Werner & Ingbar's The Thyroid: A Fundamental & Clinical Text, 9th Edition

4B.Thyroid Hormone Synthesis

Peter Kopp

Thyroid hormone biosynthesis, storage, and secretion require a series of highly regulated steps. Iodide, the rate- limiting substrate for thyroid hormone synthesis, is actively transported into thyroid follicular cells (thyrocytes) by the sodium-iodide symporter (NIS) at the basolateral membrane (Fig. 4B.1). At the apical membrane of the cells, iodide efflux into the follicular lumen is mediated, at least in part, by pendrin (PDS/SLC26A4). On the luminal side of the apical membrane, iodide is oxidized by thyroid peroxidase (TPO), a reaction that requires the presence of hydrogen peroxide (H2O2). H2O2 is generated by a calcium- dependent flavoprotein enzyme system that includes the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase THOX2. In the follicular lumen, thyroglobulin serves as matrix for the synthesis of thyroxine (T4) and triiodothyronine (T3). In a first step, TPO catalyzes the iodination of selected tyrosyl residues in thyroglobulin, a process referred to as iodination or organification. This results in the formation of mono- and diiodotyrosines (MIT, DIT). In the subsequent coupling reaction, which is also catalyzed by TPO, two iodotyrosines are coupled to form T4 or T3. Iodinated thyroglobulin is stored as colloid in the follic ular lumen. In response to demand for thyroid hormone secretion, thyroglobulin is internalized into the follicular cell by micro- and macro pinocytosis and digested in lysosomes. Subsequently, the thyronines T4(~80%) and T3 (~20%) are released into the blood stream. MIT and DIT are deiodinated by an intracellular iodotyrosine dehalogenase, and the released iodide is recycled for hormone synthesis.

FIGURE 4B.1. Thyroid hormone synthesis, secretion, and major signaling pathways in thyrocytes. AC, adenylyl cyclase; cAMP, cyclic adenosine monophosphate; DAG, diacylglycerol; DIT, diiodotyrosine; MIT, monoiodotyrosine; NIS, sodium-iodide symporter; PDS, pendrin; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; TG, thyroglobulin; TPO, thyroid peroxidase; TSH, thyrotropin; TSHR, thyrotropin receptor.

Thyroid hormone synthesis is dependent on the nutritional availability of iodine, and it is predominantly regulated by thyrotropin (thyroid-stimulating hormone, TSH). TSH binds to its cognate receptor, a member of the family of G (guanine nucleotide-binding) protein-coupled seven-transmembrane receptors, which is expressed at the basolateral membrane (Fig. 4B.2) (see section on the thyrotropin receptor in Chapters 10). Binding of TSH to its receptor leads primarily to coupling to Gα 5 and subsequent activation of adenylyl cyclase. The resulting increase in cyclic adenosine monophosphate (cyclic AMP) formation leads to phosphorylation of protein kinase A and activation of targets in the cytosol and the nucleus. The TSH-dependent cyclic AMP cascade is the major regulator of growth, differentiation, and hormone secretion of thyrocytes. At higher concentrations of TSH, stimulation of Gα 9/11 and the phospholipase C- dependent inositol phosphate Ca2+/diacylglycerol pathway activates H2O2 generation and iodination.

FIGURE 4B.2. Schematic structure of the promoter regions of the NIS (sodium-iodide symporter), Tg (thyroglobulin), and TPO (thyroid peroxidase) genes showing the sites where different transcription factors bind to the DNA. (From Dohán O, De la Vieja A, Paroder V, et al. The sodium/iodide symporter (NIS): characterization, regulation, and medical significance. Endocr Rev 2003;24:48, and Damante G, Di Lauro R. Thyroid-specific gene expression. Biochim Biophys Acta 1994;1218 255, modified with permission.)

In addition to TSH, iodide uptake is inversely regulated by the intracellular iodide concentration, and the organification process is transiently blocked by high intracellular iodide concentrations. These autoregulatory mechanisms protect the thyroid from iodide excess while ensuring adequate iodide uptake for hormone synthesis.

Previous versions of this chapter by Taurog provided comprehensive reviews of the mechanisms underlying thyroid hormone synthesis, with particular emphasis on the biochemical characteristics of thyroid peroxidase and the mechanisms of action of antithyroid drugs (1,2). Other recent detailed reviews include chapters by Gentile et al (3) and Dunn (4) in other books.

