Salvatore M. Aloj

Sequence similarity between cholera toxin and glycoprotein hormones: Implications for structure activity relationship and mechanism of action

Abstract The B chain of cholera toxin and the β subunits of thyrotropin, luteinizing hormone, human chorionic gonadotropin, and follicle-stimulating hormone are shown to have a region of sequence analogy believed to correlate with their ability to bind to receptors on cell membranes. A possible sequence analogy is also defined in the α subunits of these glycoprotein hormones and a region of the cholera toxin A 1 chain believed to be responsible for adenylate cyclase activation.

The structure and function of glycoprotein hormone receptors: ganglioside interactions with human chorionic gonadotropin.

Abstract Gangliosides inhibit the binding of 125 I-labeled human chorionic gonadotropin to rat testes membranes. The inhibition is the result of an interaction between the hormone and the ganglioside rather than the membrane and ganglioside, and the interaction with the ganglioside can be detected by fluorescence spectroscopy. In both the binding inhibition and fluorescence studies, human chorionic gonadotropin recognizes an oligosaccharide sequence on the ganglioside molecule distinct from the sequence recognized by thyrotropin.

The structure and function of glycoprotein hormone receptors: Ganglioside interactions with luteinizing hormone

Summary Gangliosides inhibit the binding of 125 I-labeled luteinizing hormone to rat testis membranes. The inhibition is the result of an interaction between the hormone and the ganglioside rather than the membrane and ganglioside, and the interaction with ganglioside can be detected by fluorescence spectroscopy. In both the binding inhibition and fluorescence studies, luteinizing hormone recognizes an oligosaccharide sequence on the ganglioside molecule distinct from that of thyrotropin, human chorionic gonadotropin, and cholera toxin.

The rates of dissociation and reassociation of the subunits of human chorionic gonadotropin

Abstract A large change in quantum yield of the fluorescent probe 1,8-anilinonaphthalene sulfonate is produced when it combines with the glycoprotein hormone, human chorionic gonadotropin. A method of analyzing for the hormone in the presence of its subunits has been developed based on the finding that the subunits have no effect on 1,8-anilinonaphthalene sulfonate fluorescence. Quantitative rates of dissociation and recombination can be obtained with very small concentrations of hormone since fluorescence measurements are fast and sensitive. The effects of temperature, pH, and urea concentration on the rate of human chorionic gonadotropin dissociation have been measured. The rates of recombination of subunits have been studied as a function of temperature, pH, and KCl concentration. Human chorionic gonadotropin is stable in water to pH 12 and pH 4.5 at 37 °C.

The binding of thyrotropin to liposomes containing gangliosides

Multilamellar liposomes containing mixed brain gangliosides specifically bind 125I-labeled bovine thyrotropin. Ganglioside-free liposomes do not bind the hormone. The amount of hormone bound correlates with the molar ratio gangliosides: phospholipid. The binding of 125I-labeled thyrotropin to gangliosides containing liposomes resembles the binding to thyroid plasma membranes; it is characterized by relatively high affinity, is competed for by the cold hormone, and is decreased by increasing ionic strength and temperature.

Rates of dissociation and recombination of the subunits of bovine thyrotropin.

Abstract The rates of dissociation and recombination of the subunits of bovine thyrotropin have been measured under a variety of conditions using the fluorescence probe 1,8-anilinonaphthalenesulfonate. The method is based on the fact that the native hormone strongly enhances the fluorescence of 1,8-anilinonaphthalenesulfonate whereas the subunits have very little effect. The hormone can be easily dissociated into subunits, either in dilute acid (pH m ) urea solutions at pH 8.O. The rate of dissociation is first order with time and increases strongly with increasing temperature. The hormone is very stable in alkali, showing little tendency to dissociate below pH 12. After dissociation in acid, the subunits can be recombined between pH 7 and 9 at a rate which increases with increasing temperature and subunit concentration. The recombination is intermediate between first and second order suggesting a two-step mechanism: association of the subunits followed by a first-order refolding process in which the subunits acquire the tertiary structure characterisitc of the native hormone. Difference absorption measurements indicate that the dissociation is accompanied by the exposure of a substantial fraction of the 16 tyrosine residues to the more polar aqueous environment, suggesting major conformational changes in one or both subunits.

