The Six N-linked Carbohydrates of the Lutropin/Choriogonadotropin Receptor Are Not Absolutely Required for Correct Folding, Cell Surface Expression, Hormone Binding, or Signal Transduction

David P. Davis, Tim G. Rozell, Xuebo Liu and Deborah L. Segaloff

Department of Physiology and Biophysics The University of Iowa College of Medicine Iowa City, IA 52242


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Using two separate methods, we have determined that all six potential sites for N-linked glyco-sylation on the rat lutropin/choriogonadotropin receptor (rLHR) contain carbohydrates. The functional roles of the carbohydrates were analyzed initially through the use of two nonglycosylated receptor mutants rLHR(N77,152,173,269,277,291Q) and rLHR(N77,152,269,277,291Q;T175A). Although Western blot analyses demonstrated both mutant recep-tors to be stably expressed, little or no hCG binding activity could be detected in detergent solubilized extracts of 293 cells expressing either nonglycosylated LHR mutant. Although this loss of hCG binding was concluded to be due to misfolding, it was unknown whether this misfolding was due to the absence of carbohydrates or to the multiple amino acid substitutions that had been introduced into the polypeptide. To differentiate between these possibilities, hCG binding assays were performed with nonglycosylated receptors obtained after tunicamycin treatment of cells expressing the wild-type rLHR. Even though these wild-type receptors were confirmed to be devoid of all N-linked carbohydrates by Western blots, they were found to bind hCG with a normal high affinity. In addition, tunicamycin-derived, nonglycosylated LHRs were present at the cell surface and exhibited a phenotype consistent with mature receptors due to their capability to mediate hCG-stimulated cAMP production as well as bind oLH with high affinity. These results indicate that the loss of high affinity hormone binding by rLHR(N77,152,173,269,277,291Q) and rLHR(N77,152,269,277,291Q;T175A) is simply due to the collective amino acid substitutions rather than to the absence of carbohydrates. Therefore, N-linked carbohydrates are not absolutely required for the proper folding of the rLHR into a mature receptor capable of binding hormone and signaling. These results are in marked contrast to the follitropin receptor (FSHR), a very similar receptor which has been shown to strictly require N-linked carbohydrates for folding of the nascent protein.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The three glycoprotein hormone receptors comprising the TSH receptor (TSHR), the FSHR and the LHR share several structural similarities. All three receptors are glycoproteins containing a large extracellular N-terminal domain with several consensus sequences for N-linked glycosylation (1). Numerous studies have shown these extracellular domains to be sufficient for high affinity binding of their respective hormones (2, 3, 4, 5, 6, 7, 8, 9, 10, 11). The N-terminal domains are followed by a seven-transmembrane helical motif ending with an intracellular C-terminus. All three glycoprotein hormone receptors have been shown to transduce hormone binding via coupling to the heterotrimeric Gs and Gq or other G proteins that activate phosholipase C (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). Because the FSHR and LHR have fundamental reproductive roles in both sexes, there has been a steadfast interest on the mechanisms by which these receptors bind hormone and transduce this binding to activate the appropriate effectors.

The functional significance of N-linked glycosylation with respect to the FSHR and LHR has been addressed in multiple studies. It has recently been demonstrated that removal of N-linked carbohydrates from the mature, wild-type rFSHR does not affect FSH binding (9, 23). However, our studies have also shown that prevention of rFSHR glycosylation by mutagenesis or tunicamycin treatment resulted a nonglycosylated rFSHR devoid of FSH binding activity (9). It was further shown that hormone binding could only be maintained if the nascent rFSHR was glycosylated on at least one of its two glycosylated sites. We concluded from these studies that instead of being directly involved in hormone binding, N-linked rFSHR glycosylation is required to ensure the correct folding of the nascent receptor. Once this conformation has been attained, the N-linked rFSHR carbohydrates are no longer required with respect to hormone binding.

Studies addressing the role of N-linked LHR carbohydrates have, however, provided conflicting views of their function. For example, some reports have suggested that N-linked LHR carbohydrates are not required for hormone binding (2, 24, 25, 26, 27), while others have suggested a requirement of these receptor carbohydrates with respect to hormone binding and/or correct folding of the nascent LHR (28, 29). The present report is, therefore, derived from an attempt to explain the conflicting conclusions that have surrounded the analyses of N-linked LHR carbohydrates, while at the same time providing a more complete understanding of their functional significance. Our data demonstrate that unlike the structurally similar rFSHR, the rLHR is heavily glycosylated and yet does not seem to rely on its carbohydrates to the same degree as the rFSHR.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Analysis of the rLHR cDNA previously identified six consensus sequences for N-linked glycosylation within the extracellular, N-terminal domain of the receptor located at amino acids 77, 152, 173, 269, 277, and 291 (30). To identify which sites are actually glycosylated, we created two sets of rLHR glycosylation mutants (series I and II) listed in Table 1Go. The mutant receptors comprising series I allowed the individual retention of each glycosylation consensus sequence while the other five potential sites were disrupted by mutagenesis. For example, rLHR(N152,269,277,291Q;T175A) has the consensus sequence at Asn-77 maintained, while the other five consensus sequences at Asn’s 152, 173, 269, 277, and 291 have been disrupted by substitution of the essential Asn or Ser/Thr residue at each site. To determine which of the series I receptor mutants are glycosylated, detergent-solubilized extracts prepared from 293 cells expressing these receptor mutants were first incubated in the presence or absence of PNGaseF (an enzyme that removes N-linked carbohydrates from glycoproteins, see Ref.31), followed by resolution on SDS-PAGE gels under reducing conditions. After transfer to PVDF membranes, the receptors were detected by probing with a previously described anti-rLHR antibody (designated anti-LHRO2; see Refs. 32 and 33). As shown in Fig. 1Go, the wild type rLHR (lanes 2 and 11) migrates as two distinct broad bands of 89 and 68 kDa which have been shown to correspond to the mature and immature forms of the receptor, respectively (4, 34). Lanes 3 and 12 demonstrate that after PNGaseF treatment, the wild type receptor migrates as a single band of 59 kDa. This PGNaseF treatment (15 h at 37 C with 32 U/ml PNGaseF) or one utilizing even higher concentrations of enzyme does not seem to remove all N-linked carbohydrates from the wild type rLHR since the molecular mass of the PNGaseF-treated wild type rLHR is larger than the nonglycosylated mutant rLHR(N77,152,173,269,277,291Q) (compare lanes 3 and 4). Nonetheless, although the PGNaseF treatment was unable to remove all the N-linked carbohydrates from the wild type rLHR, as shown below, it could efficiently remove N-linked carbohydrates from the mutant rLHRs containing fewer potential sites of N-linked glycosylation. Each of the series I rLHR mutants (each containing only one potential site for N-linked glycosylation) were tested for their sensitivity to PNGaseF. Looking first at rLHR(N152,269,277,291Q;T175A), which can only be glycosylated at the potential Asn-77 site, this mutant migrates as a single band with a mass of 53 kDa before PNGaseF treatment (lane 6). After PNGaseF treatment, its mass is reduced to 51 kDa (lane 7). These data demonstrate that Asn-77 of the rLHR must contain N-linked carbohydrate. A similar examination of the other mutant receptors in this series demonstrated that they all exhibit a reduction in their molecular mass upon PNGaseF treatment. As such, the results from the series I glycosylation mutants suggest that all six of the rLHR consensus sites normally have an N-linked carbohydrate attached (see Fig. 1Go, Gels A and B).


