Proc. Natl. Acad. Sci. USA
Vol. 76, No. 10, pp. 5143-5147, October 1979 Cell Biology
Synthesis and processing of human chorionic gonadotropin subunits in cultured choriocarcinoma cells (human choriogonadotropin precursors/mannose core/carbohydrate processing/JAR cells)
RAYMOND W. RUDDON, CHARLOTTE A. HANSON, AND NANCY J. ADDISON Biological Markers Program, National Cancer Institute-Frederick Cancer Research Center, Frederick, Maryland 21701
Communicated by James V. Neel, June 13,1979
Pulse and pulse-chase experiments have ABSTRACT identified the presence of partially glycosylated precursors of the a and ,B subunits of human chorionic gonadotropin (hCG) in cultured JAR choriocarcinoma cells. The a subunit precursor has an apparent molecular weight (by sodium dodecyl sulfate/polyacrylamide gel electrophoresis) of 18,000 (compared to 22,000 for fully processed a subunit); the , subunit precursor has an apparent molecular weight of 24,000 (fully processed, 34,000). Both of these precursors appear to have an intracellular half-life of at least 1 hr and to contain the mannose core but not the terminal carbohydrate sequences. Fully processed a and , subunits do not accumulate intracellularly, indicating that further processing of the precursors is followed by rapid secretion.
Human chorionic gonadotropin (hCG; choriogonadotropin) is produced eutopically by trophoblastic malignancies and ectopically by other human cancers including carcinomas of the lung, breast, ovary, testis, and gastrointestinal tract and certain melanomas and lymphomas (1). The two subunits of hCG, a and (3, are frequently produced discordantly by tumors in vitro (2-4) and in vivo (1, 5, 6), a subunit production and secretion frequently being greater than that of the 3 subunit (2-4, 6). In addition, the prevalence of hCG-producing human cancers has been reported to be much greater when the tumor tissue itself was examined by radioimmunoassay than when only the blood was assayed: 10% of plasma samples from patients with various cancers were positive for hCG, whereas 42% of the tumor tissues from the same patients contained detectable amounts of hCG (7). The hCG subunits produced by human tumors have been shown to be heterogeneous by gel permeation chromatography (1, 8, 9) and isoelectric focusing (9), and differences in the Mr, amino acid composition, and reassociation with the 3 subunit have been observed between a subunit produced by tumors and normal placental a subunit (9, 10). These data suggest that diagnostic acuity could be increased for hCG as a biological marker of cancer, if the biochemical differences between ectopic hCG subunits and those produced by normal placenta could be determined and if the mechanisms controlling the secretion of hCG subunits from tumor cells could be established. This manuscript describes the synthesis and processing of hCG subunits in JAR choriocarcinoma cells as a model system to study these events. MATERIALS AND METHODS Radioactive Labeling of Cells. JAR choriocarcinoma cells were obtained from Roland Pattillo (Medical College of Wisconsin) and were grown at 370C in Dulbecco's modified Eagle's medium with 10% fetal calf serum (GIBCO). Confluent cell monolayers (100-mm-diameter petri dishes) were incubated for 20 min in Eagle's minimal essential medium or Dulbecco's The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisemennt" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
medium lacking amino acids or glucose. Then they were incubated with the following radioactively labeled compounds: [a5S]methionine at 100 ,uCi/ml [500-700 Ci/mmol (1 Ci = 3.7 X 1010 becquerels); New England Nuclear] in methionine-free minimal essential medium (Flow Laboratories); '4C-labeled amino acid mixture at 50 MCi/ml (Amersham) or 3H-labeled amino acid mixture at 100 ,uCi/ml (Amersham) in minimal essential medium containing 1/10th the normal amino acid concentration (Flow Laboratories, McLean, VA); L-[4,5-3H]leucine at 100 ItCi/ml (53 Ci/mmol, Amersham) in leucine-free Dulbecco's medium (Associated Biomedic Systems); D-[63H]glucosamine at 100 ,uCi/ml (20 Ci/mmol, Amersham) or D-[1-3H]mannose at 200 ,uCi/ml (5 Ci/mmol, Amersham) in glucose-free Dulbecco's medium (Associated Biomedic Systems, Buffalo, NY). All incubations were carried out at 370C. In some experiments, the radioactivity was "chased" by removing the labeling medium, washing the cells three times with complete medium, and incubating for various times in the presence of complete Dulbecco's medium. After incubation, the medium was removed, and the cells were washed with phosphate-buffered (pH 7.2) 0.15 M NaCi and lysed by the addition of 5 ml of phosphate-buffered saline containing 1.0% Triton X-100, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (NaDodSO4) (lysis solution), followed by sonication. Medium was brought to the concentration of lysis solution by addition of a concentrated solution. Cell lysates were clarified by centrifugation at 100,000 X g for 1 hr. The 100,000 X g pellet contained no detectable hCG subunits by NaDodSO4/polyacrylamide gel electrophoresis followed by fluorography (see below) and so was discarded. Cell lysates and medium were stored at -70°C until analyzed. Immunoabsorption. hCG-specific polypeptides in the cell lysates and media were detected with rabbit antisera directed against intact hCG, hCG a subunit, and hCG-3 subunit polypeptides. Preimmune rabbit serum was used as a control. Immunoabsorptions were carried out by incubating up to 5.0 ml of lysate or medium with 1 ,tl of immune serum (1:5000 final dilution) at 40C for 16 hr and then precipitating the immune complexes by the addition of protein A-Sepharose CL-4B (Pharmacia) followed by mixing for 2 hr at 4°C and centrifugation at 1000 X g for 15 min. The immunoprecipitates were washed three times in lysis solution and then dissolved in 0.062 M Tris-HCI, pH 6.7/1% NaDodSO4/10% (vol/vol) glycerol/ 2.5% 2-mercaptoethanol. This solution was heated at 1000C for 5 min and then analyzed by electrophoresis as indicated below. NaDodSO4/Polyacrylamide Gel Electrophoresis. This was performed by the method of Laemmli (11) utilizing 5-20% linear gradient gels in a Bio-Rad model 220 slab gel apparatus for 16 hr at 35 V. Radioactivity was visualized by fluorography by the method of Bonner and Laskey (12). The following Abbreviations: hCG, human chorionic gonadotropin (choriogonadotropin); NaDodSO4, sodium dodecyl sulfate.
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Proc. Natl. Acad. Sci. USA 76 (1979)
"4C-labeled Mr standards (New England Nuclear) were used: phosphorylase B (92,500), bovine serum albumin (69,000), ovalbumin (46,000), carbonic anhydrase (30,000), and cytochrome c (12,300). Digestion with Endoglycosidase H and D. Clarified cell lysates were incubated with endo-13-N-acetylglucosaminidases H and D (Miles) by the following procedures. Cell lysates containing approximately 1 mg of protein were diluted 1:10 into a buffer containing 0.1 M sodium citrate at pH 5.5 (for endo H reaction) or 0.15 M phosphate buffer at pH 6.5 (for endo D reaction); 0.1 unit of endoglycosidase H or D was added and the lysate was incubated for 6 hr at 370C. After incubation, the lysates were adjusted to pH 7.2 and immunoprecipitated as described above. RESULTS The relative incorporation of various radioactively labeled substrates into hCG subunits in JAR choriocarcinoma cells is depicted in Fig. 1. JAR cells were incubated for 2 hr with a "'C-labeled amino acid mixture, a 3H-4 beled amino acid mixture, [3H]leucine, or [3H]glucosamine. Cell lysates were then prepared and immunoprecipitations were carried out with anti-hCG, anti-a subunit, or preimmune rabbit serum. Two polypeptides were specifically immunoprecipitated with anti-hCG, with apparent Mrs of 18,000 and 24,000. Only the 18,000 Mr band was precipitated with anti-a subunit. Similar results were observed with each of the labeled substrates. Authentic placental hCG a and ,B subunits migrated at apparent Mrs of 22,000'and 34,000, respectively, in the same system; thus, we conjectured that the radioactively labeled polypeptides observed in this experiment were precursors of the hCG subunits. Because the Mr 18,000 polypeptide was immunoprecipitated with anti-hCG and with anti-a subunit (which is specific
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FIG. 1. Synthesis of hCG subunits in JAR cells: 2-hr labeling with various radioactive substrates. JAR cells were incubated for 2 hr with 14C-labeled a'mino acids. (50 1ACi/ml), 3H-labeled amino acids (100
[3Hjleucine (100 ttCi/ml), or [3H]glucosamine (100,/ACi/mi). ,"Ci/mil), Cell lysates were immin'oprecipitated and analyzed by NaDodISO4/ polyacrylamide gel electrophoresis followed by fluorogra~phy; 3001:5000.