Separation of the glycoprotein and ganglioside components of thyrotropin receptor activity in plasma membranes

Abstract The two components of thyroid plasma membranes known to interact with thyrotropin, i.e., a glycoprotein with specific thyrotropin binding activity and the gangliosides of the thyroid membranes, are shown to segregate differently when membranes are solubilized with lithium diiodosalicylate. Individually examined, the interaction of each component with thyrotropin exhibits a different sensitivity to salts. The data suggest that the thyrotropin receptor on the thyroid membrane is a complex which is composed of both glycoprotein and ganglioside components and that its properties are derived from each component.

Graves’ IgG stimulation of iodide uptake in FRTL-5 rat thyroid cells: A clinical assay complementing FRTL-5 assays measuring adenylate cyclase and growth-stimulating antibodies in autoimmune thyroid disease

With optimal conditions and cells maintained in the absence of thyrotropin (TSH) for 7–10 days, IgG preparations from ∼ 90% of patients with active Graves’ disease can exhibit statistically significant stimulation of cAMP levels in rat FRTL-5 thyroid cells as compared to normal controls. FRTL-5 cells maintained in the absence of TSH for 7–10 days lose their ability to take up iodide. Iodide uptake returns upon readdition of TSH over a 60-hour period via a cAMP-mediated process; thus TSH can be replaced by dibutyryl cAMP or other agents which increase cAMP levels, for example, thyroid-stimulating autoantibodies (TSAbs) from Graves’ sera. TSAb stimulation of iodide uptake requires the continued presence of TSAb over at least the first 24 hours of a 48-hour reversal period; TSH, in contrast, can be withdrawn after 5 hours and will still achieve maximal effects at 36–48 hours. Iodide uptake, measured as a 30-minute pulse at 48 hours, appears, however, to be faster with TSAb than TSH. With optimized conditions (cells depleted of TSH > 7–10 days; 3-isobytyl-1-methyl xanthine, 0.005 mM; TSAb addition for the entire 48-hour assay period; and a 30-minute pulse of 10 µM 125I-sodium iodide at 37 C), TSAb stimulation is concentration-dependent with a half-maximal activity at ∼ 10-fold lower concentrations than in the cAMP stimulation assay. In a series of 24 patients with Graves’ disease, IgGs with positive values in the cAMP assay were positive in the iodide uptake assay. Activity coefficients expressed as percent of basal in both assays correlate extremely well (r = 0.85). Using these two assays and an FRTL-5 assay measuring growth antibodies (by their ability to increase thymidine uptake), TSAbs were found in 100% of patients with active Graves’ disease and could broadly be grouped into three categories: IgGs with high stimulatory cAMP/iodide uptake activity but low to moderate effects on thymidine uptake; IgGs with moderate effects in all assays; and IgGs with low or undetectable cAMP/iodide stimulatory activity, but high thymidine uptake activity. The value of the FRTL-5 rat thyroid cells assays system for measuring Graves’ autoantibodies appears, thus, to be superior to all current systems, including human cells and slices.

Ganglioside dependent return of TSH receptor function in a rat thyroid tumor with a TSH receptor defect

The 1-8 rat thyroid tumor line with a thyrotropin and cholera toxin receptor defect and a deficiency in higher order membrane gangliosides is shown to regain both receptor functions with the in vivo resynthesis or the in vitro reconstitution of higher order gangliosides. Reconstitution was achieved by exposing primary cell cultures of the tumor to preparations of gangliosides from thyroid cells with functional thyrotropin receptor activity.

Structural changes caused by thyrotropin in thyroid cells and in liposomes containing reconstituted thyrotropin receptor.

Thyrotropin causes a rapid and significant increase in the fluorescence polarization of DPH when this hydrophobic probe is incorporated into a strain of functioning rat thyroid cells (FRTL5). This increase is ligand-specific and is not related to cAMP production. The phenomenon seems to reflect the interaction of thyrotropin with the glycoprotein component of its membrane receptor, as suggested by experiments in which thyrotropin causes increases in DPH fluorescence polarization in liposomes embedded with this receptor component but not with gangliosides. A strain of nonfunctioning rat thyroid cells (FRT), exhibiting no reactivity with monoclonal antibodies to the glycoprotein component of the thyrotropin receptor, requires two orders of magnitude higher concentrations of thyrotropin to exhibit a comparable phenomenon.