RESUMO
Thyrotropin (TSH) is a pituitary glycoprotein hormone heterodimer that binds to its G-protein coupled receptor (TSH-R) at the thyroid to promote the synthesis and secretion of thyroid hormone. Very little is known about TSH-TSH-R interactions in teleost fish. Mammalian gonadotropins have been reported to have an intrinsic ability to activate teleost fish TSH-Rs, suggesting the TSH-R in teleost fish is more promiscuous than in other vertebrates. In this study we utilized the goldfish T(4)-release response and recombinant human TSH analogs as in vivo tools to evaluate the structural constraints on hormone-receptor interactions. We found that four positively charged lysines substituted for neutral or negatively charged amino acids within positions 11-20 of the glycoprotein hormone subunit α (GSUα) significantly increased biological activity of hTSH in fish, as it does in mammals. We further found that bovine follicle stimulating hormone but not luteinizing hormone, whose GSUα subunits also contain four lysine or arginine amino acid residues in the N-terminal portion of GSUα, was thyrotropic in goldfish, suggesting gonadotropin ß subunit contributes to the heterothyrotropic activity. Though recombinant human FSH did not produce a dose-dependent increase in T(4), thyrotropic activity could be acquired with the addition of positively charged amino acids at the N-terminal portion of its GSUα, confirming the importance of the charge on those amino acids for activation of the goldfish TSH-R. These studies demonstrate that mammalian glycoprotein hormone analogs can be utilized to evaluate the conservation of receptor binding and activation mechanisms between fish and mammals.
Assuntos
Carpa Dourada/metabolismo , Receptores da Tireotropina/metabolismo , Animais , Evolução Molecular , Carpa Dourada/sangue , Gonadotropinas/sangue , Gonadotropinas/metabolismo , Humanos , Imunoensaio , Tireotropina/farmacologia , Tiroxina/sangueRESUMO
By combining evolutionary considerations, sequence comparisons and homology modeling we have designed recombinant human thyroid-stimulating hormone (hTSH) analogs with increased receptor binding and activity. The introduction of seven basic residues into the peripheral loops of hTSH resulted in up to a 50,000-fold increase in receptor binding affinity and 1300-fold increase in intrinsic activity. Such analogs are not only of potential clinical interest but can be tools to explore molecular aspects of conventional as well as nonclassical actions of glycoprotein hormones. These design strategies should be applicable to the development of novel analogs of other related hormones and growth factors with a variety of therapeutic and basic science applications, particularly for proteins that have undergone evolutionary decrease in bioactivity.
Assuntos
Desenho de Fármacos , Antagonistas de Hormônios/síntese química , Tireotropina/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Fatores de Crescimento Endotelial/metabolismo , Antagonistas de Hormônios/metabolismo , Humanos , Linfocinas/metabolismo , Conformação Proteica , Receptores da Tireotropina/metabolismo , Homologia de Sequência de Aminoácidos , Tireotropina/química , Tireotropina/metabolismo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
We report the generation of superactive analogues of human glycoprotein hormones, with potential applications in thyroid and reproductive disorders. Current biological and structural data were used to rationalize mutagenesis. The 11-20 region in the alpha-subunit with a cluster of lysine residues forms a previously unrecognized domain critical for receptor binding and signal transduction, as well as an important motif in the evolution of glycoprotein hormone activities. The gradual elimination of basic residues in the alpha-subunit coincided with the evolutionary divergence of the hominids from the Old World monkeys. By selective reconstitution of certain critical residues present in homologous nonhuman hormones we have developed human thyroid stimulating hormone and chorionic gonadotropin analogues with substantial increases in receptor binding affinity and bioactivity, thus providing a paradigm for the design of novel therapeutic protein analogues.
Assuntos
Glicoproteínas/química , Glicoproteínas/genética , Hormônios/química , Hormônios/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Biotecnologia , Linhagem Celular , Gonadotropina Coriônica/química , Gonadotropina Coriônica/genética , Primers do DNA/genética , Desenho de Fármacos , Evolução Molecular , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Engenharia de Proteínas , Tireotropina/análogos & derivados , Tireotropina/química , Tireotropina/genéticaRESUMO
Recent progress in structure-function studies of glycoprotein hormones has provided new insights into the molecular mechanisms of action of these hormones and has further supported the concept that physiological modulation of assembly, bioactivity, and clearance of these hormones is dependent on specific structural components. This review emphasizes current advances in the structure-function relationships of human TSH, which have contributed to further elucidation of common and hormone specific features within the glycoprotein hormones family. Novel strategies are now being applied to investigate the role of individual structural elements. The principks discovered in such studies are essential to understand the physiological regulation of hormone bioactivity and allow for the rational design of novel analogs with potential therapeutic applications.
