©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Asparagine-linked Oligosaccharides of the Human Chorionic Gonadotropin Subunit Facilitate Correct Disulfide Bond Pairing (*)

Weijun Feng (1) (2), Martin M. Matzuk (4)(§), Kimberly Mountjoy (1), Elliott Bedows (1) (2), Raymond W. Ruddon (1) (2) (3)(¶), Irving Boime (4)

From the (1) The Eppley Institute for Research in Cancer and Allied Diseases and Departments of (2) Pharmacology and (3) Biochemistry and Molecular Biology, The University of Nebraska Medical Center, Omaha, Nebraska 68198-6805 and the (4) Departments of Pharmacology and Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
REFERENCES

ABSTRACT

The role of asparagine (N)-linked oligosaccharide chains in intracellular folding of the human chorionic gonadotropin (hCG)- subunit was determined by examining the kinetics of folding in Chinese hamster ovary (CHO) cells transfected with wild-type or mutant hCG- genes lacking one or both of the asparagine glycosylation sites. The half-time for folding of p1 into p2, the rate-determining step in folding, was 7 min for wild-type but 33 min for lacking both N-linked glycans. The p1 p2 half-time was 7.5 min in CHO cells expressing the subunit missing the Asn-linked glycan and 10 min for the subunit missing the Asn-linked glycan. The inefficient folding of hCG- lacking both N-linked glycans correlated with the slow formation of the last three disulfide bonds (i.e. disulfides 23-72, 93-100, and 26-110) to form in the hCG--folding pathway. Unglycosylated hCG- was slowly secreted from CHO cells, and subunit-folding intermediates retained in cells for more than 5 h were degraded into a hCG- core fragment-like protein. However, coexpression of the hCG- gene enhanced folding and formation of disulfide bonds 23-72, 93-100, and 26-110 of hCG- lacking N-linked glycans. In addition, the molecular chaperones BiP, ERp72, and ERp94, but not calnexin, were found in a complex with unglycosylated, unfolded hCG- and may be involved in the folding of this form. These data indicate that N-linked oligosaccharides assist hCG- subunit folding by facilitating disulfide bond formation.


INTRODUCTION

Human chorionic gonadotropin (hCG)¹(¹) is a member of the glycoprotein hormone family and consists of two noncovalently associated subunits ( and ). It is synthesized and secreted by nonmalignant and malignant trophoblast cells (1, 2) . The hCG- subunit is a 145-amino acid protein, in which 12 cysteine residues pair to form six intramolecular disulfide bonds. The rate of disulfide bond formation in the subunit is rate-limiting in formation of the heterodimer (3, 4) . The hCG--folding intermediates have been characterized based on the order of formation of the six disulfide bonds (5, 6, 7) , and the hCG--folding pathway has been defined as: p1 p2-free p2-combined mature -combined. The conversion of p1 p2 is the rate-determining step in this pathway (3) . This folding pathway is identical in JAR (human choriocarcinoma) cells (5, 6) , in Chinese hamster ovary (CHO) cells transfected with the wild-type hCG- gene (7, 20) , and in vitro(8) . hCG- is the only mammalian protein for which the intracellular folding pathway has been defined (8, 9) .

hCG- is synthesized, folds, and assembles with the subunit in the endoplasmic reticulum (ER) (2, 3, 4) . The forms of the subunit detected within the ER contain two high mannose asparagine (Asn and Asn)-linked oligosaccharide chains (3, 10, 11). During transport through the Golgi apparatus, asparagine (N)-linked oligosaccharides are processed to form complex oligosaccharides containing galactose and sialic acid (12) , and four O-linked oligosaccharides are attached to the C-terminal extension of the hCG- sequence (13, 14, 15) . Although all the information needed to determine the final conformation of a protein exists in the polypeptide chain (16) , addition of N-linked oligosaccharides and intracellular factors such as molecular chaperones significantly affect protein folding inside cells (17, 18) .

Depending on the protein, glycans may contribute to protein conformation, stability, and binding to target cells. A number of reports demonstrate that the N-linked oligosaccharides and their processing are critical for proper glycoprotein folding and assembly (17) . Matzuk and Boime (11) have demonstrated that the two N-linked oligosaccharides of hCG- are critical for efficient assembly with the subunit and for secretion. In addition, we have demonstrated that at least one N-linked oligosaccharide is required for efficient hCG- folding in vitro(19) . However, the molecular mechanism by which the N-linked oligosaccharide chain(s) affect protein folding remains unclear.

Since proper disulfide bond formation is a critical event in the folding and maturation of functional hCG- subunits (3, 5, 6, 7, 20, 27) and altered glycosylation can cause alteration of folding and disulfide bond formation (21, 22) , we postulated that N-linked oligosaccharides are involved in hCG- folding by facilitating disulfide bond formation. To test this hypothesis, we analyzed the kinetics of intracellular folding of hCG- in CHO cells transfected with either the wild-type hCG- gene or hCG- genes mutated at N-linked glycosylation sites. We found that the two N-linked glycans increased hCG- subunit folding efficiency by facilitating formation of the last three disulfide bonds (23-72, 93-100, and 26-110) to form. We also observed that inefficiently folded, unglycosylated hCG- was degraded to a core fragment-like protein. However, in the presence of the subunit, unglycosylated folded efficiently. In addition, the slow folding process of unglycosylated hCG- allowed us to detect molecular chaperone-hCG- complexes that may be involved in hCG- folding.


