From the
Wild-type human lysozyme (hLZM) is secreted when expressed in
mouse L cells, whereas misfolded mutant hLZMs are retained and
eventually degraded in a pre-Golgi compartment (Omura, F., Otsu, M.,
Yoshimori, T., Tashiro, Y., and Kikuchi, M.(1992) Eur. J. Biochem. 210, 591-599). These misfolded mutant hLZMs are associated
with protein disulfide isomerase (Otsu, M., Omura, F., Yoshimori, T.,
and Kikuchi, M.(1994) J. Biol. Chem. 269, 6874-6877).
From the observation that this degradation is sensitive to cysteine
protease inhibitors, such as N-acetyl-leucyl-leucyl-norleucinal and N-acetyl-leucyl-leucyl-methioninal, but not to the serine
protease inhibitors, 1-chloro-3-tosylamido-7-amino-2-heptanone and (p-amidinophenyl)methanesulfonyl fluoride, it was suggested
that some cysteine proteases are likely responsible for the degradation
of abnormal proteins in the endoplasmic reticulum (ER). ER-60 protease
(ER-60), an ER resident protein with cysteine protease activity (Urade,
R., Nasu, M., Moriyama, T., Wada, K., and Kito, M.(1992) J. Biol.
Chem. 267, 15152-15159), was found to associate with
misfolded hLZMs, but not with the wild-type protein, in mouse L cells.
Furthermore, denatured hLZM is degraded by ER-60 in vitro,
whereas native hLZM is not. These results suggest that ER-60 could be a
component of the proteolytic machinery for the degradation of misfolded
mutant hLZMs in the ER.
Secretory and membrane proteins, which are translocated as
unstructured polypeptides into the luminal space of the endoplasmic
reticulum (ER)
ER-60 protease (ER-60) was first purified from
the ER of rat liver and was shown to be a cysteine
protease(25) . This protein has 98% homology in amino acid
sequence to rat phosphoinositide-specific phospholipase C
Human
lysozyme (hLZM) is a monomeric secretory protein with four disulfide
bonds. When expressed in mouse L cells, misfolded mutant hLZMs fail to
be secreted, and they are retained and degraded in a pre-Golgi
compartment(17) . Previously PDI was shown to associate with
misfolded mutant hLZM in vivo and is thought to be involved in
the quality control system for secretory proteins(28) . In this
report, the degradation of misfolded hLZMs is shown to be sensitive to
cysteine protease inhibitors, N-acetyl-leucyl-leucyl-norleucinal (ALLN) and N-acetyl-leucyl-leucyl-methioninal (ALLM), but not to a
trypsin inhibitor, 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK) or
a serine protease inhibitor (p-amidinophenyl)-methanesulfonyl
fluoride (APMSF). Coupled with the results that the ER-60 protein is
chemically cross-linked to misfolded mutant hLZMs in vivo and
that ER-60 degrades the reduced and denatured form of hLZM, but not the
native form in vitro, ER-60, we suggest, is involved in the
pre-Golgi degradation of misfolded hLZMs.
Rabbit
anti-ER-60 antibody was raised by immunization with purified rat
ER-60F(25) . Rabbit anti-hLZM and anti-bovine PDI antibodies
were prepared as described previously(28) .
The mutant hLZMs, C128A, and L15G/G16D/M17S are neither
folded correctly nor secreted, and consequently they are degraded in a
pre-Golgi compartment(17, 28) . To investigate what
kinds of proteases are responsible for the degradation, mouse L cells
expressing these mutants were treated with several protease inhibitors,
respectively, in the presence of brefeldin A, which prevents protein
export from the ER (). TLCK, which inhibits trypsin and
related proteases, APMSF, a serine protease inhibitor, and
phosphoramidon, a metalloprotease inhibitor, did not affect the
degradation at all. In contrast, ALLN and ALLM, inhibitors of cathepsin
L, cathepsin B, and the calpains, slowed in vivo degradation
of C128A and L15G/G16D/M17S. However, both leupeptin, which is known to
inhibit both serine and cysteine proteases, and E-64, a specific
inhibitor of cysteine proteases, were ineffective. This can be
explained by their low membrane permeability. Diamide was also
effective in the inhibition of proteolysis assay, probably due to its
ability to change the redox conditions of the ER. It has been reported
that the redox potential plays an important role in the pre-Golgi
degradation, where some putative cysteine proteases are
essential(30) . These observations suggest the possibility that
the misfolded mutant hLZMs were degraded by cysteine proteases in a
pre-Golgi compartment.
