The Carboxyl-Terminal Region Is a Determinant for the Intracellular Behavior of the Chorionic Gonadotropin ß Subunit: Effects on the Processing of the Asn-Linked Oligosaccharides

Mesut Muyan1 and Irving Boime

Department of Molecular Biology & Pharmacology Washington University School of Medicine St. Louis, Missouri 63110


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The placental hormone human CG (hCG) consists of two noncovalently linked {alpha}- and ß-subunits similar to the other glycoprotein hormones LH, FSH, and TSH. These heterodimers share a common {alpha} subunit but differ in their structurally distinct ß subunits. The CGß subunit is distinguished among the ß subunits by the presence of a C-terminal extension with four serine-linked oligosaccharides (carboxyl terminal peptide or CTP). In previous studies we observed that deleting this sequence decreased assembly of the truncated CGß subunit (CGß114) with the {alpha}-subunit and increased the heterogeneity of the secreted forms of the uncombined subunit synthesized in transfected Chinese hamster ovary (CHO) cells. The latter result was attributed to alterations in the processing of the two N-linked oligosaccharides. To examine at what step this heterogeneity occurs, the CGß and CGß114 genes were transfected into wild-type and mutant CHO cell lines that are defective in the late steps of the N-linked carbohydrate-processing pathway. We show here that removal of the CTP alters the processing of the core mannosyl unit of the subunit to complex forms at both glycosylation sites and that the oligosaccharides contain polylactosamine. Although it has been presumed that there is little intramolecular interaction between the CTP and the proximal domains of the subunit, our data suggest that the CTP sequence participates in the folding of the newly synthesized subunit, which is manifest by the posttranslational changes observed here.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human CG (hCG), is a member of the glycoprotein hormone family, which includes the pituitary hormones LH, FSH, and TSH (1, 2, 3, 4, 5). They are heterodimers containing two nonidentical subunits, {alpha} and ß (1, 2, 3, 4, 5). Within an animal species, the amino acid sequences of the {alpha} subunits are identical, and, although the ß subunit determines the biological specificity of the hormones, there is significant amino acid sequence similarity among them (1, 2, 3, 4, 5). This is readily apparent for the LHß and CGß subunits as they share >80% sequence identity, which is presumably responsible for the similar biological activity of the corresponding dimers (1, 2, 3, 4, 5). CGß is distinguished among the ß subunits by the presence of a C-terminal extension with four O-linked oligosaccharides (5) designated CTP. Sequence analysis showed that a 1-bp deletion and 2-bp insertion in the CGß gene relative to the LHß coding sequence accounts for the extended open reading frame of CGß (5). Several studies have suggested that this extension plays no significant role in the overall conformation of the subunit (6, 7, 8), but it is associated with maintaining the prolonged half-life of hCG compared with the other hormones (9). However, our previous studies demonstrated that the intracellular behavior of the glycoprotein hormone ß subunits involves an interaction between regions in the amino and carboxyl ends of the subunit (10), suggesting the CTP contributes to the maturation of the newly synthesized subunit. This hypothesis was supported by the observation that truncation of CGß at residue 114 (CGß114) decreased its ability to form heterodimers and that CGß114 exhibited marked heterogeneity compared with the wild-type (WT) subunit when secreted from transfected Chinese hamster ovary cells (CHO) cells (11). The latter was attributed to modifications of the Asn-linked oligosaccharides reflecting a change in the structure of the subunit devoid of the CTP (11). This observation may, in part, explain the basis for the synthesis of hormone-specific oligosaccharides; since the amino acid sequence of the common {alpha} subunit is identical among the glycoprotein hormones, the specificity of N-linked oligosaccharide processing is linked to the ß subunit (2, 3, 4).