EXPERIMENTAL PROCEDURES

Cell Culture

CHO cells transfected with the wild-type or mutated hCG- genes alone, or co-transfected with the glycoprotein hormone gene (11) , were maintained in Ham's F-12 medium supplemented with 5% fetal bovine serum, penicillin (100 units/ml), streptomycin (100 µg/ml), and glutamine (2 mM) (23). The mutated hCG- genes contained mutations Asn Gln at the Asn and/or Asn codons of the two N-linked glycosylation consensus sequences (11) . The terminology used here is: CHO WT or CHO WT for CHO cells transfected with wild-type hCG- genes with or without co-transfection of the gene, respectively; CHO Asn(1+2) and CHO Asn(1+2) for CHO cells transfected with hCG- genes mutated at both N-linked glycosylation sites with or without co-transfection of the gene, respectively; CHO Asn1 or CHO Asn2 for CHO cells transfected with hCG- genes mutated at the Asn or Asn glycosylation sites, respectively.

Biosynthetic Labeling

CHO cells were metabolically labeled as described previously (20) . Briefly, 100-mm Petri dishes of 90% confluent CHO cells were starved in cysteine- or leucine-free and serum-free Dulbecco's modified Eagle's medium for 30 min. These cells were then pulse-labeled for 5 min with 300-400 µCi/ml L-[S]cysteine (1100 Ci/mmol; DuPont NEN) or L-[4,5-³H]-leucine (60 Ci/mmol; DuPont NEN) in serum-free Dulbecco's modified Eagle's medium (Life Technologies, Inc.) lacking cysteine or leucine, washed with phosphate-buffered saline (pH 7.4), and chased for the times indicated in the text. Then, cells were rinsed with cold phosphate-buffered saline and lysed in 5 ml of phosphate-buffered saline (pH 8) containing detergents (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate), protease inhibitors (20 mM EDTA and 2 mM phenylmethanesulfonyl fluoride), and 50 mM iodoacetic acid to alkylate-free sulfhydryl groups of folding intermediates to prevent further disulfide bond formation or rearrangement.

Immunoprecipitation of Cell Lysates

The cell lysates were immunoprecipitated with an anti-hCG- polyclonal antibody (1:1000), which recognizes all forms of the hCG- subunit, for 16 h at 4 °C, and the immune complexes were precipitated with protein A-Sepharose (Sigma) as described previously (3) .

Separation of hCG--folding Intermediates by SDS-Polyacrylamide Gel Electrophoresis (PAGE)

The protein A-Sepharose-hCG- immune complexes were eluted with 2 concentrated SDS-PAGE sample buffer (125 mM Tris-HCl, 2% SDS, 20% glycerol, and 40 µg/ml bromphenol blue) and analyzed by 5-20% gradient, nonreducing SDS-PAGE (24) as described previously (20) .

Determination of Kinetics of hCG- in Vivo Folding

[S]Cysteine-labeled hCG- folding intermediates (p1 and p2) were visualized by exposing to x-ray film. The integrated optical density (IOD) values of each band on autoradiograms were quantitated and analyzed using a BioImage 110S image analyzer and Whole Band software (Millipore). These bands, representing the hCG--folding intermediates (p1 or p2), were measured as the product of the band area (mm²) and optical density (OD) as calibrated with a Kodak standard 21-step gray-scale wedge (Eastman Kodak Co.). To evaluate folding efficiency of hCG-, we compared the IOD of folded p2 with that of p1. For instance, if the values of IOD of p2 and p1 were 0.80 and 0.20, respectively, this indicated that 80% (0.8/(0.8+0.2)) of p1 converted to p2, and 20% of p1 remained unfolded. Therefore, the folding efficiency at this time point was 80%. Each time point served as its own control in that the ratio of p2/(p1 + p2) was obtained for each gel lane. The t reported here for folding of p1 to p2 in CHO cells transfected with the wild-type gene was slightly longer than that previously reported (7 versus 5 min) (20) because a longer pulse time (5 versus 3 min) was used here to incorporate sufficient counts/min for tryptic peptide mapping.

Purification of hCG--folding Intermediates

The protein A-Sepharose beads containing hCG- immune complexes were eluted with 6 M guanidine hydrochloride (pH 3) (Sequanal grade; Pierce Chemical Co.) for 16 h at room temperature. Eluates were purified using Vydac 300-Å C reversed-phase high performance liquid chromatography (HPLC) with elution by an acetonitrile gradient as described previously (6) . The fractions containing hCG-p1 or -p2 subunits were collected and concentrated by Speed-Vac concentrator.

Tryptic Digestion and Separation of Tryptic Peptides on HPLC

Purified [S]cysteine-labeled hCG--folding intermediates were digested with diphenylcarbamyl chloride-treated trypsin. Trypsin-digested samples were loaded onto a Brownlee 10-µm C RP-300 reversed-phase column (Brownlee/ABI) eluted with an acetonitrile gradient as described previously (6, 7) . The profile of peaks generated indicated which hCG- disulfide bonds had not formed.

Amino Acid Sequencing

[S]Cysteine- or [³H]leucine-labeled peptides were concentrated to less than 50 µl using a Speed-Vac concentrator. Each sample was loaded onto a polybrene-coated, trifluoroacetic acid-treated cartridge filter (Applied Biosystems) and sequenced using a pulsed liquid protein sequencer (Applied Biosystems model 477A). The phenylthiohydantoins obtained from each cycle of the Edman degradation were collected and analyzed by liquid scintillation counting to determine the positions of radiolabeled residues in each peptide.