1000 cpm was applied to the gels. All antiserum dilutions were The 14C-labeled Mr standards (lane 13), represented by the dark bands from top to bottom: were! phosphorylase B (92,500), bovine serum albumin (69,000), ovp~lbumin (46,000), carbonic anhydrase (30,000), and cytochrome c (12,300). The positions of the Mr 18,000 and 24,000 bands are indicatedl by the arrows. Lanes 1-3 (14C):. precipitation with anti-hCG (lane 1), anti-a subunit (lane 2), or preimmune serum (lane 3). Lanes 4-6 (3H): precipitation with anti-hCG (lane 4), anti-a subunit (lane 5), or preimmune serum (lane 6). Lanes 7-9 ([3H]leucine): anti-hCG (lane 7), anti-a subunit (lane 8), preimmune serum (lafe 9). Lanes 10-12 ([3H]glucosamine): anti-hCG (lane 10), anti-a subunit (lane 11), preimmune serum (lane 12). Lane 13: Mr markers.
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FIG. 2. Incorporation of [3H]mannose into hCG subunit precursors. JAR cells were incubated with [3H]mannose (200 MCi/ml) for 30 min and chased for 5, 10, 15, 30, and 60 min. Cell lysates were immunoprecipitated with rabbit anti-hCG (1:5000 dilution). Approximately 3000 cpm was applied to the gels. Lanes: 1, 0 chase; 2, 5-min chase; 3, 10-min chase; 4, 15-min chase; 5, 30-min chase; 6, 60-min chase; 7, Mr markers; 8, 25I-labeled hCG a subunit, 9, 25I-labeled hCG f3 spbunit.
for frep a subunit) whereas the Mr 24,000 polypeptide was immunoprecipitated only with anti-hCG, it was postulated that the Mr 18,000 band was an a subunit precursor and the Mr 24,000 band was a f3 subunit precursor. Because hCG 3 subunit contains 12 leucine residues whereas hCG a subunit contains 4, the observation that more [3H]leucine appeared to be incorporated into the Mr 24,000 band (lane 7) is consistent with the idea that the band was an hCG 3 subunit form. The Mr 18,000 and 24,000 polypeptides incorporated [3H]glucosamine, suggesting that they were at least partially glycosylated. Additional evidence for this was obtained by a pulse-chase experiment in which JAR cells wpre incubated with [3H]mannose for 30 min and then chased for 5, 10, 15, 30, and 60 min followed by immunoprecipitation with anti-hCG (Fig. 2). Both Mr 18,000 and 24,000 bands were labeled, again suggesting that the precursor forms of the subunits contained at least the mannose core carbohydrate units. The precursors appeared to have a relatively long half-life and did not appear to be chased into fully processed hCG aPo ,3 subunits, suggesting that rapid secretion followed further processing of the precursors. Other pulse-chase experiments confirmed this (Fig. 3). JAR cells were pulsed for 1 hr with 14C-labe1ed amino acids and chased for 1, 3, 5, and 24 hr. Both the medium and cells were collected and immunoprecipitated with anti-hCG. The hCG ubunits secreted into the medium migrated like authentic placental hCG a and f3 subunits. By immunoprecipitation there was no detectable secretion of subunits into the medium during the 1-hr pulse (lane 1), and the labeled subunits secreted into the medium appeared to be stable for at least 24 hr. In the cells, both putative precursors were labeled and did not appear to be significantly diminished in specific activity until after the 1-hr chase. Again, however, fully processed a or f subunits did not accumttlpate intracellularly. In a similar experiment, 0.1 mM
Proc. Natl. Acad. Sci. USA 76 (1979)
Cell Biology: Ruddon et al.
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10 11 7 8 9 6 FIG. 3. Pulse-chase labeling with 14C-labeled amino acids of hCG subunits in JAR cells and medium. JAR cells were pulsed for 1 hr with a 14C-labeled amino acid mixture (50 MCi/ml) and chased for 1, 3, 5, and 24 hr. All immunoprecipitations were done with anti-hCG (1: 5000); 3500-13,000 cpm was applied to the gels. Lanes 1-5 (medium): labeling medium after 1-hr pulse (lane 1), 1-hr chase (lane 2), 3-hr chase (lane 3), 5-hr chase (lane 4), 24-hr chase (lane 5). Lanes 6-10 (cells): no chase (lane 6), 1-hr chase (lane 7), 3-hr chase (lane 8), 5-hr chase (lane 9), 24-hr chase (lane 10). Lane 11, Mr markers.
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phenylmethanesulfonyl fluoride (a protease inhibitor) was added to the lysis solution in which the cells were harvested. Neither the addition of inhibitor nor incubation of cell lysates for up to 24 hr before immunoprecipitation altered the pattern of hCG subunit bands observed on gel electrophoresis. Thus, it is highly unlikely that protease activity contributed to the results obtained. By radioimmunoassay, the amounts of the hCG subunits in the cells appeared to maintain a steady-state level, whereas the amount in the medium increased over time as expected (Table 1). Four- to 5-fold more a subunit than : subunit was produced and secreted. Because little of the fully processed subunits accumulated in the cells, the majority of the material identified by radioimmunoassay intracellularly must have been the pre-
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FIG. 4. Short pulse labeling of hCG subunit precursors in JAR cells. Cells were pulsed for 2, 5, 10, 15, and 30 min with [35S]methionine (100 1ACi/ml). Cell lysates were immunoprecipitated with antihCG (1:5000), and 700-9000 cpm was applied to the gels. Lanes: 1, 2-min pulse; 2,5-min pulse; 3, 10-min pulse; 4, 15-min pulse; 5, 30-min pulse; 6, Mr markers; 7, '251-labeled hCG a subunit; 8, 125I-labeled hCG # subunit.
