150-kDa oxygen-regulated protein (ORP150) functions as a novel molecular chaperone in MDCK cells

Yoshio Bando1,2, Satoshi Ogawa1,2, Atsushi Yamauchi3, Keisuke Kuwabara4, Kentaro Ozawa1,2, Osamu Hori1,2, Hideki Yanagi5, Michio Tamatani1,2, and Masaya Tohyama1,2

1 Department of Anatomy and Neuroscience and 4 First Department of Medicine, Osaka University Graduate School of Medicine, Suita City 565-0871; 3 Division of Nephrology, Department of Medicine, Osaka Rosai Hospital, Sakai, Osaka 591-8025; 5 HSP Research Institute, Kyoto Research Park, Shimogyo-Ku, Kyoto 600-8813; and 2 Core Research for Evolutional Science and Technology, Japan Science and Technology, Kawaguchi City 332-0012, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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To assess the participation of the 150-kDa oxygen-regulated protein (ORP150) in protein transport, its function in Madin-Darby canine kidney (MDCK) cells was studied. Exposure of MDCK cells to hypoxia resulted in an increase of ORP150 antigen and increased binding of ORP150 to GP80/clusterin (80-kDa glycoprotein), a natural secretory protein in this cell line. In ORP150 antisense transformant MDCK cells, GP80 was retained within the endoplasmic reticulum after exposure to hypoxia. Metabolic labeling showed the delay of GP80 maturation in antisense transformants in hypoxia, whereas its matured form was detected in wild-type cells, indicating a role of ORP150 in protein transport, especially in hypoxia. The affinity chromatographic analysis of ORP150 suggested its ability to bind to ATP-agarose. Furthermore, the ATP hydrolysis analysis showed that ORP150 can release GP80 at a lower ATP concentration. These data indicate that ORP150 may function as a unique molecular chaperone in renal epithelial cells by facilitating protein transport/maturation in an environment where less ATP is accessible.

hypoxia; energy metabolism; renal epithelium; ischemia; adenosine 5'-triphosphate kinetics


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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CELLS PRODUCE A SET of proteins in various intracellular components when exposed to altered environmental conditions that may threaten the cell's survival (38, 39). Among these stress-induced proteins, those located in the endoplasmic reticulum (ER) are induced mainly by glucose starvation and oxygen deprivation (14) and are referred to as glucose-regulated proteins (GRPs) and oxygen-regulated proteins (ORPs), respectively (7). The overlap that exists among the constituents of these two categories suggests that some environmental alterations induce a similar crisis in the ER, whose function could be maintained by the expression of these specific proteins. In fact, the stresses that regulate the expression of GRPs/ORPs, which are referred to as ER stresses, are caused by the accumulation of unfolded/immature proteins in the ER and the decelerated protein traffic via the ER (15, 28).

Another aspect of the stress proteins induced in the ER is their behavior as molecular chaperones, i.e., functional proteins that assist the maturation and transport of unfolded secretory proteins either within the ER or from the ER to the Golgi apparatus in an ATP-dependent manner (14). The 78-kDa glucose-regulated protein (GRP78), for instance, was first identified as a stress protein in transformed fibroblasts (27) and was later shown to be identical to B cell immunoglobulin-binding protein (22), a molecular chaperone that facilitates the secretion and maturation of immunoglobulin. Other stress proteins induced in the ER, including GRP94 (21), ERp72 (20), and FKBP13 (3), also share this functional property of participating in protein transport in an ATP-dependent manner, in addition to their well-preserved structural similarities, i.e., an ATPase motif and protein-binding domain.

The 150-kDa oxygen-regulated protein (ORP150) was first purified and cloned as a novel stress protein induced by oxygen deprivation in rat cultured astrocytes (10). Its amino acid sequence indicates that ORP150, like other ORPs/GRPs in the ER, shares homologous components, including a protein-binding domain and the ER retention signal (8), suggesting its possible participation in the protein transport/maturation from the ER to the Golgi apparatus. Furthermore, the dominant expression of ORP150 in cultured astrocytes (10) and human monocytes (32) in deep and prolonged hypoxia, as well as the expression of ORP150 in the core area in experimental brain ischemia (19), suggest the necessity for cells to activate the pathway mediated by ORP150 in extreme conditions.

To clarify the properties of ORP150, upon which the cellular protein-transport system becomes dependent in the extreme conditions, we examined the role of this molecular chaperone in the protein transport of Madin-Darby canine kidney (MDCK) cells. The data obtained demonstrate that ORP150, due to its higher affinity to ATP, may assist the maturation of secretory protein in conditions where less ATP is accessible.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Cell culture and achievement of hypoxia. MDCK cells were provided by the Japanese Cancer Research Resources Bank (Tokyo, Japan). MDCK cells were maintained in DMEM (20 mM glucose; Nikken, Kyoto, Japan) and supplemented with 10% FCS and penicillin/streptomycin equilibrated with 5% CO2-95% air at 37°C. After they reached confluence, the cells were plated (~106 cells/cm2) on culture dishes and then transferred to a hypoxic chamber (Coy Laboratories, Ann Arbor, MI) as described previously (23). The oxygen tension in the medium was monitored with a blood gas analyzer (ABL-2; Radiometer, Copenhagen, Denmark). The viability of the cells during the experiments was assessed with multiple parameters, including lactate dehydrogenase (LDH) release, the exclusion of trypan blue dye, and morphological criteria.

Transfection of the ORP150 sense/antisense vector to MDCK cells. To construct the ORP150 sense/antisense vector, a fragment encoding almost the entire human ORP150 cDNA, as well as some 3'-untranslated sequence (86-3093; see Ref. 8), was inserted in the vector pCAGGS (generously provided by T. Miyazaki, Osaka University Medical School) in either sense or antisense orientation (24). Ten micrograms of vector, with or without ORP150 sense/antisense, were transfected into MDCK cells using the lipofection method (Tfx-50 reagents; Promega, Madison, WI). The selection and maintenance of the neoresistant transfectants were performed in the presence of G418 (2 mg/ml, Sigma, St. Louis, MO). After 14 days, single colonies were resuspended and grown in 96-well plates at a density of about one cell per well. Several cell lines were isolated, all of which were maintained in the presence of G418 (1 mg/ml). The cells were switched to the G418-free medium 24 h before the experiments.

