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Molecular Endocrinology, doi:10.1210/me.2007-0051
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Molecular Endocrinology 21 (10): 2551-2564
Copyright © 2007 by The Endocrine Society

Rapid Maturation of Glycoprotein Hormone Free {alpha}-Subunit (GPH{alpha}) and GPH{alpha}{alpha} Homodimers

Jean-Michel Krause1, Peter Berger1, Jordi Roig, Vinod Singh and Wolfgang E. Merz

Heidelberg University Biochemistry Center (BZH) (J.-M.K., J.R., V.S., W.E.M.), 69120 Heidelberg, Federal Republic of Germany; Institute for Biomedical Aging Research, Austrian Academy of Sciences (P.B.), Innsbruck A6020, Austria; and Hormone Biochemistry Laboratory, Institute of Self Organizing Systems and Biophysics (V.S.), North-Eastern Hill University, Permanent Campus, Shillong 793022, Meghalaya, India

Address all correspondence and requests for reprints to: Wolfgang E. Merz, Ph.D., Heidelberg University Biochemistry Center (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, Federal Republic of Germany. E-mail: wolfgang.merz{at}bzh.uni-heidelberg.de.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The dynamics of glycoprotein hormone {alpha}-subunit (GPH{alpha}) maturation and GPH{alpha}{alpha} homodimer formation were studied in presence (JEG-3 choriocarcinoma cells) and absence (HeLa cells) of hCGß. In both cases, the major initially occurring GPH{alpha} variant in [35S]Met/Cys-labeled cells carried two N-glycans (Mr app = 22 kDa). Moreover, a mono-N-glycosylated in vivo association-incompetent GPH{alpha} variant (Mr app = 18 kDa) was observed. In JEG-3 cells the early 22-kDa GPH{alpha} either associated with hCGß, or showed self-association to yield GPH{alpha}{alpha} homodimers, or was later converted into heavily glycosylated large free GPH{alpha} (Mr app = 24 kDa). Micro-preparative isolation of intracellular GPH{alpha}{alpha} homodimers of JEG-3 cells and their conversion by reduction revealed that they consisted of 22-kDa GPH{alpha} monomers and not of large free GPH{alpha}. In HeLa cells, the large free GPH{alpha} variant was not observed, whereas GPH{alpha}{alpha} homodimers were present. Intracellularly, early GPH{alpha}{alpha} homodimers (35 kDa) and late variants (JEG-3: 44 kDa, HeLa: 39 kDa) were found. Both cell types secreted 45 kDa GPH{alpha}{alpha} homodimers. Large free GPH{alpha} and GPH{alpha}{alpha} homodimers were more rapidly sialylated than hCG {alpha}ß-heterodimers indicating a sequestration mechanism in the secretory pathway. In GPH{alpha}{alpha} homo- as well as hCG {alpha}ß-heterodimers the subunit interaction site, located on loop 2 of GPH{alpha} (amino acids 33–42), became immunologically inaccessible indicating similar spatial orientation of GPH{alpha} in both types of dimers. The studies demonstrate the formation, in vivo dynamics of GPH{alpha}{alpha} homodimers, and the pathways of the cellular metabolism of variants of GPH{alpha}, monoglycosylated GPH{alpha} and large free GPH{alpha}.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GLYCOPROTEIN HORMONES such as placental human chorionic gonadotropin (hCG) are composed of a common {alpha}-subunit, designated as hCG{alpha} or glycoprotein hormone {alpha}-subunit (GPH{alpha}) and a hormone-specific ß-subunit (hCGß) (1, 2). Physiologically, hCG is predominantly produced by syncytiotrophoblastic cells of the placenta; however, the free subunits as well as hCG itself also occur ectopically synthesized by nonmalignant tissues, trophoblastic, and nontrophoblastic tumors (3, 4, 5, 6, 7). Most tumors and hCG-producing cell lines show an excessive production of GPH{alpha} (8, 9). Moreover, hCG and in particular GPH{alpha} is produced in the reproductive tract of men (10) and in cases of chronic hypergastinemia (11).

It was hypothesized that free GPH{alpha} could have biological activities of its own in decidualization by supporting endometrial prolactin biosynthesis and release synergistically with progesterone (12, 13). Additionally, GPH{alpha} seems to exert trophic effects on the development of the rat pituitary (14), which possibly explains why differentiation of lactotrophes was specifically blocked by antisera against GPH{alpha} (15). Recently, a connection between the differentiation of human prostate stromal cells and the production of GPH{alpha} was shown (16).

GPH{alpha} occurs in numerous molecular variants, for example GPH{alpha} as part of the hCG {alpha}ß-heterodimers, the large free heavily glycosylated GPH{alpha}, monoglycosylated GPH{alpha}, and GPH{alpha}{alpha} homodimers. The formation as well as functional and diagnostic significance of these variants is still not clear. Large free GPH{alpha} (apparent molecular mass, Mr app = 24 kDa) is unable to associate with ß-subunits (17). This was attributed to an altered amino acid (aa) composition or to additional O-glycosylation at GPH{alpha}-Thr39 (discussed in Ref. 18). However, subunit association takes place in the endoplasmic reticulum (ER), whereas the O-glycans are attached later in the Golgi apparatus a fact that argues against the role of the Thr39 O-glycan as an obstacle for subunit association. Detailed studies of the N-linked glycan structures showed that large free GPH{alpha} contained a higher carbohydrate content of increased complexity. This observation accounts for its higher apparent molecular mass and for its inability to associate with the glycoprotein hormone ß-subunits (18, 19, 20).

The glycan residues of the hCG subunits, in particular those of GPH{alpha}, have different relevance for the physical stability of GPH{alpha}, for intramolecular interactions between the hCG subunits, the stability of hCG {alpha}ß-heterodimers, and for folding, secretion, and receptor activation (21, 22, 23, 24, 25). GPH{alpha} possesses two N-glycosylation sites, at Asn52 and Asn78 (26, 27). During hCG biosynthesis, the GPH{alpha} peptide loop L2 including the glycan residue attached to Asn52 passes through the tethered seat-belt structure formed by hCGß (25, 28). The GPH{alpha} Asn52 glycan directly interacts with hCGß residues Tyr59, Val62, Phe64, Ala83, and Thr97 and is located in a highly ordered environment as concluded from the crystal structure of hCG (29, 30). However, as observed on the basis of 1H- and 13C-NMR spectroscopy, the region of aa 33–58 of isolated GPH{alpha} seems to form a large disordered loop in solution except a small helical region (31, 32). The Asn52 glycan including the GlcNAc-1 is highly flexible and seems to influence neither the subunit structure nor the stabilization of hCG {alpha}ß-heterodimers (25). However, the Asn52 glycan possibly plays an important role in inducing and stabilizing a conformational change of hCG upon receptor binding (25) and seems to interact directly with the LH/hCG receptor via a hypothetical lectin-like domain of the receptor (33). Computer simulations of the structural dynamics of GPH{alpha} also showed a high flexibility of aa GPH{alpha}33–55 (34). In contrary, the GlcNAc-1 of Asn78 glycan shows 30 intra-subunit 1H-NOEs (Nuclear Overhauser Effects) with protons of the subunit amino acid residues (31, 35), indicating close glycan-protein interactions which result in restricted flexibility of this region (36).

