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• W1985016823 abstract "To study the influence of disulfide bridge formation on the assembly of the subunits of human chorionic gonadotropin in JEG-3 choriocarcinoma cells, dithiothreitol (DTT) was used to create a reducing milieu in the endoplasmic reticulum (ER)in vivo. In the presence of 5 mm DTT during pulse-chase experiments all of the β-subunit precursors observed in unperturbed cells (pβ0, pβ1, pβ2, and β*) collapsed into the pβ0 form. The reducing milieu of the ER was reoxidized in less than 5 min after removal of DTT from the medium. DTT markedly increased the half-life of the pβ0 precursor from 8.8 to 65.2 min. Under reoxidation conditions, the β-subunit precursors folded back from pβ0 in less than 5 min. In unperturbed JEG-3 cells, the α-subunit was present in both fully glycosylated and monoglycosylated precursor (pre-α) forms. The attachment of the second N-linked glycan residue of the α-subunit was accelerated in the presence of DTT, and consequently pre-α-subunit was missing from the DTT-treated cultures. The formation of αβ-dimers appeared to be at least partially independent of the oxidation state in the ER. The αβ-dimer was present under conditions in which disulfide bridge formation was prevented by exposure to 5 mm DTT before and during the pulse period. This clearly suggests that the human chorionic gonadotropin subunits may acquire association-competent conformations even when no disulfide bridge formation has taken place. To study the influence of disulfide bridge formation on the assembly of the subunits of human chorionic gonadotropin in JEG-3 choriocarcinoma cells, dithiothreitol (DTT) was used to create a reducing milieu in the endoplasmic reticulum (ER)in vivo. In the presence of 5 mm DTT during pulse-chase experiments all of the β-subunit precursors observed in unperturbed cells (pβ0, pβ1, pβ2, and β*) collapsed into the pβ0 form. The reducing milieu of the ER was reoxidized in less than 5 min after removal of DTT from the medium. DTT markedly increased the half-life of the pβ0 precursor from 8.8 to 65.2 min. Under reoxidation conditions, the β-subunit precursors folded back from pβ0 in less than 5 min. In unperturbed JEG-3 cells, the α-subunit was present in both fully glycosylated and monoglycosylated precursor (pre-α) forms. The attachment of the second N-linked glycan residue of the α-subunit was accelerated in the presence of DTT, and consequently pre-α-subunit was missing from the DTT-treated cultures. The formation of αβ-dimers appeared to be at least partially independent of the oxidation state in the ER. The αβ-dimer was present under conditions in which disulfide bridge formation was prevented by exposure to 5 mm DTT before and during the pulse period. This clearly suggests that the human chorionic gonadotropin subunits may acquire association-competent conformations even when no disulfide bridge formation has taken place. endoplasmic reticulum human chorionic gonadotropin dithiothreitol Dulbecco's modified Eagle's medium post-nuclear supernatant polyacrylamide gel electrophoresis Secretory and membrane glycoproteins of eukaryotic cells are co-translationally translocated into the lumen of the endoplasmic reticulum (ER)1 from where they travel to Golgi complex on the secretory pathway and to other destinations. Recently, it became evident that the transport of proteins out of the ER is limited by a unique “quality control” system that involves recognition and retention of misfolded or misassembled proteins. If further attempts of acquiring the correct folding fail, these proteins may be directed into a degradation pathway (1.Hurtley S.M. Helenius A. Annu. Rev. Cell Biol. 1989; 5: 277-307Crossref PubMed Scopus (773) Google Scholar, 2.Klausner R.D. New. Biol. 1989; 1: 3-8PubMed Google Scholar, 3.Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3563) Google Scholar, 4.Hammond C. Helenius A. Curr. Opin. Cell Biol. 1995; 7: 523-529Crossref PubMed Scopus (586) Google Scholar). In the case of oligomeric proteins, the correct formation of disulfide bonds plays an important role in the assembly of secretory and membrane proteins (5.Machamer C.E. Doms R.W. Bole D.G. Helenius A. Rose J.K. J. Biol. Chem. 1990; 265: 6879-6883Abstract Full Text PDF PubMed Google Scholar, 6.Voorberg J. Fontijn R. Calafat J. Janssen H. van-Mourik J.A. Pannekoek H. J. Cell Biol. 1991; 113: 195-205Crossref PubMed Scopus (112) Google Scholar, 7.Segal M.S. Bye J.M. Sambrook J.F. Gething M.J. J. Cell Biol. 1992; 118: 227-244Crossref PubMed Scopus (70) Google Scholar), which in turn determines stability, intracellular transport, maturation, and function. The disulfide bonds are generated through oxidation in the ER. The ER lumen is unique among the various compartments in the eukaryotic cells because it provides an oxidizing environment for the disulfide bond formation with the help of the protein disulfide isomerase that promotes the disulfide bond formation (8.Freedman R.B. Cell. 1989; 57: 1069-1072Abstract Full Text PDF PubMed Scopus (366) Google Scholar, 9.Noiva R. Lennarz W.J. J. Biol. Chem. 1992; 267: 3553-3556Abstract Full Text PDF PubMed Google Scholar). Recently, it was demonstrated that the co-translational disulfide bond formation, folding, and oligomerization of proteins within the ER can be reversibly inhibited by the addition of the disulfide bridge disrupting agent dithiothreitol (DTT) to living cells (10.Braakman I. Hoover-Litty H. Wagner K.R. Helenius A. J. Cell Biol. 1991; 114: 401-411Crossref PubMed Scopus (248) Google Scholar, 11.Braakman I. Helenius J. Helenius A. EMBO J. 1992; 11: 1717-1722Crossref PubMed Scopus (329) Google Scholar, 12.Braakman I. Helenius J. Helenius A. Nature. 1992; 356: 260-262Crossref PubMed Scopus (221) Google Scholar). Interestingly, upon the removal of DTT, the disulfide bond formation, folding via normal ER folding intermediates, and oligomerization seems to take place (10.Braakman I. Hoover-Litty H. Wagner K.R. Helenius A. J. Cell Biol. 1991; 114: 401-411Crossref PubMed Scopus (248) Google Scholar, 11.Braakman I. Helenius J. Helenius A. EMBO J. 1992; 11: 1717-1722Crossref PubMed Scopus (329) Google Scholar, 12.Braakman I. Helenius J. Helenius A. Nature. 1992; 356: 260-262Crossref PubMed Scopus (221) Google Scholar, 13.Chanat E. Weiss U. Huttner W.B. Tooze S.A. EMBO J. 1993; 12: 2159-2168Crossref PubMed Scopus (132) Google Scholar, 14.de Silva A. Braakman I. Helenius A. J. Cell Biol. 1993; 120: 647-655Crossref PubMed Scopus (90) Google Scholar, 15.Lodish H.F. Kong N. J. Biol. Chem. 1993; 268: 20598-20605Abstract Full Text PDF PubMed Google Scholar, 16.Tatu U. Braakman I. Helenius A. EMBO J. 1993; 12: 2151-2157Crossref PubMed Scopus (113) Google Scholar, 17.Opstelten D.J. de Groote P. Horzinek M.C. Vennema H. Rottier P.J. J. Virol. 1993; 67: 7394-7401Crossref PubMed Google Scholar). Moreover, DTT does not inhibit the transport within the secretory pathway (13.Chanat E. Weiss U. Huttner W.B. Tooze S.A. EMBO J. 1993; 12: 2159-2168Crossref PubMed Scopus (132) Google Scholar, 17.Opstelten D.J. de Groote P. Horzinek M.C. Vennema H. Rottier P.J. J. Virol. 1993; 67: 7394-7401Crossref PubMed Google Scholar). Human chorionic gonadotropin (hCG), a glycoprotein hormone, is composed of two noncovalently linked and glycosylated α- and β-subunits (18.Pierce J.G. Parsons T.F. Annu. Rev. Biochem. 