Human testis/sperm-specific histone H2B (hTSH2B). Molecular cloning and characterization

Human sperm, unlike the sperm of other mammals, contain replacement histones with unknown biological functions. Here, we report the identification of the novel human gene coding for a testis/sperm-specific histone H2B (hTSH2B). This variant histone is 85% homologous to somatic H2B and has over 93% homology with the testis H2B of rodents. Using genomic PCR, two genetic alleles of hTSH2B were found in the human population. The hTSH2B gene is transcribed exclusively in testis, and the corresponding protein is also present in mature sperm. We expressed recombinant hTSH2B and identified this protein with a particular H2B subtype expressed in vivo. The subnuclear distribution of H2B variants in sperm was determined using biochemical fractionation and immunoblotting. The H2B variant associated with telomere-binding activity () was solubilized by Triton X-100 or micrococcal nuclease extraction, whereas hTSH2B was relatively tightly bound in nuclei. Immunofluorescence showed that hTSH2B was concentrated in spots located at the basal nuclear area of a subpopulation (20% of cells) of mature sperm. This fact may be of particular importance, because the hTSH2B "positive" and "negative" sperm cells may undergo significantly different decondensation processes following fertilization.     

Cloning of the human thyrotropin beta-subunit gene and transient expression of biologically active human thyrotropin after gene transfection

A 17 kilobase pair fragment of DNA containing the human TSH (hTSH) beta-subunit gene was isolated from a human leukocyte genomic library. Using a 621 base pair human CG alpha-subunit cDNA and a 2.0 kilobase pair genomic fragment of hTSH beta containing both coding exons, we constructed hCG alpha and hTSH beta expression vectors containing either the early promoter of simian virus 40 or the promoters of adeno-associated virus. Cotransfection of two adeno-associated virus vectors, each containing one subunit of hTSH, together with a plasmid containing the adenovirus VA RNA genes produced hTSH as well as free human alpha- and TSH beta-subunits in an adenovirus transformed human embryonal kidney cell line (293). The levels of protein expression in this system were 10- to 100-fold greater than that found in a simian virus transformed monkey kidney cell line (COS) using vectors containing the early promoter of simian virus 40. The hTSH synthesized in 293 cells was glycosylated as indicated by complete binding to concanavalin A-Sepharose but was larger in apparent molecular weight than a standard hTSH preparation on gel chromatography suggesting an altered glycosylation pattern. However, it was immunologically and biologically indistinguishable from two pituitary hTSH standards in an immunoradiometric and in vitro iodide trapping assay, respectively.     

Monoclonal antibody approach to the relationship between immunological structure and biological activity of thyrotropin

In order to locate the domains involved in the biological activity of TSH and to get some insight in the relationship between immunological and biological properties of TSH, 24 monoclonal antibodies (mAb) to 11 different antigenic regions of hTSH were tested for both binding to hTSH and inhibition of hTSH stimulation of adenylate cyclase in human thyroid membranes. These mAb were also investigated for binding to bovine TSH (bTSH), and interference with bTSH binding to the receptor and stimulation of adenylate cyclase. Radioiodinated human TSH (hTSH) was incubated with increasing concentrations of mAb. Maximum hTSH binding by the various mAb ranged from 15-75% and was not related to the apparent affinity of the mAb for hTSH. Maximum inhibition by the mAb of hTSH stimulation of adenylate cyclase ranged from 3-92%. As compared to the antigenic map of hTSH, it was observed that mAb reacting with the same antigenic regions might display varying inhibition of hTSH. Nevertheless, it was clearly shown that the most potent inhibitors of hTSH stimulatory activity interacted with epitopes located on the alpha- and beta-subunits or expressed only by holo hTSH. Only 11 of the 24 mAb cross-reacted significantly with bTSH. Seven exhibited the same inhibition of hTSH and bTSH stimulatory activity; the four remaining mAb rather than to inhibit adenylate cyclase stimulation as observed with hTSH, did not interfere or even increased adenylate cyclase stimulation by bTSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

The production of active human thyroid-stimulating hormone from alpha and beta mRNAs in Xenopus laevis oocytes

Active human thyroid-stimulating hormone (hTSH) was produced by Xenopus laevis oocytes following injection of an mRNA mixture of hTSH beta and alpha subunits synthesized by T3 RNA polymerase. Some of the hTSH molecules were secreted into the medium, while others remained in the cells. The active molecules consisted of alpha and beta subunits and were in highly glycosylated form. The Xenopus laevis oocyte-produced hTSH stimulated the rat thyroid cell line FRTL-5 to produce and secrete the cyclic AMP as does authentic hTSH.     

