Dissociation of Early Folding Events from Assembly of the Human Lutropin ß-Subunit

Mesut Muyan1, Raymond W. Ruddon2, Sheila E. Norton, Irving Boime and Elliott Bedows

Departments of Molecular Biology and Pharmacology and Obstetrics and Gynecology (M.M., I.B.) Washington University School of Medicine St Louis, Missouri 63110


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human LH of the anterior pituitary is a member of the glycoprotein hormone family that includes FSH, TSH, and placental CG. All are noncovalently bound heterodimers that share a common {alpha}-subunit and ß-subunits that confer biological specificity. LHß and CGß share more than 80% amino acid sequence identity; however, in transfected Chinese hamster ovary (CHO) cells, LHß assembles with the {alpha}-subunit more slowly than does hCGß, and only a fraction of the LHß synthesized is secreted, whereas CGß is secreted efficiently. To understand why the assembly and secretion of these related ß-subunits differ, we studied the folding of LHß in CHO cells transfected with either the LHß gene alone, or in cells cotransfected with the gene expressing the common {alpha}-subunit, and compared our findings to those previously seen for CG. We found that the rate of conversion of the earliest detectable folding intermediate of LH, pß1, to the second major folding form, pß2, did not differ significantly from the pß1-to-pß2 conversion of CGß, suggesting that variations between the intracellular fates of the two ß-subunits cannot be explained by differences in the rates of their early folding steps. Rather, we discovered that unlike CGß, where the folding to pß2 results in an assembly-competent product, apparently greater than 90% of the LH pß2 recovered from LHß-transfected CHO cells was assembly incompetent, accounting for inefficient LHß assembly with the {alpha}-subunit. Using the formation of disulfide (S-S) bonds as an index, we observed that, in contrast to CGß, all 12 LHß cysteine residues formed S-S linkages as soon as pß2 was detected. Attempts to facilitate LH assembly with protein disulfide isomerase in vitro using LH pß2 and excess urinary {alpha}-subunit as substrate were unsuccessful, although protein disulfide isomerase did facilitate CG assembly in this assay. Moreover, unlike CGß, LHß homodimers were recovered from transfected CHO cells. Taken together, these data suggest that differences seen in the rate and extent of LH assembly and secretion, as compared to those of CG, reflect conformational differences between the folding intermediates of the respective ß-subunits.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The glycoprotein hormones, LH, human (h) CG, FSH, and TSH, are noncovalently bound heterodimers consisting of a common {alpha}-subunit and a distinct ß-subunit that confers biological specificity (1). Human LHß of the anterior pituitary and placental hCGß are the most closely related ß-subunits of this family, apparently having evolved from the same ancestral gene (2). LH- and hCGß share more than 80% sequence identity, including 12 conserved cysteine residues that form 6 intramolecular disulfide (S-S) bonds (1). The structural similarities between LH and hCG account for the fact that they bind the same receptor and elicit the same biological response (3). However, despite these similarities, LHß and hCGß subunits exhibit dramatic differences in their rates of secretion as monomer and assembly with the common {alpha}-subunit. hCGß is secreted and assembles with the {alpha}-subunit quantitatively, whereas the secretion and assembly of LHß are inefficient. These intracellular characteristics of the subunits are observed in transfected Chinese hamster ovary (CHO) cells (4, 5), mouse C-127 mammary tumor cells (6), and somatotrope and corticotrope-derived GH3 and AtT-20 cells (7, 8), respectively. Previous studies have shown that the hCGß subunit undergoes multiple maturation steps characterized by the formation of intramolecular S-S bonds to attain an assembly-competent conformation (9, 10, 11). Thus, variations in the rate and/or extent of folding of the LHß subunit could be responsible for its inability to assemble and be secreted efficiently.

The reported S-S bond pairing of the ovine LHß subunit between Cys residues 34–88, 38–57, 9–90, 23–72, 93–100, and 26–110 (12) is the same as that observed during the hCGß kinetic folding pathway (10, 11, 13). However, the crystal structure of secreted hCGß (14, 15) reveals S-S bonds formed between Cys residues 38–90 and 9–57 rather than between Cys residues 38–57 and 9–90 and implies that a S-S bond rearrangement occurs during the folding or processing of hCGß. That the positions of the cysteine residues in hCGß and LHß are conserved (1) and both ß-subunits assemble with a common {alpha}-subunit suggest that the folding steps leading to formation of assembly-competent ß-subunits are similar and that disulfide bond formation could be used as an index of LHß folding, as it has been used for hCGß folding. We have previously shown that folding of hCGß from an early detectable precursor, pß1, to an assembly-competent intermediate, pß2, and assembly of hCG pß2 with the common {alpha}-subunit, can be monitored by hCGß S-S bond formation (for recent reviews see Refs. 16, 17, 18). The pairing order of these six hCGß S-S bonds is the same whether wild-type CGß folds in human choriocarcinoma (JAR) cells (10), where the hCGß gene is eutopically expressed, in transfected CHO cells (11), or in vitro using purified hCGß subunit as substrate (13). Intracellular hCGß folding occurs in the presence or absence of the {alpha}-subunit (11).

