The human anti-thyroid peroxidase autoantibody repertoire in Graves' and Hashimoto's autoimmune thyroid diseases

Human anti-thyroid peroxidase (TPO) autoantibodies (aAb) are generated during autoimmune thyroid diseases (AITD). Within recent years, increasing knowledge of the TPO-specific aAb repertoire, gained mainly by the use of combinatorial library methodology, has led to the cloning and sequencing of around 180 human anti-TPO aAb. Analysis of the immunoglobulin (Ig) variable (V) genes encoding the TPO aAb in the ImMunoGeneTics database (IMGT) (http://imgt.cines.fr) reveals major features of the TPO-directed aAb repertoire during AITD. Heavy chain VH domains of TPO-specific aAb from Graves' disease patients preferentially use D proximal IGHV1 genes, whereas those from Hashimoto's thyroiditis are characterized more frequently by IGHV3 genes, mainly located in the middle of the IGH locus. A large proportion of the anti-TPO heavy chain VH domains is obtained following a VDJ recombination process that uses inverted D genes. J distal IGKV1 and IGLV1 genes are predominantly used in TPO aAb. In contrast to the numerous somatic hypermutations in the TPO-specific heavy chains, there is only limited amino acid replacement in most of the TPO-specific light chains, particularly in those encoded by J proximal IGLV or IGKV genes, suggesting that a defect in receptor editing can occur during aAb generation in AITD. Among the predominant IGHV1 or IGKV1 TPO aAb, conserved somatic mutations are the hallmark of the TPO aAb repertoire. The aim of this review is to provide new insights into aAb generation against TPO, a major autoantigen involved in AITD.     

Human anti-thyroid peroxidase single-chain fragment variable of Ig isolated from a combinatorial library assembled in-cell: insights into the in vivo situation

In an attempt to explore the natural variable heavy and light chain (VH/VL) pairing of autoantibodies involved in Graves' disease, we constructed a phage-displayed Ab library obtained by in-cell PCR of thyroid-infiltrating cells. We report here the molecular cloning and characterization of human single-chain fragment variable regions (scFv) specific for thyroid peroxidase (TPO) generated from this library. On the basis of the nucleotide sequences, three different scFvs were obtained (ICA1, ICB7, and ICA5). All were encoded by genes derived from the VH1 and Vlambda1 gene families. Using BIACORE for epitope mapping and kinetic analysis, we showed that these scFvs exhibited high affinity (Kd = 1 nM) for TPO and recognized three different epitopes. The biological relevance of these scFvs as compared with serum anti-TPO autoantibodies was assessed by competition studies. Sera from all the 29 Graves' disease patients tested were able to strongly inhibit (60-100%) the binding of the 3 scFvs to TPO. These data demonstrate that the in-cell PCR library generated human anti-TPO scFvs that retained the VH/VL pairing found in vivo and that the different epitope specificities defined by these scFvs overlapped with those found in the sera of patients with autoimmune thyroid disease.     

High-level production and secretion of a mouse-human chimeric Fab fragment with specificity to human carcino embryonic antigen in Escherichia coli

A high-level secretion system for the production of mouse-human chimeric antibody 21B2 (MHC 21B2) Fab fragment specific for human carcino embryonic antigen (hCEA) in Escherichia coli has been constructed. The genes encoding a light chain and an Fd fragment (a variable region and the CH₁ domain of a heavy chain) of a mouse-human chimeric antibody were directly fused to the signal peptide of the E. coli ompF gene sequence. E. coli cells containing expression vectors in which each of the two genes are located downstream of a separate tac promoter were able to secrete the light chain and Fd fragment as two of their major cellular proteins. The signal peptides were efficiently removed from the primary products by post-translational processing, although they formed insoluble aggregates, possibly in the periplasm. In high-cell-density culture experiments using a jar fermentor, the amount of light chain and Fd fragment produced was at levels of up to 2.88 g/l and 1.28 g/l culture, respectively. By optimizing the conditions that encourage correct folding, formation of disulphide bonds, and association of the light chain with the Fd fragment, we have established a procedure that can purify, refold, and combine aggregated products to electrophoretically homogeneous Fab fragment with a yield of approximately 47%. Fab fragment produced in this manner shows essentially the same antigen-binding activity and specificity to hCEA as the parental mouse antibody 21B2 (MoAb 21B2). Correspondence to: T. Shibui     

