0013-7227/79/1053-0581$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 105, No. 3 Printed in U.S.A.
Human Chorionic Gonadotropin a-Subunit from Cultured Choriocarcinoma (JEG) Cells: Comparison of the Subunit Secreted Free with That Prepared from Secreted Human Chorionic Gonadotropin* ROBERT BENVENISTE, JILL LINDNER, DAVID PUETT, AND DAVID RABIN Departments of Medicine and Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
ABSTRACT. The cultured human choriocarcinoma cell line, JEG-clone 3, secretes both biologically active hCG (JEG-hCG) and substantial quantities of free a-subunit (JEG-a). This study is concerned with a comparative characterization of the a-subunits of three distinct origins: 1) standard hCG-a obtained after dissociation of urinary (pregnancy) hCG, 2) JEG-a freely secreted by the JEG-3 cells, and 3) JEG-hCG-a obtained after dissociation of secreted JEG-hCG. Comparison of these a-subunits was made by gel exclusion chromatography, isoelectric focusing, and ability to form a radioreceptor assay-active hCG when incubated with standard hCG-/? subunit. The freely secreted JEG-a exhibits an apparent molecular weight larger than that of standard hCG-a and JEG-hCG-a, whose apparent molecular weights are similar. The majority of JEG-a is represented by a component with acidic isoelectric pH (pi 4.8), and this
component is virtually absent in standard hCG-a and JEG-hCGa. When incubated with /?-subunit, JEG-hCG-a forms a radioreceptor assay-active hCG almost to the same extent as does standard hCG-a, unlike JEG-a which is unable to do so. Thus, no major differences appear between the two a-subunits that were associated with a /?-subunit [either in urinary (pregnancy) hCG or in JEG-hCG]. In contrast, different characteristics were found in freely secreted JEG-a. Our results indicate that two different types of a-subunit can be obtained from the JEG-3 cells: an a-subunit which is "normal" and incorporated into JEGhCG and another a-subunit which is freely secreted. Further understanding of the structural and chronological relation between these two different a-subunits obtained from the JEG-3 cells should prove valuable in the unraveling of hCG biosynthesis. (Endocrinology 105: 581, 1979)
H
UMAN CG is a placental glycoprotein composed of two noncovalently associated a- and /?-subunits (1). hCG and the three glycoprotein hormones from the pituitary (LH, FSH, and TSH) each contain a specific /?-subunit and an a-subunit which is identical, or almost so, among the four hormones (2-4). Each subunit is glycosylated (5), and one of the major difficulties in the chemical characterization of these hormones is related to variation in the carbohydrate content. Such variations may be a consequence of incomplete biosynthesis or partial degradation during excretion or isolation (6). Therefore, an important criterion of purity, namely charge homogeneity, is impeded by such carbohydrate differences. Despite these difficulties, major progress has been made in glycoprotein hormone chemistry, and the
amino acid sequence of each subunit has been elucidated (7-9). The exact nature of the biosynthesis of glycoprotein hormones is unknown. Several groups of investigators have directed their efforts toward elucidating the biosynthesis of the a-subunit. Pioneering in this area, the groups of Vaitukaitis (10), Franchimont (11), and Rosen and Weintraub (12) observed the secretion of free a-subunit by placenta and by human malignant tumors. In 1973, our group reported the presence of free a-subunit in nonpregnant subjects not harboring malignancy (13,14), and we provided indirect evidence that the circulating a-subunit was not the product of peripheral dissociation of the complete hormone (15). More direct demonstration for the nonperipheral origin of circulating a-subunit was provided by Kourides et al. (16). These results led us to consider the presence in the pituitary of a separate pool of a-subunit which was released under the influence of releasing factors LHRH (17,18) or TRH (14,16,19). The report by Weintraub et al. (20) of ectopic and isolated production of a-subunit by a gastric carcinoid tumor suggested the separate biosynthesis of a-subunit. Direct evidence for separate a-subunit biosynthesis was ob-
Received February 5, 1979. Address all correspondence and requests for reprints to: Dr. Robert Benveniste, Gynecological Endocrinology Laboratories, Department of Obstetrics and Gynecology, Michael Reese Medical Center, University of Chicago, Chicago, Illinois 60616. * This work was supported by the NIH (Research Grants HD-10128 and AM-15838, Center Grant HD-05797, and Biomedical Research Support Grant RR-05424) and the American Cancer Society (Research Grant IN-25P-4).
