changed active-site residues and lacking propeptides and N-glycosylation, expressed in human embryonic kidney 293T cells Takayuki Tsukuba1, Shinobu Ikeda2, Kuniaki Okamoto2, Yoshiyuki Ya
Trang 1changed active-site residues and lacking propeptides and N-glycosylation, expressed in human embryonic kidney 293T cells
Takayuki Tsukuba1, Shinobu Ikeda2, Kuniaki Okamoto2, Yoshiyuki Yasuda1, Eiko Sakai2,
Tomoko Kadowaki1, Hideaki Sakai2and Kenji Yamamoto1
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,
the intracellular localization of cathepsin E appears to vary with cell type [3–5] In antigen-presenting cells such as macrophages and microglia, cathepsin E is mainly found in the endosomal compartment as a mature form which is N-glycosylated mostly with com-plex-type oligosaccharides [6] In erythrocytes [7–9], gastric cells [10–13], renal proximal tubule cells [13], and osteoclasts [14], cathepsin E is exclusively confined
Keywords
aspartic proteinase; cathepsin E; mutation;
processing; sorting
Correspondence
K Yamamoto, Department of
Pharmacology, Graduate School of Dental
Science, Kyushu University, Higashi-ku,
Fukuoka 812-8582, Japan
Fax: +81 92 642 6342
Tel: +81 92 642 6337
E-mail: kyama@dent.kyushu-u.ac.jp
(Received 10 October 2005, revised 7
November 2005, accepted 14 November
2005)
doi:10.1111/j.1742-4658.2005.05062.x
To study the roles of the catalytic activity, propeptide, and N-glycosylation
of the intracellular aspartic proteinase cathepsin E in biosynthesis, process-ing, and intracellular traffickprocess-ing, we constructed various rat cathepsin E mutants in which active-site Asp residues were changed to Ala or which lacked propeptides and N-glycosylation Wild-type cathepsin E expressed
in human embryonic kidney 293T cells was mainly found in the LAMP-1-positive endosomal organelles, as determined by immunofluorescence 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.
Trang 2to 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-cance remains elusive Given the variability in cellular
localization, we decided that the roles of the catalytic
activity, propeptides, and N-glycosylation of
cathep-sin E in its procescathep-sing, maturation, and trafficking
must be of particular importance
In this study, to understand the molecular basis of
the processing and intracellular trafficking of
cathep-sin E, we constructed a variety of mutants as well as
wild-type enzyme These included mutants with Asp
residues in the two active-site DTG motifs changed to
Ala residues and mutants lacking the propeptide,
puta-tive ER-retention sequence, and N-glycosylation of this
protein These mutant proteins, as well as wild-type
cathepsin E, were expressed in human embryonic
kidney (HEK)-293T cells We report that the catalytic
activity, propeptide, ER-retention sequence, and
N-gly-cosylation of this protein are all essential for
process-ing, maturation, and trafficking
Results
Expression and cellular localization of recombinant
rat cathepsin E expressed in HEK-293T cells
In this study, we used HEK-293T cells to express
wild-type cathepsin E and its mutants and to follow the
processing, maturation, and trafficking of these
pro-teins, as these cells have no detectable endogenous
cathepsin E To establish the expression system, we
transfected rat cathepsin E cDNA (wild-type) into
HEK-293T cells and analyzed the molecular forms of
the expressed proteins by SDS⁄ PAGE under reducing
conditions followed by immunoblotting with polyclonal
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 [35S]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-sensitive high-mannose-type oligosaccharide chains, whereas the latter possesses endoglycosidase H-resist-ant complex-type oligosaccharide chains (Fig 6A) The 42-kDa mature form in the cells was time-dependently increased, and the 46-kDa pro-cathepsin E was almost completely converted into the 42-kDa mature cathep-sin E after a 24-h chase These procescathep-sing events were strongly inhibited by the fungal metabolite brefeldin A
Fig 1 Immunoblot analysis of the cell extract of HEK-293T cells expressing wild-type cathepsin E HEK-293T cells were transfected with rat cathepsin E cDNA The cell lysate was subjected to SDS ⁄ PAGE under reducing conditions followed by immunoblotting with antibody to either mature cathepsin E (A) or pro-cathepsin E (B).