ER-60 is known as one of the ER resident
cysteine proteases, and its proteolytic activity is inhibited in
vitro by ALLN, ALLM, leupeptin, and E-64, but not by TPCK, TLCK,
phenylmethanesulfonyl fluoride, or a metalloprotease inhibitor, o-phenanthroline (31). It degrades bovine serum albumin, PDI,
and calreticulin, but not casein or carboxylesterase E1, in
vitro(25) . To investigate the possible involvement of
ER-60 in the degradation of mutant hLZMs, we first examined whether
hLZM can be a proteolytic substrate for ER-60 in vitro. As it
is highly difficult to purify the misfolded mutant hLZMs, wild-type
hLZM denatured with 7.6 M urea and 50 mM DTT was used
as a substrate. ER-60 did not degrade the intact wild-type hLZM, but it
degraded the denatured form in vitro (Fig. 1). It is
conceivable that the denatured hLZM was a favorable substrate for this
proteolysis, because the degradative sites of hLZM were exposed to the
protease by the denaturation. This result might mimic the intracellular
degradation of misfolded hLZM proteins to some extent.
Mouse L cells expressing the
C128A protein and the L15G/G16D/M17S protein were pretreated with 5
µg/ml of brefeldin A for 30 min, pulse-labeled for 30 min with L-[
We thank Dr. M. Ikehara for his encouragement and Dr.
T. Hayano and Dr. T. Yoshimori for valuable discussions relevant to
this work.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)at the beginning of their
synthesis, are folded and in some cases, assembled into oligomeric
complexes before transport to the cis-Golgi compartment. Some
ER resident proteins, such as immunoglobulin heavy chain binding
protein (BiP/GRP78)(1, 2) , protein disulfide isomerase
(PDI)(3, 4) , peptidyl prolyl cis-trans-isomerase(5, 6) , and calnexin (p88,
IP90)(7, 8, 9) , assist in the folding of some
polypeptides. There also seems to be a quality control system for
proteins which are destined for export from the ER. When polypeptides
are folded or assembled incorrectly, they are retained in the ER and
are degraded before they reach the Golgi apparatus(10) . It is
plausible that there are regulatory mechanisms by which cells eliminate
abnormal proteins, as well as mechanisms for correct folding or
assembly of the polypeptides in the ER. The retention of abnormal
proteins in the ER might be caused by their interactions with molecular
chaperones(11) , and the degradation is probably accomplished by
ER resident proteases. Various substrates for this degradation have
been characterized (10-17), and some of them were found to have
signals for degradation in their primary
sequences(18, 19, 20) . However, information
about the relationship between the proteases and the substrates has
been rather indirect and sometimes inconsistent. The degradation of the
T-cell receptor
-chain, the CD3
-subunit, and
3-hydroxy-3-methylglutaryl-coenzyme A reductase is sensitive to some
cysteine protease inhibitors (21, 22) and that of the
immunoglobulin
-chain is sensitive to some serine protease
inhibitors(23) . Furthermore, there are two distinct pathways in
the degradation of the H2 subunit of the asialoglycoprotein
receptor(24) . These results reflect the likely fact that
multiple proteases are responsible for the degradation of abnormal
proteins in the ER.
(ERp61,
GRP58, Q-2, HIP-70) which possesses two sets of a thioredoxin-like
sequences, consisting of Cys-Gly-His-Cys, and also a Gln-Glu-Asn-Leu
sequence which may serve as the ER retention signal at its carboxyl
terminus(25, 26) .
(
)Q-2 has an
insulin reduction activity in vitro, but lacks
phosphoinositide-specific phospholipase C
activity(27) .
The role of ER-60 in vivo has been elusive so far.
Materials
L-[S]Methionine
(>800 Ci/mmol) was purchased from the Hungarian Academy of Science
(Budapest, Hungary), and protein A-Sepharose CL-4B was obtained from
Pharmacia LKB Biotechnology Inc. (Uppsala, Sweden).
Dithiobis(succinimidylpropionate) (DSP) was purchased from Pierce
(Rockford, IL); leupeptin, E-64, and phosphoramidon were from Peptide
Institute (Osaka, Japan); and ALLN and ALLM were from Boehringer
Mannheim (Mannheim, Germany). Diamide was purchased from Sigma, TLCK
from Aldrich, and brefeldin A and APMSF were from Wako Pure Chemical
Industries, Ltd. (Osaka, Japan). The 3,3`-diaminobenzidine substrate
kit was obtained from Vector Laboratories (Burlingame, CA),
Polyvinylidene difluoride (PVDF) membrane was from Millipore (Bedford,
MA). All other chemicals were of the best grade available.
Cell Lines
The cell lines expressing the wild-type
and the mutant hLZMs were generated by stable transfection of mouse L
cells, using an expression vector described previously(28) .
Mutant hLZMs C128A (Cys-128 Ala) and L15G/G16D/M17S (Leu-15
Gly, Gly-16
Asp, Met-17
Ser) were used for the
analysis of their intracellular behaviors.
