Gonadotropin receptors, unlike the thyrotropin receptor (TSHR), are not cleaved into disulfide-linked A- and B-subunits, nor do they shed A-subunits. Heavily glycosylated TSHR A-subunits initiate or amplify responses leading to stimulating TSHR-autoantibodies and Graves' hyperthyroidism.To investigate the possibility that mice immunized with luteinizing hormone receptor (LHR) would develop functional antibodies, we constructed adenoviruses expressing the rat-LH holoreceptor (LHR-Ad) and an LHR A-subunit equivalent (LHR-289-Ad). Female BALB/c mice were immunized with high doses (10(11) particles) of LHR-Ad, LHR-289-Ad, or control (Con)-Ad. Sera were tested using LHR-expressing eukaryotic cells for antibody binding by flow cytometry and for bioactivity by measuring cyclic adenosine monophosphate (cAMP) stimulation.Elevated serum binding to LHR cells in some LHR-Ad and LHR-289-Ad immunized mice was not specific for LHR-expressing cells. Moreover, sera lacked bioactivity, consistent with unchanged serum estradiol and ovary histology. The difference between rat and mouse LHR-ectodomains is relatively small (3% at the amino-acid level). In contrast, despite amino-acid identity, immunization of mice with adenovirus expressing membrane-bound mouse thyroid peroxidase (TPO), but not soluble mouse TPO ectodomain, elicited strong TPO-specific antibodies.Our investigations provide insight into antibody responses to self-antigens. First, antibodies are induced to large self-antigens like mouse-TPO when membrane bound. Second, lesser amino acid homology between the immunogen and mouse protein (91% vs. 97% for the human-TSHR and rat-LHR, respectively) favors antibody induction. Finally, from previous studies demonstrating the immunogenicity of the highly glycosylated human TSHR A-subunit versus our present data for the nonimmunogenic less glycosylated rat LHR, we suggest that the extent of glycosylation contributes to breaking self-tolerance.
Parenthesis is an additional element independent of the core sentence structure, which is a very common linguistic phenomenon in Uyghur and Chinese.It not only has various forms, rich semantics, but also has important communicative functions.They make the discourse vivid, rich, fluent, with a strong appeal.We should attach importance to it in language research and practice.Based on this point, this paper reveals the similarities and differences in the textual functions of Chinese and Uygur parentheses through a comparative study.Generally speaking, parentheses are widely used in Chinese and Uyghur languages.No matter in classification, features or functions, parentheses in the two languages are more common than individual, with almost no differences.
An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Abstract The glycoprotein hormone receptor hinge region connects the leucine-rich and transmembrane domains. The prevalent concept is that the hinge does not play a significant role in ligand binding and signal transduction. Portions of the hinge are redundant and can be deleted by mutagenesis or are absent in certain species. A minimal hinge will be more amenable to future investigation of its structure and function. We, therefore, combined and progressively extended previous deletions (Δ) in the TSH receptor (TSHR) hinge region (residues 277–418). TSHRΔ287–366, Δ287–371, Δ287–376, and Δ287–384 progressively lost their response to TSH stimulation of cAMP generation in intact cells, consistent with a progressive loss of TSH binding. The longest deletion (TSHRΔ287–384), reducing the hinge region from 141 to 43 amino acids, totally lost both functions. Surprisingly, however, with deletions extending from residues 371–384, constitutive (ligand-independent) activity increased severalfold, reversing the suppressive (inverse agonist) effect of the TSHR extracellular domain. TSHR-activating point mutations I486F and I568T in the first and second extracellular loops (especially the former) had reduced activity on a background of TSHRΔ287–371. In summary, our data support the concept that the TSHR hinge contributes significantly to ligand binding affinity and signal transduction. Residues within the hinge, particularly between positions 371–384, appear involved in ectodomain inverse agonist activity. In addition, the hinge is necessary for functionality of activating mutations in the first and second extracellular loops. Rather than being an inert linker between the leucine-rich and transmembrane domains, the TSHR hinge is a signaling-specificity domain.
Graves disease is directly caused by thyroid-stimulating autoantibodies (TSAb's) that activate the thyrotropin receptor (TSHR). We observed upon flow cytometry using intact cells that a mouse mAb (3BD10) recognized the TSHR ectodomain with a glycosidylphosphatidylinositol (ECD-GPI) anchor approximately tenfold better than the same ectodomain on the wild-type TSHR, despite the far higher level of expression of the latter. The 3BD10 epitope contains the N-terminal cysteine cluster critical for TSAb action. Consequently, we hypothesized and confirmed that TSAb (but not thyrotropin-blocking autoantibodies [TBAb's]) also poorly recognize the wild-type TSHR relative to the ECD-GPI. Despite poor recognition by TSAb of the holoreceptor, soluble TSHR A subunits (known to be shed from surface TSHR) fully neutralized autoantibody-binding activity. These data indicate that the epitope(s) for TSAb's, but not for TBAb's, are partially sterically hindered on the holoreceptor by the plasma membrane, the serpentine region of the TSHR, or by TSHR dimerization. However, the TSAb epitope on the soluble A subunit is freely accessible. This observation, as well as other evidence, supports the concept that A subunit shedding either initiates or amplifies the autoimmune response to the TSHR, thereby causing Graves disease in genetically susceptible individuals.