Trang 3(Fig 2B), which is known to inhibit export of newly
synthesized proteins from the ER to the Golgi complex
[17] and also to cause reversible redistribution of Golgi
resident proteins into the ER [18] The strong
inhibi-tion of the processing of pro-cathepsin E into the
mature enzyme by brefeldin A indicates that the
pro-cessing and maturation of this protein occur in the
post-Golgi compartment In addition, this agent
pre-vented the secretion of subspecies of the precursor,
probably mediated by an alternative secretory pathway
at the exit from the ER
Available evidence indicates that internal
acidifica-tion of some compartments in the vacuolar system,
such as endosomes and lysosomes, is important in
bio-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 protein LAMP-1 and the ER-associated molecular chaperon Bip, respectively In agreement with the data
of the pulse–chase analysis, the results indicate that the proteolytic maturation of pro-cathepsin E occurs in the endosomal acidic compartment
Importance of the catalytic activity of cathepsin E
in acid-dependent autoactivation
In vitro studies have shown that pro-cathepsin E is rapidly converted into mature cathepsin E by a brief acid treatment [9,23] However, whether the catalytic activity of cathepsin E is essential for its processing and maturation in vivo has not been elucidated To throw light on this, we constructed an active-site mutant by changing Asp residues in the two active-site motifs (DTG) to Ala residues using site-directed muta-genesis (D98A⁄ D283A) This mutant protein was expressed in HEK-293T cells We confirmed that the D98A⁄ D283A mutant had no catalytic activity on either protein or synthetic substrates, as described in our previous study, in which the single mutants D98A and D283A, as well as the double mutant, exhibited
no catalytic activity in vitro [24] The autoprocess-ing capability of this mutant protein was analyzed
by pulse–chase experiments with [35S]methionine (Fig 4) In contrast with wild-type cathepsin E, the D98A⁄ D283A mutant in the cells was neither proc-essed nor matured even after a 24-h chase period, but stably remained as a 46-kDa precursor, indicating that 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)1brefeldin A (B) or 0.5 l M
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.
Trang 4Role of the propeptides of cathepsin E in
processing and maturation
Recent studies have suggested that the propeptides of
aspartic proteinases are necessary for correct folding
and cellular sorting [25–27] and that activation of their
zymogens is initiated by a dramatic conformational rearrangement of the zymogen propeptides [28,29] This process is often triggered by acidic pH, resulting
in the proteolytic removal of propeptides [29] More recently, using chimeric DNAs encoding the cathep-sin E propeptide fused to mature cathepcathep-sin D tagged
Fig 3 Immunofluorescence microscopy of wild-type cathepsin E and its mutants in the transfected HEK-293T cells (A) At 48 h after post-transfection with wild-type cathepsin E cDNA in HEK-293T cells, the cells were fixed and allowed to react with antibodies to mature rat cathepsin E, Bip, or LAMP-1 After being washed, the cells were incubated for 1 h with fluorescein isothiocyanate-conjugated or tetra-methylrhodamine isothiocyanate-conjugated secondary antibodies, and then visualized by confocal laser microscopy Wild-type cathepsin E is found mainly in the LAMP-1-positive endosomal organelles and partly in the Bip-positive ER (B, C) The subcellular localization of the deletion mutants lacking the propeptide (Dpro) (B) and the ER-retention motif (DER ret.) expressed in HEK-293T cells was analyzed under the same conditions as described in (A) Both mutants were exclusively confined to the ER, but not to the LAMP-1-positive organelles.