To determine whether differences in the folding pattern between the hCG- and LH- ß-subunits could account for the intracellular behavior of LHß, we studied LHß biosynthesis in CHO cells transfected with the human LHß gene alone, or with the human glycoprotein hormone {alpha}-subunit. We report here that, although the early folding steps for LHß and hCGß occur with similar kinetics in CHO cells, LH pß2, in contrast to hCG pß2, had minimal ability to assemble with the common {alpha}-subunit. Greater that 90% of the LH pß2 synthesized was assembly incompetent for at least 8 h after biosynthesis. Therefore, the rate-limiting step in the attainment of LHß assembly competence does not appear to be the pß1-to-pß2 conversion step, but rather, the maturation of the LH pß2 folding intermediate.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Early Events in hLHß Folding
We have previously shown that the assembly and secretion of the LH ß-subunit with the {alpha}-subunit is markedly less efficient than that of the hCG ß-subunit in transfected CHO cells (4, 5), mouse mammary tumor C-127 cells (6), and somatotrope-derived GH-3 and AtT-20 (7, 8) cells. These findings suggest that differences in the rate and extent of formation of assembly-competent forms could explain the differences in the intracellular fate of these two closely related ß-subunits. To examine this issue, CHO cells transfected with only the LHß gene were pulse labeled for 5 min with [35S]Cys and chased with unlabeled medium (Fig. 1Go). After a 5-min pulse and a 0-min chase, two major forms of LHß were detected by nonreducing SDS-PAGE (Fig. 1AGo). The more slowly migrating of these two forms (Mr = 30,000) apparently represents the LHß homodimer (LH ß/ß). There was no apparent precursor-product relationship in the formation of LH ß/ß since large amounts of the homodimer, representing equivalent percentages of total LHß seen at later chase times, were recovered after chase periods of 0 min (Figs. 1Go, A and B). Also observed at 0 min of chase was an early form of LHß that migrated with approximately half the apparent mol wt of LH ß/ß, termed pß1 (Mr = 17,000). LH pß1 converted to a second intermediate, pß2 (Mr = 24,000), with a t1/2 of 7–8 min (Fig. 1AGo), demonstrating that conversion of LHß folding intermediates pß1 to pß2 was analogous to the conversion of hCG pß1 to pß2, which we have previously demonstrated occurs with a t1/2 of 4–5 min (10, 11).



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Figure 1. Pulse-Chase Kinetics of Early LHß Folding Events

Panel A, CHO cells expressing LHß, but not the {alpha}-subunit, were pulse labeled for 5 min with [35S]Cys and chased for 0, 5, 15, or 30 min as indicated, precipitated with polyclonal antisera against LHß, and assayed by nonreducing SDS-PAGE. Panels B and C represent the same experiment performed with CHO cells expressing both LHß and the {alpha}-subunit at an {alpha}/ß subunit ratio of 0.4 (panel B) or of 1.6 (panel C). Arrows to the right indicate the positions (from bottom to top) of LH pß1, LH{alpha}, LH pß2, and LH ß/ß (homodimer). Panels D–F show the electrophoretic patterns of LH subunits derived from aliquots of the samples shown in panels A–C, but run under reducing SDS-PAGE. Panel D, CHO cells expressing LHß, but not the {alpha}-subunit. Panel E, CHO cells expressing both LHß and the {alpha}-subunit at an {alpha}/ß subunit ratio of 0.4. Panel F, CHO cells expressing both LHß and the {alpha}-subunit at an {alpha}/ß subunit ratio of 1.6. Positions of LH{alpha} and LHß are indicated to the right of panel F. Quantitation of gel images was obtained from fluorograph images by BioImage analysis as described in Materials and Methods. Numbers to the left indicating mol wt markers (MW) are (from top): ovalbumin (Mr = 45,000), carbonic anhydrase (Mr = 29,000), and {alpha}-lactalbumin (Mr = 14,200).

 
To assess whether the presence of the {alpha}-subunit would affect the kinetics of formation of LHß folding intermediates, we examined cells expressing both LHß and the {alpha}-subunit. It should be noted (see Fig. 1Go) that the intracellular {alpha}-subunit migrates with a Mr of 19,000 in either nonreducing (panels B and C) or reducing gels (panels E and F). On the other hand, nonreduced LH pß1 (panels A–C) and reduced LHß (panels D–F) migrate more rapidly than the {alpha}-subunit, while LH pß2 (panels A–C) migrates more slowly than {alpha}. At an {alpha}/ß-subunit ratio of either 0.4 (Fig. 1BGo) or 1.6 (Fig. 1CGo), the t1/2 of conversion of LH pß1 to pß2 was not altered. Conversion of LH pß1 to pß2 involves the formation of S-S bonds since these forms collapse to a single band under reducing conditions (Fig. 1DGo–F). These findings demonstrate that the rate of conversion of LH pß1 to pß2 did not differ significantly from that of hCGß, even in the presence of {alpha}-subunit, and suggest that the differences between the intracellular fates of the two ß-subunits cannot be explained by differences in the rate of their early folding events.

