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.     

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.     

Only a portion of the small seatbelt loop in human choriogonadotropin appears capable of contacting the lutropin receptor

Twenty residues of the human choriogonadotropin (hCG) beta-subunit that are wrapped around alpha-subunit loop 2 like a "seatbelt" stabilize the heterodimer and enable the hormone to distinguish lutropin (LHR), follitropin, and thyrotropin receptors. The N-terminal portion of the seatbelt contains a small disulfide-stabilized loop needed for heterodimer assembly and is thought to mediate hCG-LHR interactions. To test the latter notion, we compared the LHR binding and signal transduction activities of hCG analogs in which the alpha-subunit C terminus (alphaCT) was cross-linked to residues in the small seatbelt loop. Analogs having an intersubunit disulfide between a cysteine in place of alphaCT residue alphaSer-92 and cysteines substituted for loop residues betaArg-94, betaArg-95, or betaSer-96 had high activities in LHR binding and signaling assays despite the fact that both portions of the hormone are thought to be essential for hCG activity. Use of a larger probe blocked hormone activity when the alphaCT was cross-linked to cysteines in place of residues betaArg-95 and betaAsp-99, but not to cysteines in place of residues betaArg-94, betaSer-96, or betaThr-97. This suggested that the side chains of residues betaArg-95 and betaAsp-99, which face in the same outward direction from the heterodimer, are nearer than the others to the LHR interface. The finding that residue 95 can be cross-linked to small alphaCT probes without eliminating hormone activity indicates its side chain does not participate in essential LHR contacts. We suggest that contacts between the small seatbelt loop and the LHR, if any, involve its backbone atoms and possibly the side chain of residue betaAsp-99.     

Glycoprotein hormone assembly in the endoplasmic reticulum: III. The seatbelt and its latch site determine the assembly pathway

Vertebrate glycoprotein hormone heterodimers are stabilized by a strand of their beta-subunits known as the "seatbelt" that is wrapped around loop 2 of their alpha-subunits (alpha2). The cysteine that terminates the seatbelt is "latched" by a disulfide to a cysteine in beta-subunit loop 1 (beta1) of all vertebrate hormones except some teleost follitropins (teFSH), wherein it is latched to a cysteine in the beta-subunit NH(2) terminus. As reported here, teFSH analogs of human choriogonadotropin (hCG) are assembled by a pathway in which the subunits dock before the seatbelt is latched; assembly is completed by wrapping the seatbelt around loop alpha2 and latching it to the NH(2) terminus. This differs from hCG assembly, which occurs by threading the glycosylated end of loop alpha2 beneath the latched seatbelt through a hole in the beta-subunit. The seatbelt is the part of the beta-subunit that has the greatest influence on biological function. Changes in its sequence during the divergence of lutropins, follitropins, and thyrotropins and the speciation of teleost fish may have impeded heterodimer assembly by a threading mechanism, as observed when the hCG seatbelt was replaced with its salmon FSH counterpart. Whereas wrapping is less efficient than threading, it may have facilitated natural experimentation with the composition of the seatbelt during the co-evolution of glycoprotein hormones and their receptors. Migration of the seatbelt latch site to the NH(2)-terminal end of the beta-subunit would have facilitated teFSH assembly by a wraparound mechanism and may have contributed also to its ability to distinguish lutropin and follitropin receptors.     

Glycoprotein hormone assembly in the endoplasmic reticulum: II. Multiple roles of a redox sensitive beta-subunit disulfide switch

All three human glycoprotein hormone heterodimers are assembled in the endoplasmic reticulum by threading the glycosylated end of alpha-subunit loop two (alpha2) beneath a disulfide "latched" strand of the beta-subunit known as the "seatbelt." This remarkable event occurs efficiently even though the seatbelt effectively blocks the reverse process, thereby stabilizing each heterodimer. Studies described here show that assembly is facilitated by the formation, disruption, and reformation of a loop within the seatbelt that is stabilized by the most easily reduced disulfide in the free beta-subunit. We refer to this disulfide as the "tensor" because it shortens the seatbelt, thereby securing the heterodimer. Formation of the tensor disulfide appears to precede and facilitate seatbelt latching in most human choriogonadotropin beta-subunit molecules. Subsequent disruption of the tensor disulfide elongates the seatbelt, thereby increasing the space beneath the seatbelt and the beta-subunit core. This permits the formation of hydrogen bonds between backbone atoms of the beta-subunit cystine knot and the tensor loop with backbone atoms in loop alpha2, a process that causes the glycosylated end of loop alpha2 to be threaded between the seatbelt and the beta-subunit core. Contacts between the tensor loop and loop alpha2 promote reformation of the tensor disulfide, which explains why it is more stable in the heterodimer than in the uncombined beta-subunit. These findings unravel the puzzling nature of how a threading mechanism can be used in the endoplasmic reticulum to assemble glycoprotein hormones that have essential roles in vertebrate reproduction and thyroid function.     