Trang 5with haemagglutinin at the C-terminus and encoding
the cathepsin D propeptide fused to mature
cathep-sin E, we have demonstrated that the propeptide of
cathepsin E, likewise those of other aspartic
protei-nases, plays an important role in the correct folding,
maturation, and targeting of this protein to its final
destination [30] To establish further the role of the
propeptide in the processing and maturation of
cathep-sin E, we constructed a mutant lacking the propeptide
(Leu23-Phe58) and expressed it in HEK-293T cells
The transfected cells were pulse-labeled with
[35S]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
C
Fig 4 Pulse–chase analysis of the active-site mutant with Asp
resi-dues in the two active-site motifs changed to Ala resiresi-dues (A)
Schematic representation of the structures of wild-type cathepsin E
and its active-site mutant Asp residues in the two active-site
con-sensus motifs (98DTG100 and 273DTG275) are substituted by Ala
residues in the mutant (B, C) HEK-293T cells expressing wild-type
cathepsin E (B) and the active-site mutant (C) were pulse-labeled
with [35S]methionine ⁄ cysteine for 30 min and chased for the times
indicated The cell lysate and the culture medium were
immuno-precipitated with antibodies to mature rat cathepsin E The
immunoprecipitates were analyzed by SDS ⁄ PAGE under reducing
conditions followed by fluorography.
C B A
Fig 5 Pulse–chase analysis of the mutants lacking the propeptide and ER-retention sequence (A) Schematic representation of the structures of the mutants lacking the propeptide and most of the ER-retention sequence (B, C) The transfected cells expressing the mutants lacking the propeptide (Dpro) (B) and the ER-retention sequence (DER-ret.) (C) were pulse-labeled with [ 35 S]methio-nine ⁄ cysteine for 30 min and chased for the times indicated The cell lysate and the culture medium were immunoprecipitated with antibodies to mature rat cathepsin E The immunoprecipitates were analyzed by SDS ⁄ PAGE under reducing conditions followed by fluo-rography The arrows indicate degradation products of the DER-ret mutant.
Trang 6was 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 [35S]methionine for 30 min and chased with
unlabeled methionine for 24 h The intracellular and
extracellular cathepsin E were immuoprecipitated with
antibodies to mature rat cathepsin E, and the
immuno-precipitates were treated with endoglycosidase H and
then analyzed by SDS⁄ PAGE followed by
fluorogra-phy (Fig 6A) Both newly synthesized pro-cathepsin E
(30-min pulse) and the fully matured enzyme (24-h
chase) in the cells were sensitive to endoglycosidase H
A decrease in the molecular mass of 2–2.5 kDa
resul-ted, indicating the presence of at least a single
high-mannose-type oligosaccharide chain In contrast, the
extracellular cathepsin E molecules at 30-min pulse
labeling and after 24-h chase were resistant to
endo-glycosidase H, indicating that they were secreted after
modification with complex-type oligosaccharides
Cathepsin D, like other lysosomal enzymes, is
phos-phorylated in a portion of the oligosaccharide chains
during passage through the Golgi complex [34,35] This
process is considered to be important for the recognition
of cathepsin D by mannose 6-phosphate receptors and
its delivery to the prelysosomal compartment To
deter-mine whether the oligosaccharides of cathepsin E are
phosphorylated, the transfected cells were labeled with
[32P]phosphate for 12 h, and then wild-type cathepsin E
as well as endogenous cathepsin D was
immunoprecipi-tated by antibodies specific for each enzyme (Fig 6B)
Whereas the oligosaccharide chains of cathepsin D
were, at least in part, clearly phosphorylated, and this
phosphorylation had disappeared after endoglycosidase
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 cathcath-epsin 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 under reducing conditions followed by fluorography.