RESUMO
The glycoprotein hormones TSH, CG, LH, and FSH are heterodimers consisting of a hormone-specific beta-subunit and a common alpha-subunit. The aim of the present study was to investigate the role of the carboxy terminus of the common alpha-subunit (amino acids Tyr89-His90-Lys91-Ser92), which has been shown to be important for human (h) CG and hFSH, for the activity of hTSH. Successive truncations of the alpha-carboxy terminus by site-directed mutagenesis revealed a stepwise reduction of bioactivity occurring at residues alpha Ser92 and alpha His90 to 64% and 13%, respectively. This contrasts with previous findings for hCG and hFSH, where loss of bioactivity occurred in a single step with the deletion of alpha Lys91 but alpha Ser92 was not important. The decreased bioactivities of the hTSH alpha-truncation mutants were reflected by concomitant reductions of cAMP production, thyroid hormone synthesis and cell growth and were accompanied by a loss of receptor binding. Substitution of residues alpha Lys91 or alpha His90 with either a hydrophobic or a bulkier residues resulted in a reduction of receptor binding and signal transduction, indicating that the alpha-carboxy terminus of hTSH may interact with the TSH receptor in a tight contact area. Conversely, substitution of alpha His90 with smaller residues enhanced bioactivity. In addition, the integrity of the alpha-carboxy terminus was essential for hTSH expression. Thus, we showed common and different roles of the alpha-carboxy-terminal residues for the glycoprotein hormones. The unique role of alpha Ser92 in hTSH activity explains the evolutionary constraint to preserve the alpha-carboxy-terminal Ser92 in all glycoprotein hormones.
Assuntos
Tireotropina/química , Sequência de Aminoácidos , Animais , Divisão Celular , Células Cultivadas , AMP Cíclico/biossíntese , Humanos , Dados de Sequência Molecular , Ratos , Receptores da Tireotropina/fisiologia , Proteínas Recombinantes , Transdução de Sinais , Relação Estrutura-Atividade , Tri-Iodotironina/metabolismoRESUMO
Amino acid residues 33-44 of the common alpha-subunit of the glycoprotein hormones have been implicated in heterodimerization as well as high affinity receptor binding of human (h) CG. In the present study, we compared the role of specific amino acids within this region for glycoprotein hormone heterodimer formation, using a transient transfection system to coexpress different mutant alpha-subunit constructs with the beta-subunit of either hTSH, hCG, or hFSH. Our results identified a crucial role for alpha Pro38 in the heterodimer expression of hTSH as well as hFSH, similiar to what had been described for hCG. In contrast, alpha Ala38, which had been critical for hCG, was not essential for hTSH heterodimer expression and less important for hFSH, whereas alpha Phe33 and alpha Arg35 appeared uniquely important for hFSH. Furthermore, we assessed the role of these residues for bioactivity and receptor binding of hTSH. Mutation of the surface-exposed residues alpha Arg42-Ser43-Lys44, which form part of a unique alpha-helical structure, to Ala42-Ala43-Ala44, decreased TSH receptor binding using porcine thyroid membranes as well as rat FRTL-5 cells. Residues alpha Phe33 and alpha Arg35, in contrast, were not important for high affinity binding of hTSH. In the signal transduction of hTSH, alpha Ala36 was necessary for efficient growth induction in FRTL-5 cells but not for cAMP production in either FRTL-5 cells or Chinese hamster ovary cells expressing the human TSH receptor (JP09). Similarly, residues alpha Arg42-Ser43-Lys44 were more important for hTSH-mediated induction of cell growth than cAMP production. Mutating alpha Arg35 to Ala reduced cAMP induction but not receptor binding of hTSH. In summary, using site-directed mutagenesis, we identified a domain, residues 33-44 of the common alpha-subunit, important in heterodimer expression, receptor binding, and activation of hTSH. The comparison of the relative roles of specific amino acids within this region in hTSH with hCG and hFSH highlights previously unrecognized differences in the structural requirements for heterodimer expression among the members of the glycoprotein hormone family. Moreover, our findings revealed a novel role for residues alpha 33-44 in triggering different postreceptor events, suggesting that cAMP production and growth promotion may, at least in part, be dissociable functions of hTSH.
