Evidence that type II 5'-deiodinase is not a selenoprotein.

THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 266, No. 21, Issue of July 25, pp. 13477-13460,1991 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Communication

Printed in U.S.A.

Evidence That Type I1 5’-Deiodinase Is Not a Selenoprotein” (Received for publication, March 21, 1991)

Marjorie SafranS, Alan P. Farwell, and Jack L. Leonard From the Molecular Endocrinology Laboratory, University of Massachusetts Medical School, Worcester, Massachusetts 01655

Brain type I1 5’-iodothyronine deiodinase and liver type I 5’-iodothyronine deiodinase activities are dethat creased in rats fed a Se2+-deficient diet suggesting both enzymes are Se2+-dependentproteins. Since serum thyroxine (T4) concentrations are twice normal in theSe2+-deficientanimals, it isunclear whether the Se2+deficiency or the increased circulating T4 account for the decrease in thebrain enzyme. In order to separate these two possibilities, the effects of Se2+on 5’deiodinase in glial cells (type 11) and LLC-PKl cells (type I) were examined. LLC-PK1 and glial cells were grown in serum-free defined medium containing 0, 1 pM, 10 nM, and 40 nM Se2+for 3-5 days or in medium 24 h. Deiodinase isozymes were containing for determined by measuringcatalyticactivityand by quantification of the BrAc[12’I]T4affinity-labeled substrate binding subunits.Se2+deficiency was confirmed by measuring the activity of the selenoprotein, glutathione peroxidase. Se2+ caused aconcentration-dependent increase in glutathione peroxidase activityin I enzyme, but had both cell types, as well as in the type no effect on the type I1 enzyme. LLC-PK1 cells contained multiple 7SSe2+-labeledproteins including the 27-kDa substrate binding subunit of the type I 5’deiodinase. Glial cells contained seven 75Se2+-labeled proteins ranging in size from 12 to 62 kDa, none of which corresponded to the type I1 substrate binding subunit. These data show that, unlike the type I enzyme, the typeI1 enzyme does not contain aselenocysteine orselenomethionine, further emphasizing the differences between these twoisozymes.

T4’is the principal product of the thyroidgland; it functions

* This work wassupported by Grants DK38772 and DK32520 from the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health and by National Institutes of Health Clinical Investigator Award DK02005. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed Division of Endocrinology, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655. Tel.: 508-856-3609;Fax: 508-8564572. The abbreviations used are: T , thyroxine; Ts, 3,5,3’-triiodothyronine; rT3, 3,3’,5’-triiodothyronine;5’-D, iodothyronine 5”deiodinase; 5’-D-I, type I iodothyronine 5”deiodinase; 5’-D-11, type I1 iodothyronine 5”deiodinase; DMEM, Dulbecco’smodifiedEagle’s medium; F-12, Ham’s F-12 medium; BrAcT4, N-bromoacetyl-L-thyroxine; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; TEMED, N,N,N’,N’-tetramethylethylenediamine; PTU, 6-n-propyl-2-thiouracil; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; SBS, substrate binding subunit.