1981; 50: 465-495Crossref PubMed Google Scholar). It is synthesized by the trophoblast cells of the placenta as well as by malignant trophoblast cells and tumors of various origins (19.Pattillo R.A. Gey G.O. Cancer Res. 1968; 28: 1231-1236PubMed Google Scholar, 20.Braunstein G.D. Bridson W.E. Glass A. Hull E.W. McIntire K.R. J. Clin. Endocrinol. Metab. 1972; 35: 857-862Crossref PubMed Scopus (52) Google Scholar, 21.Braunstein G.D. Vaitukaitis J.L. Carbone P.P. Ross G.T. Ann. Intern. Med. 1973; 78: 39-45Crossref PubMed Scopus (456) Google Scholar, 22.Vaitukaitis J.L. J. Clin. Endocrinol. Metab. 1973; 37: 505-514Crossref PubMed Scopus (87) Google Scholar, 23.Vaitukaitis J.L. Annu. Clin. Lab. Sci. 1974; 4: 276-280PubMed Google Scholar, 24.Story M.T. Hussa R.O. J. Clin. Endocrinol. Metab. 1980; 50: 1057-1061Crossref PubMed Scopus (22) Google Scholar, 25.Hussa R.O. Endocr. Rev. 1980; 1: 268-294Crossref PubMed Scopus (118) Google Scholar, 26.Cosgrove D.E. Campain J.A. Cox G.S. Biochim. Biophys. Acta. 1989; 1007: 44-54Crossref PubMed Scopus (41) Google Scholar, 27.Beastall G.H. Cook B. Rustin G.J. Jennings J. Ann. Clin. Biochem. 1991; 28: 5-18Crossref PubMed Scopus (29) Google Scholar, 28.Bagshawe K.D. Acta Oncol. 1992; 31: 99-106Crossref PubMed Scopus (55) Google Scholar, 29.Hoermann R. Berger P. Spoettl G. Gillesberger F. Kardana A. Cole L.A. Mann K. Clin. Chem. 1994; 40: 2306-2312Crossref PubMed Scopus (30) Google Scholar). Both subunits are transcribed from separate genes and assembled post-translationally in the ER. JEG-3 choriocarcinoma cells not only secrete hCG but also an excess of free α- and a minor quantity of β-subunit (26.Cosgrove D.E. Campain J.A. Cox G.S. Biochim. Biophys. Acta. 1989; 1007: 44-54Crossref PubMed Scopus (41) Google Scholar). The α- and β-subunits are synthesized via precursors. Five β-subunit intermediates have been characterized (30.Huth J.R. Mountjoy K. Perini F. Bedows E. Ruddon R.W. J. Biol. Chem. 1992; 267: 21396-21403Abstract Full Text PDF PubMed Google Scholar, 31.Huth J.R. Perini F. Lockridge O. Bedows E. Ruddon R.W. J. Biol. Chem. 1993; 268: 16472-16482Abstract Full Text PDF PubMed Google Scholar). These intermediates represent discrete steps in the folding process that are apparently coupled with the formation of individual disulfide bonds (32.Bedows E. Huth J.R. Suganuma N. Bartels C.F. Boime I. Ruddon R.W. J. Biol. Chem. 1993; 268: 11655-11662Abstract Full Text PDF PubMed Google Scholar). The disulfide bond formation seems to take place post-translationally (30.Huth J.R. Mountjoy K. Perini F. Bedows E. Ruddon R.W. J. Biol. Chem. 1992; 267: 21396-21403Abstract Full Text PDF PubMed Google Scholar). No attempt has yet been made to understand the effect of DTT on the biosynthesis of α- and β-hCG subunitsin vivo. Here, we communicate our investigations on the effect of prevention of the disulfide bridge formation in vivo by the use of DTT on the association of the α- and β-subunits. Moreover, we have studied the effect of reduction and reoxidation on the N-glycosylation of the α-subunit as well as on the maturation of the β-subunit. JEG-3 cells (American Type Culture Collection, Manassas, VA) monolayer cultures were maintained in DMEM medium (Sigma), containing 10% (v/v) fetal calf serum (Linaris Corp., Bettingen, Germany). The medium was supplemented with 3.7 g/liter sodium bicarbonate, 100 IU/ml penicillin, and 100 μg/ml streptomycin (Linaris Corp.). Confluent cell monolayers grown in 25-cm2plastic flasks (Nunc GmbH, Wiesbaden-Biebrich, Germany) were used for pulse labeling and chase experiments. Confluent (<95%) JEG-3 cells grown at 37 °C in DMEM containing 10% (v/v) fetal calf serum were used for the pulse labeling and chase experiments. The cells, kept for 30 min in deficient medium (DMEM lacking cysteine and methionine) were pulse labeled (for time, see “Results”) with 100 μCi/ml [35S]Met/Cys mixture (Amersham Pharmacia Biotech) and