Western Blot Analysis

To detect the presence of proteins that co-immunoprecipitated with unglycosylated -folding intermediates, cell lysates derived from six 100-mm Petri dishes CHO Asn(1+2) cells were immunoprecipitated with anti-hCG-, and the hCG- subunits were eluted from protein A-Sepharose beads with 6 M guanidine (pH 3.0) for 16 h with rotation at room temperature and purified by C reversed-phase HPLC (as described above). The 80-95-min and 100-115-min fractions (termed C1 and C2, respectively) were high molecular weight complexes containing unglycosylated hCG-. These complexes were separated by SDS-PAGE under reducing conditions (24) , and the separated proteins were transferred to Immobilon poly(vinylidne fluoride) transfer membranes (Millipore) in a Trans-Blot cell (Bio-Rad) at 480 mA for 1-2 h at 4 °C. The membranes were immunoblotted with either rat anti-BiP (1:500), rabbit anti-ERp72 (1:100), rabbit anti-ERp94 (1:100), or rabbit anti-calnexin (1:2000) overnight at 4 °C with gentle shaking. Membranes were washed several times with buffer containing 20 mM Tris, 150 mM NaCl, 1% non-fat milk, and 0.2% Tween (pH 7.4) and incubated with anti-rat or -rabbit IgG peroxidase conjugate (1:1000, Sigma) for 30 min at 4 °C. Enhanced chemiluminescence (ECL Western blotting kit, Amersham Corp.) was used to identify the blotted proteins. Rat anti-BiP polyclonal antibody was provided by Dr. David Bole (University of Michigan, Ann Arbor, MI). Rabbit anti-ERp72 (against the 16 C-terminal amino acids of murine ERp72) and rabbit anti-ERp94 (against the 16 C-terminal amino acids of murine ERp94) were provided by Dr. Michael Green (St. Louis University Medical Center, St. Louis, MO). The rabbit anti-calnexin (against the C-terminal 19 amino acids of canine calnexin) was provided by Dr. Ari Helenius (Yale University, New Haven, CT). Rat and rabbit nonimmune sera (Sigma) were used as controls for specificity.

RESULTS

Kinetics of Intracellular Folding of hCG- Glycosylation Mutants

To determine what role N-linked oligosaccharide chains play in intracellular hCG- folding, CHO cells transfected with wild-type hCG- gene (WT) or mutated hCG- genes (Asn1, Asn2, or Asn(1+2)) were pulse-labeled for 5 min with [S]cysteine and chased for the times indicated in Fig. 1. The [S]cysteine-labeled hCG--folding intermediates (p1 or p2) were immunoprecipitated with polyclonal anti-hCG- and analyzed by nonreducing SDS-PAGE (Fig. 1, A and B). With increasing chase time, p1 synthesized in CHO cells transfected with wild-type or mutated genes folded into their corresponding p2 forms. To evaluate the folding efficiency, the IOD of each band on the autoradiograms was quantitated. The extent of p1 conversion to p2 was calculated (p2/(p1 + p2)) at each chase time and plotted in Fig. 1C. Based on the calculated linear initial folding rate, the half-times for the conversion of p1 to p2 were 7, 7.5, and 10 min for CHO WT, CHO Asn1, and CHO Asn2, respectively. These data indicate that hCG- lacking the Asn-linked oligosaccharide had marginally decreased folding efficiency inside cells, compared to wild-type hCG- and hCG- lacking the Asn-linked oligosaccharide. However, the t of conversion of unglycosylated p1 to p2 was 33 min, demonstrating that at least one N-linked glycan is required for efficient hCG- folding in CHO cells.


Figure 1: Kinetics of intracellular folding of wild-type and glycosylation mutants of hCG-. CHO cells transfected with wild-type or glycosylation mutants of hCG- genes were pulse-labeled with [S]cysteine for 5 min and chased for the indicated times. hCG- subunits with different numbers of N-linked oligosaccharides were immunoprecipitated and analyzed by 5-20% gradient SDS-PAGE under nonreducing conditions (see ``Experimental Procedures''). Left lanes, Panels A and B: C-labeled molecular weight markers (Sigma): carbonic anhydrase (M = 29,000), and -lactalbumin (M = 14,000). Panel A, CHO-WT, CHO cells transfected with wild-type hCG- genes and CHO Asn(1+2), CHO cells transfected with hCG- genes mutated at both N-linked (Asn and Asn) glycosylation sites; Panel B, CHO Asn1, CHO cells transfected with hCG- genes mutated at the Asn-linked glycosylation site and CHO Asn2, CHO cells transfected with hCG- genes mutated at the Asn-linked glycosylation site. Arrows indicate the respective positions of p1 (p1 lacking both N-linked glycans), p1 (p1 lacking either N-linked glycan), p2 (p2 lacking both N-linked glycans), and p2 (p2 lacking either N-linked glycan). Panel C, the intensities of each band on A and B were quantitated with a BioImage analyzer as described under ``Experimental Procedures.'' The folding efficiencies were calculated by dividing the integrated intensities of p2 by that of the integrated intensities of p2 plus p1 for each lane (see ``Experimental Procedures''). , p1 with both Asn-linked glycans; , p1 lacking Asn-linked glycan; , p1 lacking Asn-linked glycan; , p1 lacking both Asn-linked glycan.