The interactions between the glycan residues and the protein part are of particular interest because they influence subunit folding, maturation, and also the reaction with components of the ER quality control machinery. We describe here, the dynamics during the folding and processing steps of the various GPH{alpha} variants and GPH{alpha}{alpha} homodimers in JEG-3 cells (hCGß present) and HeLa cells (missing hCGß). In contrast to hCGß GPH{alpha} has the tendency to form firmly linked GPH{alpha}{alpha} homodimers obviously using the same GPH{alpha} domain as inter-subunit contact area in the homodimer that is involved in binding of hCGß in hCG {alpha}ß-heterodimers. GPH{alpha} homodimer formation seems to take place between two free nonassociated 22-kDa GPH{alpha} molecules and neither by association of monoglycosyl-GPH{alpha}, nor of large free GPH{alpha} (24 kDa). During biosynthesis, the GPH{alpha}{alpha} homodimers as well as the large free GPH{alpha} are sequestered from the hCG {alpha}ß-heterodimers and are processed in the secretory pathway in a fast-track manner resulting in early sialylation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Dynamics of GPH{alpha} Maturation in Presence of hCGß
The antibodies used and the presumptive localization of the corresponding immunological epitopes are depicted in Fig. 1Go. All antibodies used are dependent on the correct molecular conformation of recognized mature epitopes, i.e. they do not react with completely unfolded subunits. However, it is unknown to what extent they recognize partially folded structures, which are expected to occur during folding and maturation of the hCG subunits. The dynamic development of GPH{alpha} variants in JEG-3 cells during the course of a pulse-chase experiment is shown in Fig. 2Go. After immunoprecipitation of the radioactively labeled proteins in the cell lysates with GPH{alpha}-specific monoclonal antibodies (mAbs) and separation by SDS-PAGE an intensive signal for GPH{alpha} with an apparent molecular mass (Mr app) of 22 kDa (nonreduced Fig. 2Go, upper panels) and a smaller GPH{alpha} variant (Mr app = 18 kDa) were observed (0 min chase, lanes 1 and 2). This variant represents a mono-N-glycosylated GPH{alpha} (as indicated below). It decreased with increasing chase time and was undetectable in the culture medium (see panel B). A mAb directed against hCGß (code INN-hCG-22; epitope ß2) detected coprecipitated GPH{alpha}, a faint 28- to 30-kDa double band of hCGß (lane 3), and traces of some material with higher apparent molecular mass. In the first 60 min of chase the GPH{alpha} variant with a Mr app = 22 kDa decreased to 21 kDa in the nonreduced form (not shown) and increased again to 22 kDa before secretion, most likely due to processing of the glycan residues in the ER and the Golgi apparatus. A further intracellular GPH{alpha} variant (Mr app = 24 kDa) appeared during the first hour of chase that was also secreted into the culture medium. In the same time interval intracellular 35- and 44-kDa GPH{alpha} variants were observed (lanes 4 and 6). They were precipitated by a mAb (code: INN-FSH-179) directed against epitope {alpha}3* presumably located on the top of peptide loop L3 but not by a mAb INN-hCG-80 directed against the subunit interaction site (epitope {alpha}6, aa GPH{alpha}33–42; see lanes 5 and 7). The asterisk in {alpha}3* refers to the reaction of the mAb INN-FSH-179 with a structure of epitope {alpha}3 in comparison to the reaction of the {alpha}3-specific mAb INN-FSH-13. This mAb selectively recognizes free GPH{alpha} but not GPH{alpha} assembled in hCG {alpha}ß-heterodimers. As described below, the 35-kDa band represents a mixture of comigrating immature GPH{alpha}{alpha} homodimers and mature hCGß coprecipitated from hCG {alpha}ß-heterodimers which appeared with different schedules. The 44-kDa species represented mature GPH{alpha}{alpha} homodimers (see below).


Figure 1
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  		Fig. 1. Localization of a Selection of Immunological Epitopes on the Three-Dimensional Structure of GPH{alpha} (A) and hCGß (B) as Recognized by Monoclonal Antibodies

Residues depicted in the space-fill mode represent amino acids that contribute substantially to a particular immunological epitope but do not necessarily represent the entire extension of the epitope. The models were created by means of Rasmol using the x-ray data for hCG (29 ). H- and -OH stands for the amino- and carboxyl termini of the modified hCG subunits used for establishment of the x-ray structure. The Asn residues that serve as linkage sites for the N-glycans are shown in magenta. The diacetyl-chitobiosyl units (GlcNAc)2 representing the first two sugar residues of the N-glycans linked to the respective Asn residues are shown in cyan. A, Different mAbs were used to investigate the dynamic development of GPH{alpha} variants: INN-hFSH-73 (epitope {alpha}1), INN-hFSH-100 (epitope {alpha}2), INN-hFSH-98 (epitope {alpha}2), INN-hFSH-13 (epitope {alpha}3), INN-hFSH-179 (epitope {alpha}3*), INN-hFSH-132 (epitope {alpha}4), INN-hFSH-158 (epitope {alpha}5), INN-hCG-80 (epitope {alpha}6) (68 69 ). B, The immunological epitope cluster on the tips of peptide loops 1 and 3 of the hCGß subunit comprising epitopes ß2, ß4, and ß5 is shown. HCGß was investigated by mAb INN-hCG-22 (epitope ß2) and an affinity purified polyclonal anti-hCGß rabbit antiserum (Materials and Methods).

 

Figure 2
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  		Fig. 2. The Dynamics of GPH{alpha} Variants and GPH{alpha}{alpha} Homodimers

JEG-3 cells were labeled with a 10-min [35S]Met/Cys pulse and chased for the given time. A, Lysates were immunoprecipitated with mAbs recognizing either all GPH{alpha} variants (pan-GPH{alpha} mAb code: INN-hFSH-179 directed against epitope {alpha}3* on peptide loop 3; lanes 1, 4, 6, 8, and 10) or a mAb selectively recognizing only free, i.e. nonassembled GPH{alpha} via the subunit interaction site (INN-hCG-80, epitope {alpha}6, aa hCG{alpha}33–42, lanes 2, 5, 7, 9, and 11). Variants of hCGß were immunoprecipitated with a mAb (INN-hCG-22, reacting with epitope ß2 located on top of the 2 adjacent peptide loops 1 and 3; lanes 3 and 12) or with an affinity-purified polyclonal anti-hCGß (lane 13). The immunopurified proteins were separated via SDS-PAGE using nonreducing (upper panel) and reducing conditions (lower panel). The secreted GPH{alpha} variants contained in the cell culture medium immunoprecipitated with the antibodies indicated (B). For fluorography, the electrophoreses gels were exposed to x-ray films for 15 d. Besides the prominent 22-kDa GPH{alpha} band a monoglycosyl variant (Mr app = 18 kDa) was present intracellularly at the beginning (lanes 1, 2, 4, and 5) and disappeared later. The large free GPH{alpha} monomer (Mr app = 24 kDa), intracellular immature GPH{alpha}{alpha} (Mr app = 35 kDa) and intracellular mature GPH{alpha}{alpha} homodimers (Mr app = 44 kDa) emerged with time. Mature GPH{alpha}{alpha} homodimers showed a Mr app = 45 kDa after secretion into the culture medium. The pan-GPH{alpha} mAb coprecipitated also hCGß originating from hCG {alpha}ß-heterodimers (Mr app = 35 kDa).