Role of pß2 in LH-ß Assembly
To ensure that the extent of heterodimer formation was not limited by the amount of {alpha}-subunit present in the following experiments, CHO cells overexpressing the common {alpha}-subunit relative to the LHß subunit (i.e. at an {alpha}-/ß-subunit ratio of 1.6) were used. The extent of LH heterodimer formation was assessed by immunoprecipitation with subunit-specific antisera. Precipitation of the {alpha}-subunit with ß-antiserum, or the precipitation of the ß-subunit with {alpha}-antiserum, indicates heterodimer formation. Following pulse labeling, LH heterodimer precipitated with either {alpha}- or ß-antisera contains radiolabeled (newly synthesized) {alpha}-subunit during initial chase periods; however, very little radiolabeled LHß associated with labeled {alpha}-subunit is detected when the heterodimer is precipitated with {alpha}-antiserum (4, 5, 6). This is presumably due to the presence of a stable, preexisting nonradiolabeled intracellular pool of assembly-competent LHß subunit that accumulates because nascent LHß requires time to become assembly competent (4, 5, 6).

To identify all forms of LH subunits present in our cells, we performed Western blot analysis under nonreducing conditions of CHO cell lysates expressing both the LH {alpha}- and ß-subunits (Fig. 2Go). Panel A shows that when polyclonal antiserum to the {alpha}-subunit was used, two bands appeared: a band of Mr = 19,000 was seen when intracellular lysates were probed ({alpha}-int; lane A1), while a heterogeneous band typical of secreted {alpha}-subunit (Mr = 22,000–26,000) was detected in the medium ({alpha}-sec; lane A2). When polyclonal antiserum to the LH ß-subunit was used, multiple bands appeared (panel B). In lysates, the most slowly migrating band (panel B, lane 1) is LH ß/ß homodimer, while a single LHß band was detected as mature secreted LHß (ß-sec) (panel B, lane 2). In addition to LH ß/ß, lysates contained two bands that migrated at Mr = 22,000 and Mr = 20,000 (Fig. 2BGo, lane 1, designated pß2-U (upper), and pß2-L (lower), respectively]. Both of these bands are likely to be pß2 forms, based on their apparent mol wts (pß1 migrates at Mr = 17,000 (Fig. 1Go, A–C)).



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Figure 2. Western Blot Analysis of CHO Cells Expressing LH{alpha} and -ß Subunits

CHO cells expressing both {alpha}- and LH ß-subunits were probed for the presence and gel migration loci of the LH{alpha} and -ß bands. Intracellular lysates (lanes A1 and B1) or overnight serum-free conditioned medium (lanes A2 and B2) were run on 5–20% acrylamide gradient gels under nonreducing conditions and transferred onto polyvinylidene fluoride membranes. The membranes were then probed with either polyclonal antiserum to the {alpha}-subunit (panel A) or polyclonal antiserum to the LH ß-subunit (panel B). Identified in panel A, lane 1, was intracellular {alpha}-subunit ({alpha}-int), and in panel A, lane 2, heterogeneous forms of secreted {alpha}-subunit ({alpha}-sec). In panel B, lane 1, three forms of LHß were detected: (from top to bottom) LH ß/ß homodimer; pß2-U (upper, the more slowly migrating of the LH-pß2 forms), and pß2-L (lower, the more rapidly migrating of the LH-pß2 forms). Panel B, lane 2, shows the position of secreted LHß (ß-sec).

 
When CHO cells expressing only the LHß subunit were pulse labeled for 5 min and chased for 30 min to 8 h, LHß antiserum precipitated the pß2-U and pß2-L folding intermediates and LH ß/ß (Fig. 3AGo). The more predominant of the two pß2 folding intermediates, pß2-U, migrated more slowly than the lower band, pß2-L, and contained more than 90% of the radiolabeled LH pß2. When CHO cells expressing both the LHß subunit and the {alpha}-subunit were pulse labeled and precipitated with LHß antiserum (Fig. 3BGo), a small amount of pß2-L, was detected. The appearance of LH pß2-L is clearer after a 60-min chase, when changes in the migration rate of the {alpha}-subunit, presumably due to processing of the N-linked oligosaccharides, allows pß2-L to be more readily distinguished from {alpha}. At these later chase times, {alpha}-subunit secretion occurred, which reduced the amount of intracellular {alpha}-subunit detected. Figure 3CGo shows that the minor folding form, pß2-L, associates with the {alpha}-subunit. Here, CHO cells expressing both the LHß subunit and the {alpha}-subunit were labeled and immunoprecipitated with antiserum against the {alpha}-subunit. Under these conditions the rapidly migrating LH pß2-L, but not LH pß2-U, was detected at chase times of 1–4 h as {alpha}-subunit processing proceeded, indicating that pß2-L was not a spurious {alpha}-subunit band. Further verification that pß2-L was a form of LHß was obtained when we immunoprecipitated CHO cell lysates of cells expressing only the {alpha}-subunit with {alpha}-antisera and failed to detect its presence (Fig. 3DGo). Taken together, these data suggest that a form of LH pß2 representing less than 10% of the total pß2 in the cell was the only form of LH pß2 competent to associate with the {alpha}-subunit and provide an explanation for why LHß assembly is inefficient.