Immunoblotting (Western blot analysis). To examine the expression level of ORP150 antigen in the MDCK cells, we performed an immunoblotting analysis using rabbit anti-human ORP150 antibody (32). In brief, either wild-type or antisense transformant cells (~106 cells/cm2) were exposed to hypoxia for the indicated period. The cells were washed with ice-cold PBS and then were lysed in PBS containing Nonidet P-40 (NP-40; 1%). After the determination of the protein concentration, ~3 µg of protein extract were separated by 7.5% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) paper, and incubated at 37°C for 1 h in Tris · HCl-buffered saline containing BSA (5 mg/ml), Tween 20 (0.1%), and rabbit anti-human ORP150 antibody (5 µg/ml). The binding site of primary antibody was further detected by the anti-rabbit IgG monoclonal antibody labeled with alkaline phosphatase (1 µg/ml; Boehringer Mannheim, Mannheim, Germany).

Northern blot analysis. To assess the expression of ORP150 transcripts, we performed a Northern blot analysis using a 32P-labeled probe comprised of a partial human ORP150 cDNA sequence (1123-1758 bp; see Ref. 8). Total RNA was extracted from MDCK cells (~5 × 107 cells/cm2) by the modified acid-guanidium-phenol-chloroform method with a commercial reagent (ISOGEN; Nippon Gene), as described (9). RNA was separated by electrophoresis on a 1.2% agarose/formamide gel and transferred overnight to nylon membranes (Hybond-N; Amersham, Arlington Heights, IL). The membrane was prehybridized for 2 h at 42°C in hybridization buffer containing formaldehyde (50%), saline-sodium citrate (SSC; 5%), Denhardt's solution (0.5%), SDS (0.1%), sodium phosphate (50 mM), and heat-denatured salmon sperm DNA (100 µg/ml). ORP150 partial cDNA was radiolabeled with [alpha -32P]dCTP by the random hexamer procedure (5). After hybridization overnight at 42°C in hybridization buffer containing radiolabeled cDNA probe (5 ng/ml), the filters were washed three times at 50°C for 30 min in SSC (2×)/ SDS (0.5%), exposed to X-ray film (Kodak, Rochester, NY), and subjected to autoradiography. The levels of ORP150 mRNA were evaluated by comparison with those of beta -actin mRNA.

Analysis of high-energy adenine nucleotides. Measurement of cellular ATP was performed as described (34). Either wild-type or antisense transformant MDCK cells (5 × 105 cells) were exposed for 0-48 h to hypoxia. After washing cells three times with PBS, cellular adenine nucleotides were extracted with perchloric acid (1 M) and deproteinized by rapid centrifugation through a layer of bromododecane (Wako Chemicals, Osaka, Japan). The supernatant was brought to neutral pH with KOH (1 M) and was subjected to reversed-phase HPLC (Microsorb C18, 0.4 × 15 cm; Rainin Instruments). The column elute was monitored at an optical density of 254 nm, and the nucleotide concentration was calculated from the corresponding peak area using ChromatoPack CR-4A (Shimazu Seiki, Kyoto, Japan). At each time point, energy charge was calculated as described previously (34). Extraction of adenine nucleotides from hypoxic cells was performed inside the hypoxic chamber.

Immunohistochemical analysis in MDCK cells. The cellular distribution of the 80-kDa glycoprotein (GP80) was immunohistochemically determined, as described previously (18). In brief, wild-type and antisense transformant MDCK cells plated separately on chamber slides (~104 cells/cm2) were either exposed to hypoxia or maintained in normoxia for 24 h and then were fixed in PBS containing Triton X-100 (0.1%) and paraformaldehyde (4%). After being washed with ice-cold PBS two times, the cells were blocked with nonimmune goat or rabbit serum for 2 h at room temperature and then were incubated with either goat anti-GP80 (5 µg/ml), anti-calnexin monoclonal antibody (0.5 µg/ml; StressGen, Victoria, BC, Canada), or rabbit anti-protein disulfide isomerase polyclonal antibody (5 µg/ml; generously provided by Dr. S. Akagi, Kansai Medical University, Osaka, Japan; see Ref. 1) overnight at 4°C, followed by the detection of the binding sites of the primary antibody by FITC, Cy2 or Cy3-conjugated secondary antibody (1 µg/ml; Amersham).

Immunoprecipitation. The ability of ORP150 to bind to GP80 was assessed by immunoprecipitation using anti-ORP150 antibody, as described previously (11). In brief, MDCK cells (~5 × 106 cells/cm2) were exposed to hypoxia for the indicated period. The cells were washed three times with ice-cold PBS and lysed in ice-cold PBS containing NP-40 (1%), phenylmethylsulfonyl fluoride (PMSF; 1 mM), and EDTA (5 mM). After the incubation with protein G-agarose (1 mg/ml; GIBCO-BRL, Paisley, Scotland) for 1 h at 4°C to remove any material that binds nonspecifically to protein G, lysates (1 ml) were incubated with rabbit anti-human ORP150 antibody (5 µg/ml) and rotated overnight at 4°C. The mixtures were then incubated with protein G-agarose (2 mg/ml) for 2 h at 4°C, followed by centrifugation at 2,000 revolutions/min for 10 min. The immunoprecipitants were washed with ice-cold PBS containing NP-40 (0.1%), resuspended in nonreducing Laemmli's buffer (13), and heated to 95°C for 3 min. The mixture was then centrifuged, and the supernatant was applied to SDS-PAGE (10%). Electrophoresed proteins were transferred to a PVDF membrane, followed by staining with goat anti-GP80 antibody (5 µg/ml). The binding site of primary antibody was further detected by the anti-goat IgG polyclonal antibody labeled with alkaline phosphatase (1 µg/ml; Chemicon, Temecula, CA).

Pulse-chase analysis (metabolic labeling). To assess the participation of ORP150 in the protein transport/maturation of GP80, we performed a pulse-chase analysis as described previously (11). In brief, wild-type and antisense transformant MDCK cells (~106 cells) were maintained in methionine-free MEM (GIBCO-BRL) for 3 h under either normoxic or hypoxic conditions. The cells were then radiolabeled for 2 h with [35S]methionine (100 µCi/ml). After the removal of [35S]methionine, the cells were further maintained in DMEM, chased up to 1 h, and harvested for immunoprecipitation. Both the culture supernatant and cell pellets were used for immunoprecipitation by the anti-GP80 antibody (5 µg/ml) as described above and then subjected to autoradiography.

