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J Ruf

Publications and source records attributed to J Ruf.

At least 37 records · Page 2Linked to original sources
PubMed1999

Multicenter study on TGPO autoantibody prevalence in various thyroid and non-thyroid diseases; relationships with thyroglobulin and thyroperoxidase autoantibody parameters.

OBJECTIVE: TGPO autoantibodies (aAbs) that bind simultaneously to thyroglobulin (Tg) and thyroperoxidase (TPO) are present in the serum of patients with autoimmune thyroid diseases (AITD) and have been found to differ from monospecific Tg and TPO aAbs. To obtain further insights on the prevalence defined as the rate of occurrence and significance of TGPO aAbs in a large population, we carried out a collaborative study involving 15 European teams. METHODS: Serum samples from 3122 patients with various thyroid and non-thyroid diseases and normal subjects were assayed using a novel TGPO aAb detection kit. This test was designed so that TGPO aAbs are trapped between the Tg-coated solid phase and the soluble TPO labeled with a radioiodinated monoclonal antibody. RESULTS: Only three out of the 220 normal subjects (prevalence of 1.4%) were found to have positive TGPO aAb levels, which were mainly observed in the patients with AITD: the group of patients suffering from Hashimoto's thyroiditis had a TGPO aAb prevalence of 40.5% (n=437 patients), those with Graves' disease, a prevalence of 34.6% (n=645) and those with post-partum thyroiditis, 16.0% (n=243). Among the non-AITD patients with positive TGPO aAb levels, the TGPO aAb prevalence ranged from 20.7% among those with thyroid cancer (n=246) to 0% among those with toxic thyroid nodules (n=47). Among the patients with non-thyroid diseases, the TGPO aAb prevalence ranged from 9.8% in the case of Biermer's pernicious anemia (n=78) to 0% in that of premature ovarian failure (n=44). It is worth noting that the groups showing the highest TGPO aAb prevalence also contained the patients with the highest TGPO aAb titers. Statistical comparisons between the TGPO aAb prevalences in the various groups showed that TGPO aAb could be used as a parameter to distinguish between the groups of Hashimoto's and Graves' patients and between the women with post-partum thyroiditis and the post-partum women with only Tg and/or TPO aAb established during early pregnancy. Unexpectedly, the correlations between TGPO aAbs and Tg and TPO aAbs were found to depend mainly on the assay kit used. CONCLUSION: High TGPO aAb titers are consistently associated with AITD but the reverse was not found to be true. TGPO aAbs are a potentially useful tool, however, for establishing Hashimoto's diagnosis, and would be worth testing in this respect with a view to using them for routine AITD investigations.

Autoantibodies↗
PubMed1998

A conformational B-cell epitope on the C-terminal end of the extracellular part of human thyroid peroxidase.

To investigate the B-cell autoimmune epitopes on human thyroid peroxidase (TPO), we generated proteolytic peptides by enzymatic hydrolysis of TPO in nondenaturing and nonreducing conditions. The hydrolysate was chromatographed on a reverse phase column. We eluted a material immunoreactive with both a TPO monoclonal antibody recognizing a linear epitope (mAb47, amino acid 713-721) and TPO autoantibodies (aAb) from patients. The aAb immunoreactivity, but not that of mAb47, was lost after reduction. Western blots after electrophoresis without reduction showed that the aAb and mAb47 were immunoreactive with a 66-kDa band and that aAb identified a doublet at 20 kDa. For electrophoresis under reducing conditions, the 66-kDa band resolved into two peptides of 40 and 26 kDa, whereas the doublet at 20 kDa remained unchanged. None of these reduced peptides was immunoreactive with aAb, whereas the 40-kDa peptide was immunoreactive with mAb47. The 40-kDa peptide extends from amino acid 549 to 933 of TPO, and its last 192 amino acids overlap the autoimmune 20-kDa peptide. After iodine labeling, the 20-kDa peptide lost its immunoreactivity. We conclude that the C-terminal end of the extracellular part of TPO, which includes all the tyrosine residues of the 20-kDa peptide, contains at least one conformational B-cell epitope involved in autoimmune thyroid diseases.

