Biomedical subjects

G D Chazenbalk

Publications and source records attributed to G D Chazenbalk.

At least 37 records · Page 2Linked to original sources

PubMed1997

Evidence that the thyrotropin receptor ectodomain contains not one, but two, cleavage sites.

TSH receptor (TSHR) cleavage into two subunits (A and B) was explored using two new mammalian cell lines expressing the recombinant receptor; 1) TSHR-10,000 CHO cells overexpressing the TSHR; 2) TSHRmyc cells with a c-myc epitope inserted at residues 338-349. Immunoprecipitation or immunoblotting of TSHR-10,000 cells with mAb to either the A subunit or the B subunit revealed multiple forms of the TSHR: 1) uncleaved receptors of approximately 115 kDa and approximately 100 kDa with complex carbohydrate and high mannose carbohydrate, respectively; 2) two subunit TSHR with an approximately 62 kDa A subunit containing complex carbohydrate. The A subunit was approximately 35 kDa after enzymatic deglycosylation (predicted C-terminus near residue 330). The nonglycosylated B subunit was evident primarily as an approximately 42 kDa band (predicted N terminus near residue 380). The sum of the A and B subunit polypeptide backbones was smaller than the predicted size of the TSHR, a polypeptide backbone (84.5 kDa), raising the possibility that an approximately 5-kDa polypeptide fragment was excised during intramolecular cleavage. This hypothesis was supported by data obtained with the TSHRmyc cells. Thus, mAb to the c-myc epitope and to amino acid residues 22-35 (mAb A10) were equally effective in detecting the single chain forms of the TSHR in these cells. However, the 35 kDa, deglycosylated A subunit was clearly visible on immunoprecipitation with mAb A10 to the TSHR amino terminus, but not with the anti-myc mAb, indicating loss of the c-myc epitope at residues 338-349. Further, even though the A subunit was not detected in TSHRmyc cells with anti-myc mAb, 125I-TSH cross-linking to the cell surface showed similar A subunit expression in TSHRmyc and wild-type TSHR expressing cells. In summary, our study provides a surprising and novel finding for G protein-coupled receptors. Contrary to the prevailing concept of one cleavage site in the TSHR, we present evidence that there are, in fact, two such sites. The TSHR, like insulin, may release a C peptide during intramolecular cleavage into two subunits.

Animals↗

PubMed1997

Thyrotropin receptor autoantibodies in serum are present at much lower levels than thyroid peroxidase autoantibodies: analysis by flow cytometry.

Using Chinese hamster ovary (CHO) cells that express high numbers of TSH receptor (TSHR) on their surface, we studied the feasibility of detecting directly by flow cytometry the binding of autoantibodies in patients' sera to the native TSHR. After using a serum (BBI) with high potency in the TSH binding inhibition (TBI) assay to establish the protocol, we studied an additional 38 sera: 10 without TBI activity (1-4.2% inhibition), 10 with moderately high TBI values (17.3-39.4% inhibition), 10 with high TBI levels (52-95.1% inhibition), 4 from normal individuals without autoimmune thyroid disease, and 4 from patients with systemic lupus erythematosus. We observed that a number of sera, including some without thyroid autoantibodies, contain antibodies against unknown antigens on CHO cells. Preadsorption with untransfected CHO cells before addition to the TSHR-10,000 cells eliminated or greatly reduced this nonspecific background. None of the sera from normal individuals, subjects with negative TBI values, or patients with systemic autoimmunity generated a positive signal on flow cytometry with TSHR-10,000 cells relative to the signal on untransfected cells. Remarkably, only 4 of 21 TBI-positive sera (including BBI) unequivocally recognized the TSHR on flow cytometry. In contrast, when thyroid peroxidase (TPO) autoantibodies in the same sera were studied using CHO cells overexpressing TPO on their surface, all 20 sera with TPO autoantibodies clearly elicited positive net fluorescence relative to untransfected cells. Study of the potent serum, BBI, revealed similar fluorescence (approximately 250 U) for TPO autoantibodies and TSHR autoantibodies at dilutions of 1:1000 and 1:10, respectively. Thus, by flow cytometry, the titer of TPO autoantibodies in the BBI serum is about 100-fold higher than that for TSHR autoantibodies in the same serum. In conclusion, the present data provide the strongest support for the idea that TSHR autoantibodies in the sera of patients with autoimmune thyroid disease are present at much lower levels than are TPO autoantibodies. This finding has important implications for the diagnostic detection of TSHR autoantibodies and for understanding the pathogenesis of Graves' disease.

