graduate School of Biomedical Sciences, Nagasaki University, Japan
Cathepsin E (EC 3.4.23.24) is an intracellular aspartic
proteinase of the A1 family, which consists of two
identical subunits of 42 kDa (reviewed in [1,2]). Evi-
dence suggests that it is initially synthesized as a pre-
proenzyme and is targeted to the correct destination
after proteolytic processing and carbohydrate modifi-
cation. Differing from the definite localization of the
analogous lysosomal aspartic proteinase, cathepsin D,

microscopy. Consistently, pulse–chase analysis revealed that the initially
synthesized pro-cathepsin E was processed to the mature enzyme within
a 24 h chase. This process was completely inhibited by brefeldin A and
bafilomycin A, indicating its transport from the endoplasmic reticulum
(ER) to the endosomal acidic compartment. Mutants with Asp residues
in the two active-site consensus motifs changed to Ala and lacking the
propeptide (Leu23-Phe58) and the putative ER-retention sequence
(Ser59-Asp98) were neither processed nor transported to the endosomal
compartment. The mutant lacking the ER-retention sequence was rapidly
degraded in the ER, indicating the importance of this sequence in correct
folding. The single (N92Q or N324D) and double (N92Q ⁄ N324D) N-glyco-
sylation-deficient mutants were neither processed into a mature form nor
transported to the endosomal compartment, but were stably retained in the
ER without degradation. These data indicate that the catalytic activity,
propeptides, and N-glycosylation of this protein are all essential for its pro-
cessing, maturation, and trafficking.
Abbreviations
DMEM, Dulbecco’s modified Eagle’s medium; ER, endoplasmic reticulum; HEK, human embryonic kidney; LAMP, lysosome-associated
membrane protein.
FEBS Journal 273 (2006) 219–229 ª 2005 The Authors Journal compilation ª 2005 FEBS 219
to the plasma membrane. The enzyme from erythro-
cytes [15] is N-glycosylated mostly with complex-type
oligosaccharide chains, whereas that from gastric cells
[16] has high mannose-type oligosaccharides. Cathep-
sin E is also detected in the ER and Golgi complex in
various cell types, including gastric cells [12,13], human
M cells [5], Langerhans cells, and interdigitating reti-
culum cells [4]. However, fundamental information on
the biosynthesis, processing, and intracellular traffick-
ing of this protein as well as its physiological signifi-

antibodies to mature rat cathepsin E or rat pro-cathep-
sin E (Fig. 1). The transfected cells gave two intense
bands with apparent molecular masses of 46 and
42 kDa with antibodies to mature cathepsin E. Anti-
bodies to pro-cathepsin E, however, reacted with only
the 46-kDa form, indicating that the 46-kDa and
42-kDa bands are pro-cathepsin E and mature cathep-
sin E, respectively.
To follow the biosynthesis and processing of wild-
type cathepsin E, the transfected cells were labeled
with [
35
S]methionine for 30 min and chased in com-
plete Dulbecco’s modified Eagle’s medium (DMEM)
containing unlabeled methionine for different periods
of time up to 24 h. At the end of the chase, cells and
culture media were collected separately. The cell lysate
and culture medium were subjected to immunopreci-
pitation with antibodies to mature cathepsin E, and
the immunoprecipitates were then analyzed by SDS ⁄
PAGE followed by fluorography (Fig. 2A). After
30-min pulse labeling, cathepsin E was observed
mainly as a 46-kDa precursor with a small amount of
the 42-kDa mature form. A small amount of the pro-
enzyme was released into the culture medium as a
48-kDa form and accumulated during the chase
period. The difference in the molecular mass between
the extracellular and intracellular proforms is probably
due to the difference in their carbohydrate modifica-
tions, because the former has endoglycosidase H-

