Elimination of disulfide bonds affects assembly and secretion of the human chorionic gonadotropin beta subunit

Human chorionic gonadotropin (hCG) consists of two noncovalently joined alpha and beta subunits similar to the other glycoprotein hormones. To study the function of the individual disulfide bonds in subunit assembly and secretion, site-directed mutagenesis was used to convert the 12 cysteine (Cys) residues in the beta subunit of hCG to either alanine or serine. Both cysteines of proposed disulfide pairs were also mutated. These mutant hCG beta genes were transfected alone or together with the wild-type alpha gene into Chinese hamster ovary cells. Only 3-10% assembly could be achieved with derivatives containing single Cys mutations at positions 26, 110, 72, and 90, whereas no assembly was detected with the other 8 mutants. However, double mutations of pairs 26-110 or 23-72 showed increased dimer formation (11 and 36%, respectively). The secretion rate of individual mutants varied significantly. Whereas the Cys-23 and 72 mutants were secreted normally (t1/2 = 140-190 min), the Cys-26 mutant was secreted faster (t1/2 = 70 min), and the other 9 mutants were secreted slower (t1/2 = 280-440 min); mutations of both Cys at 26 and 110 caused much faster secretion (t1/2 = 34 min). Although the secretion rate of these mutants differed, they were quantitatively recovered in the medium except for mutant Cys-88, Cys-23-72, and Cys-34-88 (40, 55, and 10% secreted, respectively). Thus, interruption of any disulfide bond in the hCG beta subunit alters the structure sufficiently to block dimer formation and in some cases slow secretion, although the stability for most of the mutant hCG beta subunits is not greatly affected. The data indicate that interruption of any hCG beta disulfide bond generates different structural forms that are unable to assemble with the alpha subunit, and that the structural requirements for stability and assembly are different.     

Disulfide bonds Cys(9)-Cys(57), Cys(34)-Cys(88) and Cys(38)-Cys(90) of the beta-subunit of human chorionic gonadotropin are crucial for heterodimer formation with the alpha-subunit: experimental evidence for the conclusions from the crystal structure of hCG

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The alpha- and beta-subunits of hCG are highly cross-linked internally by disulfide bonds that seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. This paper describes the results of our studies on the role of the disulfide bonds of hCG-beta in heterodimer formation with the alpha-subunit. Six disulfide peptides incorporating each of the six disulfide bonds of hCG-beta were screened, along with their linear counterparts, for their ability to competitively inhibit the recombination of alpha- and beta-subunits. The disulfide peptides Cys (9-57), Cys (34-88) and Cys (38-90) were found to inhibit the alpha/beta recombination whereas the remaining three disulfide peptides viz. Cys (23-72), Cys (26-110) and Cys (93-100) did not exhibit any inhibition activity. Interestingly, none of the linear peptides could inhibit the alpha/beta recombination. Results clearly demonstrate that the disulfide bonds Cys(9)-Cys(57), Cys(34)-Cys(88) and Cys(38)-Cys(90) of the beta-subunit of hCG are crucial for heterodimer formation with the alpha-subunit thus providing experimental confirmation of the conclusions from the crystal structure of the hormone.     

The glycoprotein alpha-subunit is critical for secretion and stability of the human thyrotropin beta-subunit

TSH is a member of a family of heterodimeric glycoprotein hormones which have a common alpha-subunit but differ in their hormone-specific beta-subunit. To study the posttranslational processing and assembly of human TSH, eukaryotic expression vectors were constructed that contained either the human TSH beta gene only or both the TSH beta and alpha-genes. These vectors were transfected into Chinese hamster ovary cells and stable cell lines synthesizing TSH beta or TSH dimer were isolated. The kinetics of secretion of TSH beta and the rate of assembly of TSH dimer were compared to the known secretion and assembly of human LH and human CG. In the absence of the alpha-subunit, CG beta is secreted efficiently, but TSH and LH beta-subunits are slowly degraded intracellularly (t1/2 approximately equal to 6 h) and less than 10% is secreted into the medium. In the presence of the alpha-subunit CG beta was also secreted efficiently as dimer but only 50% of the LH beta appeared in the medium as LH dimer. However, unlike LH beta, the alpha-subunit efficiently combines with TSH beta since greater than 95% was secreted as TSH dimer. Thus, the determinants for human TSH beta secretion and assembly are unique from the other human glycoprotein hormone beta-subunits.     

