Hormone-induced conformational change of the purified soluble hormone binding domain of follitropin receptor complexed with single chain follitropin.

Human follicle-stimulating hormone receptor (hFSHR) belongs to family I of G protein-coupled receptors. FSHR extracellular domain (ECD) is predicted to have 8-9 alphabeta or leucine-rich repeat motif elements. The objective of this study was to identify elements of the FSHR ECD involved in ligand binding. Preincubation of recombinant hFSHR ECD with rabbit antisera raised against synthetic peptides of hFSHR ECD primary sequence abolished follitropin binding primarily in the region of amino acids 150-254. Accessibility of hFSHR ECD after hormone binding, captured by monoclonal antibodies against either ECD or FSH, was decreased for the region of amino acids 150-220 but additionally for amino acids 15-100. Thus, when hFSH bound first, accessibility of antibody binding was decreased to a much larger extent than if antibody was bound first. This suggestion of a conformational change upon binding was examined further. Circular dichroism spectra were recorded for purified single chain hFSH, hFSHR ECD, and hFSHR ECD-single chain hFSH complex. A spectral change indicated a small but consistent conformational change in the ECD.FSH complex after hormone binding. Taken together, these data demonstrate that FSH binding requires elements within the leucine-rich repeat motifs that form a central region of hFSHR ECD, and a conformational change occurs upon hormone binding.

Reversible immunoneutralization of human follitropin receptor.

Investigations of the human Follitropin receptor (hFSHR) have failed to identify the tertiary structure that forms the active hormone-receptor interaction site which is essential to develop an immunocontraceptive based upon the receptor. To identify such a domain of hFSHR, an immunoneutralizing monoclonal antibody (mAb) 106-105 (IgG2b) was generated. Flow cytometry tested whether mAb 106-105 recognized native hFSHR. The epitope of mAb 106-105 was mapped by Western blot and by peptide ELISA. Inhibition of hFSH binding and bioactivity was determined by radioreceptor assay and by cAMP production, respectively. MAb 106-105 bound native hFSHR through an epitope including residues 300-315. MAb 106-105 completely blocked hormone binding to receptor and cAMP production by Y1-R cells expressing hFSHR. These effects were completely reversible by increasing the concentration of hFSH. Coincubation of this antibody with peptide D300-F315 blocked antibody activity. These data demonstrate that a discrete linear hFSHR epitope is a target for interference with hormone activity. These results further demonstrate that antibody binding to the extracellular domain (ECD) of hFSHR and subsequent bioactivation can be modulated through a domain specific hindrance, offering a reversible immunoneutralizing target.

Human follicle stimulating hormone receptor variants lacking transmembrane domains display altered post-translational conformations.

Variant splicing of gonadotropin receptor mRNA commonly occurs, however expression of receptor protein variants and their trafficking has yet to be studied in detail. To determine receptor variant trafficking and intracellular processing in mammalian cells, the intracellular fate of intentionally truncated variants of human follicle stimulating hormone receptor (hFSH-R) expressed in CHO cells was examined. Monoclonal antibodies (mAbs) were made against the hFSH-R's extracellular domain (ECD) expressed in insect cells. Four mAbs 106.156, 106.290, 106.318, and 106.263 were chosen as probes. Epitope mapping using synthetic peptides, and truncated hFSH-R variants revealed that mAb 106.156 bound to ECD residues 183-220, while mAbs 106.318, 106.290, 106.263 bound ECD residues 300-331. Immunofluorescence microscopy showed that mAbs 106.318 and 106.156 stained the surface of fixed, intact CHO cells expressing wild type hFSH-R. However, following cell permeabilization all four antibodies stained hFSH-R in Golgi and endoplasmic reticulum. Permeabilized cells expressing truncated variants ECD213 and ECD254 showed staining accumulated in the endoplasmic reticulum/nuclear envelope continuum. ECD335/His was found to accumulate in extended endoplasmic reticulum (ER). The ER location of ECD335/His was confirmed by double labeling experiments with concanavalin A and ECD mAb. Glycosidase digestion followed by Western blot analysis show ECD213 and ECD335/His to be glycosylated, but not ECD254. Both glycosylated truncated hFSH-R variants were sensitive to peptide-N-glycanase F and endoglycosidase H but insensitive to neuraminidase indicating that these variants possess high mannose type oligosaccharides. Thus truncated hFSH-R variants do not reach the medial or trans Golgi where high mannose oligosaccharides are trimmed and sialic acid is added. These data suggest that the conformation the ECD of the wild type receptor is different from the ECD alone expressed in the endoplasmic reticulum. This information suggests that the ECD serves two distinct roles; the first is to bind FSH and the other is likely to contact the endodomain of the receptor, which presumably leads to activation of the endodomain for signal transduction.

