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Basil Rapoport

Publications and source records attributed to Basil Rapoport.

At least 37 records · Page 2Linked to original sources
PubMed2004

Why measure thyroglobulin autoantibodies rather than thyroid peroxidase autoantibodies?

Autoantibodies to thyroglobulin (TgAb) and thyroid peroxidase (TPOAb) are of immunoglobulin G (IgG) class and have high affinities for their respective autoantigens. Both autoantibodies are markers of thyroid autoimmunity and they can be measured by a variety of assays. From the clinical perspective, TgAb are less prevalent than TPOAb and less useful than TPOAb for prediction of thyroid dysfunction. Moreover, TgAb interfere with Tg measurements to monitor metastases in thyroid cancer. However, increasing evidence suggests that these TgAb provide a surrogate for Tg. In terms of disease pathogenesis, Tg has been suggested to play a role in Graves' ophthalmopathy. Pending further studies, TgAb epitopes could distinguish between individuals who are euthyroid or who have clinical disease. A final, intriguing reason for measuring and characterizing TgAb is the interest these autoantibodies have rekindled in their autoantigen. It is conceivable that Tg polymorphisms, combined with the explosive mix of iodine, TPO and H2O2 necessary for thyroid hormone synthesis, inadvertently provide the trigger for the autoimmune thyroid response.

Autoantibodies↗
PubMed2004

Evidence that the thyrotropin receptor protease is membrane-associated and is not within lipid rafts.

The thyrotropin receptor (TSHR) cleaves to a variable extent within the ectodomain into a ligand-binding A subunit linked by disulfide bonds to the largely transmembrane B subunit. To obtain insight into this variability, we examined the extent of cleavage of TSHR ectodomains tethered to the plasma membrane by different means: (1) the wild-type, serpentine region, (2) a glycosylphosphatidylinositol (GPI) anchor, and (3) a single CD8alpha transmembrane region. For this purpose, we covalently cross-linked(125)I-TSH to the TSHR ectodomain expressed on the surface of intact cell monolayers. The extent of cleavage of the CD8alpha-tethered ectodomain was similar to the wild-type TSHR (approximately 50%) whereas the same ectodomain with a GPI anchor remained almost entirely (approximately 90%) uncleaved. These findings have three possible implications. First, differential cleavage of the TSHR ectodomain depending on its attachment to the plasma membrane suggests that the TSHR protease is membrane-associated and is not a soluble (secreted or shed) protease. Second, because GPI-anchored proteins (unlike CD8alpha) segregate in membrane lipid rafts, the TSHR protease appears not to be associated with lipid rafts. Finally, the similar extent of cleavage of the wild-type TSHR and the CD8alpha (not the GPI) tethered ectodomain supports the concept that the wild-type TSHR resides largely outside lipid rafts.

Animals↗
PubMed2004

Thyroid stimulation does not require antibodies with identical epitopes but does involve recognition of a critical conformation at the N terminus of the thyrotropin receptor A-subunit.

Whether monoclonal antibodies with thyroid-stimulating activity [thyroid-stimulating antibody/antibodies (TSAb)] from immunized animals are identical to human autoantibodies in Graves' disease is unknown. Here, we compared properties of a monoclonal hamster TSAb (MS-1) with human autoantibodies. The epitopes of neither MS-1 nor human autoantibodies can be determined by peptide scanning, indicating their conformational nature. A property of human TSAb is that their epitope is partially obscured on the TSH holoreceptor on the cell surface relative to the TSH receptor (TSHR) ectodomain tethered to the membrane by a glycosylphosphatidyl inositol anchor. On flow cytometry, as for human autoantibodies, MS-1 preferentially recognized the glycosylphosphatidyl inositol-anchored ectodomain vs. the TSH holoreceptor on Chinese hamster ovary cells. Also, as with human autoantibodies, only A-subunits with the active (but not the inactive) conformation adsorbed MS-1 binding activity. This difference localizes antibody binding to a cysteine-rich region at the TSHR N terminus. Remarkably, active TSHR A-subunit more effectively ( approximately 40-fold) neutralized human autoantibodies than it did MS-1. Therefore, MS-1 interacts less well than autoantibodies with the free A-subunit. In summary, we provide evidence that TSAb need not have identical epitopes. However, the TSAb epitope does appear to require involvement of the highly conformational N terminus of the A-subunit.

Animals↗
PubMed2004

Affinity-enrichment of thyrotropin receptor autoantibodies from Graves' patients and normal individuals provides insight into their properties and possible origin from natural antibodies.