N-Linked Oligosaccharides Facilitate Formation of Disulfide Bonds in Later Steps of the hCG--folding Pathway

Since the evidence shown above demonstrated that N-linked oligosaccharides were involved in hCG- subunit folding, we examined whether this effect was due to changes in disulfide bond formation. To test this hypothesis, a strategy of identifying the disulfide bonds formed in each folding intermediate was undertaken (6) . Fig. 2illustrates the hCG- tryptic peptides arranged according to the disulfide bond assignments of Mise and Bahl (25) . We employed this strategy because our intracellular kinetic folding data (8, 26) and data from analysis of the intracellular folding of mutated at each of the six disulfide bonds (7, 27) strongly suggest that disulfide bonds 38-57 and 9-90 form early in the folding pathway. Since the crystal structure of mature, native hCG- indicates that the disulfide bonds 9-57 and 38-90 are present (28, 29) , it is likely that there are disulfide bond rearrangements in the hCG--folding pathway.


Figure 2: hCG- disulfide-linked tryptic peptides. Shown are the disulfide bond linked peptides generated following trypsin digestion of hCG- using the disulfide bond assignments by Mise and Bahl (25) and confirmed by intracellular and in vitro folding kinetics (7, 26). Seven tryptic peptides are linked by six disulfide bonds in mature hCG-. C reversed-phase HPLC peaks of tryptic peptides (Fig. 10) that have been previously identified (6, 7) were renumbered and indicated above for nine of the peptides. Cystine or leucine residues are shown in bold. Peptides or fragments of peptides missing in the proteolytic degradation product of unglycosylated (d) are shadowed.



The extent of formation of each disulfide bond was evaluated by quantitating the amount of hCG- tryptic peptides released from the remaining disulfide-linked hCG- polypeptides (6) . For instance, when hCG- is trypsinized under nonreducing conditions, no peptides containing free thiols would be released if all the disulfide bonds are formed. However, if, for example, the disulfide bond between Cys and Cys was unformed, a peptide containing Cys with a free thiol would be released from the remainder of the disulfide-linked polypeptides. Thus, the percent of the released peptide represents the extent of unformed disulfide bond between Cys and Cys.

CHO WT and CHO Asn(1+2) mutant cells were pulse-labeled for 5 min with [S]cysteine and chased for the times shown in Fig. 3 . Unformed thiols were trapped by alkylation with iodoacetate at the time of cell lysis (thereby generating a carboxymethyl derivative of nondisulfide-linked Cys residues). The p1- and p2-folding intermediates were purified by immunoprecipitation and C reversed-phase HPLC. The purified intermediates were digested with trypsin and the peptides analyzed by C reversed-phase HPLC to quantitate the amount of cysteine residues 9, 57, 72, 88, 100, and 110 that were not disulfide linked in each folding intermediate (6, 7) . The percent of unformed disulfide bonds involving each of the six disulfide bonded cysteines of was plotted according to the order in which they form (Fig. 3, A F). Compared to CHO WT, disulfide bonds 23-72, 93-100, and 26-110 were predominately unformed in p1 lacking both N-linked oligosaccharides (Fig. 3, D-F). The lag in conversion of p1 to p2 for the Asn(1+2) mutant may be due to less efficient formation of the 23-72 bond since conversion of p1 to p2 requires this bond (6, 7) . Furthermore, 70% of disulfide bond 93-100 and 95% of disulfide bond 26-110 remained unformed in unglycosylated p2 even after a 120 min chase (Fig. 3, E and F). These results suggest that N-linked oligosaccharides facilitate formation of the three late-forming disulfide bonds (23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 93, 94, 95, 96, 97, 98, 99, 100, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110) . The data also suggest that the p2 formed by the folding of unglycosylated p1 contained unstable 93-100 and 26-110 disulfide bonds because they were less completely formed in p2 than in p1 (Fig. 3, E and F). This was unexpected and was the opposite of wild-type p2, which has more complete formation of these bonds than wild-type p1 (Fig. 3, E and F).


Figure 3: Kinetics of disulfide bond formation in wild-type hCG- and in glycosylation mutants. CHO WT and CHO Asn(1+2) cells were pulse-labeled with [S]cysteine for 5 min and chased for the indicated times. hCG- subunits were immunoprecipitated with anti-hCG- and protein A-Sepharose and purified on C reversed-phase HPLC. The purified [S]cysteine-labeled hCG-p1 and -p2 were digested with trypsin and analyzed by C reversed-phase and ion-exchange HPLC (see ``Experimental Procedures''). The percent of unformed bonds for each of the six disulfide bonds of were calculated as described in the text, and data are shown for WT p1 (), WT p2 (), Asn(1+2) p1 (), and Asn(1+2) p2 (). The disulfide bonds indicated at the top of each panel are arranged in the order in which they form (A-F) (6, 7).



The Subunit Stimulates Folding and Disulfide Bond Formation of Subunit Lacking N-Linked Oligosaccharides

Previous studies in our laboratory showed that completion of the last disulfide bond (26-110) occurs after assembly with the subunit (26) and that the subunit increases the rate and extent of hCG heterodimer assembly (20). This is consistent with the crystallographic structure which reveals that formation of the disulfide bond 26-110 of forms a seat belt around the subunit after heterodimer assembly occurs (28). These data imply that the subunit assists hCG- folding by facilitating disulfide bond formation. To examine whether the subunit can increase folding of the unglycosylated subunit, CHO cells containing the Asn(1+2) mutant gene were co-transfected with the gene.