A follicle-stimulating hormone receptor ecto-domain epitope that is a target for receptor immunoneutralization yet does not affect ligand contact and activation.

The follicle-stimulating hormone receptor (FSHR) large extracellular domain suggests that interaction of ligand with receptor is likely to be complex. Residues 265-296 of the FSHR are part of a sequence primarily nonhomologous with other glycoprotein hormone receptors. A reasonable hypothesis is that this sequence of the FSHR plays a role in binding FSH. Flow cytometry studies of this region revealed that antibody X179 against peptide R265-S296 binds to human FSHR expressed by CHO cells and can be competed against by preincubating the cells with hFSH. These results suggested that the region corresponding to residues 265-296 in the extracellular domain of the FSHR is involved in binding to hormone. To test this hypothesis 10 scanning alanine mutants of rFSHR at the 265-296 epitope were generated, and the binding characteristics of these mutants were studied. Their affinity constants for 125I-hFSH did not deviate greatly from that of wild-type FSHR, in which some mutants exhibited an approximately two- to threefold reduction in Ka compared to wild-type receptor, and no mutation abolished signal transduction. These results lead to rejection of the hypothesis that this region contains residues critical for conveying hormone specificity and receptor-dependent hormone action.

Characterization of human cellular gamma-glutamyl hydrolase.

A previously identified cDNA encoding a human gamma-glutamyl hydrolase was expressed in a baculovirus system. The expressed protein had molecular mass of 37 kDa. Treatment of the protein with PNGase F produced a protein of molecular mass of 30 kDa, indicating that the protein contained asparagine-linked glycosylation. Sequence analysis of the expressed protein indicated that a 24-amino-acid signal peptide had been removed. A polyclonal antibody to the expressed enzyme was used in Western blot analysis of partially purified lysates of HL-60 promyeloid leukemia cells and MCF-7 breast cancer cells. The HL-60 and MCF-7 enzymes appeared as two closely spaced bands with a molecular mass of 37 kDa. Treatment of the HL-60 enzyme with PNGase F produced a protein with a molecular mass of 30 kDa. The activities of the expressed enzyme and the enzyme from HL-60 cells were similar on methotrexate polyglutamates. Methotrexate-gamma-Glu is a poor substrate for the human enzyme relative to methotrexate gamma-Glu2-5. During hydrolysis of methotrexate-gamma-Glu4, all possible pterin-containing cleavage products (methotrexate and methotrexate-gamma-Glu1-3) appear. The results demonstrated that the human enzyme cleaves both the ultimate and penultimate gamma-linkages of methotrexate polyglutamates. Glutamate was released as either glutamic acid or gamma-Glu2. Longer chain species of gamma-Glun>2 were not observed. Inhibition by iodoacetic acid suggested that both the expressed enzyme and the HL-60 enzyme may contain a catalytically essential cysteine. These results indicate that the identified cDNA encodes the intracellular gamma-glutamyl hydrolase found in a variety of human tumor cells and that the baculovirus-expressed enzyme is a suitable model for further structural and enzymatic studies.

The human follitropin alpha-subunit C terminus collaborates with a beta-subunit cystine noose and an alpha-subunit loop to assemble a receptor-binding domain competent for signal transduction.