Immunochemical mapping of antigenic regions on the human thyrotropin beta-subunit by antipeptide antibodies

To study antigenic sites present in the beta-subunit of human thyrotropin (hTSH), we produced site-specific antibodies directed against synthetic peptides analogous to the 1-18, 44-59, and 85-112 regions of the thyrotropin beta-subunit. The hTSH beta(1-18) peptide-carrier conjugate elicited antisera capable of binding to both radiolabeled hTSH and its beta-subunit whereas antibodies elicited against the hTSH beta(44-59) peptide-carrier conjugate bound only to the peptide. Thus, the NH2-terminal region of hTSH beta appears to be accessible at the surface of the hormone whereas the hTSH beta(44-59) region may be poorly accessible. Two monoclonal antipeptide antibodies that bound to 125I-hTSH beta, designated as TS01 and TS02, were selected after immunization with the hTSH beta(85-112) peptide-carrier conjugate. The antigenic site recognized by TS01 was located on the eight COOH-terminal(105-112) amino acid residues. TS02 antibody bound to an antigenic region included within Cys95 and Cys105. Both antigenic sites appeared to be more accessible on the free hTSH beta than on the hormone. Immunoblots performed on various preparations containing TSH revealed that TS02 antibody detected the beta-subunit from both the human and bovine species but not the rat TSH beta. Under reducing conditions, a low molecular weight material was identified in hTSH beta, likely caused by intrachain nicking.     

High-affinity and specific recognition of human thyroid stimulating hormone (hTSH) by in vitro-selected 2'-amino-modified RNA.

RNA sequences containing 2'-amino pyrimidines that bind with high-affinity to human thyroid stimulating hormone (hTSH) were isolated from a random sequence library by an in vitro selection-amplification procedure. A representative RNA ligand (T-15) has an equilibrium dissociation constant (Kd) of 2.5 nM for its interaction with hTSH and can discriminate between other members of the glycohormone family; no detectable binding was observed at low micromolar concentrations of hCG (human chorionic gonadotropin), while measured Kd values for the interactions with hLH (human leutinizing hormone) and hFSH (human follicle stimulating hormone) were > 1 microM and approximately 0.2 microM, respectively. The detection of hTSH in a dot blot assay with radiolabeled T-15 RNA was demonstrated.     

The asparagine-linked oligosaccharides at individual glycosylation sites in human thyrotrophin.

The asparagine-linked carbohydrate structures at each of the three glycosylation sites of human thyrotrophin were investigated by 400 MHz 1H-NMR spectroscopy. Highly purified, biologically active human thyrotrophin (hTSH) was dissociated into its subunits hTSH alpha (glycosylated at Asn 52 and Asn 78) and hTSH beta (glycosylated at Asn 23). The alpha-subunit was further treated with trypsin which gave two glycopeptides that were subsequently separated by reverse-phase HPLC and identified by amino acid sequence analysis. The oligosaccharides were liberated from hTSH alpha glycopeptides and from intact hTSH beta by hydrazinolysis, and were fractionated as alditols by anion-exchange and ion-suppression amine-adsorption HPLC preparatory to structural analysis. The N-glycans present on hTSH were mainly diantennary complex-type structures with a common Man alpha 1-3 branch that terminated with 4-O-sulphated GalNAc. The Man alpha 1-6 branch displayed structural heterogeneity in the terminal sequence, with chiefly alpha 2-3-sialylated Gal and/or 4-O-sulphated GalNAc. The relative amounts of the two major complete diantennary oligosaccharides and their core fucosylation differed according to glycosylation site; the sulphated/sialylated diantennary oligosaccharide was most abundant at the two sites on the alpha-subunit, whereas the disulphated, core-fucosylated oligosaccharide was more plentiful on the beta-subunit. Some interesting structural features, not previously reported for the N-glycans of hTSH, included 3-O-sulphated galactose (SO4-3Gal) and peripheral fucose (Fuc alpha 1-3GlcNAc) in the Man alpha 1-6 branch of some diantennary structures; the former suggests the presence of a hitherto uncharacterized galactose-3-O-sulphotransferase in thyrotroph cells of the human anterior pituitary gland.     