Animals↗

PubMed1997

The human thyrotropin (TSH) receptor in a TSH binding inhibition assay for TSH receptor autoantibodies.

Seven years after the molecular cloning of the human TSH receptor (TSHR), the porcine TSHR remains in general use in the TSH binding inhibition (TBI) assay for autoantibodies to the TSHR. We compared porcine and recombinant human TSHR in two types of TBI assays: one using intact Chinese hamster ovary cells expressing the recombinant human TSHR on their surface, and the other using soluble receptors extracted from these cells with detergent. In the intact cell TBI assay, monolayers expressing large numbers of TSHR were less effective than cells expressing few receptors. These findings are consistent with the very low concentration of TSHR autoantibodies in serum. Binding of [125I]human TSH was about 5-fold lower than that of [125I]bovine TSH to the intact cells. Nevertheless, TBI values with the two ligands were similar for most sera. However, a few sera produced greater inhibition of human than of bovine TSH binding. In the solubilized human TSHR TBI assay, in contrast to the intact cell TBI assay, cells expressing very large number of TSHR were an excellent source for detergent extraction of soluble human TSHR, but only if the cells were extracted while still on the dish and not after scraping. A 10-cm diameter dish of cells provided TSHR for 100-200 replicate determinations when substituted for solubilized porcine TSHR in a commercial TBI kit. TBI values in serum from 30 individuals with suspected Graves' disease correlated closely when tested with solubilized human and porcine TSHR (r = 0.954; P < 0.001). However, 2 sera that were negative with the porcine TSHR were positive with the human TSHR. TBI and thyroid-stimulating activity in these sera correlated weakly regardless of whether the TBI used human or porcine TSHR. These findings open the way to a practical TBI assay using recombinant human TSHR.

Animals↗

PubMed1996

Is receptor cleavage into two subunits necessary for thyrotropin action?

Unlike the wild-type thyrotropin (TSH) receptor, the chimeric TSH-LH/CG receptor TSH-LHR-14 does not cleave into two subunits when cross-linked to [125I]TSH on the surface of intact cells. Immunoblotting of TSH-LHR-14 in whole cell homogenates demonstrated that only a single chain receptor was detected under reducing conditions. TSH-LHR-14, like the A subunit of another chimeric receptor (TSH-LHR-10) that does cleave into two subunits, was almost entirely resistant to endoglycosidase H, indicating that it contains predominantly complex carbohydrate. The fact that TSH-LHR-14 reaches the cell surface where it binds TSH with high affinity and transduces a signal indicates that receptor cleavage into two subunits is not a prerequisite for TSH action.

Animals↗

PubMed1996

Evidence for negative cooperativity among human thyrotropin receptors overexpressed in mammalian cells.

The complementary DNA for the human TSH receptor (TSHR) translated region was amplified in the genome of stably transfected Chinese hamster ovary (CHO) cells using a dihydrofolate reductase minigene. Immunoprecipitation of TSHR in whole cells precursor-labeled with [35S]methionine and [35S]cysteine revealed an approximately 10-fold increase in TSHR expression in cells stabilized in 10,000 nM methotrexate (TSHR-10,000 cells) compared to cells with the same gene not subjected to amplification (TSHR-0 cells). Similarly, [125I]TSH cross-linking to the surface of intact CHO cells revealed a progressive increase in TSH-binding sites with dihydrofolate reductase minigene amplification, with a 12.8-fold increase in TSHR in TSHR-10,000 vs. TSHR-0 cells. Based on the known number of TSHR expressed by TSHR-0 cells, TSHR-10,000 express approximately 1.9 x 10(6) TSHR on their surface. Two ligand-TSHR complexes were evident under reducing conditions, representing the single chain holoreceptor of about 115 kDa and a dissociated A subunit of about 60 kDa. In the absence of TSH, basal cAMP levels in TSHR-10,000 cells were greater than those in TSHR-0 cells (6-fold in isotonic medium and 18.5-fold in hypotonic medium), indicating that the unliganded TSHR has significant constitutive activity. We assessed the kinetics of TSH binding to CHO cells overexpressing the TSHR using [125I]TSH in the presence of increasing concentrations of unlabeled TSH as well as by attempted saturation with labeled ligand. Surprisingly, in contrast to TSHR-0 cells (Kd = approximately 5 x 10(-10) M), we observed progressively lower affinities for TSH binding by TSHR-800 cells (Kd = approximately 10(-9) M) and TSHR-10,000 cells (Kd = approximately 2 x 10(-9) M). In summary, we report a high level of expression of TSHR in CHO cells and confirm the high constitutive activity of the TSHR in the absence of ligand as well as the binding of TSH to the single subunit, uncleaved TSHR. Moreover, we found that a high level of expression is associated with apparent negative cooperativity among the TSHR in terms of their affinity for ligand.