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Table 1. Rat LHR Mutants Used to Determine Sites of N-Linked Glycosylation

 


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Figure 1. Identification of rLHR N-linked Glycosylation Sites Using rLHR Series I Mutants

Nonclonal, stably transfected 293 cells expressing the empty pcDNAI/neo vector (lanes 1 and 10), the wild type rLHR (lanes 2, 3 and 11, 12), rLHR(N77,152,173,269,277,291Q) (lanes 4, 5 and 13, 14), rLHR(N152,269,277,291Q;T175A) (lanes 6 and 7), rLHR(N77,269,277,291Q;T175A) (lanes 8 and 9), rLHR(N77,152,269,277,291Q) (lanes 15 and 16), rLHR(N77,152,277,291Q;T175A) (lanes 17 and 18), rLHR(N77,152,269,291Q;T175A) (lanes 19 and 20), or rLHR(N77,152,269,277Q;T175A) (lanes 21 and 22) were detergent solubilized. Equal amounts of protein were incubated 15 h at 37 C in the presence or absence of 32 U/ml PNGaseF (noted as ± PNGaseF, respectively). After this incubation, the samples were resolved under reducing conditions on two 8% SDS-PAGE gels, transferred to PVDF membranes, and probed with anti-rLHRO2 antibody (32, 33). The N-linked glycosylation consensus sequence that was maintained for a particular mutant receptor is noted above each receptor.

 
To further substantiate the above conclusion, we also examined the rLHR glycosylation pattern utilizing the series II glycosylation receptor mutants listed in Table 1Go. Unlike series I, series II receptor mutants were constructed such that only one or two glycosylation consensus sequences were disrupted within each mutant rLHR. For example, the mutant rLHR(N77Q) would prevent glycosylation at Asn-77 while not affecting glycosylation at any of the other five sites. The series II mutant receptors were expressed in 293 cells and extracted by detergent solubilization as above. However, before resolution by SDS-PAGE and subsequent Western blotting, these samples were digested with N-chlorosuccinimide (NCS). This reagent cleaves proteins after tryptophan residues (35), which theoretically should cause the rLHR to be digested into six fragments. The largest of these fragments, consisting of amino acids 1–307, contains all six potential sites for N-linked glycosylation and could be detected using the anti-LHRO2 antibody. The NCS digestion increased the ratio of carbohydrate relative to protein in the total mass of this rLHR fragment, thereby providing a sharper resolution of the potential differences in molecular masses between the mutants and the wild type rLHR. After NCS digestion, the wild type receptor was found to migrate as a broad band of 50 kDa (lanes 1 and 12 of Fig. 2Go). With the exception of rLHR(N173Q), and rLHR(N291Q) (lanes 5 and 10), all the mutants were expressed at levels comparable to cells expressing with wild type rLHR. Although rLHR(N291Q) became more visible on longer exposures of the film, rLHR(N173Q) could never be detected, suggesting that it was not stably expressed. It is interesting to note that the deglycosylated rLHR mutant, in which all glycosylation consensus site asparagines were substituted with alanines, was stably expressed (lanes 4, 5, 13, and 14). We can only speculate that one or more of the other substitutions in the multiple mutant somehow compensates for the conformational changes made by the N173Q substitution, which now allow the protein to escape rapid degradation. As shown below, the lack of expression of rLHR(N173Q), however, did not preclude our ability to determine the potential addition of carbohydrate to Asn-173. It has previously been demonstrated that one can disrupt the consensus sequence for N-linked glycosylation by either deleting or substituting the Asn residue to which the carbohydrate is attached, or alternatively, by deleting or substituting the Ser/Thr residue that is essential for hydrogen bonding during the transfer of the precursor oligosaccharide (36, 37, 38, 39). By creating rLHR(T175A), we could then examine whether the lack of expression of rLHR(N173Q) was due to the potential lack of carbohydrate at Asn-173 or due to mutation of Asn-173. As shown in lane 6 of Fig. 2Go, rLHR(T175A) was stably expressed, confirming that the lack of expression of rLHR(N173Q) was due to substitution of Asn-173 for Gln and not to the potential absence of carbohydrate at that site. As shown in Fig. 2Go, all of the series II mutants containing only a single disrupted consensus site exhibited a molecular mass less than the wild type receptor. Consistent with this result, those mutants with two disrupted consensus sites ((rLHR(N77,152Q) and rLHR(N269,277Q)) exhibited an even greater decrease in molecular mass than the rLHR mutants with a single disrupted site. Therefore, as with the series I rLHR glycosylation mutants, the results from the series II rLHR glycosylation mutants clearly demonstrate the N-linked glycosylation of all six consensus sites present on the rLHR.