Metabolic Labeling of Proteins
The cells were
grown for 24 h in 60-mm diameter dishes (1.0 10
cells/dish) in
-modification of Eagle's minimum
essential medium supplemented with 10% fetal calf serum and were washed
with methionine-free medium. For pulse-chase experiments, the cells
were preincubated with 5 µg/ml brefeldin A for 30 min,
pulse-labeled with 100 mCi/ml L-[
S]methionine for 30 min, and chased
in complete culture medium containing 20 mM unlabeled
methionine. Protease inhibitors (100 µg/ml) were supplemented in
the chase where indicated. The chase was terminated by placing the
cells on ice. Labeled cells were washed with ice-cold
phosphate-buffered saline (PBS) and solubilized in lysis buffer
containing 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris/HCl (pH 7.5), 2 mM APMSF, and 200 µg/ml soybean
trypsin inhibitor. After the cell debris removed by centrifugation, the
lysates were processed for immunoprecipitations. For experiments with
the cross-linker, the cells were labeled with 100 mCi/ml L-[
S]methionine for 1 h. The labeled
cells were washed with ice-cold PBS, then treated with or without 1
mM DSP in PBS at 0 °C for 30 min. After excess
cross-linker was neutralized with 2 mM glycine, the labeled
cells were solubilized in the lysis buffer described above.
Immunoprecipitation and
Electrophoresis
Immunoprecipitation with rabbit anti-hLZM
antibody was performed as described(17) . To identify ER-60, the
cross-linked samples were immunoprecipitated twice, first with
anti-hLZM antibody and then with anti-ER-60 antibody. The first
immunoprecipitate was obtained by boiling with 2% SDS and 400 mM DTT as described previously(28) . After dilution, the
second immunoprecipitation was carried out using anti-ER-60 antibody
adsorbed to protein A-Sepharose CL-4B. To detect the association of PDI
and ER-60, the cross-linked samples were immunoprecipitated twice,
first with anti-PDI (or anti-ER-60) antibody and then with anti-ER-60
(or anti-PDI) antibody, as described above. The precipitates were
boiled for 10 min in SDS-polyacrylamide gel electrophoresis (PAGE)
sample buffer supplemented with 100 mM DTT, followed by
electrophoresis on 13% SDS-polyacrylamide gels.
Assay of Proteolytic Degradation in
Vitro
Wild-type hLZM was expressed in Saccharomyces
cerevisiae and purified as described previously(29) .
Wild-type hLZM was denatured by 7.6 M urea and 50 mM DTT at 25 °C. The denaturation of hLZM was confirmed by
checking the loss of lytic activity using Micrococcus lysodeikticus as a substrate (data not shown). ER-60 was purified from rat liver
as described previously(25) . Denatured hLZM was diluted
22.5-fold by addition to the following reaction mixture. Wild-type (1
µg) or denatured hLZM (2 µg) was incubated with purified rat
ER-60 (1 µg) in a buffer containing 10 mM bis-Tris (pH
6.3) and 100 mM -mercaptoethanol at 37 °C for 3 h.
The reaction products were analyzed by SDS-PAGE.
Immunoblot Analysis
Proteins separated by SDS-PAGE
were electrophoretically blotted onto a PVDF membrane and then
immunostained with specific polyclonal antibodies and the
3,3`-diaminobenzidine substrate kit.
Figure 1:
Proteolytic degradation of hLZMs by
ER-60 in vitro. Wild-type hLZM (1 µg of protein) (lanes 1 and 2) and denatured hLZM (2 µg) (lanes 3 and 4) were incubated with (lanes 2 and 4) and without (lanes 1 and 3)
ER-60 (1 µg) in the presence of 100 mM -mercaptoethanol and 10 mM bis-Tris (pH 6.3) at 37
°C for 3 h. The samples were analyzed by
SDS-PAGE.
We previously
demonstrated that misfolded mutant hLZMs were associated with PDI in vivo(28) . PDI and ER-60 share similarity in their
amino acid sequences. Thus we examined whether the proteins
cross-linked with misfolded mutant hLZM contained ER-60 as well as PDI,
by the immunological methods described under the ``Experimental
Procedures.'' As shown in Fig. 2, the anti-rat ER-60
antibody did not cross-react with purified bovine PDI, which has 95%
homology in amino acid sequence with mouse PDI, and the anti-bovine PDI
antibody did not cross-react with rat ER-60, which has 96% homology
with mouse ER-60.(
)The ER-60 protein was found
to be co-precipitated with the misfolded mutant hLZMs, C128A and
L15G/G16D/M17S, but not with wild-type hLZM, in the presence of the
membrane-permeable cross-linker DSP (Fig. 3a). Using a
similar immunological procedure, the association of ER-60 with PDI was
also detected in the cells expressing mutant hLZM L15G/G16D/M17S (Fig. 3b). These data suggest that ER-60 and PDI
associate with each other via the misfolded hLZM proteins. These
proteins probably control the quality of newly synthesized proteins in
the ER. ER-60 and PDI might occasionally interact with the target
proteins, in a similar manner to the actions of calnexin and BiP on
misfolded G proteins(32) . Furthermore it can be thought that
the ER-60 and PDI proteins directly associate with each other. Indeed,
the association of these proteins was also detected in
non-hLZM-expressing cells. The ER-60 and PDI proteins might associate
with each other to assist protein folding in the ER. In the case of a
correctly folded protein, the association between ER-60 and a protein
and/or the association among ER-60, PDI, and a protein might be brief.