Assuntos
Mutagênese Sítio-Dirigida , Tireotropina/química , Tireotropina/genética , Sequência de Aminoácidos , Animais , Células CHO/metabolismo , Divisão Celular/genética , Células Cultivadas , Gonadotropina Coriônica/química , Gonadotropina Coriônica/metabolismo , Cricetinae , AMP Cíclico/biossíntese , Humanos , Modelos Moleculares , Conformação Proteica , Ratos , Receptores da Tireotropina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Glândula Tireoide/citologia , Glândula Tireoide/metabolismo , Tireotropina/metabolismoRESUMO
TSH, a dimeric glycoprotein hormone, has two N-linked complex-type oligosaccharide chains on the alpha-subunit and one on the beta-subunit. The oligosaccharide chains of TSH are important for the expression of hormonal activity, but the contribution of those on each of the subunits to the activity is not clear. In the current study we have determined the relative importance of the oligosaccharide chains of TSH subunits using a recently reported method of enzymatic deglycosylation. The alpha- and beta-subunits of bovine TSH were deglycosylated with endoglycosidase-F and recombined to obtain differentially deglycosylated TSH. The derivatives showed no differences in their receptor-binding activities. The in vitro bioactivity of these derivatives was assessed by measuring adenylyl cyclase activity in bovine thyroid membranes and stimulation of cAMP production and growth in FRTL-5 cells. In the adenylyl cyclase assay, deglycosylation of the alpha-subunit alone had a more profound effect on the activity [maximal stimulatory activity (Vmax), 13% that of alpha.beta) than when the beta-subunit alone was deglycosylated (Vmax, 50% that of alpha.beta). In the FRTL-5 assays, removal of carbohydrate from TSH alpha, but not the beta-subunit, caused a 2- to 3-fold increase in the concentration required for half-maximal stimulation, with minimal change in the apparent Vmax. The adenylyl cyclase assay in bovine membranes was more sensitive to carbohydrate removal than the assays of rat FRTL-5 cells, in which the derivatives with lower activity were able to stimulate cAMP and growth to near-maximal levels, albeit at 3-fold higher concentrations. These results indicate that the carbohydrate chains in both subunits of TSH, particularly those in the alpha-subunit, are important in hormone action. In contrast to previous reports, our study shows that the beta-subunit plays a role in signal transduction.
Assuntos
Subunidade alfa de Hormônios Glicoproteicos/química , Oligossacarídeos/química , Tireotropina/química , Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Animais , Ligação Competitiva , Bovinos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , AMP Cíclico/biossíntese , Subunidade alfa de Hormônios Glicoproteicos/metabolismo , Subunidade alfa de Hormônios Glicoproteicos/farmacologia , Glicosilação , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/farmacologia , Ratos , Receptores da Tireotropina/metabolismo , Relação Estrutura-Atividade , Glândula Tireoide/citologia , Tireotropina/metabolismo , Tireotropina/farmacologiaRESUMO
The recombinant human TSH (rhTSH) with highly sialylated oligosaccharide chains showed higher in vivo bioactivity and a lower MCR than the predominantly sulfated pituitary human TSH (phTSH). The aim of the present study was to investigate the role of terminal carbohydrate residues in organ distribution and metabolic clearance of TSH using an in vivo rat model. The different 125I-labeled TSH preparations with distinct carbohydrate composition were injected i.v. At various time points (5-180 min) after bolus TSH injection, blood, liver, kidney, spleen, lung, heart, and thyroid samples were collected. TSH uptake was determined by trichloroacetic acid precipitation of [125I]TSH in the organ homogenates. The rhTSH (solely sialylated) was distributed predominantly to the kidneys 5, 15, and 30 min after injection. In contrast, phTSH (sulfated/sialylated) and bovine TSH (bTSH; solely sulfated) were cleared predominantly by the liver (at 5 min), with a later renal phase of clearance (at 30 min). Asialo-rhTSH was cleared by the liver with only minor involvement of other organs. The early liver uptake (at 5 min) was proportionally highest for the asialo-rhTSH and bTSH preparations and lowest for rhTSH, which correlated inversely with the serum levels and the degree of sialylation. Blockade of the N-acetylgalactosamine (GalNAc) sulfate receptors by injection of bovine LH resulted in a significant decrease in liver uptake of phTSH. Similarly, liver uptake of asialo-rhTSH was significantly inhibited by injection of asialo-fetuin. Thus, phTSH and bTSH preparations containing sulfated oligosaccharide chains are cleared at least in part by the GalNAc sulfate-specific receptors in the liver. In contrast, rhTSH with highly sialylated oligosaccharides in both subunits accumulates predominantly in the kidneys, even at the early phase of clearance, indicating that sialylated glycoprotein hormones escape from specific receptor-mediated clearance mechanisms in the liver. These data indicate that terminal sialic acid and GalNAc sulfate residues, each to a different extent, determine glycoprotein hormone distribution and thereby plasma level, which as we have shown previously is a major factor in determining the in vivo potency of TSH.