We used purified recombinant TSH receptor (TSHR) antigen prepared in mammalian cells to affinity-enrich TSHR autoantibodies from Graves' patients' IgG. Autoantibody enrichment, assayed by TSH binding inhibitory activity, was 20- to 1000-fold. Thyroid-stimulating antibody activity enrichment, although more difficult to quantitate, was comparable. TSHR-autoantibody approximate affinities for the holoreceptor assessed indirectly by TSH binding inhibition were 4-27 x 10(-9) m, an underestimate because 100% TSHR autoantibody purity was not attained. Consistent with previous data for serum, highly enriched TSHR autoantibodies in three of four patients showed lambda light chain bias. However, in contrast to expectations, antigen-enriched IgG was skewed primarily toward IgG2 and IgG3, subclasses associated with polysaccharides and microorganisms, respectively. Subclass depletion studies on antigen-enriched IgG indicated that TSHR autoantibodies were predominantly IgG1 and, surprisingly, IgG4. As controls, we affinity-enriched pooled IgG from normal individuals on TSHR antigen. This enriched IgG had detectable TSH binding inhibitory activity, although with lower specific activity than, and lacking the thyroid stimulatory activity of, Graves' IgG. Moreover, these natural IgG class autoantibodies largely recognized the same conformational variation in the TSHR N-terminal region as disease-associated TSHR autoantibodies. These studies suggest that TSHR autoantibodies may arise from natural autoantibodies, possibly by class switching from cross-reacting antibodies to microorganisms.

Antibodies↗
PubMed2004

Human monoclonal thyroglobulin autoantibodies: epitopes and immunoglobulin genes.

Autoantibodies to thyroglobulin (TgAbs) are common markers of thyroid autoimmunity, but relatively few human monoclonal TgAbs have been described. From a panel of 64 human monoclonal TgAbs (isolated from a thyroid-disease derived combinatorial Ig gene library), we selected seven with unique genetic features for detailed characterization. These TgAbs preferentially recognize native (not denatured) Tg, like serum autoantibodies. Most have high affinities for Tg (dissociation constant 10(-10) to 10(-9) m). Their light (L) chain Ig genes are not unusual, but four of the five heavy (H) chain genes are new. Moreover, one H chain belongs to the small VH2 family, not previously reported for autoantibodies to Tg or thyroid peroxidase. The TgAbs inhibit the binding to Tg of the thyroid donor's serum autoantibodies, indicating epitopic overlap. Competition analysis (surface plasmon resonance) shows that the TgAbs recognize overlapping epitopes in an immunodominant region on the Tg dimer ( approximately 660 kDa). Two major and several minor epitopic regions were defined, each associated with a particular H + L chain combination. In conclusion, our TgAb panel provides novel information regarding the repertoire of H chain genes encoding human TgAbs as well as the relationship between the H chains and the epitopes recognized on this major thyroid autoantigen.

Amino Acid Sequence↗
PubMed2003

Thyrotropin receptor knockout mice: studies on immunological tolerance to a major thyroid autoantigen.

Graves' disease involves a breakdown in self-tolerance to the TSH receptor (TSHR). Central T cell tolerance is established by intrathymic deletion of immature T lymphocytes that bind with high affinity to peptides from autoantigens (like the TSHR) expressed ectopically in the thymus. In TSHR-knockout mice, tolerance cannot be induced to the TSHR, which should, therefore, be a foreign antigen for these animals. To test this hypothesis, TSHR-knockout mice and wild-type controls were vaccinated (three injections) with TSHR DNA or control DNA. TSHR antibodies, measured by ELISA, binding to TSHR-expressing eukaryotic cells, and TSH binding inhibition, developed in approximately 60% of TSHR-knockout mice, not significantly different from 80% in the wild-type mice. Antibody levels were also comparable in the two groups, and both strains recognized the same immunodominant linear antibody epitope at the amino terminus of the TSHR. Splenocyte responses to TSHR protein in culture, measured as interferon-gamma production, were similar in TSHR-knockout and wild-type mice. Moreover, T cells from both strains recognized the same two epitopes from a panel of 29 synthetic peptides encompassing the TSHR ectodomain and extracellular loops. This lack of difference in immune responses in TSHR-knockout and wild-type mice is unexpected and is contrary to observations in other induced animal models of autoimmunity. The importance of our finding is that the TSHR may not be similar to other model proteins used to define the concept of central immune tolerance.