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Figure 3. Pulse-Chase Kinetics of LH-ß Assembly and Secretion

Panel A, CHO cells expressing LHß, but not the {alpha}-subunit, were pulse labeled for 5 min with [35S]Cys and chased for the times indicated, and intracellular or secreted material was precipitated with polyclonal antiserum against LHß and assayed by nonreducing SDS-PAGE. Identified were LH ß/ß, and two LH pß2 bands designated pß2-U and pß2-L. In addition, a small amount of LHß subunit (ß-sec) was found in the chase medium after 8 h. Panel B, CHO cells expressing both LHß and the {alpha}-subunit at an {alpha}/ß subunit ratio of 1.6 were labeled, chased, and immunoprecipitated with antisera against LHß as in panel A. All bands seen in Fig. 3AGo were also recovered in panel B, and in addition, intracellular {alpha}-subunit ({alpha}) and heterogeneous secreted {alpha}-subunit ({alpha}-sec) were detected. Panel C, CHO cells expressing both LHß and the {alpha}-subunit at an {alpha}/ß subunit ratio of 1.6 were labeled and chased as described for panel A and immunoprecipitated with antisera against the {alpha}-subunit. In addition to {alpha}-int and {alpha}-sec, intracellular LH pß2-L was detected, along with secreted LH-ß. Panel D, CHO cells expressing the {alpha}-subunit only were labeled and chased as described in panel A and immunoprecipitated with antiserum against the {alpha}-subunit. Note the absence of the Mr = 22,000 band of LH pß2-L. The locus at which pß2-U migrates is indicated in brackets (as [pß2-U]) in panels C and D.

 
Role of S-S Bond Formation in hLH-ß Folding
To determine why LHß undergoes such inefficient conversion to assembly-competent pß2, we used formation of S-S bonds as an index of the extent of LHß folding. We have previously shown that the conversion of hCG pß1 to assembly-competent pß2 can be monitored by ß-subunit S-S bond formation (10, 11). If any of the S-S bonds fail to form, an [35S]Cys-containing peptide is released from the disulfide-linked core following trypsin digestion, and these peptides can be detected by reversed-phase HPLC (10, 11, 13, 19, 20, 21). Moreover, these HPLC-derived tryptic maps of hCGß reveal which S-S bonds are unformed. Since there is sufficient sequence identity between the hCGß and LHß subunits, we generated tryptic maps from both HPLC-purified LH pß2 and hCG pß2. When we compared these tryptic maps (Fig. 4Go), we failed to detect any peptides released from the S-S-linked core material of LH pß2 (Fig. 4AGo). The radioactivity eluting in fractions 4–6 (Fig. 4AGo) coincides with the void volume of the column and does not contain LHß peptides when rechromatographed and thus appears to be free [35S]Cys or some other degradation product (Refs. 10, 11 and data not shown). This suggests that all of the LHß cysteine residues were paired as soon as pß1-to-pß2 conversion was detected. By contrast, in hCGß, peptides 96–104 (peaks 1a and b) and 105–114 (peak 2) are released from assembly-competent pß2 (Fig. 4BGo; and Refs. 10, 11, 13). The release of these two peptides indicates that S-S bonds 93–100 and 26–110 remain unformed when hCG pß1-to-pß2 conversion occurs (10, 11, 13), which is consistent with the observation that these two bonds form coincident with or shortly after hCGß assembly with the {alpha}-subunit (10). In the case of LHß, however, it appears that all free thiols were converted to S-S bonds as LHß folded from pß1 to pß2.



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Figure 4. Tryptic Maps of Glycoprotein Hormone ß-Subunits

Panel A shows the HPLC-derived peptide map produced following tryptic cleavage of CHO cell LH pß2 recovered after a pulse labeling period of 5 min with [35S]Cys and a chase of 6 min. The radioactive material that eluted in fractions 4–6 is void volume material that does not contain LHß peptides. The broad peak eluting between 85 and 102 min represents disulfide-linked (core) peptides of LHß. Panel B shows the tryptic maps of hGC pß2 generated under conditions identical to those described in Fig. 4AGo. In addition to the disulfide-linked core peptides, this profile reveals a doublet (peaks 1a and 1b), corresponding to peptide 96–104 (unformed disulfide bond 93–100) and peak 2, containing peptide 105–114 (unformed disulfide bond 26–110) (10 11 ). These data show that [35S]Cys-labeled peptides analogous to those released from the S-S-linked core of hCG pß2 are not released from the S-S-linked core of LH pß2 and suggest that all thiols are disulfide linked in LH pß2 as soon as it is detectable.

