Mary R. Pixley

Cystine knot of the gonadotropin alpha subunit is critical for intracellular behavior but not for in vitro biological activity.

The common α subunit of glycoprotein hormones contains five disulfide bonds. Based on the published crystal structure, the assignments are 7–31, 59–87, 10–60, 28–82, and 32–84; the last three comprise the cystine knot, a structure also seen in a variety of growth factors. Previously, we demonstrated that the efficiency of secretion and the ability to form heterodimers by α subunits bearing single cysteine residue mutants in the cystine knot were significantly reduced. These results suggested that the cystine knot is critical for the intracellular integrity of the subunit. To assess if the presence of the free thiol affected the secretion kinetics, we constructed paired cysteine mutants of each disulfide bond of the α subunit. The secretion rate for these monomers was comparable with wild type except for the α-10–60 mutant, which was 40% lower. The recovery of the α7–31 and α59–87 mutants was greater than 95%, whereas for the cystine knot mutants, it was 20–40%. Co-expression of the wild-type chorionic gonadotropin β subunit with double cysteine mutants did not enhance the recovery of α mutants in the media. Moreover, compared with wild-type, the efficiency of heterodimer formation of the α10–60 or α32–84 mutants was less than 5%. Because subunit assembly is required for biological activity, studies on the role of these disulfide bonds in signal transduction were not possible. To bypass the assembly step, we exploited the single chain model, where the α and β subunits are genetically fused. The recovery of secreted tethered gonadotropins bearing mutations in the cystine knot was increased significantly. Although dimer-specific monoclonal antibodies discriminated the conformation of single chain α10–60 and α32–84 mutants from the native heterodimer, these mutants were nevertheless biologically active. Thus, individual bonds of cystine knot are important for secretion and heterodimer formation but not for in vitro bioactivity. Moreover, the data suggest that the native heterodimer configuration is not a prerequisite for receptor binding or signal transduction.

CHARACTERIZATION OF THE O-GLYCOSYLATION SITES IN THE CHORIONIC GONADOTROPIN BETA SUBUNIT IN VIVO USING SITE-DIRECTED MUTAGENESIS AND GENE TRANSFER

Human chorionic gonadotropin (CG) is a member of a family of glycoprotein hormones which are heterodimers containing two nonidentical subunits: a common α and a hormone-specific β subunit. One of the distinguishing features of the CGβ subunit is the presence of four serine acceptors clustered within the last 25 amino acids. We previously demonstrated that this carboxyl-terminal region is important for maintaining its biologic half-life, and when the sequence was genetically fused to either the common α or follitropin β subunits, O-glycosylation was observed. Because this carboxyl-terminal sequence is located at the end of the subunit, we considered this region a convenient in vivo model for studying O-linked glycosylation in domains containing multiple serine recognition sites. A CGβ gene was engineered in which the N-linked sites were inactivated to eliminate background from those carbohydrate groups. Using this construct, we made a series of truncation and amino acid substitutions of acceptor serines, and these mutants were transfected into Chinese hamster ovary cells. O-Glycosylation was determined by [3H]glucosamine incorporation and glycanase sensitivity of the products on SDS-polyacrylamide gels. We show that the O-linked sites comprise independent repetitive regions in which each acceptor serine has a recognition signal bounded by the next carboxy acceptor serine within four to five amino acids. It is also apparent that recognition of one site is not dependent on the glycosylation of another acceptor. Amino acid mutations in the acceptor regions demonstrated the importance of proline as a necessary feature for O-linked recognition in the CGβ sequence.

The Biologic Action of Single-chain Choriogonadotropin Is Not Dependent on the Individual Disulfide Bonds of the β Subunit

Disrupting disulfide loops in the human chorionic gonadotropin β subunit (CGβ) inhibits combination with the α subunit. Because the bioactivity requires a heterodimer, studies on the role of disulfide bonds on receptor binding/signal transduction have previously been precluded. To address this problem, we bypassed the assembly step and genetically fused CGβ subunits bearing paired cysteine mutations to a wild-type α (WTα) subunit. The changes altered secretion of the single-chain mutants which parallel that seen for the CGβ monomeric subunit. Despite conformational changes in CG disulfide bond mutants (assayed by gel electrophoresis and conformationally sensitive monoclonal antibodies), the variants bind to the lutropin/CG receptor and activated adenylate cyclase in vitro The data show that the structural requirements for secretion and bioactivity are not the same. The results also suggest that the extensive native subunit interactions determined by the cystine bonds are not required for signal transduction. Moreover, these studies demonstrate that the single-chain model is an effective approach to structure-activity relationships of residues and structural domains associated with assembly of multisubunit ligands.

Expression of biologically active fusion genes encoding the common α subunit and either the CGβ or FSHβ subunits: role of a linker sequence

Abstract The gonadotropin/thyrotropin hormone family is characterized by a heterodimeric structure composed of a common α subunit non-covalently linked to a hormone-specific β subunit. The conformation of the heterodimer is essential for controlling secretion, hormone-specific post-translational modifications and signal transduction. Structure-function studies of FSH and the other glycoprotein hormones are often hampered by mutagenesis induced defects in subunit combination. Thus, the ability to overcome the limitation of subunit assembly would expand the range of structure activity relationships that can be performed on these hormones. Here we converted the FSH heterodimer to a single chain by genetically fusing the carboxyl end of the FSHβ subunit to the amino end of the α subunit in the presence or absence of a natural linker sequence. In the absence of the CTP linker, the secretion rate was decreased over three fold. (The CTP sequence is the last 28 amino acids of the CGβ sequence and contains four serine-linked oligosaccharides). Unexpectedly however, receptor binding/signal transduction was unaffected by absence of the linker. Molecular modelling of the tethers lacking the linker sequence show that the alignment of the α β domains in the single chain differ substantially from that seen in the heterodimer. These data show that the single chain FSH was secreted efficiently and is biologically active and that the conformation determinants required for secretion and biologic activity are not the same.