©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Possible Role of ER-60 Protease in the Degradation of Misfolded Proteins in the Endoplasmic Reticulum (*)

Mieko Otsu (1)(§), Reiko Urade (2), Makoto Kito (2), Fumihiko Omura (1)(¶), Masakazu Kikuchi (1)(**)

From the (1)Protein Engineering Research Institute, 6-2-3, Furuedai, Suita, Osaka 565 and the (2)Research Institute for Food Science, Kyoto University, Uji, Kyoto 611, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Secretory and membrane proteins, which are translocated as unstructured polypeptides into the luminal space of the endoplasmic reticulum (ER)()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.

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 (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.

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.


EXPERIMENTAL PROCEDURES

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.

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) .

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.


RESULTS AND DISCUSSION

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.


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

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-[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.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Biomolecular Engineering Research Institute, 6-2-3, Furuedai, Suita, Osaka 565, Japan. Tel.: 81-6-872-8208; Fax: 81-6-872-8219.

Present address: Institute for Fundamental Research, Suntory Ltd., Osaka 618, Japan.

**
Present address: Dept. of Bioscience and Technology, Faculty of Science and Engineering, Ritsumeikan University, Shiga 525-77, Japan.

The abbreviations used are: ER, endoplasmic reticulum; PDI, protein disulfide isomerase; hLZM, human lysozyme; ALLN, N-acetyl-leucyl-leucyl-norleucinal; ALLM, N-acetyl-leucyl-leucyl-methioninal; TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone; APMSF, (p-amidinophenyl)methanesulfonyl fluoride; DSP, dithiobis(succinimidyl propionate); PVDF, polyvinylidene difluoride; PBS, phosphate-buffered saline; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.

R. Urade, T. Oda, H. Ito, S. Utsumi, T. Moriyama, and M. Kito, manuscript in preparation.

M. Otsu, R. Urade, M. Kito, T. Hayano, and M. Kikuchi, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. M. Ikehara for his encouragement and Dr. T. Hayano and Dr. T. Yoshimori for valuable discussions relevant to this work.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.