processing pathway for hCG subunits, JAR cells were pulsed for 2, 5, 10, 15, and 30 min with [35S]methionine and the cell lysates were immunoprecipitated with anti-hCG (Fig. 4). The Mr 18,000 band was the first to appear, and it was not evident until 5 min after the initiation of the pulse. The Mr 24,000 band appeared faintly at 10 min and clearly at 15 min. Although this suggested that the subunit precursor was more rapidly synthesized than the this may be due to the fact that hCG subunit contains three methionine residues whereas hCG fl a
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To determine if there were short-lived intermediates in the Table 1. Level of hCG a and # subunits in JAR cells and medium during a pulse-chase experiment Chase time, ng/107 cells ( hr a Cells 1 28.4 95.6 3 108.5 27.3 5 104.8 20.5 11 20.0 93.8 Medium 1 27.4 5.8 97.8 14.9 110.9 19.9 46.9 275.0 Cells and medium were collected at various times during a pulsechase experiment and analyzed by radioimmunoassay with antiserum specific for free a subunit or hCG fl/hCG (the anti-# serum did not distinguish between free hCG /3 subunit and intact hCG). 3 5 11
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FIG. 5. Identification of putative hCG a and /B subunit precursors by competition with excess unlabeled hCG a and subunits. JARcells were pulsed for 1 hr with "4C-labeled amino acids (50 ACi/ml). Immediately before immunoprecipitation, excess unlabeled hCG a (260 pmol) or hCG ,B (140 pmol) subunit was added per ml of cell lysate. All antisera were diluted 1:5000; 2000-5000 cpm was applied to the gels. Arrows indicate the Mr 18,000 and 24,000 bands. Lane 1, Mr markers. Lanes 2-4 (controls): anti-hCG (lane 2), anti-a subunit (lane 3), anti-f subunit (lane 4). Lanes 5-7 (with excess subunit): antihCG (lane 5), anti-a subunit (lane 6), anti-f, subunit (lane 7). Lanes 8-10 (with excess subunit): anti-hCG (lane 8), anti-a subunit (lane 9), anti-,B subunit (lane 10). a
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Proc. Natl. Acad. Sci. USA 76 (1979)
Table 2. Effect of tunicamycin on the secretion of hCG subunits by JAR cells ng/107 cells Tunicamycin, a ,Ug/ml 0 73.8 0 389.4 67.2 1 317.4 5 65.4 418.8 50.4 149.4 15 Cells were incubated at 370C in the presence of various concentrations of tunicamycin for 16 hr. An aliquot of the medium was then collected and assayed for hCG a subunit and hCG-f3/hCG by radio-
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subunit contains only one methionine. An additional band was seen at Mr 15,000, which suggests that an additional intermediate of one of the hCG subunits was present. To demonstrate further that the postulated precursor forms were a and /3 subunit precursors, competition experiments were done using an excess of unlabeled hCG a and /3 subunits (Fig. 5). The cells were pulsed for 1 hr with 14C-labeled amino acids, and cell lysates were immunoprecipitated with anti-hCG, anti-a subunit, or anti-: subunit rabbit antiserum. Precipitation of the putative a subunit precursor was inhibited by excess unlabeled a subunit (lanes 5 and 6) but not by excess : subunit (lanes 8 and 9). Precipitation of the putative /3 subunit precursor was diminished by excess unlabeled / subunit (lanes 8 and 10). Because the subunit precursors appeared to be partially glycosylated, it was of interest to examine the effects of the drug tunicamycin, which blocks the formation of the lipid-carbohydrate carrier complex involved in the formation of asparagine-linked N-glycosidic bonds (13, 14). JAR cells were exposed to tunicamycin at 1, 5, or 15 Ag/ml for 16 hr and then pulsed for 1 hr with [a5S]methionine. Doses of tunicamycin up to S ,gg/ml for 16 hr did not appear to inhibit the synthesis or secretion of hCG subunits (Table 2). Immunoprecipitations were carried out with anti-hCG, anti-a subunit, anti-f subunit, or preimmune serum (Fig. 6). In the control samples (lanes 1-3), anti-hCG precipitated the Mr 24,000 and 18,000 polypeptides, whereas anti-a subunit brought down the Mr 18,000 band and the band at Mr 15,000.
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FIG. 6. Effect of tunicamycin on processing of hCG subunits in JAR cells. Cultures were incubated in the presence of medium without drug or with drug at 1 or 5 ,ug/ml for 16 hr. Cells were then pulsed for 1 hr with [35S]methionine, lysed, and immunoprecipitated with various sera (1:5000 dilutions); 3000-13,000 cpm was applied to the gels. Arrows indicate the Mr 18,000 and 24,000 bands. Lanes 1-3 (controls): anti-hCG (lane 1), anti-a subunit (lane 2), preimmune serum (lane 3). Lanes 4-7 (tunicamycin, 1 ,ug/ml): anti-hCG (lane 4), anti-a subunit (lane 5), anti-fl subunit (lane 6), preimmune serum (lane 7). Lanes 8-11 (tunicamycin, 5 ,ug/ml): anti-hCG (lane 8), anti-a subunit (lane 9), anti-f subunit (lane 10), preimmune serum (lane 11).
FIG. 7. Effects of endoglycosidases H and D on hCG subunit precursors synthesized in JAR cells. Cells were pulsed for 1 hr with [35S]methionine, lysed, incubated for 6 hr with endoglycosidases, and immunoprecipitated with anti-a subunit (1:5000). Approximately. 10,000 cpm was applied to the gels. Arrows indicate the Mr 18,000 and 15,000 bands. Lanes: 1, control + buffer (pH 5.5); 2, endoglycosidase H; 3, control + buffer (pH 6.5); 4, endoglycosidase D; 5, Mr markers.
None of these bands was precipitated by preimmune serum. With tunicamycin at 1 gg/ml, glycosylation did not appear to be comple.tely inhibited; however, a new band, immunoprecipitated by anti-hCG and by anti-a subunit, appeared at Mr 12,000. After incubation with tunicamycin at 5 ,ug/ml, which completely inhibited glycosylation based on incorporation of [3H]glucosamine (data not shown), only the Mr 12,000 band appeared in the anti-a subunit immunoprecipitate (lane 9). Moreover, precipitation of the Mr 12,000 polypeptide was inhibited by excess unlabeled a subunit (data not shown). This strongly suggested that the Mr 12,000 band was unglycosylated a subunit peptide. Because the a subunit contains two asparagine-linked carbohydrate units, the three bands observed after incubation with tunicamycin at 1 ,tg/ml may represent a mixture of the unglycosylated peptide (Mr 12,000 band), an intermediate that contained one of the two mannose core units (Mr 15,000 band), and a species that contained both mannose cores (Mr 18,000 band). It should also be noted that, after incubation with tunicamycin at 5 ,ug/ml, the Mr 24,000 /3 subunit precursor band was not present, but both anti-hCG and anti-f subunit precipitated a polypeptide at Mr 15,000 (Fig. 6, lanes 8 and 10). This is close to the expected Mr of the / subunit apoprotein (15). Further evidence that the Mr 18,000 a subunit precursor contained high-mannose core carbohydrate units was obtained by digestion of clarified cell lysates with endoglycosidase H, an enzyme that cleaves mannose oligosaccharide units from asparagine residues of the polypeptide backbone (16). Immunoprecipitation of the digested lysates with anti-a subunit resulted in the appearance of the Mr 15,000 band only (Fig. 7, lane 2). It is not clear why digestion with endoglycosidase H only produced this band and not the Mr 12,000 band of the a subunit precursor. Perhaps only one of the two asparaginelinked oligosaccharide units was sterically available for digestion in the precursor form, or perhaps one of the oligosaccharide units had been further processed such that it was a poor substrate for endoglycosidase H (16). The partially glycosylated Mr 18,000 a subunit precursor was not digested by endoglycosidase D, an enzyme that cleaves side chain-free complex type glycopeptides but not high-mannose glycopeptides (17). These results are consistent with the idea that the Mr 18,000 a subunit precursor contained high-mannose core oligosaccharide units.