Search for the autoantibody immunodominant region on thyroid peroxidase: epitopic footprinting with a human monoclonal autoantibody locates a facet on the native antigen containing a highly conformational epitope

Autoantibodies to thyroid peroxidase (TPO) are the hallmark of the humoral autoimmune response in human autoimmune thyroiditis (Hashimoto's thyroiditis). The majority of TPO autoantibodies in individual patients' sera interact with a restricted immunodominant region on TPO. Although this region can be mapped, previous studies have failed to localize its position on the TPO molecule. We, therefore, used a footprinting approach that can localize a highly conformational, discontinuous epitope on a very large molecule. Extensive biotinylation ( approximately 15 biotins/molecule protein) of lysine residues on the surface of purified, native TPO resulted in loss of multiple tryptic cleavage sites, as determined by analysis of tryptic polypeptide fragments on reverse-phase HPLC. TPO was then complexed with a monoclonal human autoantibody Fab (TR1.9) before biotinylation. After dissociation from TR1.9, TPO was recovered by gel filtration. A trypsin site, previously observed to be lost after TPO biotinylation, was restored when biotinylation was performed on the TPO-TR1.9 complex. The epitope-protected lysine (K) was present in a 30-aa TPO fragment that, by N-terminal sequencing, was found to be K713. Altered recognition by TR1.9 of a TPO-myeloperoxidase chimeric molecule involving this region supported the epitope protection data. In conclusion, we provide the first identification of an amino acid residue (K713) comprising part of an epitope within the TPO immunodominant region. This focal residue localizes the facet on the large, highly complex TPO molecule that contains the immunodominant region and provides the basis for rational guided mutagenesis studies to more fully characterize this region.     

抗破伤风类毒素单抗可变区基因的克隆及在大肠杆菌中表达的三种工程抗体

The heavy and light chain variable region genes of anti-tetanus toxoid (TT) antibody and the heavy chain Fd genes were amplified and cloned through RT-PCR from mouse hybridoma cells. The sequences of VH and VK were determined. Fd gene fragments were expressed in E. coli. The ELISA results indicated that the expressed Fd showed antigen binding activity but was nonspecific. Furthermore, through SOE and PCR techniques, the VH and VK gene fragments together with ScFv linker were assembled into single chain antibody (ScFv) gene fragment. While together with human heavy chain CH 1 gene fragment and Fab linker, they were assembled into chimeric Fab gene fragment. The two assembled gene fragments were separately inserted into phagemid pHEN 1, which was a fd-based vector containing gene 3 encoding the minor coat protein. In presence of helper phage M 13-VCS the anti-TT phage-ScFv or phage-Fab were displayed on the surface of phage particles respectively. Results from phage-ELISA indicated that both phage antibodies were TT-specific.     

抗破伤风类毒素单抗可变区基因的克隆及在大肠杆菌中表达的三种工程抗体

我们采用RT-PCR,从小鼠杂交瘤细胞中扩增并克隆了抗破伤风类毒素(TT)抗体轻、重链可变区,重链Fd区基因,测定了其VH、Vk序列。并在大肠杆菌中表达了Fd片段,ELISA分析的结果表明Fd片段具有抗原结合的能力,但特异性很差。进一步采用SOE,和PCR技术,将VH、VK基因与ScFv连接片段组装成单链抗体(ScFv)基因片段,以及将人重链CH1和Fab基因连接片段组装成Fab基因片段。将它们分别插入含噬菌体fd外壳蛋白3基因的phagem-id pHEN 1中,在辅助噬菌体M 13-VCS作用下,噬菌体表面表达了抗TT的噬菌体单链抗体(phage-ScFv)与噬菌体Fab(phage-Fab),经ELISA检测,表明它们都能与TT特异结合。     

Autoantibodies to human thyroid peroxidase in immunoassay and immunoaffinity chromatography

A biochemical system of radioimmunoassay of human thyroid peroxidase (TPO) with the use of specific autoantibodies was developed for the first time. This system includes lyophilized preparations of human autoantibodies to TPO, radioiodinated TPO, standards prepared from pure TPO, and the solid-phase protein A as a precipitating agent. An analytical system was used to study the TPO distribution in fractions of thyroid tissue homogenate. Differences in TPO concentrations in mitochondria, microsomes, and supernatant fluid depending on thyroid pathology and tissue storage conditions were found. The behavior of immobilized anti-TPO-autoantibodies in immunoaffinity chromatography of this microsomal antigen was studied.     