IODIDE UPTAKE INTO THE THYROID

Under physiological conditions, the thyroid iodide concentration is 20 to 40 times higher than the serum iodide concentration, and iodide uptake occurs against a cell-to-plasma electrochemical gradient of about -40 mV (5). The dependence of iodide uptake on a sodium gradient created by Na+,K+-adenosine triphospatase led to the prediction that iodide uptake is mediated by NIS (5,6,7,8,9), a prediction confirmed by the cloning of the symporter, which mediates iodide uptake at the basolateral membrane (8,9,10). The properties of NIS are discussed in more detail in the preceding section of this chapter.

The Gene and Protein Structure of the Sodium-Iodide Symporter

The ability of thyroid cells to concentrate iodide was recognized as early as 1896 (11), a century before the cloning of NIS (10,12). Human NIS is encoded by a single-copy gene with 15 exons that is located on chromosome 19p13 (13). NIS, officially designated as Solute Carrier 5A (SLC5A), belongs to a family of transporters that requires an electrochemical sodium gradient as the driving force for solute transport. Human NIS is a 643-amino-acid protein, which is thought to have 13 transmembrane domains with an extracellular amino terminus and an intracellular carboxy terminus (14,15). Mature human NIS is a glycoprotein of ~108 kDa (15). Partial or total deglycosylation does not impair the activity, stability, or membrane targeting of NIS (15).

Functional Characterization of the Sodium-Iodide Symporter

Consistent with findings in FRTL-5 cells (7), the Michaelis-Menten constant (Km) of NIS expressed in Xenopus oocytes is ~36 µM (10). Electrophysiological studies in oocytes demonstrate that NIS is electrogenic, resulting in an inwardly directed influx of positive charge upon addition of iodide to the extracellular perfusing solution (16). The inward steady-state current is due to the influx of sodium, and the stoichiometry of sodium to iodide is 2:1 (16). Based on kinetic results, it is thought that sodium binds to NIS before iodide, and that this is followed by a simultaneous transport of both ions (16).

Both perchlorate and thiocyanate cause rapid release of intracellular iodide across the basolateal membrane if organification is blocked by propylthiouracil (PTU) (17,18). This phenomenon is the basis for the perchlorate discharge test, which is used to evaluate intrathyroidal iodide organification (18). These anions inhibit iodide uptake through competitive inhibition (16). It is, however, controversial whether perchlorate itself serves as a substrate for NIS. In contrast to thiocyanate, perchlorate does not elicit an inward current, suggesting that perchlorate is not transported by NIS despite its potent inhibitory action on iodide uptake (16,19,20). This conclusion contrasts with studies using tetrahedral oxyanions in transfected cells, which suggest that NIS may mediate uptake of perchlorate (21).

Regulation of Iodide Uptake and Sodium-Iodide Symporter Function and Expression

TSH, acting through the cyclic AMP pathway, stimulates iodide accumulation in the thyroid (22,23). The increase in iodide uptake is the consequence of increased transcription of NIS and posttranscriptional stimulation of NIS activity. TSH up-regulates NIS mRNA and protein expression in vivo and in vitro (14,24,25,26). Consistent with an expression pattern that is not restricted to the thyroid, the structure of the promoter region of the NIS gene differs from those of the Tg (thyroglobulin) and TPO genes (Fig. 4B.2) (27,28,29).

TSH also modulates NIS protein turnover; in the presence of TSH, the half-life of NIS in FRTL-5 thyroid cells is ~5 days, in its absence it decreases to ~3 days (30). Aside from increasing NIS synthesis, TSH also regulates posttranslational events such as the subcellular distribution of NIS, specifically the targeting to and retention of NIS in the basolateral membrane. In the absence of TSH, NIS remains in the cell, and little reaches the plasma membrane.