FSH is a member of the pituitary/placental glycoprotein hormone family including luteinizing hormone, thyroid-stimulating hormone, and chorionic gonadotropin. These heterodimeric hormones share a common alpha-subunit and a highly homologous but distinct beta-subunit. The determinant loop of the FSH beta-subunit acts both as a specificity discriminator and as an essential receptor-binding site. The three-dimensional structure of hCG illustrates the proximity of the determinant loop to the carboxyl-terminal residues of the common alpha-subunit. Thus, site-directed mutagenesis was used to mak high-resolution substitutions at this carboxyl-terminal locus. The effects of those substitutions were studied. Twelve single mutations and one composite mutation were made of the region of hFSH alpha 74-92, each residue substituted by alanine. Side chain replacement in this region of FSH proved to be detrimental to binding. hFSH with mutations of either alpha S85A, alpha T86A, alpha K91A, or alpha S92A only retained 10% or less of the hFSH receptor-binding activity, while compared to these, mutants alpha H79A, alpha Y88A, and alpha Y89A retained slightly more binding activity. The single mutant alpha F74A and composite mutant alpha V76A/E77A binding activity was reduced to half of that of wild-type (WT) hFSH. In contrast, mutations of either alpha K75A, alpha T80A, alpha H83A, or alpha H90A did not adversely affect receptor binding, demonstrating the specificity of observed effects. The hFSH and mutant hormones were tested in an in vitro bioassay for stimulation of progesterone production. Those mutations that did not affect receptor binding (alpha K75A, alpha T80A, alpha H83A, and alpha H90A) did not affect signal transduction, measured by progesterone responses. After comparison of wild-type and mutant hFSH activities determined in radioreceptor assays (ID50) and in vitro bioassays (ED50), it became evident that signal transduction correlated with receptor binding.

Protection of rats against oxygen toxicity by tracheal administration of plasmid DNA: role of endogenous tumor necrosis factor.

Recombinant plasmid DNA alone or in conjunction with a gene delivery system has been used increasingly for in vivo gene transfer. However, little is known about the direct biological effects of plasmid DNA. In this study, we demonstrated that tracheal administration of a number of plasmid DNA protected rats against oxygen toxicity. This protection required the presence of intact plasmid DNA, was not due to endotoxin contamination, and was not shared by salmon testis DNA. The plasmid DNA-induced protection against oxygen toxicity was associated with the production of tumor necrosis factor (TNF) and the enhancement of pulmonary Mn-superoxide dismutase (MnSOD), CuZnSOD, and glutathione peroxidase activities. Coadministration of plasmid DNA and anti-TNF antibody (but not nonspecific IgG) partially abolished the protective effect and reduced the pulmonary MnSOD activity, suggesting that the plasmid DNA-induced oxygen tolerance was in part mediated by the endogenous TNF and MnSOD. In view of these observations and the known immunostimulatory effects of bacterial DNA, caution should be exercised in interpreting the results of in vivo gene transfer using recombinant plasmid DNA.

Transfection of a manganese-containing superoxide dismutase gene into hamster tracheal epithelial cells ameliorates asbestos-mediated cytotoxicity.

To determine if overexpression of manganese-containing SOD (MnSOD) alters cell sensitivity to asbestos, an expression cassette containing murine MnSOD cDNA was cotransfected with pSV2neo, a plasmid conferring resistance to the antibiotic G418, into a diploid cell line of hamster tracheal epithelial (HTE) cells. Pools of G418-resistant transfectants were characterized by Southern and Northern blot analyses and enzyme activity assays. Although increases in MnSOD gene copies in individual cell pools ranged from approximately 7- to 86-fold in comparison to cells transfected with pSV2neo alone, steady-state levels of MnSOD mRNA were increased only by 1.4-to 2.3-fold. Despite modest increases in MnSOD mRNA, significant elevations in MnSOD enzyme activity were observed in pools of G418-resistant cells. MnSOD-transfected cell lines were more resistant to the cytotoxic effects of crocidolite asbestos using a sensitive colony-forming efficiency (CFE) assay. These data show that MnSOD has a direct role in cell defense against asbestos-induced cytotoxicity, an oxidant-dependent process.

Overexpression of manganous superoxide dismutase (MnSOD) in pulmonary endothelial cells confers resistance to hyperoxia.

