The Molecular Biology of Thyroid Peroxidase: Cloning,Expression and Role as Autoantigen in AutoimmuneThyroid Disease*SANDRA M. McLACHLAN AND BASIL RAPOPORTThyroid Molecular Biology Unit, Veterans Administration Medical Center, San Francisco, and University ofCalifornia, San Francisco, California 94121
I. IntroductionII. Molecular Cloning of TPOIII. Abnormal and Variant Expression of the TPO GeneIV. Regulation of TPO Gene Expression in Thyroid CellsV. Expression of Recombinant hTPOVI. Molecular Studies on TPO as an Autoantigen in Autoim-
mune Thyroid DiseaseA. IntroductionB. Assays for TPO autoantibodiesC. Epitopes on hTPO recognized by B cells
1. Intact TPO2. TPO tryptic fragments3. Recombinant hTPO fragments4. hTPO cDNA random fragment libraries5. Perspective
D. Human monoclonal TPO autoantibodiesE. Definition of T cell epitopes on hTPOF. Inheritance of the ability to produce TPO autoanti-
bodiesVII. Conclusions
I. Introduction
THYROID peroxidase (TPO) is the primary enzymeinvolved in thyroid hormone synthesis, catalyzing
iodide oxidation, iodination of tyrosine residues, andcoupling of iodotyrosines to generate the iodothyroninesT3 and T4 (1). It is a membrane-bound glycoprotein witha heme prosthetic group. In addition to the pivotal phys-iological role of TPO, there was strong immunologicalevidence, even before its molecular cloning, that thisenzyme was the elusive "thyroid microsomal antigen" inautoimmune thyroid disease (discussed below).
The molecular cloning of the complementary DNA fora protein provides powerful new opportunities for study-ing protein structure and function at an unprecedented
Address requests for reprints to: Sandra M. McLachlan, Ph.D., andBasil Rapoport, M.B., Veterans Administration Medical Center, Thy-roid Molecular Biology Unit (HIT), 4150 Clement Street, San Fran-cisco, California 94121.
* This work has been supported by NIH Grants DK-36182 and EY-09498.
level of detail . In t he pas t decade, th ree of the four majorthyroid-specific proteins have been cloned, namely thy-roglobulin (TG) (2-4), TPO (see below), and the TSHreceptor (5-8). Molecular cloning of the thyroid iodidetransporter may occur soon (9). As might be expected,these advances have been followed by an explosion ofinformation regarding fundamental aspects of thyroidpathophysiological function. The goal of this review is tosummarize and interpret new information on TPO thathas accumulated since its molecular cloning.
II. Molecular Cloning of TPO
In 1986, the isolation and characterization by nucleo-tide sequencing of a cDNA clone [1.3 kilobases (kb)],representing part of the full-length pig TPO cDNA (3.1kb), was reported (10). This cDNA clone was obtainedby screening a pig thyroid cDNA library with oligonucle-otide probes based on the amino acid sequence of anumber of tryptic fragments (10) of highly purified pigTPO (11). During the following year, the complete nu-cleotide and derived amino acid sequences of pig TPO(926 amino acid residues) were reported (12). Also in1987, three groups independently reported the nucleotidesequence for human TPO (hTPO) cDNA, which codesfor a protein of 933 amino acids (Fig. 1)(13—15). Infor-mation on the cDNA sequence of rat TPO in FRTL5thyroid cells has subsequently become available (16,17).
The TPO protein has a hydrophobic signal peptide atits amino terminus and a hydrophobic region with thecharacteristics of a transmembrane domain near thecarboxyl terminus. These data are consistent with TPObeing a membrane-associated protein. Five potential gly-cosylation sites are present in the extracellular region(Fig. 1). There is evidence for at least one disulfide bondin the extracellular region creating a closed loop in thisregion of the protein (18,19). When compared with otherperoxidases of known structure, Kimura and Ikedo-Saito(20) have suggested that histidine 407 or, less likely,
192
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FlG. 1. Amino acid sequence of hTPO.The underlined amino acids representthe five potential N-linked glycosylationsites, as determined by the motif N-X-S/T (where x represents any aminoacid). The boxed area indicates the pu-tative membrane spanning region (143).The single amino acid code is used.
histidine 414 is the proximal heme binding site in TPO,consistent with the observations of Taurog et al. (19)with respect to tryptic fragments of hTPO. Comparativedata with sheep and mouse prostaglandin endoperoxidesynthase suggest that histidine 414 in hTPO is the axialheme ligand of this enzyme (21).
The single gene for TPO is located on chromosome 2(2pter-pl2) in man (13, 22) and chromosome 12 in themouse (17). In an heroic study, Kimura et al. (23) char-acterized the genomic structure of the hTPO gene, in-cluding the nucleotide sequences at all exon-intron junc-tions and approximately 32 kb of intronic and 5'-flankingregions. The gene contains 17 exons and 16 introns andextends over approximately 150 kb. hTPO is 42% ho-mologous with granulocyte myeloperoxidase, indicatinga common ancestral origin (23, 24). Exon-intron junc-tions 3-11 in hTPO correspond closely to junctions 2-11in myeloperoxidase (which contains an additional intronin this region) (23). In addition to homology with otherperoxidases, there is less homology with the epidermalgrowth factor-low density lipoprotein receptor familiesand C4b-j82 glycoprotein (24). These areas of homologyare localized to certain areas of TPO, suggesting thatthey were incorporated into the TPO gene during evo-lution.
III. Abnormal and Variant Expression of the TPOGene
Absent or abnormal TPO function has long been rec-ognized as a rare cause of thyroid dysfunction or goiter.There are excellent and comprehensive reviews on thissubject before the molecular cloning of TPO (1, 25). Tobriefly summarize this information, both quantitativeand qualitative abnormalities in TPO functional activityhave been described. Among the qualitative TPO abnor-malities are, 1) defective heme binding due to a defect inthe apoenzyme or prosthetic group, 2) the existence of aputative TPO inhibitor, 3) possible abnormalities in theintracellular location of TPO and, 4) an abnormality inthe ability of TPO to bind substrate.
We are at an early stage in understanding the molec-ular basis for abnormalities in the biochemical functionof TPO. Many of these TPO abnormalities are inherited.For this reason, the identification of disease-linked re-striction fragment length polymorphisms would be ofgreat value. A number of restriction fragment lengthpolymorphisms have been identified in the hTPO gene(26-30), but none have thus far been clearly linked toeither TPO dysfunction or to the predisposition to de-velop autoimmune thyroid disease. In one patient withdeficient TPO activity, the messenger RNA for TPO wascompletely absent (29).
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194 MCLACHLAN AND RAPOPORT Vol. 13, No. 2
Although abnormal TPO genes have yet to be reportedin patients with TPO functional deficiency, the molecu-lar cloning of the cDNA for TPO from FRTL5 rat thyroidcells (16) indicates that one TPO allele in this commonlyused cell line codes for a defective enzyme. Thus, inFRTL5 cells a point mutation (G to A) at the splicedonor site of exon 7 leads to aberrant splicing with theretention of a 54 base pair (bp) segment of the seventhintron (Fig. 2) (31). Within this retained intron is an in-frame stop codon, which would severely truncate (by71%) the protein and generate a nonfunctional enzyme.The second TPO allele in FRTL5 cells appears to benormal, and another explanation must therefore existfor the lack of TPO enzymatic activity in these cells (31).In contrast, immunoreactiue TPO is expressed on thesurface of FRTL5 cells (32).
The presence in thyroid follicular cells of differentlysized TPO mRNA species has been recognized for anumber of years. In addition to the normal 3.1 kb hTPOmRNA transcript (designated hTPO-1), Kimura et al.(13) reported and characterized the presence in Graves'thyroid tissue of another TPO mRNA species (hTPO-2)with a 171 bp deletion (nucleotides 1670-1840) withinthe 10th exon (Fig. 3). hTPO-2 mRNA, which is alsopresent in normal thyroid tissue (33), is likely to arise
FIG. 2. Upper panel, The molecular abnormality in the TPO gene inone allele of the FRTL5 rat thyroid cell line (31). The nucleotides inexons are shown in capital letters; those in introns are shown in lowercase letters. The point mutation (g to a) that abolishes the splice donorsite at the seventh exon/intron junction is shown by an arrow. As aresult of this abnormal splice donor site, translation continues throughthe intronic sequence until the stop (tga) that is underlined.Lower panel, Schematic representation of the two TPO alleles inFRTL5 cells. The upper horizontal line depicts the abnormal alleledescribe above. The lower line depicts the second allele that does nothave the point mutation. None of the cDNA clones obtained for thesecond allele were full length. All clones isolated began between nu-cleotide residues 581-683. Presumably, despite a full length clone notbeing obtained, this allele is normal and codes for the immunologicallyintact TPO expressed on the surface of FRTL5 cells.
by alternate splicing (13) and would code for a proteinof 876 amino acids, 57 amino acids shorter than thenormal protein.
Because purified TPO protein is typically visualizedon gel electrophoresis as a doublet of ~107 kilodaltons(kD) and -100 kD (34, 35), it has been suggested thatthese two forms of TPO are coded for by the hTPO-1and hTPO-2 mRNA species, respectively. However,there is no direct evidence to support this attractivehypothesis. Indeed, there is evidence to the contrary.When full-length hTPO cDNA corresponding to hTPO-1 mRNA is expressed in Chinese hamster ovary (CHO)cells, an hTPO protein doublet is still detected on West-ern blot analysis (36). Alternative explanations for theenigmatic hTPO doublet include partial proteolyticcleavage and/or varying degrees of glycosylation of thewild-type protein (37). Resolution of this issue may re-quire the direct physico-chemical analysis of the 107 kDand 100 kD TPO forms. Until it is known whether ornot hTPO-2 is a functionally active enzyme, its physio-logical significance will remain undetermined.
Although the hTPO-1 and hTPO-2 variants of hTPOmRNA have been the primary focus of attention, thereare other hTPO mRNA species present in thyroid tissue(Fig. 3). Zanelli et al. (38) have reported the eliminationby alternate splicing of 130 nucleotides in exon 16 ofhTPO. Because of a shift in the reading frame, a car-boxyl-terminal extension would lead to an hTPO variantof 929 residues, almost the same length as wild-typehTPO. This variant mRNA transcript is reported tocomprise 40-50% of hTPO mRNA in four differentGraves' thyroids (38).