In addition to TSH, iodide accumulation and organification are directly regulated by iodide itself (31,32,33). Negative regulation of the expression of NIS mRNA occurs in thyroid tissue of dogs fed moderate doses of iodide for 2 days (34). High doses of iodide block thyroid hormone synthesis through inhibition of the organification process, the Wolff-Chaikoff effect (31). This transient blockade of organification is dependent on the intracellular iodide concentration (31,32). The escape from the acute Wolff-Chaikoff effect involves a decrease in iodide transport, which leads to intracellular iodide concentrations that are too low to maintain the inhibitory effect (35). At the molecular level, an iodide-induced down-regulation of NIS expression, possibly an increase in NIS protein turnover, and a decrease in NIS activity contribute to this complex auto regulatory phenomenon induced by an increase in intracellular iodide concentrations (34,36,37,38). The down-regulation of NIS expression and activity is thyroid-specific, independent of TSH, and may be associated with formation of inactive NIS dimers (39). Thyroglobulin down-regulates NIS transcription (40,41), as do several cytokines, including transforming growth factor-β, tumor necrosis factor-α, interleukin-1, and interferon-γ (9,42).

Mutations in the Sodium-Iodide Symporter Gene

Congenital hypothyroidism can be caused by developmental defects of the thyroid, collectively referred to as thyroid dysgenesis, or by inborn errors of one of the steps required for thyroid hormone synthesis, referred to as thyroid dyshormonogenesis (see Chapters 48 and see section on congenital hypothyroidism in Chapters 75) (43,44,45). Among patients with thyroid dyshormonogenesis, a few have an iodide-trapping defect (46). The thyroid gland (and other tissues that contain NIS) does not concentrate iodine; therefore, thyroid and salivary uptake of radioiodine is very low, and the saliva/serum radioiodide ratio is very low (46). The thyroid may be normal in size at birth, but it enlarges soon thereafter unless the infant is given high doses of iodine or T4. After the NIS gene was cloned, several patients with hypothyroidism caused by this defect were found to be homozygous or compound heterozygous for inactivating NIS mutations (8,9,47). Some of the mutations decrease NIS function by substituting key functional amino acid resi dues (48), and others lead to misfolding and retention of NIS in intracellular compartments (see the preceding section of this chapter) (49).

IODIDE EFFLUX FROM THE THYROID

Compared with the mechanisms mediating iodide uptake at the basolateral membrane, iodide efflux at the apical membrane is less well characterized (Fig. 4B.1). Iodide efflux is stimulated by TSH in both poorly polarized FRTL-5 cells (50) and polarized porcine thyrocytes (51,52). In primary porcine thyrocytes grown in a transwell system, bidirectional measurements indicate that TSH stimulates iodide efflux at the apical membrane, while efflux in the basal direction does not change (52). This rapid effect of TSH facilitates the transport of iodide into the follicular lumen.

Electrophysiological studies performed with inverted plasma membrane vesicles suggest the existence of two apical channels for iodide efflux (53). One of these channels has a high permeability and specificity for iodide (Km ~70 µM), and the second channel has a lower affinity for iodide (Km ~33 mM) (53). However, the identity of these channels has not been established at the molecular level. The demonstration of iodide transport by the anion channel pendrin, together with the phenotype in patients with Pendred's syndrome, suggest that pendrin is one of the channels promoting apical iodide efflux (54,55,56,57,58). Whether the recently identified channel referred to as human apical iodide transporter (hAIT/SLC5A8) is involved in apical iodide efflux (59) or transport of other anions in thyrocytes is not known.

Cloning of the Pendrin (PDS/SLC26A4) Gene

Pendred's syndrome is an autosomal recessive disorder defined by sensorineural deafness, goiter, and impaired iodide organification (60,61,62). After Pendred's syndrome was linked to chromosome 7q22–31.1 (63,64), the PDS gene, now designated as SLC26A4, was cloned (54). The PDS/ SLC26A4 gene encompasses 21 exons and contains an open reading frame of 2,343 base pairs (Fig. 4B.3). The SLC26A family contains several transporters of sulfate or other anions and the motor protein prestin (SLC26A5), which is expressed in outer hair cells (Table 4B.1) (65,66,67). The genes encoding pendrin, prestin (SCLC26A5), and DRA/ CLD (down-regulated in adenoma/congenital chloride diarrhea, SLC26A3) are located in close vicinity on chromosome 7q21–31 and have a very similar genomic structure, suggesting a common ancestral gene.

FIGURE 4B.3. Chromosomal location and structure of the PDS/SLC26A4 gene and current model of the secondary structure of the pendrin (PDS) protein. (From Everett LA, Glaser B, Beck JC, et al. Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS). Nature Genet 1997; 17:411; Everett LA, Green ED. A family of mammalian anion transporters and their involvement in human genetic diseases. Hum Mol Genet 1999;8:1883; and Royaux IE, Suzuki K, Mori A, et al. Pendrin, the protein encoded by the Pendred syndrome gene (PDS), is an apical porter of iodide in the thyroid and is regulated by thyroglobulin in FRTL-5 cells. Endocrinology 2000;141:839, modified with permission.)

