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UDP-Glucose:Glycoprotein Glucosyltransferase Associates with Endoplasmic Reticulum Chaperones and Its Activity Is Decreased in Vivo by the Inhalation Anesthetic Halothane Hamid R. Amouzadeh,*,† Mohammed Bourdi,† Jackie L. Martin,†,‡ Brian M. Martin,§ and Lance R. Pohl† Molecular and Cellular Toxicology Section, Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1760, Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21287, and Clinical Neuroscience Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892 Received August 1, 1996X
Halothane causes an idiosyncratic hepatitis that is thought to result, in part, from immune reactions against one or more lumenal endoplasmic reticulum (ER) proteins that have been covalently modified by the trifluoroacetyl chloride metabolite of halothane. In this study, we have identified a 170 kDa protein target of halothane in the liver of rats. The 170 kDa protein was first detected when proteins in lysates of hepatocytes from halothane-treated rats were immunoprecipitated with antisera against several resident ER proteins. This 170 kDa protein was found to be associated with other protein targets of halothane, including protein disulfide isomerase, a protein disulfide isomerase isoform, a 59 kDa carboxylesterase, and 78 kDa glucoseregulated protein. Immunoblotting with antiserum directed against the trifluoroacetylated hapten indicated that the 170 kDa protein was trifluoroacetylated. Based upon its subcellular localization, molecular mass, N-terminal amino acid sequence, and antigenicity, the trifluoroacetylated 170 kDa protein was identified as UDP-glucose:glycoprotein glucosyltransferase (UGGT), a lumenal ER protein that is thought to have a role in the folding of N-linked glycoproteins. Moreover, treatment of rats with halothane caused a 44% decrease in the activity of liver microsomal UGGT, and at least 36% of the change in the activity of the enzyme could be due to a decrease in the level of the protein. The results suggest that the function of UGGT in folding of N-linked glycoproteins may be affected by other resident ER proteins or xenobiotics such as halothane.
Introduction The inhalation anesthetic halothane (CF3CHClBr) is oxidatively metabolized by cytochromes P450 in the liver to a trifluoroacetyl chloride (CF3COCl) reactive metabolite, which covalently binds to several liver endoplasmic reticulum (ER)1 proteins (1). A number of the trifluoroacetylated (TFA) proteins have been isolated from liver microsomes of halothane-treated rats and identified. The 57, 58, 59, 63, 80, 82, and 100 kDa proteins have been identified as protein disulfide isomerase (PDI) (2), a PDI isoform (3), a carboxylesterase (4), calreticulin (5), 72 kDa endoplasmic reticulum protein (ERp72) (6), 78 kDa glucose-regulated protein (GRP78) (7), and 94 kDa glucose-regulated protein (GRP94) (8), respectively. It * Corresponding author at the Molecular and Cellular Toxicology Section, National Institutes of Health, Building 10, Room 8N110, Bethesda, MD 20892-1760. Phone 301 402 7223, Fax 301 480 4852, E-mail,
[email protected]. † Molecular and Cellular Toxicology Section, NIH. ‡ The Johns Hopkins Medical Institutions. § Clinical Neuroscience Branch, NIH. X Abstract published in Advance ACS Abstracts, December 15, 1996. 1 Abbreviations: ER, endoplasmic reticulum; TFA, trifluoroacetylated; PDI, protein disulfide isomerase; ERp, endoplasmic reticulum protein; GRP, glucose-regulated protein; UGGT, UDP-glucose glycoprotein:glucosyltransferase; PVDF, poly(vinylidene difluoride); TFAp170, TFA 170 kDa protein; DOC, sodium deoxycholate; EDTA, (ethylenedinitrilo)tetraacetic acid, disodium salt, dihydrate; HPLC, high pressure liquid chromatography; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin.
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is thought that these covalently modified proteins cause an idiosyncratic hepatitis in susceptible individuals by immunopathological mechanisms (1, 9, 10). This hypothesis is supported, in part, by the presence of antibodies, specifically in the sera of the halothane hepatitis patients, that react with one or more of these TFAproteins (11). Many of these halothane hepatitis patients also have antibodies that react with the native forms of these proteins (11). Recently, we studied the trafficking of the TFA proteins in primary cultures of hepatocytes isolated from halothane-treated rats in order to understand how they might come in contact with the immune system. The half-lives of the TFA proteins and the unmodified forms of these proteins were found to be similar (12), indicating that covalent modification by TFA did not affect the processing of the lumenal ER protein targets of halothane. Moreover, all of these proteins, except GRP94, appeared to be degraded in the lysosomes or other acidic compartments. However, it was not clear whether the functions of these lumenal ER proteins are affected by either halothane or its trifluoroacetyl chloride metabolite. In this study, we found that several of the previously characterized lumenal ER protein targets of the trifluoroacetyl chloride metabolite of halothane were associated with a trifluoroacetylated 170 kDa protein. This 170 kDa protein was identified as UDP-glucose:glycoprotein glucosyltransferase (UGGT), a lumenal ER protein that
This article not subject to U.S. Copyright.