Trang 7N-glycosylation null mutant of cathepsin E
(N92Q⁄ N374D) was less stable to temperature and pH
than glycosylated cathepsin E, although the catalytic
properties of the mutant were equivalent to those of
the wild-type enzyme [15] In this study, we performed
pulse–chase experiments with HEK-293 cells expressing
N-glycosylation mutants The results show that two
single N-glycosylation mutants (N92Q and N324D),
as well as the double N-glycosylation mutant
(N92Q⁄ N374D), were stably retained in the cells, but
not processed to the mature form even after a 24-h
chase period (Fig 7) Immunofluorescence microscopy
indicated that all of the N-glycosylation mutants were
exclusively confined to the Bip-positive ER
compart-ment (data not shown) Therefore, our data indicate
that N-glycosylation of cathepsin E plays an important
role in its processing, maturation, and trafficking to
the appropriate destination in the cells, but is not
necessarily essential for its correct folding
Discussion
To assess the roles of the catalytic activity, propep-tides, and N-glycosylation of cathepsin E in its processing, maturation, and targeting to its final des-tination, we constructed wild-type rat cathepsin E and
a variety of its mutants, and successfully expressed them in heterologous HEK-293T cells The results provide the first experimental evidence that the cata-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
a precursor form [31] Similarly, rat cathepsin E expressed in normal rat kidney cells and monkey Cos 1 cells is exclusively confined to the ER (T Tsukuba, T Kadowaki and K Yamamoto, unpublished work)
Meanwhile, human cathepsin E expressed in Chinese hamster ovary cells is found in various subcellular com-partments such as the vacuolar endolysosomal system, the ER, and the cytosol [37] The present results thus indicate that HEK-293T cells are useful for studying the biosynthesis, processing, maturation, and trafficking
to the correct destination of cathepsin E
Secondly, the mutant lacking the catalytic activity of cathepsin E in the transfected cells failed to be conver-ted to the mature form, indicating that the catalytic
A
B
C
D
Fig 7 Pulse–chase analysis of the mutants lacking N-glycosylation.
(A) Schematic representation of the structures of the mutants
lack-ing slack-ingle (N92Q or N324D) and double N-glycosylation sites
(N92Q ⁄ N324D) Asn residues in the two potential N-glycosylation
sites (92NFT94 and 324NVT326) are substituted by Gln and ⁄ or Asp
residues in the mutants (B–D) HEK-293 cells expressing each
mutant as well as wild-type cathepsin E were pulse-labeled with
[ 35 S]methionine ⁄ cysteine for 30 min and chased with
nonradiolabe-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.
Trang 8activity 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
the importance of the propeptide in the processing,
maturation, and trafficking of this protein However,
the cathepsin E mutant lacking the putative
ER-retent-ion sequence was rapidly degraded in the ER,
suggest-ing that the ER-retention sequence is necessary for
correct folding In addition, this mutant did not
undergo processing, maturation, and trafficking to the
endosomal compartment
Finally, we have shown that intracellular wild-type
cathepsin E was N-glycosylated with
high-mannose-type oligosaccharides, as demonstrated by
endoglycosi-dase H sensitivity, whereas the extracellular enzyme
had endoglycosidase H-resistant oligosaccharides
Pre-vious studies have shown that modification of
oligosac-charide chains of cathepsin E differs between cell
types Therefore, it is difficult to explain the
process-ing, maturation, and targeting of cathepsin E by the
nature of its oligosaccharide chains We thus
construc-ted three N-glycosylation mutants and expressed them
in HEK-293T cells to determine the importance of
N-glycosylation in the processing, maturation, and
trafficking of this protein Pulse–chase analysis and
immunofluorescence microscopy revealed that neither
single (N92Q, N324D) nor double (N92Q⁄ N374D)
N-glycosylation mutants were processed into the
mature forms, but were stably retained in the ER
with-out degradation after a 24-h chase period As all of
the N-glycosylation mutants were converted into the
active enzyme on acidification (data not shown), each
the oligosaccharide chain appears to be not necessarily
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