 
Intracellular immature 22-kDa GPH{alpha} was almost equally recognized by {alpha}3* and {alpha}6 epitope-specific mAbs (see lanes 4–7). However, from the culture medium significantly less of the 22-kDa GPH{alpha} variant was precipitated or could be not detected by the free GPH{alpha}-specific mAb (detecting epitope {alpha}6) (Fig. 2BGo, lanes 9 and 11). In contrast, a pan-GPH{alpha} mAb directed against epitope {alpha}3* (code INN-hFSH-179) as well as hCGß-specific mAbs and polyclonal antibodies were able to coprecipitate the secreted 22-kDa GPH{alpha} variants indicating that the bulk of this material consisted of the GPH{alpha} part of hCG {alpha}ß-heterodimers (see also below).

hCG {alpha}ß-Hetero- and GPH{alpha}{alpha} Homodimers
The desorption of the immune complexes from the Protein A-agarose during immunopurification caused the complete dissociation of all immune complexes as well as of immature and mature hCG {alpha}ß-heterodimers. In contrary, GPH{alpha}{alpha} homodimers were not dissociated under these conditions and appeared in JEG-3 cells initially as immature 35-kDa variants that were then converted into 44-kDa variants before secretion (Fig. 2AGo, lanes 6 and 8). In the culture medium, the GPH{alpha}{alpha} homodimers were detectable as 45-kDa variants (Fig. 2BGo, lanes 8 and 10, upper panels). They dissociated into monomers when the samples were separated in SDS-PAGE under reducing conditions (Fig. 2BGo, lanes 8 and 10, lower panels; see also below).

hCG {alpha}ß-heterodimers immunoprecipitated by the hCGß-specific antibodies were dissociated in the subsequent desorption step into coprecipitated GPH{alpha} variants (Mr app = 22 kDa) and hCGß (Mr app = 35 kDa) (Fig. 2Go, lanes 3, 12, and 13). hCGß appeared in a number of molecular variants of which a 28- to 30-kDa double band and a 35-kDa variant were the most prominent (see below). The 35-kDa variant corresponded to sialylated mature hCGß, which first appeared after 40 min of chase (37). In analogy hCGß contained in hCG {alpha}ß-heterodimers was coprecipitated by the pan-GPH{alpha} mAb (INN-hFSH-179, epitope {alpha}3*). This suggested that the 35-kDa band may contain two molecules comigrating in SDS-PAGE, immature GPH{alpha}{alpha} homodimers (predominantly present at chase times < 30 min) and coprecipitated mature hCGß from hCG {alpha}ß-heterodimers (at chase times > 40 min). However, in experiments with a short pulse and chase times below 40 min the 35-kDa band contained only GPH{alpha}{alpha} homodimers (see below).

Immature GPH{alpha}{alpha} homodimers and mature hCGß could be discriminated not only by the different time-course of their appearance but also by reaction with subunit-specific antibodies. The evidence for the nature of the 35-kDa GPH{alpha}{alpha} homodimer band was verified by micro-preparative isolation by means of the Whole Gel Eluter, characterization in Western blots, and dissociation into the monomers by reduction (Fig. 3Go). Pulse-labeled JEG-3 cell lysates were incubated with the mAb against epitope a3* (INN-hFSH-179). The immunoprecipitated proteins served as starting material for the micro-preparative isolation of single bands by means of the Whole Gel Eluter after SDS-PAGE (1-mm gel, 50-mm slot filled with sample). After isolation in the Whole Gel Eluter the fractions were concentrated and applied to SDS-PAGE for detection. Early synthesized GPH{alpha} variants (Mr app = 22 kDa), GPH{alpha}{alpha} homodimers (Mr app = 35 kDa) and coprecipitated immature 28 kDa hCGß variant were detectable in unconcentrated starting material. They were present as single bands, except the 18 kDa variant of which the concentration was too low. Reduction of the 35-kDa band (Fig. 3BGo, lanes 1 and 2) resulted in a shift to the 22-kDa GPH{alpha} variant (Fig. 3AGo, lanes 1 and 2) strongly indicating that the 35-kDa variant solely represented GPH{alpha}{alpha} homodimers. As expected, the coprecipitated 28-kDa hCGß variant was shifted to a Mr app of 25 kDa in response to reduction (compare Fig. 3Go, A and B, lane 3). The smear above the 22-kDa GPH{alpha} band in lane 4 (panels A and B) is probably a first intimation of the large free GPH{alpha}. In Western blots (C and D) the 22- and the 35-kDa bands could be clearly identified as GPH{alpha} and GPH{alpha}{alpha} homodimer variants (Fig. 3CGo, lanes 4 and 5 and 1 and 2), whereas the 28- to 30-kDa double band reacted only with the mAb directed against hCGß (INN-hCG-22, epitope ß2; panel D, lanes 1–3). Signal intensities in the Western blots were higher than those of the radioactively labeled proteins due to detection in Western blots of the entire intracellular pools of the hCG subunits (early immature up to late mature forms). In contrast, in pulse-labeling experiments only newly synthesized hCG subunit variants are detected. Therefore, the material in lanes 1 and 2 of panel D, that represents coprecipitated hCGß originated from nonradioactive hCG {alpha}ß-heterodimers already present in the cells at the time of pulse-labeling, is not visible in panels A and B.


Figure 3
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  		Fig. 3. Identification of Intracellular GPH{alpha}, hCGß, and GPH{alpha}{alpha} Homodimer Variants

JEG-3 cells were pulse-labeled for 10 min and chased 5 min. The lysate was immunoprecipitated with a mAb directed against all GPH{alpha} variants (code: INN-FSH-179, directed against epitope {alpha}3*). This unconcentrated material that served as origin for subsequent separations is shown on the right hand site of panels A–D (designated as starting material). Immunoprecipitated proteins were separated in SDS-PAGE applying micro-preparative nonreducing conditions (1 mm polyacrylamide gel, sample slot of 50 mm width). Individual bands were isolated by means of electroelution using the Whole Gel Eluter and collected in fractions. The fractions were concentrated (500 µl Vivaspin concentrators) and applied to SDS-PAGE (lanes 1–5) under reducing (A) and nonreducing conditions (B), respectively, to monitor the radioactively labeled proteins by fluorography (A and B). In parallel, the fractions were separated by SDS-PAGE under nonreducing conditions and blotted to polyvinylidene difluoride membranes to identify the isolated single proteins by means of the Western blot technique (C and D). Western blot signals were visualized by means of an ECL enhancer kit (C and D). GPH{alpha} variants were detected in the Western blot by reaction with a mAb recognizing all GPH{alpha} variants [INN-FSH-179 (1:3000 dilution; panel C)]. Variants of hCGß coprecipitated from hCG {alpha}ß-heterodimers were visualized by reaction with a mAb detecting all hCGß variants [INN-hCG-22 MCA; epitope ß2; 1 µg/ml) as first antibody, see panel D)]. The second antibody was horseradish peroxidase-labeled antimouse IgG (1:20,000 dilution). Exposure time of the Western blot to the films was 10 min. The stronger bands in the Western blots (C and D) compared with the radioactive signals (A and B) are due to detection of the total intracellular pool of subunit variants by Western blotting but detection of only newly synthesized variants by pulse-labeling. The GPH{alpha} variants precipitated with the pan-GPH{alpha} mAb (code: INN-FSH-179) in the nonreduced samples (B) were the 18-kDa monoglycosyl subunit (monogly-GPH{alpha}), the prominent 22-kDa GPH{alpha} (two N-linked glycans) and the immature 35-kDa GPH{alpha}{alpha} homodimers. When reduced the GPH{alpha}{alpha} dissociated into its monomers (compare lanes 1 and 2 in A and B). The anti-GPH{alpha} mAb precipitated also hCGß variants (Mr app = 28 and 30 kDa) from hCG {alpha}ß-heterodimers.