Amino Acid Sequence↗
PubMed1998

Comparative mapping of cloned human and murine antithyroglobulin antibodies: recognition by human antibodies of an immunodominant region.

Antibodies (Ab) to thyroglobulin (Tg) are common in patients with autoimmune thyroid diseases, but it is currently unclear how Tg Ab are involved in the pathology of autoimmune thyroid disease. We have previously reported the isolation of immunoglobulin G (IgG)kappa and IgGlambda Fab from phage display combinatorial libraries from the cervical lymph node of a single Hashimoto's thyroiditis patient with a high anti-Tg titer. Sequence analysis of these Fab indicated a restricted heavy chain usage with a nonrestricted light chain usage, with Fab inhibiting the binding of patient Tg Ab by between 39% and 79%. Comparative mapping of nine each of these IgGkappa and IgGlambda Fab, and the patient serum from whom the Fab were derived, is described here, using a panel of 10 murine monoclonal antibodies (Mab) to human thyroglobulin (hTg). The Fab interacted principally with mAb defining the overlapping antigenic domains I and IV, previously characterized as the region recognized by the majority of patient serum Tg Ab. Tg Ab from serum of the patient from whom the Fab were derived were also directed at this region, suggesting that the Fab are representative of the Tg Ab present in this patient.

Animals↗
PubMed1996

Idiotypic study of a bispecific thyroglobulin and thyroperoxidase monoclonal antibody.

We have previously established that thyroperoxidase (TPO), a major thyroid antigen involved in autoimmune thyroid diseases, interacts with an idiotype present on human and mouse antibodies directed to thryoglobulin (TG), another thyroid autoantigen. In order to characterize the TPO-reactive idiotype, we selected a TG monoclonal antibody (mAb J7 B49.15) which bound to TPO and cross-reacted with human bispecific TG and TPO autoantibodies (TGPO aAb) for both TG and TPO binding. The TPO-reactive structure of the mAb J7 B49.15 was present on the F(ab')2, located next to the TG binding site and dependent on the association of the heavy and light chains. We found that mAb J7 B49.15 shared a TPO-reactive idiotypic structure with TG mAb from the same cluster of TG reactivity. All the mAb from this cluster were directed to an immunodominant region of TG, recognized by TG aAb. Natural anti-idiotypes from pooled normal human IgG used as a therapeutic intravenous preparation inhibited the TPO binding to mAb J7 B49.15. By homologous immunization in BALB/c mice, mAb J7 B49.15 induced an antiserum with both TG and TPO reactivities. Whereas TG reactivity decreased as early as day 14 post-immunization, TPO reactivity remained at a plateau value from day 21 to day 42. The TG and TPO reactive antisera were shown to inhibit the binding of mAb J7 B49.15 to TG and TPO, respectively. We concluded that mAb J7 B49.15 reacted with TPO through an interspecies idiotype which appeared conformational and related to the epitopic specificity of the mAb. Interestingly, this idiotype would carry the internal image of a TG structure able to induce, in homologous system, a TG antibody response followed by a TPO response without the need of any immunizing antigen.

Animals↗
PubMed1996

Antibody-dependent cell-mediated cytotoxicity in autoimmune thyroid disease: relationship to antithyroperoxidase antibodies.

Thyroid antibody-dependent cell-mediated cytotoxicity (ADCC) has been reported in autoimmune thyroid disease, and its relationship with antithyroperoxidase antibodies (TPOAb) questioned. We studied the effect of highly purified human thyroperoxidase (TPO) on thyroid ADCC activity elicited by serum from patients with autoimmune thyroid disease. ADCC promoted by a pool of Graves' disease sera could be inhibited by the addition of TPO in a dose-dependent manner. TPO at 40 micrograms/mL decreased the ADCC observed in the presence of this serum pool by 50%. In the presence of 40 micrograms/mL TPO, ADCC was significantly reduced (P < 0.0005) from 39.6 +/- 10.6% (mean +/- SD) to 14.0 +/- 12.9% for the 18 Graves' disease sera tested and from 39.1 +/- 10.5% to 6.1 +/- 1.7% for the 16 thyroiditis sera tested. Purified thyroglobulin had no effect. Immunoaffinity-purified TPOAb could mediate ADCC in a dose-dependent manner, whereas purified antithyroglobulin antibodies could not. Three TPOAb-positive, but ADCC-negative, sera appear to contain an activity able to protect thyroid cells from ADCC. This protective effect is also observed on human fibroblasts. In conclusion, TPO is the major antigen involved in thyroid ADCC.