chased (for time, see “Results”) in the DMEM containing 5 mm excess of Met and Cys in the presence or absence of 5 mm DTT (as indicated below). After incubation, the medium was removed, and the cells were chilled on ice and incubated for 5–10 min with ice cold phosphate-buffered saline (10 mm sodium phosphate, pH 7.2, 150 mm NaCl) containing 40 mm N-ethylmaleimide to prevent the rearrangement of -S-S- bonds by a blockade of the free thiol groups. The cells were again washed three times with the ice-cold phosphate-buffered saline and lysed with 50 mm Tris-HCl lysis buffer, pH 7.6, containing 200 mm NaCl, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 1.0% (w/v) Nonidet P-40, 20 mm N-ethylmaleimide, 20 mm EDTA, and 2 mm phenylmethylsulfonyl fluoride. A post-nuclear supernatant (PNS) was prepared by centrifugation of the lysate at 19,900 × g for 5 min (Biofuge 15, Heraeus, Osterode, Germany). To reduce the nonspecific coprecipitation, the PNS was precleared by shaking with protein A Staphylococcus aureuscells (Sigma; 100 μl cell suspension/1500 μl lysate) for 30 min at 4 °C. The S. aureus cells were pelleted for 5 min at 19,900 × g (Biofuge 15), and the supernatant was used for sequential immunoprecipitation by using two different anti-hCG antibodies as given below. Two different polyclonal antibodies against the hCG subunits were used in immunoprecipitation of cell lysate. The various β-subunit forms were purified by using an antibody (G10, kindly provided by Dr. E. Bedows, Omaha, NE) that recognizes all forms of free β and β-subunit precursors but does not cross-react with the α-subunit (33.Beebe J.S. Krzesicki R.F. Norton S.E. Perini F. Peters B.P. Ruddon R.W. Endocrinology. 1989; 124: 1613-1624Crossref PubMed Scopus (12) Google Scholar). The fraction of the α-subunit being associated with the β-subunit in a dimer, however, is coprecipitated with this antibody. After depleting the PNS of the β-subunit, their precursors, and the αβ-dimer, the supernatant of the same PNS was used for subsequent immunoprecipitation with a goat anti-α-hCG antibody (34.Hilf G. Merz W.E. Biochem. Biophys. Res. Commun. 1989; 159: 26-33Crossref PubMed Scopus (7) Google Scholar, 35.Merz W.E. Erlewein C. Licht P. Harbarth P. J. Clin. Endocrinol. Metab. 1991; 73: 84-92Crossref PubMed Scopus (34) Google Scholar). The immunoprecipitation with each of the antibodies was carried out for 2 h at 4 °C. The immune complexes were collected on the protein A-agarose beads (Roche Molecular Biochemicals) and washed three times with the lysis buffer and once with 20 mm Tris-HCl buffer, pH 6.8. The immune complexes were eluted by the addition of elution buffer (20 mm Tris-HCl, pH 6.8, containing 1% SDS) and heating of the samples for 1 min in a boiling water bath. Subsequently, the samples were centrifuged at 19,900 × g for 15 min, and aliquots of the supernatant were mixed with the equal volume of the nonreducing sample buffer (100 mm Tris-HCl, pH 6.8, containing 4% (w/v) SDS, 0.2% (w/v) bromphenol blue, and 20% (v/v) glycerol) and reducing sample buffer (containing 10% (v/v) 2-mercaptoethanol), respectively. The samples were separated on SDS-PAGE (Mini-Protean II, Bio-Rad). In all gels, the14C-labeled molecular weight marker (Rainbow, Amersham Pharmacia Biotech) was run together with the samples. It contained myosine (M r = 220,000), phosphorylase b (M r = 97, 400), bovine serum albumin (M r = 66,000), ovalbumin (M r = 46,000), carbonic anhydrase (M r = 30,000), trypsin inhibitorM r = 21,500), lysozyme (M r = 14,300), aprotinin (M r = 6,500), insulin chain B (M r = 3,400), and insulin chain A (M r = 2,350) as molecular weight markers. Proteins were precipitated by incubation of the polyacrylamide gels in 20% (w/v) trichloroacetic acid. After two washings (20 min each) in dimethyl sulfoxide, the gels were incubated in 22% (w/v) 2,5 diphenyloxazole (dissolved in dimethyl sulfoxide) for 90 min with gentle shaking. After several washings with water, the gels were transferred to Whatman 3MM paper and dried in a gel dryer (Bio-Rad). The gels were exposed at −80 °C to x-ray film (Fuji RX) in the presence of an intensifying screen. The quantitative evaluation of the x-ray films was performed by laser densitometry (2202 Ultro scan, LKB) and computer-supported calculation of the band intensities of the fluorograms, or by the Kodak Digital Science one-dimensional system (Amersham Pharmacia Biotech). In some experiments, cells treated with 5 mm DTT prior to (5–40 min) as well as during the pulse (15 min) were lysed at the end of the pulse. The lysate was purified as described above, however, using three different monoclonal antibodies (INN-hCG-45, INN-hCG-53, and INN-hCG-55, kindly provided by Dr. P. Berger, Innsbruck, Austria) directed against epitopes that are exposed on hCG but not on the free subunits to assess the immunologic properties of the αβ-dimer formed in the presence of DTT. Three cultures of JEG-3 cells grown in 75-cm2 flasks were treated 45 min in Met/Cys-deficient DMEM medium (Sigma) prior to labeling with 88.3 MBq [35S]Met/Cys per flask for 45 min followed by 45 min of chase. The cells were rinsed five times with ice-cold phosphate-buffered saline containing each of 5 mm Met and Cys. Cell lysis, preparation of the post-nuclear supernatant, and preabsorption with the protein A S. aureus cells was performed as described above. 2 ml of the lysate were applied to a Sephadex G150 column (0.75 × 100 cm) equilibrated with 50 mm Tris-HCl buffer, pH 7.5, containing 0.05% (w/v) SDS, 20 mm EDTA, 10 mm N-ethylmaleimide, 0.02% (w/v) Nonidet-P40, 2 mm phenylmethylsulfonyl fluoride, and 0.1% (w/v) bovine serum albumin. The column was run in a fast protein liquid chromatography system (Amersham Pharmacia Biotech) at a flow rate of 5 ml/h. Fractions of 750 μl were collected, and 20 μl of aliquot of each fraction was counted in a Tricarb 2450 scintillation counter (Canberra Packard, Dreieich, Germany). The pooled fractions (see below) were purified by immunoprecipitation and analyzed by SDS-PAGE as described above except that boiling of the unreduced samples was omitted. The electrophoresis was performed in duplicate. One gel for the preparation of a fluorogram. The other gel was exposed at 4 °C to a x-ray film (Fuji RX). The bands were excised, and the radioactive material was eluted by breaking up the gels in to small pieces with a glass rod and incubation overnight in reducing sample buffer as well as a freezing-thawing cycle. The eluted proteins were analyzed by SDS-PAGE. The pulse-chase kinetics of the hCG-β-subunit, their intermediates, and the α-subunit contained in αβ-dimers is shown in Fig. 1. The band pattern is very similar as observed by others in the case of JAR cells (see below). At least four different precursor forms of the mature β-subunit can be discerned, designated as pβ0, pβ1, pβ2, and β*. The pβ0 form disappeared completely within the first 15 min of chase (Fig. 1 A). The pβ1 and pβ2 forms show higher apparent molecular weights (M r = 30,300 and 32,600, respectively) than pβ0 (M r = 25,200) in the SDS-PAGE under nonreducing conditions. Upon reduction of the samples prior to electrophoresis, all the β-subunit intermediates collapse into one band (Fig. 1 B) with the same apparent molecular weight as the pβ0 band (M r = 25,200). This suggests that the different electrophoretic mobilities of the precursors under unreduced conditions display differences in the number of disulfide bridges formed. Small amounts of a β-subunit with an apparent molecular weight of 