An attempt to analyze various thyroid stimulators by the receptor assay using hTSH radioimmunoassay.

In an attempt to analyze thyroid stimulators in serum we developed an assay procedure using hTSH radioimmunoassay (RIA) in combination with receptor competition. The principle of this method is the determination by RIA of hTSH displaced by other thyroid stimulators from a thyroidal receptor preparation which previously bound unlabelled hTSH. Practically 4 microunits of hTSH were bound with human or bovine receptor, and then hTSH displaced by addition of test serum (0.1 ml) or samples dissolved in serum (0.1 ml) was measured by RIA. This assay can determine the thyroid stimulators other than hTSH in serum that has the displacement activity of 0.5-4.0 microunits of hTSH in the useful range, such as mU/ml level of bovine TSH or rat TSH. Cholera toxin that has the thyroid stimulating activity like TSH also showed the displacement of the bound hTSH. This assay is not applicable for the human serum with more than 5 microunits/ml of TSH, because the assay value is over estimated by the free hTSH derived from the test serum. On the other hand, eighteen sera with high LATS activity and 42 sera with negative LATS activity from patients with untreated hyperthyroidism did not show any displacement. This might be due to the lower binding activity of LATS with hTSH receptor or the lower sensitivity of this assay method. Although it is difficult to use this assay clinically because of its low sensitivity, increased TSH in animal serum can be determined by this assay. The principle of this method may be also useful for examining the receptor binding of other peptide hormone that can be determined by an RIA method.     

Production of human thyroid-stimulating hormone in Chinese hamster ovary cells

Human thyroid-stimulating hormone (hTSH) has been produced in Chinese hamster ovary (CHO) cells co-transformed with two plasmids: one carrying the alpha subunit cDNA with mouse dihydrofolate reductase gene and the other carrying hTSH beta subunit cDNA. Each cDNA was driven to expression under the control of SV40 early promoter. hTSH and its alpha subunit were secreted into culture media, and their secretion increased with exposure of the cells to increasing concentrations of methotrexate. Gel filtration analysis revealed that the molecular size of the hTSH was the same as that of natural hTSH. Furthermore, the CHO cell-produced hTSH elevated the cyclic AMP level in the rat thyroid cell line FRTL-5 in the same manner as natural hTSH does.     

Bioengineering of human thyrotropin superactive analogs by site-directed "lysine-scanning" mutagenesis. Cooperative effects between peripheral loops

We have previously engineered the first superactive analogs of human thyrotropin (hTSH) by using a novel design strategy. In this study, we have applied homology comparisons focusing on the alphaL3 loop of the common alpha-subunit of human glycoprotein hormones. Seven highly variable amino acid residues were identified, and charge-scanning mutagenesis revealed three previously unrecognized modification permissive domains and four gain-of-function lysine substitutions. Such gain-of-function mutations were hormone- and receptor-specific and dependent on location and basic charge. Cooperativity of individual substitutions was established in double and triple lysine mutants. In combinations of the most potent alphaL3 loop analog with two previously characterized loop analogs, a higher degree of cooperativity for the alphaL3 loop analog compared with both the alphaL1 loop analog and the hTSH-betaL3 loop analog was observed. We demonstrated that spatially distinct regions of the common alpha-subunit contribute differentially to the interaction of hTSH with its receptor and that combinations of two modified loops on the same and on opposite sides of the hTSH molecule display similar increases in in vitro biopotency. In addition, combination of all three superactive loops showed cooperativity in receptor binding and activation resulting in the most potent hTSH superactive analog described to date.     

Engineering a potential antagonist of human thyrotropin and thyroid-stimulating antibody