 
When the 26–110 S-S bond of uncombined hCGß is formed, the subunit cannot assemble with the common {alpha}-subunit (13) because this is the last S-S bond to form during hCGß folding (10, 11, 13) and forms a "seatbelt" (14) that stabilizes native hCG following heterodimer assembly; preformation of the 26–100 bond inhibits CGß assembly with the {alpha}-subunit (13). We, therefore, examined whether the 26–110 S-S bond of LHß had already formed, preventing LH assembly. To do this, we used an in vitro assembly assay (13). We have previously demonstrated that protein disulfide isomerase is capable of reducing the 26–110 and 93–100 S-S bonds of hCGß, thereby enhancing its in vitro assembly with the {alpha}-subunit under appropriate redox conditions (13). Similarly, we determined whether protein disulfide isomerase was capable of enhancing LH assembly. Using HPLC-purified pß2 (see Materials and Methods) derived from CHO cells expressing either the LHß or CGß subunits, we examined the ability of the respective pß2 subunits to assemble with a large molar excess of purified urinary {alpha}-subunit in vitro (Fig. 5Go). Under conditions where hCG pß2 efficiently assembled with the {alpha}-subunit (Fig. 5AGo; also Ref. 13), no heterodimer containing LH pß2 was detected (Fig. 5BGo). This result supports the hypothesis that there is some structural constraint other than reduction of the 26–110 S-S bond in LH pß2 that must be overcome before the LHß subunit can form heterodimer. Conceivably, these constraints might make the LHß 26–110 S-S bond insusceptible to protein disulfide isomerase.



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Figure 5. In Vitro Assembly of Glycoprotein Hormone pß2-Subunits with {alpha}-Subunits

[35S]Cys-labeled LH pß2 and hCG pß2 were purified by HPLC as described in Materials and Methods and tested for their respective abilities to assemble with a vast molar excess of unlabeled urinary {alpha}-subunit. Assays were performed in the presence of glutathione to maintain appropriate redox conditions as previously described (Ref. 13; and Materials and Methods), and protein disulfide isomerase (Takara) was included in each reaction. Incubations were carried out at 37 C, and reactions were stopped by the addition of iodoacetate at the times indicated. Samples were analyzed by nonreducing SDS-PAGE at 4 C followed by autoradiography. The data reveal that LH pß2 (panel B) was assembly incompetent under assay conditions (13 ) where hCG pß2 (panel A) was assembly competent. (The faint band that appears at Mr = 30,000 at all time points of panel B likely reflects minor contamination of the LH pß2 substrate with LH ß/ß.)

 
As mentioned above, when lysates of CHO cells expressing LHß were precipitated with LHß antiserum, a band migrating with a Mr = 30,000 was seen by nonreducing SDS-PAGE (Fig. 1Go, A and B, and Fig. 3Go, A and B). There appeared to be no precursor-product relationship between LH ß/ß and other LHß folding intermediates as large amounts of the homodimer representing equivalent percentages of total LHß seen at later chase times were recovered following chase periods of 0 min (Fig. 1Go, A and B). These forms persisted within the cell for chase periods of up to 8 h (Fig. 3Go, A and B). Moreover, since LH ß/ß comigrated with LHß monomer (Mr = 17,000) under reducing conditions (Fig. 1Go, D and E), it appears that LH ß/ß is formed by intermolecular S-S bonds. For these reasons, and because the tryptic digestion of LH ß/ß produced a peptide map similar to those seen when intermolecular S-S bond containing homodimers and multimers of hCG-ß were analyzed (Ref. 19 and data not shown), we have concluded that this material represents a homodimeric form of the LHß subunit.

High levels of LH ß/ß were detected in LHß transfected CHO cells either lacking (Figs. 1AGo and 3AGo) or underexpressing (Fig. 1BGo) the {alpha}-subunit relative to LHß ({alpha}/ß subunit ratio = 0.4). However, when the {alpha}-subunit was overexpressed ({alpha} subunit ratio = 1.6), very little LH ß/ß was seen, especially at early chase times (Figs. 1CGo and 3BGo). Thus, it appears that the {alpha}-subunit is affecting the kinetics of formation and the amount of LH ß/ß formed. LH ß/ß was not detected in the media following chase periods of up to 8 h (Figs. 3Go, A and B), demonstrating that, like unassembled LHß subunits (Fig. 3AGo), secretion of LH ß/ß was inefficient.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although hCGß and hLHß subunits share extensive amino acid sequence identity (1), their kinetics and extent of assembly and secretion in a variety of cell lines, which include pituitary-derived GH3 and AtT-20 cells (4, 5, 6, 7, 8), differ significantly. Here we addressed why these two closely related molecules have different fates by comparing the folding kinetics of LHß to that of hCGß. The folding pathway of hCGß has been well studied (16, 17); it folds efficiently from an early detectable precursor, pß1, to an assembly-competent intermediate, pß2.