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BENVENISTE ET AL.
582
tained from studies using cell-free protein-synthesizing systems (21, 22). Taken together, these studies support the notion that the understanding of a-subunit biosynthesis should ultimately help to unravel the biosynthesis of glycoprotein hormones. During the past 2 yr, we have directed our attention to the study of hCG-a secreted free by cultured human choriocarcinoma JEG-3 cells. The JEG cells have retained several secretory characteristics of the normal trophoblastic cell, including its ability to secrete human placental lactogen, progesterone, and biologically active hCG (23). As previously reported, the JEG-3 cells secrete a relatively large quantity of free a-subunit (JEG-a) which is heterogeneous and has different chromatographic properties (as judged by gel exclusion and electrofocusing) than standard (pregnancy urine) hCG-a (24). We then turned our attention to defining the characteristics of the a-subunit contained within the JEG-3 cell hCG (JEG-hCG) and to compare this subunit with the characteristics of both freely secreted JEG-a and standard hCG-a. These studies form the basis of the present report. Materials and Methods Cell cultures The JEG cells (clone number 3) were generously supplied by Dr. P. Kohler (University of Arkansas, Little Rock, AR). Cells were grown in 250-ml plastic flasks in 25 ml Dulbecco's Modified Eagle Medium enriched with 10% fetal calf serum at 37 C in a 90% air-10% CO2 atmosphere. Subcultures were made by scraping the cell sheet, with subsequent plating at 1:10. Under these conditions, confluency was attained in about 5 days and represented about 6 mg cellular protein/flask. Thereafter, the medium was changed and replaced with Dulbecco's Modified Eagle Medium-0.1% bovine serum albumin (BSA) supplemented with 1 mM dibutyryl cAMP. The rationale underlying this latter procedure was to decrease the protein concentration due to fetal calf serum and stimulate with the cyclic nucleotide both hCG and hCG-a secretion by the JEG-3 cells (25, 26). The medium corresponding to 36-h incubation was collected, centrifuged at 5500 X g for 20 min at 4 C to eliminate a small number of floating cells and debris, aliquoted, and stored at -20 C.
Kndo • l!)79 Vol 105 • No.)
hCG and hCG-a preparations served as radioiodinated tracers, standards, and immunogens in the rabbit. Antisera were obtained according to the procedure of Vaitukaitis et al. (31). Iodination of tracers was performed according to the chloramine-T method (32). RIAs and RRA were performed with methods previously described (33, 34). Gel exclusion chromatography Cell medium was dialyzed against distilled water, lyophilized, and reconstituted to a 4-ml sample with phosphate-buffered saline (PBS), pH 7.5. The recovery of hCG and hCG-a, determined by RIAs, was greater than 90% after this procedure. Samples were applied to an exclusion chromatography system composed of two Sephadex G-50- and G-75-coupled columns (1.6 x 90 cm each) and developed with PBS-0.05% BSA. Trace quantities of radioiodinated markers were added to the sample: human immunoglobulin G ([12TJIgG) to determine the void volume, standard hCG-a (['2'T]a), and sodium iodide (12r>I~) to determine the salt peak. The elution position of each component was calculated as the percent elution volume between the void volume and the salt peak. Recovery after gel filtration, as assessed by RIA, was consistently greater than 80%. To dissociate JEG-hCG (i.e. the hCG secreted by the cells), samples were concentrated by ultrafiltration in an Amicon cell (Amicon Corp., Lexington, MA) using a UM-5 membrane (mol wt cut-off, 5000) and then incubated for 24 h at 4 C in 5 M urea pretreated with Rexyn 1-300 ion exchanger (Bio-Rad Laboratories, Richmond, CA). Exclusion chromatography of ureatreated samples was performed on a Sephadex G-100 column (1.5 X 90 cm) developed with a buffer containing 0.05% BSA and 1 M urea. Electrofocusing An LKB 8100 apparatus (LKB Instruments, Inc., Rockville, MD) of 110 ml capacity was used for electrofocusing. Samples were introduced with the low and high density sucrose gradient, 5% and 50%, respectively, to which was added a 2% ampholine solution (pH 3.5-10). The column was refrigerated by circulating cold water, and electrofocusing was carried out for 20 h at constant power supplied by an LKB power supply (model 2103). Two-milliliter fractions were then collected. An internal radioactive marker [3H]oleic acid and the measured pH served as references for the electrofocused samples. Before RIA, each fraction obtained after electrofocusing was dialyzed (2000 mol wt cut-off tubing) to eliminate ampholines.