Disulfide bond rearrangement during formation of the chorionic gonadotropin beta-subunit cystine knot in vivo

The intracellular kinetic folding pathway of the human chorionic gonadotropin beta-subunit (hCG-beta) reveals the presence of a disulfide between Cys residues 38-57 that is not detected by X-ray analysis of secreted hCG-beta. This led us to propose that disulfide rearrangement is an essential feature of cystine knot formation during CG-beta folding. To test this, we used disulfide bond formation to monitor progression of intracellular folding intermediates of a previously uncharacterized protein, the CG-beta subunit of cynomolgous macaque (Macaca fascicularis). Like its human counterpart hCG-beta with which it shares 81% identity, macaque (m)CG-beta is a cystine knot-containing subunit that assembles with an alpha-subunit common to all glycoprotein hormone members of its species to form a biologically active heterodimer, mCG, which, like hCG, is required for pregnancy maintenance. An early mCG-beta folding intermediate, mpbeta1, contained two disulfide bonds, one between Cys34 and Cys88 and the other between Cys38 and Cys57. The subsequent folding intermediate, mpbeta2-early, was represented by an ensemble of folding forms that, in addition to the two disulfides mentioned above, included disulfide linkages between Cys9 and Cys57 and between Cys38 and Cys90. These latter two disulfides are those contained within the beta-subunit cystine knot and reveal that a disulfide exchange occurred during the mpbeta2-early folding step leading to formation of the mCG-beta knot. Thus, while defining the intracellular kinetic protein folding pathway of a monkey homologue of CG-beta, we detected the previously predicted disulfide exchange event crucial for CG-beta cystine knot formation and attainment of CG-beta assembly competence.     

Threading of a glycosylated protein loop through a protein hole: implications for combination of human chorionic gonadotropin subunits

Chorionic gonadotropin (hCG) is a heterodimeric placental glycoprotein hormone essential for human reproduction. Twenty hCG beta-subunit residues, termed the seatbelt, are wrapped around alpha-subunit loop 2 (alpha 2) and their positions "latched" by a disulfide formed by cysteines at the end of the seatbelt (Cys 110) and in the beta-subunit core (Cys 26). This unique arrangement explains the stability of the heterodimer but raises questions as to how the two subunits combine. The seatbelt is latched in the free beta-subunit. If the seatbelt remained latched during the process of subunit combination, formation of the heterodimer would require alpha 2 and its attached oligosaccharide to be threaded through a small beta-subunit hole. The subunits are known to combine during oxidizing conditions in vitro, and studies described here tested the idea that this requires transient disruption of the latch disulfide, possibly as a consequence of the thioredoxin activity reported in hCG. We observed that alkylating agents did not modify either cysteine in the latch disulfide (Cys 26 or Cys 110) during heterodimer formation in several oxidizing conditions and had minimal influence on these cysteines during combination in the presence of mild reductants (1--3 mM beta-mercaptoethanol). Reducing agents appeared to accelerate subunit combination by disrupting a disulfide (Cys 93--Cys 100) that forms a loop within the seatbelt, thereby increasing the size of the beta-subunit hole. We propose a mechanism for hCG assembly in vitro that depends on movements of alpha 2 and the seatbelt and suggest that the process of glycoprotein hormone subunit combination may be useful for studying the movements of loops during protein folding.     