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.     

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.     

The polypeptide part of human chorionic gonadotrophin affects the kinetics of alpha 6-sialylation of its N-linked glycans but does not alter the branch specificity of CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase.

Human chorionic gonadotrophin (hCG) is a heterodimeric glycoprotein hormone consisting of an alpha- and a beta-subunit, both containing two N-linked, complex-type glycans. Using this hormone as a model glycoprotein, the influence of its polypeptide part on the activity and specificity of bovine colostrum CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase (alpha 6-sialyltransferase) was investigated. Initial rates of sialic acid incorporation into the desialylated glycans of hCG alpha and hCG beta in the heterodimer were higher with the alpha-subunit. This appeared to be due to a higher V which, together with a slightly lowered affinity (higher Km), resulted in a higher kinetic efficiency of the sialyltransferase for the glycans of this subunit. By contrast, the kinetic parameters did not differ significantly when the subunits were in the free form, indicating that the differences in the kinetics of sialylation found for the subunits in the heterodimeric state were not caused by the differences in N-linked carbohydrate structures of the subunits. It is proposed that these effects are due to conformational constraints which the polypeptide moieties put on the glycan chains upon dimerization. Furthermore, it was investigated whether the polypeptide of hCG would interfere with the sialyltransferase so as to alter the branch specificity of the enzyme. 1H-NMR spectroscopy (400 MHz) of the glycan chains, alpha 6-sialylated in vitro, showed that the enzyme highly prefers the galactosyl residue at the Gal beta 1----4GlcNAc beta 1----2-Man alpha 1----3Man branch for attachment of the first mol of sialic acid into the diantennary glycans of desialylated hCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycoprotein hormone assembly in the endoplasmic reticulum: I. The glycosylated end of human alpha-subunit loop 2 is threaded through a beta-subunit hole

Glycoprotein hormone heterodimers are stabilized by their unusual structures in which a glycosylated loop of the alpha-subunit straddles a hole in the beta-subunit. This hole is formed when a cysteine at the end of a beta-subunit strand known as the "seatbelt" becomes "latched" by a disulfide to a cysteine in the beta-subunit core. The heterodimer is stabilized in part by the difficulty of threading the glycosylated end of the alpha-subunit loop 2 through this hole, a phenomenon required for subunit dissociation. Subunit combination in vitro, which occurs by the reverse process, can be accelerated by removing the alpha-subunit oligosaccharide. In cells, heterodimer assembly was thought to occur primarily by a mechanism in which the seatbelt is wrapped around the alpha-subunit after the subunits dock. Here we show that this "wraparound" process can be used to assemble disulfide cross-linked human choriogonadotropin analogs that contain an additional alpha-subunit cysteine, but only if the normal beta-subunit latch site has been removed. Normally, the seatbelt is latched before the subunits dock and assembly is completed when the glycosylated end of alpha-subunit loop 2 is threaded beneath the seatbelt. The unexpected finding that most assembly of human choriogonadotropin, human follitropin, and human thyrotropin heterodimers occurs in this fashion, indicates that threading may be an important phenomenon during protein folding and macromolecule assembly in the endoplasmic reticulum. We suggest that the unusual structures of the glycoprotein hormones makes them useful for identifying factors that influence this process in living cells.     

Role of the invariant aspartic acid 99 of human choriogonadotropin beta in receptor binding and biological activity

The four human glycoprotein hormones are heterodimers that contain a common alpha subunit and a hormone-specific beta subunit. Within this hormone family, 23 amino acid sequences from 11 mammalian species are available. There are 19 invariant amino acid residues in the beta subunits, 12 of which are Cys that form six disulfide bonds. Of the remaining seven conserved amino acid residues, we have investigated the role of an Asp which occurs at position 99 in human choriogonadotropin beta (hCG beta). Site-directed mutagenesis was used to replace hCG beta Asp99 with three residues, Glu, Asn, and Arg, and to prepare an inversion double mutant protein, Arg94----Asp and Asp99----Arg. The cDNAs were placed in a eukaryotic expression vector, and the plasmids were transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for bovine alpha. Radioimmunoassays demonstrated that the mutant forms of hCG beta were capable of subunit assembly to the same extent as hCG beta wild type. The heterologous heterodimers were assayed in vitro using transformed mouse Leydig cells (MA-10) by competitive inhibition of 125I-hCG binding and stimulation of progesterone production. The gonadotropins containing Glu and Asn were active, although the potency was less than that associated with the hCG beta wild type-containing gonadotropin. In contrast, the Arg99-containing mutant protein and the inversion mutant protein Asp94/Arg99 were devoid of activity. Thus, in hCG beta Asp99 can be substituted with certain residues without total loss of function, although replacement with a positively charged residue leads to an inactive heterodimer. The primary role of Asp99 in hCG beta seems to involve, either directly or indirectly, receptor recognition.     