36,900 (β*) emerged at a chase time of 30 min. The apparent molecular weight of this precursor form in the SDS-PAGE is almost identical with that of the mature hCG-β-subunit; however, β* showed an apparent molecular weight of 26,500 upon reduction. Moreover, the results also indicate that the observed β-subunit intermediates represent ER forms of the β-subunit that have not entered into a Golgi compartment where the O-glycan residues are attached. After this has taken place an apparent molecular weight of 37,500 is reached and maintained also in the presence of reducing agents (βmature). At the end of the pulse period (Fig.1 A, lane 2), a significant fraction of the α-subunit was coprecipitated by the anti-β antibody (G10), indicating that an αβ-dimer has already formed. The free α-subunit is not precipitated with G10. Experiments to find out the efficient DTT concentration needed to reduce the disulfide bridges of the hCG subunits in JEG-3 cells were performed in a range of 0.1- 20 mm DTT. A 5 mm concentration of DTT turned out to be completely sufficient to obtain the same picture of the subunit bands in the SDS-PAGE as after complete reduction of the sample with 1.3 m β-mercaptoethanol prior to electrophoresis (data not shown). We, therefore used a concentration of 5 mm DTT in the subsequent experiments. The cells were pulse labeled in the absence of DTT and subsequently chased in the presence of 5 mm DTT up to 240 min. The pulse labeled cells showed the presence of α-subunit (contained in αβ-dimers) and β-subunit intermediates (Fig. 2,lane 2). The pβ0 form was the prominent intermediate in the presence of DTT. Within a period of 5 min in the presence of DTT in the chase medium, a distinctly higher intensity of the pβ0 form (36.6 ± 13.0% (n = 4)versus 12.8 ± 1.2% (n = 5) in the absence of DTT; intensity of all β forms = 100%) was observed. Moreover, the pβ1 intermediate was missing in the presence of DTT. In a further series of experiments, the DTT treatment was performed during the pulse (15 min) and the first 15 min of chase. Fig.3 shows the results of a representative experiment. The reduced β-subunit form (pβ0) was the only β precursor observed as long as DTT was present. Remarkably, an α-subunit band was also visible in the anti-β precipitated samples (lanes 1 and 2), indicating the presence of αβ-dimer even under the reducing conditions in vivo. After 5 min of chase in a DTT-free medium, the β-subunit precursors seemed to be recovered and reoxidized (Fig. 3 A, lane 3). The band pattern seems to be the same as observed in the unperturbed cells. The supernatants of the samples preabsorbed with anti-β antibody (which removes αβ-dimers) were immunoprecipitated with an antibody that recognizes free α-subunit. No free α-subunit could be detected in the presence of DTT (Fig. 3 B, lanes 1 and2). This is due to the fact that the anti-α antibody used does not react with the completely reduced α-subunit. 2V. Singh and W. E. Merz, unpublished data. Within 5 min of DTT-free chase, the free α-subunit (α not contained in the αβ-dimer) had regained a conformation that was recognized by the anti-α antibody (Fig. 3 B, lane 3). The decrease of the intracellular α-subunit concentration in the later chase time (≥60 min) was due to the export of the free α-subunit via the secretory pathway into the culture medium (Fig. 3 C). Besides the mature free β-subunit (βm), a small amount of a β-subunit form with a lower apparent molecular weight (βf) was also secreted from DTT-treated cells (Fig.3 C). It might represent an immature β- or a degraded β-subunit. Gel filtration chromatography of the cell lysates of unperturbed JEG-3 cells was used to separate αβ-dimers from free subunits (Fig.4 A). The fractions were pooled as indicated and