Published 1997 by the American Chemical Society
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Figure 1. Immunoprecipitation of proteins in lysates of hepatocytes with protein-specific antisera. Primary cultures of hepatocytes were prepared 16 h after treatment of rats with halothane (10 mmol/kg, ip). Cells were pulsed with [35S]methionine overnight and chased for 4 days. Cell lysates were collected daily, and proteins were immunoprecipitated with 57 kDa (PDI), 58 kDa (PDI isoform), 59 kDa (carboxylesterase), 82 kDa (GRP78), 63 kDa (calreticulin), 80 kDa (ERp72), and 100 kDa (GRP94) antisera and analyzed by SDS-PAGE and fluorography. (A) Antisera that immunoprecipitated a 170 kDa protein; (B) antisera that did not immunoprecipitate a 170 kDa protein.
glucosylates glucose-free, high mannose-type oligosaccharides (13) and is thought to have a role in the folding of newly synthesized N-linked glycoproteins (14). Since halothane caused a significant decrease in the activity of UGGT, the results suggest that halothane or possibly other xenobiotics may cause toxicity, at least in part, by disrupting the folding of proteins in the ER.
Experimental Procedures Animals. Male Sprague-Dawley rats (Taconic, Germantown, NY) of similar age and weight (175-200 g) were maintained in our animal facility according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (15). Materials. The following reagents were purchased commercially: halothane (Halocarbon Labs, Inc., Hackensack, NJ); goat anti-rabbit IgG-peroxidase conjugated antibody (Boehringer-
Mannheim, Indianapolis, IN); BCA protein assay reagents (Pierce, Rockford, IL); poly(vinylidene difluoride) (PVDF) membrane (0.45 µm, Immobilon, Millipore, Bedford, MA); UDP-[14C]glucose (302 mCi/mmol), [35S]methionine (1018 Ci/mmol), and ECL enhanced chemiluminescence reagents (Amersham, Arlington Height, IL); deoxynojirimycin and thyroglobulin (Sigma Chemical Co., St. Louis, MO); DEAE-Sepharose (Pharmacia, Piscataway, NJ); 2.5 cm GF/F glass microfiber filters (Whatman, Hillsboro, OR); broad range (6.5-200 kDa) molecular weight standards (Bio-Rad, Hercules, CA); and Protogel and Hydrofluor scintillation cocktail (National Diagnostics, Atlanta, GA). Immunoprecipitation of a 170 kDa Protein with ER Protein Targets of Halothane. Following a previously described procedure (12), primary cultures of hepatocytes were prepared from male Sprague-Dawley rats 16 h after treatment with halothane (10 mmol/kg, ip). The cells were pulsed for approximately 12 h with [35S]methionine to label newly syn-
Halothane Inhibits Glycoprotein Glucosyltransferase thesized native proteins and then chased for 4 days. Cell lysates were collected daily and proteins were either immunoprecipitated with protein-specific antisera and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) (7% acrylamide gels) and fluorography or analyzed by immunoblotting with TFA antiserum to measure levels of 35Slabeled native and trifluoroacetylated proteins, respectively. Purification of TFA 170 kDa Protein (TFA-p170). The procedure for extraction and purification of TFA proteins from the lumen of rat liver ER has been described previously (5). Briefly, livers from halothane-treated rats (16 h after treatment) were homogenized in a solution of 100 mM Tris acetate, 1 mM EDTA, and 250 mM sucrose (pH 7.5), and microsomes were prepared by differential centrifugation and were extracted with a solution of 0.1% (w/v) sodium deoxycholate (DOC), 10 mM TrisHCl, and 0.1 mM EDTA (pH 7.5). The extract was placed onto a DEAE-Sepharose anion exchange column that was equilibrated with a solution of 10 mM Tris-HCl (pH 7.5), containing 0.1 mM EDTA. TFA-p170 was eluted from the column at approximately 0.3 M sodium chloride and was further purified on a hydroxyapatite HPLC column (HCA, 7.6 mm × 10 cm, Rainin, Woburn, MA) that was equilibrated with 10 mM sodium phosphate (pH 6.8), at 1 mL/min for 10 min. The sample was injected onto the column, and proteins were eluted with a sodium phosphate linear gradient of 10-350 mM sodium phosphate (pH 6.8). Fractions were collected and were analyzed by SDS-PAGE and immunoblotting. TFA-p170 was eluted from the column at approximately 250 mM sodium phosphate. N-Terminal Sequence Analysis of TFA-p170. A sample of the pooled HPLC fractions containing TFA-p170 was electroblotted onto PVDF membrane, and the band corresponding to TFA-p170 was cut from the membrane and was used for N-terminal amino acid sequence analysis as