 
Monoglycosyl GPH{alpha}
N-glycans of the intracellular hCG subunit variants could be removed completely when digested with peptide N-glycanase F (PNGase F) in the presence of ß-mercaptoethanol or dithiothreitol. In SDS-PAGE performed under reducing conditions the mature 35-kDa hCGß variant shifted to 26 kDa caused by the enzymatic release of the N-glycans (de-N-gly-hCGß; O-glycans still present). The reduced immature hCGß variants (Mr app = 25 kDa) were converted to a band of 18 kDa (Fig. 4Go, lane 2). The reduced 22-kDa GPH{alpha} variants (GPH{alpha}{alpha} homodimers dissociated into monomers) showed a shift to 15 kDa. However, when the enzymatic deglycosylation was carried out in the absence of a reducing agent the hCGß variants with Mr app = 35 kDa and Mr app = 25 kDa (Mr app of reduced hCGß) remained undigested whereas one N-glycan residue was removed by the peptide N-glycanase from the 22-kDa GPH{alpha} variant yielding a monoglycosyl GPH{alpha} variant with a Mr app of 18 kDa (lane 3). Redigestion with PNGase F under reducing conditions of these subunit forms after isolation of individual bands with the Whole Gel Eluter (lanes 5 and 6) caused a shift of the partially deglycosylated GPH{alpha} Mr app = 18-kDa variant to the fully deglycosylated Mr app = 15-kDa subunit (lane 7). Redigestion of isolated immature hCGß (Mr app = 25 kDa, reduced) resulted in the Mr app = 18-kDa variant devoid of both N-linked carbohydrate moieties (lane 4). The 18-kDa GPH{alpha} variant as observed above during biosynthesis was also shifted to the 15-kDa deglycosylated GPH{alpha} in response to PNGase F digestion indicating that the 18-kDa variant represents a monoglycosyl GPH{alpha} (data not shown).


Figure 4
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  		Fig. 4. Partial and Complete Enzymatic Deglycosylation of Intracellular hCG Subunit Variants

JEG-3 cells were labeled for 45 min with [35S]Met/Cys and chased for 10 min to obtain a broad spectrum of subunit variants. Immunoprecipitation of mature secretion-competent hCG and hCGß variants was carried out with an immunopurified polyclonal anti-hCGß antibody. All samples were separated by means of SDS-PAGE under reducing conditions irrespective of the conditions used for the enzymatic deglycosylation. Radioactive signals were visualized by fluorography. Immunopurified samples were digested with PNGase F in presence (lane 2) or absence (lane 3) of 1.3 mM ß-mercaptoethanol (A). Lane 1, undigested control. Digestion in the presence of ß-mercaptoethanol converted the hCG subunits almost completely into the N-deglycosylated forms (lane 2). In particular, digestion with PNGase F caused a shift of the mature, secretion-competent free hCGß variant from a Mr app = 35 to 26 kDa (de-N-gly-hCGß; N-glycans removed, O-linked glycans still present). Immature hCGß (not yet equipped with the O-glycan residues) shifted from 25 kDa (Mr app of reduced hCGß) to 18 kDa (degly-hCGß), the GPH{alpha} variants from 22 to 15 kDa. Enzymatic digestion in the absence of a reducing agent led to partial deglycosylation (lane 3). The hCGß variants (35-kDa mature hCGß, 25-kDa immature hCGß) were resistant against digestion under these conditions. However, digestion of the GPH{alpha} induced a shift of the bulk of the subunit from 22 to 18 kDa (monogly-GPH{alpha}). The individual bands obtained by partial digestion in absence of ß-mercaptoethanol (lane 3) were subsequently isolated by means of the Whole Gel Eluter (B, lanes 5 and 6). The isolated bands were redigested with PNGase F in the presence of ß-mercaptoethanol (lanes 4 and 7). Thereby, the immature 25-kDa hCGß variants were converted into an 18-kDa variant devoid of the N-linked carbohydrate residues (degly-hCGß). The isolated 18-kDa monoglycosylated GPH{alpha} (lane 6) yielded deglycosylated 15-kDa GPH{alpha} when redigested under reducing conditions (degly-GPH{alpha}, lane 7).

 
The monoglycosyl 18-kDa GPH{alpha} variant was sensitive to digestion with endoglycosidase H, indicating that it represented a mannose-rich form that had not reached the medial Golgi. Moreover, at 5 min of pulse-labeling all GPH{alpha} variants (nonassembled as well as those already associated with hCGß) and hCGß itself were still endoglycosidase H-sensitive (data not shown) and have not reached the medial Golgi at that time point. The apparent molecular masses of the endoglycosidase H-digested subunits were slightly larger (15.7 kDa for GPH{alpha}- and 18.7 kDa for hCGß) than in the case of digestion with PNGase F due to the fact that one N-acetyl-glucosaminyl residue per glycan residue (two/subunit) remained on the protein backbone after endoglycosidase H digestion.

GPH{alpha} Maturation and GPH{alpha}{alpha} Homodimer Formation in the Absence of hCGß
In HeLa cells large amounts of the GPH{alpha} are expressed in response to stimulation with butyrate, whereas little or no hCGß is synthesized. Our attempts to immunoprecipitate radioactively labeled material with monoclonal or polyclonal anti-hCGß-specific antibodies failed (data not shown). HeLa cells were used to determine which GPH{alpha} variants are present when hCG {alpha}ß-heterodimer formation is prevented due to the absence of the hCGß-subunit. Labeling kinetics were performed with [S35]Met/Cys pulses (1, 2, 5, 10, 15, 30 min). Chase intervals at each pulse condition of 5, 15, 30, and 60 min were applied. Representative parts of these experiments are depicted in Fig. 5Go. Four different intracellular GPH{alpha} variants with Mr apps of 18, 22, 35, and 39 kDa were detected (SDS-PAGE under nonreducing conditions). Interestingly, the large free GPH{alpha} (Mr app = 24 kDa) as observed in JEG-3 cells was absent in HeLa cells. The 35- and 39-kDa variants were sensitive to reduction and collapsed into bands of 22 kDa. The 35- and 39-kDa bands appear to be immature GPH{alpha}{alpha} homodimer variants. The 39-kDa signal appeared late during the chase interval suggesting that the 39-kDa GPH{alpha}{alpha} homodimers in HeLa cells represent a presecretion form of mature GPH{alpha}{alpha} homodimers like the 44-kDa variants in JEG-3 cells. In both cell types GPH{alpha}{alpha} homodimers were secreted into the culture medium as 45-kDa variants. The GPH{alpha} secreted by HeLa cells showed a Mr app of 25 kDa (Fig. 5CGo, nonreduced). The 45-kDa GPH{alpha}{alpha} homodimers were converted into 25-kDa GPH{alpha} monomers in response to reduction (see Fig. 5CGo, reduced). Differences in the Mr apps of intracellular and GPH{alpha} variants secreted by HeLa cells vs. the variants found in the case of JEG-3 cells seem to be due to differences in glycosylation. This was suggested by the observation that in tunicamycin-treated cultures a N-glycan-free GPH{alpha} with a Mr app of 15 kDa was the only species found when analyzed under reducing conditions (data not shown). A direct quantitative comparison of the radiolabeled bands between GPH{alpha}{alpha} homodimers in JEG-3 and HeLa cells was not possible. However, increased synthesis of the GPH{alpha}{alpha} homodimers in HeLa cells indicates that it is dependent on the concentration of the free 22-kDa GPH{alpha}. This hypothesis was supported by a significant decrease in the concentration of GPH{alpha}{alpha} homodimers in hCGß-transfected HeLa cells (data not shown).