However, when the folding is not completed, this association might be
more stable. This relatively stable association, which was detected
with the misfolded mutant hLZMs, might activate the degradative
function of ER-60. Taken together, these results suggest that ER-60 is
involved in the fate of misfolded proteins in cooperation with PDI.
Figure 2:
Western blot analysis of PDI and ER-60.
After SDS-PAGE resolution of purified bovine PDI and rat ER-60, the
proteins were stained with Coomassie Brilliant Blue R-250 (a)
or transferred onto PVDF membranes and then immunostained with
anti-bovine PDI antibody (b) or anti-rat ER-60 antibody (c), as described under ``Experimental
Procedures.''
Figure 3:
Coprecipitation of misfolded hLZMs and
ER-60 (a) and coprecipitation of PDI and ER-60 (b). a, labeled mouse L cells expressing the L15G/G16D/M17S protein
were treated with DSP. The cell lysate were immunoprecipitated with
anti-hLZM antibody. After the precipitates were boiled with 2% SDS and
400 mM DTT, the sample was immunoprecipitated with either
anti-ER-60 antibody (lane 2) or non-immune rabbit serum (lane 5). Lane 1 shows the precipitates that are the
same as in lane 2, but without the DSP treatment. The
reactivity of the cross-linked protein to the anti-ER-60 antibody was
abolished with an excess of unlabeled rat ER-60 (lane 3), but
not with unlabeled bovine PDI (lane 4). Lanes 6 and 7 show the precipitates obtained by the treatment with
anti-ER-60 (lane 6) and anti-PDI antibodies (lane 7),
respectively, for both the first and second antibodies. The star on lane 7 shows the degraded product of PDI. Lanes 8 and 9 are the same as lane 2, except that
expressing wild-type hLZM (WT) and parental L cells (C), respectively, were used. Lanes 10 and 11 are the same as lanes 1 and 2, respectively,
except that cells expressing hLZM C128A were used. The numbers on the left indicate molecular mass (in kDa). b,
labeled cells expressing the L15G/G16D/M17S protein were treated with
DSP, lysed, and immunoprecipitated with anti-PDI antibody. The
precipitates were boiled with SDS and DTT and then immunoprecipitated
by either the anti-ER-60 antibody (lane 3) or pre- immune
rabbit serum (lane 6). Lane 2 shows the same
precipitate as lane 3, but without treatment by DSP. The
reactivity of the cross-linked protein was competed with an excess of
unlabeled ER-60 (lane 4), but not by unlabeled bovine PDI (lane 5). In lanes 8-11, anti-ER-60 antibody
was used as the first antibody, and anti-PDI antibody was used as the
second. Lane 8 shows the precipitates in the absence of DSP. Lane 9 shows the case in the presence of DSP. Lanes 10 and 11 were the same as lane 9, except that
unlabeled PDI and ER-60 were added, respectively. Lane 12 shows the control with pre-immune rabbit serum as the second
antibody. Lanes 1 and 7 show the control precipitates
obtained using anti-ER-60 (lane 1) and anti-PDI antibody (lane 7), respectively, for both the first and second
antibodies. The star on lane 7 shows the degraded
product of PDI.
In this report, we verified the proteolytic activity of ER-60
against denatured hLZM in vitro and the association of ER-60
with misfolded hLZMs in vivo. These observations obtained in vivo and in vitro are complementary and begins to
explain the possibility that the ER-60 protein acts as a protease in
the ER. These results and our previous report (28) offer a
probable mechanism, that misfolded hLZM proteins are recognized by PDI
and/or ER-60 and eventually are subjected to the proteolytic machinery,
in which the ER-60 protein is now implicated. This is the first report
to show candidates of proteases related to ER degradation in
vivo. Much remains to be understood about the mechanism of quality
control in the ER; however, our results suggest roles for PDI and ER-60
in the degradation of misfolded proteins in the ER.
Table: Effect of various protease inhibitors on the
degradation of misfolded mutant hLZMs
S]methionine, and chased for 4 h
with complete medium supplemented with 20 mM unlabeled
methionine, in the presence of 100 µg/ml of each protease
inhibitor, if needed. The percentage inhibition was calculated on the
basis of the inhibition results without treatment.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.