TABLE 4B.1. SELECTED MEMBERS OF THE SOLUTE CARRUIER FAMILY 26A


Gene Symbol

Alternative Gene Symbols

Protein

Major Expression

Anion Transport/Function

Chromosome Location

Human Disease


SCL26A2

DTDST

Diastrophic dysplasia

Ubiquitous

Sulfate
Chloride
Oxalate

5q32-q33.1

Diastrophic dysplasia
Achondrogenesis IB
Atelosteogenesis II

SCL26A3

DRA or CLD

Down-regulated in adenoma

Ileum
Colon
Seminal vesicle

Sulfate
Chloride
Bicarbonate
Hydroxide
Oxalate

7q21–31

Congenital chloride diarrhea

SCL26A4

PDS

Pendrin

Inner ear
Thyroid
Kidney

Iodide
Chloride
Bicarbonate
Hydroxide
Formate

7q21–31

Pendred's syndrome
Enlarged vestibular aqueduct
Recessive deafness

SCL26A5

PRESP

Prestin

Outer hair-cells

Motor protein

7q21–31

Sensorineural deafness


CLD, chloride diarrhea; DFNB4, autosomal recessive deafness 4; DTDST, diastrophic dysplasia sulfate transporter; SCL, Solute Carrier.

Protein Structure of Pendrin

Pendrin is a highly hydrophobic membrane protein consisting of 780 amino acids (Fig. 4B.3) (54). It contains a conserved, albeit slightly variant sulfate transport motif. Initially, pendrin was thought to have 11 transmembrane domains with an intracellular amino terminus and an extracellular carboxy terminus (54), but more recent studies suggest there are 12 transmembrane domains and an intracellular carboxy terminus (68). The intracellular location of both the amino terminus and the carboxy terminus has been confirmed by the analysis of pendrin fusion proteins (58). Pendrin is a glycoprotein with three putative extracellular N-glycosylation sites (68,69). In extracts of human thyroid membranes studied under denaturing conditions, pendrin is a single molecular species of ~110 to 115 kDa; deglycosylation leads to a reduction in molecular mass to ~85 kDa (69).

 

Expression and Regulation of Pendrin

Pendrin mRNA, assessed by Northern analysis, is primarily detected in the thyroid (54), but small amounts of both pendrin mRNA and protein are in the endolymphatic system of the inner ear (70,71), β-intercalated cells of the renal cortical collecting duct (72,73,74), syncytiotrophoblast cells of the placenta (75), endometrium (76), and mammary glands of pregnant and lactating mice (77). Very low levels of pendrin mRNA have also been found in lung, prostate, and testis (78).

Immunolocalization studies reveal that pendrin is located at the apical membrane of thyrocytes (68,79). The immu nostaining is heterogeneous within and among normal thyroid follicles (79). In thyroid tissue from patients with Graves' disease, immunostaining revealed higher pendrin protein expression than in normal thyroid tissue, suggesting a correlation between pendrin abundance and increased iodide organification (68,80). In hyperfunctioning thyroid adenomas, the levels of pendrin mRNA were normal, but immunostaining and Western blot analysis revealed more pendrin, as compared with normal thyroid tissue (79,81). In follicular adenomas, the levels of pendrin mRNA and protein were similar to those in normal thyroid tissue (69,82). In hypofunctioning adenomas, the levels of pendrin mRNA were normal, but the pendrin content of the adenomas, as detected by immunostaining, was highly variable (79,81). In FRTL-5 cells, low concentrations of thyroglobulin, but not TSH, insulin, interferon-α, or iodide, increased pendrin mRNA levels (68). This contrasts with the negative effect of thyroglobulin on the levels of expression of the NISTPOTg, and TSH receptor genes (40,41).

In differentiated thyroid carcinomas, pendrin mRNA and protein expression were low (69,79,80,83) and did not correlate with NIS expression (83,84). Pendrin mRNA was also scarce in thyroid cancer cell lines (68). Consistent with this low or absent expression, aberrant hypermethylation of the promoter region of the PDS gene was found in the majority of thyroid cancers (85).