hCGß folds via the following kinetic pathway (19):


(where the numbers above the arrows indicate the S-S bonds that form at each folding step), but the crystal structure of secreted hCGß (14, 15) reveals S-S bonds formed between Cys residues 38–90 and 9–57 rather than between Cys residues 38–57 and 9–90. This implies that a S-S bond rearrangement occurs during the folding or processing of the hCGß subunit. In any case, assembly of hCG occurs coincident with the formation of S-S bond 93–100 (t1/2 = 8–15 min; Refs. 10, 11) and before the formation of S-S bond 26–110 (t1/2 = 20–25 min; Refs. 10, 11). Both of these events occur shortly after the conversion of hCG pß1 to pß2.

By contrast, conversion of LH pß1 to pß2 did not produce an assembly-competent subunit. Rather, for LHß, the folding and assembly steps appeared to require additional conformational changes, possibly involving S-S bond rearrangement before becoming assembly competent. In contrast to hCGß, all 12 LHß cysteine residues appeared to be involved in S-S linkages as soon as pß1-to-pß2 conversion was detected. This conclusion was based on the observation that no peptides were released from LHß S-S linked core protein following tryptic digestion (Fig. 4AGo). While we cannot rule out the possibility that tryptic cleavage sites were buried due to collapse of the hydrophobic domains of the LHß subunit, this seems unlikely because the purified form of LHß subjected to trypsin had been highly denatured by exposure to SDS and 6 M guanidinium hydrochloride during the extraction and purification procedures before trypsin treatment.

It is not clear why LHß folds and assembles differently from hCGß. While the two molecules share extensive homology, they have very different C-terminal amino acid sequences (1). hCGß has a 31-amino acid hydrophilic C-terminal peptide that contains four O-linked glycans. Unlike hCGß, LHß possesses a seven-amino acid hydrophobic C terminus. The hydrophobicity conferred by the LHß C-terminal heptapeptide, together with hydrophobic amino acid residues localized at the amino terminus of the molecule, appear capable of serving as nucleation sites for LHß aggregation soon after the nascent polypeptide is synthesized (4, 5). This is consistent with previous studies showing that an interaction of the hydrophobic LHß C terminus with other LHß residues is critical in delayed secretion and assembly of the LHß subunit (5). This hypothesis is also supported by the detection of a homodimeric species of LHß, LH ß/ß. There is apparently no precursor-product relationship between the LH ß/ß form and the assembly-competent subunit since it was detected following a 0-min chase. LH ß/ß was detected intracellularly in all CHO cell clones, regardless of the expression level of LHß (data not shown) following chase periods of up to 8 h. Because of this intracellular stability, LH ß/ß may represent a dead-end nonproductive product or an assembly-incompetent storage form of LHß, a fraction of which can be rescued when sufficient amounts of {alpha}-subunit are present to drive assembly.

By decreasing LH ß/ß formation (aggregation), and facilitating folding and assembly, the {alpha}-subunit, when overexpressed in CHO cells, appears to be acting in a chaperone-like manner, presumably by binding to LHß subunits before they bind each other. Since LHß is not assembly competent following 0 min of chase, the {alpha}-subunit seems to bind assembly-incompetent LHß at one epitope accessible in assembly-incompetent LHß and at a different epitope, found only in assembly-competent LHß, during heterodimer formation. This mechanism explains why LHß need not be assembly competent to bind {alpha}. It is likely that the endoplasmic reticulum chaperones play a role in facilitating the folding and assembly of LHß. We previously identified hCGß-chaperone complexes that facilitate folding and assembly of CG in transfected CHO cells (22). The endoplasmic reticulum chaperones, BiP, ERp72, GRp94 (22), calreticulin, and calnexin (E. Bedows, unpublished), associate with hCGß as pß1 folds into assembly-competent pß2. Because of the large degree of amino acid identity between CGß and LHß subunits, and the hydrophobic nature of the LHß C terminus, molecular chaperone intervention likely determines how and when LHß folding intermediates proceed along their kinetic folding pathway.