Figure 5
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  		Fig. 5. Early Intracellular GPH{alpha}, GPH{alpha}{alpha} Homodimers, and Secreted Variants in the Absence of hCGß

Butyrate-stimulated HeLa cells (no hCGß expression) were pulse-labeled for 1, 2, 5, 10, 15, and 30 min with [35S]Met/Cys and chased for the time indicated (5, 15, 30, and 60 min). Representative parts of the experiment showing the intracellular GPH{alpha} variants after pulse-labeling [10 min (A) and 30 min (B)] are depicted. C, The GPH{alpha} and GPH{alpha}{alpha} homodimer variants secreted into the cell culture medium after 10-min pulse-labeling and 60 min chase are shown, separated by SDS-PAGE under nonreducing and reducing conditions. Immunoprecipitations were carried out with a mAb recognizing all GPH{alpha} variants (INN-FSH-132, epitope {alpha}4). Radioactive signals were visualized by fluorography. The intracellular GPH{alpha} variants were basically the same as in the JEG cells, an 18-kDa monoglycosyl variant that persisted up to 30 min of chase, a prominent 22-kDa form, and two GPH{alpha}{alpha} homodimer variants (35 and 39 kDa). The late precursor of secreted GPH{alpha}{alpha} homodimers was smaller in size than in the JEG-3 cells (39 vs. 44 kDa). The GPH{alpha}{alpha} homodimers secreted by HeLa cells into the culture medium had the same Mr app (45 kDa) as in JEG cells in the nonreduced form and were converted into the monomers in response to reduction.

 
Immunological Epitopes of Microheterogeneous Isoforms hCG Subunits
GPH{alpha} subunit-specific mAbs were tested for reactivity with the intracellular GPH{alpha} and GPH{alpha}{alpha} homodimer variants. Comparable amounts of early free intracellular GPH{alpha} (Mr app = 22 kDa) as well as GPH{alpha} assembled in the hCG {alpha}ß-heterodimers were immunoprecipitated from the same starting material (JEG-3 cells, 30-min pulse, 10-min chase) by a panel of 8 mAbs recognizing different epitopes on GPH{alpha} (Fig. 6AGo). Early GPH{alpha}{alpha} homodimers (Mr app = 35 kDa) were immunoprecipitated by all mAbs except the mAb specific for free GPH{alpha} (INN-hCG-80, directed against epitope {alpha}6). The ability of the anti-GPH{alpha} antibodies to react with hCG {alpha}ß-heterodimers and therefore to coprecipitate hCGß varied and was in some cases completely absent (compare lanes anti-{alpha}1, -4, -6). Interestingly, mature hCG as standard substance was detected by the mAb directed against the {alpha}4 epitope when used in different in vitro tests (data not shown). This indicates marked differences between immature and mature hCG subunits, which may be used for the differentiation of immature and mature hCG {alpha}ß-heterodimers. The various anti-GPH{alpha} antibodies were used to study the microheterogeneous isoforms of the respective intracellular GPH{alpha} variants (Fig. 6BGo). Different isoelectric points (ip) are mainly caused by the extent of sialylation that is evident when the sialic acids residues were removed by neuraminidase digestion, which converted the isoforms into the asialo proteins with ip above 8.5 (data not shown). The mAbs reproducibly reacted in principle with all microheterogeneous GPH{alpha} isoforms. The mAb specific for the free GPH{alpha} (directed against epitope {alpha}6) detected highly sialylated GPH{alpha} isoforms inferior to other mAbs. This may account for the decreased reactivity of this mAb with the intracellular vs. secreted GPH{alpha} (see Fig. 2Go, A vs. B). It is evident that the pattern of isoforms of the GPH{alpha} monomer and the GPH{alpha}{alpha} homodimers were very similar and differed from that of hCGß. The major hCGß isoforms had ip between 3.1 and 5.2. Depending on the pulse and chase intervals immature isoforms with ip around 8.9 were also observed. GPH{alpha} isoforms had ip values between 3.5 and 9.0.


Figure 6
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  		Fig. 6. Microheterogeneity of GPH{alpha} Variants, GPH{alpha}{alpha} Homodimers, and of Coprecipitated hCGß

JEG-3 cells were labeled with a 30-min pulse of [35S]Met/Cys and chased for 10 min. The lysate was immunoprecipitated in parallel with 8 mAbs directed against different epitopes on GPH{alpha} (see legend for Fig. 1AGo). Immunopurified proteins were separated on SDS-PAGE using nonreducing conditions and visualized by fluorography (A). The major 22-kDa GPH{alpha} band contained free nonassembled GPH{alpha} as well as GPH{alpha} dissociated from hCG {alpha}ß-heterodimers. The first lane (MW) shows the molecular weight standards of 14C-labeled marker proteins. The same samples as used in panel A were submitted to two-dimensional electrophoresis (B). In the first dimension, the protein samples were separated by IEF on IPG strips in the pH range between 3 and 10 under nonreducing conditions followed by SDS-PAGE into the second dimension using precast linear gradient gels 8–16%. The epitopes of the mAbs used for immunopurification are indicated ({alpha}1-{alpha}6). Visualization of the radioactively labeled proteins was performed by fluorography. As shown in A, all mAbs reacted with the 18-kDa monoglycosyl GPH{alpha}. Except for the mAb directed against the subunit interaction site (epitope {alpha}6, aa 33–42) all other mAbs recognized the GPH{alpha}{alpha} homodimers. Recognition of the hCG {alpha}ß-heterodimers differed from mAb to mAb and was strongest for the mAb directed against epitope {alpha}3* as indicated by the coprecipitation of the 28-kDa hCGß isoforms. mAbs directed against the epitopes {alpha}4 and {alpha}6 under these conditions did not recognize GPH{alpha} when associated with hCGß. In the two-dimensional electrophoreses, the subunit variants (GPH{alpha}, GPH{alpha}{alpha} homodimers, coprecipitated hCGß) showed an array of isoforms. GPH{alpha} isoforms may be divided into two groups of microheterogeneous variants with ip below 5.6 and above 6.5, respectively. The acidic group was underrepresented in those isoforms precipitated by the mAb directed against the free, unassociated GPH{alpha} (code INN-hCG-80; directed against GPH{alpha} epitope 6) as compared with those precipitated with anti-pan-GPH{alpha} mAbs, in particular when directed against epitope {alpha}3*. The patterns of coprecipitated hCGß and GPH{alpha} isoforms was different whereas the GPH{alpha} monomer and GPH{alpha}{alpha} homodimer patterns were very similar.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The rationale of the experiments presented here was to characterize the development and maturation of the GPH{alpha} variants. In particular, a panel of mAbs against different GPH{alpha} epitopes of known localization was employed for this purpose. Characterization of the molecular properties of the GPH{alpha} variants as well as their discrimination by mAbs is important with respect to the suggested biological functions of GPH{alpha} in the physiology and pathophysiology of the endometrium and the prostate as pointed out in the introduction.

N-Glycosylation, Disulfide Bridge Formation, and Subunit Folding
Folding, disulfide bridge formation and N-glycosylation are closely interdependent biosynthetic ER-located steps. In particular the sequence of disulfide bridge formation and N-glycosylation may be pivotal because of the interaction of free thiol groups and glycan residues with the complex of the thiol-disulfide oxidoreductase ERp57 and the calnexin/calreticulin lectin chaperones (for a review see Ref. 38). Improper disulfide bridge formation may be prevented by lectin chaperones recruited by a previously attached glycan (39). As well, N-glycosylation may be prevented by disulfide bridges formed before the glycan attachment (40). In the latter case, complete glycosylation could be achieved in the presence of dithiothreitol (DTT), whereas in untreated cells usage of glycan attachment sites was variable. As discussed in greater detail in the next paragraph, the problem of timing of disulfide bridge formation and usage of N-glycosylation sites exists also in the case of the GPH{alpha}. Both hCG subunits belong to the family of cystine knot growth factors (41, 42, 43). The influence of the assignment of the disulfide bridges for the folding pathway, subunit assembly and biologic activity has been published recently by several groups (44, 45, 46, 47).