The biological action of choriogonadotropin is not dependent on the complete native quaternary interactions between the subunits

Human CG (hCG) is a member of the glycoprotein hormone family characterized by a heterodimeric structure consisting of a common alpha-subunit noncovalently bound to a hormone-specific beta-subunit. The two subunits are highly intertwined and only the heterodimer is functional, implying that the quaternary structure is critical for biological activity. To assess the dependence of the bioactivity of hCG on the heterodimeric interactions, alpha- and beta-subunits bearing mutations that prevent assembly were covalently linked to form a single chain hCG. Receptor binding and signal transduction of these analogs were tested and their structural integrity analyzed using a panel of monoclonal antibodies (mAbs). These included dimer-specific mAbs, which react with at least four different epitope sites on the hormone, and some that react only with the free beta-subunit. We showed that there was significant loss of quaternary and tertiary structure in several regions of the molecule. This was most pronounced in single chains that had one of the disulfide bonds of the cystine knot disrupted in either the alpha- or beta-subunit. Despite these structural changes, the in vitro receptor binding and signal transduction of the single chain analogs were comparable to those of the nonmutated single chain, demonstrating that not all of the quaternary configuration of the hormone is necessary for biological activity.     

A nicked beta-subunit of human chorionic gonadotropin purified from pregnancy urine.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone composed of two dissimilar subunits (alpha and beta) and normally excreted in urine of pregnant women. An uncommon beta-subunit of hCG was purified from fresh early normal pregnancy urine by Sepralyte C8, resin adsorption. Sephadex G-100 column chromatography, and reverse-phase HPLC. SDS-PAGE under non-reducing conditions showed that the apparent molecular weight (39,000) of this beta-subunit was extremely similar to that of the native beta-subunit, which is known to consist of 145 amino acid residues and carbohydrates. However, SDS-PAGE, under reducing conditions, resulted in two bands with apparent molecular weights of 22,000 and 18,000, indicating that it consisted of two peptide fragments connected with disulfide bridge(s). These two peptide fragments, separated and purified from the reduced and carboxymethylated protein, were subjected to amino acid and N-terminal sequence analyses. It was found that this beta-subunit consisted of two polypeptide chains composed of residues 1-47 disulfide-bridged to residues 48-145 of the beta-subunit, which may be produced by nicking of the beta-subunit at the one site (Gly47-Val48). This beta-subunit was termed a nicked beta-subunit of hCG (N-hCG beta). It was also found that N-hCG beta was present in urine as an alpha beta dimer, indicating that an intrachain nicking of this site in the beta-subunit does not inhibit alpha beta dimer formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Sulfur shuffle: modulating enzymatic activity by thiol-disulfide interchange

The facile modulation of biological processes is an important goal of biological chemists. Here, a general strategy is presented for controlling the catalytic activity of an enzyme. This strategy is demonstrated with ribonuclease A (RNase A), which catalyzes the cleavage of RNA. The side-chain amino group of Lys41 donates a hydrogen bond to a nonbridging oxygen in the transition state for RNA cleavage. Replacing Lys41 with a cysteine residue is known to decrease the value of k(cat)/K(m) by 10(5)-fold. Forming a mixed disulfide between the side chain of Cys41 of K41C RNase A and cysteamine replaces the amino group and increases k(cat)/K(m) by 10(3)-fold. This enzyme, which contains a mixed disulfide, is readily deactivated by dithiothreitol. Forming a mixed disulfide between the side chain of Cys41 and mercaptopropyl phosphate, which is designed to place a phosphoryl group in the active site, decreases activity by an additional 25-fold. This enzyme, which also contains a mixed disulfide, is reactivated in the presence of dithiothreitol and inorganic phosphate (which displaces the pendant phosphoryl group from the active site). An analogous control mechanism could be installed into the active site of virtually any enzyme by replacing an essential residue with a cysteine and elaborating the side chain of that cysteine into appropriate mixed disulfides.     