Cellular distribution of GP80 antigen. The participation of ORP150 in protein maturation was also analyzed by Western blot of subcellular fractionation (10). In brief, either wild-type or antisense transformant cells (~5 × 108 cells) were either exposed to hypoxia for 24 h or maintained under normoxic conditions. Cells were suspended in 10 ml of buffer A [0.25 M sucrose, 10 mM HEPES/NaOH, pH 7.5, 1 mM dithiothreitol (DTT), 1 mM PMSF, and 1 µg/ml leupeptin], and the cells were cavitated at 400 psi N2 pressure for 30 min by nitrogen cavitation bomb (Kontes Glass, Vineland, NJ). After homogenization, the cell lysate was clarified by centrifugation at 10,000 g for 15 min at 4°C. The supernatant was then centrifuged and fractionated by a series of sucrose steps as follows: 38% sucrose, 30% sucrose, and 20% sucrose (all prepared in 10 mM HEPES/NaOH, pH 7.5, 1 mM DTT) at 100,000 g for 3 h at 4°C (OPTIMA TLX; Beckman). The pellet at the bottom of the tube was resuspended in 3 ml of buffer A (precipitate fraction 1). Layered fractions (fractions 2-5) were collected by puncturing the tube at the desired depth and gently withdrawing the fluid. After measurement of protein concentration, each fraction (~5 µg protein) was subjected to Western blotting using anti-GP80 antibody. Enrichment of cellular structures/organelles in subcellular fractions was identified by the presence of marker enzymes [alkaline phosphodiesterase I (plasma membrane), alpha -mannosidase II (Golgi apparatus), and dolichol-P-mannose synthetase (ER; see Ref. 36)].

Enrichment of ORP150 from hypoxic MDCK cells and chromatography on ATP-agarose. ORP150 expressed in hypoxic MDCK cells was enriched by ion-exchange chromatography as described previously (10). In brief, MDCK cells (~108 cells) were exposed to hypoxia for 24 h, and the protein extract was applied to ion-exchange chromatography (MONO-Q; Pharmacia), followed by elution in a NaCl ascending gradient (0-1.5 M). To determine the content of stress proteins (ORP150, GRP94, and GRP78) in each fraction, 5 µl of each elute were subjected to SDS-PAGE (8%), followed by Western blotting using either anti-ORP150 antibody or anti-KDEL monoclonal antibody (SPA-827; Stressgen, Victoria, Canada). This antibody identifies GRP78 and GRP94 proteins containing the KDEL signal sequence.

The fractions in which ORP150, GRP94, or GRP78 was enriched were further subjected to ATP chromatography as described (4) with minor modification. In brief, three fractions were combined and dialyzed with ATP-column equilibration buffer that contained HEPES-KOH (20 mM, pH = 7.5), KCl (200 mM), MgCl2 (10 mM), EDTA (1 mM), glycerol (30%), and NP-40 (0.1%) at 4°C overnight. The dialyzed sample was then applied on ATP-agarose (Sigma) that was packed into a 10 × 100-mm column preequilibrated with equilibration buffer. The column was then washed with one column volume of low-salt buffer containing HEPES-KOH (20 mM, pH = 7.5), MgCl2 (10 mM), EDTA (1 mM), glycerol (30%), and NP-40 (0.1%) and corrected as fallthrough. Bound proteins were eluted in steps of 10 mM MgATP in equilibration buffer (ATP Elute) and 10 mM MgATP and 2 M KCl but no glycerol. Each fraction was concentrated 10 times with an ultrafiltration membrane (Amicon; Millipore, Bedford, MA) and subjected to Western blotting.

ATP kinetics study. To further characterize the ATP affinity of ORP150, we performed an ATP kinetics study as described (31). In brief, ~5 × 106 MDCK cells exposed to hypoxia for 24 h were harvested, and the cell lysates were immunoprecipitated as described above using either anti-ORP150 antibody (5 µg/ml) or anti-KDEL monoclonal antibody (Stressgen), which each recognize both GRP94 and GRP78 (5 µg/ml; see Ref. 12). The immunoprecipitants were then incubated with ATP and MgCl2 (5 mM) at the indicated concentration overnight at 4°C. The mixture was then centrifuged, and the supernatants (~400 µl) were concentrated 20 times with an ultrafiltration membrane (Amicon; Millipore) and applied to nonreducing SDS-PAGE (10%), followed by a Western blot analysis with the anti-GP80 antibody.

The density of bands corresponding to the ER form of GP80 at the indicated concentration of ATP was assessed as the percentage of GP80 intensity released from the immunoprecipitants at 10 mM MgATP concentration according to the formula
% Total bind

 = 100 × <FR><NU>GP80 density at indicated ATP concentration</NU><DE>GP80 density at ATP (10 mM)</DE></FR>

Densitometric and statistical analysis. Where indicated, densitometric analysis was performed to standardize results of Western blots, Northern blots, and immunoprecipitation. Autoradiograms or blots were scanned with an image scanner (GT-9500; Epson, Tokyo, Japan), and the density of the corresponding bands was further analyzed with Quality One software (Bio-Rad, Tokyo, Japan). Statistic analysis was performed by using either two-way ANOVA followed by the multiple contrast analysis or one-way ANOVA followed by multiple-comparison analysis (Newman-Keuls test).


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ABSTRACT
INTRODUCTION
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Cell viability during hypoxia. Cell viability of both wild-type and antisense transformant MDCK cells, based on the absence of LDH in the medium, was well maintained for up to 48 h after the onset of hypoxia. These results were consistent with the observation that hypoxic cells continued to exclude trypan blue and remained firmly adherent to the growth substrate.

Stress-induced expression of ORP150 in MDCK cells. In the wild-type MDCK cells, the exposure to hypoxia markedly enhanced the expression of ORP150 antigen (~8-fold increase based on the densitometric analysis; Fig. 1, A and C). This stress-induced expression of ORP150 in MDCK cells was accompanied by an increase in the ORP150 message, which was confirmed by the Northern blot analysis (~20-fold increase based on the densitometric analysis; Fig. 1, B and D). These data suggest the hypoxia-mediated inducibility of ORP150 in MDCK cells, as previously reported in other cell types (8, 10, 33).