Thyrotropin (TSH) and the gonadotropins (FSH, LH, hCG) are a family of heterodimeric glycoprotein hormones composed of two noncovalently linked subunits, alpha and beta. We have recently converted the hTSH heterodimer to a biologically active single chain (hTSHbeta.CTPalpha) by fusing the common alpha-subunit to the C-terminal end of the hTSH beta-subunit in the presence of a approximately 30-amino acid peptide from hCGbeta (CTP) as a linker. The hTSHbeta.CTPalpha single chain was used to investigate the role of the N-linked oligosaccharides of alpha- and beta-subunits in the secretion and function of hTSH. Using overlapping PCR mutagenesis, two deglycosylated variants were prepared: one lacking both oligosaccharide chains on the alpha-subunit (hTSHbeta.CTPalpha(1+2)) and the other lacking the oligosaccharide chain on the beta-subunit (hTSHbeta.CTPalpha(deg)). The single chain variants were expressed in CHO cells and were secreted into the medium. hTSH variants lacking the oligosaccharide chains were less potent than hTSHbeta.CTPalpha wild-type with respect to cAMP formation and thyroid hormone secretion in cultured human thyroid follicles. Both deglycosylated variants competed with hTSH in a dose-dependent manner. The hTSHbeta.CTPalpha(1+2) variant blocked cAMP formation and thyroid hormone secretion stimulated by hTSH as well as by the antibody, thyroid-stimulating immunoglobulins, responsible for the most common cause of hyperthyroidism, Graves disease. Thus, this variant behaves as a potential antagonist, offering a novel therapeutic strategy in the treatment of thyrotoxicosis caused by Graves' disease and TSH-secreting pituitary adenoma.     

Stokes radius determination of radioiodinated polypeptide hormones by gel filtration

A simple technique for determination of the molecular (Stokes) radius of radioiodinated proteins was developed using the same column and chromatographic conditions employed in routine radioimmunoassay tracer purification. The calibration curve for five radioiodinated standard proteins presented a highly significant correlation (r = -0.996; P less than 0.001) and allowed precise molecular radius determination for labeled human growth hormone (hGH), luteotropin (hLH), follicle-stimulating hormone (hFSH), thyrotropin (hTSH), prolactin (hPRL), and corticotropin (hACTH), enabling detection of differences of the order of +/- 3%. The validity of the method was verified by determining the molecular radius of hGH in both "cold" (unlabeled standards and unknowns) and "hot" (radioiodinated standards and unknowns) systems. The technique can be applied in a very simple manner, requiring just one simple additional calibration run before Sephadex G-100 tracer purification. Furthermore, it can be applied to any protein, even when only extremely limited amounts are available. Since the standards and unknowns are labeled and chromatographed under identical conditions, potential common alterations of the molecule due to oxidation, iodine incorporation, tracer-carrier interactions, etc., are automatically corrected for.     

The role of the carboxyl-terminal 6 amino acid extension of human TSH beta subunit

The nucleotide sequence of human thyroid stimulating hormone (hTSH) gene can encode a protein of 138 amino acids. However, the mature polypeptide is lacking 6 amino acids of the carboxyl-terminus (C-terminus), suggesting posttranslational cleavage of these residues. To analyze a possible function of these 6 amino acids, we expressed two hTSH beta cDNAs with or without the 6 codons for C-terminal extension, together with alpha subunit cDNA in CHO cells, and determined the amino acid sequence of C-terminus of hTSH beta. hTSH beta propeptides without C-terminal extension were glycosylated, associated with alpha subunit and secreted, as normal propeptides were, and its heterodimer with alpha subunit showed normal TSH bioactivity in FRTL-5 bioassay. These data indicate that the 6 amino acid C-terminal extension is not necessary for the hTSH maturation in the process of the biosynthesis and for its bioactivity.     

A detailed functional and structural analysis of a major thyroid hormone inhibitory element in the human thyrotropin beta-subunit gene.

The first exon of the human thyrotropin-beta (hTSH beta) gene has been demonstrated in our laboratory to contain a major thyroid hormone inhibitory element. In order to characterize fully this element, we have performed a detailed functional and structural scanning mutational analysis of this element. Various -1192 to +37 (base pairs) bp fragments of the hTSH beta gene containing consecutive five deoxythymidine substitution mutations of the first exon were inserted into a luciferase reporter plasmid and transiently transfected into human embryonal cells (293) and stably transfected into rat pituitary cells (GH3). Two domains (domain 1 and 2) were identified by scanning mutations that were essential for function of the thyroid hormone inhibitory element: +3 to +13 bp and +28 to +37 bp. Biotinylated DNA fragments containing -12 to +43 bp of the hTSH beta gene and the identical scanning mutations demonstrate that in vitro synthesized c-erbA-beta binding is disrupted as much as 95% by mutations from -3 to +17 bp and to a lesser extent (20-30%) by mutations from +23 to +27 bp and from +33 to +43 bp. Domain 1 displayed a higher affinity for c-erbA-beta than domain 2 in avidin-biotin complex DNA-binding and gel-mobility assays. Using increasing amounts of in vitro synthesized c-erbA-beta, we were unable to demonstrate more than one protein-DNA complex in gel-mobility assays. However, using the avidin-biotin complex DNA-binding assay and the cross-linking reagent, 1,6-bismaleimidohexane, we were able to demonstrate thyroid hormone receptor dimer formation on domain 1 but not to any significant extent on domain 2. In conclusion, functional and DNA-binding studies suggest that the thyroid hormone receptor binds to two distinct regions in the first exon of the hTSH beta gene. The upstream site (domain 1) binds c-erbA-beta with higher affinity and is capable of binding c-erbA-beta as a dimer under some conditions, while the downstream site (domain 2) appears to bind a single molecule of c-erbA-beta with lower affinity. These results suggest that thyroid hormone receptor, binding to at least two sites in the first exon, act in conjunction to mediate T3 inhibition of hTSH beta expression.     