Proc. Natl. Acad. Sci. USA 76 (1979)
Cell Biology: Ru'ddon et al. DISCUSSION Processing of the a and :3 subunits in cultured JAR-choriocarcinoma cells involves intermediate, partially glycosylated species with a relatively long half-life (> 1 hr). The subunit precursor has an apparent Mr of 18,000 (compared to 22,000 for fully processed hCG a subunit) and the :3 subunit precursor has an apparent Mr of 24,000 (34,000 for processed hCG subunit). These precursors appear to contain asparagine-linked mannose core carbohydrate units, as evidenced both by incorporation of carbohydrate substrates (Figs. 1 and 2) and by their disappearance after tunicamycin treatment (Fig. 6). Further processing of these precursors must be followed by rapid secretion because fully processed hCG and :3 subunits are not detectable in the cells in pulse or pulse-chase experiments. The greater shift in Mr for the processed :3 subunit (24,000 to 34,000) than for the a subunit (18,000 to 22,000) may be due to the fact that the : subunit contains four serine-linked carbohydrate units that are not present in the a subunit (18). Thus, terminal glycosylation would be expected to result in a greater shift in apparent Mr of hCG subunit, based on NaDodSO4/polyacrylamide gel electrophoresis in which glycoproteins are known to migrate aberrantly. It is not clear why the partially glycosylated precursors accumulate in the cells whereas the fully processed subunits do not. It may be that the rate-limiting step in subunit processing occurs subsequent to the formation of the Mr 18,000 and 24,000 precursors. Such might be the case if these are the forms that are secreted into the cisternal space of the endoplasmic reticulum as they are synthesized and partially glycosylated. Based on what is known for other secreted proteins and glycoproteins, it is likely that the hCG subunits are synthesized on bound polyribosomes of the endoplasmic reticulum as precursor forms containing hydrophobic NH2-terminal signal peptides (19) which are rapidly cleaved as the NH2 termini enter the cisternal space of the endoplasmic reticulum. Subsequent processing would involve movement to the Golgi apparatus, further carbohydrate processing, sequestration into secretory granules, and secretion. Thus, the rate-limiting step in hCG subunit processing would appear to be prior to the point of the biochemical alterations that occur in the Golgi apparatus. Boime and his colleagues (15, 20) have shown that mRNA isolated from first-trimester human placenta directs the synthesis of a pre-a subunit polypeptide of Mr 14,000 and a pre-1 subunit polypeptide of Mr 18,000 in cell-free systems. The pre-a subunit polypeptide is cleaved and glycosylated in the presence of microsomal membranes, and the glycosylation step is inhibited by tunicamycin (20, 21). This cleaved, glycosylated subunit contains a sugar core consisting of N,N'-diacetylchitobiose and at least four mannose residues (20). In our experiments with intact cells, it is likely that the signal peptide has already been cleaved at the earliest pulse times used because that would be expected to be a very rapid event (19, 20, 22) and to occur either prior to or concomitant with glycosylation (20). It is also likely that the Mr 18,000 and 24,000 precursors observed in our experiments contain asparaginelinked high-mannose core oligosaccharides, based on the avid incorporation of [3H]mannose (Fig. 2), tunicamycin effects (Fig. 6), and endoglycosidase H digestion (Fig. 7). Further processing of the hCG subunits probably occurs in the Golgi network where an a-mannosidase removes extra mannose residues, and complex-type oligosaccharide biosynthesis is then completed by the addition of the outer N-acetylglucosamine residues, galactose, andsialic acid (23). The apparent stepwise addition of high-mannose-containing oligosaccharides to asparagine residues followed by subsequent processing is consistent with what is known about other glycoproteins, including IgG heavy chain (23), vesicular stomatitis virus G protein (23-25), and the a
a
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glycosylated env gene precursor (Pr 80env) of murine leukemia -.virus (26). Weintraub and Stannard (27) found results similar to ours for the processing of a subunit by mouse thyrotropic tumor cells that produce TSH. Their results indicated that the intracellular a subunit had a lower molecular weight (by NaDodSO4/polyacrylamide gel electrophoresis) than did extracellular a subunit, and their preliminary experiments using [3H]glucosamine as a precursor suggested that the conversion from lower to higher molecular weight forms of a subunit involved progressive glycosylation. The authors thank Dr. Gerald Putterman, Lucile White, and Dr. Fulvio Perini for purified hCG a and : subunits used to prepare antisera, Dr. Paul Aldenderfer and Kimberly Meade for characterization of the antisera to hCG subunits, and Sally Miller and Jo Ann Tichnell for help in preparation of the manuscript. This research was supported by the National Cancer Institute under Contract NO1-CO-75380 with Litton Bionetics, Inc. 1. Vaitukaitis, J. L., Ross, G. T., Braunstein, G. D. & Rayford, P. L. (1976) Recent Prog. Horm. Res. 32,289-331. 2. Tashjian, A. H., Jr., Weintraub, B. D., Barowsky, N. J., Rabson, A. S. & Rosen, S. W. (1973) Proc. Natl. Acad. Sci. USA 70, 1419-1422. 3. Chou, J. Y., Robinson, J. C. & Wang, S.-S. (1977) Nature (London) 268 543-544. 4. Ruddon, R. W., Anderson, C., Meade, K. S., Aldenderfer, P. H. & Neuwald, P. D. (1979) Cancer Res., in press. 5. Weintraub, B. D. & Rosen, S. W. (1973) J. Clin. Invest. 52, 3135-3142. 6. Rosen, S. W. & Weintraub, B. D. (1974) N. Engl. J. Med. 290, 1441-1447. 7. Hattori, M., Fukase, M., Yoshimi, H., Matsukura, S. & Imura, H. (1978) Cancer 42, 2328-2333. 8. Hussa, R. 0. (1977) J. Clin. Endocrinol. Metab. 44, 11541162. 9. Benveniste, R., Conway, M. C., Puett, D. & Rabinowitz, D. (1979) J. Clin. Endocrinol. Metab. 48, 85-91. 10. Weintraub, B. D., Krauth, G., Rosen, S. W. & Rabson, A. S. (1975) J. Clin. Invest. 56, 1043-1052. 11. Laemmli, U. K. (1970) Nature (London) 227, 680-684. 12. Bonner, W. M. & Laskey, R. A. (1974) Eur. J. Biochem. 46, 83-88. 13. Takatsuki, A., Arima, K. & Tamura, G. (1971) J. Antibiot. 24, 215-233. 14. Struck, D. K. & Lennarz, W. J. (1977) J. Biol. Chem. 252, 1007-1013. 15. Daniels-McQueen, S., McWilliams, D., Birken, S., Canfield, R., Landefeld, T. & Boime, I. (1978) J. Biol. Chem. 253, 71097114. 16. Tarentino, A. L., Plummer, T. H., Jr. & Maley, F. (1974) J. Buol. Chem. 249, 818-824. 17. Koide, N. & Muramatsu, T. (1974) J. Biol. Chem. 249, 48974904. 18. Bahl, 0. P., Marz, L. & Kessler, M. J. (1978) Biochem. Biophys. Res. Commun. 84,667-676. 19. Lingappa, V. R., Lingappa, J. R., Prasad, R., Ebner, K. E. & Blobel, G. (1978) Proc. Natl. Acad. Sci. USA 75,2338-2342. 20. Bielinska, M. & Boime, I. (1978) Proc. Natl. Acad. Sci. USA 75, 1768-1772. 21. Bielinska, M., Grant, G. A. & Boime, I. (1978) J. Bilo. Chem. 253, 7117-7119. 22. Habener, J. F., Potts, J. T., Jr. & Rich, A. (1976) J. Biol. Chem.
251,3893-3899. 23. Tabas, I., Schlesinger, S. & Kornfeld, S. (1978) J. Biol. Chem. 253, 716-722. 24. Hunt, L. A., Etchison, J. R. & Summers, D. F. (1978) Proc. Natl. Acad. Sci. USA 75,754-758. 25. Rothman, J. E., Katz, F. N. & Lodish, H. F. (1978) Cell 15, 1447-1454. 26. Witte, 0. N. & Wirth, D. F. (1979) J. Virol. 29, 735-7,43. 27. Weintraub, B. D. & Stannard, B. S. (1978) FEBS Lett. 92, 303-307.