The Asn(1+2)- and Asn(1+2)-expressing cells were pulse-labeled for 5 min with [S]cysteine and chased as described in Fig. 4 . The percent of each of the six disulfide bonds that was not formed was calculated as described above and plotted in the order in which they form (Fig. 4, A F). The rates of disulfide bond formation involving cysteines 9, 34, 38, 57, 88, and 90 were similar in the either presence or absence of the subunit (Fig. 4, A-C). However, the slow formation of disulfide bonds 23-72, 93-100, and 26-110 in -lacking N-linked oligosaccharides was accelerated in the presence of the subunit, suggesting that subunit facilitates formation of disulfide bonds that are completed later in the hCG--folding pathway.


Figure 4: Kinetics of disulfide bond formation in glycosylation mutants in the presence or absence of subunit. CHO cells transfected with N-linked glycosylation mutants with or without co-transfection with wild-type glycoprotein hormone gene (CHO Asn(1+2) or CHO Asn(1+2), respectively) were pulse-labeled with [S]cysteine for 5 min and chased for the indicated times. hCG- subunits were prepared as described in Fig. 3. Shown are Asn(1+2) p1 (), Asn(1+2) p2 (), Asn(1+2) p1 (), Asn(1+2) p2 ().



A Potential Role for Chaperones in Folding of Glycosylation Mutants of hCG-

When CHO cells containing WT and Asn(1+2) mutated genes were pulse-labeled for 5 min with [S]cysteine and chased for 5 min to 5 h, the hCG--folding intermediates purified on C reversed-phase HPLC showed different profiles (Fig. 5). With increasing chase time, wild-type p1 (Fig. 5, A-C) converted more efficiently into p2 than the unglycosylated p1 (Fig. 5, D-F), and two additional peaks appeared at 90 min (C1) and 110 min (C2) in the HPLC profile of the unglycosylated (Fig. 5, D-F). The C1 and C2 peaks diminished with increasing chase times. When the C1 and C2 peaks were collected and analyzed by SDS-PAGE (Fig. 6), it was observed that hCG-p1 (lacking two N-linked glycans) was complexed with a variety of high molecular weight proteins.


Figure 5: Purification of [S]cysteine labeled hCG- folding intermediates and protein complexes from CHO WT and CHO Asn(1+2) cell lysates. CHO WT and CHO Asn(1+2) cells were pulse-labeled with [S]cysteine for 5 min and chased for the indicated times. hCG- subunits were immunoprecipitated with anti-hCG- and protein A-Sepharose. Then, the [S]cysteine-labeled hCG- subunits were eluted with guanidine and analyzed by C4 reversed-phase HPLC as described under ``Experimental Procedures.'' The cell types and chase times are indicated at the top of each panel. WT-0, WT-5, or WT-15, CHO WT cells chased for 0, 5, or 15 min, respectively; Asn(1+2)-15, Asn(1+2)-60, or Asn(1+2)-300, CHO (1+2) cells chased for 15, 60, or 300 min, respectively; p1, p1 with both N-linked glycans; p2, p2 with both N-linked glycans; p1, p1 lacking both N-linked glycans; p2, p2 lacking both N-linked glycans; C1 and C2, protein complexes containing hCG-p1 and chaperone-like proteins (see Fig. 6).




Figure 6: Identification of protein complexes on reducing SDS-PAGE. The [S]cysteine-labeled p1, p1 C1, and C2 peaks (Fig. 5) were analyzed by 5-20% reducing SDS-PAGE. Lane 1, C-labeled molecular weight markers (Sigma): bovine serum albumin (M = 66,000), chicken egg albumin (M = 45,000), carbonic anhydrase (M = 29,000), and -lactalbumin (M = 14,000); lane 2, hCG-p1 (p1 with both N-linked glycans) and hCG-p1 (p1 lacking either N-linked glycan); lane 3, hCG-p1 (p1 lacking both N-linked glycans); lane 4, protein complex 1 (C1); lane 5, protein complex 2 (C2). P1-P7 represent chaperone-like proteins that were co-immunoprecipitated as part of hCG-1 complex.



To identify these proteins, the C1 and C2 peak fractions were analyzed by 5-20% gradient SDS-PAGE under reducing conditions. The separated proteins were transferred to nitrocellulose membranes and subjected to Western blot analysis using antibodies against BiP (Fig. 7A, lanes 3 and 4), ERp72 (Fig. 7B, lanes 3 and 4), and ERp94 (Fig. 7B, lanes 5 and 6). To rule out nonspecific blotting, nonimmune rat serum (Fig. 7A, lanes 1 and 2) and rabbit serum (Fig. 7B, lanes 1 and 2) were used as controls. The data in Fig. 7suggested that C1 and C2 are protein complexes of unglycosylated hCG-p1 with molecular chaperones such as BiP, ERp72, and ERp94. Western blot analysis of the C1 and C2 protein complexes indicated that they did not contain calnexin (data not shown), which might be expected since unglycosylated hCG-p1 lacks calnexin binding sites (glucose residues) (17) .