Mutational study on the roles of disulfide bonds in the beta-subunit of gastric H+,K+-ATPase

The gastric proton pump, H(+),K(+)-ATPase, consists of the catalytic alpha-subunit and the non-catalytic beta-subunit. Correct assembly between the alpha- and beta-subunits is essential for the functional expression of H(+),K(+)-ATPase. The beta-subunit contains nine conserved cysteine residues; two are in the cytoplasmic domain, one in the transmembrane domain, and six in the ectodomain. The six cysteine residues in the ectodomain form three disulfide bonds. In this study, we replaced each of the cysteine residues of the beta-subunit with serine individually and in several combinations. The mutant beta-subunits were co-expressed with the alpha-subunit in human embryonic kidney 293 cells, and the role of each cysteine residue or disulfide bond in the alpha/beta assembly, stability, and cell surface delivery of the alpha- and beta-subunits and H(+),K(+)-ATPase activity was studied. Mutant beta-subunits with a replacement of the cytoplasmic and transmembrane cysteines preserved H(+),K(+)-ATPase activity. All the mutant beta-subunits with replacement(s) of the extracellular cysteines did not assemble with the alpha-subunit, resulting in loss of H(+),K(+)-ATPase activity. These mutants did not permit delivery of the alpha-subunit to the cell surface. Therefore, each of these disulfide bonds of the beta-subunit is essential for assembly with the alpha-subunit and expression of H(+),K(+)-ATPase activity as well as for cell surface delivery of the alpha-subunit.     

Mutagenesis and gene transfer define site-specific roles of the gonadotropin oligosaccharides

Human chorionic gonadotropin (hCG), luteinizing hormone (LH), follicle-stimulating hormone and thyroid-stimulating hormone are a family of glycoprotein hormones that share a common alpha subunit but differ in their hormone-specific beta subunits. Using site-directed mutagenesis and gene-transfer, we analyzed the role of the N-linked oligosaccharides of alpha and chorionic gonadotropin (CG)beta in the secretion, assembly, and biologic activity of hCG. Absence of carbohydrate at alpha asparagine (Asn) 52 decreased combination with CG beta but did not alter monomer secretion. Absence of the alpha Asn78 oligosaccharide increased the degradation of the alpha subunit, but the presence of CG beta stabilized this alpha mutant in an efficiently formed dimer complex. Alternatively, absence of both alpha oligosaccharides slowed both secretion and dimer formation but allowed an intermediate level of alpha secreted or dimerized compared to the single-site mutants. Analysis of the CG beta glycosylation mutants revealed that absence of the Asn30 oligosaccharide, but not Asn13, slowed secretion but not assembly, whereas absence of both oligosaccharides slowed both secretion and dimer formation. Analysis of the receptor binding of the hCG glycosylation mutants showed that absence of any or all of the hCG N-linked oligosaccharides had only a minor effect on receptor affinity of the derivatives. However, the absence of alpha Asn52, but not the alpha Asn78 or the CG beta carbohydrate units, reduced the steroidogenic effect, unmasked differences in the beta oligosaccharides, and converted the deglycosylated derivatives into antagonists.     

The role of the asparagine-linked oligosaccharides of the alpha subunit in the secretion and assembly of human chorionic gonadotrophin

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common alpha subunit but differ in their hormone-specific beta subunit. Site-directed mutagenesis of the two asparagine-linked glycosylation sites of hCG alpha was used to study the function of the individual oligosaccharide chains in secretion and subunit assembly. Expression vectors for the alpha genes (wild-type and mutant) and the hCG beta gene were constructed and transfected into Chinese hamster ovary cells. Loss of the oligosaccharide at position 78 causes the mutant subunit to be degraded quickly and less than 20% is secreted. However, the presence of hCG beta stabilizes this mutant and allows approximately 45% of the subunit in the form of a dimer to exit the cell. Absence of carbohydrate at asparagine 52 does not perturb the stability or transport of the alpha subunit but does affect dimer secretion; under conditions where this mutant or hCG beta was in excess, less than 30% is secreted in the form of a dimer. Mutagenesis of both glycosylation sites affects monomer and dimer secretion but at levels intermediate between the single-site mutants. We conclude that there are site-specific functions of the hCG alpha asparagine-linked oligosaccharides with respect to the stability and assembly of hCG.     

Epitope analysis and molecular modeling reveal the topography of the C-terminal peptide of the beta-subunit of human chorionic gonadotropin.