described previously (5). Determination of UGGT Activity. The activity of UGGT in liver microsomes, prepared from pooled liver tissues of three control and three halothane-treated (10 mmol/kg) rats, was determined according to the method described previously (16). Briefly, 300 µg of microsomes was incubated in 4 mM Tris-HCl (pH 8), containing 10 mM CaCl2, 0.6% (v/v) Triton X-100, 300 µM deoxynojirimycin, 480 µM urea denatured thyroglobulin, and 5 µM UDP-[14C]glucose in a total volume of 100 µL at 37 °C. After 10 min, the reaction was stopped by filtering the mixture through glass fiber filters treated with 10% (w/v) cold trichloroacetic acid. Proteins collected on the filters were washed with 1 mL aliquots of 10% cold trichloroacetic acid (w/v) and 95% (v/v) ethanol three times each, air-dried, placed in a scintillation cocktail, and counted the next day. The mean ( SE of cpm values (n ) 4) was calculated and compared using Student t-test. A probability level of p < 0.05 was considered significant. Other Methods. Methods for raising antisera against the TFA hapten (17), PDI (2), PDI isoform (18), carboxylesterase (4), calreticulin (5), ERp72 (6), and UGGT (13) were described previously. Similar procedures were used for the preparation of GRP78 and GRP94 antisera. All of the protein antisera were found to be specific and reacted with both native and trifluoroacetylated forms of the respective proteins, as determined by immunoblotting with liver tissues from control and halothanetreated rats (data not shown). Protein concentrations were determined using BCA reagents with BSA as standard. SDSPAGE and immunoblotting were done as described previously (12). The intensities of protein bands on immunoblots were measured by laser densitometry (Molecular Dynamics Personal Densitometer, Sunnyvale, CA).
Results Association of Target Proteins of Halothane with 170 kDa Protein. Sixteen hours after rats were treated with halothane, hepatocytes were isolated, cultured, pulsed with [35S]methionine, and chased for 4 days. As reported previously (12), this procedure allowed for the simultaneous study of the cellular biology of 35S-labeled
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Figure 2. Immunoblot of lysates of hepatocytes with TFAantiserum. Primary cultures of hepatocytes were prepared 16 h after treatment of rats with halothane (10 mmol/kg, ip). Samples of cell lysates were collected daily for 4 days and were analyzed by immunoblotting with TFA antiserum and chemiluminescence detection.
native and the trifluoroacetylated proteins. When PDI, a PDI isoform, a carboxylesterase, calreticulin, ERp72, GRP78, or GRP94 kDa proteins were immunoprecipitated with protein-specific antisera from cell lysates, several other 35S-labeled native proteins coprecipitated with each of these proteins. In particular, a protein of 170 kDa coprecipitated with PDI (57 kDa), a PDI isoform (58 kDa), a carboxylesterase (59 kDa), and GRP78 (82 kDa) (Figure 1A), but not with the calreticulin (63 kDa), ERp72 (80 kDa), or GRP94 (100 kDa) (Figure 1B). Immunoblot analyses of the cell lysates with TFA antiserum showed that, in addition to the other previously identified TFA proteins, a 170 kDa protein was trifluoroacetylated (Figure 2). Identification of TFA-p170 as UGGT. It was thought that TFA-p170 might be UGGT because of the similarity of their molecular masses and subcellular localizations (13). This idea was tested by monitoring the purification of TFA-p170 from liver microsomes of halothane-treated rats with TFA and UGGT antisera. Lumenal proteins were extracted from microsomes with 0.1% DOC (5), which resulted in the enrichment of both TFA-p170 and UGGT in the extract (results not shown). After chromatography on a DEAE-Sepharose anion exchange column, TFA-p170 was further purified on a hydroxyapatite HPLC column. Both TFA and UGGT antisera appeared to recognize the same 170 kDa protein eluting from the column (Figure 3). To confirm the identity of TFA-p170 as UGGT, a sample of partially purified protein was electroblotted onto PVDF membrane and its N-terminal amino acid sequence was determined by Edman degradation. Only one sequence (NSKAIT) was detected which was identical
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Figure 3. Immunoblots of HPLC fractions with TFA and UGGT antisera. Fractions eluting from the hydroxyapatite column were analyzed by immunoblotting with TFA and UGGT antisera and chemiluminescence detection. M, microsomes.