Vol. 76, No. 10, pp. 5143-5147, October 1979 Cell Biology
Synthesis and processing of human chorionic gonadotropin subunits in cultured choriocarcinoma cells (human choriogonadotropin precursors/mannose core/carbohydrate processing/JAR cells)
RAYMOND W. RUDDON, CHARLOTTE A. HANSON, AND NANCY J. ADDISON Biological Markers Program, National Cancer Institute-Frederick Cancer Research Center, Frederick, Maryland 21701
Communicated by James V. Neel, June 13,1979
Pulse and pulse-chase experiments have ABSTRACT identified the presence of partially glycosylated precursors of the a and ,B subunits of human chorionic gonadotropin (hCG) in cultured JAR choriocarcinoma cells. The a subunit precursor has an apparent molecular weight (by sodium dodecyl sulfate/polyacrylamide gel electrophoresis) of 18,000 (compared to 22,000 for fully processed a subunit); the , subunit precursor has an apparent molecular weight of 24,000 (fully processed, 34,000). Both of these precursors appear to have an intracellular half-life of at least 1 hr and to contain the mannose core but not the terminal carbohydrate sequences. Fully processed a and , subunits do not accumulate intracellularly, indicating that further processing of the precursors is followed by rapid secretion.
Human chorionic gonadotropin (hCG; choriogonadotropin) is produced eutopically by trophoblastic malignancies and ectopically by other human cancers including carcinomas of the lung, breast, ovary, testis, and gastrointestinal tract and certain melanomas and lymphomas (1). The two subunits of hCG, a and (3, are frequently produced discordantly by tumors in vitro (2-4) and in vivo (1, 5, 6), a subunit production and secretion frequently being greater than that of the 3 subunit (2-4, 6). In addition, the prevalence of hCG-producing human cancers has been reported to be much greater when the tumor tissue itself was examined by radioimmunoassay than when only the blood was assayed: 10% of plasma samples from patients with various cancers were positive for hCG, whereas 42% of the tumor tissues from the same patients contained detectable amounts of hCG (7). The hCG subunits produced by human tumors have been shown to be heterogeneous by gel permeation chromatography (1, 8, 9) and isoelectric focusing (9), and differences in the Mr, amino acid composition, and reassociation with the 3 subunit have been observed between a subunit produced by tumors and normal placental a subunit (9, 10). These data suggest that diagnostic acuity could be increased for hCG as a biological marker of cancer, if the biochemical differences between ectopic hCG subunits and those produced by normal placenta could be determined and if the mechanisms controlling the secretion of hCG subunits from tumor cells could be established. This manuscript describes the synthesis and processing of hCG subunits in JAR choriocarcinoma cells as a model system to study these events. MATERIALS AND METHODS Radioactive Labeling of Cells. JAR choriocarcinoma cells were obtained from Roland Pattillo (Medical College of Wisconsin) and were grown at 370C in Dulbecco's modified Eagle's medium with 10% fetal calf serum (GIBCO). Confluent cell monolayers (100-mm-diameter petri dishes) were incubated for 20 min in Eagle's minimal essential medium or Dulbecco's The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisemennt" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
medium lacking amino acids or glucose. Then they were incubated with the following radioactively labeled compounds: [a5S]methionine at 100 ,uCi/ml [500-700 Ci/mmol (1 Ci = 3.7 X 1010 becquerels); New England Nuclear] in methionine-free minimal essential medium (Flow Laboratories); '4C-labeled amino acid mixture at 50 MCi/ml (Amersham) or 3H-labeled amino acid mixture at 100 ,uCi/ml (Amersham) in minimal essential medium containing 1/10th the normal amino acid concentration (Flow Laboratories, McLean, VA); L-[4,5-3H]leucine at 100 ItCi/ml (53 Ci/mmol, Amersham) in leucine-free Dulbecco's medium (Associated Biomedic Systems); D-[63H]glucosamine at 100 ,uCi/ml (20 Ci/mmol, Amersham) or D-[1-3H]mannose at 200 ,uCi/ml (5 Ci/mmol, Amersham) in glucose-free Dulbecco's medium (Associated Biomedic Systems, Buffalo, NY). All incubations were carried out at 370C. In some experiments, the radioactivity was "chased" by removing the labeling medium, washing the cells three times with complete medium, and incubating for various times in the presence of complete Dulbecco's medium. After incubation, the medium was removed, and the cells were washed with phosphate-buffered (pH 7.2) 0.15 M NaCi and lysed by the addition of 5 ml of phosphate-buffered saline containing 1.0% Triton X-100, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (NaDodSO4) (lysis solution), followed by sonication. Medium was brought to the concentration of lysis solution by addition of a concentrated solution. Cell lysates were clarified by centrifugation at 100,000 X g for 1 hr. The 100,000 X g pellet contained no detectable hCG subunits by NaDodSO4/polyacrylamide gel electrophoresis followed by fluorography (see below) and so was discarded. Cell lysates and medium were stored at -70°C until analyzed. Immunoabsorption. hCG-specific polypeptides in the cell lysates and media were detected with rabbit antisera directed against intact hCG, hCG a subunit, and hCG-3 subunit polypeptides. Preimmune rabbit serum was used as a control. Immunoabsorptions were carried out by incubating up to 5.0 ml of lysate or medium with 1 ,tl of immune serum (1:5000 final dilution) at 40C for 16 hr and then precipitating the immune complexes by the addition of protein A-Sepharose CL-4B (Pharmacia) followed by mixing for 2 hr at 4°C and centrifugation at 1000 X g for 15 min. The immunoprecipitates were washed three times in lysis solution and then dissolved in 0.062 M Tris-HCI, pH 6.7/1% NaDodSO4/10% (vol/vol) glycerol/ 2.5% 2-mercaptoethanol. This solution was heated at 1000C for 5 min and then analyzed by electrophoresis as indicated below. NaDodSO4/Polyacrylamide Gel Electrophoresis. This was performed by the method of Laemmli (11) utilizing 5-20% linear gradient gels in a Bio-Rad model 220 slab gel apparatus for 16 hr at 35 V. Radioactivity was visualized by fluorography by the method of Bonner and Laskey (12). The following Abbreviations: hCG, human chorionic gonadotropin (choriogonadotropin); NaDodSO4, sodium dodecyl sulfate.
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"4C-labeled Mr standards (New England Nuclear) were used: phosphorylase B (92,500), bovine serum albumin (69,000), ovalbumin (46,000), carbonic anhydrase (30,000), and cytochrome c (12,300). Digestion with Endoglycosidase H and D. Clarified cell lysates were incubated with endo-13-N-acetylglucosaminidases H and D (Miles) by the following procedures. Cell lysates containing approximately 1 mg of protein were diluted 1:10 into a buffer containing 0.1 M sodium citrate at pH 5.5 (for endo H reaction) or 0.15 M phosphate buffer at pH 6.5 (for endo D reaction); 0.1 unit of endoglycosidase H or D was added and the lysate was incubated for 6 hr at 370C. After incubation, the lysates were adjusted to pH 7.2 and immunoprecipitated as described above. RESULTS The relative incorporation of various radioactively labeled substrates into hCG subunits in JAR choriocarcinoma cells is depicted in Fig. 1. JAR cells were incubated for 2 hr with a "'C-labeled amino acid mixture, a 3H-4 beled amino acid mixture, [3H]leucine, or [3H]glucosamine. Cell lysates were then prepared and immunoprecipitations were carried out with anti-hCG, anti-a subunit, or preimmune rabbit serum. Two polypeptides were specifically immunoprecipitated with anti-hCG, with apparent Mrs of 18,000 and 24,000. Only the 18,000 Mr band was precipitated with anti-a subunit. Similar results were observed with each of the labeled substrates. Authentic placental hCG a and ,B subunits migrated at apparent Mrs of 22,000'and 34,000, respectively, in the same system; thus, we conjectured that the radioactively labeled polypeptides observed in this experiment were precursors of the hCG subunits. Because the Mr 18,000 polypeptide was immunoprecipitated with anti-hCG and with anti-a subunit (which is specific
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FIG. 1. Synthesis of hCG subunits in JAR cells: 2-hr labeling with various radioactive substrates. JAR cells were incubated for 2 hr with 14C-labeled a'mino acids. (50 1ACi/ml), 3H-labeled amino acids (100
[3Hjleucine (100 ttCi/ml), or [3H]glucosamine (100,/ACi/mi). ,"Ci/mil), Cell lysates were immin'oprecipitated and analyzed by NaDodISO4/ polyacrylamide gel electrophoresis followed by fluorogra~phy; 3001:5000.