Human chorionic gonadotropin (hCG) belongs to a family of heterodimeric glycoprotein hormones that share a common alpha-subunit and a hormone-specific beta-subunit. Among the gonadotropin beta-subunits, greater than 85% homology exists between lutropin (hLH)beta and hCGbeta in their first 114 amino acid residues. However, unlike hLHbeta, hCGbeta contains a 31-amino acid hydrophilic stretch at its carboxyl end (CTPbeta: C-terminal peptide). Although the crystal structure of deglycosylated hCG has been solved, the topography of CTPbeta remains unknown. In this study, we have attempted to define the topology of CTPbeta using mAb probes. We investigated three epitopes on hCGalpha, which are hidden in the hCGalphabeta dimer. However, these epitopes are not hidden in hLH, which has a similar subunit interface to that of hCG, but lacks CTPbeta. This suggested that these epitopes are not masked at the subunit interface of hLH or hCG. Hence, we hypothesized that, in the case of hCG, these epitopes are masked by the CTPbeta. Consistent with this view, several treatments of hCG that removed CTPbeta unmasked these epitopes and enhanced their reactivity with the corresponding mAbs. In order to localise the position of CTPbeta on the alpha-subunit, we used an epitope-mapping strategy [N. Venkatesh & G. S. Murthy (1997) J. Immunol. Methods 202, 173-182] based on differential susceptibility of epitopes to covalent modifications. This enabled us to predict the possible topography of CTPbeta. Further, we were also able to build a model of CTPbeta, completely independently of the epitope-mapping studies, using a homology-based modeling approach [S. Krishnaswamy, I. Lakshminarayanan & S. Bhattacharya (1995) Protein Sci. 4 (Suppl. 2), 86-97]. Results obtained from these two different approaches (epitope analysis and homology modeling) agree with each other and indicate that portions of CTPbeta are in contact with hCGalpha in the native hCG dimer.     

The carboxy-terminal region of the glycoprotein hormone alpha-subunit: contributions to receptor binding and signaling in human chorionic gonadotropin.

The glycoprotein hormones are heterodimeric and contain a common alpha-subunit, which is noncovalently associated with a hormone-specific beta-subunit. The alpha-subunit has been highly conserved throughout evolution; for example, the five amino acid residues of the carboxy-terminus, Tyr-Tyr-His-Lys-Ser-COOH, are identical in nine of the 10 available amino acid sequences. It has been shown that enzymatic removal of these five amino acid residues, while not affecting holoprotein formation, results in a heterodimer that exhibits very little, if any, binding to the CG/LH receptor. Using site-directed mutagenesis on the human alpha-subunit, we have prepared two deletion mutants, Des-(88-92)alpha and Des-(89-92)alpha, and two point mutants, where each of the two tyrosines, 88 and 89, was replaced with phenylalanine, in order to delineate more specifically the contributions of these aromatic side-chains to receptor binding. The cDNAs for wild-type hCG alpha and mutants were introduced into a pcDNAINEO expression vector, and the cDNA for hCG beta was inserted into a pRSV plasmid; both were transiently cotransfected into DUXB-11 cells. The media were collected, and RIAs showed that all mutants formed heterodimers; moreover, there was no discernable difference in subunit assembly between wild-type hCG alpha and the various mutant alpha-subunits. The gonadotropin mutants were assayed in vitro using a competitive binding assay with [125I]hCG and stimulation of progesterone production in the transformed murine Leydig cell line MA-10.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutational analysis of alpha-beta subunit interactions in the delivery of Na,K-ATPase heterodimers to the plasma membrane