Figure 4. Immunoblots of immunoprecipitates with TFA and UGGT antisera. Proteins in hepatocyte cell lysates from halothane-treated rats were immunoprecipitated with either 59 kDa carboxylesterase (lane 1) or 80 kDa ERp72 (lane 2) antiserum and were analyzed by immunoblotting with TFA and UGGT antisera.
to that determined for rat liver UGGT.2 Moreover, the 170 kDa protein that was immunoprecipitated with the 59 kDa carboxylesterase antiserum, but not with the 80 kDa ERp72 antiserum, was recognized by UGGT and TFA antisera, providing further evidence that TFA-p170 kDa was the trifluoroacetylated form of UGGT (Figure 4). Effects of Halothane on UGGT Activity. The glucosylation of denatured thyroglobulin by UGGT was reduced by 44% (p < 0.05) in liver microsomes of halothane-treated rats as compared to that in the control rats. Approximately 36% of this reduction in activity appeared to be due a 16% decrease in the level of UGGT that was found in the microsomes of halothane-treated rats (Figure 5), assuming that the polyclonal UGGT antiserum reacted equally with native and the trifluoroacetylated forms of UGGT.
Discussion In this study we have identified UGGT as a 170 kDa protein target of the trifluoroacetyl chloride metabolite of halothane. This assignment was based upon the findings that TFA-p170 had the same apparent monomeric molecular mass, subcellular localization in the ER, and N-terminal amino acid sequence as UGGT and was recognized by UGGT antiserum. It was also considered that TFA-p170 might correspond to a 170 kDa GRP that was identified in the lumen of the ER of Chinese hamster ovary cells (19). However, the 170 kDa GRP has a different N-terminal amino acid sequence than that of TFA-p170 (20). UGGT is a soluble protein of the ER that catalyzes the glucosylation of protein-linked, glucose-free, high mannose-type oligosaccharides in a Ca2+-dependent reaction (13). This reaction occurs preferentially with unfolded and/or incompletely folded N-linked glycoproteins and is thought to facilitate the folding of these proteins by allowing them to interact with the chaperones calnexin and/or calreticulin (14). Since we found that UGGT can 2 Personal communication, Dr. A. J. Parodi, Instituto de Investigaciones Bioquimicas, Fundacion Campomar, Buenos Aires, Argentina.
Figure 5. Reduction in the level of UGGT in liver microsomes of halothane-treated rats. Liver microsomes were prepared from control (C) and halothane-treated (H, 10 mmol/kg, ip) rats 16 h after treatment. Samples (20, 40, and 60 µg) of microsomes were analyzed by immunoblotting with UGGT antiserum and chemiluminescence detection. (A) Immunoblot; (B) band intensity as determined by laser densitometry of the immunoblot; control (O), halothane (b).
be associated with several soluble lumenal ER proteins in rat hepatocytes, including the known chaperones PDI (21-23) and GRP78 (23-26) as well as a PDI isoform and a 59 kDa carboxylesterase (also known as ES-10) (27) (Figure 1A), it is possible that these proteins may affect the glucosylation reaction catalyzed by UGGT. It remains to be determined whether UGGT associates with the native and/or the trifluoroacetylated forms of these proteins, because the specific protein antisera used for immunoprecipitation would bind to both forms of these proteins. Moreover, like PDI (21-23), UGGT may be a chaperone and have a role in protein folding, independent of its enzymatic activity. It was found that treatment of rats with halothane reduced the activity of UGGT in liver microsomes by 44%. Part of this effect could be due to a decrease in the level of UGGT in liver microsomes (Figure 5), which might have been caused by a reduction in the rate of synthesis
Halothane Inhibits Glycoprotein Glucosyltransferase
of UGGT, by an increase in its rate of degradation, or by a combination of both effects. It is also possible that the covalent modification of UGGT or other ER proteins by the trifluoroacetyl chloride metabolite could contribute to the loss of UGGT activity. Nevertheless, the finding that halothane decreased the activity of UGGT is important because it suggests that halothane and possibly other xenobiotics may be toxic to cells by disrupting protein folding in the ER. This could also lead indirectly to toxicity by promoting immune reactions against cryptic epitopes of unfolded, incompletely folded, and/or malfolded proteins (28). Indeed, protein folding anomalies have been implicated in the etiology of a number of human diseases (29). However, it appears that UGGT is not essential for cell viability in yeast (30), suggesting that other pathways are involved in the folding of N-linked glycoproteins. Whether UGGT is essential in protein folding in human is not known presently. Perhaps patients who develop halothane hepatitis require UGGT for protein folding. This might explain, at least in part, the rare incidence and the idiosyncratic nature of halothane hepatitis.
Acknowledgment. We would like to thank Drs. A. J. Parodi and S. E. Trombetta of Instituto de Investigaciones Bioquimicas, Fundacion Campomar, Buenos Aires, Argentina, for providing the UGGT antiserum.
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