David Ben-Menahem

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.

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.

Converting heterodimeric gonadotropins to genetically linked single chains: new approaches to structure activity relationships and analogue design.

One of the distinguishing features of the gonadotropin and thyrotropin hormone family is their heterodimeric structure; the subunits combine early in the secretory pathway and only the dimers are capable of binding to receptors. Therefore, assembly is rate limiting in the production of functional heterodimers, a problem encountered when removing the carbohydrates from one or both subunits as discussed in this review. If the heterodimers can be expressed as single chains, this might avoid mutagenesis-induced defects in secretion and combination of individual subunits for structure-function studies and analogue design. Here we discuss the feasibility of this approach for such problems.

Cytosolic Phospholipase A2α Is Targeted to the p47phox-PX Domain of the Assembled NADPH Oxidase via a Novel Binding Site in Its C2 Domain

We have previously demonstrated a physical interaction between cytosolic phospholipase A2α (cPLA2) and the assembled NADPH oxidase on plasma membranes following neutrophil stimulation. The aim of the present study was to define the exact binding sites between these two enzymes. Here we show, based on blot overlay experiments, Förster resonance energy transfer analysis and studies in neutrophils from patients with chronic granulomatous disease deficient in p67phox or p47phox, that cPLA2 specifically binds to p47phox and that p47phox is sufficient to anchor cPLA2 to the assembled oxidase on the plasma membranes upon stimulation. Blot overlay and affinity binding experiments using subfragments of cPLA2 and p47phox demonstrated that the cPLA2-C2 domain and the p47phox-PX domain interact to form a complex that is resistant to high salt. Computational docking was used to identify hydrophobic peptides within these two domains that inhibited the association between the two enzymes and NADPH oxidase activity in electro-permeabilized neutrophils. These results were used in new docking computations that produced an interaction model. Based on this model, cPLA2-C2 domain mutations were designed to explore its interaction p47phox in neutrophil lysates. The triple mutant F35A/M38A/L39A of the cPLA2-C2 domain caused a slight inhibition of the affinity binding to p47phox, whereas the single mutant I67A was highly effective. The double mutant M59A/H115A of the p47phox-PX domain caused a significant inhibition of the affinity binding to cPLA2. Thus, Ile67 of the cPLA2-C2 domain is identified as a critical, centrally positioned residue in a hydrophobic interaction in the p47phox-PX domain.

The LHβ Gene of Several Mammals Embeds a Carboxyl-terminal Peptide-like Sequence Revealing a Critical Role for Mucin Oligosaccharides in the Evolution of Lutropin to Chorionic Gonadotropin in the Animal Phyla

The expression of a previously untranslated carboxylterminal sequence is associated with the ancestral lutropin (LH) β to the β-subunit gene evolution of choriogonadotropins (CG). The peptide extension (denoted as CTP) is rich in mucin-type O-glycans and confers new hormonal properties on CG relative to the LH. Although the LHβ gene is conserved among mammals and only a few frameshift mutations account for the extension, it is merely seen in primates and equids. Bioinformatics identified a CTP-like sequence that is encrypted in the LHβ gene of several mammalian species but not in birds, amphibians, or fish. We then examined whether or not decoding of the cryptic CTP in the bovine LHβ gene (boCTP) would be sufficient to generate the LHβ species of a ruminant with properties typical to the CGβ subunit. The mutated bovine LHβ-boCTP subunit was expressed and N-glycosylated in transfected Chinese hamster ovary cells. However, unlike human (h) CGβ CTP, the cryptic boCTP was devoid of mucin O-glycans. This deficiency was further confirmed when the boCTP domain was substituted for the natural CTP in the human CGβ subunit. Moreover, when expressed in polarized Madin-Darby canine kidney cells, this hCGβ-boCTP chimera was secreted basolaterally rather than from the apical compartment, which is the route of the wild type hCGβ subunit, a sorting function attributed to the O-glycans attached to the CTP. This result shows that the cryptic peptide does not orientate CG to the apical face of the placenta, to the maternal circulation as seen in primates. The absence of this function, which distinguishes CG from LH, provides an explanation as to why the LHβ to CGβ evolution did not occur in ruminants. We propose that in primates and equids, further natural mutations in the progenitor LHβ gene resulted in the efficient O-glycosylation of the CTP, thus favoring the retention of an elongated reading frame.