Animals↗

PubMed1996

Critical relationship between autoantibody recognition and thyrotropin receptor maturation as reflected in the acquisition of complex carbohydrate.

Generation of large amounts of recombinant TSH receptor (TSHR) protein capable of recognition by TSHR autoantibodies is a goal of clinical importance. We expressed in Chinese hamster ovary cells the human TSHR ectodomain (ECD) with a carboxyl-terminus six-histidine tag. After transgene amplification, expressing clones were selected by nickel chelate chromatography in combination with [35S] methionine precursor labeling. An approximately 74-kDa protein was detected in the culture medium, and larger quantities of an approximately 68-kDa protein were found in the cell soluble fraction. Immunoblot analysis with a rabbit antiserum revealed that most of the TSHR-ECD was not secreted, but was retained within the soluble fraction of the cell. Nickel chelate chromatography was not effective in purifying significant quantities of this material. In contrast, with Concanavalin A, but not with wheat germ agglutinin, an approximately 50-fold purification of TSHR-ECD was achieved from the cell soluble fraction. Surprisingly, this affinity-enriched TSHR, containing high mannose carbohydrate, was not recognized by human TSHR autoantibodies in sera from six individuals. By ion exchange chromatography, the autoantibody-neutralizing TSHR in the cell supernatant fraction was found to be nonidentical with TSHR-ECD protein recognized by antisera from immunized animals. The present data indicate the critical relationship between autoantibody recognition and TSHR maturation as reflected in the acquisition of complex carbohydrate. Nonsecretion of the TSHR-ECD appears to be related to the specific protein rather than to the glycosylation apparatus of the host cell. Antibodies from immunized animals may be ineffective in monitoring purification of autoantigen-competent TSHR. Finally, the data explain why soluble recombinant TSHR generated in many expression systems is not recognized satisfactorily by human autoantibodies.

Animals↗

PubMed1995

Expression of the extracellular domain of the thyrotropin receptor in the baculovirus system using a promoter active earlier than the polyhedrin promoter. Implications for the expression of functional highly glycosylated proteins.

Conventional baculovirus vectors that utilize the very late polyhedrin promoter have not proved successful for expressing a thyrotropin (TSH) receptor capable of ligand and Graves' disease autoantibody binding comparable to the receptor produced in mammalian cells. Because of the clinical importance of high level expression of this protein, we reassessed the baculovirus system using a new transfer vector (pAcMP3) containing the late basic protein promoter, which functions earlier than the classical polyhedrin promoter. Maximal synthesis of the [35S]methionine-labeled TSH receptor extracellular domain, affinity-purified using a 6-histidine tag, occurred earlier (1 day after insect cell infection) than with a vector (pVL1393) containing the polyhedrin promoter. The pAcMP3-derived TSH receptor extracellular domain was larger (approximately 68 kDa) than the pVL1393-derived protein (approximately 63 kDa). Only the 68-kDa product was secreted, albeit in trace amounts detectable only by precursor labeling. Enzymatic deglycosylation reduced both 68- and 63-kDa cellular proteins to approximately 54 kDa, indicating that the pAcMP3 vector generated a protein with greater carbohydrate content. However, despite its greater degree of glycosylation, most of the 68-kDa protein remained within the cell, almost entirely in the particulate fraction. Remarkably, the trace amounts of 68-kDa receptor protein affinity-purified from the soluble cytosolic fraction of infected insect cells completely neutralized TSH receptor autoantibodies in patients' sera and partly inhibited TSH binding. In conclusion, a baculovirus vector with a promoter active earlier than the conventional polyhedrin promoter generates a more glycosylated and functional TSH receptor extracellular domain protein, albeit at low levels. These data carry important implications for the expression by baculovirus vectors of functional, highly glycosylated proteins.