Assuntos
Asparagina/fisiologia , Oligossacarídeos/metabolismo , Hipófise/metabolismo , Tireotropina/química , Tireotropina/metabolismo , Acetilglucosamina/antagonistas & inibidores , Animais , Receptor de Asialoglicoproteína , Assialoglicoproteínas/metabolismo , Asparagina/química , Metabolismo dos Carboidratos , Humanos , Rim/metabolismo , Fígado/metabolismo , Masculino , Oligossacarídeos/química , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/antagonistas & inibidores , Proteínas Recombinantes , Tireotropina/sangue , Distribuição TecidualRESUMO
Recombinant TSH is now successfully being used in clinical studies of thyroid cancer. Because of its therapeutic potential, we have constructed a longer acting analog of TSH by fusing the carboxy-terminal extension peptide (CTEP) of hCG beta onto TSH beta. When coexpressed either with alpha-subunit complementary DNA or alpha minigene in African green monkey (COS-7) and human embryonic kidney (293) cells, the chimera was fully bioactive in vitro and exhibited enhanced in vivo potency associated with a prolonged plasma half-life. The addition of 25 amino acids with 4 O-linked oligosaccharide chains did not affect the assembly and secretion of chimeric TSH. Wild-type (WT) and chimeric TSH secreted by COS-7 and 293 cells displayed wide differences in their plasma half-lives, presumably due to the presence of terminal sialic acid and SO4 on their oligosaccharide chains, respectively. Chimeric and WT TSH secreted by both cell lines demonstrated similar bioactivity in cAMP production, with some differences in [3H]thymidine incorporation. Chimeric TSH appears to be more effective in COS-7 cells than in 293 cells, as judged by growth assay. COS-7-produced chimeric TSH showed the maximum increase in half-life, indicating the importance of sialic acid in prolonging half-life and in vivo potency. Sulfation of both subunits, predominantly beta and to a lesser extent alpha, appears to be responsible at least in part for the increased metabolic clearance of WT and chimeric TSH secreted by 293 cells. Apart from its therapeutic potential, chimeric TSH produced in various cell lines can be used as a tool to delineate the roles of sulfate and sialic acid in the in vivo clearance and, thereby, the in vivo bioactivity.
Assuntos
Carboidratos/fisiologia , Gonadotropina Coriônica/análise , Fragmentos de Peptídeos/análise , Proteínas Recombinantes de Fusão/análise , Tireotropina/química , Tireotropina/farmacocinética , Animais , Sequência de Bases , Células CHO , Chlorocebus aethiops , Gonadotropina Coriônica/química , Gonadotropina Coriônica Humana Subunidade beta , Cricetinae , AMP Cíclico/metabolismo , Primers do DNA/análise , Primers do DNA/química , Primers do DNA/genética , Meia-Vida , Humanos , Rim/citologia , Rim/embriologia , Rim/metabolismo , Masculino , Metionina/análise , Metionina/metabolismo , Camundongos , Dados de Sequência Molecular , Ácido N-Acetilneuramínico , Fragmentos de Peptídeos/química , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacocinética , Ácidos Siálicos/fisiologia , Sulfatos/análise , Sulfatos/metabolismo , Timidina/análise , Timidina/metabolismo , Tireotropina/genética , TrítioRESUMO
To obtain large amounts of hTSH and to study the role of the N-linked oligosaccharides for its biological activity, hTSH was produced using recombinant baculovirus containing the human alpha-subunit and a hTSH beta-minigene, respectively, both under the control of the polyhedrin promoter. Expression in insect cells was 800-1000 ng/ml, 30-fold higher than in our optimized mammalian transient transfection system using Chinese hamster ovary (CHO) cells (20-50 ng/ml). The in vitro activity of insect-cell expressed hTSH (IC-hTSH) was increased 5-fold compared with CHO-hTSH, judged by the ability to induce cAMP production in CHO cells stably transfected with the hTSH receptor (JP09) and the rat thyroid cell line FRTL-5, as well as growth promotion in FRTL-5 cells. Lectin binding and enzymatic desialylation studies suggested that in contrast to CHO-hTSH, IC-hTSH lacked complex-type oligosaccharides terminating with sialic acid but contained predominantly high mannose-type oligosaccharides. The in vitro activity of CHO-hTSH also increased 5- to 6-fold upon treatment of the hTSH-producing cells with the oligosaccharide processing inhibitors swainsonine and castanospermine, which inhibit formation of complex, terminally sialylated oligosaccharides, and upon enzymatic desialylation. In contrast, insect cell-expression or treatment with processing inhibitors did not affect TSH receptor binding. Despite the higher in vitro activity, IC-hTSH had a much lower in vivo activity than CHO-hTSH, due to rapid clearance from the circulation. In summary, this study shows for the first time that relatively high levels of recombinant hTSH with high in vitro bioactivity can be produced in a baculovirus system. Cell-dependent glycosylation is a major factor that determines the final in vivo biopotency of recombinant glycoproteins, a finding that should be of general relevance for all insect cell-produced glycosylated proteins. Although not suitable for clinical use, highly bioactive recombinant hTSH derived from high expression in insect cells should be useful in defining structure-function relations of hormone analogs.