Amino Acid Sequence↗
PubMed2003

Low-dose immunization with adenovirus expressing the thyroid-stimulating hormone receptor A-subunit deviates the antibody response toward that of autoantibodies in human Graves' disease.

Immunization with adenovirus expressing the TSH receptor (TSHR) induces hyperthyroidism in 25-50% of mice. Even more effective is immunization with a TSHR A-subunit adenovirus (65-84% hyperthyroidism). Nevertheless, TSHR antibody characteristics in these mice do not mimic accurately those of autoantibodies in typical Graves' patients, with a marked TSH-blocking antibody response. We hypothesized that this suboptimal antibody response was consequent to the standard dose of TSHR-adenovirus providing too great an immune stimulus. To test this hypothesis, we compared BALB/c mice immunized with the usual number (10(11)) and with far fewer viral particles (10(9) and 10(7)). Regardless of viral dose, hyperthyroidism developed in a similar proportion (68-80%) of mice. We then examined the qualitative nature of TSHR antibodies in each group. Sera from all mice had TSH binding-inhibitory (TBI) activity after the second immunization, with TBI values in proportion to the viral dose. After the third injection, all groups had near-maximal TBI values. Remarkably, in confirmation of our hypothesis, immunization with progressively lower viral doses generated TSHR antibodies approaching the characteristics of autoantibodies in human Graves' disease as follows: 1) lower TSHR antibody titers on ELISA and 2) lower TSH-blocking antibody activity without decrease in thyroid-stimulating antibody activity. In summary, low-dose immunization with adenovirus expressing the free TSHR A-subunit provides an induced animal model with a high prevalence of hyperthyroidism as well as TSHR antibodies more closely resembling autoantibodies in Graves' disease.

Adenoviridae↗
PubMed2003

Does thyrotropin cleave its cognate receptor?

A recent report of major pathophysiological significance, and opposed to present concepts, is that TSH (but not MS-1, a hamster monoclonal thyroid-stimulating antibody), cleaves the single-chain TSH receptor (TSHR) on the cell surface into its two-subunit form. We reassessed the issue using two approaches. First we wished to confirm the flow-cytometric assay previously used to quantitate TSHR cleavage. We used CHO cell lines expressing large (TSHR-10,000 cells) or conventional (TSHR-0 cells) numbers of TSHR. Cells were preincubated (16 h) in either control medium or medium supplemented with TSH (5 x 10(-8) m) or MS-1 (10 microg/ml). After stringent washing to maximize removal of residual ligand, we performed flow cytometry with two antibodies, one recognizing only the single-chain TSHR, the other recognizing all (cleaved and uncleaved) TSHRs. TSH pretreatment did not appear to increase TSHR cleavage. Instead we observed ligand occupancy of the TSHR (with MS-1) or fewer receptors on the cell surface (down-regulation), particularly with the TSHR-0 cells. Second, we covalently cross-linked [125I]TSH to monolayers of these cells, an unequivocal method to determine directly the proportion of single-chain and two-subunit TSHR forms. Pretreatment of TSHR-10,000 and TSHR-0 cells with TSH had no effect on the degree of TSHR cleavage. MS-1 slightly reduced spontaneous cleavage. In conclusion, in contrast to a recent report, we show that TSH does not alter the subunit structure of its cognate receptor, and we provide insight into the difficulties associated with the flow-cytometric assay for TSHR cleavage.

Animals↗
PubMed2003

Prevention of autoantibody-mediated Graves'-like hyperthyroidism in mice with IL-4, a Th2 cytokine.

Graves' hyperthyroidism has long been considered to be a Th2-type autoimmune disease because it is directly mediated by autoantibodies against the thyrotropin receptor (TSHR). However, several lines of evidence have recently challenged this concept. The present study evaluated the Th1/Th2 paradigm in Graves' disease using a recently established murine model involving injection of adenovirus expressing the TSHR (AdCMVTSHR). Coinjection with adenovirus expressing IL-4 (AdRGDCMVIL-4) decreased the ratio of Th1/Th2-type anti-TSHR Ab subclasses (IgG2a/IgG1) and suppressed the production of IFN-gamma by splenocytes in response to TSHR Ag. Importantly, immune deviation toward Th2 was accompanied by significant inhibition of thyroid-stimulating Ab production and reduction in hyperthyroidism. However, in a therapeutic setting, injection of AdRGDCMVIL-4 alone or in combination with AdCMVTSHR into hyperthyroid mice had no beneficial effect. In contrast, coinjection of adenoviruses expressing IL-12 and the TSHR promoted the differentiation of Th1-type anti-TSHR immune responses as demonstrated by augmented Ag-specific IFN-gamma secretion from splenocytes without changing disease incidence. Coinjection of adenoviral vectors expressing IL-4 or IL-12 had no effect on the titers of anti-TSHR Abs determined by ELISA or thyroid-stimulating hormone-binding inhibiting Ig assays, suggesting that Ab quality, not quantity, is responsible for disease induction. Our observations demonstrate the critical role of Th1 immune responses in a murine model of Graves' hyperthyroidism. These data may raise a cautionary note for therapeutic strategies aimed at reversing Th2-mediated autoimmune responses in Graves' disease in humans.