synthesis, sorting, transport, and degradation of pro-
teins and other macromolecules [19–21]. When the
transfected cells were treated with bafilomycin A1, a
specific inhibitor of vacuolar-type H
+
-ATPase, the
processing and maturation of pro-cathepsin E was
completely inhibited, with a concomitant increase in
a secreted form of pro-cathepsin E in the culture
medium (Fig. 2C). Given that bafilomycin A1 effect-
ively inhibits acidification of intracellular acidic organ-
elles including endosomes, lysosomes, and phagosomes
without perturbation of the formation of intracellular
organelles and without alteration of the morphology
of vacuolar compartments, resulting in the profound
inhibition of the endosomal ⁄ lysosomal degradation of
macromolecules and targeting of lysosomal acid hydro-
lases and cholesterol to the lysosome and processing of
various secretory proteins including prohormones in
the trans-Golgi network [22], our results indicate that
acidification of intracellular acidic compartments is
necessary for the processing and maturation of pro-
cathepsin E.
To determine the intracellular localization of wild-
type cathepsin E expressed in HEK-293T cells, indirect
immunofluorescence staining was performed (Fig. 3A).
With antibodies to mature cathepsin E, the transfected
cells showed mainly punctate staining and partly
reticular staining over the whole cytoplasm, consistent
with staining of the lysosome-associated membrane

the catalytic activity is essential for the processing and
maturation of this protein.
A
B
C
Fig. 2. Pulse–chase analysis of wild-type cathepsin E expressed in
HEK-293T cells. (A) The transfected cells were metabolically labeled
with [
35
S]methionine ⁄ cysteine for 30 min and chased for the times
indicated. Cathepsin E in the cell lysate and culture medium was
immunoprecipitated with antibodies to mature rat cathepsin E, and
analyzed by SDS ⁄ PAGE under reducing conditions and fluorogra-
phy. (B, C) The transfected cells were preincubated at 37 °C for
3 h in the presence of 5 lgÆmL
)1
brefeldin A (B) or 0.5 lM bafilomy-
cin A1 (C). The cells were labeled with [
35
S]methionine ⁄ cysteine
for 30 min and chased for the times indicated in the continued
presence of the drugs. The cell lysate and the culture medium
were immunoprecipitated with antibodies to mature rat cathep-
sin E. The immunoprecipitates were analyzed by SDS ⁄ PAGE under
reducing conditions followed by fluorography.
T. Tsukuba et al. Recombinant cathepsin E and its mutants
FEBS Journal 273 (2006) 219–229 ª 2005 The Authors Journal compilation ª 2005 FEBS 221
Role of the propeptides of cathepsin E in
processing and maturation
Recent studies have suggested that the propeptides of

S]methionine for 30 min and chased for different
periods up to 24 h (Fig. 5B). In the cells, this mutant
protein was first synthesized as a 42-kDa precursor but
was neither processed nor matured even after a 24-h
chase period. Little protein was released into the med-
ium. Consistent with these data, immunofluorescence
microscopy revealed that this mutant protein was
found mostly in the Bip-positive ER compartment, but
not LAMP-1-positive organelles (Fig. 3B).
Finley & Kornfeld [31] expressed various chimeric
proteins between cathepsin E and pepsinogen in
monkey Cos 1 cells and analyzed their targeting and
subcellular localization. They showed that the amino-
acid sequence 1–48 of human mature cathepsin E,
which corresponds to 55–103 of the signal sequence
[32], appeared to be essential for the retention of cath-
epsin E in the ER. We thus examined whether this
putative ER-retention sequence is also required for the
processing, maturation, and trafficking of cathepsin E
to the appropriate destination using a mutant lacking
most of the ER-retention sequence (Ser59-Asp98).
Pulse–chase analysis revealed that this mutant protein
was initially synthesized as a 41-kDa precursor but not
processed into a mature form. Importantly, this
mutant protein was rapidly degraded in the cells dur-
ing a chase period up to 2 h without any detectable
formation of its mature form (Fig. 5C). In addition,
we found that the level of expression of this mutant
A
B