as a prohormone and mustbe converted to the active metabolite T3 by outer ring or 5’-deiodination to exert its major metabolic effects (1).Two isozymes of the iodothyronine 5‘deiodinase have been identified with different tissue distributions,substrateaffinities,sensitivitiestoPTU,andresponsestohypothyroidism(1,2).Type I 5’-D is mainly localized to the kidney, liver, and thyroid and is the main over source of circulating T3 (1).rTB is its preferred substrate T4, and enzyme activity is inhibitedby P T U (3). Type I1 5’D, on the other hand, is limited to the nervous central system, pituitary, and brown adipose tissue and catalyzes the gener(1).T4is the preferred ation of intracellular TI in these tissues substrate for 5’-D-11, and PTU has little or noeffect on this isozyme’s activity. Unlike 5”D-I which responds in parallel t o thyroid status, 5’-D-II is markedly increased with hypothyroidism and decreased with hyperthyroidism (2). Recent work from our laboratory has demonstrated that the two 5’D isozymes are of different molecular weight, are composed of multiple subunits, and contain unique substrate binding subunits (4). Animals receiving Se*+-deficient diets have been observed to have increased serum T4 and decreased serum TBconcentrations (5,6). These findings led Arthur et al. (6) to speculate that the enzyme(s) catalyzing thyroid hormone metabolism was impaired. These workers found that levels of 5”D-I were markedly decreased in the liver of Se2+-deficient rats(6) and subsequently demonstrated that the substrate binding subunit of liver 5”D-I was a selenoprotein (7). Most recently, Berry et al. (8) isolated a clone from a rat liver cDNA library which encodes a -27-kDa selenoprotein with the expected characteristics of 5”D-I. Decreased brain 5’-D-II activity was also observed in ratsfed aSe’+-deficient diet raising the possibility that it too was a Se2’-containing protein (6). However, since serum T4 concentrations were twice normal in the Se2+deficient animals, it is unclear whether the Se2+ deficiency or the increasedT4concentrations accountedfor the decrease in brain 5’-D-II activity. In order to separate thesetwo possibilities, we determined the effects of Se2+ on the synthesisof these two isozymes in cultured cells. Our studies confirm that the substrate binding subunit of 5”D-I is a selenoprotein and that 5”D-I synthesis isSe2+-dependent.Incontrast, 5’-D-11 synthesisin cyclic AMP-stimulated glial cells is unaffectedby alterations in Se2+ content. Glialcells contained seven 75Se2’-labeled proteins ranging insize from 12 to 62 kDa, noneof which corresponded to the substrate binding subunitof 5’-D-II. These data demonstrate that 5’-D-II does not contain aselenocysteine or selenomethionineinitssubstratebindingsubunit,further demonstrating thedifferences betweenthe two 5’-Disozymes. MATERIALSANDMETHODS

DMEM, F-12, antibiotics, Hanks’ balanced salt solution, and glucosewere obtained from GIBCO; supplemented bovine calf serum from Hyclone, Inc. (Logan, UT); dibutyryl cyclic AMP, hydrocortsone, and sodium selenite from Sigma; dithiothreitol from Calbiochem; acrylamide and N,N’-methylenebisacrylamide from U. S. Biochemical Corp.; and ammonium persulfate and TEMED from Bio-Rad. B ~ A C [ ’ ~ ’ I ]was T ~ synthesized as described previously (9). The purity of the BrAc[”’1]T4 was >90% as determined by reverse phase high pressure liquid chromatography analysis (10). 7sSeZ+as selenious acid (specific activity, 750-1000 Ci/g) was obtained from

