Human Transcortin Synthesis by a Cell-Free Translation of Hepatic Mrna

Endocrinol. Japon. 1985, 32 (2), 295-303

Human Transcortin Synthesis by a Cell-Free Translation of Hepatic mRNA

KAORI SUEDA, HISAO SEO AND NOBUO MATSUI

The Research Institute of Environmental Medicine Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464

Abstract

To evaluate the site of synthesis and to characterize the translated transcortin, poly (A)-containing RNA (mRNA) from human liver was translated in a cell-free system derived from rabbit reticulocyte lysate. The in vitro synthesized product was identified as transcortin by immuno-precipitation with its specific antiserum. This translated transcortin could be displaced from the antibody by unlabeled purified transcortin obtained from plasma . Furthermore, when the translation mixture was applied to a cortisol-Sepharose column, the translated transcortin was bound to the matrix in a specific manner, indicating that this product binds to cortisol. The molecular weight of the translated transcortin was estimated to be 45,700 by its mobility in sodium dodesyl sulfate polyacrylamide gel electrophoresis, while that of plasma transcortin was 53,800. The difference in molecular weight between the translated transcortin and plasma transcortin was probably due to the presence of presequence (signal peptide) in addition to the absence of carbohydrate moiety in the former. In conclusion, human liver mRNA directed the synthesis of transcortin, and the translated transcortin binds to cortisol in spite of the absence of carbohydrate moiety.

Transcortin is a binding protein specific Recently, Weiser et al. (1979) demonstrated for corticosteroid in plasma (Daughaday, in rat that liver slice, incubated in vitro, 1958; Slaunwhite and Sandberg, 1959). Since synthesized a corticosteroid binding protein protein-bound corticosteroids are inactive which might be transcortin. In addition, physiologically (Matsui and Plager. 1966; hepatic synthesis of transcortin was con- Westphal, 1971), the variation in transcortin firmed by in vitro translation of liver mRNA concentration is of great importance in the obtained from guinia pigs and rats (Perrot- modulation of glucocorticoid activity (Ma- Applanat and Milgrom, 1979; Wolf et al., tsui et al., 1979; Yamamoto et al., 1983). 1981). However, no study in human liver The site of synthesis of transcortin has has been published. been supposed to be the liver (Gala and Muldoon and Westphal (1976) reported Westphal, 1966; Doe et al., 1964), but no that enzymatic removal of sialic acid from definite proof of this had been obtained. human transcortin did not affect its cortisol- binding affinity. On the other hand, Murata Received December 14, 1984 et al. (1984) reported that translated thy- Endocrinol. Japon. 296 SUEDA et al. April 1985

roxine binding globulin (TBG) did not have rum and purified as described previously (Sueda binding activity, suggesting the importance et al., 1983). of carbohydrate moiety in the tertiary struc- ture of the biologically active TBG. 2. Extraction of RNA In this study, human liver poly (A)-con- Human liver was obtained from an autopsy taining RNA was translated in a cell-free of a 29-year-old male patient who had died of lung cancer. No metastatic lesion was observed system to confirm whether human trans- macroscopically. The liver was immediately cortin is also synthesized in the liver. The placed in ice, freed from connective tissue, then presence of transcortin in the translation chopped into small pieces, frozen in liquid N2 products was identified by immuno-precipi- and stored at-80•Ž until use. Five grams of tation using specific antiserum to human the liver pieces were pulverized and homogenized transcortin. The size and the cortisol-bind- in 10 vol of 200mM Tris-HC1 (pH 8.6), 50mM ing activity of the translated transcortin were KCI, 25mM MgCl2, 200mM sucrose, 0.5mg/ml heparine and 0.1% Triton X-100. After centri- also studied. fugation at 600•~g for 10min, the supernatant was added to 4 vol of 10mM Tris-HCI (pH 8.6), 100mM NaCl, 5mM EDTA and 1.2% SDS and the mixture was extracted with phenol-chloro- Materials and Methods form (Seo et al., 1977). The ethanol-precipitated RNA was successively washed with 2M LiCI and 1. Materials 66% ethanol in 100mM NaCl. The poly (A)- L-[3, 4, 5-3H(N)]-leucine (158.0Ci/mmol) and containing RNA (mRNA) was purified by poly [1,2, 6, 7-3H(N)]-cortiso1(91.0Ci/mmol), EN- (U)-Sepharose chromatography (Vassart et al., 3HANCE were purchased from New England 1975). The mRNA thus obtained was dissolved in sterile water at a concentration of I mg/ml Nuclear Corp. (Boston, Mass., U.S.A.). NCS and stored at-20•Ž was from Anaersham (Buckinghamshire, England). Poly(U)-Sepharose was from Pharmacia Fine Globin mRNA was also prepared from rabbit Chemicals (Uppsala, Sweden). Micrococcal nu- reticulocyte lysate (Sala-Trepat et al., 1978). clease was from Boehringer Mannheim (Mann- heim, West Germany), creatine phosphate kinase 3. Cell-free translation of liver mRNA by re- was from Sigma Chemical Company (ST. Louis, ticulocyte lysate