Functional Characterization of Pendrin

Pendrin, expressed in Xenopus oocytes and S f9 insect cells, and present in cultured thyrocytes from patients with Pendred's syndrome, is unable to transport sulfate despite its homology with sulfate transporters (Fig. 4B.4)(55,86). In Xenopus oocytes, pendrin mediated uptake of chloride and iodide in a sodium-independent manner (55). The apical localization of pendrin in thyrocytes (68,79), together with its ability to transport iodide in oocytes and the impaired iodide organification found in patients with Pendred's syndrome (55), suggested a possible role in iodide transport into the follicular lumen (65,68). Functional studies in transfected cells subsequently demonstrated that the protein can indeed mediate iodide efflux (56,57). Further evidence that pendrin mediated apical iodide efflux was obtained in a model system with polarized Madin-Darby canine kidney (MDCK) cells expressing NIS and pendrin (Fig. 4B.5) (58). Consistent with the partial organification defect present in patients with Pendred's syndrome, naturally occurring mutations of pendrin lead to impaired apical transport of iodide into the follicular lumen of thyroid follicles (56,58,87).

FIGURE 4B.4. Time course of anion transport by pendrin in Xenopus oocytes injected with PDS/SLC26A4 complementary RNA. Pendrin, in contrast to the rat sulfate transporter Sat1, is unable to promote uptake of (35S)-Na2SO4 (a), but does promote uptake of (125I)-tetramethylammonium iodide (b) and (36C)-tetramethylammonium chloride (c). (From Scott DA, Wang R, Kreman TM, et al. The Pendred syndrome gene encodes a chloride-iodide transport protein. Nature Genet 1999; 21:440, with permission.)

FIGURE 4B.5. Transport of iodide by cells expressing NIS, pendrin (PDS), or both. Polarized MDCK cells expressing sodium-iodide symporter (NIS), pendrin, or NIS and pendrin growing in a bicameral cells were exposed to a solution containing (125I)-iodide in the lower chamber. There was a large increase in intracellular iodide in the cells expressing NIS. In cells expressing NIS and pendrin, the intracellular iodide content was low, and there was a large increase in iodide transport into the upper chamber. Cells expressing only pendrin accumulated little iodide in the intracellular compartment, but iodide did reach the upper chamber in increased amounts, as compared with wild-type MDCK cells. (From Gillam MP, Sidhaye A, Lee EJ, et al. Functional characterization of pendrin in a polarized cell system: evidence for pendrin-mediated apical iodide efflux. J Biol Chem 2004;279: 13004, with permission.)

The function of pendrin is not limited to iodide transport. In Xenopus oocytes, pendrin acts as a chloride/formate exchanger (88). In transfected HEK-293 cells, pendrin facilitates exchange of chloride with bicarbonate, hydroxide, and formate (72). Perfused renal tubules isolated from alkali-loaded normal mice secrete bicarbonate, whereas tu bules from alkali-loaded Pds-/- mice do not, confirming that pendrin is a chloride/base exchanger (73). A physiological role for pendrin in bicarbonate secretion is also suggested by its differential regulation by acid-base and electrolyte status (89,90). In mice, acid-loading results in reduction of the pendrin content in cortical-collecting duct cells, and it is relocalized from the apical membrane to the cytosol (89,90). Conversely, bicarbonate loading results in an increase in pendrin in the membranes of these cells (90). Based on the enlargement of the endolymphatic system in patients with Pendred's syndrome and Pds null mice (91,92), pendrin is thought to be involved in anion and fluid transport and maintenance of the endocochlear potential in the inner ear (71,91,92).

Pendred's Syndrome and Its Allelic Variants

Pendred's syndrome was first recognized by Vaughan Pendred in 1896 (see Chapters 48) (93). The incidence of Pendred's syndrome is thought to be as high as 7.5 to 10 in 100,000 people, and it is probably the most common form of syndromic deafness, accounting for about 10% of all patients with hereditary deafness (94,95).