Adult↗
PubMed1996

Majority of thyroid peroxidase autoantibodies in patients with autoimmune thyroid disease are directed to a single TPO domain.

Murine monoclonal antibodies (mAb) produced against native human thyroid peroxidase (TPO) are powerful tools for analyzing the autoantibody (Aab) epitopes on TPO. Binding sites of thirteen mAbs cover all or most antigenic regions on TPO. We determined the competition between Aabs from 75 AITD patients and 13 mAbs in binding to TPO. Autoantibodies recognize predominantly the TPO area close or identical to mAb#9 epitope. All sera tested inhibited this mAb binding by 92.9 +/- 14.8 (mean +/- SD), range from 69-100%. AITD patients' sera with low Aabs titer up to 1/2,000 inhibited mAb#9 binding to TP0 by 85 +/- 11.5% (mean +/- SD) and did not influence remaining mAbs binding to TPO. With elevated Aab levels the inhibition of other mAbs binding was higher, but never exceeded 35%. The amount of Aabs yielding 50% inhibition of mAbs binding was lowest for mAb#9. In order to obtain this degree of inhibition for other mAbs 5 to 25 times more Aabs were needed. Our results demonstrate that the majority of autoantibodies in sera of patients with AITD recognize a single immunodominant region on the TPO mapped by mAb#9. They account for about 80-90% of serum TPO autoantibodies. The autoimmune response to other regions on TPO molecule is directed to several other epitopes, but represents quantitatively a minority of autoantibodies. This response intensifies with increasing Aabs level in the serum.

Adolescent↗
PubMed1995

Identification of the thyroid Na+/I- cotransporter as a potential autoantigen in thyroid autoimmune disease.

The thyroid gland is the target of several autoimmune diseases. Specific thyroid proteins have been identified as autoantigens associated with these diseases (e.g. thyroperoxidase, thyroglobulin and the thyrotrophin (TSH) receptor). In this paper, we report that the serum of a patient suffering from Hashimoto's thyroiditis, autoimmune gastritis and rheumatoid arthritis was able to inhibit the chronic TSH-induced I- uptake of dog thyrocytes in culture, even at a 1:1000-fold dilution, without affecting their 86Rb+ uptake. This blocking activity is rare as 147 sera (from patients positive for antibodies to the thyroid microsomes and the gastric parietal cell antigen, patients with Sjögren's syndrome, patients with a high titre of microsomal antibodies and low or negative for antibodies to thyroperoxidase, and patients with a high titre of microsomal antibodies and frank hypothyroidism) were negative when tested for their ability to inhibit I- uptake. Subsequently we tested 20 murine monoclonal antibodies previously obtained by immunizing mice with a crude human thyroid membrane preparation, which were all negative when tested against thyroglobulin and thyroperoxidase. One of the monoclonal antibodies displayed a 50% inhibition of the chronic TSH-induced 125I- uptake of dog thyrocytes without affecting the 86Rb+ uptake of the cells. Immunoglobulins purified from the ascite fluid by affinity chromatography on a protein A cellulose column had the same characteristics. Taken together, the data suggest that thyroidal 125I- uptake can be inhibited by antibodies, that autoantibodies in the patient's serum are most probably responsible for the observed inhibition and therefore that the Na+/I- cotransporter is probably an autoantigen.

Animals↗
PubMed1994

Induction of thyrotropin receptor (TSH-R) autoantibodies and thyroiditis in mice immunised with the recombinant TSH-R.