The dynamics of the molecular GPH{alpha} variants in the course of the subunit maturation is mainly dominated by N-glycosylation processing and to a lesser extent by the folding state as shown for hCGß. Although GPH{alpha} folds very rapidly, with a half-life of approximately 90 sec, hCGß showed a t1/2 of 5 min for folding (48). In contrast to hCG {alpha}ß-heterodimers, which have to pass through complex folding events, large free GPH{alpha} and GPH{alpha}{alpha} homodimers, seem to get the approval by the quality control system of the ER much earlier. This was concluded from the fact that 10 min before the start of secretion, large free GPH{alpha} and GPH{alpha}{alpha} homodimers were already sialylated (see Fig. 6Go) in contrast to the subunits the hCG {alpha}ß-heterodimers (our manuscript in preparation). As shown here, after a short pulse early GPH{alpha} variants in JEG-3 cells consisted of an 18-kDa GPH monoglycosylated GPH{alpha} variant, a GPH{alpha} variant (Mr app = 22 kDa) released by dissociation from hCG {alpha}ß-heterodimers and a molecular species designated as free nonassembled GPH{alpha} initially also showing a Mr app of 22 kDa. This free nonassembled GPH{alpha} is likely to be the ancestor of the large free GPH{alpha} and GPH{alpha}{alpha} homodimers. Only very early 22-kDa GPH{alpha} variants were still sensitive to reduction indicating that disulfide bridge formation was not completed at that time point. The rapid folding and disulfide bridge formation in case of GPH{alpha} was also evident from the low amount of free thiol groups accessible to the in vivo covalent modification with [3H]N-ethylmalimide. This was in contrast to hCGß, which folded through at least eight intermediates characterized by a high extent of free cysteine residues, i.e. a low number of already linked disulfide bridges at the time point regarded (our manuscript in preparation).

Monoglycosyl GPH{alpha}
The 18-kDa GPH{alpha} variant had only one of the two N-glycan residues transferred cotranslationally. This endoglycosidase H-sensitive variant is expressed in JEG-3 and HeLa cells only as an early intermediate which was absent at the time-point of secretion (Figs. 2Go and 5Go), as well as in first trimester placenta tissue (49). The 18-kDa GPH{alpha} variant seemed to be indistinguishable from PNGase F-treated intracellular GPH{alpha} (see Fig. 4Go). It was previously shown that this enzyme selectively removes the carbohydrate moiety at Asn52 of purified GPH{alpha} (25, 35), which seems to be attributed to the increased accessibility of the Asn 52 vs. the Asn78-linked glycan.

It is not known which GPH{alpha} Asn-X-Thr sequon is unoccupied in the case of the 18-kDa GPH{alpha} variant. However, some considerations argue for the hypothesis that the Asn 52 sequon is not used. Bioinformatical studies have shown that the efficiency for the usage of Asn-X-Thr is not equally frequent along the sequence of a protein showing a distinct drop at the fifth decile and after the eighth decile and being influenced by flanking sequences (50). Furthermore, downstream sequences have an impact on glycosylation efficacy (51). Moreover, in vivo folding of the nascent protein and the glycosylation may compete and result in posttranslational N-glycosylation (52). After prevention of disulfide bridge formation in the ER in vivo by addition of 5 mM DTT to JEG-3 cells the half-life of the 18-kDa GPH{alpha} was decreased from 33 min to 3 min (53). This suggests that the disulfide bridge formation may influence the usage of the sequon at position 52 perhaps due to the formation of the adjacent disulfide bridges Cys 59-Cys87, Cys60-Cys10.

The association of monoglycosyl GPH{alpha} with hCGß to yield hCG {alpha}ß-heterodimers seems to be unfavorable in vivo because monoglycosyl GPH{alpha} was not coprecipitated by hCGß-specific antibodies (data not shown). This is in agreement with results indicating that the in vivo association of mutated GPH{alpha} with hCGß is less efficient when the Asn52 glycan is missing as compared with a mutant without a Asn78 glycan, which had no effect on subunit association (21). This effect does not seem to exist in the case of in vitro association of a degly-Asn52 GPH{alpha} variant with hCGß (21) and also when equine LH subunits were used (54). These different results indicate that the association of immature, incompletely folded subunits in vivo is not completely congruent with the recombination of mature subunits in vitro.

The monoglycosyl GPH{alpha} decreased with time and was absent in the protein secreted into the culture medium. In HeLa cells, the decrease seemed to be slower than in JEG-3 cells. The disappearance of this variant was explained either by degradation (21, 70). However, in the publications no degradation products were shown. Moreover, in the presence of lactacystin, an inhibitor of the proteasome-dependent degradation, no change in the band pattern was observed (data not shown). A further suggestion was the posttranslational transfer of the second N-linked glycan residue (18, 49). It was shown that the oligosaccharyltransferase has excess to proteins for at least 1 h after complete translation to perform posttranslational N-glycosylation (55). In HeLa cells, the capacity to perform posttranslational N-glycosylation might be more constrained in comparison to JEG-3 cells that could explain the differences in the persistence of the monoglycosyl GPH{alpha}.

Large Free GPH{alpha}
Large free 24-kDa GPH{alpha} was a prominent variant in JEG-3 cells (Fig. 2Go) and absent in HeLa cells (Fig. 5Go). For that reason, it seems to be unlikely that monoglycosyl GPH{alpha} (Mr app = 18 kDa) present in JEG-3 and HeLa cells is the precursor of large free GPH{alpha} (Mr app = 24 kDa) as suggested previously (18). Large free GPH{alpha}, which appeared in JEG cells between 30 and 60 min of chase, is characterized by its inability to associate with hCGß (19, 56). In addition to the biantennary carbohydrate structures, which are prevalent in assembled GPH{alpha} of hCG, large free GPH{alpha} carries in part triantennary and tetraantennary glycan structures synthesized in the Golgi. These account for the higher apparent molecular weight of the large free GPH{alpha} (18, 57, 58).

Subunit Homodimerization
Dimerization seems to be a common characteristic of the members of the cystine knot protein family, implicating homo- as well as heterodimerization (41, 42). The correct formation of the cystine-knot consensus disulfide bridge cluster seems to be mandatory for heterodimerization as shown for GPH{alpha} (45) as well as for homodimerization like in case of the muc2 rat mucin protein (59). As we show here, GPH{alpha} forms GPH{alpha}{alpha} homodimers in vivo, whereas in JEG-3 cells no hCGßß homodimerization was found. These observations agree with sedimentation equilibrium studies of bovine lutropin subunits showing that the formation of homodimers was weakly exothermic for bovine LH{alpha} and endothermic for bovine LHß (60). However, it cannot be excluded that the missing hCGßß homodimer formation is due to the low concentration of free hCGß in JEG-3 cells (9). In purified preparations of recombinant hCGß as well as in standard preparations, hCGßß homodimers were found (61). Moreover, recombinant hCGßß homodimers consisting of two hCGß molecules linked in tandem have been constructed and expressed in CHO cells (62). However, it remains unclear whether nongenetically engineered hCGßß homodimers are really synthesized in vivo or are artifacts formed from hCGß monomers during isolation and purification.