To characterize the posttranslational modification affected by the truncation, the CGß and the CGß114 genes were transfected into WT and mutant CHO cell lines, which are defective in the late steps of the N-linked carbohydrate processing pathway, and the products were probed with ß subunit-specific antiserum. Because defined glycoprotein intermediates accumulate, these mutant cell lines provide a model for elucidating at what step the changes in heterogeneity occurs in the truncated subunit. We show that removal of the carboxyl-terminal region of the CGß subunit alters terminal processing of the core glycosyl units of the subunit, and the major modification is the formation of polylactosamine-containing structures. The data also suggest that the carboxyl-terminal region is involved in the folding of the CGß subunit, reflected by changes in the processing of the N-linked carbohydrates.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Removal of the 31-amino acid carboxy-terminal region (carboxy-terminal peptide or CTP) from CGß resulted in a variant (CGß114) secreted from transfected CHO cells that was much more heterogeneous than the WT subunit. We attributed this to altered processing of the N-linked oligosaccharides (11). The initial steps in the processing of N-linked carbohydrates is the trimming of the high-mannose oligosaccharide unit to Man5GlcNAc2 (12, 13). Further modification of this intermediate is catalyzed by the Golgi enzyme N-acetylglucosaminyl transferase I, which is a pivotal step in the synthesis of fully processed N-linked oligosaccharides (12, 13). Because the heterogeneity of the glycoproteins primarily occurs at the processing of the core glycan unit to complex forms (2, 4, 14, 15), we examined whether the changes in the N-linked oligosaccharides of the secreted CGß114 occur at the Man5GlcNAc2 step. The WT CGß and the mutant CGß114 genes were transfected into the CHO mutant cell line 15B, which lacks the Golgi enzyme N-acetylglucosaminyl transferase I and, thus, synthesizes glycoproteins bearing only the Man5GlcNAc2 core (16). If the heterogeneity of CGß114 is related to altered processing beyond this point, CGß114 secreted from 15B cells should exhibit little or no heterogeneity compared with the form secreted from WT-CHO cells. CGß synthesized in WT-CHO cells displays two forms reflecting the presence of one (N1) or two (N2) N-linked oligosaccharides (Ref. 17 and Fig. 1Go, lane 1) and CGß114 exhibits the heterogeneity reported previously (lane 3). The migration of CGß114 differs from CGß because of the absence of the CTP and its associated O-linked oligosaccharides (11). Compared with WT-CHO cells (lane 3), CGß114 secreted from 15B cells is more homogeneous; i.e. the ladder of bands associated with the N2 form of the wild type is absent in CGß114 (lane 4). As expected, the CGß-secreted subunit from 15B cells was likewise homogeneous (lane 2). That the ß subunits secreted from 15B cells bear only noncomplex oligosaccharides was shown by their quantitative digestion with endoglycosidase H (data not shown). This enzyme hydrolyzes high mannose and hybrid N-linked, but not complex, oligosaccharides (18). These results show that removal of the carboxy terminus of CGß alters the processing of the variant beyond the Man5GlcNAc2 step.



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Figure 1. Analysis of the Secreted Forms of the CGß and CGß114 Subunits from the WT-CHO and the Glycosylation Mutant 15B Cells

Cells were labeled overnight with 25 µCi/ml 35S-labeled Pro-Mix, and the media were immunoprecipitated with the ß antiserum, subjected to SDS-PAGE, and autoradiographed. The migration of the CGß species bearing two (N2) and one (N1) asparagine-linked oligosaccharides in the electropherograms is indicated. The numbers 34 and 31 correspond to the molecular weights of the N2 (34,000) and N1 (31,000) forms of the CGß subunit; the 24 refers to 24,000, the mol wt of the dimer form of the {alpha} subunit (see Ref. 39).

 
Because heterogeneity of glycoproteins is often due to variations in sialic acid content, we treated CGß or CGß114 secreted from WT-CHO cells with neuraminidase (Fig. 2AGo) (19). If the heterogeneity of CGß114 is due to differences in sialic acid content, removal by neuraminidase should lead to a more homogeneous form compared with the untreated subunit. Although CGß and CGß114 are neuraminidase sensitive, as evident by the faster migrating electrophoretic forms compared with the untreated samples, the heterogeneity of CGß114 is unchanged (lane 4). These results suggest that the modified oligosaccharides in CGß114 are not due to alterations in the sialic acid content. Further support for this conclusion is provided by studies with the mutant CHO cell line, 1021 (20). These cells are deficient in the Golgi transport of CMP-sialic acid and, consequently, synthesize glycoproteins devoid of terminal sialic acid residues (20). Similar to that seen for neuraminidase-treated CGß114, the electrophoretic pattern of the variant is still observed (panel B, lane 8). As expected, the subunits recovered from 1021 cells were not sensitive to neuraminidase (data not shown). It is curious that CGß114 secreted from 1021 cells is much more heterogeneous than the form secreted from WT-CHO cells (compare lanes 6 and 8); even WT CGß subunit synthesized in 1021 cells is more heterogeneous than that normally observed for the subunit recovered from WT cells (lanes 5 and 7). It should be noted that the experimental approaches using neuraminidase and 1021 cells to delineate sialic acid contribution to the heterogeneity of the subunits are biologically distinct: neuraminidase removes sialic acid from already synthesized protein whereas protein secreted from 1021 cells reflects the consequence of inhibiting intracellular sialic acid addition. Thus, in the case of CGß and CGß114 synthesized in 1021 cells, the presence or absence of sialic acid may affect the extent of the modification (see Discussion).