1000 cpm was applied to the gels. All antiserum dilutions were The 14C-labeled Mr standards (lane 13), represented by the dark bands from top to bottom: were! phosphorylase B (92,500), bovine serum albumin (69,000), ovp~lbumin (46,000), carbonic anhydrase (30,000), and cytochrome c (12,300). The positions of the Mr 18,000 and 24,000 bands are indicatedl by the arrows. Lanes 1-3 (14C):. precipitation with anti-hCG (lane 1), anti-a subunit (lane 2), or preimmune serum (lane 3). Lanes 4-6 (3H): precipitation with anti-hCG (lane 4), anti-a subunit (lane 5), or preimmune serum (lane 6). Lanes 7-9 ([3H]leucine): anti-hCG (lane 7), anti-a subunit (lane 8), preimmune serum (lafe 9). Lanes 10-12 ([3H]glucosamine): anti-hCG (lane 10), anti-a subunit (lane 11), preimmune serum (lane 12). Lane 13: Mr markers.
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FIG. 2. Incorporation of [3H]mannose into hCG subunit precursors. JAR cells were incubated with [3H]mannose (200 MCi/ml) for 30 min and chased for 5, 10, 15, 30, and 60 min. Cell lysates were immunoprecipitated with rabbit anti-hCG (1:5000 dilution). Approximately 3000 cpm was applied to the gels. Lanes: 1, 0 chase; 2, 5-min chase; 3, 10-min chase; 4, 15-min chase; 5, 30-min chase; 6, 60-min chase; 7, Mr markers; 8, 25I-labeled hCG a subunit, 9, 25I-labeled hCG f3 spbunit.
for frep a subunit) whereas the Mr 24,000 polypeptide was immunoprecipitated only with anti-hCG, it was postulated that the Mr 18,000 band was an a subunit precursor and the Mr 24,000 band was a f3 subunit precursor. Because hCG 3 subunit contains 12 leucine residues whereas hCG a subunit contains 4, the observation that more [3H]leucine appeared to be incorporated into the Mr 24,000 band (lane 7) is consistent with the idea that the band was an hCG 3 subunit form. The Mr 18,000 and 24,000 polypeptides incorporated [3H]glucosamine, suggesting that they were at least partially glycosylated. Additional evidence for this was obtained by a pulse-chase experiment in which JAR cells wpre incubated with [3H]mannose for 30 min and then chased for 5, 10, 15, 30, and 60 min followed by immunoprecipitation with anti-hCG (Fig. 2). Both Mr 18,000 and 24,000 bands were labeled, again suggesting that the precursor forms of the subunits contained at least the mannose core carbohydrate units. The precursors appeared to have a relatively long half-life and did not appear to be chased into fully processed hCG aPo ,3 subunits, suggesting that rapid secretion followed further processing of the precursors. Other pulse-chase experiments confirmed this (Fig. 3). JAR cells were pulsed for 1 hr with 14C-labe1ed amino acids and chased for 1, 3, 5, and 24 hr. Both the medium and cells were collected and immunoprecipitated with anti-hCG. The hCG ubunits secreted into the medium migrated like authentic placental hCG a and f3 subunits. By immunoprecipitation there was no detectable secretion of subunits into the medium during the 1-hr pulse (lane 1), and the labeled subunits secreted into the medium appeared to be stable for at least 24 hr. In the cells, both putative precursors were labeled and did not appear to be significantly diminished in specific activity until after the 1-hr chase. Again, however, fully processed a or f subunits did not accumttlpate intracellularly. In a similar experiment, 0.1 mM
Proc. Natl. Acad. Sci. USA 76 (1979)
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10 11 7 8 9 6 FIG. 3. Pulse-chase labeling with 14C-labeled amino acids of hCG subunits in JAR cells and medium. JAR cells were pulsed for 1 hr with a 14C-labeled amino acid mixture (50 MCi/ml) and chased for 1, 3, 5, and 24 hr. All immunoprecipitations were done with anti-hCG (1: 5000); 3500-13,000 cpm was applied to the gels. Lanes 1-5 (medium): labeling medium after 1-hr pulse (lane 1), 1-hr chase (lane 2), 3-hr chase (lane 3), 5-hr chase (lane 4), 24-hr chase (lane 5). Lanes 6-10 (cells): no chase (lane 6), 1-hr chase (lane 7), 3-hr chase (lane 8), 5-hr chase (lane 9), 24-hr chase (lane 10). Lane 11, Mr markers.
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phenylmethanesulfonyl fluoride (a protease inhibitor) was added to the lysis solution in which the cells were harvested. Neither the addition of inhibitor nor incubation of cell lysates for up to 24 hr before immunoprecipitation altered the pattern of hCG subunit bands observed on gel electrophoresis. Thus, it is highly unlikely that protease activity contributed to the results obtained. By radioimmunoassay, the amounts of the hCG subunits in the cells appeared to maintain a steady-state level, whereas the amount in the medium increased over time as expected (Table 1). Four- to 5-fold more a subunit than : subunit was produced and secreted. Because little of the fully processed subunits accumulated in the cells, the majority of the material identified by radioimmunoassay intracellularly must have been the pre-
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FIG. 4. Short pulse labeling of hCG subunit precursors in JAR cells. Cells were pulsed for 2, 5, 10, 15, and 30 min with [35S]methionine (100 1ACi/ml). Cell lysates were immunoprecipitated with antihCG (1:5000), and 700-9000 cpm was applied to the gels. Lanes: 1, 2-min pulse; 2,5-min pulse; 3, 10-min pulse; 4, 15-min pulse; 5, 30-min pulse; 6, Mr markers; 7, '251-labeled hCG a subunit; 8, 125I-labeled hCG # subunit.