RIA and radioreceptor assay (RRA)
Recombination of subunits
Purified hCG and subunits were prepared from a crude (pregnancy urine) hCG preparation (Organon Pharmaceuticals, West Orange, NJ) according to standard procedures, including anion exchange and gel exclusion chromatography (27, 28). The purified hCG preparation thus obtained exhibited a potency by RIA and RRA closely resembling that of the CR 115 purified hCG preparation (generously supplied by the National Pituitary Agency). hCG was the source of subunit preparations after dissociation with either concentrated urea or guanidine hydrochloride and isolation by chromatography (29, 30). Purified
The ability of the a-subunit to recombine with the /?-subunit was evaluated by RRA (35). Two micrograms of a-subunit (quantitated by RIA) of three different origins were individually tested for their ability to form in vitro active hCG (as measured by RRA) when mixed in 1 ml PBS-0.05% BSA with a 5-fold excess of hCG-/? subunit (CR 119-3, NIAMDD). Standard hCG, individual subunit preparations, and the mixed a- and ^-subunits were incubated for different lengths of time, ranging from 0-24 h, at 37 C, and then serially diluted in order to assess their individual potencies in the RRA.
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FREE AND ASSOCIATED hCG-a
583
Results The elution profile obtained after chromatography of concentrated JEG-3 cell media on two Sephadex G-50and G-75-coupled columns is shown in Fig. 1; the upper panel displays the elution pattern of hCG secreted by the cells (JEG-hCG), and the lower panel shows the pattern of a-subunit secreted free (JEG-a). The JEGhCG and JEG-a peaks were separately pooled, concentrated, and rechromatographed in order to minimize cross-contamination. Figure 2 shows the elution profiles obtained after chromatography of JEG-hCG when an identical volume from each fraction was assayed by hCG RIA {upper panel) and hCG-a RIA {lower panel). Comparison of the two elution patterns thus obtained indicates that the contamination of JEG-hCG by free JEGa is
Vol. 105, No. 3 Printed in U.S.A.
Human Chorionic Gonadotropin a-Subunit from Cultured Choriocarcinoma (JEG) Cells: Comparison of the Subunit Secreted Free with That Prepared from Secreted Human Chorionic Gonadotropin* ROBERT BENVENISTE, JILL LINDNER, DAVID PUETT, AND DAVID RABIN Departments of Medicine and Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
ABSTRACT. The cultured human choriocarcinoma cell line, JEG-clone 3, secretes both biologically active hCG (JEG-hCG) and substantial quantities of free a-subunit (JEG-a). This study is concerned with a comparative characterization of the a-subunits of three distinct origins: 1) standard hCG-a obtained after dissociation of urinary (pregnancy) hCG, 2) JEG-a freely secreted by the JEG-3 cells, and 3) JEG-hCG-a obtained after dissociation of secreted JEG-hCG. Comparison of these a-subunits was made by gel exclusion chromatography, isoelectric focusing, and ability to form a radioreceptor assay-active hCG when incubated with standard hCG-/? subunit. The freely secreted JEG-a exhibits an apparent molecular weight larger than that of standard hCG-a and JEG-hCG-a, whose apparent molecular weights are similar. The majority of JEG-a is represented by a component with acidic isoelectric pH (pi 4.8), and this
component is virtually absent in standard hCG-a and JEG-hCGa. When incubated with /?-subunit, JEG-hCG-a forms a radioreceptor assay-active hCG almost to the same extent as does standard hCG-a, unlike JEG-a which is unable to do so. Thus, no major differences appear between the two a-subunits that were associated with a /?-subunit [either in urinary (pregnancy) hCG or in JEG-hCG]. In contrast, different characteristics were found in freely secreted JEG-a. Our results indicate that two different types of a-subunit can be obtained from the JEG-3 cells: an a-subunit which is "normal" and incorporated into JEGhCG and another a-subunit which is freely secreted. Further understanding of the structural and chronological relation between these two different a-subunits obtained from the JEG-3 cells should prove valuable in the unraveling of hCG biosynthesis. (Endocrinology 105: 581, 1979)
H