Adenoviridae↗
PubMed2003

A major role for non-major histocompatibility complex genes but not for microorganisms in a novel murine model of Graves' hyperthyroidism.

The etiology of Graves' disease is multifactorial. We investigated the role of genetic and environmental factors on the susceptibility to Graves' hyperthyroidism using a new murine model. Intramuscular injection of recombinant adenovirus expressing the thyrotropin receptor (AdCMVTSHR) induces Graves'-like hyperthyroidism (thyrotropin receptor [TSHR] antibodies, elevated thyroxine, and diffuse goiter) in more than 50% of female BALB/c mice. The relative contributions of major histocompatibility complex (MHC) and non-MHC genes on the susceptibility to hyperthyroidism were studied by immunizing BALB/c (H-2d), BALB.K (H-2k), and DBA/2J (H-2d) mice with AdCMVTSHR. Hyperthyroidism developed in approximately 50% of BALB/c and BALB.K mice but only 5% of DBA/2J mice, indicating a major role for non-MHC genes in disease development. The effect of environmental microorganisms was evaluated by comparing disease incidence in BALB/c mice maintained in pathogen-free conditions versus those in nonsterile, conventional housing, as well as by coadministering microorganism components (Escherichia coli lipopolysaccharide or yeast zymosan A) as adjuvants with AdCMVTSHR. Neither type of exposure to environmental pathogens influenced disease induction. In conclusion, non-MHC genes, but not infectious organisms, play a major role in the etiology of this novel murine model of Graves' disease.

Adenoviridae↗
PubMed2003

Thyrotropin receptor-DNA vaccination of transgenic mice expressing HLA-DR3 or HLA-DQ6b.

Graves' disease in Caucasians is associated with the major histocompatibility (MHC) antigen HLA-DR3. One approach to studying the role of susceptibility genes involves the use of mice that lack murine MHC and instead express human HLA antigens. Although Graves' disease does not arise spontaneously in animals, thyrotropin receptor (TSHR) antibodies can be induced in mice by vaccination with TSHR-DNA in a plasmid. In the present study, we characterized TSHR antibodies and thyroiditis developing in HLA-DR3 transgenic mice vaccinated with TSHR-DNA. As controls, we used mice transgenic for HLA-DQ6b, an MHC antigen rarely associated with Graves' disease. We observed that approximately 30% of DR3-, but none of DQ6b-transgenic mice, developed TSHR antibodies detectable by enzyme-linked immunosorbent assay (ELISA). The cysteine-rich amino terminal peptide was the dominant linear antibody epitope in DR3 mice, as in other strains vaccinated with TSHR-DNA. Sera from some vaccinated DR3 mice were positive on flow cytometry using intact cells expressing the TSHR, demonstrating recognition of the native TSHR on the cell surface. Although none of the these mice had thyroid stimulating antibodies or were hyperthyroid, a few developed lymphocytic infiltration of the thyroid. These data, together with information for other mouse strains, demonstrate that MHC (human and murine) and non-MHC genes contribute to the outcome of TSHR-DNA vaccination and indicate the potential value of DR3 transgenic mice for dissecting immune responses to the TSHR.

Animals↗
PubMed2003

The thyrotropin receptor autoantigen in Graves disease is the culprit as well as the victim.