Figure 7: Identification of chaperone-like proteins by Western blotting analysis. Cell lysates from six 100-mm Petri dishes of CHO Asn(1+2) cells were immunoprecipitated with anti-hCG- and protein A-Sepharose. Then, the nonradioactive-labeled hCG- subunits were eluted with guanidine and purified by C reversed-phase HPLC. The C1 (80-95th min) and C2 (100-115th min) fractions were collected and analyzed by 5-20% gradient SDS-PAGE under reducing conditions. The protein bands were transferred from acrylamide gels to nitrocellulose membranes and immunoblotted with specific antibodies indicated below or preimmune IgG (rat or rabbit). Secondary peroxidase-conjugated antibodies and enhanced chemiluminescence reagents were used to detect the chaperone proteins (see ``Experimental Procedures''). The molecular weight was determined based on prestained molecular weight markers (Bio-Rad): bovine serum albumin (M = 97,200), ovalbumin (M = 50,000), carbonic anhydrase (M = 35,100), soybean trypsin inhibitor (M = 29,700), lysozyme (M = 21,900). Panel A, lanes 1 (C1) and 2 (C2) were immunoblotted with preimmune rat IgG; lanes 3 (C1) and 4 (C2) were immunoblotted with rat anti-BiP. Panel B, lanes 1 (C1) and 2 (C2) were immunoblotted with preimmune rabbit IgG; lanes 3 (C1) and 4 (C2) were immunoblotted with rabbit anti-ERp72; lanes 5 (C1) and 6 (C2) were immunoblotted with rabbit anti-ERp94.



Production of Core Fragment-like Protein

In the absence of the subunit, unglycosylated hCG- is still secreted as free subunit with t of 5 h (t of wild-type hCG- = 2.5 h) (11) . However, our results indicated that unglycosylated p2 subunits contain more unformed 93-100 and 26-110 disulfide bonds than the wild-type p2 (Fig. 3, E and F). Even after a 24-h chase, 60% of the 93-100 and 95% of 26-110 disulfide bonds still were not formed in the subunit that remained inside the cells (data not shown). However, with chase times longer than 5 h, a new peak (d) appeared on the HPLC profiles (Fig. 8, C-F). In order to identify this new peak, CHO Asn(1+2) cells were pulse-labeled with [S]cysteine and chased for 15-24 h. The cell lysates were immunoprecipitated with anti-hCG- and purified on C reversed-phase HPLC. The d fractions were collected and analyzed by SDS-PAGE under either reducing (Fig. 9A) or nonreducing (Fig. 9B) conditions. The results indicated that d migrated faster than unglycosylated p1 (p1) (compare to Fig. 1) and was composed of two disulfide-linked polypeptides. To further identify this apparent subunit degradation product, d was reduced and digested with trypsin and tryptic peptides analyzed on C reversed-phase HPLC. In comparison with the tryptic peptide map of unglycosylated p2 (p2) (Fig. 10A), four peaks (1, 5, 8, and 12) were missing (Fig. 10B) and two new peaks (3 and 9) were generated. Previous studies (6) had identified peaks 1, 5, 8, and 12 as peptides , , , and , respectively (). Since d lacked these four peaks, the data suggested that the , , , and peptides were missing or at least not intact in d. Moreover, amino acid sequence data indicated that new peaks 3 and 9 were peptides that contained [S]cysteine at positions 3 (Fig. 10E) or 6 and 8 (Fig. 10F), respectively. Since peptides and were missing and since Gln and Leu are putative proteolytic cleavage sites in hCG- (30) , the most likely explanation is that peaks 3 and 9 represent peptides and . This is consistent with the finding that peak 3 contained [S]cysteine at cycle 3 and peak 9 contained [S]cysteine at cycles 6 and 8 (see Fig. 2 ). Thus, we concluded that peaks 3 and 9 represent peptides generated by proteolytic degradation of p2.


Figure 8: Identification of [S]cysteine-labeled hCG- proteolytic products from CHO Asn(1+2) cell lysates. CHO Asn(1+2) cells were pulse-labeled with [S]cysteine for 5 min and chased for periods of 0-24 h. hCG- subunits were immunoprecipitated and analyzed by C reversed-phase HPLC as described above. Panels A-F represent the HPLC profiles of the [S]cysteine-labeled CHO Asn(1+2) cell lysates chased for 0, 1, 5, 10, 15, and 24 h, respectively. p1, p1 lacking both N-linked glycans; p2, p2 lacking both N-linked glycans; C1 and C2, protein complexes containing hCG-p1 and chaperone-like proteins; d, proteolytic form(s) of the p2; , mature hCG- with four O-linked glycans.




Figure 9: Identification of the proteolytic hCG- substrate (d) on SDS-PAGE. The [S]cysteine-labeled d peak fractions (Fig. 8F) were analyzed by either nonreducing (Panel A, lane 2) or reducing (Panel B, lane 2) SDS-PAGE. Panels A and B, lanes 1, [C]-labeled molecular weight markers. d represents proteolytic form(s) of the p2. df1 and df2 represent the disulfide bond-linked fragments of d.