Assuntos
Proteínas Recombinantes/biossíntese , Tireotropina/biossíntese , Animais , Baculoviridae/genética , Células CHO , Cricetinae , Glicosilação , Humanos , Lectinas/metabolismo , Masculino , Taxa de Depuração Metabólica , Camundongos , Ratos , Receptores da Tireotropina/metabolismo , Spodoptera , Tireotropina/farmacologiaRESUMO
Among glycoprotein hormone receptors the TSH receptor (TSHR) is the most susceptible to constitutive activation by mutations in various regions of the molecule, including mutations in the extracellular domain (ECD) and extracellular loops of the transmembrane domain (TMD). To understand the role of the ECD in TSHR activation we have tested several TSHR constructs with major deletions of the ECD. Previous studies reported very low expression of such truncated glycoprotein hormone receptors, which prevented reliable assessment of their ligand-binding and basal constitutive activities. We have eliminated this problem using TSHR tagged at its N-terminus with a hemagglutinin tag (HA) recognized by the HA-specific monoclonal antibody. Based on such quantitation the TSHR deletion mutant missing 386 N-terminal amino acid residues, constituting 98% of the entire ECD, showed 4-7 fold higher normalized basal activity compared to activity of the corresponding wild-type (WT) TSHR construct. This increase in basal activity was significantly inhibited by linking the common alpha-subunit of glycoprotein hormones at the N-terminus of the truncated TSH receptor. The role of a hypothetical activating fragment (409-418) in TSHR activation was further studied using peptides and mutagenesis of charged residues. This study provides important evidence supporting the "two-state" model of TSHR activation and the potential role of proteolytic cleavage for receptor activation. Accordingly, the mechanism of hormone-induced receptor activation is dependent, at least in part, on the elimination of inhibitory interactions within the receptor. Such intra-molecular inhibition of TSHR may include electrostatic interactions between the ECD and extracellular loops of TMD. Moreover, the truncated, constitutively active receptors described herein provide new insights valuable in the design of TSHR antagonists.