Not to be confused with the two foregoing hTPOmRNA variants, which are very similar in size (between2.9 kb and 3.1 kb in length), Nagayama et al. (39)observed other hTPO mRNA species of widely varyingsize (4.0, 2.1, and 1.7 kb) in all 14 human thyroid cellcultures examined. The transcripts were relatively abun-dant, and their levels increased with TSH stimulation.These findings were intriguing because other laboratoriesusing human thyroid cells or human thyroid tissue fromdifferent species did not observe TPO mRNA transcriptsof these sizes (13,40,41). However, the molecular cloningof the 2.1 kb and 1.7 kb hTPO mRNA species (hTPO Iand hTPO II, respectively) (Fig. 3) has confirmed thepresence of these transcripts in human thyroid cells andtissue (42). The elucidation of their molecular structuresalso provides the answer as to why they were not detectedin other studies. The hTPO I mRNA contains exons 1-6 followed by the 5'-end of intron 6. hTPO II containsexons 1-5 and an unidentified tract of 558 bases (presum-ably intronic) at its 3'-end. Studies in which hTPOmRNA I and II were undetectable used cDNA probesfrom regions downstream of exon 6 whereas Nagayama
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May, 1992 MOLECULAR BIOLOGY OF TPO 195
3 kbI
FIG. 3. Summary of variations in TPOmRNA transcripts so far identified inhuman thyroid cells. See text for details.hTPO-1 is the wild-type TPO mRNA.In hTPO-2, the 10th exon is deleted (13).hTPO-Zanelli lacks exon 16 (38). hTPO-I and hTPO-II contain only exons 1-7and 1-6, respectively (42).
hTPO-1 •Wild-type '
hTPO-2
hTPOZanelli
hTPO-l
hTPO-II
Exons 1-17
3048 bp
Exon 10
3048
Exon 16
3036
Exons 1-7 Intron 62710 2840
1 654
Exons 1-6 Intron ?
486
et al. (39) used a cDNA probe corresponding to the 5'-end of hTPO. The hTPO I and hTPO II transcriptswould code for proteins of 225 and 174 amino acids,respectively. However, these proteins (if expressed) arelikely to be nonfunctional because they lack the putativefunctional region of the molecule coded for by exons 8-10 (20, 23).
It is becoming clear that thyroid cells contain multipleTPO mRNA transcripts of varying size rather than tran-scribing only hTPO-1 and hTPO-2 mRNA. The physio-logical or pathophysiological significance of any of thetranscripts, other than the wild-type (hTPO-1), is un-known, and it is likely that the majority lack enzymaticfunction. Without knowing the stability or transcriptionrates for each TPO mRNA species, it is not possible toestimate their proportional production rates. Finally, theabundant variants of TPO mRNA in human thyroidtissue are an unusual finding, even for a gene of the sizeof TPO. The reason for this observation is presentlyunknown.
IV. Regulation of TPO Gene Expression inThyroid Cells
Rapid progress is being made in understanding themechanism underlying the tissue-specific expression ofTPO. Thyroid-specific enhancer elements have beenidentified in the 5'-flanking regions of the human andrat TPO genes. In the hTPO gene, this enhancer elementis located 5.5 kb upstream of the transcriptional startsite (43). In contrast, the rat TPO gene exhibits multiplethyroid-specific regulatory elements within the first 145bp of the 5'-flanking region (44). A suppressor elementmay be present further upstream in the rat gene. Tissue-specific expression of both human and rat TPO genes isinduced by binding to cis-acting DNA elements of a
Stimulation of the thyroid gland by TSH increasesTPO bioactivity (46, 47), apparently via a process in-volving new protein synthesis (48). TSH stimulationincreases the levels of TPO mRNA in dog thyroid pri-mary cell cultures (49) with kinetics similar to those ofTSH on TPO enzymatic activity in the same system(50). Subsequent studies with FRTL5 rat (17, 51, 52) andhuman (39, 41) thyroid cells have supported this obser-vation and have also shown that the stimulatory effectof TSH stimulation is mimicked by cAMP and agentsthat increase intracellular cAMP levels. Unlike the genefor TG, TPO gene expression is not greatly dependenton the co-presence of insulin or insulin-like growth factorI (17, 53). Phorbol esters (41), interferon-7 (54), inter-leukin 1-a, and interleukin 1/9 (55) all reduce the stimu-latory effect of TSH on TPO mRNA levels in culturedhuman thyrocytes. Whether or not cytokine modulationof thyroid differentiated function is of pathophysiologicalimportance remains to be determined.
Despite the consensus that TSH raises the steady statelevel of TPO in thyroid cells, the underlying mechanismfor this stimulatory effect is controversial. An increasein mRNA levels for a specific gene can result from: 1)increased transcriptional activity, and/or 2) stabilizationof its mRNA. In FRTL5 rat thyroid cells, TSH stimu-lation does not increase transcription of the TPO gene(17, 51). Support for this observation is the unrespon-siveness to TSH of the hTPO promoter when transfectedinto FRTL5 cells (56). In contrast, TSH stimulation indog thyroid cells (53) and slices (57) rapidly increasesthe transcriptional rate of TPO mRNA. This stimulatoryeffect of TSH occurs even in the presence of epidermalgrowth factor, which, itself, has a suppressive effect onTPO mRNA expression (53). Further, the activity of thehTPO promoter, when transfected into dog thyroid cells,
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196 MCLACHLAN AND RAPOPORT Vol. 13, No. 2
is greatly enhanced by TSH in a cAMP-dependent man-ner (58). The effect of TSH on TPO gene transcriptionin thyroid cells is much more rapid (1 h) (53) than theincrease in steady state TPO mRNA levels (days) (49).However, because of the difference in species (dog vs.FRTL5 cells) and methodological conditions, this tem-poral difference in the response to TSH should be inter-preted with caution.
Overall, there is no question that the TSH-inducedincrease in TPO functional activity in the thyroid glandis mediated, at least in part, by an increase in the steadystate level of the mRNA for TPO. What is confusing isthe discrepancy between dog thyroid tissue and FRTL5cells in terms of the mechanism by which this increasein TPO mRNA level is attained. In the case of dogthyroid tissue the mechanism is transcriptional whereasin FRTL5 cells the mechanism is nontranscriptional andpresumably involves stabilization of the TPO mRNA, ashas been shown for other mRNA species in thyroidfollicular cells (59, 60). Whether or not this difference isspecies-related (dog us. rat) is unknown. Clearly, dogthyroid slices or primary cell cultures are a more phys-iological model than an immortalized cell line which,although relatively well differentiated, may have funda-mental nuclear regulatory alterations. It is also not yetknown whether the same nontranscriptional regulationof the TPO gene observed in rat thyroid cells in cultureapplies to the rat thyroid in vivo. It will be fascinating,and of potential heuristic importance, if the regulationof expression of a particular gene shifts from transcrip-tional to posttranscriptional in association with cell im-mortalization.
V. Expression of Recombinant hTPO
The availability of large amounts of purified TPO isessential for future advances in understanding the en-zymatic function of TPO at a molecular level and also inunderstanding the pathogenetic role of TPO in autoim-mune thyroid dysfunction (see below). Before the molec-ular cloning of TPO, thyroid tissue was the only sourcefor the purification of this enzyme. Very large amountsof thyroid tissue are required. For example, 1500 g humanthyroid tissue were needed to purify ~1 mg of a largetryptic fragment of the TPO protein (19). The generationof monoclonal antibodies to pig and human TPO hasgreatly improved the technique for TPO purification(61). Nevertheless, the supply of human thyroid tissue islimited, and it is difficult, even with hTPO monoclonalantibody affinity purification, to totally separate TPOfrom other thyroid antigens, especially highly abundantTG.
The molecular cloning of human and pig TPO has ledto the ability to generate large amounts of enzymatically
active TPO uncontaminated with other thyroid antigens.Different host cells are available for the generation ofrecombinant proteins, including mammalian and insectcells as well as yeast and bacteria. Eukaryotic cells offerthe advantage of a normal or near-normal glycosylationpattern. Thus far, hTPO has been stably introduced intothe genome of CHO cells, and this continually growingcell line provides an unlimited and uniform source offunctionally active protein (36, 62). TPO is expressed onthe surface of the CHO cells and is also present in themicrosomes of these cells (36). In addition, TPO cDNAhas been expressed in Hep G2 cells using vaccinia virusas a vector (63). The potential use of this system inunderstanding the functional activity of TPO remains tobe explored. More recently, Kendler et al. (64) reportedexpression of immunoreactive hTPO in Sf-9 insect cellsusing recombinant baculovirus.
Mutagenesis of hTPO cDNA has facilitated the pro-duction of recombinant hTPO protein. Thus, a majordifficulty associated with the purification of TPO fromeukaryotic cells is that the protein is membrane-associ-ated. A typical protocol for TPO purification from thy-roid tissue involves treatment of a cellular particulatefraction with detergent as well as limited proteolyticdigestion (65-68). This procedure releases a large, solublefragment of TPO with full enzymatic activity. The mo-lecular basis for the membrane association of TPO be-came evident after its molecular cloning and determina-tion of its derived amino acid sequence (Fig. 1). A hydro-phobic region near the carboxyl terminus of hTPO(amino acids 846-870) was a putative membrane-span-ning region, anchoring the protein to the plasma mem-brane. Confirmation of this possibility was obtained bythe introduction of stop codons immediately upstream ofthe putative transmembrane domain (69). RecombinanthTPO, when truncated at its carboxyl terminus to alength of 848 amino acids, is a soluble protein secretedinto the culture medium and retains enzymatic activity.
An additional modification by which to facilitate thegeneration of recombinant hTPO in eukaryotic cells hasbeen to amplify the number of copies of the hTPOtransgenome in CHO cells. This has been accomplishedusing expression vectors containing the dihydrofolatereductase (dhfr) gene linked in tandem with the hTPOtranscriptional unit. Exposure of transfected cells toprogressively increasing concentrations of methotrexateselects cells with increasing copies of both the dhfr andhTPO genes. Although the use of this approach has notbeen uniformly successful with the full length, mem-brane-associated hTPO protein (62, 70), the truncated,secreted form of hTPO has been greatly amplified (70).The reason for the variable success with the membrane-bound form of TPO is unknown. One possibility is thatvery high concentrations of TPO may be toxic to cultured
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May, 1992 MOLECULAR BIOLOGY OF TPO 197
cells and exert a negative selective pressure. In contrast,the secreted TPO is diluted in the culture medium anddoes not accumulate to very high levels if the medium isharvested at regular intervals.
These technical advances, namely, overexpression ofthe truncated form of TPO and/or potentially high levelsof TPO production in a baculovirus system, provide areadily available source of TPO for future structural andimmunological studies.