13477

Content Different Se2+

13478

of 5”Deiodinase Isozymes

Dr. Curtis Zinn, University of Missouri Research Reactor Facility, Columbia, MO. Culture Conditions-LLC-PK, cells were obtained from the American Tissue Culture Collection and have previously been shown to be a good source of 5”D-I (11).Glial cells were prepared from neonatal rat brains as previously described (12) and used during passages 2-6. When stimulated with dibutyryl cyclic AMP, these cells exhibit 5’D-I1 activity which demonstrates all the properties observed in vivo (13, 14). Bothcell types were grown either in DMEM containing 5% (LLC-PK1)or 10% (glial) supplemented bovine calf serum as previously described (growth media) (4). For studies examining the effect of Se’+ on 5’-D activity, cells were grownto near confluence in growth media. After washing the monolayers once with Hanks’ buffered salt solution, medium wasthen changed to defined medium containing 040 nM Se“. LLC-PK, defined medium contained DMEM:F-12 (1:l) with 15 mM HEPES (pH 7.4), 14 mM sodium bicarbonate, 25 rg/ml transferrin, 10 pg/ml insulin, 0.1 p M hydrocortisone, 1 nM T3, 10 microunits/ml vasopressin, 10 nM cholesterol (15). Glial defined medium contained DMEM:F-12 (1:l) with 15 mM HEPES (pH 7.4), 14 mM sodium bicarbonate, 0.1% glucose, 50 nM hydrocortisone, 100 nM putrescine, 0.5 pg/ml prostaglandin FZn,50 pg/ml insulin, 0.1 pg/ ml basic fibroblast growth factor (16). Maximal levels of 5’-D-I1 were induced in glial cells prior to affinity labeling by stimulation for 16 h with 1 mM dibutyryl cyclic AMP and 100 nM hydrocortisone. Cells in growth medium were changed to serum-free defined medium for 8 h prior to stimulation (14). 5 ’ - 0 Actiuity and Content-5”D activity was measured using the iodide release method. 5”D-I was determined in the presence of 10 p M rTZ3 and 20 mM dithiothreitol (3). 5’-D-IIactivity was determined in the presence of 2 nM rTB,20 mM dithiothreitol, and 1 mM PTU (17). 5’-D content was determined by quantification of the affinitylabeled substrate binding subunit (5”D-SBS) (4, 11, 14). In brief, cells were affinity-labeled by incubating the monolayers at 37 “C for 20 min with 2.7nM BrAc[’*’I]T4(specific activity, -2200 Ci/mmol) in the absence (glial) or presence (LLC-PK,) of 0.5 mg/ml digitonin. Cell pellets were resuspended in lysis buffer composed of 10 mM HEPES (pH 7.0), 10 mM dithiothreitol, 0.1 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride, sonicated, and added to SDS-PAGE sample buffer to achieve final concentrations of 50 mM Tris-HC1, pH 6.8, 10% glycerol (v/v), 10 mg/ml SDS, 140 mM mercaptoethanol, and 20 pg/ml bromphenol blue. The affinity-labeled proteins were separated by 12.5% SDS-PAGE gels (18), and radioautographs were analyzed using scanning densitometry. Glutathione Peroxidase Actiuity-Glutathione peroxidase activity was measured using anadaptation of the method of Beutler as described by Chada et al. (19). Confluent monolayers of glial and LLC-PK1 cells were washed free of medium with 2 X 5 ml of20mM potassium phosphate buffer (pH 7.4) containing 150 mM NaCl. The cell pellets were collected, resuspended in the same buffer, lysed by sonication, and kept on ice until used. Assay tubes contained 100 pg of protein, 0.05% Triton X-100,0.2 mM NADPH, 2 mM GSH, and 1 unit/ml glutathione reductase in a total volume of 1 ml. Glutathione peroxidase activity was determined from the oxidation of NADPH in the presence of 0.35 mM t-butyl hydroperoxide monitored spectrophotometrically at 340 nm. 7sSe2+ Labeling of LLC-PK1 and Glial Cells-Cells were allowed to grow to confluence prior to labeling with %e2’. %e2+ (2 nM) was added to LLC-PK, cells for 24 h. Glial cells were transferred to serum-free medium containing 2 nM 7’sez+(4 X lo6 cpm/ml) for 8 h and then stimulated with dibutyryl cyclic AMP and hydrocortisone for an additional 16 h as described above. 75Se2+-labeled monolayers were washed twicewith iced 20 mM potassium phosphate buffer (pH 7.4) containing 150 mM NaC1. The cell pellets were collected, and the labeled proteins were reduced with SDS-PAGE sample buffer, denatured by heating, and analyzed by SDS-PAGE as described for affinity labeled cells above. RESULTS

To examine the effect of Se2+ on glutathione peroxidase, 5”D-I and 5’-D-11, cells were cultured in defined medium containing 0, 1 pM, 10 nM, and 40 nM Se2+for 3-5 days. In preliminary experiments, glutathione peroxidase activity was linear with respect to protein up to 150 pg of protein/tube in both cell types. Therefore, 100 pg of cell protein was used in all subsequent assays. Glutathione peroxidase activity in cells grown in medium containing 40 nM Se2+averaged 231 & 12

(mean f S.E.) and 117 f 17 nmol NADPH oxidized.min”. mg protein” in LLC-PKl and glial cells, respectively. As shown in Fig. 1, Se2+supplementation caused a dosedependent increase in glutathione peroxidase activity in both cell types. Glutathione peroxidase activity increased from 0 to 10 nM Se2+and plateaued between 10 and 40 nM Se2+. Similarly, Se2+ supplementationresulted in a concentrationdependent increase in both 5”D-I activity and in thecellular content of the affinity-labeled type I 5’-D-SBS (Fig. L4). In contrast, theabsence or presence of Se2+ hadno effect on 5’D-I1 activity or the content of affinity-labeled type I1 5’-DSBS (Fig. 1B). In order to identify selenoproteins, confluent monolayers of LLC-PK1 and dibutyryl cAMP stimulated glial cells were presence of 2 nM 75Se2’and thelabeled cultured for 24 h in the proteins identified by SDS-PAGE. 75Se2’ labeling of LLCPK1 cells identified multiple proteins, including a 27-kDa protein which has previously been recognized as thetype I 5’D-SBS by affinity labeling (Fig. 2 A ) . Dibutyryl cAMP stimulated glial cells, on the other hand, contained seven 76Sez+labeled proteins of 61.5,53.5,21.2,20.2,15.7, 13, and 11.9 kDa, none of which corresponded to the type I1 5”D-SBS (Fig. 2B). Dibutyryl cyclic AMP induced the 29-kDa, affinitylabeled glial protein but failed to result in the appearance of a 29-kDa selenoprotein. To demonstrate further that the type I1 5’D-SBS was not a selenoprotein, dibutyryl cAMP stimulated glial cells were labeled with 40 nM 75Se2+, and cell proteins were seperated by SDS-PAGE. Figure 3 shows the distribution of 75Se2+incorporated into labeled cell proteins. The glial cells incorporated 269,000 f 41,000 cpm 75Se2+/mgcell protein (mean f S.E., n = 3), and theamount of 75Se2C in the region corresponding to the type I1 5’D-SBS accounted for < 0.2% of the total counts incorporated. In contrast, 10% of 75Se2+ was incorporated into the type I 5’D-SBS in LLC-PK1 cells as judged by scanning densitometry. DISCUSSION