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Figure 2. Analysis of Sialic Acid in CGß114

Panel A shows the effects of neuraminidase (neura) treatment of the CGß and CGß114 subunits secreted from WT-CHO cells. Precipitates were incubated without (-) (lanes 1 and 3) or with (+) (lanes 2 and 4) enzyme for 16 h at 37 C. Panel B displays the secretion of CGß and CGß114 subunits from WT-CHO (lanes 5 and 6) and the mutant 1021 cells (lanes 7 and 8). The labeling and immunoprecipitation are described in the legend of Fig. 1Go.

 
A modification that results in the heterogeneity of Asn-linked oligosaccharides in several glycoproteins is the addition of poly-N-acetyllactosamine (PL). These structures are composed of repeating disaccharide units [Galß (1, 2, 3, 4)->GlcNAc (1, 2, 3)]n attached to the Man3GlcNAc2 core (21, 22). To assess whether the oligosaccharides on CGß114 contain this modification, immunoprecipitates of secreted CGß or CGß114 from CHO-WT cells were treated with endo-ß-D-galactosidase (endo-ß-gal) (21, 22), which hydrolyzes ß-galactosyl linkages in the N-acetyllactosamine moiety (23) (Fig. 3Go). The WT CGß subunit was resistant (lane 2), but CGß114 was digested by the enzyme as indicated by the disappearance of the protein-ladder compared with the untreated subunit (lane 4). Both subunits secreted from 1021 cells were sensitive to the enzyme (lanes 6 and 8) and, as expected, the enzyme had no effect on the subunits derived from 15B cells (lanes 10 and 12). The secreted forms of glycosylation variants of CGß114, in which either of the two N-linked glycosylation sites at Asn-13 and -30 were mutated, also exhibited the characteristic polylactosamine modification (data not shown). These data imply that alterations of the CGß114 oligosaccharides are due, to poly-N-acetyllactosamine addition to the mannose core of the immature N-linked carbohydrates at both glycosylation sites.



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Figure 3. Endo-ß-Galactosidase (endo-ß-gal) Treatment of the CGß and CGß114 Subunits Secreted from the WT-CHO and the Mutant 1021 and 15B Cells

WT-CHO (lanes 1–4), 1021 (lanes 5–8), and 15B (lanes 9–12) cells expressing the CGß and CGß114 subunits were labeled, and medium samples were immunoprecipitated as described in legend of Fig. 1Go. Precipitates were incubated overnight at 37 C in the absence (-) (lanes 1, 3, 5, 7, 9, and 11) or presence (+) (lanes 2, 4, 6, 8, 10, and 12) of endo-ß-gal and subjected to SDS-PAGE.

 
Assembly of CGß114 with {alpha} and the Heterogeneity of Asn-Linked Oligosaccharides
The quaternary structure, i.e. the heterodimeric configuration, of multisubunit glycoproteins influences the processing pattern of their N-linked oligosaccharides (24, 25, 26, 27). To examine whether the electrophoretic heterogeneity of CGß114 is altered when combined with the {alpha} subunit, WT-CHO cells coexpressing the {alpha} and CGß114 subunits were labeled overnight, and equal aliquots of media were precipitated with the {alpha} and ß antisera (Fig. 4Go). The mature N2 form of CGß114 comigrates with the {alpha} subunit (11), and thus electrophoretic analysis of the heterodimeric forms is difficult (lanes 1 and 2). To examine only the combined forms of the subunits, samples were subjected to multiple immunoprecipitation and electrophoresis. The first step was to capture the dimer with {alpha} antiserum since only the dimer form of ß will be precipitated. The proteins are then resolved by SDS-PAGE to separate the labeled subunits from the antiserum; the electrophoresis was performed under nonreducing conditions to minimize a loss in immunoreactivity of the subunits to the subsequent precipitation. After eluting the dimer/uncombined subunits (arrow), the mixture was boiled to ensure complete dissociation of the heterodimer. The ß antiserum was then used in the next step. This approach permits analysis of only the dimer form of ß subunit. These precipitates were then treated with the endo-ß-gal and electrophoresed under reducing conditions. The data show that the heterodimeric forms of CGß114 are relatively homogeneous (lane 3) and resistant to endo-ß-gal digestion (lane 4). Based on the earlier experiments, we predicted that uncombined CGß114 would be heterogeneous. To test this point the supernate obtained after precipitation with {alpha} antiserum described in lane 1 of panel A was precipitated again with ß antiserum (ß -super). As expected the uncombined CGß114 displayed the characteristic heterogeneity (lane 5) and was sensitive to the enzyme (lane 6). These results show that assembly modifies the processing of the PL-modified N-linked carbohydrates of the CGß114 subunit.