VI. Molecular Studies on TPO as an Autoantigenin Autoimmune Thyroid Disease
A. Introduction
Autoantibodies of immunoglobulin G (IgG) classagainst the thyroid "microsomal" antigen (71) and TG(72) are characteristically present in patients with au-toimmune thyroid disease. Microsomal antibodies arelikely to be of greater pathogenetic importance than anti-TG for a number of reasons. First, the majority of Hash-imoto patients have microsomal antibodies whereas anti-TG are sometimes absent or present in only smallamounts (73). Second, the levels of microsomal antibod-ies correlate with the active phase of the disease (74, 75).Third, unlike anti-TG, microsomal antibodies may di-rectly damage thyroid cells by activating the complementcascade (71). Microsomal antibodies have also beenshown to damage thyroid cells in vitro by an antibody-dependent cell cytotoxic mechanism involving K cells(76, 77).
Before the molecular cloning of TPO, there was strongimmunological evidence that the thyroid microsomal an-tigen was, in fact, TPO. Thus, 1) there was an excellentcorrelation between microsomal antibody titers and anti-TPO activity in sera, and 2) microsomal antibodies wereobserved to interact with purified TPO (34, 78-80). Con-firmation of this identity was obtained by the molecularcloning of the thyroid microsomal antigen. This goal wasattained by screening a thyroid cDNA expression librarywith a monoclonal antibody raised against the microso-mal antigen. The cDNA nucleotide sequence of the mi-crosomal antigen (40) was highly homologous to that ofthe recently cloned pig TPO cDNA (10). Similar conclu-sions were obtained after the cloning of the microsomalantigen from a thyroid cDNA library by screening withHashimoto's thyroiditis sera containing microsomal an-tibodies (24).
B. Assays for TPO autoantibodies
In recent years, there has been a proliferation of assaysfor anti-TPO antibodies in the sera of patients withautoimmune thyroid disease using both recombinant (81,82) and nonrecombinant (83, 84) TPO. The data ob-
tained demonstrate the superior sensitivity and specific-ity of anti-TPO assays compared with microsomal anti-body assays. Anti-TPO assays are presently supplantingthose for the thyroid microsomal antigen. The termmicrosomal antigen will fade from clinical use as has theterm "long acting thyroid stimulator" (LATS) for TSHreceptor antibodies. Because of the greater availabilityand uniformity of recombinant TPO, this material islikely to be used in the majority of assays in the future.The truncated, soluble form of TPO containing the ex-tracellular region of TPO and lacking the transmem-brane and intracytoplasmic regions of the protein (seeabove) is recognized by anti-TPO antibodies in patients'sera to the same extent as the full length, membrane-associated form of TPO. This observation is consistentwith the observation that the microsomal antigen isexpressed on the surface of human thyroid cells (85) andthat recombinant TPO is expressed on the surface ofCHO cells transfected with the TPO gene (36, 62). Rec-ognition by patients' sera, together with its ease of pro-duction, may make the truncated form of hTPO thechoice of antigen in future diagnostic anti-TPO assays.
C. Epitopes on hTPO recognized by B cells
Recognition by the immune system of autoantigens,similar to exogenous antigens, usually involves both Blymphocytes and T lymphocytes. However, the specificregions of an antigen recognized by antibodies (B cellepitopes) may differ from the epitopes on the sameantigen recognized by T cells (86). B cell epitopes aregenerally conformational (dependent on three-dimen-sional structure) (87, 88). On the other hand, T cellepitopes are short, linear peptide fragments presented bymajor histocompatibility (MHC) molecules to the T cellreceptor (Fig. 4). Because different approaches have beenused to investigate B and T cell epitopes on TPO, theresults of these investigations will be considered sepa-rately.
The nature of the interaction between TPO and serumTPO autoantibodies has been widely investigated andhas led to a confusing literature. In interpreting thesestudies it is necessary to consider whether or not appro-priate controls are included. Antibodies directed againstantigens other than TPO may bind with low affinity toTPO. Such antibodies may be more abundant in Hashi-moto sera, which are frequently hypergammaglobuli-nemic (89). This type of interaction may be the expla-nation for the apparent "cross-reactivity" of autoanti-bodies from patients with autoimmune thyroid diseasefor TPO and TG (90). Indeed, because of nonspecificinteractions, it was previously reported that the thyroidmicrosomal antigen is an epitope on the TSH receptor(91).
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198 MCLACHLAN AND RAPOPORT Vol. 13, No. 2
T-CELL EPITOPES
NP7 (535-551)
B-CELL EPITOPE
PROTEINANTIGEN
IMMUNOGLOBULIN
(igG)
FIG. 4. Schematic representation of the difference between T cell andB cell epitopes on a hypothetical protein. The cylinders represent a-helices. B6 and NP7 represent short, linear T cell epitopes on TPO(139), as depicted on this hypothetical model. A B cell epitope, recog-nized by the Fab fragment of IgG, comprises discontinuous loops of thepolypeptide chain.
Studies attempting to identify the number and locationof disease-associated B cell epitopes on TPO may begrouped into four categories based on the form of TPOstudied.
1. Intact TPO. All patients with antibodies against thethyroid microsomal antigen also recognize nonreducedTPO (36), indicating that some of the epitopes on TPOare expressed on the native, folded protein. Because thesedata were obtained by Western blot analysis with recom-binant hTPO expressed in nonthyroidal cells, the possi-bility of recognition of non-TPO antigens (such as TG)could be excluded. Under nonreducing conditions, anti-TPO (or antimicrosomal) antibodies interact with a pro-tein of ~200 kD, approximately twice the size of hTPO(35, 36, 92-94), suggesting that in its native state TPOmay exist as a dimer, or in association with anothermolecule of similar size. Recognition of TPO by auto-antibodies is decreased when the antigen is treated withreducing agents (36, 95), indicating that the integrity ofat least some epitopes is dependent on the three-dimen-sional conformation of the protein. These observationsare consistent with those obtained for microsomal anti-bodies that generally show a marked reduction in theability to bind to thyroid microsomal antigen treatedwith a reducing agent (93, 96, 97). Some patients' serareact well with TPO even under reducing conditions (36,93,98), suggesting that some TPO epitopes are not highlyconformational (see below). However, because of the
possibility of partial protein renaturation during West-ern blot analysis, this procedure is not ideal for drawingconclusions as to whether or not antibodies are interact-ing with linear or conformational epitopes.
Other studies involving the intact protein have alsobeen informative in discriminating between differentTPO epitopes. Thus some, but not all, TPO autoanti-bodies inhibit TPO enzymatic activity (95, 99, 100), andthere are differences among autoantibodies in their cross-reactivity with other related peroxidases such as myelo-peroxidase, lactoperoxidase, and horseradish peroxidase(101). Ruf et al. (102) mapped the interaction of a panelof 13 mouse monoclonal antibodies (MAb) raised againsthTPO to four antigenic domains (102). Cross-competi-tion of these MAb with human TPO autoantibodiessuggest that the latter recognize only two of these anti-genic domains. Consequently, the human autoantibodyresponse to TPO is more restricted than that of themouse after immunization with hTPO.
Overall, using the foregoing approaches, it has beensuggested that there may be as many as six differentantigenic domains on TPO with which human autoanti-bodies interact (95). The precise number can only beresolved when the TPO epitopes are identified at a moredetailed level. The presence of six antigenic domains onTPO would contrast with the human autoimmune re-sponse to the much larger molecule of TG which appearsto have only two to three antigenic areas on each of thetwo identical 330 Kd polypeptides (103,104).
2. TPO tryptic fragments. In theory, the use of TPOprotein fragments, rather than the intact molecule, is apreferable approach to defining epitopes for hTPO auto-antibodies. Indeed, limited tryptic digestion as part ofthe purification procedure for pig or human TPO gen-erates a number of polypeptide fragments that have beenused for this purpose. The 93 kD extracellular region ofhTPO and a 36 kD fragment (amino terminus at aminoacid 564) are recognized by human autoantibodies, evenunder reducing conditions (19). Porcine TPO is recog-nized less well than hTPO by autoantibodies (96). How-ever, using pig TPO tryptic fragments, IgG in sera ofpatients with autoimmune thyroid disease displayed mul-tiple recognition patterns, suggesting the existence ofseveral epitopes for TPO autoantibodies (98).
3. Recombinant hTPO fragments. Polypeptide fragmentsoffer a more precise approach to defining autoantibodyepitopes on hTPO. A segment of hTPO termed C2(amino acid residues 590-675 combined as a fusion pro-tein with 116 kD 0-galactosidase) (Fig. 5) was reportedto contain an epitope recognized by 95% of Hashimoto'sthyroiditis sera (24). In a subsequent, much larger, studyusing the C2 hTPO fusion protein in an enzyme-linkedimmunosorbent assay, IgG in the sera of 63% of patients
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May, 1992 MOLECULAR BIOLOGY OF TPO 199
Amino acids oMembrane
800n 933
450 500
B CELL EPITOPES
Trypsin
C2
C21
R3
R3a
R3b
R3c
600 650
Linear epitoperegion
700 750 800
Linear epitope
5641
5771
577
4561Cla-Bam
MAb #47
1"677
J750
1845
1845
FIG. 5. Summary of autoimmune thyroid disease-associated B cellepitopes described on hTPO. All epitopes are situated between aminoacid 456 and 845. The latter is the junction with the plasma membrane.Trypsin represents a tryptic fragment of highly purified hTPO (100).C2, C21 (105, 106), and R3, R3a, R3b, and R3c (107) are polypeptidefragments of hTPO expressed as bacterial fusion proteins. Cla-Bam isClal-BamHl fragment of hTPO also expressed as a bacterial fusionprotein (108). MAb 47 is the epitope on hTPO recognized by mouseMAb 47 (112). This epitope overlaps an epitope recognized by someTPO antibodies present in Hashimoto's serum (102).
with hTPO antibodies, as well as 9% of normal individ-uals without hTPO autoantibodies, reacted with C2(105). There is more limited recognition by patients' seraof another recombinant hTPO fragment (C21; aminoacids 651-750) (106) (Fig. 5).
Another study analyzed a series of TPO polypeptidefragments fused to 26 kD glutathione S-transferase usingthe more discriminating Western blot technique. Someautoimmune thyroid disease sera clearly reacted withhTPO fragment R3 (amino acid residues 577-845), aswell as with some internal fragments within R3 (107).Specific reactivity with other hTPO fragments was sug-gested, but the data are less convincing. Preliminaryevidence suggests autoantibody recognition of a Clal-BamHl fragment of hTPO (amino acid residues 456-631)(108) (Fig. 5).