Se2+has long been known to be a necessary trace element. Multiple selenium-containing polypeptides have been identified in mammalian tissues (20,21).In most, if not all, of these proteins Se2+is incorporated co-translationally as either selenocysteine or selenomethionine (21). The first selenoprotein to be well described in mammals was glutathione peroxidase, a ubiquitous antioxidant enzyme important in neutrophil function (22). Animals receiving a Se2’-deficient diet show poor growth and increased susceptibility to infection (23). Some, but not all, of the biological changes observed in Se2+deficient animals can be explained by the loss of glutathione peroxidase activity alone. In particular, a27.4-kDa selenoprotein has been identified in rat liver which appears to be the substrate binding subunit of the type I 5’-D (7). This observation along with other studies by Arthur et al. (6) demonstrating that hepatic5”D-I activity is decreased in Se2+deficient animals suggests that 5”D-I is a selenoprotein. This proposal has been confirmed by the recent work of Berry et al. (8) who identified a cDNA from a rat liver cDNA library which encodes a 27-kDa selenoprotein with all the features of the type I 5”D. Since 5“D-I has been shown to have a M, = -55,000 (4),the enzyme is likely to be a homodimer. In the current study, we have exploited well-characterized cell culture models for the two isozymes of 5’deiodinase to evaluate the possibility that 5’D-I1 is also a selenoprotein. Since the amount of 5’D-I in LLC-PK1 cells approximately equals the amount of 5’D-I1 in dibutyryl cAMP stimulated glial cells (2-5 pmol enzyme/mg cell protein (11,14)), direct

Different Se2+Content of 5"Deiodinase Isozymes

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comparisons of the isozymes can be made. We have confirmed that 5'D-I from kidney is a selenoprotein becauseenzyme activity and affinity labeling of the type I 5'D-SBS is decreased in LLC-PK, cells grown in Se" deficient medium and these cells contained a 27-kDa "Se"-labeled protein. In contrast, neither5'D-I1 activity nor the contentof affinity-labeled type I1 5'D-SBS of glial cells was affected by Se". Although glial cells contained seven selenoproteins,there were none of M , corresponding to the typeI1 5'D-SBS. Less than 0.2% of "'Se'+ incorporated into glial cells is present in proteins of 27-30 kDa. These dataagree with the observation that rat brain lacks selenoproteins in the 26-32-kDa range (20). Although 5'D-I1 has a holoenzyme size of 199 kDa, the identity of its other subunits is unknown (4). From these data, we can clearly state that the substrate binding subunit of 5'DI1 is not a selenoprotein. In addition, the lack of effect of selenium deficiency on 5'D-I1 activity suggests that Se2+ is not important in the holoenzyme as well. Other than glutathioneperoxidase (24 kDa), therole of the remaining selenoproteins present in these cells has not been established. Although a 55-kDa BrA~['~"I]T,-labeled protein is present in LLC-PK, andglial cells, it has been shown to be unrelated to either5'D-I (11)or 5'D-I1 (14). Thus the55-kDa "Se"-labeled protein in glial cells is to unrelated 5'D but may

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Different Se2+Content of 5”Deiodinase Isozymes