Molecular cloning of the human thyrotropin-beta subunit gene

Genomic DNA fragments that carried a gene for human thyrotropin-beta (hTSH beta) subunit were isolated. Nucleotide sequence analysis of the gene showed that the hTSH beta subunit precursor consists of 138 amino acid residues. There is an N-terminal sequence of 20 amino acids as a signal peptide, followed by 112 amino acids, whose sequence is in agreement with that known for the secretory form of hTSH beta subunit. This is followed by an additional stretch of 6 hydrophobic amino acids, which may be eliminated post-translationally. The coding region is separated by an intron of about 460 bp. Genomic Southern blot hybridization analysis suggested that the hTSH beta gene is a unique single copy gene.     

In vitro responses of luteinizing rat granulosa cells to human thyroid-stimulating hormone

The effect of human thyroid-stimulating hormone (hTSH) on progesterone (P4) secretion during initial luteinization and subsequent prolactin (Prl)-mediated steroidogenesis by cultured rat granulosa cells was studied. Granulosa cells, obtained from pregnant mare's serum gonadotropin (PMSG)-treated immature female rats, were preincubated for 1, 3, 6, 12, or 24 h in control medium lacking added hormones or in medium containing 1.0 microgram/ml human chorionic gonadotropin (hCG) or hTSH, and maintained subsequently for 6 days in medium containing 1.0 microgram/ml bovine (bPrl). Indices of luteotropic stimulation were provided by: 1) elevated P4 concentrations determined by radioimmunoassay of spent media samples; and 2) cytoplasmic lipid accumulation assessed by osmium tetroxide staining following fixation after 7 days of culture. Progesterone levels in media from cultures exposed to hCG for 24 h were twofold higher than control cultures, whereas those in media from cultures preincubated in hTSH for 24 h were fourfold higher than control levels. Cultures preincubated in 1.0 microgram/ml hCG for as little as 1 h and then maintained for 6 days in Prl secreted significantly more P4 than did control cultures also maintained with Prl for 6 days. Cultures preincubated in hTSH required a 24-h exposure before a significant increase in Prl-mediated P4 secretion was observed. Intensity of cytoplasmic osmiophilia correlated directly with P4 concentration. These results suggest that: 1) hTSH has the ability to promote P4 secretion during initial luteinization and to regulate subsequent Prl-mediated steroidogenesis by cultured rat granulosa cells; and 2) the mechanism by which hTSH stimulates Prl-mediated P4 secretion in this model system may differ from that of hCG.     

The Superagonistic Activity of Bovine Thyroid-stimulating Hormone (TSH) and the Human TR1401 TSH Analog Is Determined by Specific Amino Acids in the Hinge Region of the Human TSH Receptor