Absorption with intact TPO or with thyroid micro-somes would provide information on the relative propor-tions of autoantibodies recognizing these regions of TPOand would also confirm the specificity of these interac-tions. Nevertheless, these investigations with hTPOpolypeptide fragments (both recombinant and nonre-combinant) from several independent groups indicatethat there is an epitopic "hot spot" between amino acids590 and 767 (Fig. 5).
4. hTPO cDNA random fragment libraries. Another ap-proach to defining B cell epitopes at a more detailed levelinvolves immunoscreening of an expression library madesolely from small cDNA fragments of a single gene (109).An epitope is evident from the area of overlap amongmultiple clones selected by the antibody (109). Thisapproach has the potential of defining the minimal sizeof an epitope, approximately 16-18 amino acids (87).
Using this technique, an hTPO cDNA library express-ing large numbers (~106) of random hTPO polypeptidefragments (66-166 amino acids in length) was screenedwith sera containing IgG-class hTPO autoantibody ac-tivity (110). None of 13 patients' sera with TPO autoan-tibody activity reacted with these hTPO peptide frag-ments. In contrast, a mouse monoclonal hTPO antibody,MAb 20.10 (111), generated against an internal, hydro-phobic region of the denatured molecule, recognized aseries of overlapping hTPO fragments. Sequencing of thecDNA coding for these hTPO fragments allowed theprecise definition of the MAb 20.10 epitope to aminoacids 266-281 (110).
The same cDNA fragment library was screened with apanel of 13 mouse MAb to undenatured hTPO (102) (seeabove). Twelve MAb did not interact with the hTPOfragments, and therefore their epitopes could not bedetermined. However, the epitope for one MAb, no. 47,was mapped to amino acids 713-721 (112) (Fig. 5). hTPOMAb 47 is the only one in the panel that recognizesintact hTPO under both denaturing and reducing con-ditions (102, 112, 113), suggesting that its epitope islinear and on the surface of the globular protein. Mostimportant, because hTPO autoantibodies compete forMAb 47 binding to TPO (102), the epitope for hTPOMAb 47 is likely to overlap with an epitope recognizedby some hTPO autoantibodies. Using a similar approachwith a different vector and screening with a particularlypotent serum from a Hashimoto patient, Libert et al.(114) mapped an hTPO epitope to the identical region(amino acids 710-722) (Fig. 5). In addition, these studiesnarrowed the C2 epitope (24) to hTPO amino acids 590-622 (Fig. 5).
5. Perspective. To date, two epitopes for hTPO autoanti-bodies appear to have been identified (Fig. 5). Both liewithin the tryptic fragment described by Yokoyama etal. (100). The narrowest dimensions of one epitope areamino acids 713-721. This epitope is recognized by MAb47 (112), has been localized to amino acids 710-722within the C21 fragment (114), and also lies within hTPOfragments R3 and R3b (107). A second hTPO epitope,less precisely defined, lies between amino acids 590-622(114). This epitope is within the C2 (24), R3 and R3a(107), and Cla-Bam (108) hTPO fragments.
In view of the consensus for the definition of the above
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200 MCLACHLAN AND RAPOPORT Vol. 13, No. 2
epitopes, it is useful to consider why there has beenvariability among different studies in autoantibody rec-ognition of hTPO fragments generated by the cDNArandom fragment library approach. A combination oftwo major factors may contribute to this variability.First, autoantibody heterogeneity may play a role. Thereis strong evidence that autoantibodies to hTPO are het-erogeneous: 1) Microsomal antibodies are principally ofsubclass IgGx and IgG4, with lesser amounts of IgG2 andvery small amounts of IgG3 (115-117). Extrapolatingfrom these studies, there are variable proportions ofsubclasses IgGi_4 among TPO autoantibodies in differentpatients. 2) The affinities of TPO autoantibodies ofdifferent IgG subclasses are variable (117). 3) There aredata suggesting that TPO autoantibodies of different IgGsubclasses may interact with different regions of TPO(118). As discussed above, 4) some, but not all, TPOautoantibodies interact with reduced TPO, lactoperoxi-dase, myeloperoxidase, or horseradish peroxidase and 5)some, but not all, TPO autoantibodies inhibit TPO en-zymatic activity. The proportion of each of these heter-ogeneous components differs in individual patients.
Second, there are technical differences among the var-ious studies. For example, the 0-galactosidase component(~10 kD) in the X-Zap vector used to screen the cDNAfragment library in the studies of Finke and co-workers(110, 112) is much smaller than the 116 kD full-lengthi8-galactosidase molecule in the X-gtll (24) and pUEXl(114) libraries used to express C2. The level of proteinexpression in plaques by X-Zap may also be less than inX-gtll plaques. Western blot analysis involving the useof a large concentration of hTPO polypeptide, as reportedfor R3 and R3b (107) (there are no Western blot datareported for C2), provides a more sensitive and specificdetection system than plaque screening or enzyme linkedimmunosorbent assay. The absence of glycosylation ofhTPO fragments produced in bacteria is an unlikelyexplanation for the poor recognition of TPO epitopes byautoantibodies because enzymatic deglycosylation ofhTPO (119) or pig TPO (120) does not reduce antigenic-ity of the protein.
Now the important question is whether or not theepitopes at amino acids 713-721 and 590-622 representa major proportion of the epitopes recognized by hTPOautoantibodies. These two epitopes are linear becausethey are recognized in small polypeptide fragments ofhTPO. Extensive evidence from other antigen/antibodysystems indicates that the majority of epitopes recog-nized by antibodies are discontinuous or nonlinear (87,88). Consequently, and as originally suggested (110), webelieve that the majority of hTPO epitopes recognizedby sera from patients with autoimmune thyroid diseaseare discontinuous and highly conformational (Fig. 4).Support for this concept is the decreased antigenicity of
intact hTPO after denaturation. We suggest that theautoantibodies that recognize linear epitopes at aminoacids 713-721 and 590-622 are of low affinity and/orabundance. A low autoantibody affinity could renderplaque detection more difficult in an expression cDNAlibrary system (see above).
In view of our hypothesis that the majority of epitopesrecognized by anti-TPO antibodies in patients' sera arediscontinuous, and not amenable to identification by thegeneration of recombinant or synthetic polypeptides, weanticipate that it will be difficult to determine all of theB cell epitopes involved in autoimmune thyroid diseaseby currently used techniques.
D. Human monoclonal TPO autoantibodies
To overcome the problems of heterogeneity of hTPOautoantibodies in patients' sera, human monoclonal an-tibodies derived from the B lymphocytes of patients withautoimmune thyroid disease are likely to be an importanttool in defining the hTPO B cell epitopes. Unfortunately,production of IgG class human monoclonal antibodies toa variety of antigens is extremely difficult. This has beenthe experience with Epstein-Barr virus immortalizationof B cells and/or fusion with an immortal cell line (121).The same difficulties have been encountered in produc-ing monoclonal thyroid autoantibodies (122), even whenusing B cells isolated from thyroid tissue that sponta-neously secrete greater amounts of thyroid autoantibod-ies than B cells from lymph nodes or blood (reviewed inRef. 123).
Because of these problems, very few IgG class humanmonoclonal autoantibodies to TG or TPO have beenproduced. A human-mouse hybrid, produced by fusingHashimoto thyroid lymphocytes with a mouse myeloma,secreted hTPO autoantibody of subclass IgG3, but thecell line was unstable (118). An IgGi subclass antibodywith an affinity comparable to serum TPO autoantibod-ies was produced subsequently and appears to be stable(124).
Recently a technique has been developed that mayovercome the difficulties associated with conventionalhuman monoclonal antibody production. This approachinvolves cloning portions of the IgG genes and expressingthem as Fab fragments in bacteria (125). The immuno-globulin heavy (H) and light (L) chain genes in a B cellare formed by the combination of a number of smallergenes located on the same chromosome (reviewed in Ref.126)(Fig. 6). The variable (V) region of the heavy chainis formed by the combination of one of ~200 V genes,one of ~20 diversity (D) genes, and one of six joining (J)genes. This VDJ unit is then joined to the constantregion of the heavy chain. Similarly, the light (L) chain
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May, 1992 MOLECULAR BIOLOGY OF TPO 201
KAPPA LIGHT CHAIN LOCI (CHROMOSOME 2)
B CELL-LIGHT CHAINGERMLINE GENE
B CELL - MUTATED LIGHT CHAIN GENE
HEAVY CHAIN LOCI (CHROMOSOME 14)
H u A r
HH1-- 200 GENES 20 GENES 6 GENES
B CELL • HEAVY CHAINGERMLINE GENE
B CELL - HEAVY CHAIN MUTATED GENE
FIG. 6. Organization of human immunoglobulin genes. Upper panel,Genes coding for the K-light chain (chromosome 2 in man). A /c-lightchain gene is formed by the combination of one of ~100 variable (V)genes and one of ~six joining (J) genes. Subsequently this VJ unit iscombined with the K-constant (C) region gene. Lower panel, Genescoding for the heavy chain (chromosome 14 in man). A heavy chaingene is formed by the combination of one of ~200 V genes, one of ~ 20diversity (D) genes, and one of six J genes (although not in this order).This VDJ unit then combines with the constant region gene of IgM(Cfi). At a later stage of an immune response, the same VDJ unit may"switch" to another heavy chain constant region gene (c), for exampleIgG.
Antibody diversity for a multitude of different antigens is generatedas follows: 1) Combination of different heavy and light chains; 2)Combination of different V and J genes in individual light chains, anddifferent V, D, and J genes in individual heavy chains; 3) Imprecisejoining of light chain VK to JK and of heavy chain VH to D and D toJH; 4) Finally, somatic mutations in the V and J genes of the lightchains and the V, D, and J genes of the heavy chain.
is formed from one of ~100 V genes and one of six Jgenes, as illustrated for the /(-chain (Fig. 6).
This information has been utilized in the techniquementioned above (125) for the generation of hTPO-specific, monoclonal Fab fragments in bacteria. A cDNAlibrary generated from a Graves' thyroid gland, whichalso contains cDNA transcribed from infiltrating B cells,was used as a template in the polymerase chain reactionwith oligonucleotide primers designed to include thevariable regions of the heavy or the light chains (Fig. 7).These DNA fragments were used to construct separateheavy and light chain cDNA libraries. Subsequently,random pairs of heavy and light chains from these twolibraries were used to construct a new "combinatorial"
library. Each phage in the library is capable of synthe-sizing a different IgG Fab fragment.