The intracellular behavior of these two gonadotropin ß-subunits may reflect their respective biological roles. Secretion of hCG from the placenta is primarily constitutive to maintain the corpus luteum, whereas secretion of LH from the pituitary is pulsatile and regulated by LHRH levels (for a review see Ref. 23). Since unassembled LHß is not secreted efficiently, the ability of the pituitary to build up stores of free LHß may assist secretion of large quantities of the hormone from the anterior pituitary during the LH surge before ovulation (4). There remain several unanswered questions about the mechanisms for the selective retention of LHß in the endoplasmic reticulum. Cellular factors including chaperone association (24, 25, 26) and the presence of intracellular retention signals (27) may influence how quickly secretory proteins such as LH are allowed to exit the endoplasmic reticulum. It will be important to explore how these factors differentially control the folding, assembly, and secretion of the glycoprotein hormone ß-subunits with their common {alpha}-subunit.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
CHO cells transfected with wild-type hLHß (4) or hCGß (5, 11) genes alone or cotransfected with the wild-type glyco-protein hormone {alpha} gene, were grown in F-12 medium supplemented with 5% FBS, the neomycin analog G-418 (GIBCO, Grand Island, NY), and antibiotics (19, 28).

Metabolic Labeling of Cells with Radioactive Substrates
CHO cells grown to 90–95% confluency in 100-mm plastic dishes were pulse labeled for the times indicated in the text with L-[35S]cysteine (~1100 Ci/mmol; Du Pont-New England Nuclear, Boston, MA), at a concentration of 200–300 µCi/ml, in serum-free medium lacking cysteine (19). All pulse incubations were carried out as described previously (19), and the cells were incubated for the chase times indicated in the text. Cells were harvested by rinsing with cold PBS and immediately lysed in 5 ml PBS containing detergents (1.0% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS); protease inhibitors (20 mM EDTA and 2 mM phenylmethanesulfonyl fluoride); and 50 mM iodoacetic acid (pH 8.0), to trap the free sulfhydryl groups of the ß folding intermediates. Cell lysates were incubated 20–30 min at 22 C in the dark, followed by disruption through a 22-ga needle (three times), centrifuged for 1 h at 100,000 x g, and immunoprecipitated (see below) or frozen at -70 C for further use.

Immunoprecipitation of Cell Lysates and Culture Media
The immunoreactive forms of LHß or hCGß were immunoprecipitated with a rabbit (4) or goat (19) polyclonal antiserum that recognizes the folding intermediates of both ß-subunits. Because of the sequence identity between them, efficient precipitation of both subunits was observed. All immunoprecipitations were carried out for 16 h at 4 C with rotation in the dark. Immune complexes were precipitated with Protein A-Sepharose (Sigma Chemical Co., St. Louis, MO) and prepared for SDS-PAGE or reversed-phase HPLC as described below.

SDS-PAGE, Fluorography, and Western Blot Analysis
Radiolabeled LH or CG forms that adsorbed to Protein A-Sepharose beads were eluted with 2 x concentrated SDS gel sample buffer (125 mM Tris-HCl, pH 6.8, containing 2% SDS, 20% glycerol, and 40 µg/ml bromophenol blue). Samples run under reducing conditions were boiled for 4 min in sample buffer containing 2% ß-mercaptoethanol, while samples run under nonreducing conditions were boiled for 4 min in sample buffer lacking ß-mercaptoethanol. The washed samples, including the Protein A-Sepharose beads, were applied to polyacrylamide gradient slab gels (5–20%) that were run by the method of Laemmli (29). Gels were rinsed in water, dried in vacuo on filter paper, and exposed to x-ray film. Fluorographs were photographed with a Kodak CCD (charged caption device) camera (BioImage 110S System, Genomic Solutions Inc., Ann Arbor, MI). Quantitation of gel images was obtained by transferring photographed fluorograph images to a Sun SPARCstation 1+ computer and analyzed using BioImage Whole Band software and printed on a Seiko Instruments USA Inc. (San Jose, CA) CH-5504 color printer.

Western blot analysis was performed using aliquots (50 µl) of media or cell lysates from CHO cells expressing LHß and the common {alpha}-subunit following a 24-h incubation in conditioned media lacking serum and were resolved by SDS-PAGE on a 5–20% gradient gel under nonreducing conditions. Gels were transferred to nitrocellulose membranes and probed with either {alpha}-antiserum or hCGß antiserum. Proteins were detected by the Tropix (Bedford, MA) chemiluminescent detection system.

Purification of ß-Subunit Folding Intermediates and HPLC Analysis
The hCGß and LHß folding intermediates pß1 and pß2 were purified by a two-step process (immunoprecipitation followed by C4 reversed-phase HPLC) as described by Huth et al. (10). Briefly, pß1 and pß2 were immunoprecipitated from cell lysates with polyclonal antisera, and immunocomplexes were precipitated with Protein A-Sepharose beads. To dissociate precipitated immunocomplexes, pellets were treated with 6 M guanidine-HCl (pH 3) (Pierce, Rockford, IL; sequencing grade) for 16 h at room temperature with 100 µg of myoglobin (Sigma) as carrier. Following low-speed centrifugation to remove Protein-A Sepharose beads, the guanidine eluates were injected onto a Vydac 300 Å C4 reversed-phase column (Hesparia Separations Group, Hesparia, CA) equilibrated with 0.1% trifluoroacetic acid (TFA) and eluted using an acetonitrile gradient as previously described (10). Fractions containing LHß or hCGß forms were concentrated by vacuum centrifugation and pooled for tryptic analysis.