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Figure 2. Identification of rLHR N-linked Glycosylation Sites Using rLHR Series II Mutants

293 cells transiently expressing the empty pcDNAI/neo vector (lane 11), the wild type rLHR (lanes 1 and 12), rLHR(N77Q) (lane 2), rLHR(N152Q) (lane 3), rLHR(N77,152Q) (lane 4), rLHR(N173Q) (lane 5), rLHR(T175A) (lane 6), rLHR(N269Q) (lane 7), rLHR(N277Q) (lane 8), rLHR(N269,277Q) (lane 9), or rLHR(N291Q) (lane 10) were detergent solubilized. Equal amounts of protein were incubated 2 h at 25 C in the presence of 50 mM N-chlorosuccinimide. After this incubation, the samples were resolved under reducing conditions on a 10% acrylamide SDS-PAGE gel, transferred to PVDF membranes, and probed with anti-rLHRO2 antibody.

 
The possible requirement of N-linked rLHR carbohydrates with respect to correct folding of the nascent receptor and acquisition of hormone binding has been investigated previously by ourselves and others through the use of receptor mutants in which consensus sites for N-linked glycosylation were either individually or collectively disrupted (25, 28, 40, 41). We reported earlier that normal hCG binding by a nonglycosylated receptor mutant could be maintained provided the asparagine residue at 173 was not substituted with a glutamine (41). These results suggested that N-linked rLHR carbohydrates are not required for proper folding and subsequent hCG binding. However, we have since discovered the mutant receptor thought to be rLHR(N77,152,269,277,291Q;T175A) actually contained two intact glycosylation con-sensus sequences at Asn-77 and Asn-152. As such, this mutant was really rLHR(N269, 277,291Q;T175A). Therefore, a correct rLHR(N77,152,269,277,291Q;T175A) was created as well as a nonglycosylated rLHR(N77,152,175,269,277,291Q), and both were determined to be nonglycosylated by Western blotting (data not shown). Hormone-binding assays were performed with detergent-solubilized extracts of cells expressing either the two nonglycosylated receptor mutants or rLHR(N269,277,291Q;T175A). The results summarized in Table 2Go show that rLHR(N269,277,291Q;T175A) bound hCG with a slightly reduced affinity compared with the wild type rLHR. In marked contrast, however, there was no detectable hCG-binding activity observed in detergent extracts of cells expressing either nonglycosylated rLHR mutant. These results correct our previously reported conclusions and demonstrate that the nonglycosylated mutant receptor rLHR-(N77,152,269,277,291Q;T175A) does not bind hCG.


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Table 2. Lack of hCG Binding Activity by Nonglycosylated rLHR Mutants

 
The lack of hCG binding observed with the nonglycosylated mutants rLHR(N77,152,269,277,291Q;T175A) and rLHR(N77,152,173,269,277,291Q) markedly contrasts with the hCG binding activity seen with rLHR-(N269,277,291Q;T175A), in which only the glycosylation consensus sequences at Asn’s 77 and 152 were maintained. These data suggest that carbohydrates at Asn’s 77 and/or 152 might be essential for maintaining hCG binding activity. To test this hypothesis, rLHR(N77,152Q) was created and stably expressed in 293 cells. The N-linked carbohydrate content of this mutant was again analyzed by Western blots. Lane 4 of Fig. 3Go demonstrates that rLHR(N77,152Q) migrates as a single band with a molecular mass between that of the immature wild type rLHR and the PNGaseF-treated, deglycosylated wild type receptor (lanes 2 and 3, respectively). The decreased mass of rLHR-(N77,152Q) was expected due to the absence of N-linked carbohydrates at Asn’s 77 and 152. Upon PNGaseF treatment, a further reduction in the size of rLHR(N77,152Q) occurred (lane 5) due to removal of carbohydrates present at the remaining four glycosylation sites. To determine the binding affinity of rLHR(N77,152Q) for hCG, competition binding assays were performed with detergent-solubilized extracts of cells expressing this mutant. As seen in Table 2Go, rLHR(N77,152Q) bound hCG with a slightly reduced affinity relative to the wild type receptor. This small decrease in affinity cannot explain the more drastic loss in hCG binding affinity observed with either rLHR(N77,152,173,269,277,291Q) or rLHR(N77,152,269,277,291Q;T175A). Therefore, the maintenance of the glycosylation consensus sites at Asn’s 77 and 152 does not seem to be required for high-affinity hCG hormone binding. These data also suggest that the inability to bind hormone by either of the nonglycosylated rLHR mutants is more likely due to the disruption of these rLHR mutants’ peptide backbone rather than the prevention of glycosylation at Asn’s 77 and 152.



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Figure 3. Analysis of rLHR(N77,152Q) N-linked Carbohydrate Content

Detergent-solubilized extracts of 293 cells stably expressing the empty pcDNAI/neo vector (lane 1), wild type rLHR (lanes 2 and 3), or rLHR(N77,152Q) (lanes 4 and 5) were incubated for 15 h at 37 C in the presence or absence of 32 U/ml PNGaseF (denoted as ± PNGaseF treatment, respectively). The samples, adjusted to contain equal amounts of protein, were then resolved under reducing conditions on a 7% SDS-PAGE gel, transferred to a PVDF membrane, and probed with an anti-rLHR antibody.