UMAN CG is a placental glycoprotein composed of two noncovalently associated a- and /?-subunits (1). hCG and the three glycoprotein hormones from the pituitary (LH, FSH, and TSH) each contain a specific /?-subunit and an a-subunit which is identical, or almost so, among the four hormones (2-4). Each subunit is glycosylated (5), and one of the major difficulties in the chemical characterization of these hormones is related to variation in the carbohydrate content. Such variations may be a consequence of incomplete biosynthesis or partial degradation during excretion or isolation (6). Therefore, an important criterion of purity, namely charge homogeneity, is impeded by such carbohydrate differences. Despite these difficulties, major progress has been made in glycoprotein hormone chemistry, and the
amino acid sequence of each subunit has been elucidated (7-9). The exact nature of the biosynthesis of glycoprotein hormones is unknown. Several groups of investigators have directed their efforts toward elucidating the biosynthesis of the a-subunit. Pioneering in this area, the groups of Vaitukaitis (10), Franchimont (11), and Rosen and Weintraub (12) observed the secretion of free a-subunit by placenta and by human malignant tumors. In 1973, our group reported the presence of free a-subunit in nonpregnant subjects not harboring malignancy (13,14), and we provided indirect evidence that the circulating a-subunit was not the product of peripheral dissociation of the complete hormone (15). More direct demonstration for the nonperipheral origin of circulating a-subunit was provided by Kourides et al. (16). These results led us to consider the presence in the pituitary of a separate pool of a-subunit which was released under the influence of releasing factors LHRH (17,18) or TRH (14,16,19). The report by Weintraub et al. (20) of ectopic and isolated production of a-subunit by a gastric carcinoid tumor suggested the separate biosynthesis of a-subunit. Direct evidence for separate a-subunit biosynthesis was ob-
Received February 5, 1979. Address all correspondence and requests for reprints to: Dr. Robert Benveniste, Gynecological Endocrinology Laboratories, Department of Obstetrics and Gynecology, Michael Reese Medical Center, University of Chicago, Chicago, Illinois 60616. * This work was supported by the NIH (Research Grants HD-10128 and AM-15838, Center Grant HD-05797, and Biomedical Research Support Grant RR-05424) and the American Cancer Society (Research Grant IN-25P-4).
581
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 23:14 For personal use only. No other uses without permission. . All rights reserved.
BENVENISTE ET AL.
582
tained from studies using cell-free protein-synthesizing systems (21, 22). Taken together, these studies support the notion that the understanding of a-subunit biosynthesis should ultimately help to unravel the biosynthesis of glycoprotein hormones. During the past 2 yr, we have directed our attention to the study of hCG-a secreted free by cultured human choriocarcinoma JEG-3 cells. The JEG cells have retained several secretory characteristics of the normal trophoblastic cell, including its ability to secrete human placental lactogen, progesterone, and biologically active hCG (23). As previously reported, the JEG-3 cells secrete a relatively large quantity of free a-subunit (JEG-a) which is heterogeneous and has different chromatographic properties (as judged by gel exclusion and electrofocusing) than standard (pregnancy urine) hCG-a (24). We then turned our attention to defining the characteristics of the a-subunit contained within the JEG-3 cell hCG (JEG-hCG) and to compare this subunit with the characteristics of both freely secreted JEG-a and standard hCG-a. These studies form the basis of the present report. Materials and Methods Cell cultures The JEG cells (clone number 3) were generously supplied by Dr. P. Kohler (University of Arkansas, Little Rock, AR). Cells were grown in 250-ml plastic flasks in 25 ml Dulbecco's Modified Eagle Medium enriched with 10% fetal calf serum at 37 C in a 90% air-10% CO2 atmosphere. Subcultures were made by scraping the cell sheet, with subsequent plating at 1:10. Under these conditions, confluency was attained in about 5 days and represented about 6 mg cellular protein/flask. Thereafter, the medium was changed and replaced with Dulbecco's Modified Eagle Medium-0.1% bovine serum albumin (BSA) supplemented with 1 mM dibutyryl cAMP. The rationale underlying this latter procedure was to decrease the protein concentration due to fetal calf serum and stimulate with the cyclic nucleotide both hCG and hCG-a secretion by the JEG-3 cells (25, 26). The medium corresponding to 36-h incubation was collected, centrifuged at 5500 X g for 20 min at 4 C to eliminate a small number of floating cells and debris, aliquoted, and stored at -20 C.