Screening this Graves' combinatorial library with ra-diolabeled hTPO led to the detection and cloning of ahuman Fab fragment (SP2) that binds specifically tohTPO (127). This Fab fragment is an IgGi with a /c-lightchain and has a high affinity for TPO (~10~10 M) com-parable with that of patients' hTPO autoantibodies (84).The VH and VL genes of SP2 appear to be moderatelymutated forms of germline gene families VHI and VKI,91.2% and 89.6%, respectively. Due to very low homologywith reported D region nucleotide sequences, it is difficultto assign the D region of SP2 heavy chain to a particulargene family. The heavy chain J segment is a JH3 thatappears to be truncated at its 5'-end. The light chain Jsegment is a JK2. It is not known if this particular heavyand light chain combination reflects the in vivo situation.However, because of the extremely high affinity of SP2for TPO, this may well be the case.
Antibodies recognizing the same epitope as SP2 arepresent in the sera of all 11 patients with autoimmunethyroid disease so far investigated (127a). More impor-tant, this type of TPO autoantibody is abundant, repre-senting 30-70% of serum TPO autoantibodies in individ-ual patients. Since SP2 does not recognize fusion pro-teins generated by a TPO cDNA fragment library (seeabove) and does not recognize denatured TPO, it appearsthat the SP2 epitope is conformational, unlike the linearepitopes described above.
Analysis of SP2 provides the first characterization ata molecular level of a hTPO antibody associated withautoimmune thyroid disease. The use of SP2 and othersuch autoantibodies will be invaluable in defining thespectrum of hTPO B cell epitopes in autoimmune thyroiddisease. This approach also provides the opportunity tostudy the genes coding for thyroid autoantibodies.
E. Definition of T cell epitopes on hTPO
The production of TPO autoantibodies is dependenton help provided by antigen-specific T lymphocytes. Tcells with reactivity for the thyroid microsomal antigen(128-130) or hTPO (131) have been demonstrated. How-ever, the specific T cell epitopes involved were not iden-tified. As mentioned above, the epitopes for T cells arenot necessarily the same as those for B cells. Unlike Bcell epitopes, T cell epitopes are produced by proteolyticdigestion of the antigen (hTPO), after which the peptidefragments are displayed within a groove on the surfaceof class II MHC molecules expressed on the surface ofantigen presenting cells (132). Recognition of this pep-tide-MHC complex is restricted to a subset of T cellswith receptors specific for a particular MHC subtype,e.g. DR3. The specificity of the interaction is further
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202 MCLACHLAN AND RAPOPORT Vol. 13, No. 2
THYROID TISSUE LYMPHOCYTES
cDNA LIBRARY
PCR
Vh D Jh CM Ch2 Ch3
(HEAVY CHAIN LIBRARY)
Vk Jk Ck
(LIGHT CHAIN LIBRARY)
(COMBINATORIAL LIBRARY)
SCREEN FOR EXPRESSION OF 125 I- TPO BINDING
FIG. 7. Molecular cloning and expression in bacteria of a humanautoantibody Fab fragment with specificity for hTPO (127). A cDNAlibrary from Graves' thyroid tissue, infiltrated with B lymphocyteswhich have rearranged their immunoglobulin genes (Fig. 6), was usedas template in the polymerase chain reaction (PCR). Heavy chain DNAfragments were generated using oligonucleotide primers upstream ofthe heavy chain V gene and downstream of the first domain of theconstant region of IgG (Chi). Light chain DNA fragments were gen-erated using oligonucleotide primers upstream of the K-V gene anddownstream of the K-constant region (C). These DNA fragments wereused to construct separate heavy and light chain cDNA libraries in thevector Immunozap. Subsequently, an IgG-/c combinatorial cDNA li-brary was constructed from random pairs of heavy and light chaingenes. The library was then screened for the expression of Fab frag-ments with specificity for 125I-labeled recombinant TPO.
increased by the peptide fragment within the MHCgroove. Antigen presenting cells include monocyte/mac-rophages, dendritic cells, and B cells themselves. Thyroidcells, under the influence of cytokines, in particularinterferon-7, are induced to express class II MHC mol-ecules and thereby become antigen presenting cells (133,134). Whether or not "aberrant" MHC expression onthyrocytes is a primary or secondary event in the autoim-mune process remains controversial.
Analysis of the amino acid composition common to T-cell epitopes in general has led to the development ofalgorithms valuable in predicting the regions of a proteinmost likely to be presented as T cell epitopes (135,136).Consistent with their situation within the MHC cleft, Tcell epitopes typically have a-helical structures. Eluci-dation of the primary amino acid sequence of hTPOprovided the first opportunity to define the T cell epi-topes on hTPO that are involved in thyroid autoimmu-nity.
A series of hTPO peptides based on the Berzofsky andRothbard (135, 136) algorithms have been used to ex-
amine the proliferative response of T cells isolated frompatients with autoimmune thyroid disease. Variable re-sults have been obtained using primary cultures of hu-man lymphocytes. In one study, none of these peptideselicited significantly higher responses than controlswhen tested with primary cultures of T cells from blood,thyroid, and lymph nodes of patients with autoimmunethyroid disease (118, 137). More recently, Tandon et al.(138) have reported proliferative responses by blood lym-phocytes to three TPO peptides (amino acids 415-432,439-457, and 463-481). Although the response was sta-tistically higher in patients compared with controls,these differences were small and were only observed in23-37% of the 30 patients studied. As yet, there is noreport on the isolation of T cell clones with specificityfor these peptides.
In addition to primary lymphocyte cultures, studieshave also been performed using T cell lines and clonesgenerated by interleukin-2 and anti-CD3 expansion ofactivated intrathyroidal T cells from a single patient.Two of a series of TPO synthetic peptides were found tostimulate T cell proliferation (139). These peptides (NP-7 and B6; hTPO amino acid residues 535-551 and 632-645, respectively) are presented in association with MHCantigens DP2/DQ6 and DQ2, respectively.
It is clear that the identification of T cell epitopes,such as NP-7 and B6, represents only the beginning of along process in defining all the hTPO T cell epitopesinvolved in thyroid autoimmunity. These epitopes arelikely to be heterogeneous, as evidenced by the diversityin the single patient studied thus far and will make thetherapeutic use of hTPO peptides in autoimmune thyroiddisease very difficult (139). A peptide corresponding to apotential T cell epitope common to hTPO and TG (basedon a computer analysis of the primary amino acid se-quences of these two proteins) (140) did not induce Tcell proliferation in primary lymphocyte cultures (137)or with T cell clones from the single patient studied(139). In addition to performing similar studies on manymore patients, it will be most important to determinewhether or not the T cell clones isolated are capable ofdriving B cells to produce specific hTPO antibodies.
F. Inheritance of the ability to produce TPOautoantibodies
It has recently been found that the ability to produceTPO autoantibodies is inherited as an autosomal domi-nant trait in women with incomplete penetrance in men(141, 142). The location and nature of the gene or genecluster responsible for this inheritance have not yet beenidentified. A number of candidate genes have been ex-cluded, notably those coding for MHC antigens (141). Ifand when the responsible gene(s) is identified, predis-
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May, 1992 MOLECULAR BIOLOGY OF TPO 203
posed individuals could be identified before the clinicalemergence of the disease.
VII. Conclusions
The molecular cloning of TPO has already led to amuch greater understanding of the molecular structureand pathophysiological role of this pivotal enzyme inthyroid cellular function. It is anticipated that in thenext few years the active site(s) of the enzyme will bedefined. In addition, genetic abnormalities in the TPOgene underlying the organisation defect in some pa-tients with goiter, with or without hypothyroidism, willalso be determined. Identification of the B cell and Tcell epitopes against which the autoimmune response isdirected may provide new immunological approaches tothe therapy of autoimmune thyroid disease. Such infor-mation will ultimately elucidate the origin of man's au-toimmune response to TPO.