Characteristics of immunoaffinity chromatography and a new method for isolation of human thyroid peroxidase from subcellular fractions of thyroid gland

A system for quantitative determinations of human thyroid peroxidase (TPO) in biological fluids has been obtained, based on the use of enzyme-linked immunosorbent assay. The immunochemical properties of TPO were studied under variable conditions and a new method for isolating the protein from the microsomes, mitochondria, and cytosol of thyroid glands of patients with diverse thyroid diseases was developed. The procedure involves solubilization of subcellular fractions with detergents, their sonication, two sequential runs of chromatography (on sorbents with immobilized monoclonal antibodies against TPO and goat anti-human immunoglobulin antibodies), treatment with ribonuclease, and dialysis. Highly purified preparations of intact TPO and a product of its limited trypsinolysis are expected to be used as research tools and components of high-sensitivity immunoassays.     

Characteristics of immunoaffinity chromatography and a new method for isolation of human thyroid peroxidase from subcellular fractions of thyroid gland

A system for quantitative determinations of human thyroid peroxidase (TPO) in biological fluids has been obtained, based on the use of enzyme-linked immunosorbent assay. Immunochemical properties of TPO were studied under variable conditions, and a new method for isolating the protein from microsomes, mitochondria, and cytosol of thyroid glands of patients with diverse thyroid diseases was developed. The procedure involves solubilization of subcellular fractions with detergents, their sonication, two sequential runs of chromatography (on sorbents with immolbilized monoclonal antibodies against TPO and goat anti-human immunoglobulin antibodies), treatment with ribonuclease, and dialysis. Highly purified preparations of intact TPO and a product of its limited trypsinolysis are expected to be used as research tools and components of high-sensitivity immunoassays.     

Mapping of a linear autoantigenic epitope within the human thyroid peroxidase using recombinant DNA techniques.

Autoantibodies directed against the thyroid peroxidase (TPO), the thyroid microsomal antigen, are widely used to diagnose human autoimmune thyroid disease. A cloned 3.088 kb cDNA coding for the entire mature human TPO was isolated from a cDNA library derived from a pathological thyroid gland of a Graves' disease patient and used further to generate a so-called TPO epitope cDNA library in order to map linear autoantigenic epitopes involving a recombinant molecular biology approach. The TPO epitope cDNA library consisting of randomly fragmented cDNA sequences inserted in the expression vector pGEX-2T was expressed in Escherichia coli and screened with characterized anti-TPO autoantisera from Hashimoto's disease patients. All the sera were positively tested with a purified thyroid microsomal antigen fraction (TMA/TPO). Only about 1% of examined autoantisera were able to recognize bacterial expressed recombinant TPO representing sequential antigenic determinants. A corresponding autoantigenic epitope with 61 amino acids in length was located at the C-terminus of human TPO.     

Light chain determines the binding property of human anti-dsDNA IgG autoantibodies

We have previously prepared human anti-double-stranded (ds) DNA IgG Fab clones using phage-display technology. Nucleotide sequence analysis of genes of immunoglobulin (Ig) heavy and light chain variable regions in these Fab clones suggested that the DNA-binding activity of the clones depended on light chain usage. To confirm the role of the light chain in antibody binding to DNA, we constructed in the present study's new recombined Fab clones by heavy and light chain shuffling between the original anti-dsDNA Fab clones. Clones constructed by pairing Fdgamma fragments with the light chain from a high DNA-binding clone showed high DNA-binding activities, whereas other constructed clones using light chains from low DNA-binding clones showed low DNA-binding activities. Our results indicate that light chains in anti-dsDNA antibodies can determine the DNA-binding activity of the antibodies. Ig chain shuffling of phage-display antibodies may be useful for investigating the molecular mechanisms for antigen-antibody binding of human autoantibodies.     