Two groups of 10 Balbc by Jico male mice were immunised on days 0, 15 and 35, with the extra cellular domain (ECD) of the human thyrotropin receptor (TSH-R) expressed as a fusion protein in bacteria (group 1) or with the maltose binding protein (MBP) fusion partner alone (group 2). Blood was obtained on days 0, 22, 32, 42 and 49 and samples from the individual animals pooled for each group. Serum and immunoglobulin (IgG) preparations were tested, using CHO cells expressing the human TSH-R (JP26 and JP09) for thyroid stimulating (TSAb); thyroid blocking (TBAb) and thyrotropin binding inhibiting (TBII) activities. Neither serum nor IgGs were found to contain TSAb at any time point. TBII activity was present in the serum of both groups on day 32 and in group 1 only on day 49; when the test was performed on IgGs, only the MBP-ECD day 49 preparation remained significantly positive for TBII (p < 0.005). Significant TBAb activity was present in both the serum and IgG of group 1 day 49 (p < 0.005) and to a lesser extent on 42 (p < 0.02). Following the second immunisation (day 15) both groups and had decreased circulating T4 levels (p < 0.05) when compared with day 0 in each case. Group 2 were unaffected by the third immunisation on day 35 but the MBP-ECD group again had significantly decreased T4 levels (p < 0.02) compared with MBP day 49 and (p < 0.03) when compared with MBP-ECD day 0. Histological examination of thyroids from group 1 animals revealed extensive vascularisation and an atypical lymphoblastoid infiltration which was not observed in control mice. These preliminary results indicate that care is required in interpreting data since a non-receptor antigen was shown to decrease circulating thyroxine and serum from these animals had apparent TBII like activity. However, the results obtained with the IgGs suggest that receptor autoantibodies can be induced by immunising with the human TSH-R, in addition, the immunised mice show histological evidence for the development of thyroiditis.

Animals↗
PubMed1994

Bispecific thyroglobulin and thyroperoxidase autoantibodies in patients with various thyroid and autoimmune diseases.

Recent evidence indicates that thyroid autoimmune disorders are associated with the presence of circulating autoantibodies (aAb) with dual specificity for thyroglobulin (TG) and thyroperoxidase (TPO). The question of whether these aAb, called TGPO aAb, are of clinical relevance compared to TG and TPO aAb remains to be determined. The availability of purified preparations of human TG and TPO allowed the development of a specific and sensitive RIA for TGPO aAb in serum. In the present study, we compared levels of aAb that cross-react with both TG and TPO (TGPO aAb) and total TG and TPO aAb levels, respectively, in sera from 84 normal controls and 226 patients with various thyroid and autoimmune diseases, including nontoxic goiter (n = 50), toxic nodular goiter (n = 13), thyroid carcinoma (n = 20), primary idiopathic myxedema (n = 15), postpartum thyroiditis (n = 11), Hashimoto's thyroiditis (n = 38), pernicious anemia (n = 27), rheumatoid arthritis (n = 19), and insulin-dependent diabetes mellitus (n = 33). In addition, 16 patients with Hashimoto's thyroiditis were studied before therapy and after more than 3 months of treatment with L-T4. It was shown that TGPO aAb were generally, but not always, present in the serum of patients with Hashimoto's thyroiditis, which also contained TG and TPO aAb. In contrast, TGPO aAb were undetectable in normal controls (excepting a few cases reaching borderline levels) as well as in sera from the majority of the other patients tested. Selecting sera positive for TGPO and either TG or TPO aAb, a statistically significant correlation was found between TGPO and TG (n = 26; P < 0.005), but not TPO aAb. Interestingly, the TGPO aAb level significantly decreased in patients with Hashimoto's thyroiditis after hormonal therapy (P < 0.05), some of them shifting from TGPO aAb positive before treatment to negative after treatment. In conclusion, TGPO aAb determination distinguishes Hashimoto's patients from patients with either thyroid and/or autoimmune diseases. The specific presence of TGPO aAb in a subset of Hashimoto's patients and their variation during T4 therapy remain to be understood. This could give a clue to mechanisms of autoimmune thyroid disease.