processing pathway for hCG subunits, JAR cells were pulsed for 2, 5, 10, 15, and 30 min with [35S]methionine and the cell lysates were immunoprecipitated with anti-hCG (Fig. 4). The Mr 18,000 band was the first to appear, and it was not evident until 5 min after the initiation of the pulse. The Mr 24,000 band appeared faintly at 10 min and clearly at 15 min. Although this suggested that the subunit precursor was more rapidly synthesized than the this may be due to the fact that hCG subunit contains three methionine residues whereas hCG fl a
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To determine if there were short-lived intermediates in the Table 1. Level of hCG a and # subunits in JAR cells and medium during a pulse-chase experiment Chase time, ng/107 cells ( hr a Cells 1 28.4 95.6 3 108.5 27.3 5 104.8 20.5 11 20.0 93.8 Medium 1 27.4 5.8 97.8 14.9 110.9 19.9 46.9 275.0 Cells and medium were collected at various times during a pulsechase experiment and analyzed by radioimmunoassay with antiserum specific for free a subunit or hCG fl/hCG (the anti-# serum did not distinguish between free hCG /3 subunit and intact hCG). 3 5 11
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FIG. 5. Identification of putative hCG a and /B subunit precursors by competition with excess unlabeled hCG a and subunits. JARcells were pulsed for 1 hr with "4C-labeled amino acids (50 ACi/ml). Immediately before immunoprecipitation, excess unlabeled hCG a (260 pmol) or hCG ,B (140 pmol) subunit was added per ml of cell lysate. All antisera were diluted 1:5000; 2000-5000 cpm was applied to the gels. Arrows indicate the Mr 18,000 and 24,000 bands. Lane 1, Mr markers. Lanes 2-4 (controls): anti-hCG (lane 2), anti-a subunit (lane 3), anti-f subunit (lane 4). Lanes 5-7 (with excess subunit): antihCG (lane 5), anti-a subunit (lane 6), anti-f, subunit (lane 7). Lanes 8-10 (with excess subunit): anti-hCG (lane 8), anti-a subunit (lane 9), anti-,B subunit (lane 10). a
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Table 2. Effect of tunicamycin on the secretion of hCG subunits by JAR cells ng/107 cells Tunicamycin, a ,Ug/ml 0 73.8 0 389.4 67.2 1 317.4 5 65.4 418.8 50.4 149.4 15 Cells were incubated at 370C in the presence of various concentrations of tunicamycin for 16 hr. An aliquot of the medium was then collected and assayed for hCG a subunit and hCG-f3/hCG by radio-
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subunit contains only one methionine. An additional band was seen at Mr 15,000, which suggests that an additional intermediate of one of the hCG subunits was present. To demonstrate further that the postulated precursor forms were a and /3 subunit precursors, competition experiments were done using an excess of unlabeled hCG a and /3 subunits (Fig. 5). The cells were pulsed for 1 hr with 14C-labeled amino acids, and cell lysates were immunoprecipitated with anti-hCG, anti-a subunit, or anti-: subunit rabbit antiserum. Precipitation of the putative a subunit precursor was inhibited by excess unlabeled a subunit (lanes 5 and 6) but not by excess : subunit (lanes 8 and 9). Precipitation of the putative /3 subunit precursor was diminished by excess unlabeled / subunit (lanes 8 and 10). Because the subunit precursors appeared to be partially glycosylated, it was of interest to examine the effects of the drug tunicamycin, which blocks the formation of the lipid-carbohydrate carrier complex involved in the formation of asparagine-linked N-glycosidic bonds (13, 14). JAR cells were exposed to tunicamycin at 1, 5, or 15 Ag/ml for 16 hr and then pulsed for 1 hr with [a5S]methionine. Doses of tunicamycin up to S ,gg/ml for 16 hr did not appear to inhibit the synthesis or secretion of hCG subunits (Table 2). Immunoprecipitations were carried out with anti-hCG, anti-a subunit, anti-f subunit, or preimmune serum (Fig. 6). In the control samples (lanes 1-3), anti-hCG precipitated the Mr 24,000 and 18,000 polypeptides, whereas anti-a subunit brought down the Mr 18,000 band and the band at Mr 15,000.
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FIG. 6. Effect of tunicamycin on processing of hCG subunits in JAR cells. Cultures were incubated in the presence of medium without drug or with drug at 1 or 5 ,ug/ml for 16 hr. Cells were then pulsed for 1 hr with [35S]methionine, lysed, and immunoprecipitated with various sera (1:5000 dilutions); 3000-13,000 cpm was applied to the gels. Arrows indicate the Mr 18,000 and 24,000 bands. Lanes 1-3 (controls): anti-hCG (lane 1), anti-a subunit (lane 2), preimmune serum (lane 3). Lanes 4-7 (tunicamycin, 1 ,ug/ml): anti-hCG (lane 4), anti-a subunit (lane 5), anti-fl subunit (lane 6), preimmune serum (lane 7). Lanes 8-11 (tunicamycin, 5 ,ug/ml): anti-hCG (lane 8), anti-a subunit (lane 9), anti-f subunit (lane 10), preimmune serum (lane 11).
FIG. 7. Effects of endoglycosidases H and D on hCG subunit precursors synthesized in JAR cells. Cells were pulsed for 1 hr with [35S]methionine, lysed, incubated for 6 hr with endoglycosidases, and immunoprecipitated with anti-a subunit (1:5000). Approximately. 10,000 cpm was applied to the gels. Arrows indicate the Mr 18,000 and 15,000 bands. Lanes: 1, control + buffer (pH 5.5); 2, endoglycosidase H; 3, control + buffer (pH 6.5); 4, endoglycosidase D; 5, Mr markers.
None of these bands was precipitated by preimmune serum. With tunicamycin at 1 gg/ml, glycosylation did not appear to be comple.tely inhibited; however, a new band, immunoprecipitated by anti-hCG and by anti-a subunit, appeared at Mr 12,000. After incubation with tunicamycin at 5 ,ug/ml, which completely inhibited glycosylation based on incorporation of [3H]glucosamine (data not shown), only the Mr 12,000 band appeared in the anti-a subunit immunoprecipitate (lane 9). Moreover, precipitation of the Mr 12,000 polypeptide was inhibited by excess unlabeled a subunit (data not shown). This strongly suggested that the Mr 12,000 band was unglycosylated a subunit peptide. Because the a subunit contains two asparagine-linked carbohydrate units, the three bands observed after incubation with tunicamycin at 1 ,tg/ml may represent a mixture of the unglycosylated peptide (Mr 12,000 band), an intermediate that contained one of the two mannose core units (Mr 15,000 band), and a species that contained both mannose cores (Mr 18,000 band). It should also be noted that, after incubation with tunicamycin at 5 ,ug/ml, the Mr 24,000 /3 subunit precursor band was not present, but both anti-hCG and anti-f subunit precipitated a polypeptide at Mr 15,000 (Fig. 6, lanes 8 and 10). This is close to the expected Mr of the / subunit apoprotein (15). Further evidence that the Mr 18,000 a subunit precursor contained high-mannose core carbohydrate units was obtained by digestion of clarified cell lysates with endoglycosidase H, an enzyme that cleaves mannose oligosaccharide units from asparagine residues of the polypeptide backbone (16). Immunoprecipitation of the digested lysates with anti-a subunit resulted in the appearance of the Mr 15,000 band only (Fig. 7, lane 2). It is not clear why digestion with endoglycosidase H only produced this band and not the Mr 12,000 band of the a subunit precursor. Perhaps only one of the two asparaginelinked oligosaccharide units was sterically available for digestion in the precursor form, or perhaps one of the oligosaccharide units had been further processed such that it was a poor substrate for endoglycosidase H (16). The partially glycosylated Mr 18,000 a subunit precursor was not digested by endoglycosidase D, an enzyme that cleaves side chain-free complex type glycopeptides but not high-mannose glycopeptides (17). These results are consistent with the idea that the Mr 18,000 a subunit precursor contained high-mannose core oligosaccharide units.