Animals↗

PubMed1995

Genetic alterations in thyroid hyperfunctioning adenomas.

Thirty-seven thyroid autonomously hyperfunctioning adenomas were screened for mutations in the TSH receptor (TSHR), G alpha s (gsp), and ras genes. Polymerase chain reaction-amplified fragments of the TSHR C-terminal part (exon 10), the G alpha s (exons 8 and 9), and the three ras genes were obtained from the genomic DNA extracted from 37 tumors and their adjacent normal tissues and were studied by direct nucleotide sequencing and hybridization with synthetic probes. A point mutation in the third intracellular loop (codon 623) of the TSHR was found in 3 of 37 adenomas studied. This mutation codes for a change (Ala to Ser) in the TSHR structure and is somatic and heterozygotic. Constitutive activation of the TSHR was demonstrated by an increase in basal cAMP levels after transfection of Chinese hamster ovary cells with a mutated Ser623-TSHR complementary DNA. Nine gsp[00ae]MDRV[00af]- and one ras-activating mutations were also detected. No simultaneous alteration of the studied genes was present. Thus, in hyperfunctioning thyroid adenomas, our data suggest that a mutational activation of the TSHR and gsp genes may play a tumorigenic role through constitutive activation of the cAMP pathway.

Adenoma↗

PubMed1994

Cleavage of the thyrotropin receptor does not occur at a classical subtilisin-related proprotein convertase endoproteolytic site.

The human thyrotropin receptor (TSHR) undergoes proteolytic cleavage closely upstream to amino acid 317. Between residues 261 and 313 are three clusters of positively charged amino acids, arginines (Arg) and lysines (Lys), which are potential subtilisin-related proprotein convertase sites. We used oligonucleotide-directed mutagenesis to perform conservative amino acid substitutions within these regions (Arg or Lys to glutamine, Gln). Chinese hamster ovary cells stably transfected with mutant receptor cDNA TSHR-CS1 (Gln261) and TSHR-CS3 (Gln312, Gln313) bound radiolabeled TSH with an affinity similar to the wild-type TSHR. Mutant cDNA TSHR-CS2 (Gln290, Gln291) and TSHR-CS4 (Gln261, Gln290, Gln291, Gln312, Gln313) did not express a protein on the cell surface capable of specific TSH binding. After covalent cross-linkage of radiolabeled TSH to TSHR-CS1 and TSHR-CS3, the mutant receptors dissociated into two subunits under reducing conditions. The most prominent cluster of basic amino acids in the TSHR extracellular region (residues 287-293) was studied in a second series of mutations designed to eliminate the classical proprotein convertase sites in this region and yet be compatible with TSHR function. All three mutant receptors, TSHR-CS5 (Gln290), TSHR-CS6 (Gln291), and TSHR-CS7 (Gln291, Gln293) bound TSH with an affinity similar to that of wild type, and none of these amino acid substitutions prevented proteolytic cleavage of the extracellular domains of the TSHR. Thus, cleavage of the TSHR extracellular domain does not involve a classical subtilisin-related proprotein convertase cleavage site, raising the possibility that TSHR cleavage occurs after processing and trafficking of the protein to the plasma membrane.

Amino Acid Sequence↗

PubMed1994

Recombinant thyroid peroxidase-specific autoantibodies. II. Role of individual heavy and light chains in determining epitope recognition.