Graves disease, a common organ-specific autoimmune disease affecting humans, differs from all other autoimmune diseases in being associated with target organ hyperfunction rather than organ damage. Clinical thyrotoxicosis is directly caused by autoantibodies that activate the thyrotropin receptor (TSHR). The etiology of Graves disease is multifactorial, with nongenetic factors playing an important role. Of the latter, there is the intriguing possibility that the molecular structure of the target antigen contributes to the development of thyroid-stimulatory autoantibodies (TSAb's). Among the glycoprotein hormone receptors, only the TSHR undergoes intramolecular cleavage into disulfide-linked subunits with consequent shedding of some of the extracellular, autoantibody-binding A subunits. Functional autoantibodies do not arise to the noncleaving glycoprotein hormone receptors. Recently, TSAb's were found to preferentially recognize shed, rather than attached, A subunits. Here we use a new adenovirus-mediated animal model of Graves disease to show that goiter and hyperthyroidism occur to a much greater extent when the adenovirus expresses the free A subunit as opposed to a genetically modified TSHR that cleaves minimally into subunits. These data show that shed A subunits induce or amplify the immune response leading to hyperthyroidism and provide new insight into the etiology of Graves disease.

Adenoviridae↗
PubMed2003

Targeted restoration of cleavage in a noncleaving thyrotropin receptor demonstrates that cleavage is insufficient to enhance ligand-independent activity.

Two unusual features of the TSH receptor (TSHR) ectodomain are its intramolecular cleavage at the cell surface into disulfide-linked subunits and its constraint of ligand-independent (constitutive) activity inherent to the serpentine region. Whether ectodomain cleavage alters the level of TSHR constitutive activity is an important unanswered question. To address this issue, we used a TSHR engineered so as not to undergo spontaneous cleavage into subunits (deletion of amino acid residues 317-366 and GQE(367-369)NET substitution). Into this noncleaving TSHR (termed TSHR-D1-NET), we introduced thrombin recognition motifs (termed Thr 6 and Thr 18) at the site of spontaneous cleavage. Treatment of intact Chinese hamster ovary cells expressing TSHR-D1-NET-Thr 6 and -Thr 18 with thrombin induced cleavage into A and B subunits, as determined by (125)I-TSH covalent cross-linking. Nevertheless, constitutive activity of the thrombin-cleaved TSHR was unaltered. The level of TSHR constitutive activity was, therefore, fully dissociated from intramolecular cleavage into subunits. Trypsin treatment of the same cells expressing the noncleaving TSHR also generated disulfide-linked A and B subunits but, in contrast to thrombin, enhanced TSHR constitutive activity. Therefore, the activating effect of trypsin appears to involve clipping at an additional, as-yet unidentified, site. In summary, our data demonstrate that TSHR cleavage is, by itself, insufficient to reduce TSHR ectodomain constraint on ligand-independent constitutive activity. These data are consistent with other evidence that A subunit shedding consequent to TSHR cleavage is a critical factor in enhancing TSHR constitutive activity.

Animals↗
PubMed2003

The cysteine-rich amino terminus of the thyrotropin receptor is the immunodominant linear antibody epitope in mice immunized using naked deoxyribonucleic acid or adenovirus vectors.

Experimental Graves' disease is more effectively produced by immunization approaches involving in vivo TSH receptor (TSHR) expression than by conventional immunization with TSHR protein and adjuvant. Unlike conformational epitopes that are extremely difficult to define, linear epitopes can be readily assessed using synthetic peptides. TSHR linear epitopes are well characterized in conventionally immunized animals, but there is no information for animals vaccinated with TSHR DNA in plasmid or adenovirus vectors. We used synthetic peptides to characterize linear epitopes in mice immunized by in vivo expression of TSHR DNA. TSHR adenovirus-injected mice had higher antibody levels than TSHR DNA-vaccinated mice. However, the dominant peptide recognized in both groups was the TSHR cysteine-rich N terminus (residues 22-41). Sera from TSHR adenovirus-immunized (but not TSHR DNA-vaccinated) mice interacted to a lesser extent with peptides encompassing residues 352-401, which include the region deleted following TSHR cleavage as well as the ectodomain juxta-membrane region. Although antibodies characterized using synthetic peptides are probably TSH blockers or nonfunctional, stimulating antibodies may recognize linear components in a conformational epitope. The cysteine-rich TSHR N terminus is functionally important in the action of stimulating TSHR autoantibodies in humans. The immunodominance of the same region in immunized mice suggests that this region may also be immunodominant in humans.

Adenoviridae↗
PubMed2003

Evidence that the C terminus of the A subunit suppresses thyrotropin receptor constitutive activity.