 
There are two possible explanations that would account for the lack of hCG-binding activity seen with the nonglycosylated receptor mutants (rLHR-(N77,152,173,269,277,291Q) and rLHR(N77,152,269,277,291Q;T175A). One possibility is that, as with the structurally similar rFSHR (9), N-linked rLHR carbohydrates may be necessary for the nascent receptor to attain a conformation enabling it to bind hormone with high affinity. Alternatively, the loss of hCG-binding activity by rLHR(N77,152,173,269,277,291Q) and rLHR-(N77,152,269,277,291Q;T175A) may be due to the multiple amino acid substitutions introduced into the polypeptide rather than to the lack of N-linked carbohydrates. To differentiate between the above hypotheses, we created a nonglycosylated receptor population by tunicamycin treatment of cells expressing the wild type rLHR and analyzed their ability to bind hCG. rLHR(wt-1) cells were cultured for 3 days in the absence or presence of 10 µg/ml of tunicamycin and then detergent solubilized. Results documenting the absence of carbohydrates on the rLHR isolated from tunicamycin-treated cells are shown in Fig. 4Go. In this experiment we tested the susceptibility of rLHR solubilized from tunicamycin-treated rLHR(wt-1) cells and untreated rLHR(wt-1) cells to PNGaseF and to endoglycosidase H (endoH), a glycosidase that specifically removes high mannose-containing carbohydrates from proteins (36). We chose to utilize two distinct endoglycosidases in this experiment to determine by two independent means whether or not carbohydrates were present on the tunicamycin-derived rLHR. Looking at the results from untreated cells first, as has been shown before (4, 34), endoH treatment causes a decrease in molecular mass of the immature form of the rLHR only, and PGNaseF treatment results in a decrease in the molecular masses of both the immature and mature forms of the receptor to a single 58 kDa species. As shown in Fig. 4Go, the rLHR from tunicamycin-treated cells is resistant to both glycosidases, suggesting that this receptor species is non-glycosylated. The molecular mass of the rLHR from tunicamycin-treated cells (51 kDa), however, is somewhat larger than that of the nonglycosylated mutant rLHR(N77,152,173,269,277,291Q) (49 kDa) and is comparable to that of rLHR(N77,152,173,277,291Q), which contains a single N-linked carbohydrate at Asn-269. One possible explanation for this discrepancy is that the rLHR from tunicamycin-treated cells is indeed still partially glycosylated, but the PGNaseF and endoH treatments are not removing the remaining carbohydrates. We consider this possibility unlikely, however, due to the observation that the same glycosidase treat-ments can clearly show a decrease in mass in rLHR(N77,152,173,277,291Q), a mutant containing only a single N-linked carbohydrate at Asn-269 and which remains trapped intracellularly in an immature form (Figs. 1Go and 5Go). Furthermore, concentrations of tunicamycin as high as 50 µg/ml did not cause any further reduction in mass (data not shown). Therefore, we conclude that the more likely reason why the rLHR from tunicamycin-treated cells migrates with a slightly higher mass on SDS gels, as compared with the nonglycosylated mutant rLHR(N77,152,173,269,277,291Q), is that the polypeptide backbone of the rLHR from tunicamycin-treated cells is unaltered whereas rLHR-(N77,152,173,269,277,291Q) contain six amino acid substitutions.



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Figure 4. Tunicamycin treatment prevents N-linked glycosylation of the rLHR

Clonal rLHR(wt-1) cells stably expressing the wild type rLHR were cultured for 3 days in the absence (wt) or presence (wt + tunic) of 10 µg/ml tunicamycin. Nonclonal 293 cells stably expressing the empty cDNAI/neo vector (neo), the nonglycosylated mutant rLHR(N77,152,175,269,277,291Q) (all CHO sites mutated), or the mutant rLHR(N77,152,277,291Q;T175 A) (all CHO sites mutated except N269) were used as controls. Detergent-solubilized extracts of cells were then treated with or without 300 mU/ml endoH or 32 U/ml PNGaseF for 15 h at 37C.). The samples, adjusted to contain equal amounts of protein, were resolved under reducing conditions on a 7% SDS-PAGE gel, transferred to a PVDF membrane, and probed with an anti-rLHR antibody.

 


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Figure 5. Western Blots of Tunicamycin-Treated Cells Expressing the Full-Length or Truncated rLHR when Comparing Equal Amounts of Binding Activity

The cell lines rLHR(wt-1) and rLHR (t338) (gels A and B, respectively) were cultured for 3 days in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of 10 µg/ml tunicamycin. The cells were then detergent solubilized, and hCG competition binding assays were performed to calculate the amount of hCG- binding activity per milligram of total protein. Equal quantities of hCG-binding activity for each sample were resolved on a 7% SDS-PAGE gel under reducing conditions after incubation at 37 C for 15 h in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 32 U/ml PNGaseF. The samples were transferred to a PVDF membrane and probed with an anti-rLHR antibody.

 
After determining the tunicamycin treatment conditions necessary for preventing N-linked glycosylation of the wild type rLHR, we then assayed the ability of these tunicamycin-derived, nonglycosylated receptors to bind hCG. Due to the possible effect of tunicamycin treatment on cell surface localization of the rLHR, hCG-binding assays were performed with detergent-solubilized cell extracts. As shown in Table 3Go, the tunicamycin-derived, nonglycosylated rLHR bound hCG with an affinity similar to that of the control rLHR solubilized from nontreated cells. To ensure that the hCG-binding activity present in the tunicamycin-treated rLHR(wt-1) cells was not due to a small population of partially glycosylated rLHR contaminating the nonglycosylated rLHR, the experiment shown in the top panel of Fig. 5Go was performed. As before, rLHRs were solubilized from control and tunicamycin-treated rLHR(wt-1) cells. The samples were incubated with or without PGNaseF, run on SDS gels, and analyzed by Western blotting. However, in this experiment the samples were adjusted to contain equal amounts of hCG-binding activity before the PGNaseF incubations. The rationale for doing so was as follows. Assume the 51 kDa rLHR from tunicamycin-treated cells was contaminated with an imperceptibly small amount of larger rLHR containing carbohydrate, and it was this small amount of higher molecular mass receptor that contained binding activity. If an equal amount of binding activity as compared with rLHR from untreated cells was then loaded onto the gel, one would expect to see this larger sized band, and it should be of comparable intensity as the PGNaseF-treated rLHR from untreated cells. The 51-kDa nonglycosylated band would, in turn, be expected to be of much greater intensity than the PGNaseF-treated rLHR from untreated cells. However, as shown in Fig. 5Go, equal band intensities were observed for both the PGNaseF-treated rLHR from untreated cells and the tunicamycin-derived nonglycosylated receptor samples (compare lanes 3 and 4 with lane 2). These results argue against the existence of a small amount of glycosylated rLHR in the sample derived from tunicamycin-treated cells and further show that the hCG-binding activity of the rLHR from tunicamycin-treated cells is indeed due to the nonglycosylated 51 kDa receptor species. One can conclude, therefore, that N-linked glycosylation of the rLHR does not seem to be absolutely required for the nascent receptor to fold into a conformation capable of binding hCG with high affinity. As such, these data further suggest that the lack of hCG binding by the nonglycosylated receptor mutants (rLHR-(N77,152,173,269,277,291Q) and rLHR(N77,152,269,277,291Q;T175A) is the result of the multiple amino acid substi-tutions rather than the absence of N-linked carbohy-drates on these mutants.