In JEG-3 as well as in HeLa cells secreted GPH{alpha}{alpha} homodimers (Mr app = 45 kDa) were directly generated from intracellular immature forms with apparent molecular masses of 35 kDa that were converted into 44 kDa (JEG-3) or 39 kDa (HeLa) variants before secretion (Figs. 2Go, 3Go, and 5Go). Recently 35- and 44-kDa variants have been observed in human seminal fluid of healthy probands (10) and a 45-kDa variant in choriocarcinoma patients (63). Large free GPH{alpha} (Mr app = 24 kDa) is unlikely to be the precursor of the GPH{alpha}{alpha} homodimers because it is absent in HeLa cells, whereas GPH{alpha}{alpha} homodimers were synthesized in considerable amounts (Fig. 5Go). Furthermore, the immature intracellular GPH{alpha}{alpha} homodimers found in JEG-3 as well as HeLa cells dissociated into 22-kDa GPH{alpha} by reduction and not into 24-kDa large free GPH{alpha} (Figs. 3Go and 5Go), again indicating that the latter is not the precursor of GPH{alpha}{alpha} homodimers. In the Golgi the free GPH{alpha} (Mr app = 22 kDa) is converted into the large free GPH{alpha} (Mr app = 24 kDa) by addition of further sugar residues. Homoassociation of free 22-kDa GPH{alpha} seems to occur already before that step. Immature GPH{alpha}{alpha} homodimers (Mr app = 35 kDa) appeared before the formation of large free GPH{alpha} variants (Fig. 2AGo, lane 1: 0 min chase; Fig. 3Go, A and B, lanes 1, 2: 5 min chase). GPH{alpha}{alpha} homodimer variants were recognized by all mAb directed against GPH{alpha} except mAb INN-hCG-80 (Fig. 6Go), which exclusively recognizes nonassembled GPH{alpha} via epitope {alpha}6 comprising the amino acid residues hCG{alpha}33–42 (see Fig. 1Go). This region seems to be an essential part of the contact interface between the subunits of heterodimeric hCG (64). Thus, the spatial orientation of the two GPH{alpha} molecules in the homodimer seems to be similar as the subunits in hCG {alpha}ß-heterodimers. Association of monomers is obviously much tighter in GPH{alpha}{alpha} homodimers than in the case of the hCG {alpha}ß-heterodimers because the latter is completely dissociated during the immunopurification procedure, whereas the GPH{alpha}{alpha} homodimers are stable under these conditions. It is only dissociated by reduction under more stringent conditions, e.g. as applied for SDS-PAGE with reduced samples (see Materials and Methods), which suggests that the GPH{alpha} monomers are either linked by disulfide bridge formation or held together otherwise by strong intermolecular bonds.

Sialylation indicates that the proteins have reached Golgi compartments with high sialyltransferase activities like the trans Golgi and the trans-Golgi network. The large free GPH{alpha} as well as the GPH{alpha}{alpha} homodimers showed predominantly isoforms with low ip (see Fig. 6Go), which were much less abundant or even missing in GPH{alpha} contained in the hCG {alpha}ß-heterodimer isoforms at the same time point (our manuscript in preparation). This indicates that the large free GPH{alpha} as well as the GPH{alpha}{alpha} homodimers were sequestered in the secretory pathway from hCG{alpha}ß heterodimer and reached the sites of sialylation much earlier than the hCG {alpha}ß-heterodimers.

In conclusion, almost completely folded GPH{alpha} associates with a highly immature hCGß to yield hCG {alpha}ß-heterodimers. Neither monoglycosyl nor large free GPH{alpha} seems to be competent to associate with hCGß. Appearance of formation of large free GPH{alpha} seems to be cell-specific because it was formed in JEG-3 cell but not in HeLa cells. Excessive GPH{alpha} is the basis for the formation of GPH{alpha}{alpha} homodimers. GPH{alpha}-subunits in homodimers are associated in an orientation that renders the aa residues hCG{alpha}33–42 ({alpha}6-epitope) of both subunits to become inaccessible. The pattern of sialylation of the different GPH{alpha} variants suggests sequestration of large free GPH{alpha} and GPH{alpha}{alpha} homodimers from hCG {alpha}ß-heterodimers in the secretory pathway. It remains to be determined whether GPH{alpha}{alpha} homodimers show any biological activity or if the biological activities attributed the GPH{alpha} as described above are in fact caused by the presence of homodimers. Interestingly, in case of hCGßß homodimers growth promoting effects by exerting antiapoptotic effects have been shown recently (65).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
All experiments were reproduced several times.

Cell Culture
JEG-3 cells (American Type Culture Collection, Manassas, VA) monolayer cultures were maintained in DMEM (Sigma, Taufkirchen, Germany), containing 10% (vol/vol) fetal calf serum (FCS; Linaris Corp., Bettingen, Germany). The medium was supplemented with 3.7 g/liter sodium bicarbonate, 100 IU/ml penicillin, and 100 µg/ml streptomycin (Linaris Corp., Bettingen, Germany). Subconfluent cell monolayers were grown in petri dishes (94- or 35-mm diameter), six- or four-well plates (Nunc GmbH, Wiesbaden-Biebrich, Germany) depending on the design of the pulse-chase experiment. The cell counts were 8 x 105–1 x 106 cells/well in the six-well plates and 5 x 104–1 x 105 cells/well in the four-well plates. The JEG-3 cells used for the experiments described in this paper were pretreated overnight with 250 µM 8-bromo-cAMP before the start of the experiment to increase the amount of synthesized proteins. Some experiments were carried out without this pretreatment as controls. In all cases examined, no quantitative changes were observed in comparison to 8-bromo-cAMP-treated cell cultures. HeLa cells (ATCC) monolayer cultures were grown in RPMI medium (Sigma), supplemented with 10% FCS. HeLa cells were stimulated before the start of experiment for 15–20 h with 5 mM sodium butyrate (Sigma) to increase the synthesis of GPH{alpha} (66). Generally, cell cultures were kept in the incubator (Heraeus, Osterode, Germany) at 37 C at 95% humidity in a 5% CO2 atmosphere. When short pulse times (1–5 min) were applied, for facilitated handling cells were kept outside the incubator during the pulse on a thermo-regulated metal plate adjusted to a temperature which provided 37 C in the cultures.

Radioactive Labeling and Cell Lysate Preparation
Cells were kept for 30 min in deficient medium (DMEM, lacking cysteine and methionine) without FCS before the pulse with 300 µCi/ml [35S]Met/Cys mixture (Amersham Buchler, Braunschweig, Germany). For the chase DMEM containing 10% FCS was used as culture medium. Further treatment and cell lysis was performed as described elsewhere (67).

Immunopurification
A panel of different mouse monoclonal antibodies (mAbs) directed against GPH{alpha} (68, 69) and a mAb against hCGß (code: INN-hCG-22, epitope ß2) were used for immunoprecipitation of cell lysates. Epitope characteristics of the antibodies (64) are presented in Fig. 1Go. In some experiments an in-house affinity-purified polyclonal anti-hCGß rabbit antiserum was used. Purification was carried out by passage of the ammonium sulfate-precipitated desalted {gamma}-globulin fraction through a column of highly purified in-house agarose-linked GPH{alpha} (linked via the carbohydrate part to Hz-Affi-Gel (Bio-Rad Laboratories, Munich, Germany). Immunoprecipitation with the antibodies was carried out for 1 h at 37 C under gentle rotation. The immune complexes were bound to Protein-A-agarose beads (Sigma) by incubation for 2 h at room temperature, then washed three times with a wash buffer [same composition as lysis buffer omitting sodium dodecyl sulfate (SDS)] and once with 10 mM Tris-EDTA buffer, pH 6.8. Elution of the immune complexes from the Protein-A-agarose was performed with a solution of 6 M urea in 100 mM glycine pH 3.0 at ambient temperature. This elution procedure resulted in a much lower background in the SDS-PAGE than with SDS containing elution buffers.