The TSH receptor (TSHR), unlike the LH receptor (LHR), has considerable ligand-independent adenylyl cyclase activity, a feature of pathophysiological importance. The TSHR ectodomain partially suppresses constitutive activity, an effect reversed by trypsin treatment of intact cells. Localizing the functional site of trypsin action would provide insight into how the TSHR ectodomain exerts its constraint. For this purpose, we examined the effect of trypsin on intact cells expressing a series of modified TSHR. Trypsin did not increase cAMP production by a chimeric TSH-LH receptor involving substitution of TSHR residues 261-418 (the ectodomain C terminus). In contrast, with the wild-type TSHR, trypsin enhanced constitutive activity despite mutation of the following potential tryptic cleavage sites [arginine (R) and lysine (K) residues]: 1) K565, K651, K660 in the extracellular loops of the serpentine region; 2) B subunit juxtamembrane residues K371, K401, K415; 3) A subunit residues R310, R312, K313. We previously excluded K337 and K339 from being implicated in TSHR tryptic activation. By exclusion, only one R/K cluster remains as a possible target for the functional effect of trypsin, namely K287, K290, K291, and R293. Mutation of this cluster is incompatible with TSHR cell surface expression. However, tryptic clipping at this locus would reproduce a previously demonstrated structural effect of trypsin on the TSHR, removal of about a 2-kDa polypeptide fragment extending downstream from the locus to the C terminus of the A subunit. Taken together, these data suggest that the C terminus of the A subunit functions as a suppressor of TSHR constitutive activity.

Animals↗
PubMed2003

Thyroglobulin-thyroperoxidase autoantibodies are polyreactive, not bispecific: analysis using human monoclonal autoantibodies.

Autoantibodies (Ab) to thyroglobulin (Tg) and to thyroid peroxidase (TPO) are reported to share common epitopes, and an assay for bispecific TgPOAb has been developed that may distinguish between different clinical presentations of thyroid autoimmunity. We sought to clone TgPOAb from an Ig gene combinatorial library constructed from B cells infiltrating the thyroid of a patient with TgPOAb. As described for isolating serum TgPOAb, we panned the phage display library by alternating from Tg- to TPO-coated ELISA wells. After panning, the library was enriched for TgPO-binding phage. Of 526 clones tested for expressed Ab, most were negative; 3 clones were specific for Tg, and 5 clones specifically recognized TPO. Antibody from a single clone, encoded by a non-Tg, non-TPO Ig heavy chain gene, bound both Tg and TPO (TgPO activity). However, this antibody also bound equally well to nonthyroid antigens. In conclusion, enrichment for Tg- and TPO-binding phage was largely attributable to phage specific for either Tg or TPO. This finding, albeit from a single patient, questions previous observations of serum TgPOAb prepared by affinity chromatography. Combined with the isolation of a polyreactive monoclonal antibody, our data provide powerful evidence against shared, cross-reactive epitopes on 2 major thyroid autoantigens.

Antibodies, Monoclonal↗
PubMed2002

Localization of the thyroid peroxidase autoantibody immunodominant region to a junctional region containing portions of the domains homologous to complement control protein and myeloperoxidase.

Thyroid peroxidase (TPO) autoantibody epitopes are largely restricted to an immunodominant region (IDR) on the extracellular region of the native molecule. Localization of the IDR has been a longstanding and difficult goal. The TPO extracellular region comprises a large myeloperoxidase-like domain, linked to the plasma membrane by two smaller domains with homology to complement control protein (CCP) and epidermal growth factor (EGF), respectively. Recent studies have focused on the CCP- and EGF-like domains as the putative location of the TPO autoantibody IDR. To address this issue, we attempted to express on the surface of transfected cells native TPO in which the CCP- and EGF-like domains were deleted, either together or individually. We used a quartet of human monoclonal autoantibodies that define the TPO IDR, as well as polyclonal TPO autoantibodies in patients' sera, to detect these mutated TPO molecules by flow cytometry. The combined CCP/EGF-like domain deletion did not produce a signal with TPO autoantibodies but did not traffic to the cell surface. In contrast, both monoclonal and polyclonal autoantibodies recognized TPO with the juxtamembrane EGF-like domain deleted equally as well as the wild-type TPO on the cell surface. TPO with the CCP-like domain deleted expressed normally on the cell surface, as determined using the polyclonal mouse antiserum. Nevertheless, this modified TPO molecule was recognized very poorly by both the human monoclonal autoantibodies and the polyclonal autoantibodies in patients' sera. In conclusion, we have clearly excluded the juxtamembrane EGF-like domain as being part of the IDR. In contrast, a component of the CCP-like domain does contribute to the IDR. These data, together with findings from other studies, localize the TPO autoantibody IDR to the junction of the CCP-like domain and the much larger myeloperoxidase-like domain on TPO.

Animals↗