The beta-subunit of the Na,K-ATPase is required to deliver functional alpha beta-heterodimers to the plasma membrane (PM) of baculovirus-infected insect cells. We have investigated the molecular determinants in the beta-subunit for the assembly and delivery processes. Trafficking of both subunits was analyzed by Western blots of fractionated membranes enriched in endoplasmic reticulum (ER), Golgi, and PM. Heterodimer assembly was evaluated by co-immunoprecipitation, and enzymatic activity was measured by ATPase assay. Elimination of enzymatic activity by D369A point mutation of the alpha-subunit had no effect on the compartmental distribution of the Na,K-ATPase, demonstrating that enzymatic functioning is not a prerequisite for PM delivery. Replacement of all three N-glycosylation site asparagines with glutamines produced no effect on the delivery to the PM or the activity of the enzyme, but increased susceptibility to degradation was observed. Analysis of beta-subunits in which the disulfide bonds were removed through substitution reveals that the bridges are important for PM targeting but not for assembly of the heterodimer. Assembly is supported by beta-subunits with greatly truncated extracellular domains. The presence of the amino-terminal domain and transmembrane segment is sufficient for assembly and PM delivery. Intermediate length truncated beta-subunits and some disulfide bridge substitution mutants assemble with the alpha-subunit but are not able to exit the ER. We conclude that there are different and separable requirements for the assembly of Na,K-ATPase heterodimer complexes, exit of the dimer from the ER, delivery to the PM, and catalytic activity of the dimer.     

Site-directed mutagenesis of the human chorionic gonadotropin beta-subunit: bioactivity of a heterologous hormone, bovine alpha-human des-(122-145)beta

Human CG contains an alpha-subunit, common to the pituitary glycoprotein hormones, and a hormone-specific beta-subunit, but unlike the pituitary beta-subunits, hCG beta is characterized by an O-glycosylated carboxy-terminal extension. A mutant beta-subunit, des-(122-145)hCG beta, was prepared using site-directed mutagenesis, and the pRSV expression plasmids were transfected into Chinese hamster ovary cells that produce the bovine alpha-subunit (b alpha). The mutant beta-subunit binds to b alpha, and the heterologous gonadotropin, b alpha-des-(122-145)hCG beta, was capable of stimulating steroidogenesis in cultured Leydig tumor cells (MA-10) to the same extent as standard hCG. When compared with the heterologous gonadotropin, b alpha-hCG beta wild type, the hybrid hormone with the truncated hCG beta exhibited equal potency, within the accuracy of the RIAs used to determine hormone concentrations, and gave a similar time course of steroidogenesis. Interestingly, these transformed Leydig cells do not distinguish between the steroidogenic potencies (as measured by progesterone production) of hCG and human LH (hLH) as do some preparations of normal rodent Leydig cells (as measured by testosterone production). However, the MA-10 cells were able to distinguish hCG from hLH based on their cAMP response; the latter produced a greater response at both maximal and submaximal gonadotropin concentrations. The two expressed heterologous gonadotropins were equipotent in their abilities to stimulate cAMP and gave similar time courses of cAMP accumulation in MA-10 cells. Thus, the carboxy-terminal extension of hCG beta is not required for association with the alpha-subunit nor for functional receptor binding, as judged by cAMP accumulation and progesterone production in MA-10 cells.     

Inhibins and activins: chemical properties and biological activity

The long-sought, nonsteroidal, gonadal inhibitor of the secretion of FSH has been isolated, characterized, and the primary structure in several species (human, porcine, bovine, murine) has been deduced. Inhibins are proteins consisting of two subunits (18-kDa alpha- and 14-kDa beta-subunits) linked by disulfide bridges and two forms of inhibins were observed in human, porcine, and murine, but only one in bovine. Each form of inhibin (A and B) has a common alpha-subunit, but a highly homologous, distinct beta-subunit (beta A and beta B). The beta-subunits and the alpha-subunit are linked to form inhibins A and B which exert an inhibitory effect on basal FSH secretion, but the dimer formed by either two beta A-subunits or two distinct beta A- and two beta B-subunits (homoactivin-A and activin, respectively) possess FSH-stimulating activity. Inhibin secreted in response to FSH from the pituitary originates primarily from the granulosa cells of the ovary and the Sertoli cells of the testes, thus demonstrating a reciprocal feedback relationship.     