Use of protein knobs to characterize the position of conserved alpha-subunit regions in lutropin receptor complexes

Efforts to identify the manner in which human choriogonadotropin (hCG) contacts lutropin receptors (LHR) have been stymied by the complex structure of the hormone and the likelihood that it contacts the receptor at multiple sites. During studies of hCG assembly in mammalian cells, we found that addition of a cysteine to the long disordered beta-subunit COOH terminus (betaCT) enabled it to become cross-linked by a disulfide to cysteines that are substituted for residues in loop alpha2 or in the alpha-subunit COOH terminus (alphaCT). This created a "knob" on the alpha-subunit at the location of the cysteine. Knobs of various sizes and charges were useful for probing surfaces of the alpha-subunit thought previously to contact the LHR. Attachment of the betaCT to residues in loop alpha2 facing loops beta1 and beta3 reduced hormone activity only a few fold revealing that this surface does not participate in essential high affinity receptor contacts, a finding inconsistent with our earlier view of the hCG-LHR complex. In contrast, this approach showed that the opposite surface of loop alpha2 appeared to be nearer the receptor interface. Although attachment of knobs to portions of the alphaCT reduced hormone activity substantially, this finding was difficult to interpret. As discussed, this procedure should be adapted readily to other proteins and may facilitate the introduction of fluorophores, enzymes, or other reagents at specific sites on protein surfaces. It may also permit one to cross-link proteins or to obscure specific protein surfaces during the development of "Trojan Horse" therapeutics.     

The glycoprotein hormones: recent studies of structure-function relationships

The structural features of the heterodimeric glycoprotein hormones (LH, FSH, TSH, and hCG) are briefly reviewed. Removal of carbohydrate chains does not reduce binding of the hormones to membrane receptors, but markedly reduces biological responses. The glycopeptides from the hormone do not reduce binding of native hormone to receptors but do reduce biological responses. Newer data concerned with replication of different regions of the peptide chains of these molecules using synthetic peptides are reviewed and presented. These studies indicate that two regions on the common alpha subunit are involved with receptor binding of the LH, hCG, and TSH molecules. These regions are alpha 26 to 46 and alpha 75-92. Two synthetic disulfide loop peptides from the hCG beta subunit beta 38-57 and beta 93-100 also block binding of hCG to its receptor. In addition, the beta 38-57 peptide stimulates testosterone production by Leydig cells. These data indicate that glycoprotein hormone binding to plasma membrane receptors involves a discontinuous site on the hormone that spans both the alpha and beta subunits, and that the alpha subunit sites are similar for several hormones.     

Site-directed mutagenesis defines a domain in the gonadotropin alpha-subunit required for assembly with the chorionic gonadotropin beta-subunit.

hCG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that share a common alpha-subunit, but differ in their hormone-specific beta-subunits. Using site-directed mutagenesis and gene transfer, we studied the region in the common alpha-subunit that has been implicated in the assembly with the beta-subunits. The wild-type or mutated alpha-gene was cotransfected into Chinese hamster ovary cells with the wild-type hCG beta gene. Deletion of the sequence Pro38-Thr39-Pro40 or a change in Tyr37 or Thr39 in the alpha-subunit eliminated or reduced combination with the beta-subunit. Deletion of the sequence Leu41-Arg42-Ser43 had little effect on hCG dimer formation. Disruption of the disulfide bone in the carboxyl end of the subunit did not affect assembly, which suggests that the disulfide bond of Cys59 and Cys87 is not critical for dimer formation. Based on our data and the previously published results from several laboratories, the region encompassed by amino acids 37-40 is a key determinant in initiating and maintaining alpha:beta assembly.     

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.     

Localization of residues that confer antibody binding specificity using human chorionic gonadotropin/luteinizing hormone beta subunit chimeras and mutants.

The glycoprotein hormones are a family of conserved heterodimeric proteins which share a common alpha subunit but differ in their hormone-specific beta subunits. We used chimeras of human chorionic gonadotropin (hCG) and luteinizing hormone (hLH) beta subunits to identify residues which enable monoclonal antibodies (mAb) to distinguish the two hormones. The LH beta-CG beta chimeras appeared to fold similar to hCG beta, since they combined with hCG alpha and, depending on their sequences, were recognized by hCG-selective mAbs. Amino acid residues Arg8-Arg10,Gly47-Ala51, and Gln89-Leu92 form a major epitope region and appear to be adjacent to each other on the surface of hCG beta. Gly47-Ala51 and Gln89-Leu92 are recognized by dimer-specific mAbs while Arg8-Arg10 is recognized by mAbs which have highest affinity for the free beta subunit. These observations suggest that the conformation of this region of the beta subunit changes when the alpha and beta subunits combine. Residues which are C-terminal of Asp112 form a second epitope domain. mAbs to the third domain distinguish hCG beta and hLH beta by the presence of Asn77 in hCG beta and can be detected after hCG binds to receptors. These findings were used to develop a model of hCG beta which predicts the locations of these residues and their positions relative to the alpha subunit and receptor interfaces.