Assuntos
Antitireóideos/farmacologia , Espaço Extracelular/metabolismo , Receptores da Tireotropina/antagonistas & inibidores , Receptores da Tireotropina/metabolismo , Animais , Antitireóideos/química , Células COS , Desenho de Fármacos , Espaço Extracelular/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Deleção de Genes , Humanos , Mutação/genética , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , Receptores da Tireotropina/genéticaRESUMO
The biological significance of glycosylation variants of pituitary glycoprotein hormones remains controversial because of the indirect methods usually employed to determine carbohydrate composition or structure as well as the use of unreliable biological/immunological ratio to determine bioactivity. We have previously characterized recombinant human TSH (rhTSH) secreted by Chinese hamster ovary cells attached to microcarrier beads in a large scale bioreactor after stable transfection of hCG alpha and hTSH beta minigenes. In the present study rhTSH has been used as a model to determine structure-function relationships of different isoforms of glycoprotein hormones. We have now produced greater than 200 mg rhTSH using a hollow fiber bioreactor. The highly purified rhTSH produced in the hollow fiber bioreactor (rhTSH-N) as well as rhTSH commercially produced in a large scale bioreactor (rhTSH-G) were quantitated by immunoassays, receptor binding assay, and amino acid analysis and further characterized by a variety of physico-biochemical methods, including chromatofocusing and carbohydrate analysis. rhTSH-G, rhTSH-N, as well as pituitary human TSH (phTSH) have been separated by chromatofocusing on a Mono P column into several isoforms with different pI values. Compositional analysis of the fractions showed higher sialic acid content in the more acidic rhTSH-G fractions. phTSH acidic isoforms showed higher total sulfate and sialic acid contents than the more basic fractions. The bioactivities of various TSH isoforms based on rigorous quantitation of mass by amino acid analysis determined in three different FRTL-5 cell bioassays showed that the more basic and less sialylated fractions of rhTSH-G were more active than the more acidic fractions. In contrast to the in vitro data, highly sialylated and acidic rhTSH-G isoforms showed longer plasma half-lives and higher in vivo bioactivity than the basic forms. These results indicate that secreted rhTSH, similar to intrapituitary phTSH, exists as a mixture of charge isoforms that are related at least in part to the degree of sialylation. The degree of sialylation, highly dependent on the bioreactor production conditions, appears to be the major factor affecting the charge heterogeneity, MCR, and bioactivity of rhTSH.
Assuntos
Ácidos Siálicos/metabolismo , Sulfatos/metabolismo , Tireotropina/química , Tireotropina/isolamento & purificação , Aminoácidos/análise , Animais , Bioensaio , Células CHO , Carboidratos/análise , Linhagem Celular , Cricetinae , AMP Cíclico/biossíntese , Eletroforese em Gel de Poliacrilamida , Isomerismo , Ácido N-Acetilneuramínico , Ensaio Radioligante , Proteínas Recombinantes , Tireotropina/metabolismoRESUMO
We have gained insight into the molecular mechanism of human thyrotropin (hTSH) action through cloning of the human TSHbeta subunit gene, development of recombinant TSH and novel analogues and chimeras produced by site-directed as well as cassette mutagenesis. A variety of loss of function mutations have shown several key domains in both the alpha- and beta-subunits that are important for high-affinity ligand interaction with the receptor. In contrast the specificity of receptor interaction was shown to be determined primarily by areas within the hTSH-beta "seat-belt" region. We have also designed various gain of function mutants (superagonists) using evolutionary considerations, homology modeling, and sequence comparisons within the cystine knot growth factor superfamily. Such superagonists resulted from increasing the positive charge by introduction of lysine or arginine residues or neutralization of negatively charged residues of the peripheral hairpin loops of each subunit in various combinations. Certain superagonists increased receptor binding, in vitro and in vivo bioactivity 100- to 1000-fold, more than that achieved previously for any other known protein ligand. In vivo metabolic clearance and biologic activity could be separately modulated by alteration of TSH carbohydrate structure including production of chimeras that added sites of O-glycosylation and/or covalently linked the alpha- and beta-subunits. These data suggest that electrostatic interactions resulting from net positive charge in TSH and net negative charge in its receptor play an important role in high-affinity TSH receptor binding and signal transduction. Insights gained from the design of such novel recombinant TSH analogues and chimeras should have many diagnostic and therapeutic applications. These include the design of improved in vitro assays for thyrotropic factors as well as the design of second generation recombinant TSH analogues for the detection and treatment of thyroid cancer.
Assuntos
Tireotropina/genética , Tireotropina/fisiologia , Glicosilação , Humanos , Modelos Moleculares , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Receptores da Tireotropina/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tireotropina/químicaRESUMO
A new and simple in vivo bioassay suitable for routine testing of pituitary and recombinant TSH preparations was developed. Male Albino Swiss CF-1 mice were given T3 in their drinking water to suppress endogenous TSH. T3, 3.0 micrograms/mL, given to mice for a period of 4 days decreased plasma total T4 (TT4) level to less than 10% of the nonsuppressed (control) level. Various doses of exogenous pituitary and recombinant TSH preparations were injected intraperitoneally and blood samples were obtained from the orbital sinus 6 h later. The TT4 level, measured by radioimmunoassay, served as the assay end-point. The assay required injection of approximately 3.0 micrograms of pituitary human TSH (phTSH), 1.0 microgram recombinant human TSH (rhTSH), 0.2 microgram bovine TSH (bTSH), and 0.1 microgram rat TSH (rTSH) to attain half-maximal response. The maximal level of TT4 after TSH stimulation was similar to that observed in normal, nonsuppressed mice. The procedure developed is relatively easy to perform, economical, and, unlike earlier TSH bioassays, does not require the use of radionuclides. This bioassay showed acceptable sensitivity and reliability in structure-function studies of pituitary TSH from different species as well as rhTSH.