The first insights into the pathophysiology of Pendred's syndrome followed recognition that these patients have a partial defect in the organification of iodide (96). Despite this defect, many patients with Pendred's syndrome are clinically and biochemically euthyroid unless dietary iodine intake is low (61,62). Furthermore, the prevalence of goiter may be lower in patients with this syndrome who live in iodine-replete regions (97,98). Pds knockout mice do not have thyroid enlargement or abnormal serum thyroid hormone production (92). These findings suggest that iodide may enter the lumen of thyroid follicles independent of pendrin, either by means of another iodide-transporting channel or nonspecific anion channels assisted by the electrochemical gradient between the cytoplasm of thyrocytes and the lumen (53).

Patients with Pendred's syndrome from consanguineous families are homozygous for PDS gene mutations, whereas sporadic cases typically have compound heterozygous mutations (62). Mutations in the PDS gene display impressive allelic heterogeneity, and more than 75 mutations have already been identified (54,99,100,101,102,103,104). In many instances, the mutated pendrin protein is retained in an intracellular compartment, usually the endoplasmic reticulum (56,58,105), indicating that the mutations result in aberrant folding of the protein, thus preventing full maturation and insertion into the cell membrane. Other mutated pendrin molecules are normally inserted into the membrane but cannot normally mediate iodide efflux, suggesting that the mutations involve amino acid residues that are important for iodide transport (56).

Mutations in the PDS gene are found not only in patients with classic Pendred's syndrome, but also in patients with familial enlargement of the vestibular aqueduct (106,107), suggesting that the incidence of sensorineural hearing impairment associated with alterations in this gene may even be higher than previously thought. Limited functional studies in oocytes suggest that the mutations found in Pendred's syndrome result in complete loss of chloride and iodide transport, whereas the mutations found in nonsyndromic hearing loss have some transport activity (87). However, the same mutation may be associated with variable phenotypic expression, suggesting that other genetic and environmental factors modify the effects of the mutation (56).

THYROGLOBULIN

Thyroglobulin, a large glycoprotein dimer secreted into the follicular lumen, serves as the matrix for the synthesis of T4 and T3, and as the storage form of the hormones and iodide (see Chapters 5). When T4 and T3 are needed, thyroglobulin is taken up by the thyrocytes and digested in lysosomes, and T4 and T3 are released into the blood stream (Fig. 4B.1).

Structure of the Thyroglobulin Gene and Protein

Thyroglobulin is encoded by a single-copy gene of 270 kb located on human chromosome 8q24.2–8q24.3 (108,109,110,111). It contains 48 exons separated by introns of up to 65 kb (108,112,113). The promoter region of the gene has remarkable structural similarity with the promoter region of the TPO gene and is regulated by the transcription factors TTF-1, TTF-2, and Pax-8 (Fig. 4B.2) (28,29). The full-length human mRNA contains a 41-nucleotide 5′-untrans lated segment preceding an open reading frame of 8,307 bases, and a 3′-untranslated region ranging from 101 to 120 base pairs (114,115).

The thyroglobulin monomer consists of a 19-amino-acid signal peptide followed by 2,749 residues containing 66 tyrosine residues (115). After translation of the mRNA, thyroglobulin monomers are transported to the endoplasmic reticulum (ER), where they are folded and undergo dimerization. The dimers are then glycosylated. Mature thyroglobulin migrates to the apical membrane in small secretory vesicles and is secreted into the follicular lumen (116,117). In the follicular lumen, thyroglobulin is present as a 19S dimeric glycoprotein of 660 kDa (118).

Analysis of the primary structure of thyroglobulin for internal homology led to its division into four major regions. The type 1 repetitive region has 11 segments that contain a cysteine-rich consensus sequence CWCV(D), a sequence found in many proteins (119,120,121). These segments may bind and inhibit cysteine proteases, a feature that could play a role in the processing and degradation of thyroglobulin (see later in the chapter) (119,120). In the mature protein lacking the signal peptide, these segments are located between amino acids 12 and 1191 and amino acids 1492 and 1546. The type 2 repetitive region, composed of three elements, is located between amino acids 1437 and 1484. The type 3 repetitive region is characterized by five elements between residues 1584 and 2168. The carboxy-terminal region of the thyroglobulin monomer, encompassing residues 2192 to 2716, shares remarkable homology with acetylcholinesterase (121,122). Overall, this structure has been interpreted to indicate the possibility of a convergent origin of the Tg gene from different ancestral DNA sequences (123).