References1. DeGroot LJ, Niepomniszcze H 1977 Biosynthesis of thyroid hor-
mone: basic and clinical aspects. Metabolism 26:665-7182. Mercken L, Simons M, Swillens S, Massaer M, Vassart G 1985
Primary structure of bovine thyroglobulin deduced from the se-quence of its 8,431-base complementary DNA. Nature 316:647-651
3. Malthiery Y, Lissitzky S 1985 Sequence of the 5'-end quarter ofthe human-thyroglobulin messenger ribonucleic acid and of itsdeduced amino-acid sequence. Eur J Biochem 147:53-58
4. Di Lauro R, Obici S, Condliffe D, Ursini VM, Musti A, MoscatelliC, Avvedimento VE 1985 The sequence of 967 amino acids at thecarboxyl-end of rat thyroglobulin. Eur J Biochem 148:7-11
5. Nagayama Y, Kaufman KD, Seto P, Rapoport B 1989 Molecularcloning, sequence and functional expression of the cDNA for thehuman thyrotropin receptor. Biochem Biophys Res Commun165:1184-1190
6. Libert F, Lefort A, Gerard C, Parmentier M, Perret J, LudgateM, Dumont JE, Vassart G 1989 Cloning, sequencing and expres-sion of the human thyrotropin (TSH) receptor: evidence forbinding of autoantibodies. Biochem Biophys Res Commun165:1250-1255
7. Misrahi M, Loosfelt H, Atger M, Sar S, Guiochon-Mantel A,Milgrom E 1990 Cloning, sequencing and expression of humanTSH receptor. Biochem Biophys Res Commun 166:394-403
8. Frazier AL, Robbins LS, Stork PJ, Sprengel R, Segaloff DL, ConeRD 1990 Isolation of TSH and LH/CG receptor cDNAs fromhuman thyroid: regulation by tissue specific splicing. Mol Endo-crinol 90:1264-1276
9. Vilijn F, Carrasco N 1989 Expression of the thyroid sodium/iodidesymporter in Xenopus laevis oocytes. J Biol Chem 264:11901-11903
10. Magnusson RP, Gestautas J, Seto P, Taurog A, Rapoport B 1986Isolation and characterization of a cDNA clone for porcine thyroidperoxidase. FEBS Lett 208:391-396
11. Rawitch AB, Taurog A, Chernoff SB, Dorris ML 1979 Hog thyroidperoxidase: physical, chemical and catalytic properties of thehighly purified enzyme. Arch Biochem Biophys 194:244-257
12. Magnusson RP, Gestautas J, Taurog A, Rapoport B 1987 Molec-ular cloning of the structural gene for porcine thyroid peroxidase.J Biol Chem 262:13885-13888
13. Kimura S, Kotani T, McBride OW, Umeki K, Hirai K, NakayamaT, Ohtaki S 1987 Human thyroid peroxidase: complete cDNA andprotein sequence, chromosome mapping, and identification of twoalternately spliced mRNAs. Proc Natl Acad Sci USA 84:5555-5559
DeGroot LJ, Rapoport B 1987 Molecular cloning of the comple-mentary deoxyribonucleic acid for human thyroid peroxidase. MolEndocrinol 1:856-861
15. Libert F, Ruel J, Ludgate M, Swillens S, Alexander N, Vassart G,Dinsart C 1987 Complete nucleotide sequence of the humanthyroperoxidase-microsomal antigen cDNA. Nucleic Acids Res15:6735
16. Derwahl M, Seto P, Rapoport B 1989 Complete nucleotide se-quence of the cDNA for thyroid peroxidase in FRTL5 rat thyroidcells. Nucleic Acids Res 17:8380
17. Isozaki O, Kohn LD, Kozak CA, Kimura S 1989 Thyroid peroxi-dase: rat cDNA sequence, chromosomal localization in mouse,and regulation of gene expression by comparison to thyroglobulinin rat FRTL-5 cells. Mol Endocrinol 3:1681-1692
18. Yokoyama N, Taurog A 1988 Porcine thyroid peroxidase: Rela-tionship between the native enzyme and an active, highly purifiedtryptic fragment. Mol Endocrinol 2:838-844
19. Taurog A, Dorris ML, Yokoyama N, Slaughter C 1990 Purificationand characterization of a large, tryptic fragment of human thyroidperoxidase with high catalytic activity. Arch Biochem Biophys278:333-341
20. Kimura S, Ikedo-Saito M 1988 Human myeloperoxidase andthyroid peroxidase, in two enzymes with separate and distinctphysiological functions, are evolutionarily related members of thesame gene family. PROTEINS: Structure, Function, and Genetics3:113-120
21. DeWitt DL, El-Harith EA, Kraemer SA, Andrews MJ, Yao EF,Armstrong RL, Smith WL 1990 The aspirin and heme-bindingsites of ovine and murine prostaglandin endoperoxide synthases.J Biol Chem 265:5192-5198
22. de Vijlder JJM, Dinsart C, Libert F, Geurts van Kessel A, BikkerH, Bolhuis PA, Vassart G 1988 Regional localization of the genefor thyroid peroxidase to human chromosome 2pter-pl2. Cytoge-net Cell Genet 47:170-172
23. Kimura S, Hong YS, Kotani T, Ohtaki S, Kikkawa F 1989Structure of the human thyroid peroxidase gene: comparison andrelationship to the human myeloperoxidase gene. Biochemistry28:4481-4489
24. Libert F, Ruel J, Ludgate M, Swillens S, Alexander N, Vassart G,Dinsart C 1987 Thyroperoxidase, an auto-antigen with a mosaicstructure made of nuclear and mitochondrial gene modules.EMBO J 6:4193-4196
25. Lever EG, Medeiros-Neto GA, DeGroot LJ 1983 Inherited disor-ders of thyroid metabolism. Endocr Rev 4:213-239
26. Massaro G, Libert F, Vassart G, Dinsart C 1989 RFLP's detectedat 2 pter-pl2 with a thyroid peroxidase cDNA probe, TPO3(McKusick no. 27450). Nucleic Acids Res 17:2155
27. Bikker H, Bolhuis PA, Vassart G, Libert F, Massaro G, de VijlderJJM 1989 EcoRl RFLP in the human thyroid peroxidase (TPO)gene on chromosome 2. Hum Genet 82:95
28. Mangklabruks A, Cox N, DeGroot LJ 1991 Genetic factors inautoimmune thyroid disease analyzed by restriction fragmentlength polymorphisms of candidate genes. J Clin EndocrinolMetab 73:236-244
29. de Vijlder JJM, Bikker H, Vulsma T 1990 DNA polymorphismsin the TPO gene, hereditary defects in iodide organification. In:Carayon P, Ruf J (eds) Thyroperoxidase, Thyroid Autoimmunity.John Libbey & Company Ltd, London, pp 149-155
31. Derwahl M, Seto P, Rapoport B 1990 An abnormal splice donorsite in one allele of the thyroid peroxidase gene in FRTL5 ratthyroid cells introduces a premature stop codon: association withthe absence of functional enzymatic activity. Mol Endocrinol4:793-799
32. Chiovato L, Vitti P, Lombardi A, Ceccarelli P, Cucchi P, MarcocciC, Carayon P, Pinchera A 1988 Studies on the mechanism re-sponsible for thyrotropin-induced expression of microsomal/per-oxidase antigen in FRTL5 cells. Endocrinology 123:1140-1146
33. Elisei R, Vassart G, Ludgate M 1991 Demonstration of the exist-ence of the alternatively spliced form of thyroid peroxidase innormal thyroid. J Clin Endocrinol Metab 72:700-702
34. Czarnocka B, Ruf J, Ferrand M, Carayon P, Lissitzky S 1985
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 June 2014. at 22:14 For personal use only. No other uses without permission. . All rights reserved.
204 MCLACHLAN AND RAPOPORT Vol. 13, No. 2
Purification of the human thyroid peroxidase and identificationas the microsomal antigen in thyroid diseases. FEBS Lett109:147-152
35. Kajita Y, Morgan D, Parkes AB, Rees Smith B 1985 Labellingand immunoprecipitation of thyroid microsomal antigen. FEBSLett 187:334-338
36. Kaufman KD, Rapoport B, Seto P, Chazenbalk GD, MagnussonRP 1989 Generation of recombinant, enzymatically-active, humanthyroid peroxidase and its recognition by antibodies in the seraof patients with Hashimoto's thyroiditis. J Clin Invest 84:394-403
37. Furmaniak J, Kiso Y, Morteo C, Bradley K 1991 Analysis ofcarbohydrate residues on human thyroid peroxidase (TPO). AnnEndocrinol (Paris) 52:75 (Abstract)
38. Zanelli E, Henry M, Charvet B, Malthiery Y 1990 Evidence foran alternate splicing in the thyroperoxidase messenger from pa-tients with Graves' disease. Biochem Biophys Res Commun170:735-741
39. Nagayama Y, Yamashita S, Hirayu H, Izumi M, Uga T, IshikawaN, Ito K, Nagataki S 1989 Regulation of thyroid peroxidase andthyroglobulin gene expression by thyrotropin in cultured humanthyroid cells. J Clin Endocrinol Metab 68:1155-1159
40. Seto P, Hirayu H, Magnusson RP, Gestautas J, Portmann L,DeGroot LJ, Rapoport B 1987 Isolation of a cDNA clone for thethyroid microsomal antigen: homology with the gene for thyroidperoxidase. J Clin Invest 80:1205-1208
41. Collison KS, Banga JP, Barnett PS, Kung AWC, McGregor AM1989 Activation of the thyroid peroxidase gene in human thyroidcells: effect of thyrotrophin, forskolin and phorbol ester. J MolEndocrinol 3:1-5
42. Nagayama Y, Seto P, Rapoport B 1990 Characterization, bymolecular cloning, of smaller forms of thyroid peroxidase messen-ger ribonucleic acid in human thyroid cells as alternatively splicedtranscripts. J Clin Endocrinol Metab 71:384-390
43. Kikkawa F, Gonzalez FJ, Kimura S 1990 Characterization of athyroid-specific enhancer located 5.5 kilobase pairs upstream ofthe human thyroid peroxidase gene. Mol Cell Biol 10:6216-6224
44. Francis-Lang H, Price M, Martin U, Di Lauro R1990 The thyroidspecific nuclear factor, TTF-1, binds to the rat thyroperoxidasepromoter. In: Carayon P, Ruf J (eds) Thyroperoxidase, ThyroidAutoimmunity. John Libbey & Company, Ltd, London, pp 25-32
45. Mizuno K, Gonzalez FJ, Kimura S 1991 Thyroid-specific enhan-cer-binding protein (T/EBP): cDNA cloning, functional charac-terization, and structural identity with thyroid transcription fac-tor TTF-1. Mol Cell Biol 11:4927-4933
46. Nagataki S, Uchimura H, Masuyama Y, Nakao K 1973 Thyrotro-pin and thyroidal peroxidase activity. Endocrinology 92:363-371
47. Yamamoto K, DeGroot LJ 1974 Peroxidase and NDPH-cyto-chrome C reductase activity during thyroid hyperplasia and in-volution. Endocrinology 95:600-612
48. Nagasaka A, Hidaka H 1980 Quantitative modulation of thyroidiodide peroxidase by thyroid stimulating hormone. Biochem Bio-phys Res Commun 96:1143-1149
49. Chazenbalk G, Magnusson RP, Rapoport B 1987 TSH stimulationof cultured thyroid cells increases steady state levels of the mes-senger RNA for thyroid peroxidase. Mol Endocrinol 1:913-917