Role of the beta 93-100 determinant loop sequence in receptor binding and biological activity of human luteinizing hormone and chorionic gonadotropin

The intercysteine loop sequence (93-100) in the beta-subunit has been postulated to be important for receptor binding and specificity in the glycoprotein hormones, LH and human CG (hCG). To demonstrate this directly, and to characterize the structural features essential for activity, we prepared a series of synthetic peptides and analogs incorporating this determinant loop region. Peptides were assayed for inhibition of labeled hCG binding to ovarian membrane receptors and stimulation of testosterone production in Leydig cells. Peptides with the native (93-100) sequence from hCG and hLH inhibited hCG binding half maximally at 2.18 and 2.62 x 10(-4) M, respectively, while the sequence from FSH was inactive. Isosteric substitution of Ala for Cys resulted in an inactive peptide, indicating that the (93-100) disulfide bridge is essential for activity. Optimal binding activity requires at least one net positive charge among the side chains, as shown by loss of activity in hybrid analogs with neutral or negative charges conferred by progressive replacement of Arg by Asp at 94 and 95 or by introduction of Asp at 96 and 97. Despite binding to receptors, the native sequence did not promote testosterone production at doses up to 10(-2) M. This contrasts with a second receptor binding sequence, beta (38-57) that activates testosterone production. There are differences between the (93-100) and (38-57) loop sequences in their chemical and physical properties, biological activity and antigenicity. While the cumulative evidence suggests that they associate with counterpart sites in alpha-subunit to form a topographical binding domain in the whole hormone, our results suggest that each sequence may contribute in different ways to activation of postreceptor events.     

Alternatively folded choriogonadotropin analogs. Implications for hormone folding and biological activity

Most heterodimeric proteins are stabilized by intersubunit contacts or disulfide bonds. In contrast, human chorionic gonadotropin (hCG) and other glycoprotein hormones are secured by a strand of their beta-subunits that is wrapped around alpha-subunit loop 2 "like a seatbelt." During studies of hCG synthesis in COS-7 cells, we found that, when the seatbelt was prevented from forming the disulfide that normally "latches" it to the beta-subunit, its carboxyl-terminal end can "scan" the surface of the heterodimer and become latched by a disulfide to cysteines substituted for residues in the alpha-subunit. Analogs in which the seatbelt was latched to residues 35, 37, 41-43, and 56 of alpha-subunit loop 2 had similar lutropin activities to those of hCG; that in which it was latched to residue 92 at the carboxyl terminus had 10-20% the activity of hCG. Attachment of the seatbelt to alpha-subunit residues 45-51, 86, 88, 90, and 91 reduced lutropin activity substantially. These findings show that the heterodimer can form before the beta-subunit has folded completely and support the notions that the carboxyl-terminal end of the seatbelt, portions of alpha-subunit loop 2, and the end of the alpha-subunit carboxyl terminus do not participate in lutropin receptor interactions. They suggest also that several different architectures could have been sampled without disrupting hormone activity as the glycoprotein hormones diverged from other cysteine knot proteins.     

Mapping the receptor binding regions of human chorionic gonadotropin (hCG) using disulfide peptides of its beta-subunit: possible involvement of the disulfide bonds Cys(9)-Cys(57) and Cys(23)-Cys(72) in receptor binding of the hormone.

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The alpha- and beta-subunits of hCG are highly cross-linked internally by disulfide bonds which seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. The purpose of this study was to delineate the role of the disulfide bonds of hCGbeta in receptor binding of the hormone. Six disulfide peptides incorporating each of the six disulfide bonds of hCGbeta were synthesized and screened, along with their linear counterparts, for their ability to competitively inhibit the binding of [125I] hCG to sheep ovarian corpora luteal LH/CG receptor. Disulfide peptide Cys (9-57) was found to be approximately 4-fold more potent than the most active of its linear counterparts in inhibiting radiolabeled hCG from binding to its receptor. Similarly, disulfide peptide Cys (23-72) exhibited receptor binding inhibition activity, whereas the constituent linear peptides were found to be inactive. The results suggest the involvement of the disulfide bonds Cys(9)-Cys(57) and Cys(23)-Cys(72) of the beta-subunit of hCG in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hCG.     