Most thyroid peroxidase (TPO) autoantibodies in man recognize closely associated epitopes in two domains (A and B) on TPO. These epitopes were defined by recombinant monoclonal human autoantibodies expressed as antigen-binding fragments [F(ab)]. Only five heavy (H) and light (L) chain gene combinations encoded 34 F(ab), all of which have high affinity (Kd, approximately 10(-10) M) for TPO. We, therefore, investigated the roles of H and L chain genes in TPO domain recognition in two ways. First, we created hybrid F(ab) by forced recombination of H and L chain genes from 4 F(ab) recognizing the A or B domains. These hybrid F(ab) proteins, expressed in bacteria, bound extremely poorly (or not at all) to TPO, even at concentrations more than 100-fold higher than those required for detection of TPO binding by the original F(ab). Nucleotide sequencing of the cDNA as well as gel electrophoresis of the expressed proteins confirmed that poor hybrid F(ab) binding to TPO was not the result of cloning artifacts. Therefore, contrary to prevailing views on combinatorial libraries, we found no tolerance for H and L chain cross-combinations in high affinity TPO binding. These observations strengthen the likelihood that the H and L chain combinations from combinatorial libraries reflect those of TPO autoantibodies in vivo. In a second approach to examine the roles of H and L chains in TPO binding, we focused on three original F(ab) with similar L chains (encoded by KL012-like germline genes) and similar H chains (encoded by V1-3B-like germline genes), but different diversity (D) regions. All F(ab) bound predominantly to TPO domain A, as observed previously for a F(ab) with a KL012 L chain and a different H chain. Conversely, a F(ab) with a V1-3B-like H chain but a different L chain (A') bound to TPO domain B. These data indicate that the L chain plays a major role in defining TPO epitope recognition.

Amino Acid Sequence↗

PubMed1994

Studies on homologous desensitization of the thyrotropin receptor in 293 human embryonal kidney cells.

It is well known that the TSH receptor (TSHR) undergoes homologous desensitization. That is, prolonged stimulation of thyroid cells with TSH attenuates the cAMP response to subsequent TSH stimulation. However, the existence of homologous desensitization of the recombinant TSHR expressed in nonthyroidal eukaryotic cells is controversial. In the present studies, therefore, we first investigated whether or not the TSHR was desensitized by TSH in 293 human embryonal kidney cells, a cell line in which the LH/CG receptor (LH/CGR) is reported to undergo homologous desensitization. The wild type (wt) TSHR and the wt-LH/CGR stably expressed in 293 cells bound to their respective hormones with high affinity and produced a dose-dependent intracellular cAMP response to hormone stimulation. Pretreatment of cells expressing the TSHR or the LH/CGR with their respective hormones attenuated the cAMP response to subsequent hormone stimulation without down-regulation of the receptors, demonstrating that the TSHR, as well as the LH/CGR, undergoes homologous desensitization in 293 cells. With this cell type expressing mutant TSHRs, we then studied some aspects of the molecular mechanism of TSHR desensitization and compared our data to those obtained with the beta-adrenergic receptor (beta-AR), which is widely regarded as the prototype for receptor desensitization. We cotransfected the wt-TSHR and a chimeric receptor consisting of the LH/CGR extracellular ligand binding domain with the TSHR transmembrane/cytoplasmic signal transducing region. These two receptors have distinct hormone specificities but share common signal regulatory mechanisms. We observed that, like the beta-AR, only hormone-occupied receptor is likely to be involved in homologous desensitization. On the other hand, studies with a truncated TSHR indicated that, in contrast to the beta-AR, the serine/threonine-rich region in the carboxyl two thirds of the cytoplasmic tail of the TSHR is not involved in homologous desensitization.

Animals↗

PubMed1993

Lack of promiscuity in autoantigen-specific H and L chain combinations as revealed by human H and L chain "roulette".