Treatment of cells or organisms with agents that increase the expression of MnSOD confers resistance to certain types of oxidative damage. However, since these treatments also affect other cellular systems with antioxidant capabilities, the role of MnSOD remains uncertain. To better determine whether increased MnSOD expression confers increased resistance to oxidant stress, a eukaryotic expression vector harboring a mouse MnSOD cDNA was constructed. Bovine lung microvessel endothelial cells were co-transfected with the MnSOD expression vector and pSV2-neo, which contains the neor gene and provides a dominant selectable marker. Control clones were generated by transfecting the cells with psV2-neo alone. Stably transfected cell lines were selected and cell lines overexpressing MnSOD were confirmed by Northern blotting, immunoblot analysis, and activity gels. The activities of copper/zinc superoxide dismutase, catalase, and glutathione peroxidase were examined, and no increase in activity of any of these enzymes was detected. Cells were exposed to hyperoxic challenge by treatment with 95% O2 and 5% CO2 for 24 h. Viability was assessed by a clonogenic assay. The cell lines that overexpressed MnSOD showed a twofold increase in survival compared to control cells. These results demonstrate a significant resistance to hyperoxia induced oxidative stress in endothelial cells overexpressing MnSOD.

Identification of amino acids in the C-terminal region of human follicle-stimulating hormone (FSH) beta-subunit involved in binding to human FSH receptor.

Recent analyses of human FSH (hFSH) using antipeptide antibodies, monoclonal antibodies, and chimeric constructions of hCG/hFSH strongly suggest that the C-terminal region, including residues 81-100 of the hFSH beta-subunit, is involved in subunit association as well as hFSH heterodimer binding and/or activation of receptor. To test this hypothesis, site-directed mutagenesis was used to generate five triple alanine mutants of the C-terminal region of hFSH beta: Q81, H83, G85; K86, D88, S89; D90, S91, T92; D93, T95, V96; and R97, G98, L99. The baculovirus-infected insect cell system was used for expression. High Five cells were infected with virus harboring either delta hFSH beta complementary DNA (cDNA) or wild-type hFSH beta (hFSH beta wt) cDNA and coinfected with virus containing hFSH alpha cDNA. After infections, media were assayed for FSH using a heterodimer-specific enzyme-linked immunosorbent capture assay. All delta hFSH beta subunits formed heterodimers with hFSH alpha wt subunit and were secreted in the medium. These results suggest, for all five mutants, that side chains of amino acids substituted with alanine had no significant role in subunit association. The FSHs delta hFSH and hFSHwt were tested in a RRA, using cell lines that express the hFSH receptor, to determine if there were any changes in binding activity. Similarly, delta hFSH and hFSHwt were compared for receptor activation by measuring the levels of progesterone production in an in vitro FSH bioassay. delta hFSH-(93-96) exhibited minimal binding activity and no detectable steroidogenic activity. delta hFSH-(97-99) showed reduced binding affinity compared with that of hFSHwt, whereas the binding potency and bioactivity of the remaining delta hFSH were comparable to those of hFSHwt. These data demonstrate that within the hFSH beta-(81-99) region, FSH receptor-binding sites are contained within the sequence 93-99.

Expression of human catalase in acatalasemic murine SV-B2 cells confers protection from oxidative damage.

Reactive oxygen species have been implicated in aerobic organisms as causative agents in damage to DNA, proteins, and lipids. Catalase is a major enzyme in the defense against such oxidant damage. To determine whether increased catalase expression confers greater resistance to oxidant stress, a eukaryotic expression vector harboring a human catalase cDNA clone was constructed. Acatalasemic murine fibroblasts were then co-transfected with that catalase expression vector and pSV2-neo, and successfully transfected cells were identified by their ability to grow in the presence of geneticin. Clones that contained integrated copies of the catalase expression vector were identified by Polymerase Chain Reaction (PCR) analysis. Stably transfected geneticin-resistant cell lines that overexpressed catalase in potentially positive cell lines were confirmed by catalase enzyme assays. To examine the physiological relevance of catalase overexpression, cells were exposed to oxidant stresses (hydrogen peroxide and hyperoxia), and survival rates were determined. Results demonstrated a significant resistance to oxidative stress in cells overexpressing catalase when compared to controls. These transfected cell lines will provide important models for further evaluation of the role of catalase in protecting cells against the toxic effects of oxygen-derived free radicals and their derivatives.