Adolescent↗
PubMed1994

Overexpression of the extracellular domain of the thyrotrophin receptor in bacteria; production of thyrotrophin-binding inhibiting immunoglobulins.

The availability of high affinity antibodies to the human TSH receptor (TSHR) would help in defining its functional domains, but this requires the production of pure receptor as immunogen. We have expressed the extracellular domain (ECD) of the TSHR (residues 21-414) as a fusion protein with maltose-binding protein (MBP) in Escherichia coli, using the pMAL-cR1 vector. The major protein in an electrophoretically separated, crude bacterial lysate had a molecular mass of 89 kDa, in agreement with the size predicted for the MBP-ECD fusion product. Its identity was confirmed by Western blotting in which it was recognized by two polyclonal antibodies to synthetic peptides of the TSHR and an anti-MBP. Following purification on an amylose column, 15 mg pure MBP-ECD per litre of culture were produced, which was 5% of the total bacterial protein. Following extensive dialysis in a buffer which produces slight denaturation, MBP-ECD was cleaved with factor Xa. The identity of each protein was confirmed by Western blotting. To investigate the possibility of using the fusion protein as an immunogen we produced rabbit polyclonal antibodies to the ECD which were able to produce immunofluorescent staining of Chinese hamster ovary cells that expressed the TSHR, and revealed a protein of 95 kDa in Western blots of the same cells, in addition to a protein of 55 kDa. Only the protein of 55 kDa was detected in Western blots of human thyroid membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP-Binding Cassette Transporters↗
PubMed1994

Autoantibodies and monoclonal antibodies directed to an immunodominant antigenic region of thyroglobulin interact with thyroperoxidase through an interspecies idiotype.

We investigated whether thyroglobulin (TG) autoantibodies (aAb) cross-react with thyroperoxidase (TPO) through an idiotypic structure using pooled normal human IgG (NhlgG) as a natural anti-idiotype reagent. Affinity-purified TG aAb from pooled IgG of patients with autoimmune thyroid disease were chromatographed on Sepharose-bound NhlgG. About one fourth of the loaded material bound to and eluted from the coupled gel. Eluted TG aAb were found reactive to TG and TPO and their TPO but not TG binding was strongly inhibited by molar excess of NhlgG. These TG aAb appeared to be mainly directed to an immunodominant TG antigenic region defined by TG monoclonal antibodies (mAb) from a single cluster of reactivity. These TG mAb were also found to recognize TPO and their binding to TPO but not TG was inhibited by molar excess of NhlgG as already observed with TG aAb. Taken together, these results indicated that TPO interacts with an idiotype present on human TG aAb and mouse TG mAb displaying a similar epitopic specificity; this interspecies idiotype is recognized by anti-idiotype antibodies present in NhlgG. Our results suggest that thyroid autoimmunity can be envisaged, at least in part, as a disturbance in interconnected idiotypic networks.

Adult↗
PubMed1994

Microparticle-enhanced nephelometric immunoassay of anti-thyroid peroxidase autoantibodies in thyroid disorders.

Crude thyroid peroxidase extracted from human thyroid microsomes was covalently bound onto polyacrylic and polyfunctional copolymerized microparticles. We observed agglutination of the thyroid peroxidase-microparticle conjugate with 13 monoclonal antibodies (mAbs) specific for epitopes on four different antigenic domains of human thyroid peroxidase (TPO; EC 1.11.1.7), after addition of anti-mouse immunoglobulins. We quantified agglutination by measuring with a specially designed nephelometer the light scattered by the conjugates. This allowed us to develop a microparticle-enhanced nephelometric immunoassay for human anti-TPO autoantibodies (aAbs) with defined epitopic specificity, based on the ability of aAbs to inhibit mAb-induced agglutination. Applied to patients with autoimmune thyroid diseases, this assay confirmed the polyclonality of anti-TPO aAbs and their preferential reactivity toward epitopes located on the A and B antigenic domains of the TPO molecule. The same specificities seem to be present in patients with Hashimoto thyroiditis or Graves disease.