U.S.A.). Molecular weight standards for sodium The rabbit reticulocyte lysate was prepared dodesyl sulfate (SDS)/polyacrylamide gel elec- according to the method of Palmiter et al. (1973), trophoresis were from Bio-Rad Laboratories and treated with naicrococcal nuclease to reduce

(Richimond, Calif., U.S.A.). Cortisol-Sepharose endogenous mRNA activity (Pelham and Jackson, (21-deoxy-21-(ƒÖ-aminohexyl) aminocortisol-agar- 1976). The protein synthesizing system in a final ose) was prepared according to the method of volume of 0.1ml contained 80ƒÊl of treated re-

Chan et al. (1977). Ultrogel AcA 44 was pur- ticulocyte lysate, 10mM creatine phosphate, 5ƒÊg chased from LKB (Bromma, Sweden). Anti- of creatine phosphate kinase, 15ƒÊM of each 19 human transcortin antibody was raised in a L-amino acids required for protein synthesis (ex- rabbit by immunization with purified transcortin cept leucine), 10ƒÊCi of 3H-leucine, 100mM KCI

(Sueda et al., 1983). In order to remove non- and 0.1 to 0.5ƒÊg of mRNA. Incubation was specific antibodies, the antiserum was incubated performed at 30•Ž for 1h, and the reaction was with pooled human serum (v/v; 1: 1) at 37•Ž for stopped by cooling to 0•Ž and adding 0.1ml of

30 min and centrifuged at 3,000•~g for 15 min the solution containing 2% deoxycholic acid, 2% and the supernatant was used as a specific anti- Triton X-100 and 0.2% unlabeled leucine. transcortin antibody. Anti-human albumin antiserum (rabbit), and rabbit gamma globulin (Frac- 4. Assay of mRNA activity tion II) were obtained from Miles Laboratories, Activity of mRNA was estimated by the

Inc. (Eckerhalt, Indiana, U.S.A.). Anti-rabbit amount of newly synthesized product. Newly gamma-globulin antibody was raised in a goat. synthesized total protein was precipitated with Human transcortin was isolated from pooled se- trichloroacetic acid (TCA) as reported by Shimke Vol.32, No.2 SYNTHESIS OF HUMAN TRANSCORTIN in vitro 297 et al. (1974). Translated transcortin and albumin formed by a modification of the procedure of were immuno-precipitated from the translation Rosner and Bradlow (1971). Pass-through fraction was collected (fraction 1) mixture using a double antibody technique , and the column (Pasteels et al., 1971). The TCA precipitate and was washed with 1ml of 0.05M phosphate buffer, immuno-precipitates were digested in 1ml of pH 7.0, containing 0.1M NaCl (fraction H). Then, NCS at 50•Ž and radioactivity was measured. the column was eluted with I ml of 0.01M

phosphate buffer, pH 7.0, containing 200 ƒÊg/ml of

5. SDS-polyacrylamide gel electrophoresis of cortisol (fraction 110, and finally with I nil of 0.05M phosphate buffer pH 7.0, containing 200 translation products The purified immuno-precipitate from the tig/ml of cortisol and 0.1M NaCl (fraction IV).

translation mixture was dissolved in 50ƒÊl sample Radioactivities of TCA precipitate and of the buffer (10mM Tris-HCl buffer pH 6.8, 2% SDS, immuno-precipitate by anti-transcortin antiserum 10% glycerol (v/v) and 1% 2-mercaptoethanol), were measured in each fraction.