Figure 10: Separation and identification of tryptic peptides of reduced [S]cysteine-labeled hCG-p2 or -d. [S]Cysteine-labeled hCG-p2 and -d were reduced and digested with trypsin. The tryptic peptides were separated on C reversed-phase HPLC (see ``Experimental Procedures''). Panel A, tryptic peptide HPLC profile of reduced [S]cysteine-labeled hCG-p2. Every peak that contains a known specific peptide (6, 7) is numbered in the order that it appears (Table I). Panel B, tryptic peptide HPLC profile of reduced [S]cysteine-labeled hCG-d The peaks 3 and 9 were identified by amino acid sequencing (Panels E and F). Panel C, ion-exchange HPLC analysis of peak 4 generated in Panel A showing that peak 4 contains two co-eluted pepetides ( and ). Panel D, ion-exchange HPLC analysis of peak 4 generated in Panel B showing that peak 4 contains only peptide. Panel E, amino acid sequencing of peak 3 generated in Panel B showing that the third amino acid from the N-terminal end peptide is [S]cysteine-labeled cysteine. Panel F, amino acid sequencing of peak 9 generated in Panel B showing that the sixth and eighth amino acids from the N-terminal of the peptide are [S]cysteine-labeled cysteines.



To separate peptides (4a) and (4b) that co-migrate on C reversed-phase HPLC (6, 7) , the peaks 4 of Fig. 10, A and B, were separated on ion-exchange HPLC. As can be seen in Fig. 10C, peak 4 from the p2 contained both 4a and 4b peaks, indicating that both peptides and were present. However, only peak 4a was present in the d (Fig. 10D), indicating was missing in d.

Since the C-terminal of hCG- contains leucine but not cysteine residues, CHO Asn(1+2) cells were labeled with [³H]leucine to see if the C-terminal of was present in d. [³H]leucine-labeled hCG-p2 and -d were digested with trypsin and analyzed as above. Peaks 2 (), 5 (), 6 (), 8 (), 10 (), and 12 () found in p2 were not found in d (data not shown). The missing 2, 6, and 10 peaks suggested that d does not contain and . The lack of peak 12 and peak 4b further support the conclusion that d did not contain intact and . The absence of peak 8 and the presence of peak 9 indicated, as above, that was clipped into and . Due to the absence of cysteine and leucine residues in , the [S]cysteine- and [³H]leucine-labeled hCG- would not detect whether was present in d. Since, however, and peptides were missing, it is likely that was missing in d as well.

Taken together, these results indicate that a portion of hCG-p2 is degraded proteolytically into d lacking and . This strongly suggests that d is a corelike fragment containing only peptides and (30) .

DISCUSSION

The data reported here indicate that the t of folding of hCG-p1 lacking both Asn- and Asn-linked glycans into p2 is significantly longer (t = 33 min) than that of wild-type p1 (t =7 min). However, most of the mutant p1 can slowly fold into the corresponding p2 as seen after a 90 min chase (Fig. 1C). We have previously reported that when wild-type or glycosylation mutants of hCG- are folded in vitro, 60% of wild-type p1 (two N-linked glycans), 60% of p1 lacking the Asn-linked glycan, 40% of p1 lacking the Asn-linked glycan, and 10% of hCG-p1 lacking both Asn- and Asn-linked glycans, were able to fold into the corresponding p2 (19) . The discrepancy in the extent of folding of the various subunit types in vitro and in vivo may be due to more optimal conditions such as a favorable redox potential and the presence of molecular chaperones that increase hCG--folding efficiency inside cells.

It should be noted that folding of the subunit and its assembly with the subunit are completed within the ER while the subunit contains high mannose type N-linked oligosaccharides. Processing of the N-linked glycans to complex oligosaccharides and addition of O-linked oligosaccharides occur in the Golgi after assembly and translocation from the ER. Also, unglycosylated secreted from CHO cells transfected with genes mutated in the Asn codons of the N-linked glycan consensus sequences does become O-glycosylated (data not shown). All of the disulfide bonds are formed in when it is secreted as dimer from CHO cells cotransfected with the Asn mutant and genes. This was demonstrated by the absence of trypsin-releasable peptides digested under nonreducing conditions by the methods previously described (6, 8) .

Huth et al.(26) have suggested that hCG- does not fold by a simple sequential pathway and that folding occurs independently in different domains of the molecule. The disulfide bonds of the putative domain(s) involving amino acids 1-90 of hCG- form in a discrete order. However, the later forming disulfide bonds 93-100 and 26-110 begin to form before the complete formation of the early forming disulfide bonds that stabilize the amino acid 1-90 domain(s) and continue to form after complete formation of the early forming disulfide bonds. We have also shown that completion of the 26-110 disulfide bond of does not occur until after assembly (26) , and indeed, if it is preformed by complete oxidation in vitro, will not assemble with (8) . This is consistent with the crystal structure of the hCG- dimer in that the C terminus of forms a seatbelt around (28) . This could only occur if the 26-110 disulfide bond is formed after assembly.

The two N-linked (Asn and Asn) oligosaccharides may protect critical folding domains of the molecule until proper folding occurs. This may account for the deficiency of formation of disulfide bonds 93-100 and 26-110 in the absence of both N-linked oligosaccharides. The crystal structure of hydrofluoric acid-treated hCG- (28, 29) suggests that in an early folding intermediate the N-linked glycans may be located between two important folding loops that are held together by disulfide bond 23-72. The N-linked glycans may protect important hydrophobic residues in this region (such as Leu, Val, Ile, and Val) and assist disulfide 23-72 formation, which occurs before completion of disulfide bonds 93-100 and 26-110. Thus, the instability of 93-100 and 26-110 disulfide bonds may result from less efficient formation of disulfide bond 23-72.