Autoantibodies↗
PubMed1993

Thyroglobulin in medullary thyroid carcinoma: immunohistochemical study with polyclonal and monoclonal antibodies.

The presence of thyroglobulin (TG) was investigated by immunohistochemistry with one polyclonal and three distinct monoclonal anti-TG antibodies in 19 primary and 15 metastatic medullary thyroid carcinoma (MTC) cases. In two primary tumors possessing a mixed trabecular and follicular growth pattern, a significant number of tumor cells were positive with the four antibodies in all parts of the tumor; these tumors were considered to be genuine mixed follicular and parafollicular tumors. The 17 other primary tumors looked like "classical" (ie, predominantly compact) MTC. Eight contained tubular or microfollicle-like structures that were consistently TG-negative. Eight contained residual entrapped normal follicles positive with the four anti-TG antibodies. In six cases isolated foci of tumor cells reacted with polyclonal antibody or monoclonal antibody 11. In 10 cases circulating TG stained positively with polyclonal antibody in vascular channels, five cases stained positively for TG with monoclonal antibody 11, four cases stained positively for TG with monoclonal antibody 7, and three cases stained positively for TG with monoclonal antibody 1. In metastases TG was found in tumor cells in only one instance, but positive reactions were obtained in vascular channels and macrophages in several cases. We conclude from this study that there is no genuine secretion of TG in classical, compact MTC, which differs morphologically and functionally from mixed follicular and parafollicular tumors secreting both TG and thyrocalcitonin.

Antibodies, Monoclonal↗
PubMed1993

Significance of thyroglobulin antibodies cross-reactive with thyroperoxidase (TGPO antibodies) in individual patients and immunized mice.

Thyroglobulin (TG) and thyroperoxidase (TPO), both involved in thyroid hormone synthesis, represent major autoantigens in thyroid autoimmune disease. Despite numerous studies, the emergence, pathophysiological significance and role of autoantibodies to TG and TPO remain elusive. The recent identification of a new category of thyroid-specific autoantibody interacting with both TG and TPO (TGPO autoantibodies) offers a new opportunity in the study of thyroid autoimmunity. To gain a better insight into the significance of these TGPO autoantibodies, measurement in individual samples appeared necessary. The unique property of TGPO autoantibodies, simultaneous binding to TG and TPO, was used to set up a sandwich method which combined coated TG and radio-iodinated TPO. This method was found to be strictly specific for TGPO autoantibodies and sensitive enough to assay TGPO autoantibodies in serum. In humans, TGPO autoantibodies were found in most of the sera with high TG and TPO autoantibody titres, but not in sera negative for TG autoantibodies, whatever the TPO autoantibody titre. Furthermore, high TGPO autoantibody titres were found in sera strongly cytotoxic for cultured porcine thyroid cells. However, significant correlation of TGPO autoantibody titre was observed neither with TG and TPO autoantibody titres (n = 48) nor with complement-dependent cytotoxicity (n = 50). TGPO antibody assay was also performed in individual plasma of CBA/J mice immunized with either human TG (n = 6) or human TPO (n = 6). Immunization with TG induced high levels of not only TG but also TGPO antibodies, which exhibited a strong reactivity for TPO and whose binding to TG and TPO was fully inhibited by TG. In contrast, immunization with TPO induced high levels of only specific TPO antibodies accompanied by low levels of specific TG antibodies. In this case TGPO antibodies were not detected. Of note, TG- and TPO-immunized mice mounted an immune response against their own TG, but did not exhibit histological signs of thyroiditis. Large panels of TG and TPO MoAbs were also investigated with this method: 18/25 TG MoAbs and only 1/13 TPO MoAbs were found cross-reactive. Taken together, these data provide evidence that TGPO antibodies are effectively present in individual patients and TG-immunized mice, are different from specific TG and TPO antibodies, and may derive from natural B cell repertoire by autoimmune processes involving TG and not TPO.