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Table 3. High-Affinity hCG Binding to Detergent-Solubilized Extracts of Tunicamycin-Treated Cells Expressing Either Nonglycosylated Wild Type rLHR or Nonglycosylated rLHR Extracellular Domain

 
While this study was in progress, Zhang et al. (28) reported that tunicamycin treatment of insect cells expressing the rLHR extracellular domain (referred to as form B rLHR and encompassing amino acids 1–294) prevented hormone binding as detected using ligand blots. We, therefore, examined the ability of a similarly truncated rLHR to bind hCG when glycosylation was prevented by tunicamycin treatment. rLHR-t338(c1), a previously described, clonal 293 cell line stably expressing the extracellular domain (encompassing amino acids 1–338) of the wild type rLHR (4), was cultured in the presence of 10 µg/ml tunicamycin as described above for the full-length rLHR. As shown in Fig. 5Go, these tunicamycin conditions were sufficient to prevent glycosylation of rLHR(t338) as evident from the lack of any change in molecular mass upon PNGaseF treatment before SDS-PAGE resolution (compare lanes 3 and 4). Hormone-binding assays were then performed with detergent-solubilized extracts of these cells. As shown in Table 3Go, the non-glycosylated rLHR(t338) from tunicamycin-treated cells exhibited a 5-fold decrease in hCG-binding affinity compared with the glycosylated control rLHR(t338), but the binding of hCG was clearly detectable. Therefore, although the extracellular domain when expressed alone does seem to require N-linked carbohydrates to achieve the mature conformation required for normal high-affinity binding, in the absence of carbohydrates it can fold sufficiently well to bind hCG at a somewhat reduced affinity. As such, our results do not agree with those previously reported by Zhang et al. (28) (see Discussion).

The results presented thus far demonstrate that although all six sites on the rLHR contain N-linked carbohydrates, they are not absolutely required for the folding of the receptor into a conformation capable of binding hCG with high affinity. It is possible, however, that cell surface localization and/or hCG-mediated signal transduction of the nonglycosylated rLHR could be altered by the absence of N-linked carbohydrates. To examine these possibilities, the following experiments were performed. First, the presence of tunicamycin-derived, nonglycosylated rLHR at the cell surface was analyzed by hCG competition-binding experiments using intact cells. Intact cells that had been pretreated with tunicamycin bound hCG with the same high affinity as control intact cells (data not shown). However, the tunicamycin treatment decreased the number of receptors on the cell surface of the rLHR(wt-1) cells from 450,000 (42) to 21,100 (Table 4Go). This reduction in cell surface receptors was fully accountable by the reduction in total rLHR protein after tunicamycin treatment as determined by Western blotting and is presumably due to a general decrease in protein synthesis caused by the tunicamycin treatment (43). To analyze the ability of the tunicamycin-treated cells to respond to hCG with increased cAMP production, we matched them with a control, non-tunicamycin-treated wild type rLHR cell line (referred to as rLHR(wt-16)), which expresses a comparable number of cell surface receptors. The rLHR(wt-16) cells are a clonal line of 293 cells stably transfected with the same wild type rLHR cDNA used to create the rLHR(wt-1) cells. The only difference between the two cell lines is the number of cell surface rLHRs expressed. Hence, the rLHR should not be glycosylated any differently in the rLHR(wt-16) cells. As evident from the hCG dose-response curves shown in Fig. 6Go and the EC50 and Rmax values listed in Table 4Go, there was little or no decrease in the ability of the tunicamycin-derived, nonglycosylated rLHR to stimulate cAMP production in response to hCG. These results indicate that N-linked carbohydrates are not absolutely essential for the proper folding and membrane insertion of the rLHR, for its ability to bind hCG with high affinity, or for its ability to activate Gs upon the binding of hCG.


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Table 4. Human CG-Stimulated cAMP Production in Tunicamycin-Treated Cells Expressing Nonglycosylated rLHR

 


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Figure 6. hCG-Stimulated cAMP Dose-Response Curve

Tunicamycin-treated rLHR(wt-1) cells, expressing 21,100 cell surface receptors per cell, and a control rLHR(wt-16) cell line, stably expressing 13,600 cell surface wild type receptors per cell, were incubated with increasing concentrations of hCG to determine hCG-stimulated cAMP production as described in Materials and Methods. The data were calculated using the Delta Graph computer program. One representative experiment is shown.