purified by immunoprecipitation as described above. The immune complexes were eluted from the protein A-Sepharose and applied to the SDS-PAGE with and without reduction of the samples (Fig.4 B). In the unreduced samples of the pooled fractions 1 and 2, the immune complexes were visible as a high molecular weight band that did not enter into the separating gel. Moreover two other bands (M r = 52,200 and M r = 37,700) were detected in the gel (Fig. 4 B, pool 2). After reduction, the α- and β-subunit bands were visible. Whereas the α-subunit present in the pool fractions 1 and 2 was coprecipitated with the β-subunit (as αβ-dimer), the bulk of the free α-subunit was eluted from the column in the fractions of pool 3 (Fig. 4 B, right panel). The inability of the anti-β antiserum to precipitate the free α-subunits is obvious from the Fig. 4 B (left panel). This shows clearly that the α-subunit eluted in the higher molecular weight fractions of pool 1 and 2 was indeed part of an αβ-dimer complex, whereas the free α-subunit was eluted later. We cut the individual bands of the unreduced samples from the gel and separated the eluted material again in SDS-PAGE under reducing conditions to in identify the individual components of each band (Fig. 4 C). The immune complexes at the top of the gel dissociated into the same subunit band pattern as already seen in Fig. 4 B. The band withM r = 52,000 turned out to represent an αβ*-dimer, whereas the M r = 37,700 band consisted of α-pβ-dimers. In the pool fraction 3, in addition to the free α-subunit, a pre-α-band (see also below) was also clearly visible (Fig. 4 B). In the case of the αβ-dimers the pre-α-band was missing (Fig. 4 B, pool 2). In the free α-subunit fraction (after having removed αβ-dimers by immunoprecipitation), besides the regular α-subunit (M r = 20, 500), a molecular variant designated as pre-α was observed (apparent M r= 18,300; Fig. 5). It represents an α-subunit with only one of the two carbohydrate residues attached to the protein. Both α-subunit forms collapsed into one band with a molecular weight of 10,000 after digestion with peptideN-glycanase F (data not shown). Interestingly, the pre-α-subunit was missing in the DTT-treated JEG-3 cells (Fig.5 B). This seems to indicate the accelerated linkage of the second N-linked carbohydrate residue of the α-subunit when the disulfide bridges are not formed. In the case ofN-glycosylation of the β-subunit a similar process was not observed. We have studied the effect of a prevention of the disulfide bridge formation on the subunit association in hCG biosynthesis. This was achieved by shifting the oxidizing into a reducing milieu in the ER by means of DTT. The DTT concentrations used in the present investigation do not influence protein synthesis and translocation along the secretory pathway (36.Ibrahimi I. J. Cell Biol. 1987; 105: 1555-1560Crossref PubMed Scopus (5) Google Scholar). In the presence of DTT disulfide bridge formation is delayed and reinitiated post-translationally without a loss in efficiency after removal of DTT (11.Braakman I. Helenius J. Helenius A. EMBO J. 1992; 11: 1717-1722Crossref PubMed Scopus (329) Google Scholar). The hCG contains as much as 11 disulfide bridges (five in the α-subunit and six in the β-subunit). The significance of the sequence of disulfide bridge formation for the folding and the association of the subunits is not yet fully elucidated. In this context the effect of a replacement in the β-subunit of individual pairs of cysteine by alanine residues was thoroughly studied (32.Bedows E. Huth J.R. Suganuma N. Bartels C.F. Boime I. Ruddon R.W. J. Biol. Chem. 1993; 268: 11655-11662Abstract Full Text PDF PubMed Google Scholar, 41.Bedows E. Norton S.E. Huth J.R. Suganuma N. Boime I. Ruddon