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Figure 4. Assembly of CGß114 Alters Heterogeneity

To examine the fate of CGß114 when paired with the {alpha} subunit, CGß114 dimer secreted from WT-CHO cells was subjected to two immunoprecipitation steps. In the first step (lanes 1 and 2), equal aliquots of media were immunoprecipitated with either {alpha} (lane 1) or ß (lane 2) antisera (AS). The samples were electrophoresed under nonreducing conditions. The electrophoretic migration of the subunits in the CGß114 dimer is indicated. In the second step, the region containing the subunits (bracket) were eluted from the dried gel and precipitated with ß antiserum (lanes 3 and 4), to obtain only the heterodimeric form of the ß subunit, and subjected to reducing SDS-PAGE. The supernate obtained after precipitating with {alpha} antiserum shown in lane 1 was reprecipitated with ß antiserum to recover the uncombined ß subunit (ß-super; lanes 5 and 6). The precipitates were treated with endo-ß-galactosidase (egal, lanes 4 and 6).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although they arise from the processing of a common preassembled glycan unit, the mature Asn-linked oligosaccharides of secretory glycoproteins exhibit differences in the number and location of peripheral branches and terminal modifications (for review, see Refs. 2, 3, and 4). A major determinant for this specificity is encoded in the primary amino acid sequence (for review, see Refs. 28 and 29). However, these determinants are not well understood. Smith and Baenziger (30, 31) have shown that a sequence in the glycoprotein hormone subunits is associated with the recognition by a GalNAc transferase in the biosynthesis of sulfated Asn-linked oligosaccharides. From the results presented here it is evident that presence or absence of the CTP influences the processing of the Asn-linked oligosaccharides, although it is not clear how such sequences cause these posttranslational modifications. They may contain regions specifically recognized by the enzymes in the oligosaccharide pathway, or they could hinder accessibility of sites in the 114 region. Deleting the CTP might change the conformation of the truncated protein such that recognition by one or more glycosyl transferases is modified.

A critical question arises as to whether or not these alterations reflect that seen in vivo. The carbohydrate modifications observed could reflect one or more epitopes in the ß subunit that contribute to the specificity of the oligosaccharide processing in vivo. As shown here, the heterodimeric form of CGß114 does not exhibit the posttranslational changes observed for the noncombined subunit. Therefore, due to differences in the configuration of the combined subunit, assembly prevents the heterogeneity. This result is consistent with earlier investigations showing that the conformation of the heterodimer induced by the unique ß subunit is an important element in gonadotropin-specific oligosaccharide processing (2, 4, 27).

The ß subunits exhibit distinct intracellular fates (10, 25, 32, 33) and have different carboxyl ends: LHß and TSHß have stretches of hydrophobic residues at their carboxyl termini and bear biantennary oligosaccharides ending with GalNAc-linked sulfate, whereas the FSHß subunit lacking such an extension contains triantennary sugars terminating with Gal and sialic acid (14, 15). Because CHO cells lack the pituitary-specific GalNAc- and sulfo-transferases it is difficult to recapitulate the processing conditions in the gonadotrope. The use of mutant and/or chimeric subunits expressed in a pituitary cell line could provide a strong approach to define the intracellular role of the carboxyl terminus on assembly and hormone-specific processing of the N-linked oligosaccharides.

It is intriguing that the N-linked oligosaccharides of CGß114 synthesized in 1021 cells bear apparently more PL compared with the subunit secreted from WT-CHO cells. This suggests that the absence or presence of sialic acid affects the synthesis and elongation of PL. Thus, competition for the terminal Gal residue by the PL-specific glucosaminyl (21, 22, 34) and sialyl transferases could be responsible for the observed heterogeneity of CGß subunits. The CGß114 mutant may be a poor substrate for sialyl transferase and thus lack of the sialic acid cap would favor PL addition. This could also explain why the WT subunit synthesized in 1021 cells contains PL, since in the absence of sialic acid a fraction of the subunit is recognized by the glucosaminyl transferase. These data imply that altering the rate of sialic acid addition could be a determinant in the specificity of Asn-linked carbohydrate processing. It could also explain the appearance of numerous charged species that contribute to the heterogeneity observed in glycoprotein hormones purified from pituitary and placenta (2, 35).