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Fig. 1.   Induction of 150-kDa oxygen-regulated protein (ORP150) antigen (A and C) and message (B and D) in Madin-Darby canine kidney (MDCK) cells exposed to hypoxia. In A, MDCK cells (~106 cells/cm2) were exposed to hypoxia (0-48 h). Protein extracts by Nonidet P-40 (NP-40; 3 µg in each lane) were subjected to Western blotting using anti-ORP150 antibody as described in text. The migration of molecular mass markers is shown on right [triosephosphatase isomerase (32.5 kDa), aldolase (47.5 kDa), glutamic dehydrogenase (62 kDa), maltose-binding protein (MBP)-paramyosin (83 kDa), and MBP-beta -galactosidase (175 kDa)]. In B, MDCK cells (~5 × 107 cells/cm2) were exposed to hypoxia (0-12 h). Total RNA (20 µg in each lane) was then subjected to Northern blotting using a radiolabeled cDNA probe to either human ORP150 (top) or human beta -actin (bottom). Migration of ribosomal RNA is shown on right. The experiment was repeated 4 times, and a typical example is shown. Densitometric analysis was performed to quantitate the induction of ORP150 antigen (C) and message (D) by scanning the Western and Northern blots, respectively. Values are expressed as degree of increase of the density of each band at time 0, the normoxic condition (n = 4 experiments, means ± SD shown). ** P < 0.01 by the multiple-comparison analysis after ANOVA.

Binding of ORP150 to GP80. The Western blot analysis of GP80 in MDCK cells revealed the existence of four major intracellular forms of GP80, whose relative molecular mass corresponded to 35, 45, 65, and 80 kDa, along with its secretory form (~85 kDa) and higher-molecular-mass complex (~170 kDa and over; Fig. 2A). The immunoprecipitation analysis indicated the existence of GP80 immunogenicity of ~65 kDa, which corresponds to the immature form of this protein in the ER (6, 17, 35), in the immunoprecipitants prepared from MDCK cells by anti-ORP150 antibody. GP80 immunogenicity was not detected in the immunoprecipitants prepared with nonimmune IgG (Fig. 2B). The exposure of MDCK cells to hypoxia caused an increase of GP80 antigen in the immunoprecipitants prepared with the anti-ORP150 antibody (Fig. 2C). These data suggest that hypoxia slowed the traffic of GP80 through the ER, resulting in the increase of GP80 bound to ORP150 within the ER.


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Fig. 2.   Binding of ORP150 to 80-kDa glycoprotein (GP80). In A, the protein extract from MDCK cell culture (~10 µg derived from ~106 cells) was subjected to Western blotting using an anti-GP80 antibody. Each intracellular form of GP80, i.e., the 35- and 45-kDa premature forms (arrowheads), the 65-kDa endoplasmic reticulum (ER) form (open circle ), the 80-kDa Golgi form (), and the 85-kDa secretory form (black-diamond ) is indicated. In B, protein extracts from ~107 MDCK cells were immunoprecipitated with either anti-ORP150 antibody or nonimmune rabbit IgG, followed by Western blotting using the anti-GP80 antibody. In C, ~107 MDCK cells were exposed to hypoxia (0-48 h). Protein extracts (10 µg in each lane) were then subjected to immunoprecipitation with the anti-ORP150 antibody, followed by the Western blotting using the anti-GP80 antibody. Note that ~5-fold more protein was loaded in B compared with C. Experiments were repeated three times, and a typical example is shown. The migration of molecular weight markers is shown between gels A and B [lysozyme (16.5 kDa), beta -lactoglobulin A (25 kDa), triosephosphatase isomerase (32.5 kDa), aldolase (47.5 kDa), glutamic dehydrogenase (62 kDa), MBP-paramyosin (83 kDa), and MBP-beta -galactosidase (175 kDa)].

Establishment of permanent antisense transformant of ORP150 in MDCK cells. To assess the role of ORP150 in the protein transport in MDCK cells, we established a permanent transfectant of ORP150 antisense cDNA, as described previously (24). Compared with wild-type MDCK cells (Fig. 3A, lane 1) or cultures exposed to vector alone (Fig. 3A, lane 2), immunoblotting of lysates from two clones of ORP150 antisense transfectants showed lower ORP150 antigen (Fig. 3A, lanes 3-5), whereas two clones of ORP150 sense transformants displayed higher levels of ORP150 (Fig. 3A, lanes 6-7). Laser densitometry indicated a 10-fold increase in ORP150 expression in sense transfectants and ~40-fold suppression in antisense transfectants. For the experiments shown below, studies were performed in parallel with each of these cell lines, and representative results are shown.


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Fig. 3.   Baseline expression of ORP150 (A) and the induction of ORP150 (B) and 78- and 94-kDa glucose-regulated protein (GRP; C and D, respectively) in ORP150 antisense transformant MDCK cells. In A, protein extract (10 µg/lane) from either wild-type (lane 1), vector only transfected (lane 2), antisense transformant (lanes 3-5), or sense transformant MDCK cells (lanes 6 and 7) was subjected to Western blot using anti-ORP150 antibody as described in text. Migrations of molecular weight markers for A and B are shown on right in B. In B and C, an ORP150 antisense transformant cell line, cloned as described in text, was exposed to hypoxia (0-48 h). Protein extracts (10 µg in each lane) were then subjected to Western blotting using either the anti-ORP150 antibody (B) or an anti-KDEL monoclonal antibody (C). In D, wild-type MDCK cells were exposed to hypoxia (0-48 h), and protein extracts (10 µg in each lane) were then subjected to Western blotting using the anti-KDEL monoclonal antibody. Experiments were repeated four times, and typical example is shown. Migrations of molecular weight markers for C and D are shown on the right in D.

Established clones of antisense transformants, which showed a reduction of the ORP150 baseline expression (Fig. 3A), failed to induce ORP150 after the exposure to hypoxia (Fig. 3B), whereas GRP78 and GRP94, other molecular chaperones in the ER, were similarly induced (Fig. 3C), as demonstrated in wild-type MDCK cells (Fig. 3D). Northern blot analysis also exhibited no increase of ORP150 transcripts in antisense transformant cells after the exposure to hypoxia (data not shown).

Hypoxia-mediated energy depletion in MDCK cells. In both wild-type and antisense transformant cells, cellular ATP showed a time-dependent decline over 48 h of about the same magnitude, reaching 52.3 ± 2.3% of the level observed in normoxic wild-type cells (45.1 ± 8.3 nmol/mg protein). In parallel, energy charge, another parameter representing cellular energy metabolism (34), also showed significant decline (P < 0.01 by multiple-comparison analysis) in a similar manner in both wild-type and antisense transformant cells (null hypothesis was not rejected by 2-way ANOVA). These data suggest that exposure to hypoxia resulted in the depletion of cellular high-energy metabolites (Fig. 4A).