heated at 80•Ž for 5 min and analyzed on 10%

SDS-polyacrylamide gel electrophoresis (Laemli, 1970). The gel was treated with EN3HANCE Results and its radioactivity was assessed by fluorography

(Laskey and Mills, 1975) using Kodak X Omat AR film. Molecular weight standards were elec- 1. Specificity of anti-human transcortin anti- trophoresed at the same time and were stained serum with Commassie brilliant blue R. In order to examine the specificity of the antiserum, immuno-electrophoresis was 6. Binding of translated transcortin to cortisol- carried out. A single precipitin line was Sepharose formed when the anti-transcortin antiserum The translation mixture (0.4ml) was incubated was incubated with either purified plasma with cortisol-Sepharose (0.1ml of packed gel) at

4•Ž, and the mixture was transferred into a transcortin or human serum (Fig. 1-A). column (0.5•~1.0cm). Fractionation was per- Furthermore, when such imm 1.1no-elect ro-

A. staining Fig. 1. Immuno-electrophoresis of human serum by anti-transcortin antiserum Pooled serum (a) and purified transcortin (b) were mixed with a trace amount of 3H- cortisol and electrophoresed in Tris-glycine buffer, pH 8.6 for 90 min at 150 volt. Then the wells were filled with anti- whole serum antiserum (c) and B. autoradiography anti-transcortin antiserum (d). After diffusion for 24h to permit formation of precipitin arcs, the gel was washed, dried and autoradiographed (B). The gel was then stained with Com- massie Brilliant Blue R (A). Endocrinol. Japan. 298 SUEDA et el. April 1985 phoresis was carried out in the presence of were 2,671 and 2,099dpm, respectively. 3H-cortisol, radioactivity was detected only These represent 1.2% and 0.9% of TCA pre- at the precipitin line (Fig. 1-B). The spe- cipitable radioactivity. Although slight, con- cific interaction between antibody and pu- sistent decreases in the specific counts for rified transcortin was also observed in the transcortin was obtained by adding 3 and Ouchterlony double diffusion test (data not 30ƒÊg of plasma transcortin. shown). in comparison, one of the most abun- dant mRNA in the human liver, namely

2. In vitro translation of human liver mRNA albumin mRNA, was also studied. Radio- After translation of liver mRNA, the activity of immuno-precipitate specific for newly synthesized product was analyzed by anti-albumin antiserum was 15-20% of TCA

TCA precipitation and by immuno-precipi- precipitable radioactivity (data not shown). tation with anti-transcortin antiserum. The results are shown in Table 1. Radioactivity 3. Analysis of the translated transcortin by of 3H-leucine in TCA precipitate was 229,393 SDS polyacrylamide gel electrophoresis dpm (A), while that in immuno-precipitate The translation product of liver mRNA was 8,926dpm (B). When purified plasma. was precipitated by anti-transcortin anti- transcortin (30ƒÊg) was added to the trans- serum. The precipitate was applied on SDS- lation mixture, radioactivity of 3H-leucine polyacrylamide gel electrophoresis. A single in the immuno-precipitate (B) clearly de- radioactive band was observed as shown in creased from 8,926 to 6,255dpm. The ra- Fig. 2-A. No radioactive band was detected dioactivity in the immuno-precipitate ob- when normal rabbit serum was used instead tained in the presence of 30ƒÊg transcortin of anti-transcortin antiserum. Again, no appears to represent the non-specific absorp- band was observed when translation was tion, because calibration of the antibody carried out either without liver mRNA or displacement revealed that 30ƒÊg of cold with globin mRNA. When the immuno- transcortin completely displaced the radio- precipitate was electrophoresed with several active transcortin from the antibody in the molecular weight markers, the molecular present assay system. Thus, the specific weight of translated transcortin was esti- counts for transcortin (C) in the absence mated to be about 45,700•}1,600 (Mean•} and presence of 3ƒÊg of cold transcortin SD, n=3). The molecular weight of plasma transcortin determined in the same system was estimated to be 53,800•}1,000 (data Table 1. TCA, precipitable and immuno- not shown). precipitable radioactivity in the translation product of liver The immuno-precipitate by anti-albumin mRNA and influence of the antiserum presented a major band and sev- addition of plasma transcortin eral minor bands on SDS-polyacrylamide gel electrophoresis. The molelcular weight of the major one was 70,200•}600 (Fig. 2-B).