Assuntos
Bioensaio/métodos , Tireotropina/sangue , Animais , Bovinos , Masculino , Camundongos , Camundongos Endogâmicos , Hipófise/química , Radioimunoensaio , Ratos , Proteínas Recombinantes , Tiroxina/sangue , Tri-Iodotironina/farmacologiaRESUMO
The exact mechanism of anticancer activity of indomethacin still remains unclear. In view of the gathered observations and ideas, I hypothesize that the effect of indomethacin may be related--at least in certain cases--to its antagonism to gastrin/cholecystokinin family of peptides and/or to its agonistic activity to thromboxane A2 and melatonin. Future experiments in this direction are required.
Assuntos
Antineoplásicos/farmacologia , Indometacina/farmacologia , Animais , Humanos , Modelos Biológicos , Neoplasias/tratamento farmacológico , Prostaglandinas/biossínteseAssuntos
Hormônios Adeno-Hipofisários/fisiologia , Tireotropina/fisiologia , Sequência de Aminoácidos , Animais , Evolução Biológica , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Hormônios Adeno-Hipofisários/química , Hormônios Adeno-Hipofisários/uso terapêutico , Proteínas Recombinantes , Alinhamento de Sequência , Especificidade da Espécie , Relação Estrutura-Atividade , Tireotropina/química , Tireotropina/farmacologia , Tireotropina/uso terapêuticoRESUMO
Recombinant human thyrotrophin (rhTSH) contains oligosaccharides terminating in -galactose-sialic acid, and had lower metabolic clearance and higher in vivo bioactivity compared to pituitary hTSH, which has oligosaccharides terminating predominantly in -N-acetylgalactosamine-sulphate. Previous studies using complete removal of the oligosaccharide chains showed an important role for the carbohydrate in the biological activity of the hormone. In the present study, we have determined the contribution of the individual monosaccharides to hormonal activity by sequential deglycosylation of rhTSH using exoglycosidases. We have also investigated the effect of resialylation of desialylated rhTSH using sialyltransferases. Sequential removal of sialic acid, galactose or N-acetylglucosamine resulted in a > 10-fold increase in the in vitro bioactivity of rhTSH. The metabolic clearance of the derivatives was faster than that of intact hormone, but agalacto-rhTSH was cleared slower than asialo-rhTSH. However, the in vivo bioactivity decreased progressively with each monosaccharide removal. The increased cyclic AMP-stimulating activity, increased metabolic clearance and the decreased in vivo biologic activity were all reversed by resialylation of the terminal galactose residues. These results indicate that the in vitro, as well as the in vivo, bioactivities of rhTSH are modulated by terminal sialylation. The effects of sequential deglycosylation on the in vitro activity of rhTSH are different from those reported earlier for human chorionic gonadotrophin. Thus, modification of the oligosaccharides by glycosidases and glycosyltransferases can be used as a powerful tool to delineate the function of carbohydrate in glycoproteins and to engineer more potent hormone analogues with a longer half-life and/or higher bioactivity.
Assuntos
Oligossacarídeos/química , Tireotropina/química , Animais , Linhagem Celular , AMP Cíclico/biossíntese , Glicosilação , Humanos , Cinética , Estrutura Molecular , Ácido N-Acetilneuramínico , Ratos , Receptores da Tireotropina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Ácidos Siálicos/química , Relação Estrutura-Atividade , Tireotropina/metabolismo , Tireotropina/farmacologiaRESUMO
The recombinant human thyroid stimulating hormone (rhTSH) containing oligosaccharides terminated with NeuAc(alpha 2-3)Gal(beta 1-4)GlcNAc beta 1 showed higher in vivo activity and lower metabolic clearance rate (MCR) than pituitary human TSH (phTSH), which contains oligosaccharides terminating predominantly in SO(4)4GalNAc(beta 1-4)GlcNAc beta 1. To elucidate the relative contribution of the sulfated and sialylated carbohydrate chains of each subunit in the MCR and bioactivity of the hormone, the alpha and beta subunits of phTSH, rhTSH, and enzymatically desialylated rhTSH (asialo-rhTSH; asrhTSH) were isolated, their oligosaccharides were analyzed, and the respective subunits were dimerized in various combinations. The hybrids containing alpha subunit from phTSH or asrhTSH showed higher in vitro activity than those with alpha subunit from rhTSH, indicating that sialylation of alpha but not beta subunit attenuates the intrinsic activity of TSH. In contrast, hybrids with beta subunit from rhTSH displayed lower MCR compared to those with beta subunit from phTSH. The phTSH alpha-rhTSH beta hybrid had the highest in vivo bioactivity followed by rhTSH alpha-rhTSH beta, rhTSH alpha-phTSH beta, phTSH alpha-phTSH beta, and asrhTSH dimers. These differences indicated that hybrids with beta subunit from rhTSH displayed the highest in vivo activity and relatively low MCR, probably due to higher sialylation, more multiantennary structure, and/or the unique location of the beta-subunit oligosaccharide chain in the molecule. Thus, the N-linked oligosaccharides of the beta subunit of glycoprotein hormones have a more pronounced role than those from the alpha subunit in the metabolic clearance and thereby in the in vivo bioactivity. In contrast, the terminal residues of alpha-subunit oligosaccharides have a major impact on TSH intrinsic potency.