Individual H or L chains from a human autoantibody were used to search for other L or H chains that could form antigen-binding fragments, Fab, with the same specificity. The parent Fab (SP1.2) exhibits high affinity binding for thyroid peroxidase (TPO), a 107-kDa protein that is the major autoantigen in human autoimmune thyroiditis. This autoantibody "roulette," performed by using Ig H and L chain gene libraries expressed in bacteria, increased the frequency of TPO-binding clones in the new libraries. However, the frequency was still much lower than would be the case if promiscuous combinations with a variety of H or L chains were compatible with specific Ag binding. Nucleotide sequence analysis of the H and L chains of the new TPO-binding clones revealed even more restriction. Thus, with the SP1.2 H chain, all 11 new Fab utilized L chains from the same V kappa 1 family germline gene as SP1.2 itself. Similarly, five of six H chains "captured" by the SP1.2 L chain were very closely related to the SP1.2 H chain. However, one totally different H chain was isolated: SP4.6 has a VH region that differs substantially from that of SP1.2. SP4.6 also has a distinct D region, uses a different JH, and, unlike SP1.2, which is an IgG1, belongs to subclass IgG4. The affinities for TPO of SP4.6 (with the different H chain) and SP1.20 (which had the least mutated L chain germline gene) were similar to that of SP1.2 (approximately 10(-10) M). As expected, the SP1.2 and SP1.20 Fab, which have the same H chain and closely related L chains, bound to the same domain on TPO. However, a similar domain on TPO was recognized by both SP4.6 and SP1.2, despite the fact that their V, D, and J regions are quite different. This observation raises the possibility that the L chain is critical in defining epitope specificity, even in the presence of completely different D regions and nonidentical VH regions.

Amino Acid Sequence↗

PubMed1993

Human organ-specific autoimmune disease. Molecular cloning and expression of an autoantibody gene repertoire for a major autoantigen reveals an antigenic immunodominant region and restricted immunoglobulin gene usage in the target organ.

The most common organ-specific autoimmune disease in humans involves the thyroid. Autoantibodies against thyroid peroxidase (TPO) are present in the sera of virtually all patients with active disease. We report the molecular cloning of the genes for 30 high-affinity, IgG-class human autoantibodies to TPO from thyroid-infiltrating B cells. Analysis of the putative germline genes used for the TPO human autoantibodies suggests the use of only five different H and L chain combinations involving four H chains and three L chains. In addition, the same combination of H and L chains was found in multiple patients. The F(ab) proteins expressed by these genes define two major, closely associated domains (A and B) in an immunodominant region on TPO. These A and B domains contain the binding sites of approximately 80% of IgG-class TPO autoantibodies in the sera of patients with autoimmune thyroid disease. The present information permits analysis, not previously possible, of the relationship between autoantibody H and L chain genes and the antigenic domains on an autoantigen. Our data, obtained using target organ-derived autoantibodies, indicate that there is restriction in H and L chain usage in relation to the interaction with specific antigenic domains in human, organ-specific autoimmune disease.

Antibody Affinity↗

PubMed1993

The immunodominant region on human thyroid peroxidase recognized by autoantibodies does not contain the monoclonal antibody 47/c21 linear epitope.

We performed studies to determine whether the binding sites on thyroid peroxidase (TPO) of immunoglobulin antigen binding fragments (Fabs) representing more than 80% of the human autoantibody repertoire overlap with the binding site of monoclonal antibody (Mab) 47, the only Mab whose partial epitope has been defined at the amino acid level (residues 713-721). We also investigated whether these Fabs preferentially recognize native or denatured TPO. None of the Fabs, when bound to radiolabeled TPO, interfered with the ability of Mab 47 to bind to this material. In enzyme-linked immunosorbent assay experiments, the binding of TPO autoantibody Fabs SP1.5, WR1.7, TR1.8, and TR1.9 was greatly diminished by denaturation of TPO. In contrast, binding of Mab 47 was higher to denatured TPO than to intact TPO. Our studies indicate that the Mab 47/C21 epitope lies outside the immunodominant region on TPO. Further, the data confirm that the majority of epitopes for TPO autoantibodies are highly conformational (dependent on the three-dimensional structure of the native protein). Native TPO will be needed to complete the mapping of the epitopes for TPO autoantibodies as well as to determine the amino acids at the autoantibody-antigen-binding sites.

Animals↗

PubMed1992

Cloning of a member of the arrestin family from a human thyroid cDNA library.

We used the cDNA of human retinal arrestin as a probe to screen a human thyroid cDNA library. We isolated and plaque-purified one clone (hTHY-ARRX). The nucleotide sequence of the 1.8 kb cDNA insert had an open reading frame of 1227 bp coding for a protein of 409 amino acids. Northern blot analysis revealed a single transcript of 1.7 kb in human thyroid cells. There is significant homology between amino acid sequences of human thyroid arrestin and human retinal arrestin (63%) and bovine beta-arrestin (74%), respectively. The hTHY-ARRX cDNA was stably transfected into Chinese hamster ovary cells already expressing a functional human thyrotropin (TSH) receptor. The cAMP response to TSH stimulation was unaltered in these cells, and homologous desensitization to TSH stimulation was not restored. It is not presently known whether hTHY-ARRX is human beta-adrenergic arrestin or a new member of the arrestin family.