The TSH receptor (TSHR) comprises an extracellular leucine-rich domain (LRD) linked by a hinge region to the transmembrane domain (TMD). Insight into the orientation of these components to each other is required for understanding how ligands activate the receptor. We previously identified residue E251 at the LRD-hinge junction as contributing to coupling TSH binding with receptor activation. However, a single residue cannot stabilize the LRD-hinge unit. Therefore, based on the LRD crystal structure we selected for study four other potential LRD-hinge interface charged residues. Alanine substitutions of individual residues K244, E247, K250 and R255 (as well as previously known E251A) did not affect TSH binding or function. However, the cumulative mutation of these residues in varying permutations, primarily K250A and R255A when associated with E251A, partially uncoupled TSH binding and function. These data suggest that these three residues, spatially very close to each other at the LRD base, interact with the hinge region. Unexpectedly and most important, monoclonal antibody CS-17, a TSHR inverse agonist whose epitope straddles the LRD-hinge, was found to interact with residues K244 and E247 at the base of the convex LRD surface. These observations, together with the functional data, exclude residues K244 and E247 from the TSHR LRD-hinge interface. Further, for CS-17 accessibility to K244 and E247, the concave surface of the TSHR LRD must be tilted forwards towards the hinge region and plasma membrane. Overall, these data provide insight into the mechanism by which ligands either activate the TSHR or suppress its constitutive activity.
Monoclonal antibody CS-17 is a TSH receptor (TSHR) inverse agonist (suppresses constitutive activity) and a TSH antagonist. Elucidation of the CS-17 epitope will provide insight into TSHR structure and function. Present information on its epitope conflicts with recent data regarding another TSHR inverse agonist antibody. To characterize further the CS-17 epitope, we exploited the observation that CS-17 does not recognize a chimeric receptor with TSHR hinge region residues 261–289 replaced with homologous rat LH receptor residues (13 mismatches). We generated individual and double TSHR mutations corresponding to these mismatches. On flow cytometry, only T273L/R274V reduced CS-17 recognition. No mutation affected TSH-stimulated cAMP generation. Because the immunogen for CS-17 generation was highly glycosylated, we also investigated whether the glycan moiety at N198, topologically adjacent to Y195 (a previously identified epitopic component), could contribute to the CS-17 epitope. Elimination of this N-linked glycan (mutations of N198 and T200) abrogated CS-17 binding without altering TSH responsiveness. However, studies with tunicamycin suggested that these mutations affected CS-17 binding by altering the polypeptide backbone rather than eliminating the glycan moiety. TSHR residues N198 and T200, like Y195, are on the convex facet of the leucine-rich domain. In summary, the present data indicate that the discontinuous epitope of CS-17, a TSHR inverse agonist and TSH antagonist, includes a component in the hinge region as well as the convex surface of the TSHR leucine-rich domain. These findings expand our present concept of glycoprotein hormone binding and function.
AbstractBackground:The COVID-19-associated mortality rate of haemophilia patients is similar to that of the general population, but the risk of hospitalization and bleeding is higher. However, the specific impact of this infection on haemophilia patients has not been reported yet. In this study, we aimed to investigate the impact of the coronavirus disease 2019 (COVID-19) pandemic on the infection susceptibility, symptoms, drug use, and social intercourse of patients with haemophilia. Methods: A survey was distributed to a total of 265 patients with haemophilia [adult (n = 185) and pediatric patients (n = 80)] in the Fujian haemophilia therapeutic center (Fuzhou City, China) during the COVID-19 pandemic, and data were collected between January 2022 and January 2023. The impacts of SARS-CoV-2 infection on haemophilia symptoms, drug use, and social intercourse of these patients were investigated, and the association between the recovery time and disease conditions was explored in infected patients. Results: During the COVID-19 pandemic, compared with adult patients, pediatric patients had reduced social intercourse and outdoor activities because of the fear of contracting COVID-19 (85.0% vs.66.5%; P = 0.002). Bleeding events were also significantly fewer in children than in adults (61.2% vs.81.1%; P = 0.001). The SARS-CoV-2 infection rate was significantly higher in patients living in urban areas than in those living in rural areas (74.3% vs. 53.6%; P < 0.001). The duration of achieving symptomatic recovery from COVID-19 was not significantly associated with hemorrhage, type and classification of haemophilia, presence of inhibitors, complications, and vaccination status. Conclusion: Having COVID-19 infection did not significantly influence the symptoms and treatments in patients with haemophilia. Compared with adults, pediatric patients had significantly fewer bleeding events.
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