 
Recent studies from our laboratory have shown that maturation of the rLHR involved not only changes in the N-linked carbohydrates from an endoH-sensitive form to an endoH-insensitive form, but also changes in the conformation of the receptor that affect its ability to bind oLH (44). Whereas both the immature and mature forms of the rLHR bind hCG with high affinity, oLH binds the immature form of the receptor with a reduced affinity (50 nM) as compared with the mature cell surface form of the receptor (5 nM). These results demonstrate that the conformation of the immature receptor is distinct from that of the mature receptor and suggest further alterations in folding must ensue between the time of the receptor’s exit from the endoplasmic reticulum and its insertion into the plasma membrane. Since carbohydrates are thought to be the recognition sites for a chaperone protein involved in the folding of many glycoproteins, it was possible that, in the absence of carbohydrates, the tunicamycin-derived rLHR might "escape" the cell’s normal quality control system and be inserted into the plasma membrane in the immature conformation. To examine this possibility, the binding affinity for oLH was determined on intact rLHR(wt-1) cells that had either been untreated or treated with tunicamycin to generate cell surface nonglycosylated rLHRs. It was predicted that tunicamycin treatment of rLHR(wt-1) cells would result in the cell surface expression of nonglycosylated receptors with either a single high binding affinity for oLH (indicative of only mature receptors at the cell surface), a single low binding affinity for oLH (indicative of only immature receptors at the cell surface), or a composite of two binding affinities for oLH (indicative of the presence of both mature and immature receptors at the cell surface). Results of these experiments showed that the tunicamycin-derived, cell surface, nonglycosylated rLHRs bound oLH with a single high affinity (Kd = 3.31 ± 0.340), comparable to that of the glycosylated, mature receptor (Kd = 1.54 ± 0.035). These results demonstrate that the cell surface population of nonglycosylated receptors consists only of mature, fully folded receptors. We conclude from these results that N-linked rLHR carbohydrates are not required for ensuring the release of only mature receptors from the endoplasmic reticulum and their subsequent localization at the cell surface.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Using two distinct approaches, we have shown herein that all six potential N-linked rLHR glycosylation sites normally contain carbohydrates. In spite of the large extent of glycosylation of the rLHR, however, our studies show that N-linked glycosylation is not absolutely required for the correct folding and cell surface expression of the rLHR. Thus, tunicamycin treatment of wild type rLHR-expressing cells produced nonglycosylated receptors that were demonstrated to be present at the cell surface in a form capable of binding hCG with a high affinity and able to transduce hCG-mediated cAMP stimulation. Finally, using the ability of oLH to bind the immature and mature forms of the rLHR with distinct affinities, we demonstrated that the nonglycosylated rLHR at the cell surface of tunicamycin-treated cells did not arise from mislocalization of the nonglycosylated immature receptor but was instead a properly folded, mature form of the receptor. Therefore, the N-linked carbohydrates of the rLHR are not an indispensible requirement for the proper folding of the rLH. This conclusion does not a priori exclude a role for N-linked carbohydrates in the folding of the rLHR. They may, for example, aid in the kinetics of folding. Because we are measuring the steady state levels of receptor, any effects of the N-linked carbohydrates on the kinetics of this process would go unnoticed. Further experiments will be required to address this issue. Nonetheless, the results with the rLHR stand in marked contrast to the rFSHR, where the N-linked carbohydrates are essential for the steady state acquisition of a receptor pool capable of binding hormone with high affinity (9).

As mentioned above, the results on the lack of a critical need for N-linked carbohydrates for the proper folding of the rLHR stand in marked contrast to the closely related rFSHR (1, 13). Previous studies have shown that two of the three potential sites for N-linked glycosylation on the rFSHR are normally glycosylated. To attain a functional rFSHR with respect to hormone binding, at least one of the two glycosylated consensus sites must be glycosylated. This reliance on N-linked glycosylation was demonstrated to be at the level of folding since PNGaseF deglycosylated mature rFSHR-bound FSH with a normal high affinity while nonglycosylated rFSHR mutants as well as the tunicamycin-derived nonglycosylated wild type rFSHR were devoid of hormone-binding activity (9). This conclusion is in contrast to the present findings concerning the rLHR, in which carbohydrates are not essential for receptor folding. Therefore, despite the high degree of deduced amino acid identity between the FSHR and the LHR, our present results imply the existence of structural differences between these receptors that result in the observed dissimilar reliance on N-linked glycosylation. The dichotomous importance of N-linked carbohydrates in protein folding, as seen between the FSHR and LHR, has been reported with other glycoproteins. In many cases, only one to five amino acid differences have been shown to determine the need of a particular glycoprotein for its carbohydrates in proper folding (45, 46). Whether a similar scenario is also true in the case of the FSHR and LHR will require further study.

Recent evidence from our laboratory has provided further evidence to support the existence of structural variances between the rFSHR and the rLHR (47). An analogous series of rFSHR and rLHR mutants were constructed and then analyzed for their ability to correctly fold. The mutations were introduced at positions on the receptors (transmembrane loops and intracellular C-terminal tail) that are not directly involved in hormone binding so that hormone binding could be used as a tool to determine whether a particular mutant had been correctly folded. Both rFSHR and rLHR mutants were retained in the endoplasmic reticulum as demonstrated by their sensitivity to endoH. However, while the intracellularly retained rLHR mutants exhibited normal high-affinity hormone binding, the analogous intracellularly retained rFSHR mutants were unable to bind hormone. Therefore, as the rLHR exits the endoplasmic reticulum, it has already acquired a conformation allowing it to bind hCG (although it is still in an immature conformation as assessed by its decreased affinity for oLH). In contrast, as the rFSHR exits the endoplasmic reticulum, it has not yet folded into a conformation that can bind FSH even with decreased affinity. These results suggest a temporal difference in the folding of these two closely related receptors. Taken together with the absolute reliance of the rFSHR on its N-linked carbohydrates to fold properly (9) and the lack of reliance of the rLHR on its N-linked carbohydrates to fold properly, it appears as if the rLHR can fold more readily than the structurally related rFSHR.

It should be pointed out that our results on the tunicamycin-derived rLHR are in apparent disagreement with those reported by Dufau and co-workers (28). This group examined the ability of the wild type rLHR extracellular domain expressed in insect cells to fold correctly when glycosylation was prevented by culturing the cells with tunicamycin. The resulting nonglycosylated receptors were found to be incapable of hormone binding when assayed by ligand blots. It was, therefore, concluded that N-linked glycosylation is required to allow the nascent receptor to attain the conformation necessary for hormone binding (28). One possible explanation for the discrepancy between this group’s conclusions and ours was their use of a truncated form of the rLHR for their experiments. We attempted to control for this by examining the effects of tunicamycin treatment on the folding of the rLHR’s extracellular domain expressed in rLHR-t338(c1) cells. After culturing rLHR-t338(c1) cells with tunicamycin, this truncated receptor was demonstrated to be devoid of carbohydrates by Western blots. Yet, as with the full-length receptor, detergent-solubilized extracts from tunicamycin-treated rLHR-t338(c1) cells bound hCG with a high affinity. It is possible that the additional C-terminal 44 amino acids of rLHR(t338) relative to the rLHR B form used by Dufau and co-workers allows the correct folding of the nonglycosylated rLHR(t338). A more likely explanation, however, is the difference in assay techniques. Dufau and co-workers examined hormone binding by ligand blots, which requires the denatured receptor resolved by SDS-PAGE to correctly renature before it can bind hormone. This renaturation step may require the presence of carbohydrates for the rLHR to correctly refold and, therefore, prevent hormone binding by nonglycosylated receptors. Support for this hypothesis can be found in the same report by Dufau and co-workers (28). By comparing the effects of endoH and PNGaseF treatment of the rLHR B form, they concluded the proximal N-acetylglucosamine to be essential for renaturing the receptor after resolution by SDS-PAGE. This conclusion would, therefore, explain the inability of the tunicamycin-derived, nonglycosylated rLHR B form to bind hCG when assayed by ligand blots. Another earlier report by Ji et al. (24) used tunicamycin to test the functional role of the N-linked carbohydrates on the LHR. Their results showed that tunicamycin treatment of wild type rLHR-expressing cells resulted in the absence of cell surface hormone binding. These results could have been due to a loss of binding activity of nonglycosylated cell surface receptors or to the absence of cell surface nonglycosylated receptors. As shown herein, culturing cells with tunicamycin not only prevents glycosylation but also results in a reduction in receptor expression (coinciding with a decrease in total protein expression). Because the studies by Ji and co-workers were performed utilizing cells that express much lower levels of rLHR than the rLHR(wt-1) cells, it is likely that the tunicamycin treatment decreased the cell surface expression of the rLHR to undetectable levels.