Amino Acid Sequence↗

PubMed1992

Recognition by recombinant autoimmune thyroid disease-derived Fab fragments of a dominant conformational epitope on human thyroid peroxidase.

To characterize the nature of thyroid peroxidase (TPO) autoantibodies present in the sera of patients with autoimmune thyroid disease, we cloned three IgG1/kappa Fab fragments which bind 125I-TPO. This was accomplished by the molecular cloning and expression in bacteria of IgG gene fragments from B cells infiltrating the thyroid of a patient with Graves' disease. The three Fab fragments (SP2, SP4, and SP5) are coded for by a common heavy chain (VH1, D, JH3) and three related, but different, light chains (VK1, JK2). The SP Fab fragments bind specifically to TPO with high affinities (6 x 10(-11)-2 x 10(-10) M) comparable to those of serum TPO autoantibodies. TPO autoantibodies represented by the SP Fab fragments are present in all 11 patients studied, constitute a high proportion (36-72%) of serum TPO autoantibodies in individual patients and interact with a conformational epitope on TPO.

Amino Acid Sequence↗

PubMed1992

Role of amino acids 261-418 in proteolytic cleavage of the extracellular region of the human thyrotropin receptor.

Previous in vivo cross-linking studies of TSH to the recombinant TSH receptor revealed that the receptor exists at least in part as a single chain glycoprotein of approximately about 100 kilodaltons (kDa), with intramolecular disulfide bonds. TSH also binds to a 54-kDa amino-terminal fragment of the TSH receptor (cleaved up-stream of amino acid residue 317). In the present study in order to better understand the structure of the TSH receptor, we covalently cross-linked radiolabeled TSH to six TSH-LH receptor extracellular region chimeras and the wild-type TSH receptor expressed on Chinese hamster ovary cells in vivo. In these chimeras, different regions of the TSH receptor were substituted with the homologous regions of the LH receptor. When analyzed under nonreducing conditions by polyacrylamide gel electrophoresis, the TSH-cross-linked products were similar to all TSH-LH receptor chimeras and the wild-type receptor. In contrast, differences among the receptors were noted when the TSH-cross-linked products were examined under reducing conditions. With the exception of two chimeras, as noted previously with the wild-type receptor, two TSH-cross-linked products were observed, representing TSH cross-linked to a holoreceptor of about approximately 100 kDa and a fragment of the receptor of about approximately 54 kDa. However, in the two chimeras in which both domains D and E (amino acids 261-418) of the TSH receptor were substituted, only the holoreceptor and not the smaller fragment was detected. Substitution of domains ABC (amino acids 1-260) did not prevent proteolytic cleavage of the TSH receptor. In conclusion, amino acids 261-418 are necessary for proteolytic cleavage of the extracellular region of the human TSH receptor.

Endopeptidases↗

PubMed1992

Studies on the role of amino acids 38-45 in the expression of a functional thyrotropin receptor.

We previously reported that deletion or substitution of a unique eight-amino acid tract (residues 38-45) in the extracellular domain of the human TSH receptor led to the loss of specific ligand binding to the surface of transfected cells. In the present study we analyzed this region in more detail. Using site-directed mutagenesis of the TSH receptor cDNA, we substituted amino acid residues 38-45, either in three overlapping groups of four amino acids each or individually. The resultant TSH receptor mutant cDNAs were stably transfected into Chinese hamster ovary cells, and the cells were tested for their TSH-binding ability. Our data demonstrate that amino acid residues 38-40 and 42-45 in this region of the human TSH receptor can be substituted without alteration in receptor function and are, therefore, not critical in forming or maintaining the TSH-binding site. However, substitution of Cys41, either alone or together with adjacent amino acids, leads to the loss of TSH binding to its receptor. These data suggest a central role for the amino acid in position 41 in preserving the biological function of the TSH receptor.

Amino Acid Sequence↗