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Fig. 4.   Energy metabolism and the distribution of GP80 antigen in wild-type and ORP150 antisense transformant MDCK cells under hypoxia. In A, either wild-type (filled bars) or antisense transformant (open bars) MDCK cells were exposed to hypoxia (0-48 h). The content of high-energy adenine nucleotide was measured by reverse-phase HPLC as described in text. Cellular energy metabolism was expressed by ATP content (AI) and energy charge (AII). ATP content is expressed as % of the ATP content of wild-type cells at normoxic condition. In A and B, means ± SD are shown (n = 6). * P < 0.05; ** P < 0.01 by multiple-contrast analysis after 2-way ANOVA. In B, wild-type MDCK cells were either maintained in normoxic conditions (BI) or exposed to hypoxia (BIII) for 24 h. The cells were then fixed with paraformaldehyde (4%), permeabilized by Triton X-100 (0.3%) containing albumin (5%), and stained by anti-GP80 antibody. In BII and BIV, ORP150 antisense transformant MDCK cells were either maintained in normoxic conditions (BII) or exposed to hypoxia (BIV) for 24 h followed by staining by anti-GP80 antibody. In BV and BVI, antisense transformant cells exposed to hypoxia (24 h) were stained with either anti-calnexin antibody (BV) or anti-protein disulfide isomerase antibody (BVI; magnification ×400 in BI-BVI). In B, typical example is shown after the repeated experiments (4 times).

Distribution and maturation of GP80 in ORP150 antisense transformant cells. In both wild-type and antisense transformant MDCK cells, GP80 immunogenicity was distributed diffusely inside the cell under normoxic conditions (Fig. 4, BI and BII). In the antisense transformants, hypoxia resulted in a marked increase of GP80 immunointensity around the nucleus, whereas the distribution of GP80 remained unchanged in the wild-type MDCK cells (Fig. 4, BIII and BIV). The distribution of GP80 in the antisense transformants exposed to hypoxia showed a pattern comparable to that of calnexin (Fig. 4BV; see Ref. 37) and protein disulfide isomerase (Fig. 4BVI; see Ref. 1), both of which are located in the luminal side of the ER.

Pulse-chase labeling of MDCK cells showed similar time course in the elaboration of labeled GP80 to the medium in both wild-type and antisense transformant cells (Fig. 5A), whereas hypoxia caused a marked delay of the elaboration of GP80 antigen in the culture supernatants, especially in antisense cells (Fig. 5, C and E). The immunoprecipitation analysis of pulse-labeled cell lysates showed similar time course in the intracellular maturation of GP80 under the normoxic conditions (Fig. 5B). In contrast, exposure to hypoxia, which increased the ER form of GP80 in both cell types (Fig. 5D), markedly slowed the formation of the Golgi form of this tracer protein in the antisense transformant cells (Fig. 5, D and F).


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Fig. 5.   Maturation of GP80 antigen in MDCK cells. In A and B, either wild-type or antisense transformant MDCK cells were pulse labeled with [35S]methionine under normoxic conditions for 2 h and chased up to 60 min. At each time point, either culture supernatant (A) or cell lysate obtained with NP-40 (B; 100 µCi/lane in each case) was immunoprecipitated with anti-GP80 antibody and subjected to SDS-PAGE (10%), followed by autoradiography. In C and D, either wild-type or antisense transformant cells cultured in hypoxic conditions for 24 h were pulse labeled with radiolabeled tracer inside the hypoxic chamber and chased under hypoxic conditions. Cell samples were then subjected to autoradiography as described above. The ER form (~65 kDa; open circle ), Golgi form (~75 kDa; ) and secretory form (~85 kDa; black-diamond ) of GP80 are indicated. Migrations of molecular weight markers are shown on the far right. In E and F, densitometric analysis was performed to either 85-kDa secretory form in C (E) or 80-kDa matured form in D (F) in both wild-type (filled bars) and antisense transformant (open bars) cells. Values are expressed as %density of corresponding band in the wild-type cells at the time of 60 min; n = 4, means ± SD. ** P < 0.01 by multiple-contrast analysis followed by the 2-way ANOVA.

Western blot analysis of fractionated cell lysate showed the existence of the Golgi form of GP80 in the Golgi fraction in both wild-type and antisense transformants (Fig. 6A, lanes 3 and 8) under normoxic conditions. In contrast, exposure to hypoxia resulted in a marked decline of the 80-kDa band, the Golgi form of GP80 antigen in antisense transformant (Fig. 6B, lane 8, and Fig. 6C), whereas this signal can be detected in wild-type cells under hypoxia (Fig. 6B, lane 3, and Fig. 6C). These data suggest that low expression of ORP150 delayed the protein traffic from the ER to Golgi and that ORP150 participates in the maturation of GP80, especially under hypoxic conditions.


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Fig. 6.   Distribution of GP80 antigen in fractionated cell lysate. Either wild-type or antisense transformant MDCK cells were either cultured under normoxic (A) or hypoxic (B) conditions for 24 h. Cell lysates were then fractionated by the sucrose gradient as described in text, followed by Western blot analysis using anti-GP80 antibody. Each lane represents ER fraction (lanes 1-2 and 6-7), Golgi fraction (lanes 3 and 8), plasma membrane (lanes 4 and 9), and cytosol (lanes 5 and 10). ER form (~65 kDa; open circle ) and Golgi form (~80 kDa; ) of GP80 antigen are indicated. Migrations of molecular weight markers are also shown on right. Experiments were repeated 4 times, and typical example is shown. In C, densitometric analysis was performed for quantitative analysis. Photodensity of 80-kDa matured form of GP80 in Golgi fraction (no. 3) in either wild-type (filled bars) or antisense transformant (open bars) under normoxic (N) or hypoxic (H) condition was assessed by the ratio to 75-kDa (ER form) band in the same fraction; n = 4, means ± SD shown. ** P < 0.01 by multiple-comparison analysis followed by 1-way ANOVA.

Affinity of ORP150 to ATP. To clarify the role of ORP150 in relation to that of other stress proteins in the ER, especially in the hypoxic environment, the affinity to ATP of ORP150 was assessed by ATP affinity chromatography. Three fractions (nos. 9, 11, and 13 in Fig. 7B) in which ORP150, GRP94, or GRP78 was enriched by ion exchange chromatography were mixed and loaded on ATP-agarose (Fig. 8A). Western blot analysis using either anti-ORP150 antibody (Fig. 8B) or anti-KDEL monoclonal antibody (Fig. 8C) demonstrated no antigen of each protein detected in the fallthrough from the column, whereas each protein was detected in the ATP-elute (Fig. 8, B and C), suggesting that ORP150 has ATP binding affinity similar to GRP78 and GRP94.