Our results demonstrating that all six sites of the rLHR are glycosylated are also in apparent disagreement with the report by Dufau and co-workers (28). Specifically, they reported the lack of any N-linked glycosylation at the Asn-77 consensus site. As noted above, Dufau and co-workers used insect cells to express the rLHR. It is possible that the insect cells do not utilize the same sites for N-linked glycosylation that mammalian cells do. It is also possible that the conclusion of an absence of carbohydrate at Asn-99 by Dufau and co-workers may have been due to a lack of detection rather than to a true absence of carbohydrate. As demonstrated in Fig. 1Go, the individual glycosylation of Asn-77 or any of the other five sites increases the rLHR molecular mass by at most 2 kDa. Therefore, preventing glycosylation at one site may not produce a readily visible difference in molecular mass. This effect may have been further exacerbated due to the use of insect cells to express the mutant receptors. Dufau and co-workers demonstrated in the same report that insect cells do not glycosylate the rLHR to the same extent as mammalian cells, which would decrease the contribution of the carbohydrates toward the overall molecular mass of the receptor. By utilizing mammalian cells to express rLHR glycosylation mutants in the context of the full-length receptor, we have conclusively demonstrated the glycosylation of Asn-77. This is most visible by analyzing the additive contribution in molecular mass of the carbohydrates at Asn-77 and Asn-152. When glycosylation at both sites is prevented, there is a greater decrease in molecular mass relative to the individual disruption of each of these sites (compare lane 4 with lanes 2 and 3 in Fig. 2Go).

Given that all six sites of the rLHR are normally glycosylated, it might be possible to create a nonglycosylated mutant of the rLHR by disrupting all six consensus sites for N-linked glycosylation. If the mutations did not perturb the peptide backbone, one would predict that this nonglycosylated mutant should fold normally and bind hormone with high affinity because tunicamycin-derived nonglycosylated rLHR can do so. However, two separate nonglycosy-lated mutants, rLHR(N77,152,269,277,291Q;T175A) and rLHR-(N77,152,173,269,277,291Q), were unable to bind any detectable hCG when assayed in detergent-solubilized cell extracts. The present study corrects a previous report from this laboratory (41) and further suggests that the lack of binding activity of these two mutants is the result of multiple amino acid substitutions. Given that one could disrupt a consensus site for N-linked glycosylation by substituting either the Ser/Thr or Asn within the consensus sequence with any one of the remaining 19 amino acids, there are a huge number of possible nonglycosylated rLHR mutants one could construct. It may be possible, though formidable, to determine whether appropriate combinations of amino acid substitutions exist that would prevent N-linked glycosylation while at the same time not affecting the correct folding and membrane insertion of a non-glycosylated, mutant rLHR.

In summary, we have shown that, unlike the closely related rFSHR, the rLHR does not absolutely require N-linked glycosylation for folding into a mature conformation. Experiments are underway to examine the folding of these glycoproteins in more detail and the role of chaperone proteins in these processes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Construction of rLHR Glycosylation Mutants
Creation of the B series of rLHR mutants, as well as the creation of rLHR(N77,152,173,269,277,291Q) and rLHR-(N77,152,269,277,291Q;T175A), has been described previously (41). rLHR(N152,269,277,291Q;T175A) and rLHR(N77,269, 277,291Q;T175A) were constructed by splicing the corresponding fragments from the wild type pDLHR9 (41) into the EcoRV/XmnI and the XmnI/NsiI sites, respectively, of rLHR-(N77,152,269,277,291Q;T175A). rLHR(N77,152,269,277,291Q), rLHR-(N77,152,277,291Q;T175A), rLHR(N77,152,269,291Q;T175A), and rLHR(N77,152, 269,277Q;T175A) were all created by the PCR (48, 49) using rLHR(N77,152,269,277,291Q;T175A) as a template. rLHR(77,152Q) was constructed by oligonucleotide-directed mutagenesis of the pDLHR9, which had been subcloned into pALTER-1 (Promega, Madison, WI). All of the resulting rLHR mutants were cloned into the pcDNAI/neo expression vector (Invitrogen, San Diego, CA), and their identities were confirmed by dideoxy sequencing the entire region amplified by the PCR (50).

Cell Lines
Human embryonic kidney 293 (HEK293) cells (ATCC CRL 1573) were maintained at 5% CO2 in a culture medium consisting of DMEM containing 50 µg/ml gentamicin, 10 mM HEPES, and 10% newborn calf serum. Transfections were performed using the CaPO4 method as described previously (41). For assays utilizing transient rLHR expression, cells were harvested 48 h posttransfection. Nonclonal cell lines stably expressing the wild type or mutant rLHR, or the empty pcDNA/neo expression vector, were prepared as previously described (9) by selection with 700 µg/ml G418 (GIBCO, Grand Island, NY). These nonclonal cell lines, as well as the clonal cell lines rLHR(wt-1) (42), rLHR(wt-16) (44), and rLHR-t338(c1) (4) were maintained in culture medium containing 700 µg/ml G418.