Structural studies on equine glycoprotein hormones. Amino acid sequence of equine chorionic gonadotropin beta-subunit

The complete amino acid sequence of the beta-subunit of equine chorionic gonadotropin (eCG beta) has been established by both automated Edman and manual 5-dimethylaminonaphthalene-1-sulfonyl-Edman degradations. Specific fragments were produced by cleavage with Staphylococcus aureus V8 protease, trypsin, or dilute HCl. For the sequence analyses of the heavily glycosylated COOH-terminal portion, a chemical deglycosylation procedure with trifluoromethanesulfonic acid was employed. The peptide chain of eCG beta consists of 149 amino acid residues. Five or more oligosaccharide chains are attached to the protein, 1 unit linked by an N-glycosidic bond to asparagine at residue 13 and four or more units linked by O-glycosidic bonds to serine or threonine at residues in the COOH-terminal portion. The carbohydrate-bearing hydroxy amino acids have not yet been rigorously established. As compared to the beta-subunits of the pituitary gonadotropin hormones, lutropin, follitropin, and thyrotropin, eCG beta possesses a glycosylated COOH-terminal extension of about 30 amino acid residues, as does the human chorionic gonadotropin beta-subunit (hCG beta). When the comparison is restricted inside the disulfide bond-containing core (residues 1-110), the beta-subunit of eCG is highly homologous to hCG beta (66%). On the other hand, although the overall structural features closely resemble each other, much less homology exists in the COOH-terminal extensions of eCG beta and hCG beta.     

Intracellular folding pathway of human chorionic gonadotropin beta subunit.

Few experimental models have been used to investigate how proteins fold inside a cell. Using the formation of disulfide bonds as an index of conformational changes during protein folding, we have developed a unique system to determine the intracellular folding pathway of the beta subunit of human chorionic gonadotropin (hCG). Three folding intermediates of the beta subunit were purified from [35S]cysteine-labeled JAR choriocarcinoma cells by immunoprecipitation and by reverse-phase high performance liquid chromatography (HPLC). To identify unformed disulfide bonds, nonreduced folding intermediates were treated with trypsin to liberate non-disulfide-bound, [35S]cysteine-containing peptides from the disulfide-linked peptides. Released peptides were purified by HPLC and identified by amino acid sequencing. The amount of a peptide that was released indicated the extent of disulfide bond formation involving the cysteine in that peptide. Of the six disulfide bonds in hCG-beta, bonds 34-88 and 38-57 form first. The rate-limiting event of folding involves the formation of the S-S bonds between cysteines 23 and 72 and cysteines 9 and 90. Disulfide bond 93-100, the formation of which appears to be necessary for assembly with the alpha subunit of the hCG heterodimer, forms next. Finally, disulfide bond 26-110 forms after assembly with the alpha subunit, suggesting that completion of folding of the COOH terminus in the beta subunit occurs after assembly with the alpha subunit.     

Glycoprotein hormone assembly in the endoplasmic reticulum: IV. Probable mechanism of subunit docking and completion of assembly

The unique structures of human choriogonadotropin (hCG) and related glycoprotein hormones make them well suited for studies of protein folding in the endoplasmic reticulum. hCG is stabilized by a strand of its beta-subunit that has been likened to a "seatbelt" because it surrounds alpha-subunit loop 2 and its end is "latched" by an intrasubunit disulfide bond to the beta-subunit core. As shown here, assembly begins when parts of the NH(2) terminus, cysteine knot, and loops 1 and 3 of the alpha-subunit dock reversibly with parts of the NH(2) terminus, cystine knot, and loop 2 of the hCG beta-subunit. Whereas the seatbelt can contribute to the stability of the docked subunit complex, it interferes with docking and/or destabilizes the docked complex when it is unlatched. This explains why most hCG is assembled by threading the glycosylated end of alpha-subunit loop 2 beneath the latched seatbelt rather than by wrapping the unlatched seatbelt around this loop. hCG assembly appears to be limited by the need to disrupt the disulfide that stabilizes the small seatbelt loop prior to threading. We postulate that assembly depends on a "zipper-like" sequential formation of intersubunit and intrasubunit hydrogen bonds between backbone atoms of several residues in the beta-subunit cystine knot, alpha-subunit loop 2, and the small seatbelt loop. The resulting intersubunit beta-sheet enhances the stability of the seatbelt loop disulfide, which shortens the seatbelt and secures the heterodimer. Formation of this disulfide also explains the ability of the seatbelt loop to facilitate latching during assembly by the wraparound pathway.