It has been widely accepted that the CTP sequence does not contribute significantly to the overall secondary/tertiary structure of the CGß subunit. This was based on the following observations: 1) The native hCGß subunit or the synthetic CTP peptide competed at the same molarity with radiolabeled hCG for monoclonal antibodies to the CTP (6, 7); 2) the immunoreactivity of the denatured or native CGß subunit to these monoclonal antibodies is comparable (6, 7); 3) heterodimers bearing the CGß114 have the same receptor-binding affinity as the WT CG dimer (9); 4) the CTP sequence could be fused to the FSHß subunit without significantly affecting signal transduction of the chimera (8); and 5) x-ray diffraction failed to resolve the carboxy-terminal region (36, 37). There is sufficient evidence, however, to support the hypothesis that the CTP sequence interacts with other domains within the subunit and ultimately participating in the overall folding of the subunit. Studies using the LHß and CGß subunit chimeras showed a cooperativity between the amino and carboxyl ends of the subunits (10). Because the carboxyl domains in those chimeras included residues 87–145, it was not possible to restrict conclusions to the CTP sequence. Our current data that deleting the CTP sequence alters processing of the oligosaccharides, and reduces heterodimer formation compared with CGß (11), support the hypothesis that the CTP with its attached O-linked oligosaccharides is an interactive domain within the native subunit.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Cell culture media and reagents were prepared by the Washington University Center for Basic Research. Dialyzed calf serum, the neomycin analog G418, and Immunoprecipitin were purchased from GIBCO-BRL (Grand Island, NY). Endoglycosidase H and neuraminidase were purchased from Boehringer-Mannheim (Indianapolis, IN). Peptide-N-glycosidase F was purchased from New England BioLabs (Beverly, MA). Endo-ß-gal was obtained from V-Labs (Covington, LA). Rabbit polyclonal antiserum against the {alpha} or the CGß subunit was prepared in this laboratory. [35S]Pro-Mix and [35S]cysteine were purchased from Amersham (Arlington Heights, IL) and ICN (Costa Mesa, CA), respectively.

Plasmid Construction, Transfection, and Selection of Stable Clones
The construction of mammalian expression plasmids pM2CGß and pM2CGß114 (truncated at amino acid 114) were described previously (10, 17). The mutation was within a BglII and BamHI fragment, which was inserted into the unique BamHI site of the expression vector, pM2 (9). Transfection of the WT-CHO cells and the mutant cell lines 15B (16) and 1021 (20) with CGß subunit, with or without the {alpha} subunit gene, and the isolation of stable clones were described (10, 11, 38).

Cell Labeling and Immunoprecipitation
CHO clones were grown in 12-well plates to near confluency before labeling. Cells were washed once with PBS and with Ham’s F-12 labeling medium [minus methionine and/or cysteine] and labeled overnight (16 h) in this medium containing 25 µCi/ml of 35S-labeled Pro-Mix or [35S] cysteine. Samples were precleared with normal rabbit serum. Samples were precipitated with CGß antiserum, resolved on SDS-PAGE, and autoradiographed. Treatment of samples with endoglycosidase H, peptide-N-glycosidase F, and neuraminidase was according to Keene et al. (38). Samples were treated with endo-ß-gal according to the supplier’s instruction. Briefly, immunoprecipatates were dissolved in 50 µl of 50 mM sodium acetate buffer (pH 5.5) containing 1% ß-mercaptoethanol, boiled for 3 min, and centrifuged. The supernates were incubated at 37 overnight (18 h) with 2.5 mU of endo-ß-gal.


    ACKNOWLEDGMENTS
 
We are grateful to Drs. Alison Jackson, Jacques Baenziger, Diana Blithe, David Ben-Menahem, and Vicenta Garcia-Compayo for their critical reading of the manuscript and to Dr. Susan Daniels-McQueen for her technical advice. We thank Mary Wingate for her invaluable assistance in preparing the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Irving Boime, Department of Molecular Biology & Pharmacology, 660 South Euclid Avenue, Campus Box 8103, St. Louis, Missouri 63110.

1 Present address: The University of Rochester Medical Center, Department of Biochemistry, 601 Elmwood Avenue, Rochester, New York 14642. Back

Received for publication December 1, 1997. Revision received February 5, 1998. Accepted for publication February 10, 1998.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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