4. Cortisol-binding activity of the translated transcortin Cortisol-binding activity of translated transcortin was examined by using cortisol- Sepharose. After translation product was

(B) total imnauno-precipitable count incubated with cortisoi-Sepharose, the mix- (C) specific immuno-precipitable count ture was transferred to a colum, from which Vol.32, No.2 SYNTHESIS OF HUMAN TRANSCORTIN in vitro 299 four fractions were obtained. As shown on precipitate with anti-transcortin antiserum, Table 2, radioactivity in TCA precipitate though it decreased as elution progressed , rapidly decreased as elution progressed. In its percentages in TCA precipitable radio- the case of radioactivity of the immuno- activity markedly increased in fractions III

A B

Fig. 2. Analysis of translated transcortin by SDS-polyacrylamide gel electrophoresis- Fluorography of 3H-leucine Translation product of human liver mRNA in the reticulocyte lysate was immuno- precipitated with specific antiserum and subjected to SDS- polyacrylamide gel electrophoresis (Laemli, 1970). Ra- dioactivity was examined by fluorography (Laskey and Mills, 1975). The molecular weight of the immuno-precipitate was com- pared with that of myosin (molecular weight; 200,000), β-galactosidase (130,000), phos- phorylase B (94,000), bovine serum albumin (68,000), IgG heavy chain (50,000), ovalbumin (45,000) and carbonic anhydrase (30,000). A; the immuno-precipitate with anti-transcortin antiserum B; the immuno-precipitate with anti-albuhain antiserumj Endocrinol. Japon. 300 SUEDA et al. April 1985

Table 2. Binding of translated transcortin human liver rnRNA contained a protein or

to cortisol-Sepharose proteins which were precipitated by anti- albumin antiserum. The precipitate showed one major band and several minor bands on SDS-polyacrylamide gel electrophoresis. The molecular weight of the major product

(70,200•}600) was concordant with that of pre-proalbuinin reported previously in the rat (Strauss et al., 1977). It was not clear whether the minor bands might represent the materials precipitated by the contami- nants in anti-albumin antiserum or the pre- maturely terminated peptides. In addition, albumin specific radioactivity represented 15-20% of TCA precipitable radioactivity, which is consistent with previous data in rats (Keller and Taylor, 1976 ; Peters and Peters, 1972). and IV. Since these fractions were eluted Using this in vitro translation system, it by the cortisol containing buffer, antiserum was demonstrated that translation products precipitable radioactivity is considered to of human liver mRNA contained a protein have been bound to cortisol-Sepharose in a which was precipitated by anti-transcortin competitive manner with cortisol. antiserum. The specificity of anti-transcortin To ascertain the specificity of the bind- antiserum was ascertained by Ouchterlony ing to cortisol-Sepharose, the translation double diffusion test and immuno-electro- mixture of globin. mRNA was treated in phoresis (Fig. 1). The binding of translated the same manner. Despite the fact that product to the anti-transcortin antibody TCA precipitable radioactivity of globin could be displaced by purified plasma trans- mRNA was 5 times more than that of liver cortin (Table 2). mRNA, the radioactivity of the immuno- The molecular weight of translated trans- precipitate was less than that of liver mRNA cortin was calculated to be 45,700 from its in all fractions. The radioactivity in immuno- mobility on SDS-polyacrylamide gel electro- precipitate decreased in parallel with that phoresis, white that of plasma transcortin in the TCA precipitate, the former was less was 53,800 in the same condition. Since

than 0.1% of the latter in all fractions, and glycosylation of nascent protein does not no increases in the percentage in fractions occur in the cell free system used, the dis- III and IV were observed. crepancy in the molecular weight might

partly be attributed to the lack of carbohydrate in translated transcortin. The mo- Discussion lecular weight of the deglycosylated poly-