R.W. J. Biol. Chem. 1994; 269: 10574-10580Abstract Full Text PDF PubMed Google Scholar). This methodology is of great value. However, at least theoretically the possibility cannot be excluded that the elimination of single or even more disulfide bridges might have an impact on the conformation of the subunit as well as on the folding and association with the α-subunit. Reduction and reoxidation studies of the wild-type subunits in vivo by the use of DTT provides an independent way to study the interdependences between disulfide bridge formation, folding, subunit association, andN-glycosylation. In this publication, we have addressed the question whether disulfide bridge formation is an essential requirement for subunit association. To our knowledge, no experiments have been published carried out to study the in vivo effects of DTT on the hCG subunit folding pattern and subunit association. In unperturbed JEG-3 cells, the mature hCG-β-subunit is formed through well defined intermediates that seem to acquire distinct conformations that allow separation in the SDS-PAGE. Obviously these intermediates are defined by the formation of disulfide bridges (Fig. 1). These β-subunit precursors seem to resemble very closely or are even identical to the pattern observed and extensively studied in JAR cells (31.Huth J.R. Perini F. Lockridge O. Bedows E. Ruddon R.W. J. Biol. Chem. 1993; 268: 16472-16482Abstract Full Text PDF PubMed Google Scholar, 37.Martell R.E. Ruddon R.W. Endocrinology. 1990; 126: 2757-2764Crossref PubMed Scopus (12) Google Scholar, 38.Beebe J.S. Mountjoy K. Krzesicki R.F. Perini F. Ruddon R.W. J. Biol. Chem. 1990; 265: 312-317Abstract Full Text PDF PubMed Google Scholar, 39.Huth J.R. Mountjoy K. Perini F. Ruddon R.W. J. Biol. Chem. 1992; 267: 8870-8879Abstract Full Text PDF PubMed Google Scholar) and Chinese hamster ovary cells transfected with hCG subunits (32.Bedows E. Huth J.R. Suganuma N. Bartels C.F. Boime I. Ruddon R.W. J. Biol. Chem. 1993; 268: 11655-11662Abstract Full Text PDF PubMed Google Scholar, 40.Bedows E. Huth J.R. Ruddon R.W. J. Biol. Chem. 1992; 267: 8880-8886Abstract Full Text PDF PubMed Google Scholar, 41.Bedows E. Norton S.E. Huth J.R. Suganuma N. Boime I. Ruddon R.W. J. Biol. Chem. 1994; 269: 10574-10580Abstract Full Text PDF PubMed Google Scholar). We have purified the β-subunit intermediates with the same antibodies (G10) as used in the literature (30.Huth J.R. Mountjoy K. Perini F. Bedows E. Ruddon R.W. J. Biol. Chem. 1992; 267: 21396-21403Abstract Full Text PDF PubMed Google Scholar, 31.Huth J.R. Perini F. Lockridge O. Bedows E. Ruddon R.W. J. Biol. Chem. 1993; 268: 16472-16482Abstract Full Text PDF PubMed Google Scholar, 32.Bedows E. Huth J.R. Suganuma N. Bartels C.F. Boime I. Ruddon R.W. J. Biol. Chem. 1993; 268: 11655-11662Abstract Full Text PDF PubMed Google Scholar, 33.Beebe J.S. Krzesicki R.F. Norton S.E. Perini F. Peters B.P. Ruddon R.W. Endocrinology. 1989; 124: 1613-1624Crossref PubMed Scopus (12) Google Scholar, 39.Huth J.R. Mountjoy K. Perini F. Ruddon R.W. J. Biol. Chem. 1992; 267: 8870-8879Abstract Full Text PDF PubMed Google Scholar). In JAR cells, the following sequence of β-subunit intermediates, leading to a form that combines with the α-subunit, was described: pβ0 → pβ1early → pβ1late → pβ1early → pβ2free → pβ2combined-early → pβ2combined-late(30.Huth J.R. Mountjoy K. Perini F. Bedows E. Ruddon R.W. J. Biol. Chem. 1992; 267: 21396-21403Abstract Full Text PDF PubMed Google Scholar, 31.Huth J.R. Perini F. Lockridge O. Bedows E. Ruddon R.W." @default.
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• W1985016823 title "Disulfide Bond Formation Is Not Required for Human Chorionic Gonadotropin Subunit Association" @default.
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