50. Magnusson RP, Rapoport B 1985 Modulation of differentiatedfunctions in cultured thyroid cells: TSH control of thyroid per-oxidase activity. Endocrinology 116:1493-1500
51. Damante G, Chazenbalk GD, Russo D, Rapoport B, Foti D, FilettiS 1989 TSH regulation of thyroid peroxidase mRNA levels incultured rat thyroid cells: evidence for the involvement of a non-transcriptional mechanism. Endocrinology 124:2889-2894
52. Zarrilli R, Formisano S, Di Jeso B 1990 Hormonal regulation ofthyroid peroxidase in normal and transformed rat thyroid cells.Mol Endocrinol 4:39-45
53. Gerard CM, Lefort A, Libert F, Christophe D, Dumont JE, VassartG 1988 Transcriptional regulation of the thyroperoxydase geneby thyrotropin and forskolin. Mol Cell Endocrinol 60:239-242
54. Ashizawa K, Yamashita S, Nagayama Y, Kimura H, Hirayu H,Izumi M, Nagataki S 1989 Interferon-F inhibits thyrotropin-induced thyroidal peroxidase gene expression in cultured humanthyrocytes. J Clin Endocrinol Metab 69:475-477
55. Ashizawa K, Yamashita S, Tobinaga T, Nagayama Y, Kimura H,Hirayu H, Izumi M, Nagataki S 1989 Inhibition of human thyroid
peroxidase gene expression by interleukin 1. Acta Endocrinol(Copenh) 121:465-469
56. Foti D, Gestautas J, Rapoport B 1990 Studies on the functionalactivity of the promoter for the human thyroid peroxidase gene.Biochem Biophys Res Commun 168:281-287
57. Gerard CM, Lefort A, Christophe D, Libert F, Van Sande J,Dumont JE, Vassart G 1989 Control of thyroperoxidase andthyroglobulin transcription by cAMP: evidence for distinct regu-latory mechanisms. Mol Endocrinol 3:2110-2118
58. Abramowicz MJ, Vassart G, Christophe D 1990 Thyroid peroxi-dase gene promoter confers TSH responsiveness to heterologousreporter genes in transfection experiments. Biochem Biophys ResCommun 166:1257-1264
59. Wadsworth HL, Chazenbalk GD, Rapoport B 1990 Thyrotropincan increase the steady state level of a messenger ribonucleic acidspecies (glyceraldehyde-3-phosphate dehydrogenase) in thyroidcells by decreasing its rate of disappearance. Endocrinology 127:5-9
61. Nakagawa H, Kotani T, Ohtaki S, Nakamura M, Yamazaki 11985Purification of thyroid peroxidase by monoclonal antibody-as-sisted immunoaffinity chromatography. Biochem Biophys ResCommun 127:8-14
62. Hata J, Yamashita S, Yagihashi S, Kato H, Kabeno S, Hirai K,Kuma K, Kimura S, Umeki K, Kotani T, et al. 1989 Stable highlevel expression of human thyroid peroxidase in cultured Chinesehamster ovary cells. Biochem Biophys Res Commun 164:1268-1273
63. Kimura S, Kotani T, Ohtaki S, Aoyama T 1989 cDNA-directedexpression of human thyroid peroxidase. FEBS Lett 250:377-380
64. Kendler DL, Brennan V, Magnusson RP 1991 Expression ofimmunogenic human thyroid peroxidase (hTPO) in Sf-9 insectcells using recombinant baculovirus. Thyroid 1:S-21 (Abstract)
65. DeGroot LJ, Davis AM 1962 Studies on the biosynthesis ofiodotyrosines: a soluble thyroidal iodide-peroxidase tyrosine-io-dinase system. Endocrinology 70:492-504
66. Taurog A, Lothrop ML, Estabrook RW 1970 Improvements inthe isolation procedure for thyroid peroxidase: nature of the hemeprosthetic group. Arch Biochem Biophys 139:221-229
67. Alexander NM 1977 Purification of bovine thyroid peroxidase.Endocrinology 100:1610-1620
69. Foti D, Kaufman KD, Chazenbalk G, Rapoport B1990 Generationof a biologically-active, secreted form of human thyroid peroxidaseby site-directed mutagenesis. Mol Endocrinol 4:786-791
70. Kaufman KD, Foti D, Seto P, Rapoport B 1991 Overexpressionof an immunologically-intact, secreted form of human thyroidperoxidase in eukaryotic cells. Mol Cell Endocrinol 78:107-114
71. Belyavin G, Trotter WR 1959 Investigations of thyroid antigensreacting with Hashimoto sera. Lancet 1:648-652
73. Doniach D 1975 Humoral and genetic aspects of thyroid autoim-munity. Clin Endocrinol Metab 4:267-285
74. Yoshida H, Amino N, Yagawa K, Uemura K, Satoh M, Miyai K,Kumahara Y 1978 Association of serum antithyroid antibodieswith lymphocytic infiltration of the thyroid gland: studies ofseventy autopsied cases. J Clin Endocrinol Metab 46:859-862
75. Jansson R, Thompson PM, Clark F, McLachlan SM 1986 Asso-ciation between thyroid microsomal antibodies of subclass IgG-1and hypothyroidism in autoimmune postpartum thyroiditis. ClinExp Immunol 63:80-86
76. Bogner U, Schleusener H, Wall JR1984 Antibody-dependent cell-mediated cytotoxicity against human thyroid cells in Hashimoto'sthyroiditis but not Graves' disease. J Clin Endocrinol Metab59:734-738
77. McLachlan SM, Pegg CAS, Atherton MC, Middleton SL, Dick-inson A, Clark F, Rees Smith B 1987 The thyroid microenviron-ment in autoimmune thyroid disease: effects of TSH and lympho-kines on thyroid lymphocytes and thyroid cells. Acta Endocrinol(Copenh) 281:125-132
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 June 2014. at 22:14 For personal use only. No other uses without permission. . All rights reserved.
May, 1992 MOLECULAR BIOLOGY OF TPO 205
78. Portmann L, Hamada N, Heinrich G, DeGroot LJ 1985 Anti-thyroid peroxidase antibody in patients with autoimmune thyroiddisease: possible identity with antimicrosomal antibody. J ClinEndocrinol Metab 61:1001-1003
79. Kotani T, Umeki K, Matsunga S, Kato E, Ohtaki S 1986 Detectionof autoantibodies to thyroid peroxidase in autoimmune thyroiddiseases by micro-ELISA and immunoblotting. J Clin EndocrinolMetab 62:928-933
80. Mariotti S, Anelli S, Ruf J, Bechi R, Czarnocka B, Lombardi A,Carayon P, Pinchera A 1987 Comparison of serum thyroid micro-somal and thyroid peroxidase autoantibodies in thyroid disease.J Clin Endocrinol Metab 65:987-993
81. Kaufman KD, Filetti S, Seto P, Rapoport B 1990 Recombinanthuman thyroid peroxidase generated in eukaryotic cells: a sourceof specific antigen for the immunologic assay of anti-microsomalantibodies in the sera of patients with autoimmune thyroid dis-ease. J Clin Endocrinol Metab 70:724-728
82. Kendler DL, Martin A, Magnusson RP, Davies TF 1990 Detectionof autoantibodies to recombinant human thyroid peroxidase bysensitive enzyme immunoassay. Clin Endocrinol (Oxf) 33:751-760
83. Ruf J, Czarnocka B, Ferrand M, Doullais F, Carayon P 1988Novel routine assay of thyroperoxidase autoantibodies. Clin Chem34:2231-2234
84. Beever K, Bradbury J, Phillips D, McLachlan SM, Pegg C, GoralA, Overbeck W, Feifel G, Rees Smith B 1989 Highly sensitiveassays of autoantibodies to thyroglobulin and to thyroid peroxi-dase. Clin Chem 35:1949-1954
85. Khoury EL, Hammond L, Bottazzo GF, Doniach D 1981 Presenceof organspecific "microsomal" autoantigen on the surface of hu-man thyroid cells in culture: its involvement in complement-mediated cytotoxicity. Clin Exp Immunol 45:316-328
86. Livingstone AM, Fathman CG 1987 The structure of T-cell epi-topes. Annu Rev Immunol 5:477-501
90. Kohno Y, Naito N, Hiyama Y, Shimojo N, Suzuki N, TarutaniO, Niimi H, Nakajima H, Hosoya T 1988 Thyroglobulin andthyroid peroxidase share common epitopes recognized by auto-antibodies in patients with chronic autoimmune thyroiditis. JClin Endocrinol Metab 67:899-907
91. Islam MN, Tuppal R, Hawe BS, Briones-Urbina R, Farid NR1986 The thyroid "microsomal" antigen is an epitope on thethyrotropin receptor. J Cell Biochem 31:107-120
92. Hamada N, Grimm C, Mori H, DeGroot LJ 1985 Identificationof a thyroid microsomal antigen by Western blot and immuno-precipitation. J Clin Endocrinol Metab 61:120-128
93. Hamada N, Jaeduck N, Portmann L, Ito K, DeGroot LJ 1987Antibodies against denatured and reduced thyroid microsomalantigen in autoimmune thyroid disease. J Clin Endocrinol Metab64:230-238
94. Hamada N, Portmann L, DeGroot LJ 1987 Characterization andisolation of thyroid microsomal antigen. J Clin Invest 79:819-825
95. Doble ND, Banga JP, Pope R, Lalor E, Kilduff P, McGregor AM1988 Autoantibodies to the thyroid microsomal/thyroid peroxi-dase antigen are polyclonal and directed to several distinct anti-genic sites. Immunology 64:23-29
96. Nakajima Y, Howells RD, Pegg C, Davies Jones E, Rees Smith B1987 Structure activity analysis of microsomal antigen/thyroidperoxidase. Mol Cell Endocrinol 53:15-23
97. Gardas A, Domek H 1988 The effect of sulphydryl reagents onthe human thyroid microsomal antigen. J Endocrinol Invest11:385-388
98. Yokoyama N, Taurog A, Klee GG 1989 Thyroid peroxidase andthyroid microsomal autoantibodies. J Clin Endocrinol Metab68:766-773
99. Kohno Y, Hiyama Y, Shimojo N, Niimi H, Nakajima H, HosoyaT 1986 Autoantibodies to thyroid peroxidase in patients withchronic thyroiditis: effect of antibody binding on enzyme activi-ties. Clin Exp Immunol 65:534-541
100. Yokoyama N, Taurog A, Dorris ML, Klee GG 1990 Studies with
purified human thyroid peroxidase and thyroid microsomal auto-antibodies. J Clin Endocrinol Metab 70:758-765
101. Banga JP, Doble N, Tomlinson RWS, Odell E, McGregor AM1989 Thyroid microsomal/thyroid peroxidase autoantibodiesshow discrete patterns of cross-reactivity to myeloperoxidase lac-toperoxidase and horseradish peroxidase. Immunology 67:197-204
102. Ruf J, Toubert M, Czarnocka B, Durand-Gorde J, Ferrand M,Carayon P 1989 Relationship between immunological structureand biochemical properties of human thyroid peroxidase. Endo-crinology 125:1211-1218
103. Roitt IM, Campbell PN, Doniach D 1958 The nature of the thyroidauto-antibodies present in patients with Hashimoto's thyroiditis(lymphadenoid goitre). Biochem J 69:248-257