rography. The arrows indicate degradation products of the DER-ret.
mutant.
T. Tsukuba et al. Recombinant cathepsin E and its mutants
FEBS Journal 273 (2006) 219–229 ª 2005 The Authors Journal compilation ª 2005 FEBS 223
was low compared with wild-type-cathepsin E and
other mutant proteins. It was found in the Bip-positive
ER compartment, but not in LAMP-1-positive organ-
elles (Fig. 3C). These results strongly suggest that the
putative ER-retention sequence is absolutely required
for the correct folding, processing maturation, and tar-
geting of cathepsin E to the endosomal compartment.
Characterization of N-linked oligosaccharide
chains of cathepsin E
We have previously demonstrated that cathepsin E
from human erythrocyte membranes [9,15] and rat
microglia [6] is N-glycosylated with complex-type
oligosaccharides, whereas the enzyme from rat spleen
[9,33] and rat stomach [16] has high-mannose-type
oligosaccharide chains. These results suggest that the
nature of the N-glycosylation of cathepsin E varies
with cell type or its cellular localization. We thus ana-
lyzed the nature of the oligosaccharide chains of rat
cathepsin E in the transfected cells and then assessed
the role of N-glycosylation in its folding, processing,
maturation, and subcellular trafficking. HEK-293T
cells expressing wild-type cathepsin E were pulse-labe-
led with [
35
S]methionine for 30 min and chased with
unlabeled methionine for 24 h. The intracellular and

H treatment, wild-type cathepsin E was not phosphoryl-
ated at all, suggesting that this may serve to sort cathep-
sin E from lysosomal enzymes.
Role of the N-glycosylation of cathepsin E
in processing and maturation
To determine the role of N-glycosylation in the correct
folding, processing, maturation, and subcellular traf-
ficking of cathepsin E, we constructed mutants lack-
ing one or two N-glycosylation sites by site-directed
mutagenesis. We have previously shown that the
A
B
Fig. 6. Characterization of oligosaccharide chains of wild-type cath-
epsin E and endogenous cathepsin D. (A) HEK-293 cells expressing
wild-type cathepsin E were pulse-labeled with [
35
S]methionine ⁄
cysteine for 30 min and chased for 24 h. The cell lysate and
the culture medium were immunoprecipitated with antibodies to
mature rat cathepsin E. The immunoprecipitates were then incuba-
ted at 37 °C for 18 h with or without endoglycosidase H. The
mixtures were analyzed by SDS ⁄ PAGE under reducing condi-
tions followed by fluorography. (B) The transfected cells were
pulse-labeled with [
32
P]orthophosphate for 12 h. The cell lysate and
the culture medium were immunoprecipitated with antibodies to
either mature rat cathepsin E or rat cathepsin D. The immuno-
precipitates were incubated at 37 °C for 18 h with or without
endoglycosidase H. The mixtures were analyzed by SDS ⁄ PAGE

lytic activity, propeptide, ER-retention motif, and
N-glycosylation are all essential for its processing,
maturation, and intracellular trafficking to the endo-
somal compartment. This conclusion is based on sev-
eral lines of evidence. First, the pulse–chase analysis
showed that the wild-type enzyme in the transfected
cells was processed from pro-cathepsin E to the
mature form within a 24-h chase period. Consistent
with this finding, immunofluorescence microscopy
revealed that wild-type cathepsin E was found mostly
in LAPM-1-positive organelles and significantly in the
ER. Given that cathepsin E in rat microglia is mainly
localized in endosome-like vacuoles distinct from typ-
ical lysosomes, as determined by immunoelectron
microscopy [6] and that the localization of this
enzyme in mouse microglia is consistent with that of
LAMP-2-positive organelles [36], wild-type cathep-
sin E in the transfected cells is probably targeted to
the endosomal compartment. However, the processing
of wild-type cathepsin E in the transfected cells
appears to be slower than that of natural cathepsin E
in rat microglia [6]. This is probably due to the dif-
ference in the level of expression of cathepsin E
between the transfected cells and the primary cultured
microglia. In contrast, none of the mutants were
processed or targeted to the endosomal compartment.
Interestingly, the intracellular localization of recom-
binant cathepsin E appears to vary with the cell type
used. In mouse L cell and monkey Cos 1 cells, the
expressed human cathepsin E is localized in the ER as