Proc. Natl. Acad. Sci. USA 76 (1979)
Cell Biology: Ru'ddon et al. DISCUSSION Processing of the a and :3 subunits in cultured JAR-choriocarcinoma cells involves intermediate, partially glycosylated species with a relatively long half-life (> 1 hr). The subunit precursor has an apparent Mr of 18,000 (compared to 22,000 for fully processed hCG a subunit) and the :3 subunit precursor has an apparent Mr of 24,000 (34,000 for processed hCG subunit). These precursors appear to contain asparagine-linked mannose core carbohydrate units, as evidenced both by incorporation of carbohydrate substrates (Figs. 1 and 2) and by their disappearance after tunicamycin treatment (Fig. 6). Further processing of these precursors must be followed by rapid secretion because fully processed hCG and :3 subunits are not detectable in the cells in pulse or pulse-chase experiments. The greater shift in Mr for the processed :3 subunit (24,000 to 34,000) than for the a subunit (18,000 to 22,000) may be due to the fact that the : subunit contains four serine-linked carbohydrate units that are not present in the a subunit (18). Thus, terminal glycosylation would be expected to result in a greater shift in apparent Mr of hCG subunit, based on NaDodSO4/polyacrylamide gel electrophoresis in which glycoproteins are known to migrate aberrantly. It is not clear why the partially glycosylated precursors accumulate in the cells whereas the fully processed subunits do not. It may be that the rate-limiting step in subunit processing occurs subsequent to the formation of the Mr 18,000 and 24,000 precursors. Such might be the case if these are the forms that are secreted into the cisternal space of the endoplasmic reticulum as they are synthesized and partially glycosylated. Based on what is known for other secreted proteins and glycoproteins, it is likely that the hCG subunits are synthesized on bound polyribosomes of the endoplasmic reticulum as precursor forms containing hydrophobic NH2-terminal signal peptides (19) which are rapidly cleaved as the NH2 termini enter the cisternal space of the endoplasmic reticulum. Subsequent processing would involve movement to the Golgi apparatus, further carbohydrate processing, sequestration into secretory granules, and secretion. Thus, the rate-limiting step in hCG subunit processing would appear to be prior to the point of the biochemical alterations that occur in the Golgi apparatus. Boime and his colleagues (15, 20) have shown that mRNA isolated from first-trimester human placenta directs the synthesis of a pre-a subunit polypeptide of Mr 14,000 and a pre-1 subunit polypeptide of Mr 18,000 in cell-free systems. The pre-a subunit polypeptide is cleaved and glycosylated in the presence of microsomal membranes, and the glycosylation step is inhibited by tunicamycin (20, 21). This cleaved, glycosylated subunit contains a sugar core consisting of N,N'-diacetylchitobiose and at least four mannose residues (20). In our experiments with intact cells, it is likely that the signal peptide has already been cleaved at the earliest pulse times used because that would be expected to be a very rapid event (19, 20, 22) and to occur either prior to or concomitant with glycosylation (20). It is also likely that the Mr 18,000 and 24,000 precursors observed in our experiments contain asparaginelinked high-mannose core oligosaccharides, based on the avid incorporation of [3H]mannose (Fig. 2), tunicamycin effects (Fig. 6), and endoglycosidase H digestion (Fig. 7). Further processing of the hCG subunits probably occurs in the Golgi network where an a-mannosidase removes extra mannose residues, and complex-type oligosaccharide biosynthesis is then completed by the addition of the outer N-acetylglucosamine residues, galactose, andsialic acid (23). The apparent stepwise addition of high-mannose-containing oligosaccharides to asparagine residues followed by subsequent processing is consistent with what is known about other glycoproteins, including IgG heavy chain (23), vesicular stomatitis virus G protein (23-25), and the a
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glycosylated env gene precursor (Pr 80env) of murine leukemia -.virus (26). Weintraub and Stannard (27) found results similar to ours for the processing of a subunit by mouse thyrotropic tumor cells that produce TSH. Their results indicated that the intracellular a subunit had a lower molecular weight (by NaDodSO4/polyacrylamide gel electrophoresis) than did extracellular a subunit, and their preliminary experiments using [3H]glucosamine as a precursor suggested that the conversion from lower to higher molecular weight forms of a subunit involved progressive glycosylation. The authors thank Dr. Gerald Putterman, Lucile White, and Dr. Fulvio Perini for purified hCG a and : subunits used to prepare antisera, Dr. Paul Aldenderfer and Kimberly Meade for characterization of the antisera to hCG subunits, and Sally Miller and Jo Ann Tichnell for help in preparation of the manuscript. This research was supported by the National Cancer Institute under Contract NO1-CO-75380 with Litton Bionetics, Inc. 1. Vaitukaitis, J. L., Ross, G. T., Braunstein, G. D. & Rayford, P. L. (1976) Recent Prog. Horm. Res. 32,289-331. 2. Tashjian, A. H., Jr., Weintraub, B. D., Barowsky, N. J., Rabson, A. S. & Rosen, S. W. (1973) Proc. Natl. Acad. Sci. USA 70, 1419-1422. 3. Chou, J. Y., Robinson, J. C. & Wang, S.-S. (1977) Nature (London) 268 543-544. 4. Ruddon, R. W., Anderson, C., Meade, K. S., Aldenderfer, P. H. & Neuwald, P. D. (1979) Cancer Res., in press. 5. Weintraub, B. D. & Rosen, S. W. (1973) J. Clin. Invest. 52, 3135-3142. 6. Rosen, S. W. & Weintraub, B. D. (1974) N. Engl. J. Med. 290, 1441-1447. 7. Hattori, M., Fukase, M., Yoshimi, H., Matsukura, S. & Imura, H. (1978) Cancer 42, 2328-2333. 8. Hussa, R. 0. (1977) J. Clin. Endocrinol. Metab. 44, 11541162. 9. Benveniste, R., Conway, M. C., Puett, D. & Rabinowitz, D. (1979) J. Clin. Endocrinol. Metab. 48, 85-91. 10. Weintraub, B. D., Krauth, G., Rosen, S. W. & Rabson, A. S. (1975) J. Clin. Invest. 56, 1043-1052. 11. Laemmli, U. K. (1970) Nature (London) 227, 680-684. 12. Bonner, W. M. & Laskey, R. A. (1974) Eur. J. Biochem. 46, 83-88. 13. Takatsuki, A., Arima, K. & Tamura, G. (1971) J. Antibiot. 24, 215-233. 14. Struck, D. K. & Lennarz, W. J. (1977) J. Biol. Chem. 252, 1007-1013. 15. Daniels-McQueen, S., McWilliams, D., Birken, S., Canfield, R., Landefeld, T. & Boime, I. (1978) J. Biol. Chem. 253, 71097114. 16. Tarentino, A. L., Plummer, T. H., Jr. & Maley, F. (1974) J. Buol. Chem. 249, 818-824. 17. Koide, N. & Muramatsu, T. (1974) J. Biol. Chem. 249, 48974904. 18. Bahl, 0. P., Marz, L. & Kessler, M. J. (1978) Biochem. Biophys. Res. Commun. 84,667-676. 19. Lingappa, V. R., Lingappa, J. R., Prasad, R., Ebner, K. E. & Blobel, G. (1978) Proc. Natl. Acad. Sci. USA 75,2338-2342. 20. Bielinska, M. & Boime, I. (1978) Proc. Natl. Acad. Sci. USA 75, 1768-1772. 21. Bielinska, M., Grant, G. A. & Boime, I. (1978) J. Bilo. Chem. 253, 7117-7119. 22. Habener, J. F., Potts, J. T., Jr. & Rich, A. (1976) J. Biol. Chem.
251,3893-3899. 23. Tabas, I., Schlesinger, S. & Kornfeld, S. (1978) J. Biol. Chem. 253, 716-722. 24. Hunt, L. A., Etchison, J. R. & Summers, D. F. (1978) Proc. Natl. Acad. Sci. USA 75,754-758. 25. Rothman, J. E., Katz, F. N. & Lodish, H. F. (1978) Cell 15, 1447-1454. 26. Witte, 0. N. & Wirth, D. F. (1979) J. Virol. 29, 735-7,43. 27. Weintraub, B. D. & Stannard, B. S. (1978) FEBS Lett. 92, 303-307.