104. Nye L, Pontes de Carvalho LC, Roitt IM 1980 Restrictions in theresponse to autologous thyroglobulin in the human. Clin ExpImmunol 41:252-263
105. Ludgate M, Mariotti S, Libert F, Dinsart C, Piccolo P, Santini F,Ruf J, Pinchera A, Vassart G 1989 Antibodies to human thyroidperoxidase in autoimmune thyroid disease: studies with a clonedrecombinant complementary deoxyribonucleic acid epitope. J ClinEndocrinol Metab 68:1091-1096
106. Elisei R, Mariotti S, Swillens S, Vassart G, Ludgate M 1990Studies with recombinant autoepitopes of thyroid peroxidase:Evidence suggesting an epitope shared between the thyroid andthe gastric parietal cell. Autoimmunity 8:65-70
107. Banga JP, Barnett PS, Ewins DL, Page M, McGregor AM 1990Mapping of autoantigenic epitopes on recombinant thyroid per-oxidase fragments using the polymerase chain reaction. Autoim-munity 6:257-268
108. Maastricht J, Koenig RJ, Kaplan MM, Thompson N, Baker JrJR, Localization of an epitope in thyroid peroxidase (TPO) rec-ognized by Hashimoto's patients (HP) autoantibodies. Programof the 73rd Annual Meeting of The Endocrine Society, Washing-ton, DC, 1991, p 315 (Abstract 1140)
109. Mehra V, Sweetser D, Young RA 1986 Efficient mapping ofprotein antigenic determinants. Proc Natl Acad Sci USA 83:7013-7017
110. Finke R, Seto P, Rapoport B 1990 Evidence for the highly con-formational nature of the epitope(s) on human thyroid peroxidasethat are recognized by sera from patients with Hashimoto's thy-roiditis. J Clin Endocrinol Metab 71:53-59
111. Portmann L, Fitch FW, Havran W, Hamada N, Franklin WA,DeGroot LJ 1988 Characterization of the thyroid microsomalantigen, and its relationship to thyroid peroxidase, using mono-clonal antibodies. J Clin Invest 81:1217-1224
112. Finke R, Seto P, Ruf J, Carayon P, Rapoport B 1991 Determi-nation at the molecular level of a B-cell epitope on thyroidperoxidase likely to be associated with autoimmune thyroid dis-ease. J Clin Endocrinol Metab 73:919-921
113. Czarnocka B, Ruf J, Ferrand M, Carayon P 1990 Immunochemicalproperties of hTPO. In: Carayon P, Ruf J (eds) Thyroperoxidase,Thyroid Autoimmunity. John Libbey & Company Ltd, London,pp 59-67
114. Libert F, Ludgate M, Dinsart C, Vassart G 1991 Thyroperoxidase,but not the thyrotropin receptor, contains sequential epitopesrecognized by autoantibodies in recombinant peptides expressedin the pUEX vector. J Clin Endocrinol Metab 73:857-860
115. Parkes AB, McLachlan SM, Bird P, Rees Smith B 1984 Thedistribution of microsomal and thyroglobulin antibody activityamong the IgG subclasses. Clin Exp Immunol 57:239-243
116. McLachlan SM, Feldt-Rasmussen U, Young ET, Middleton SL,Blichert-Toft M, Siersboek-Nielsen K, Date J, Carr D, Clark F,Rees Smith B 1987 IgG subclass distribution of thyroid autoanti-bodies: a 'fingerprint' of an individual's response to thyroglobulinand thyroid microsomal antigen. Clin Endocrinol (Oxf) 26:335-346
117. Weetman AP, Black CM, Cohen SB, Tomlinson R, Banga JP,Reimer CB 1989 Affinity purification of IgG subclasses and thedistribution of thyroid auto-antibody reactivity in Hashimoto'sthyroiditis. Scand J Immunol 30:73-82
118. Fukuma N, Sarsero D, Furmaniak J, Pegg CAS, McLachlan SM,Rees Smith B B 1990 T cell epitopes on thyroid peroxidase. In:Carayon P, Ruf J (eds) Thyroperoxidase, Thyroid Autoimmunity.Colloque INSERM/John Libbey Eurotext Ltd, pp 195-201
119. Foti D, Rapoport B 1990 Carbohydrate moieties in recombinant
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 June 2014. at 22:14 For personal use only. No other uses without permission. . All rights reserved.
206 MCLACHLAN AND RAPOPORT Vol. 13, No. 2
human thyroid peroxidase: role in recognition by antithyroidperoxidase antibodies in Hashimoto's thyroiditis. Endocrinology126:2983-2988
120. Moura EG, Pazos-Moura CC, Yokoyama N, Dorris ML, TaurogA 1991 Enzymatic deglycosylation of porcine thyroid peroxidase:effects on catalytic activity and immunoreactivity. Acta Endocri-nol (Copenh) 124:107-114
121. Thompson K 1988 Human monoclonal antibodies. Immunol To-day 9:113-117
122. Fukuma N, Petersen VB, McLachlan SM, Pegg CAS, Rees SmithB 1991 Human monoclonal thyroglobulin autoantibodies of highaffinity. I. Production, characterisation and interaction with mu-rine monoclonal thyroglobulin antibodies. Autoimmunity 10:291-295
123. Rees Smith B, McLachlan SM, Furmaniak J 1988 Autoantibodiesto the thyrotropin receptor. Endocr Rev 9:106-121
124. Horimoto M, Petersen VB, Pegg C, Fukuma N, Wakabayashi N,Rees Smith B 1991 Production of a human monoclonal TPOautoantibody. Ann Endocrinol (Paris) 52:82 (Abstract)
125. Huse WD, Sastry L, Iverson SA, Kang AS, Alting-Mees M,Burton DR, Benkovic SJ, Lerner RA 1989 Generation of a largecombinatorial library of the immunoglobulin repertoire in phagelambda. Science 246:1275-1281
127. Portolano S, Seto P, Chazenbalk GD, Nagayama Y, McLachlanS, Rapoport B 1991 A human Fab fragment specific for thyroidperoxidase generated by cloning thyroid lymphocyte-derived im-munoglobulin genes in a bacteriophage lambda library. BiochemBiophys Res Commun 179:372-379
127a.Portolano S, Chazenbalk GD, Seto P, Hutchison JS, Rapoport B,McLachlan SM, 1992 Recognition by recombinant autoimmunethyroid disease-derived Fab fragments of a dominant conforma-tional epitope on human thyroid peroxidase. J Clin Invest, inpress
128. Weetman AP, Volkman DJ, Burman KD 1986 The productionand characterization of thyroid-derived T cell lines in Graves'Disease and Hashimoto's thyroiditis. Clin Immunol Immuno-pathol 39:131-142
129. Fisfalen ME, DeGroot LJ, Franklin WA, Soltani K 1988 Micro-somal antigen-reactive lymphocyte lines and clones derived fromthyroid tissue of patients with Graves' disease. J Clin EndocrinolMetab 66:776-784
130. Fisfalen M-E, Soltani K, Janiga AM, Kawakami Y, Macchia E,Quintans J, DeGroot LJ 1990 The regulatory role of human helper
T-cell clones on antithyroid antibody production by peripheral B-cells. J Clin Endocrinol Metab 71:170-178
131. Martin A, Magnusson RP, Kendler DL, Davies TF 1991 Success-ful hTPO-antigen specific human T cell cloning via hTPO trans-fected and immortalized B cell lines from patients with autoim-mune thyroid disease. Thyroid l:S-20 (Abstract)
132. Bjorkman PJ, Saper MA, Samraoui B, Bennett WS, StromingerJL, Wiley DC 1987 Structure of the human class I histocompati-bility antigen, HLA-A2. Nature 329:506-512
133. Hanafusa T, Pujol-Borrell R, Chiovato L, Russell RCG, DoniachD, Bottazzo GF 1983 Aberrant expression of HLA-DR antigen onthyrocytes in Graves' disease: relevance for autoimmunity. Lancet2:1111-1115
134. Bottazzo GF, Pujol-Borrell R, Hanafusa T 1983 Role of aberrantHLA-DR expression and antigen presentation in induction ofendocrine autoimmunity. Lancet 2:1115-1118
135. Margalit H, Spouge JL, Cornette JL, Cease KB, Delisi C, Berzof-sky JA 1987 Prediction of immunodominant helper T cell anti-genic sites from the primary sequence. J Immunol 138:2213-2229
136. Rothbard JB, Taylor WR 1988 A sequence pattern common to Tcell epitopes. EMBO J 7:93-100
137. Fukuma N, McLachlan SM, Rapoport B, Goodacre J, MiddletonSL, Phillips DIW, Pegg CAS, Rees Smith B 1990 Thyroid au-toantigens and human T cell responses. Clin Exp Immunol82:275-283
138. Tandon N, Freeman M, Weetman AP 1991 T cell responses tosynthetic thyroid peroxidase peptides in autoimmune thyroiddisease. Clin Exp Immunol 86:56-60
139. Dayan CM, Londei M, Corcoran AE, Grubeck-Loebenstein B,James RFL, Rapoport B, Feldmann M 1991 Autoantigen recog-nition by thyroid-infiltrating T cells in Graves disease. Proc NatlAcad Sci USA 88:7415-7419
140. McLachlan S, Rapoport B 1989 Evidence for a potential commonT-cell epitope between human thyroid peroxidase and humanthyroglobulin with implications for the pathogenesis of autoim-mune thyroid disease. Autoimmunity 5:101-106
141. Phillips D, McLachlan S, Stephenson A, Roberts D, Moffitt S,McDonald D, Ad'Hiah A, Stratton A, Young E, Clark F, BeeverK, Bradbury J, Rees Smith B 1990 Autosomal dominant trans-mission of autoantibodies to thyroglobulin and thyroid peroxi-dase. J Clin Endocrinol Metab 70:742-746
142. Phillips D, Prentice L, Upadhyaya M, Lunt P, Chamberlain S,Roberts DF, McLachlan S, Rees Smith B 1991 Autosomal domi-nant inheritance of autoantibodies to thyroid peroxidase andthyroglobulin—Studies in families not selected for autoimmunethyroid disease. J Clin Endocrinol Metab 72:973-975
143. Kyte J, Doolittle RF1982 A method for displaying the hydropathiccharacter of a protein. J Mol Biol 157:105-132
Cellular and Molecular Biology of the Adrenal CortexA Satellite Symposium of the 9th International Congress of Endocrinology
(Nice, Aug. 30 - Sept. 5, 1992)
The Symposium will be held at the Centre des Congres in Avignon, France, August 27 to 29, 1992. This meeting will focus on thefollowing main topics: angiotensin II receptors, mechanisms of signal transduction in the adrenal cortex, new local hormones withautocrine action in the adrenal cortex, molecular biology of steroidogenic enzymes, pathophysiology of new disorders of steroidreceptors of the adrenal cortex, growth factors and adrenal function.
Scientific Committee: A. Brownie (USA), E. Chambaz (France), A.M. Capponi (Switzerland), F. Mantero (Italy), J.M. Saez(France).
For further information, please contact the Scientific Secretary: Dr. Jose M. Saez, INSERM U 307, Hopital Debrousse, 29, RueSoeur Bouvier, 69322 Lyon Cedex 05, France (Tel: 33-78-25-18-08; FAX: 33-78-25-61-68)
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