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Fig. 7.   Enrichment of ER-located stress proteins by ion-exchange chromatography. In A, protein extract (~5 mg) was prepared from wild-type MDCK cells (~108 cells) exposed to hypoxia for 24 h and applied to ion-exchange chromatography (MONO-Q) with a linear gradient of NaCl (0-1.5 M), as described in text. The profile for optical density at 280 nm of the elute is shown in A. Each fraction was then subjected to Western blotting using either an anti-ORP150 antibody or anti-KDEL monoclonal antibody, which recognizes both GRP94 and GRP78 (B). Experiments were repeated 4 times, and typical example is shown.



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Fig. 8.   ATP-binding ability of ORP150. Three fractions, where ORP150, GRP94, and GRP78 (fractions 9, 11, and 13 in Fig. 7, respectively) were enriched, were mixed and applied to an ATP-agarose column and eluted with MgATP (10 mM) as described in the text. The total cell extract (Total), MONO-Q mixed fractions (QFraction), fallthrough of the ATP column (Fallthrough), and eluted fraction (ATP Elute), each of which corresponds to ~5 µg of protein, were separated by SDS-PAGE (8%), followed by either silver staining (A) or Western blotting using either anti-ORP150 antibody (B) or anti-KDEL monoclonal antibody (C). Experiments were repeated 4 times, and typical example is shown.

In agreement with these findings, the ATP kinetics study indicated that GP80 antigen is released from the immunoprecipitants prepared with the ORP150 antibody at lower concentrations (100-500 µM; Fig. 9, Ai) compared with those prepared with the anti-KDEL monoclonal antibody (0.5-1 mM; Fig. 9, Aii; see Ref. 12). Densitometric analysis showed a significant difference in the ATP dose dependency in the release of GP80 from the immunocomplex (P < 0.01 by 2-way ANOVA; Fig. 9B).


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Fig. 9.   Characterization of ORP150 affinity to GP80. MDCK cells exposed to hypoxia for 24 h were lysed by NP-40 (1%) and immunoprecipitated with either the anti-ORP150 antibody (Ai) or anti-KDEL monoclonal antibody (Aii). The immunoprecipitants were then incubated at 37°C in the presence of Mg2+ (5 mM) and ATP (0-10 mM), followed by centrifugation. The supernatant of the mixture was then subjected to Western blotting using the anti-GP80 antibody. The migration of the molecular weight markers is shown on right. Experiments were repeated 4 times, and typical example is shown. In B, the release of GP80 from immunoprecipitants was quantitatively assessed by densitometric analysis as described in text; n = 4, means ± SD. ** P < 0.01 by 2-way ANOVA.

These data indicate that ORP150 has a higher affinity to ATP compared with the other molecular chaperones in the ER and that ORP150, therefore, can participate in the protein transport, even under a low environmental level of ATP.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Oxygen deprivation represents a major alteration of extracellular environments in the ischemic vasculature, which drives cells to express a set of stress proteins, ORPs. This concept, the expression of hypoxia-mediated stress proteins, was initially proposed as a possible explanation of the pathophysiology for tumor survival by providing a hypothesis that the expression of ORPs enables tumor cells to survive under an anoxic environment, especially in the core region of hypovascular tumors (7). ORP150 was initially purified and cloned from cultured astrocytes, which also exhibit a marked resistance to ischemic stresses (10). The expression of ORP150 has been demonstrated not only in human tumor tissue (32) but also in the core area of cerebral ischemia in an experimental stroke model (19), and even in an atherosclerotic lesion of human aorta (33), suggesting a pivotal role of hypoxia-induced stress proteins in the maintenance of cellular metabolism in both tumor cells and the nontumor constituents of organs under oxygen deprivation. These data suggest a role of ORP150 as a stress protein that maintains cellular viability under altered conditions. In this context, we have previously demonstrated that the expression of ORP150 is essential for mammalian cells to survive chronic hypoxia (24).

The predicted amino acid sequence of ORP150 from its cDNA suggests another aspect of ORP150 as a molecular chaperone. ORP150 has several characteristics commonly observed in the molecular chaperone in the ER, including ATPase domains, protein binding site, and the retention signal in the ER (8). Consistent with these characteristics expected from the amino acid sequence, our present study demonstrates that 1) ORP150 binds to the ER form of GP80, 2) ORP150 has affinity to the ATP column, and 3) the binding of GP80 to ORP150 can be reversed by the addition of ATP. The 170-kDa glucose-regulated protein (GRP170), a homologue of ORP150 in Chinese hamster, was also characterized as a molecular chaperone by another group (16). GRP170 is reported to form a complex with immunoglobulins (16). In immune response cells, GRP170 is associated with the transporter associated with antigen processing and other molecular chaperones in the ER, including ERp72 and calnexin (29).

MDCK cells, a polarized epithelial cell line derived from dog kidney tubular epithelium, are one of the most extensively characterized model systems representing the function of transporting epithelium of renal tubules. In MDCK cells, GP80/clusterin is known as a secretory protein released after intracellular maturation (37). Exposure of MDCK cells to hypoxia, which resulted in the decline of cellular ATP content and energy charge in a similar manner in both wild-type and antisense transformant MDCK cells (Fig. 4A), caused the retention of GP80 antigen within the ER only in the antisense transformant cells (Fig. 4B). Furthermore, metabolic labeling analysis showed a marked delay in the maturation and secretion of GP80 antigen in the antisense transformants, whereas no retardation was observed under normoxic conditions (Fig. 5). Consistently, the subcellular fraction analysis indicated the failure to form GP80 in the Golgi fraction in the antisense transformant culture under hypoxia (Fig. 6). From the viewpoint of energy metabolism, oxygen deprivation results in the shift of cellular energy metabolism to anaerobic glycolysis, followed by the decline of cellular high-energy adenine nucleotide. These data suggest that the induction of ORP150 is essential to ensure protein transport/maturation, especially under hypoxic conditions.

The association of protein to the molecular chaperone can be reversed by the addition of ATP (12, 31). Thus the affinity to ATP represents a major characteristic of the molecular chaperone. Our data indicated that ORP150 has high affinity to the ATP column comparable to that of GRP78 and GRP94. Furthermore, GP80 was released from ORP150 immunoprecipitants at a lower concentration of ATP (~1/4) compared with the concentration where GP80 is released from GRP78/GRP94 immunoprecipitants (Fig. 9). These data suggest that ORP150 may assist the prompt maturation/secretion of GP80 by providing a more efficient pathway from the ER to the Golgi in MDCK cells than those mediated by other molecular chaperones in the ER due to the higher affinity of ORP150 to ATP, especially under hypoxic conditions.