led medium for the times indicated. The cell lysate and culture
medium were immunoprecipitated with antibodies to mature rat
cathepsin E. The immunoprecipitates were analyzed by SDS ⁄ PAGE
under reducing conditions followed by fluorography. (B) N92Q; (C)
N324D; (D) N92Q ⁄ N324D.
T. Tsukuba et al. Recombinant cathepsin E and its mutants
FEBS Journal 273 (2006) 219–229 ª 2005 The Authors Journal compilation ª 2005 FEBS 225
activity is necessary for processing and maturation.
Although pro-cathepsin D is capable of acid-dependent
autoactivation to yield catalytically active pseudo-cath-
epsin D [38–40], lysosomal cysteine proteinase(s) is
required to accomplish proteolytic removal of the entire
propeptide [29,41]. BACE ⁄ memapsin, a membrane-type
aspartic proteinase, is also processed by furin-like con-
vertase(s) [42–44]. In contrast, secretory-type aspartic
proteinases, such as pepsin A, chymosin and gastricsin,
are capable of acid-dependent autoactivation to yield
their mature forms without the aid of other proteinases
[45,46]. Therefore, it is of interest that cathepsin E
undergoes acid-dependent autoproteolysis to yield
mature cathepsin E in a similar manner to secretory
aspartic proteinases rather than nonsecretory enzymes.
Thirdly, whereas the cathepsin D mutant lacking its
propeptide expressed in mouse Ltk
–
cells was rapidly
degraded, probably in the ER [47], the propeptide-
deletion cathepsin E mutant in HEK-293T cells was
relatively stable in the ER but was neither processed
nor targeted to the endosomal compartment, indicating

required for correct folding but to be essential for
processing, maturation, and trafficking.
Moreover, we show for the first time that wild-type
cathepsin E is not phosphorylated on either the poly-
peptide backbone or the oligosaccharide chains. Pre-
vious studies have shown that mannose 6-phosphate
receptors participate in general in the sorting of indi-
vidual lysosomal proteins, albeit with variable effi-
ciency [35,48]. The enzyme phosphotransferase, which
transfers phosphate to the high-mannose oligosaccha-
ride chain of individual lysosomal proteins, recognizes
conformational determinants on the proenzymes [49].
The phosphorylation of cathepsin D occurs mainly at
the large oligosaccharide chain of two glycosylation
sites [50]. In agreement with previous studies, this
work indicates that cathepsin D is apparently phos-
phorylated on its oligosaccharide chains. Therefore, we
conclude that the sorting and segregation of cathep-
sin E from other secretory proteins is independent of
mannose 6-phosphate mechanisms.
Experimental procedures
Materials
[
35
S]Methionine ⁄ cysteine and Protein A–Sepharose were
purchased from Amersham Biosciences (Piscataway, NJ,
USA). Pansorbin was from Calbiochem (La Jolla, CA,
USA). The pcDNA 3.0 ⁄ Amp plasmid, DMEM, methio-
nine-free DMEM, and Opti-Mem were from Invitrogen
(Carlsbad, CA, USA). Endoglycosidase H was from Boeh-

were transfected into pcDNA 3.0 plasmid for heterologous
expression in mammalian cells.
Tissue culture and transfection
HEK-293T cells were maintained in DMEM supplemented
with 10% fetal calf serum, 50 UÆmL
)1
penicillin and
50 lgÆmL
)1
streptomycin (complete DMEM) in a 37 °C
incubator with 5% CO
2
. Wild-type cathepsin E and its
mutants were expressed in HEK-293T cells after transfec-
tion by the calcium phosphate precipitation method using
10 lg expression plasmid in a 100-mm tissue culture plate
[24]. Cells were grown to  90% confluency by overnight
incubation in complete DMEM.
Preparation of cell lysate and culture medium
The tissue culture media were collected after 48 h of transfec-
tion and subjected to centrifugation at 16 000 g for 20 min.
The supernatants were concentrated 10-fold to 500 lL ⁄ plate
using Centriprep-30 and Microcon-30 concentrators. The
cells were washed twice with NaCl ⁄ P
i
, removed from the
plates with a rubber scraper, and subjected to centrifugation
at 300 g for 5 min. The sedimented cells were suspended in
NaCl ⁄ P
i