SDS-PAGE Analysis, Western Blotting, Isolation of Single Protein Bands in the Whole Gel Eluter
Samples eluted from the Protein A-agarose were centrifuged at 15,000 x g for a few seconds. Aliquots of the supernatant were mixed with equal volumes of either nonreducing sample buffer (125 mM Tris-HCl, pH 6.8; containing 4% (wt/vol) SDS, 0.2% (wt/vol) bromophenol blue, and 20% (vol/vol) glycerol) or reducing buffer (sample buffer containing 10% (vol/vol) 2-mercaptoethanol). Only reduced samples were heated for 1 min. Separation was performed by SDS-PAGE (Mini-Protean II; Bio-Rad Laboratories). Generally, 14% gels of 0.75 mm thickness were used. 14C-labeled molecular weight markers (mixture of Rainbow markers; Amersham) were run together with samples. After electrophoresis proteins were precipitated in the gels by incubation in 20% (wt/vol) trichloroacetic acid for 20–30 min, followed by two washings (10 min each) in dimethylsulfoxide. Gels were prepared for fluorography by incubation in 2,5 diphenyloxazole dissolved in dimethylsulfoxide (22% wt/vol) followed by washings with water and drying on Whatman 3MM paper in a gel dryer (Bio-Rad). Fluorography was carried out by exposing at –80 C to x-ray film (Fuji RX; Bechthold, Heidelberg, Germany) in the presence of intensifying screens. Quantitative evaluation of the x-ray films was performed with the Kodak Digital Science (Rochester, NY) one-dimensional system using the density calibration system provided by the manufacturer.

Western blotting was performed immediately after separation of samples by transfer of proteins to polyvinylidene difluoride membranes (Roche AG, Mannheim, Germany) using the Mini Transfer-Blot cell of Bio-Rad Laboratories. The transfer buffer was a mixture of 30 mM glycine, 48 mM Tris, and 0.037% SDS in 20% methanol. Blotting was performed for 1 h at 100 V using prestained proteins as molecular weight markers (Carl Roth GmbH, Karlsruhe, Germany). The membranes were incubated in PBS (pH 7.5) containing 0.1% low-fat milk and 0.1% Tween 20 (Sigma) for 1 h to block nonspecific binding. The first antibodies were diluted in the above solution to concentrations as indicated below. Second antibodies were peroxidase-coupled antimouse Ig antibody contained in the detection kit (see below) or a {gamma}-chain-specific peroxidase-coupled goat anti-mouse Ig antibody (Sigma) in dilutions of 20,000–30,000. Signal detection was performed with the ECL kit (Amersham) and exposure to Fuji RX film (Bechthold, Heidelberg, Germany).

Micro-preparative isolation of single bands was carried out with polyacrylamide gels of 1 mm thickness and sample slots of 50-mm width for the separation in the SDS-PAGE. Afterward, gels without the stacking gels were submitted to electroelution vertical to the original running direction in a Mini Whole Gel Eluter (Bio-Rad Laboratories) according to the protocol of the manufacturer. Protein bands present in gels of 64- x 55-mm in size were separated in up to 14 fractions and harvested from the elution chambers. Individual fractions were concentrated in 500 µl Vivaspin concentrators (Mr cut-off 10,000; VivaScience, Hannover, Germany), reanalyzed by SDS-PAGE to monitor the isolated bands or run for characterization in Western blots.

Two-Dimensional Gel Electrophoresis
Isoelectric focusing (IEF) was carried out in a Protean IEF Cell (Bio-Rad Laboratories). In the second dimension, SDS-PAGE was performed in a Criterion chamber (Bio-Rad Laboratories) using precast linear gradient gels 8–16% (Bio-Rad Laboratories). For the first dimension the immunoprecipitated samples were equilibrated on 500 µl Vivaspin concentrators with 8 M urea, 2% 3-(3-cholamidopropyl)-dimethylammonio-1-propane-sulfonate, and 0.2% Biolyte (Serva, Heidelberg, Germany) with or without 50 mM DTT as indicated below. Carrier medium for the first dimension were IPG strips (ReadyStrip IPG strips; Bio-Rad Laboratories). IEF was carried out overnight using programmed volt-hours setting (50 V, 12 h; 250 V, 6 h; 5000 V, 2.5 h; 8000 V 1–2 h). After IEF, strips were either stored at –80 C or used immediately for the second dimension step after pretreatment (10 min at room temperature) with the following buffer: 375 mM Tris-HCl (pH 8.8) containing 6 M urea, 2% SDS, and 130 mM DTT. When no reduction of the disulfide bridges was desired DTT was omitted in both dimensions. In the case of reduced samples free thiol groups were alkylated by incubation with 375 mM Tris-HCl (pH 8.8) containing 6 M urea, 2% SDS, and 675 mM iodoacetamide.

Digestion with Glycosidases
For enzymatic release of glycans were equilibrated on 500 µl Vivaspin concentrators with the buffers required. Sialic acid was removed by the incubation of 20 µl of the samples with 2 mU neuraminidase of V. cholerae (EC 3.2.1.18; Sigma) for 3 h at 37 C using the following buffer: 10 mM sodium acetate (pH 7.5) containing 0.8 mM calcium chloride. N-linked carbohydrate residues were released by digestion with peptide N-glycanase F (PNGase F; peptide-N4-(N-acetyl-ß-glucosaminyl) asparagine amidase, EC 3.5.1.52; Roche, Mannheim, Germany) or endoglycosidase H (endo-ß-N-acetylglucosaminidase H; EC 3.2.1.96; Roche). Digestion with endoglycosidase H was performed in 10 mM sodium acetate (pH 5.5), using 15 mU enzyme/25 µl sample. Incubation was carried out for 3 h at 37 C. Digestion with 2 µU PNGase F/20 µl sample was carried out in presence or absence of 10 mM ß-mercaptoethanol as indicated by incubation of the samples for 3 h at 37 C. The buffer consisted of 50 mM sodium phosphate and 125 mM EDTA (pH 7.2).


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the help of Dr. N. Sampson (Innsbruck, Austria) with language editing.


   FOOTNOTES
 
This work was supported by a grant of the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (to W.E.M.; Me 545/12-1, 2), by the Austrian Science Fund (to P.B., FWF; NRN, S9307B05) and a fellowship of the Alexander von Humboldt Foundation, Bonn-Bad Godesberg (to V.S.).

In memory of Vinod Singh, who has passed away.

Disclosure Summary: The authors have nothing to disclose.

First Published Online July 3, 2007

1 J.-M.K. and P.B. contributed equally to this study. Back

Abbreviations: aa, Amino acid; DTT, dithiothreitol; ER, endoplasmic reticulum; GPH{alpha}, glycoprotein hormone {alpha}-subunit; hCG, human chorionic gonadotropin; hCGß, hormone-specific ß-subunit; IEF, isoelectric focusing; ip, isoelectric point; mAb, monoclonal antibody; PNGase F, peptide N-glycanase F; SDS, sodium dodecyl sulfate.

Received for publication January 26, 2007. Accepted for publication June 20, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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