8. Berry, M. J., Banu, L. & Larsen, P. R. (1991) Nature 3 4 9 , 438440 9. Koehrle, J., Rasmussen, U. B., Rokos, H.,Leonard, J. L. & Hesch, R. D. (1990) J. Biol. Chem. 2 6 5 , 6146-6154 10. Koehrle, J., Rasmussen, U. B., Ekenbarger, D. M.,Alex, S., Rokos, H., Hesch, R. D. & Leonard, J. L. (1990) J. Biol. Chem. 265,6155-6163 11. Leonard, J. L., Ekenbarger, D. M., Frank, S. J., Farwell, A. P. & Koehrle, J. (1991) J. Biol. Chem. 266, 11262-11269 12. Leonard, J. L. (1988) Biochem.Biophys.Res.Commun. 151, 1164-1172 13. Courtin, F., Chantoux, F. & Francon, J. (1986) Mol. Cell. Endocr. 48,167-178 14. Farwell, A. P. & Leonard, J. L. (1989) J. Biol. Chem. 264,2056120567 15. Chuman, L., Fine, L. G., Cohen, A. H. & Saier, M. H. (1982) J. Cell Biol. 9 4 , 506-510 16. Morrison, R. S. & de Vellis, J. (1981) Proc. Natl.Acad. Sci. U. S. A. 78, 7205-7209 Acknowledgment-We thank Susan Dubord for technical assist- 17. Visser, T. J., Leonard, J. L., Kaplan, M.M. & Larsen, P. R. (1982) Proc. Natl. Acad. Sci. U. S. A. 7 9 , 5080-5084 ance. 18. Laemmli, U. K. (1970) Nature 227,680-685 19. Chada, S., Whitney, C. & Newburger, P. (1989) Blood 7 4 , 2535REFERENCES 2541 J. L. & Visser, T. J. (1986) in Thyroid Hormone MetabLeonard, 1. 20. Behne, D., Hilmert, H., Scheid, S., Gessner, H. & Elger, W . olism (Hennemann, G., ed) pp. 189-229, Marcel Dekker, New (1988) Biochim. Biophys. Acta 966,12-21 York 21. Danielson, K. G. & Medina, D. (1986) Cancer Res. 46,4582-4589 2. Kaplan, M. M. (1986) in Thyroid Hormone Metabolism (Henne- 22. Forstrom, J. W., Zakowski, J. L. & Tappel, A. L. (1978) Biochemmann, G., ed) pp. 231-253, Marcel Dekker, New York istry 17,2639-2644 3. Leonard, J. L. & Rosenberg, I. N. (1980) Endocrinology 1 0 7 , 23. Wendel, A. (ed) (1989) Selenium inBiology and Medicine, Sprin1376-1383 ger-Verlag, Berlin 4. Safran, M. & Leonard, J. L. (1991) J. Biol. Chem. 2 6 6 , 323324. Visser, T. J., Frank, S. & Leonard, J. L. (1983) Mol. Cell. Endocr. 3238 33,321-327 5. Arthur, J. R., Morrice, P. C. & Beckett, G. J. (1988) Res. Vet. 25. Visser, T. J., Kaplan, M.M., Leonard, J. L. & Larsen, P. R. Sci. 45,122-123 (1983) J. Clin. Znuest. 71,991-1002 6. Beckett, G. J., MacDougall,D. A., Nicol, F. & Arthur, J. R. (1989) 26. Leonard, J. L. & Rosenberg, I. N. (1980) Endocrinology 1 0 6 , Biochem. J. 259,887-892 444-451 7. Arthur, J. R., Nicol, F. & Beckett, G. J. (1990) Biochem. J . 2 7 2 , 27. Leonard, J. L. & Visser, T. J. (1984) Biochim. Biophys. Acta787, 122-130 537-540

be a Se2+ transport protein described by others (20). The absence of a selenocysteine in the type I1 5’-D-SBS is consistent with the finding that 5’-D-11 is -100-fold less sensitive than is 5”D-I to inhibition by iodoacetate (24) and may explain the observation that PTU is unable to form the mixed disulfide necessary to inhibit 5’-D-II activity under conditions that show complete inhibition of 5”D-I (24, 25). The strong nucleophilic nature of the selenocysteine moiety explains the hyper-reactivity of 5’-D-I toward a-haloacids and the ability of PTU to protect the enzyme from inactivation by a-haloacids by forming a mixed seleno-sulfhydryl bond with the enzyme (26, 27). The finding that 5’-D-11 is not a selenoprotein and our previous work showing that the substrate binding subunits of 5”D-I and 5’-D-II are unique (4) suggest that these two isoenzymes arose independently.