4 °C for 16 h. The immunoprecipitates were mixed with
40 lL Protein A–Sepharose beads (50% gel suspension) for
3 h at 4 °C with gentle agitation. The beads were washed
3 times with 0.1% SDS ⁄ 0.1% Triton X-100 ⁄ 200 mm
EDTA ⁄ 10 mm Tris ⁄ HCl (pH 7.5), washed another 3 times
with the same buffer containing 1 m NaCl and 0.1%
sodium lauryl sarcosinate, then washed twice with 5 mm
Tris ⁄ HCl (pH 7.0). The beads were boiled for 5 min at
100 °C with 50 mL 0.1% SDS ⁄ 0.5 mm EDTA ⁄ 5% sucrose ⁄
5mm Tris ⁄ HCl (pH 8.0) with 2-mercaptoethanol.
SDS ⁄ PAGE and immunoblotting
SDS ⁄ PAGE and immunoblotting were performed as des-
cribed previously [24].
Endoglycosidase digestion
Radiolabeled immunoprecipitates were dissolved by being
boiled for 5 min at 100 °Cin10lL5mm Tris ⁄ HCl
(pH 8.0) containing 0.2% SDS. To this was added 90 lL
50 mm sodium acetate buffer (pH 6.0) containing 0.75%
Triton X-100 and 100 lgÆmL
)1
protease inhibitor cocktail
and 10 mU endoglycosidase H, and then incubated at
37 °C for 18 h. Reactions were stopped by boiling the sam-
ples in SDS ⁄ PAGE sample buffer.
Immunofluorescence microscopy
The transfected cells were grown on glass coverslips. The
cells were briefly washed with NaCl ⁄ P
i
and fixed with 3.7%
formaldehyde in NaCl ⁄ P

Boveri E, Bosi F, Cornaggia M, Capella C, et al. (1993)
Cathepsin E in antigen-presenting Langerhans and
interdigitating reticulum cells. Its possible role in anti-
gen processing. Eur J Histochem 37, 19–26.
5 Finzi G, Cornaggia M, Capella C, Fiocca R, Bosi F,
Solcia E & Samloff IM (1993) Cathepsin E in follicle
associated epithelium of intestine and tonsils: localiza-
tion to M cells and possible role in antigen processing.
Histochemistry 99, 201–211.
6 Sastradipura DF, Nakanishi H, Tsukuba T, Nishishita
K, Sakai H, Kato Y, Gotow T, Uchiyama Y & Yama-
moto K (1998) Identification of cellular compartments
involved in processing of cathepsin E in primary cul-
tures of rat microglia. J Neurochem 70, 2045–2056.
7 Yamamoto K & Marchesi VT (1984) Purification and
characterization of acid proteinase from human erythro-
cyte membranes. Biochim Biophys Acta 790, 208–218.
8 Takeda M, Ueno E, Kato Y & Yamamoto K (1986)
Isolation, and catalytic and immunochemical properties
of cathepsin D-like acid proteinase from rat erythro-
cytes. J Biochem (Tokyo) 100, 1269–1277.
9 Takeda-Ezaki M & Yamamoto K (1993) Isolation and
biochemical characterization of procathepsin E from
human erythrocyte membranes. Arch Biochem Biophys
304, 352–358.
10 Kageyama T & Takahashi K (1980) A cathepsin D-like
acid proteinase from human gastric mucosa. Purification
and characterization. J Biochem (Tokyo) 87, 725–735.
11 Muto N, Murayama-Arai K & Tani S (1983) Purifica-
tion and properties of a cathepsin D-like acid proteinase