The data also suggest that molecular chaperones are involved in the folding of unglycosylated hCG-. At least two classes of proteins, widely distributed in prokaryotes and eukaryotes, are involved in polypeptide folding (18) . These proteins include: 1) enzymes such as peptidylprolyl cis/trans-isomerase and protein disulfide isomerase that catalyze specific rate-limiting isomerization steps in protein folding; 2) binding proteins (molecular chaperones) that stabilize unfolded or partially folded structures and prevent the formation of inappropriate intra- or interchain interactions. The molecular chaperones, composed of two major families (stress-70 and stress-90) proteins in eukaryotic cells, are induced under a variety of stress conditions and function in the stabilization, translocation, and degradation of partially folded intermediates during polypeptide folding and assembly. ERp72 has been identified as an ER protein containing protein disulfide isomerase homology units (31) . BiP is a member of the stress-70 chaperone family in the ER of eukaryotic cells. It has been proposed that BiP associates transiently with unfolded or misfolded proteins to modulate protein folding and translocation of folding intermediates across the ER membrane (18) . ERp94, a member of stress-90 chaperones, appears to function with BiP to assist protein folding in the ER lumen (32) . In addition, calnexin (also called p88, IP90), an ER trans-membrane protein, also plays a chaperone-like role by binding with glucose-containing N-linked glycans of newly synthesized glycoproteins (33) .

Since intracellular folding of unglycosylated hCG- is inefficient but does occur slowly, accumulation of unfolded or misfolded hCG- might stimulate the formation of slowly dissociated unglycosylated -chaperone complexes. The C1 and C2 peaks observed in Fig. 5contain at least seven proteins with a variety of molecular weights, some of which have been identified by Western blots as the ER chaperones BiP, ERp72, and ERp94. Both the C1 and C2 peaks disappear with increasing chase time up to 5 h, during which time a greater amount of folded p2 appears (Fig. 5, D-F) and some of the d degradation product is evident as well. This strongly suggests that the complexes contained in the C1 and C2 peaks are not dead-end folding complexes targeting for degradation, but rather are chaperone- complexes that assist subunit folding. Therefore, the slower folding rate of unglycosylated may have enabled us to detect such complexes that are transiently formed in the folding pathway of wild-type . This hypothesis is currently being tested in our laboratory.

Some intracellular degradation of unglycosylated , however, eventually occurs in CHO cells ( Fig. 8and Fig. 9). This appears to be derived from p2 forms that are unstable without N-linked glycans since that is the form of mainly present after a 5-h chase when the degradation product d begins to appear (Fig. 8). There is some precedent for this based on observations with other glycoproteins. Hoe and Hunt (34) reported that human transferrin receptor lacking N-linked oligosaccharide is unable to form intermonomer disulfide bridges and undergoes site-specific proteolysis after a long period of time in the ER lumen. It was also shown that unglycosylated subunits of thyroid-stimulating hormone are 50-65% degraded intracellularly (35) . Furthermore, unglycosylated vesicular stomatitis virus G protein forms non-native intramolecular disulfide bonds and is degraded intracellularly (22) .

We have shown that the d degradation product of unglycosylated is a core-like fragment. The core fragment, which contains the and the portions of subunit linked by disulfide bonds, is a major immunoreactive component in the urine of pregnant women (30, 36) . This fragment has been considered as a partially degraded subunit of hCG produced in the kidney (37) . The C-terminal region of is susceptible to degradation and is cleaved free from a disulfide-bridged core product. Also, the region between residues 40 and 50 is highly susceptible to proteolysis (13, 30) . It seems that the N-linked glycans shield the protease-cleavage sites in hCG- or that the conformation of misfolded or unfolded unglycosylated hCG- is more accessible to endopeptidases.

  
Table: Trypsin-released peptides from reduced hCG-p2 and core fragment-like protein

The [S]cysteine- or [³H]leucine-labeled hCG-p2 and core fragment-like protein were reduced and digested with trypsin. The tryptic peptides were separated on HPLC (see ``Experimental Procedures''). The peaks were numbered in the order in which they appear in the HPLC profiles (Fig. 10, A and B). The peaks contained the peptides identified previously (6, 7) or in Fig. 10, E and F. It should be noted that the incomplete digestion at tryptic site between Arg and Leu results in the appearance of (peak 6) and that the proteolytic clip of the Leu-Ser peptide bond produces peptides and (peak 5) that are derived from (peak 8) (6, 7).



FOOTNOTES

*
This work was supported in part by National Cancer Institute Grant CA32949 (to R. W. R.), Cancer Center Support Grant P30 CA36727 to the Eppley Institute, and National Institutes of Health Grant HD23398 (to I. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Current address: Depts. of Pathology, Cell Biology, and Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030.

To whom correspondence should be addressed: The Eppley Institute for Research in Cancer and Allied Diseases, The University of Nebraska Medical Center, 600 S. 42nd St., Omaha, NE 68198-6805. Tel.: 402-559-4238; Fax: 402-559-4651.

¹
The abbreviations used are: hCG, human chorionic gonadotropin; WT, wild type hCG- gene product; Asn1, hCG- lacking Asn; Asn2, hCG- lacking Asn; Asn(1+2), hCG- lacking both Asn and Asn; CHO, Chinese hamster ovary; ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; IOD, integrated optical density.


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