In the renal tubular epithelial cells, the secretion of glycoproteins is essential to suppress the activation of complement and coagulation pathways in the luminar side of the urinary tract (2, 26). Furthermore, renal tubular cells in this portion have to maintain an extreme gradient of Na+, which makes an additional energy demand in the tubular epithelium. Morphologically, tubular epithelium in this portion is characterized by the abundance of ER-Golgi complexes (30), which suggests the necessity for the protein transport system to be protected under chronic insufficiency for ATP. Furthermore, our pilot study demonstrates native expression of ORP150 in the renal epithelium in the outer medulla, suggesting that ORP150 antigen in the rat kidney tubular epithelium is localized in the very portion where the atmospheric oxygen tension is extremely low (unpublished data).

Our data demonstrate that ORP150 may function in such circumstances that may result in the cellular energy depletion. In these situations, ORP150 may provide a more efficient pathway for protein transport from the viewpoint of energy metabolism. The failure in the induction of ORP150 leads to the accumulation of immature protein, which could eventually initiate cell death caused by the dysfunction of the ER (25). In the antisense transformants, therefore, GP80 protein remaining within the ER in an immature form may possibly cause the functional failure of the ER. In contrast, the antisense transformant MDCK cells showed an enhanced vulnerability under prolonged hypoxic conditions (when exposed to hypoxia for >48 h; data not shown) where the same mechanism might be functioning as observed in our previous study (24). Taken together, our data demonstrate that ORP150, due to its higher affinity to ATP, may assist the maturation of secretory protein in conditions where less ATP is accessible, leading to the protection of cell death by maintaining the function of ER.


    FOOTNOTES

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

Address for reprint requests and other correspondence: Y. Bando, Dept. of Anatomy and Neuroscience, Osaka Univ. Graduate School of Medicine, 2-2 Yamada-oka, Suita City, 565-0871, Japan (E-mail:ybando{at}anat2.med.osaka-u.ac.jp).

Received 23 June 1999; accepted in final form 11 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1.   Akagi, S, Yamamoto A, Yoshimori T, Masaki R, Ogawa R, and Tashiro Y. Distribution of protein disulfide isomerase in rat hepatocytes. J Histochem Cytochem 36: 1533-1542, 1988[Abstract].

2.   Brady, HR, Brenner BM, and Lieberthal W. Acute renal failure. In: The Kidney, edited by Brenner BM.. Boston, MA: Saunders, 1996, p. 1200-1252.

3.   Bush, KT, Hendrickson BA, and Nigam SK. Induction of the FK506-binding protein, FKBP13, under conditions which misfold proteins in the endoplasmic reticulum. Biochem J 303: 705-708, 1994[ISI][Medline].

4.   Dikers, T, Vollmer J, Schlenstedt G, Jung C, Sandholzer U, Zachmann K, Schlotterhhose P, Neifer K, Schmidt B, and Zimmermann R. A microsomal ATP binding protein involved in efficient protein transport into an mammalian endoplasmic reticulum. EMBO J 15: 6931-6942, 1996[Abstract].

5.   Feinberg, AP, and Vogelstein BV. A technique for radiolabelling DNA restriction endonulcease fragments to high specific activity. Anal Biochem 132: 6-13, 1983[ISI][Medline].

6.   Hartmann, K, Rauch J, Urban J, Parczyk K, Diel P, Pilarsky C, Appel D, Haase W, Mann K, Weller A, and Brant KC. Molecular cloning of gp 80, a glycoprotein complex secreted by kidney cells in vitro and in vivo A link to the reproductive system and to the complement cascade. J Biol Chem 266: 9924-9931, 1991[Abstract/Free Full Text].

7.   Heacock, CS, and Sutherland RM. Induction characteristics of oxygen regulated protein. Int J Radiat Oncol Biol Phys 12: 1287-1290, 1986[ISI][Medline].

8.   Ikeda, J, Kaneda S, Kuwabara K, Ogawa S, Kobayashi T, Matsumoto M, Yura T, and Yanagi H. Cloning and expression of cDNA encoding the human 150 kDa oxygen regulated protein, ORP150. Biochem Biophys Res Commun 230: 94-99, 1997[ISI][Medline].

9.   Ishizawa, M, Kobayashi Y, Miyamura T, and Matsuura S. Simple procedure of DNA isolation from human serum (Abstract). Nucleic Acids Res 19: 5792, 1991[ISI][Medline].

10.   Kuwabara, K, Matsumoto M, Ikeda J, Hori O, Ogawa S, Maeda Y, Kitagawa K, IMuta N, Kinoshita K, Stern D, Yanagi H, and Kamada T. Purufucation and characterization of a novel stress protein, the 150kDa oxygen regulated protein (ORP150), from cultured rat astrocytes, and its expression in ischemic mouse brain. J Biol Chem 279: 5025-5032, 1996.

11.   Kuwabara, K, Ogawa S, Matsumoto M, Koga S, Clauss M, Pinsky DJ, Lyn P, Leavy J, Witte L, Joseph-Silverstein J, Furie MB, Torcia G, Cozzolino F, Kamada T, and Stern DM. Hypoxia-mediated induction of acidic/basic fibroblast growth factor and platelet-derived growth factor in mononuclear phagocytes stimulates growth of hypoxic endothelial cells. Proc Natl Acad Sci USA 92: 4606-4610, 1995[Abstract].

12.   Kuznetsov, G, Chen LB, and Nigam SK. Several endoplasmic reticulum stress proteins,including Erp72, interact with thyroglobulin during its maturation. J Biol Chem 269: 22990-22995, 1994[Abstract/Free Full Text].

13.   Laemmli, U. Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 277: 680-685, 1970.

14.   Lee, AS. Mammalian stress response: induction of the glucose regulated protein family. Curr Opin Cell Biol 4: 267-273, 1992[Medline].

15.   Liu, H, Bowes RC, van de Water B, Sillence C, Nagelkerke JF, and Stevens JL. Endoplasmic reticulum chaperones GRP78 and calreticulin prevent oxidative stress, Ca2+ disturbances, and cell death in renal epithelial cells. J Biol Chem 272: 21751-21759, 1997[Abstract/Free Full Text].