Bovine TSH (bTSH) has a higher affinity to the human TSHR (hTSHR) and a higher signaling activity than human TSH (hTSH). The molecular reasons for these phenomena are unknown. Distinct negatively charged residues (Glu²⁹⁷, Glu³⁰³, and Asp³⁸²) in the hinge region of the hTSHR are known to be important for bTSH binding and signaling. To investigate the potential relevance of these positions for differences between bTSH and hTSH in the interaction to the hTSHR, we determined bTSH- and hTSH-mediated cAMP production of several substitutions at these three hinge residues. To examine specific variations of hTSH, we also investigated the superagonistic hTSH analog TR1401 (TR1401), whose sequence differs from hTSH by four additional positively charged amino acids that are also present in bTSH. To characterize possible interactions between the acidic hTSHR positions Glu²⁹⁷, Glu³⁰³, or Asp³⁸² and the additional basic residues of TR1401, we investigated TR1401 binding and signaling properties. Our data reveal increased cAMP signaling of the hTSHR using TR1401 and bTSH compared with hTSH. Whereas Asp³⁸² seems to be important for bTSH- and TR1401-mediated but not for hTSH-mediated signaling, the substitution E297K exhibits a decreased signaling for all three TSH variants. Interestingly, bTSH and TR1401 showed only a slightly different binding pattern. These observations imply that specific residues of the hinge region are mediators of the superagonistic activity of bTSH and TR1401 in contrast to hTSH. Moreover, the simultaneous localization of binding components in the glycoprotein hormone molecule and the receptor hinge region permits important reevaluation of interacting hormone receptor domains.It is well known that bovine TSH (bTSH)² has a higher affinity to the human TSHR (hTSHR) and a 6–10-fold higher intrinsic signaling activity than human TSH (hTSH) (1–5). Human TSH and bTSH share high amino acid sequence identity in the α-subunit (74.1%) and β-subunit (88.4%) (6). Studies involving fusion of hTSH and bTSH α- and β-subunits indicate that the higher affinity and the superagonistic cAMP activity of bTSH at the hTSHR depend primarily on amino acid sequences of the bTSH α-subunit (6). The most noticeable sequence differences between bovine and human TSH consist of four positively charged residues located in the surface-exposed loops of the α-subunit and one positively charged residue in the β-subunit of bTSH (Fig. 1). Moreover, it has previously been shown that positively charged α loop 1 (α-L1) residues are important for the high bioactivity of bTSH, and they have been implicated in receptor binding. These specific characteristics led to the generation of superagonistic hTSH analogs (6). The human TSH analog TR1401 and bTSH differ from hTSH most importantly by four additional positively charged amino acids located in close spatial proximity at the α-L1, of which three are located at identical positions in bTSH and TR1401 (Fig. 1).Open in a separate windowFIGURE 1.Sequence differences between TSH variants used in the present study. A, alignment of the α- and β-subunit of the hTSH (SwissProt: GLHA_HUMAN {"type":"entrez-protein","attrs":{"text":"P01215","term_id":"121312","term_text":"P01215"}}P01215, TSHB_HUMAN {"type":"entrez-protein","attrs":{"text":"P01222","term_id":"311033515","term_text":"P01222"}}P01222), bTSH (GLHA_BOVIN {"type":"entrez-protein","attrs":{"text":"P01217","term_id":"121310","term_text":"P01217"}}P01217, TSHB_BOVIN {"type":"entrez-protein","attrs":{"text":"P01223","term_id":"136442","term_text":"P01223"}}P01223), and the superagonistic hTSH analog TR1401. The additional positively charged residues at TR1401 and at bTSH compared with wt hTSH are boxed in blue. Sequence numbering for human TSH and human analog TR1401 without signal peptide is shown in blue. B, three-dimensional structural TSH models illustrating the spatial localization of the charge related sequence differences between the TSH variants. The TSH α-subunit is shown in gray, and the β-subunit is in orange. Positively charged residues are highlighted in blue, and the C-α atoms of additional positively charged residues compared with hTSH are highlighted by blue globes. Panel i, bovine TSH, characterized by four additional positively charged residues in the α-L1 (T11K, Q13K, P16K, and Q20K) and one positively charged residue in the β-L3 (L69R); panel ii, human TSH without positively charged residues in the α-L1 and β-L3; and panel iii, the human TSH analog TR1401 is characterized by four additional positively charged residues in the α-L1 (Q13K, E14K, P16K, and Q20K) but shows a lack of the additional positively charged residue in the β-L3.TSH binds to the large extracellular region of its receptor. The extracellular region of the TSHR consists of the leucine-rich repeat domain (LRRD), which is linked with the membrane-spanning serpentine domain by the hinge region. Recently, the binding arrangements between the homologous FSH and a part of the FSH receptor ectodomain including the LRRD (FSH receptor amino acids Cys¹⁸–Ala²⁴⁶) have been identified (7). However, the hinge region is not contained in this x-ray structure (7).In vitro data provide convincing evidence for the functional importance of the hinge region for receptor activation and TSH binding (8–22). Recently, we specified positions Glu²⁹⁷ and Glu³⁰³ in the N-terminal portion and Asp³⁸² in the C-terminal portion of the hTSHR hinge region as important for bTSH binding, suggesting that in the process of bTSH binding an extended hormone-binding site is obviously essential (18). The negative charge of positions Glu²⁹⁷ and Asp³⁸² likely interact with positively charged residues of bTSH by complementary charge-charge interaction (18).To elucidate whether these hinge residues of the hTSHR are specific for interaction with bTSH, we investigated the functional characteristics of the hTSH analog TR1401 and the native ligand hTSH. For the comparison of these two TSH variants with bTSH, we used several mutations and alanine combinations at the signaling and bTSH binding-sensitive hTSHR hinge positions Glu²⁹⁷, Glu³⁰³, and Asp³⁸². Our data indicate that the higher bioactivity of the TSH variants TR1401 and bTSH are mediated by specific charged residues of the hormone and the hinge region of the hTSHR. Our findings also support the concept that the hinge region of the TSHR is an modulator of TSH potency and efficacy.     