complex and accumulation of secretory proteins in the
endoplasmic reticulum. J Biol Chem 263, 18545–18552.
18 Doms RW, Russ G & Yewdell JW (1989) Brefeldin A
redistributes resident and itinerant Golgi proteins to the
endoplasmic reticulum. J Cell Biol 109, 61–72.
19 Mellman I, Fuchs R & Helenius A (1986) Acidification
of the endocytic and exocytic pathways. Annu Rev
Biochem 55, 663–700.
20 Tycko B & Axfield FR (1982) Rapid acidification of
endocytic vesicles containing a2-macroglobulin. Cell 28,
643–651.
21 Merion M, Schlesinger P, Brooks M, Moehring JM,
Moehring TJ & Sly WS (1983) Defective acidification of
endosomes in Chinese hamster ovary cell mutants ‘cross
resistant’ to toxins and viruses. Proc Natl Acad Sci USA
80, 5315–5319.
22 Xu H & Shields D (1994) Prosomatostatin processing in
permeabilized cells. Endoproteolytic cleavage is
mediated by a vacuolar ATPase that generate an acidic
pH in the trans-Golgi network. J Biol Chem 269,
22875–22881.
23 Athauda SBP, Takahashi T, Kageyama T & Takahashi
K (1991) Autocatalytic processing of procathepsin E to
cathepsin E and their structural differences. Biochem
Biophys Res Commun 175, 152–158.
24 Liu J, Tsukuba T, Okamoto K, Ohishi M & Yamamoto
K (2002) Mutational analysis of residues in two consen-
sus motifs in the active sites of cathepsin E. J Biochem
(Tokyo) 132, 493–499.
25 Yonezawa S, Takahashi T, Wang X, Wong RNS, Hart-

Taggart RT (1989) Human gastric cathepsin E.
Predicted sequence, localization to chromosome 1, and
sequence homology with other aspartic proteinases.
J Biol Chem 264, 16748–16753.
33 Okamoto KYuH, Misumi Y, Ikehara Y & Yamamoto
K (1995) Isolation and sequencing of two cDNA clones
encoding rat spleen cathepsin E and analysis of the acti-
vation of purified procathepsin E. Arch Biochem Biophys
322, 103–111.
34 Pohlmann RA, Waheed A, hasilik A & von Figura K
(1982) Synthesis of phosphorylated recognition marker
in lysosomal enzymes is located in the cis part of Golgi
apparatus. J Biol Chem 257, 5323–5325.
35 Kornfeld S (1992) Structure and function of the man-
nose 6-phosphate ⁄ insulinlike growth factor II receptors.
Annu Rev Biochem 61, 307–330.
36 Nishioku T, Hashimoto K, Yamashita K, Liou SY,
Kagamiishi Y, Maegawa H, Katsube N, Peters C,
von Figura K, Saftig P, et al. (2002) Involvement of
cathepsin E in exogenous antigen processing in primary
cultured murine microglia. J Biol Chem 277, 4816–4822.
37 Tsukuba T, Hori H, Azuma T, Takahashi T, Taggart
RT, Akamine A, Ezaki M, Nakanishi H, Sakai H &
Yamamoto K (1993) Isolation and characterization of
recombinant human cathepsin E expressed in Chinese
hamster ovary cells. J Biol Chem 268, 7276–7282.
38 Conner GE (1989) Isolation of procathepsin D from
mature cathepsin D by pepstatin affinity chromatogra-
phy. Autocatalytic proteolysis of the zymogen form of
the enzyme. Biochem J 263, 601–604.

-terminal amino acid sequences. Eur J Biochem
141, 261–269.
46 Foltmann B (1988) Activation of pepsinogens. FEBS
Lett 241, 69–72.
47 Conner GE (1992) The role of the cathepsin D propep-
tide in sorting to the lysosome. J Biol Chem 267, 21738–
21745.
48 Pohlmann R, Boeker MW & von Figura K (1995) The
two mannose 6-phosphate receptors transport distinct
complements of lysosomal proteins. J Biol Chem 270,
27311–27318.
49 Cuozzo JW, Tao K, Wu QL, Young W & Sahagian
GG (1995) Lysine-based structure in the proregion of
procathepsin L is the recognition site for mannose phos-
phorylation. J Biol Chem 270, 15611–15619.
50 Hasilik A & Neufeld EF (1980) Biosynthesis of lysoso-
mal enzymes in fibroblasts. Phosphorylation of mannose
residues. J Biol Chem 255, 4946–4950.
51 Kunkel TA (1985) Rapid and efficient site-specific muta-
genesis without phenotypic selection. Proc Natl Acad
Sci USA 82, 488–492.
T. Tsukuba et al. Recombinant cathepsin E and its mutants
FEBS Journal 273 (2006) 219–229 ª 2005 The Authors Journal compilation ª 2005 FEBS 229