Thyrotropin stimulation of cultured thyroid cells increases steady state levels of the messenger ribonucleic acid for thyroid peroxidase

We have examined the effect of TSH on thyroid peroxidase (TPO) mRNA levels in dog thyroid cell primary cultures. Freshly dispersed dog thyroid cells were cultured for up to 5 days in the absence or presence of 5 mU/ml bovine TSH. At the outset of culture, and at daily intervals thereafter, total cytoplasmic RNA was extracted and applied to Nytran paper using a slot-blot apparatus. A nick-translated cDNA fragment of the porcine TPO gene was used to probe these filters. Autoradiographs were quantified by densitometry. Nonspecific binding was negligible as determined using a pUC18 probe. During the first 2 days of culture, TPO mRNA levels declined irrespective of whether or not TSH was present in the medium. TSH did not affect this decline. Between 3 and 5 days of culture, TPO mRNA levels in control (no TSH) cells increased to 3 times the initial level (expressed relative to cellular DNA). However, during the same period TSH stimulated TPO mRNA levels 8-fold above the initial level. To confirm that the signal with the cDNA probe was actually that of dog TPO mRNA, cellular RNA (day 4 of culture) was subjected to Northern blot analysis using the same cDNA probe. Specific bands of 2.9 kilobases were detected corresponding to the known size of TPO mRNA in pig thyroid tissue. The signal of this 2.9 kilobase species was enhanced by TSH. In conclusion, the data indicate that chronic TSH stimulation raises steady state levels of TPO mRNA and provide an explanation, at least in part, for the mechanism by which TSH enhances TPO bioactivity in thyroid tissue.     

Distinct immunological and biochemical properties of thyroid peroxidase purified from human thyroid glands and recombinant protein produced in insect cells.

The biosynthesis of thyroid hormone from thyroglobulin is catalysed by thyroid peroxidase (TPO), an integral membrane protein. TPO is also a major autoantigen in autoimmune thyroid disease and autoantibodies to TPO are markers for disease activity. Large quantities of purified TPO are essential for elucidating its structure and understanding its role in disease activity. We describe the high yield purification of full-length recombinant human TPO from baculovirus infected insect cells and compare it to purified native TPO from human thyroid glands. In contrast to native human TPO, the human TPO produced in insect cells as a recombinant protein was insoluble and resistant to solubilisation in detergents. Reversible substitution of lysine residues with citraconic anhydride led to increased solubility of the recombinant TPO, allowing high-yield purification by monoclonal antibody chromatography. The purified enzyme preparation was shown to be TPO by its reactivity with monoclonal and polyclonal antibodies by enzyme linked immunosorbent assay and Western blotting. Both the human and recombinant purified TPO preparations also react with sera from patients with autoimmune thyroid disease, although the binding of conformational dependent autoantibodies was considerably lower to the recombinant TPO than to the native TPO. This suggests that the recombinant TPO may differ in some aspects of its tertiary structure. The purified recombinant TPO was devoid of enzyme activity, in contrast to the enzymatically active, purified human TPO preparations. Both preparations contained comparable amounts of haem (R(z)=0.269), but a shift in the Soret band of recombinant TPO (402 nm) from that of natural TPO (409 nm) indicates that the lack of enzymatic activity of the recombinant enzyme may be due to changes in the protein backbone surrounding the haem. Both the purified native and recombinant TPO, under non-denaturing conditions, show evidence of high molecular mass oligomers, although the latter preparation is prone to a greater degree of aggregation. In conclusion, our studies indicate that recombinant TPO generated in insect cells is conformationally distinct from the native TPO, is insoluble and enzymatically inactive, consistent with the difficulties associated with its purification and crystallisation.     

Purification of the human thyroid peroxidase and its identification as the microsomal antigen involved in autoimmune thyroid diseases

Human thyroid peroxidase (TPO) has been purified from thyroid microsomes by immunoaffinity chromatography using a monoclonal antibody (mAb) to TPO. The eluted material had a specific activity of 381 U/mg and exhibited a peak in the Soret region. The ratio of A411 to A280 ranged from 0.20 to 0.25. Upon SDS-polyacrylamide gel electrophoresis, the purified enzyme gave two contiguous bands in the 100 kDa region. Further, it has been demonstrated that sera with anti-microsomal autoantibodies from patients presenting Graves' or Hashimoto's thyroiditis diseases were able to bind to purified TPO and to inhibit in a dose-dependent manner the mAb binding to purified TPO. This suggests that TPO is the thyroid antigen termed to date the microsomal antigen.