16.   Lin, HY, Masso-Welch P, Di YP, Cai JW, Shen JW, and Subjeck JR. The 170-kDa glucose-regulated stress protein is an endoplasmic reticulum protein that binds immunoglobulin. Mol Biol Cell 4: 1109-1119, 1993[Abstract].

17.   Losch, A, and Koch-Brandt C. Dithiothretiol treatment of Madin-Darby canine kidney cells reversibly blocks export from the endoplasmic reticulum but does not affect vectorial targeting of secretory proteins. J Biol Chem 270: 11543-11548, 1995[Abstract/Free Full Text].

18.   Matsuo, N, Ogawa S, Imai Y, Takagi T, Tohyama M, Stern D, and Wanaka A. Cloning of a novel RNA binding polypeptide (RA301) induced by hypoxia/reoxygenation. J Biol Chem 270: 28216-28222, 1995[Abstract/Free Full Text].

19.   Matsushita, K, Matsuyama T, Nishimura H, Takaoka T, Kuwabara K, Tsukamoto Y, Sugita M, and Ogawa S. Marked, sustained expression of a novel 150-kDa oxygen regulated stress protein, in severely ischemic mouse neurons. Mol Brain Res 60: 98-106, 1998[ISI][Medline].

20.   Mazzarella, R, Srinivasan M, Haugejorden S, and Green M. ERp72, an abundant luminal endoplasmic reticulum protein, contains three copies of the active site sequence of protein disulfide isomerase. J Biol Chem 265: 1094-1101, 1990[Abstract/Free Full Text].

21.   Melnick, J, Dul JL, and Argon Y. Sequential interaction of the chaperons Bip and GRP94 with immunoglobulin chains in the endoplasmic reticulum. Nature 370: 373-376, 1994[ISI][Medline].

22.   Munro, S, and Pelham HRB An Hsp70 like Protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobin heavy chain binding protein. Cell 46: 291-300, 1986[ISI][Medline].

23.   Ogawa, S, Gerlach H, Esposito C, Macaulay AP, Brett J, and Stern D. Hypoxia modulates the barrier and coagulant function of cultured bovine endothelium. J Clin Invest 85: 1090-1098, 1990[ISI][Medline].

24.   Ozawa, K, Kuwabara K, Tamatani M, Yakatsuji K, Tsumakoto Y, Kaneda S, Yanagi H, Stern D, Ogawa S, and Tohyama M. ORP150 (150 kDa oxygen-regulated protein) suppresses hypoxia-induced apoptotic cell death. J Biol Chem 274: 6397-6404, 1999[Abstract/Free Full Text].

25.   Pahl, HL, and Baeuerle P. A Endoplasmic reticulum-induced signal transduction and gene expression. Trends Cell Biol 7: 50-55, 1997[ISI].

26.   Rosen, MK, and Silkensen J. Clusterin and the kidney. Exp Nephrol 3: 9-14, 1995[ISI][Medline].

27.   Shui, RPB, Pouyssegur J, and Pastan I. Glucose depletion accounts for the induction of two transformation sensitive membrane proteins in Rous sarcoma virus transformed chick embryo fibroblast. Proc Natl Acad Sci USA 74: 3840-3844, 1977[Abstract].

28.   Sidrauski, C, Chapman R, and Walter R. The unfold protein response: an intracellular signalling pathway with many surprising features. Trends Cell Biol 8: 245-249, 1998[ISI][Medline].

29.   Spee, P, Subjeck J, and Neefjes J. Identification of novel peptide binding proteins in the endoplasmic reticulum: ERp72, calnexin, and grp170. Biochemistry 38: 10559-10566, 1999[ISI][Medline].

30.   Tisher, CC, and Madsen KM. Anatomy of the kidney. In: The Kidney, edited by Brenner BM.. Boston, MA: Saunders, 1996, p. 3-71.

31.   Toledo, H, Carlino A, Vidal V, Redfield B, Nettleton MY, Kochan JP, Brot N, and Weissbach H. Dissociation of glucose-regulated protein Grp78 and Grp78-IgE Fc complex by ATP. Proc Natl Acad Sci USA 90: 2505-2508, 1993[Abstract].

32.   Tsukamoto, Y, Hirota S, Kuwabara K, Kawano K, Yoshikawa K, Ozawa K, Kobayashi T, Yanagi H, Kitamura Y, Tohyama M, and Ogawa S. Expression of 150 kDa oxygen-regulated protein (ORP150), a new member of the HSP70 family, in human breast cancers. Lab Invest 78: 699-706, 1998[ISI][Medline].

33.   Tsukamoto, Y, Kuwabara K, Hirota S, Ikeda J, Stern D, Yanagi H, Matsumoto M, Ogawa S, and Kitamura Y. The 150 kDa oxygen regulated protein (ORP150) is expressed in human atherosclerotic plaques and allows mononuclear phagocytes to withstand cellular stress on exposure to hypoxi and modified LDL. J Clin Invest 98: 1930-1941, 1996[Abstract/Free Full Text].

34.   Ueda, H, Hashimoto T, Furuya E, Tagawa K, Kitagawa K, Matsumoto M, Yoneda S, Kimura K, and Kamada T. Changes in aerobic and anaerobic ATP-synthesizing activities in hypoxic mouse brain. J Biochem (Tokyo) 104: 81-86, 1988[Abstract].

35.   Urban, J, Parcyzyk K, Leutz A, Kayne M, and Kondor-Koch C. Constitutive apical secretion of an 80kDa sulfated glycoprotein complex in the polarized epithelial Madin-Darby canine kidney cell line. J Cell Biol 105: 2735-2743, 1987[Abstract].

36.   Vidugiriene, J, and Menon AK. Early intermediates in glycosyl-phosphatidylinosotol anchor assembly are synthesized in the ER and located in the cytoplasmic leaflet of the ER membrane bilayer. J Cell Biol 121: 987-996, 1993[Abstract].

37.   Wada, I, Ou WJ, Liu MC, and Sheele G. Chaperone function of calnexin for the folding intermadiate of gp80, the major secretory protein in MDCK cells. J Biol Chem 269: 7464-7472, 1994[Abstract/Free Full Text].

38.   Welch, WJ. Mammalian stress response: cell physiology, structure/funciton of stress protein, and implications for medicine and disease. Physiol Rev 72: 1063-1081, 1992[Free Full Text].

39.   Wynn, RM, Davie JR, Cox RP, and Chuang DT. Molecular chaperones: heat-shock proteins, foldases, and matchmakers. J Lab Clin Med 124: 31-36, 1994[ISI][Medline].


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