[35S]Methionine⁄ cysteine and Protein A–Sepharose were purchased from Amersham Biosciences (Piscataway, NJ, USA) Pansorbin was from Calbiochem (La Jolla, CA,
methio-nine-free DMEM, and Opti-Mem were from Invitrogen (Carlsbad, CA, USA) Endoglycosidase H was from Boeh-ringer Mannheim (Mannheim, Germany) Centriprep-30 and Microcon-30 concentrators were from Millipore (Bed-ford, MA, USA) Fluorescein isothiocyanate-conjugated and tetramethylrhodamine isothiocyanate-conjugated secon-dary antibodies were from BD Bioscience (San Jose, CA, USA) Vectashield was from Vector Laboratory Inc (Bur-lingame, CA, USA) Antibodies to Bip (GRP 78) and Lamp1 were from Stressgen Bioreagents (Victoria, BC, Canada) Antibodies to rat cathepsin E and to rat cathep-sin D were raised in rabbit and purified as described previ-ously [24] Polyclonal antibodies to the synthetic peptide Ser-Gln-Leu-Ser-Glu-Phe-Trp-Lys-Ser-His-Asn-Leu-Asp-Met, which corresponds to the prosequence comprising residues Ser23-Met36 of human procathepsin E, were raised in rab-bits and purified on the peptide–Sepharose affinity column
as described previously [24)
Plasmid construction and mutagenesis
The pBluescript II SK plasmid containing full-length wild-type rat cathepsin E cDNA was described previously [15,24] Deletion mutants lacking the ER retention sequence (DER ret.) and the propeptide (Dpro) were constructed by the method of Kunkel [51] with some modifications The oligonucleotide primers used in the mutagenesuis reaction
Trang 9were as follows: primer 1, 5¢-ATGATCGAATTCACGGG
CGGCTCA-3¢ (for DER ret.); primer 2, 5¢-CAGGC
CCAAGGGGTGAGCGAGTCCTGT-3¢ (for Dpro) All
constructs were verified as correct by DNA sequence
analy-ses The cDNAs of wild-type cathepsin E and its mutants
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 37C
incubator with 5% CO2 Wild-type cathepsin E and its
mutants were expressed in HEK-293T cells after
transfec-tion by the calcium phosphate precipitatransfec-tion 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 centrifugatransfec-tion 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⁄ Pi, 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⁄ Picontaining 0.1% Triton X-100, sonicated for 1 min
at 4C, and subjected to centrifugation at 100 000 g for 1 h;
the supernatant fraction is referred to as the cell lysate
Pulse–chase analysis
The transfected cells were preincubated for 1 h at 37C in
DMEM without methionine supplemented with 10%
dia-lyzed fetal bovine serum The cells were pulse-labeled for
30 min with [35S]methionine⁄ cysteine (100 lCiÆmL)1per
dish), and then chased in fresh serum-free Opti-MEM
(1.5 mL⁄ plate) At the times indicated, the cells were
separ-ated from the medium, washed twice with NaCl⁄ Pi, lysed in
NaCl⁄ Pi containing 1% Triton X-100, 0.5% sodium
de-oxycholate, 0.02% sodium azide, and a proteinase inhibitor
cocktail, and subsequently sonicated for 1 min The
suspen-sion was centrifuged at 6500 g for 10 min to obtain the cell
lysate fraction
Immunoprecipitation
The cells and media were mixed with 40 lL Pansorbin for
1 h at 4C to prevent nonspecific binding to IgG–Protein
A beads, and then centrifuged at 6500 g for 30 min The
supernatants fractions were incubated with 15 lL anti-(rat
cathepsin E) IgG at 37C for 10 min, and then stored at
4C for 16 h The immunoprecipitates were mixed with
40 lL Protein A–Sepharose beads (50% gel suspension) for
3 h at 4C 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
100C with 50 mL 0.1% SDS ⁄ 0.5 mm EDTA ⁄ 5% sucrose ⁄
5 mm 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 100C in 10 lL 5 mm 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
37C 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⁄ Piand fixed with 3.7% formaldehyde in NaCl⁄ Pifor 10 min at room temperature The fixed cells were washed and permeabilized with 0.2% Triton X-100 in NaCl⁄ Pi The cells were incubated with the indicated primary antibodies for 1 h at room temperature Antibodies to mature rat cathepsin E, rat pro-cathepsin E, rat Bip, and rat LAMP-1 were used as primary antibodies After being washed, the cells were incubated for 1 h with fluorescein isothiocyanate-conjugated or tetramethylrhod-amine isothiocyanate-conjugated secondary antibodies After treatment with Vectashield, the cells were observed
by confocal microscopy (Leica Microsystems)
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