Characterization of nucleosomes consisting of the human testis/sperm-specific histone H2B variant (hTSH2B)

We have reported earlier the occurrence of a specific histone H2B variant in human testis and sperm. Here we have structurally characterized this protein, its association with the rest of the histone octamer, and its effects on the nucleosome structure. We show that a reconstituted octamer consisting of hTSH2B and a stoichiometric complement of histones H2A, H3, and H4 exhibits a lower stability compared to the reconstituted native counterpart consisting of H2B. In contrast, the hTSH2B containing octamers are able to form nucleosome core particles which are structurally and dynamically indistinguishable from those reconstituted with octamers consisting of only native histones. Furthermore, the presence of hTSH2B in the nucleosome does not affect its ability to bind to linker histones.     

A newly identified TSHβ splice variant is involved in the pathology of Hashimoto’s thyroiditis

Thyrotropin (TSH) is a protein that plays a key role in the control of thyroid function. TSH consists of a common α-subunit and a unique β-subunit; the latter is responsible for hormone specificity. A novel splice variant of human TSHβ was identified in 2009. To date, only the tissue distribution of the human TSHβ splice variant mRNA has been studied. Therefore, we aimed to characterize the protein translated from this splice variant. Salting-out, dialysis and concentration of serum proteins were followed by immunoprecipitation to identify the hTSHβ splice variant in serum. Stable CHO cell lines expressing the hTSHβ splice variant and V5-hTSHα were generated. After co-culture, co-immunoprecipitation was used to determine if the hTSHβ splice variant can dimerise with TSHα. We showed for the first time that the hTSHβ splice variant exists in human serum and dimerises with TSHα. To explore the relationship between the TSHβ splice variant and the pathogenesis of autoimmune thyroiditis, we assessed variations in the mRNA expression of the TSHβ splice variant in the peripheral blood leukocytes (PBLs) of Hashimoto’s thyroiditis (HT) patients using quantitative RT-PCR. We found that the mRNA expression levels of the TSHβ splice variant were higher in the PBLs of HT patients who were not undergoing prednisone therapy (n?=?10, P?<?0.0001) and in the PBLs of HT patients with a longer duration of illness (>18?months) who were undergoing prednisone therapy (n?=?5, P?=?0.023) than in those of the control group. This pattern was reversed in the PBLs of HT patients with a shorter duration of illness (<9?months) who were undergoing prednisone therapy (n?=?8, P?<?0.0001). Dexamethasone inhibition of the TSHβ splice variant mRNA expression occurred in a dose- and time-dependent manner. These results demonstrated that the TSHβ splice variant may participate in the pathogenesis of HT.     

Determination of Chinese hamster ovary cell-derived recombinant thyrotropin by reversed-phase liquid chromatography

A reversed-phase high-performance liquid chromatography (RP-HPLC) methodology for the qualitative and quantitative analysis of human thyrotropin (hTSH) in CHO cell conditioned medium and in purified preparations has been set up and validated for accuracy, precision and sensitivity. A recovery test indicated a bias of less than 2% and intra-day and inter-day quantitative determinations presented relative standard deviations (RSD) always <7%, while sensitivity was 0.2 microg (RSD=5.6%). The novel methodology was applied to the study of the best cultivation conditions and was able to detect a significant difference in retention time (t(R)) between pituitary and recombinant hTSH, probably reflecting the influence of the heterogeneity of the carbohydrate moiety on the hydrophobic properties of the molecule.