Assuntos
AMP Cíclico/metabolismo , Subunidade alfa de Hormônios Glicoproteicos/metabolismo , Subunidade alfa de Hormônios Glicoproteicos/farmacologia , Oligossacarídeos/química , Tireotropina/metabolismo , Tireotropina/farmacologia , Animais , Células CHO , Sequência de Carboidratos , Carboidratos/análise , Cricetinae , Subunidade alfa de Hormônios Glicoproteicos/química , Humanos , Substâncias Macromoleculares , Masculino , Taxa de Depuração Metabólica , Dados de Sequência Molecular , Oligossacarídeos/metabolismo , Oligossacarídeos/farmacologia , Multimerização Proteica , Ratos , Ratos Sprague-Dawley , Receptores da Tireotropina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Relação Estrutura-Atividade , Tireotropina/química , Transfecção , Tri-Iodotironina/sangue , Tri-Iodotironina/farmacocinéticaRESUMO
The human thyroid-stimulating hormone (hTSH) subunits alpha and beta are transcribed from different genes and associate noncovalently to form the bioactive hTSH heterodimer. Dimerization is rate-limiting for hTSH secretion, and dissociation leads to hormone inactivation. Previous studies on human chorionic gonadotropin (hCG) and human follicle-stimulating hormone had shown that it was possible by subunit gene fusion to produce a bioactive, single chain hormone. However, neither the stability nor the clearance from the circulation of such fused glycoprotein hormones has been studied. We show here that genetic fusion of the hTSH alpha- and beta-subunits using the carboxyl-terminal peptide of the hCG beta-subunit as a linker created unimolecular hTSH whose receptor binding and bioactivity were comparable to native hTSH. Interestingly, the fused hTSH had higher thermostability and a longer plasma half-life than either native or dimeric hTSH containing the hCG beta-subunit-carboxyl-terminal peptide, suggesting that dimer dissociation may contribute to glycoprotein hormone inactivation in vivo. In addition, we show for the first time that synthesis of hTSH as a single polypeptide chain could overcome certain mutagenesis-induced defects in hTSH secretion, therefore enabling functional studies of such mutants. Thus, in addition to prolongation of plasma half-life, genetic fusion of hTSH subunits should be particularly relevant for the engineering of novel analogs where desirable features are offset by decreased dimer formation or stability. Such methods provide a general approach to expand the spectrum of novel recombinant glycoprotein hormones available for in vitro and in vivo study.
Assuntos
Receptores da Tireotropina/metabolismo , Tireotropina/química , Animais , Células CHO , Membrana Celular/metabolismo , Cricetinae , Dimerização , Humanos , Taxa de Depuração Metabólica , Mutagênese Sítio-Dirigida , Ratos , Suínos , Tireotropina/metabolismo , Tireotropina/farmacocinéticaRESUMO
This article provides the reader with an overview of methodological strategies to investigate structure-function relationships of human thyroid-stimulating hormone (hTSH). Various aspects of hTSH production, purification, and characterization described here in more detail are not only relevant to studies on other members of the glycoprotein hormone family, but also applicable to studies of other glycosylated proteins. Knowledge of structure-function relationships of specific hTSH domains is important for a better understanding of the molecular mechanisms of its action. New insights from such studies permit the design of glycoprotein hormone analogs with specific pharmacological properties and potential clinical applications.