Tryptic Digestions and HPLC Purification of Tryptic Peptides
Nonreduced LHß or hCGß forms were digested for 16 h at 37 C in silanized polypropylene tubes containing 100 µg myoglobin, 0.03% diphenylcarbamyl chloride-treated Trypsin (Sigma), 5 mM CaCl2, and 100 mM Tris-HCl, pH 8. The digestion was continued with the addition of two sequential aliquots of 50 µg trypsin (0.06% final concentration) for 2 h. Tryptic digests of ß-subunits were injected onto a Brownlee 300 Å C8 reversed-phase column (Applied Biosystems, Foster City, CA) equilibrated with 0.1% TFA (10, 11). The column was eluted isocratically for 3 min with 0.1% TFA followed by a 0.32%/min acetonitrile gradient in 0.1% TFA for 100 min. The column was washed with 80% acetonitrile, 0.1% TFA for 5 min and then reequilibrated in 0.1% TFA. The flow rate was 1.0 ml/min. One-minute fractions were collected in silanized polypropylene tubes. Tubes into which S-S-linked peptides eluted contained 5 µg myoglobin as carrier. Samples were concentrated by vacuum centrifugation and stored at -20 C.

Identification of Peptides Following Tryptic Digestion
Fully folded hCGß contains 6 disulfide bonds and 13 Arg and Lys residues that are arranged such that all of the Cys-containing tryptic peptides remain attached to each other as a result of their covalent disulfide bridges (9, 10). If, however, particular disulfide bonds are not formed in a given hCGß folding intermediate, specific Cys-containing tryptic peptides are released from the S-S-linked CGß core. For example, if the 26–110 bond is unformed, then CGß peptide 105–114 (containing Cys-110) would be released. The pattern of tryptic-released peptides, distinguished from the disulfide-linked peptides by HPLC, reveals incomplete bond formation (10). By lysing cells in the presence of the alkylating agent iodoacetate, the Cys residues of the unformed hCGß S-S bonds are trapped. The alkylated folding intermediates are then resolved by C8 reversed-phase HPLC (10). Identification of HPLC peptide peaks was made by comparing elution times of peaks generated from wild-type CGß tryptic digests (10, 11) that had been verified by microsequencing. Amino acid sequence analysis revealed whether the Cys-containing peptides had been alkylated (indicating that the S-S bond had not been formed in the intact molecule).

In Vitro Assembly of LH- and CG{alpha} and -ß Subunits
In vitro assembly reactions were performed by a modification of the procedure described by Huth et al. (13). To generate the pß2 used in the experiment shown in Fig. 5Go, CHO cells expressing either CGß or LHß were pulse labeled for 5 min with [35S]Cys and chased with unlabeled medium for 20 min for CGß or 30 min for LHß, followed by lysis in PBS containing EDTA, phenylmethanesulfonyl fluoride, and the detergent mixture described above, but lacking alkylating agent. Respective CG and LH pß2 subunits were purified by immunoprecipitation followed by reversed-phase HPLC. HPLC-derived fractions containing radiolabeled pß2 substrate to be used in CG or LH assembly reactions were concentrated under vacuum. Each reaction contained a final concentration of 1 µM urinary hCG{alpha}, 150,000 cpm (~2 ng) [35S]cysteine-labeled pß2, 1.7 mM reduced glutathione, 0.27 mM oxidized glutathione, 1 mM EDTA, and 20 mM sodium phosphate (pH 7.8); a final concentration of 17.5 µM bovine liver protein disulfide isomerase (Takara Biochemical Inc., Berkeley, CA) was also included in the assay. The reactions were incubated at 37 C, and aliquots were withdrawn at the times indicated and terminated with a solution of 900 mM iodoacetate containing 450 mM Tris-HCl (pH 8.7). Samples were mixed with ice-cold nonreducing electrophoresis buffer and analyzed by SDS-PAGE at 4 C.


    FOOTNOTES
 
Address requests for reprints to: Dr. Elliott Bedows, The Eppley Institute For Research in Cancer and Allied Diseases, The University of Nebraska Medical Center, Omaha Nebraska 68198-6805.

This work was supported in part by NIH Grants HD-23398 and CA-32949, by American Cancer Society Institutional Grant IRG-165G and NCI Cancer Center Support Grant P30CA3627.

1 Current address: University of Rochester Medical Center, Department of Biochemistry, Box 607, 601 Elmwood, Rochester, New York 14620. Back

2 Current address: Corporate Office of Science and Technology, Johnson & Johnson, 410 George Street, New Brunswick, New Jersey 08901. Back

Received for publication April 8, 1998. Revision received June 10, 1998. Accepted for publication June 21, 1998.


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 ABSTRACT
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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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