Kndo • l!)79 Vol 105 • No.)
hCG and hCG-a preparations served as radioiodinated tracers, standards, and immunogens in the rabbit. Antisera were obtained according to the procedure of Vaitukaitis et al. (31). Iodination of tracers was performed according to the chloramine-T method (32). RIAs and RRA were performed with methods previously described (33, 34). Gel exclusion chromatography Cell medium was dialyzed against distilled water, lyophilized, and reconstituted to a 4-ml sample with phosphate-buffered saline (PBS), pH 7.5. The recovery of hCG and hCG-a, determined by RIAs, was greater than 90% after this procedure. Samples were applied to an exclusion chromatography system composed of two Sephadex G-50- and G-75-coupled columns (1.6 x 90 cm each) and developed with PBS-0.05% BSA. Trace quantities of radioiodinated markers were added to the sample: human immunoglobulin G ([12TJIgG) to determine the void volume, standard hCG-a (['2'T]a), and sodium iodide (12r>I~) to determine the salt peak. The elution position of each component was calculated as the percent elution volume between the void volume and the salt peak. Recovery after gel filtration, as assessed by RIA, was consistently greater than 80%. To dissociate JEG-hCG (i.e. the hCG secreted by the cells), samples were concentrated by ultrafiltration in an Amicon cell (Amicon Corp., Lexington, MA) using a UM-5 membrane (mol wt cut-off, 5000) and then incubated for 24 h at 4 C in 5 M urea pretreated with Rexyn 1-300 ion exchanger (Bio-Rad Laboratories, Richmond, CA). Exclusion chromatography of ureatreated samples was performed on a Sephadex G-100 column (1.5 X 90 cm) developed with a buffer containing 0.05% BSA and 1 M urea. Electrofocusing An LKB 8100 apparatus (LKB Instruments, Inc., Rockville, MD) of 110 ml capacity was used for electrofocusing. Samples were introduced with the low and high density sucrose gradient, 5% and 50%, respectively, to which was added a 2% ampholine solution (pH 3.5-10). The column was refrigerated by circulating cold water, and electrofocusing was carried out for 20 h at constant power supplied by an LKB power supply (model 2103). Two-milliliter fractions were then collected. An internal radioactive marker [3H]oleic acid and the measured pH served as references for the electrofocused samples. Before RIA, each fraction obtained after electrofocusing was dialyzed (2000 mol wt cut-off tubing) to eliminate ampholines.
RIA and radioreceptor assay (RRA)
Recombination of subunits
Purified hCG and subunits were prepared from a crude (pregnancy urine) hCG preparation (Organon Pharmaceuticals, West Orange, NJ) according to standard procedures, including anion exchange and gel exclusion chromatography (27, 28). The purified hCG preparation thus obtained exhibited a potency by RIA and RRA closely resembling that of the CR 115 purified hCG preparation (generously supplied by the National Pituitary Agency). hCG was the source of subunit preparations after dissociation with either concentrated urea or guanidine hydrochloride and isolation by chromatography (29, 30). Purified
The ability of the a-subunit to recombine with the /?-subunit was evaluated by RRA (35). Two micrograms of a-subunit (quantitated by RIA) of three different origins were individually tested for their ability to form in vitro active hCG (as measured by RRA) when mixed in 1 ml PBS-0.05% BSA with a 5-fold excess of hCG-/? subunit (CR 119-3, NIAMDD). Standard hCG, individual subunit preparations, and the mixed a- and ^-subunits were incubated for different lengths of time, ranging from 0-24 h, at 37 C, and then serially diluted in order to assess their individual potencies in the RRA.
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FREE AND ASSOCIATED hCG-a
583
Results The elution profile obtained after chromatography of concentrated JEG-3 cell media on two Sephadex G-50and G-75-coupled columns is shown in Fig. 1; the upper panel displays the elution pattern of hCG secreted by the cells (JEG-hCG), and the lower panel shows the pattern of a-subunit secreted free (JEG-a). The JEGhCG and JEG-a peaks were separately pooled, concentrated, and rechromatographed in order to minimize cross-contamination. Figure 2 shows the elution profiles obtained after chromatography of JEG-hCG when an identical volume from each fraction was assayed by hCG RIA {upper panel) and hCG-a RIA {lower panel). Comparison of the two elution patterns thus obtained indicates that the contamination of JEG-hCG by free JEGa is