Tunicamycin Treatment of rLHR(wt-1) and rLHR-t338(c1) Cell Lines
The clonal cell lines rLHR(wt-1) and rLHR-t338(c1) were plated with culture medium on dishes precoated with 0.1% gelatin [0.1% gelatin (DIFCO Laboratories, Detroit MI] in CMF PBS (GIBCO) and allowed at least 48 h to equilibrate and attach to dishes. The culture medium was then replaced with fresh culture medium containing 10 µg/ml of tunicamycin (homologs A, B, C, and D). Fresh culture medium with 10 µg/ml tunicamycin was readded 24 and 48 h later. The cells were then detergent solubilized, as described herein, 72 h after the initial tunicamycin addition.

Western Blotting of Detergent-Solubilized Cell Extracts
Detergent-solubilized cell extracts were prepared using 0.5% Nonidet P-40 in buffer A (150 mM NaCl, 20 mM HEPES, pH 7.4) as described previously (9). After solubilization, the cell extracts were subjected to incubations performed in the presence or absence of PNGaseF or NCS as noted. PNGaseF treatment before gel loading was attained by incubation of the detergent-solubilized cell extracts for 15 h at 37 C with 32 U/ml PNGaseF. Detergent-solubilized cell extracts digested with NCS before gel loading were incubated for 2 h at 25 C with 50 mM NCS in 4 M urea. With the exception of the experiments shown in Fig. 5Go, all samples were adjusted to contain equal amounts of protein before loading on the SDS gel. In Fig. 5Go, the samples were adjusted to contain equal amounts of hCG-binding activity. A reducing, 6x SDS Lammeli sample buffer (12% wt/vol SDS, 40% vol/vol glycerol, 109 mM EDTA, 1.5 M Tris/HCl, 98 mg/ml dithiothreitol, and 6% vol/vol ß-mercaptoethanol) was added to the extracts to achieve a final 1x concentration, and the samples were incubated at room temperature for 1 h. The reduced and denatured cell extracts were resolved by SDS-PAGE gel, transferred to a PVDF membrane, and probed with the anti-rLHR antibody anti-LHRO2, which was raised against a peptide corresponding to amino acids 194–207 of the rLHR (32, 33).

[125I]hCG Binding to Detergent-Solubilized Cell Extracts
Saturation[125I]hCG bindings were performed by incubating detergent-solubilized cell extracts with 100 ng/ml [125I]hCG overnight at 4 C in the absence or presence of 50 IU/ml crude hCG. Equilibrium binding constants were derived by competition [125I]hCG binding assays in which cell extracts were incubated with a subsaturating concentration of [125I]hCG (2 ng/ml) overnight at 4 C in the presence of increasing concentrations of unlabeled hCG (0–4.3 µg/ml). For both assays, the receptor-hormone complexes were separated from the unbound hormone by vacuum filtration through polyethylenimine-treated filters (51). Equilibrium binding parameters were calculated from the competition binding assays using the LIGAND computer program (52).

Determination of Cell Surface Receptor Numbers and hCG-Mediated cAMP Production
The number of cell surface receptors was determined by performing competition hCG-binding assays with intact cells. The cells were first cooled on ice for 15 min and then washed two times with cold buffer B (Waymouth’s media without sodium bicarbonate and containing 0.1% BSA). A subsaturating concentration of [125I]hCG (2 ng/ml) and increasing concentrations of unlabeled hCG (0–4.3 µg/ml) were added to the cells and incubated overnight at 4 C. The assay was completed by scraping and washing the cells to remove unbound hormone, and the resulting data were calculated using the LIGAND computer program to determine the equilibrium-binding parameters (52). Human CG-mediated stimulation of cAMP production was also performed as described (53). Briefly, cells were washed with warm buffer B containing sodium bicarbonate, preincubated with 3-isobutyl-1-methylxanthine, and then incubated for 30 min at 37 C in the presence of increasing concentrations of hCG. The cells were collected and the total cAMP determined by RIA.

Ovine LH Binding Assays with Tunicamycin-Derived, Nonglycosylated rLHR
rLHR(wt-1) cells cultured in the presence or absence of tunicamycin as described above were analyzed for oLH binding. After cooling on ice for 15 min, the cells were washed two times with cold buffer B, and a subsaturating concentration (2 ng/ml) of [125I]hCG was added. Increasing concentrations of unlabeled hCG (0–4.3 µg/ml) or unlabeled oLH (0–105 µg/ml) were added to the cells and incubated overnight at 4 C. The assay was completed and the LIGAND computer program used to analyze whether the resulting data best fit a one- or two-site ligand-binding model as described previously (44).

Hormones and Supplies
Highly purified hCG (CR-127) was kindly provided by the National Hormone and Pituitary Agency of the NIDDK (NIH) and was iodinated as described previously (54). NP-40, crude hCG, and tunicamycin were obtained from Sigma (St. Louis, MO) PNGaseF was purchased from Boehringer Mannheim (Indianapolis, IN). PVDF membranes were obtained from Bio-Rad (Richmond, CA) and the enhanced chemiluminescence (ECL) detection kit was obtained from Amersham (Arlington Heights, IL). Tissue culture plasticware and reagents were from Corning (Corning, NY) and GIBCO (Grand Island, NY), respectively.


    ACKNOWLEDGMENTS
 
We thank Julie Jacquette for expert technical assistance and Dr. Mario Ascoli for critical reading of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Deborah L. Segaloff, Department of Physiology, The University of Iowa College of Medicine, Iowa City, Iowa 52242.

These studies were supported by NIH Grant HD-28970 (to D.L.S.). The services and facilities provided by the Diabetes and Endocrinology Research Center of the University of Iowa (supported by Grant DK-25295) are also gratefully acknowledged. D.L.S. is a recipient of NIH Research Career Award HD-00968.

Received for publication January 3, 1997. Revision received February 13, 1997. Accepted for publication February 18, 1997.


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