peptide of plasma transcortin was calculated The poly (A)-containing RNA (niRNA) to be 39,800, assuming that the carbohy- was purified from human liver obtained at drate content of transcortin is 26.1% as autopsy and translated in a cell-free reticulo- reported by Muldoon and Westphal (1967). cyte lysate system. The fidelity of the trans- Therefore, translated transcortin seemed lation system was ascertained by albumin to be slightly larger than the deglycosylated mRNA activity. Translation products of transcortin. Vol.32, No.2 SYNTHESIS OF HUMAN TRANSCORTIN in vitro 301 Recently a number of studies have de- that serum transcortin concentration was low monstrated that the products of cell-free in the patient with liver cirrhosis , trans- translation of secretory proteins are larger cortin has been suggested to be synthesized than native secreted forms and have signal in human liver (Doe et al., 1964). 1-Iowever, sequences (15-30 amino acids residue ex- no definite proof of this has been presented tentions at NH2-terminal) (Lingappa and so far. The results of the present study Blobel, 1980). Since transcortin is also a strongly suggest that the human liver is the secretory protein and the molecular weight site of transcortin synthesis. of the translated transcortin was larger than the assumed molecular weight of deglycosylated transcortin, it is probable that the Acknowledgements translated transcortin is a precursor form having a signal peptide. The authors wish to thank Dr. Y. Ariyoshi The steroid-binding activity of translated (Aichi-ken Cancer Center) for his fruitful discussion and collaboration. transcortin was examined using cortisol- This work was supported in part by the Naito Sepharose. The pass-through fraction and Foundation Research Grand for 1981 and a fraction eluted with a buffer without cortisol Grant-in-Aid for Scientific Research (No. 58570997) contained some radioactivity that was pre- from the Japanese Ministry of Education, Sci- cipitated by anti-transcortin antiserum. The ence and Culture. amount of this radioactivity comprises 1-3% of total TCA precipitable radioactivity. On the other hand, the percentage of immuno- References precipitable radioactivity in TCA precipitable Chan, D. W., M. Sharma and W. R. Slaunwhite radioactivity reached approximately 30% in Jr. (1977). The chemistry of human transcortin; fractions eluated with cortisol containing Improved affinity matrices for the purification buffer. Such specific binding for cortisol- of transcortin. Arch. Biochem. Biophys. 182, Sepharose was not seen in the translation 197-202. Daniels-McQueen, S., D. McWilliams, S. Birken, product of globin mRNA. These results indicate that the translated transcortin has R. Canfield, T. Landefeld and I. Boime (1978). Identification of mRNAs encoding the a and cortisol-binding activity. Thus, the presence βsubunits of human choriogonadotropin.Ｊ. of the signal peptide and the absence of Biol. Ch8m. 253, 7109-7114. carbohydrate moiety does not interfere with Daughaday, W. H. (1958). Binding of corti- the cortisol-binding activity of transcortin. costeroids by plasma protein. III. The binding The presence of transcortin has been re- of human plasma and plasma protein fractions. ported in kidney (Feldman et al., 1976), J. Clin. Invest. 37, 511-518. brain and pituitary (Koch et al., 1976: Dekloet, E. R. and B. S. McEwen (1976). A putative glucocorticoid receptor and transcortin- Dekloet and McEwen, 1976), muscle (Mayer like macromolecule in pituitary cytosol. Biochen7. et al., 1975), lung (Giannopoulos, 1976) and Biophys. Acta. 421, 115-123. uterus (Milgrom and Baulien, 1970), in ad- Doe, R. P., R. Fernandez and U. S. Seal (1964). dition to the liver. It is not known whether Measurement of corticosteroid-binding globulin these proteins are synthesized in situ or in man. J. Clin. Endocr. 24, 1029-1039. transported there by circulation. Hepatic Feldman, D., .1. W. Funder and I. S. Edelman synthesis of transcortin was confirmed by (1973). Evidence for a new class of corti- costerone receptors in the rat kidney. Endo- in vitro translation using liver mRNA ob- crinology 92, 1429-1441. tained from guinea pigs and rats (Perrot- Gala, R. R. and U. Westphal (1966). Further Applanat and Milgrom, 1979; Wolf et al., studies on the corticosteroid-binding globulin 1981). From clinical observation showing in the rat; proposed endocrine control. Endo- Endocrinol. Japon. 302 SUEDA et al. April 1985

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