Animals↗
PubMed1992

Sequence of the complete cDNA and the 5' structure of the human sucrase-isomaltase gene. Possible homology with a yeast glucoamylase.

The complete sequence of the 6 kb cDNA and the 5' genomic structure are reported for the gene coding for the human intestinal brush border hydrolase sucrase-isomaltase. The human sucrase-isomaltase cDNA shows a high level of identity (83%) with that of the rabbit enzyme, indicating that the protein shares the same structural domains in both species. In addition to the previously reported homology with lysosomal alpha-glucosidase, the sucrase and isomaltase subunits also appear to be homologous to a yeast glucoamylase. A 14 kb human genomic clone has been isolated which includes the first three exons and the first two introns of the gene, as well as 9.5 kb 5' to the major start site of transcription. The first exon comprises 62 bp of untranslated sequence and the second starts exactly at the initiation ATG codon. Typical CAAT and TATA boxes are seen upstream of the first exon. A genetic polymorphism is described which involves a PstI site in the second intron. Southern blotting, sequencing and mRNA studies indicate that the structures of the sucrase-isomaltase gene and its mRNA are unaltered in the two human colon cancer cell lines Caco-2 and HT-29 in comparison with normal human small intestine.

Amino Acid Sequence↗
PubMed1992

Tyrosine iodination and iodotyrosyl coupling of the N-terminal thyroid hormone forming site of human thyroglobulin modulate its binding to auto- and monoclonal antibodies.

The present work was aimed at studying the interaction of autoantibodies (aAb) and monoclonal antibodies (mAb) with the N-terminal thyroid hormone forming site of human thyroglobulin (TG). Obtained by CNBr treatment of TG, the peptide (22 kDa) containing the complete major hormonogenic site of human TG was purified in three forms according to the degree of iodination and iodotyrosine coupling: the native, poorly iodinated form (n-22K), the iodinated form containing iodotyrosine but not hormone residues (i-22K) and the form containing thyroid hormone (t-22K). We report that aAb from some patients with autoimmune thyroid diseases showed significant binding to both iodinated 22 kDa forms. Furthermore, a detailed study using mAb evidenced that iodination and coupling induced changes in the antigenicity of the molecule, some occurring without direct implication of iodine or thyroid hormones. The 22 kDa peptide appears as an interesting model to study the antigenic changes induced by the structural modifications in the course of thyroid hormone synthesis. This observation could be relevant to the etiopathogenic process of thyroid autoimmune diseases.

Antibodies, Monoclonal↗
PubMed1992

Effects of deglycosylation of human thyroperoxidase on its enzymatic activity and immunoreactivity.

Thyroid peroxidase (TPO) is a glycoprotein enzyme which catalyses the iodination of thyroglobulin and the coupling of iodinated tyrosines. Human TPO (hTPO) is the microsomal antigen recognized by the autoantibodies in the serum of patients with autoimmune thyroid disease. An active detergent-solubilized immunoaffinity-purified hTPO was deglycosylated, either by peptide N-glycosidase F (PNGase F) or by endo-beta-N-acetylglucosaminidase H (endo H), and the enzymatic activity and immunoreactivity of the native and deglycosylated forms were compared. Electrophoretic controls and affinoblotting with concanavalin A showed that deglycosylation was not total and that it was more pronounced with endo H than with PNGase F. The enzymatic activity of hTPO was inhibited by endo H deglycosylation, but not by PNGase F deglycosylation; this inhibition was not due to aggregation and/or insolubilization of the molecule subsequent to deglycosylation. Immunoreactivity was monitored by enzyme-linked immunosorbent assay (ELISA) with 13 mouse monoclonal antibodies, rabbit polyclonal antibodies and antibodies from serum of patients with Hashimoto's thyroiditis. In contrast with enzymatic activity, immunoreactivity was not modified or was slightly enhanced (with four monoclonal antibodies) by deglycosylation. The results indicate that strong, if not total, deglycosylation induces a modification of the tertiary structure of hTPO, which affects the enzymatic site but does not modify markedly the epitopes implicated in the recognition of the molecule by the antibodies tested.

Amidohydrolases↗