The maturation of thyroglobulin is controlled by several molecular chaperones such as BiP, GRP94, Erp72, and calnexin (see Chapters 5) (124). Thyroglobulin monomers contain 20 potential glycosylation sites; among them, 16 are known to be glycosylated (4,125), and about 10% of the molecular weight is accounted for by carbohydrates. Other secondary modifications of the protein include sulfation and phosphorylation (126,127). Consensus sequences required for tyrosine sulfation are present at most of the hormonogenic sites within thyroglobulin, and sulfation may play a role in hormone formation (128).

Mutations in the Thyroglobulin Gene

Recessive mutations in the Tg gene have been identified in several animal species and humans with goiter and overt or subclinical hypothyroidism (see Chapters 48) (129,130,131). The patients typically have very low serum thyroglobulin concentrations (132). Most have had homozygous inactivating mutations in the TG gene (130,131); rare patients have had different mutations in each allele (compound heterozygous genotype) (133). Some of these mutations result in synthesis of thyroglobulin molecules that are retained in the endoplasmic reticulum and thus fall into the class of ER storage diseases (124,134,135).

THYROID PEROXIDASE

In order to serve as an iodinating agent, iodide must be oxidized to a higher oxidation state, a step that is dependent on the presence of H2O2 and is catalyzed by TPO. TPO, a glycoprotein with a prosthetic heme group, is located in the apical membrane. In addition to catalyzing the oxidation of iodide, TPO is also essential for the incorporation of iodide into tyrosine residues in thyroglobulin (organification) and coupling of the iodotyrosines to form T4 and T3 (Fig. 4B.1).

The purification and the biochemical properties of TPO were discussed in detail in the previous versions of this chapter by Taurog (1,2). The cloning of TPO and its role as an autoantigen in autoimmune thyroid disease, which is discussed in Chapters 14, have been reviewed by McLachlan and Rapoport (136).

The Gene and Protein Structure of Thyroid Peroxidase

The human TPO gene is located on chromosome 2pter-p12 (137,138), spans about 150 kb, and consists of 17 exons separated by 16 introns (139). The first cDNAs encoding TPO were isolated from human and porcine cDNA libraries (137,140,141). The full-length human TPO cDNA encodes a protein of 933 amino acids (137). In addition to the full-length mRNA encoding full-length TPO (TPO1), several shorter transcripts of unknown biological importance have been identified (137,142,143). The most abundant alternative transcript lacks 171 nucleotides, secondary to deletion of exon 10, which encompasses codons 533 to 590 (TPO2) (137,144). The encoded protein lacks enzymatic activity (144). In immunoblot analyses, TPO appears as a doublet of 110 and 105 kDa, a phenomenon that is not explained by translation of TPO2 (145).

The amino terminus of TPO is located in the lumen of thyroid follicles, and the extracellular domain forms a loop created by two (human) or one (porcine) intramolecular disulfide bonds, which is followed by a single membrane-spanning domain in close proximity to its carboxy terminus (146,147). Introduction of a stop codon immediately upstream of the putative transmembrane domain (amino acids 846 to 870) results in a soluble protein that is secreted into the medium and retains enzymatic activity (148). Human TPO has five potential glycosylation sites, and about 10% of its weight is carbohydrate (2). The location and nature of the N-linked oligosaccharide units have been determined only for porcine TPO (149).

The prosthetic heme group, a bis-hydroxylated heme that is distinct from the heme b (protoporphyrin IX) found in many other hemoproteins, is covalently bound to glutamine 399 and aspartate 238 of the apoprotein (150). Amino-acid side chains directly linked to the iron atom or positioned in its immediate proximity are critical for modulating enzyme reactivity and specificity. These side chains are provided by two histidine residues located on opposite sides of the heme moiety, a proximal histidine coordinately linked to the iron center, and a distal histidine located in proximity of the peroxide-binding pocket (150,151,152). Based on sequence alignment with myeloperoxidase (MPO), the distal histidine in TPO is located at position 239, and the proximal histidine at position 494 (150,152). The distal histidine and a nearby arginine residue at position 396 are thought to be involved in the formation of compound I, a two-electron oxidized form of TPO (see later in chapter) (150,152). TPO2 is enzymatically inactive (144), presumably because it lacks asparagine 579, which forms a stabilizing hydrogen bond with the proximal histidine 494 (150,152).

All mammalian peroxidases belong to the same gene family (152). TPO has a high degree of sequence similarity with MPO (140,153,154,155) and other mammalian peroxidases (156,157,158). The first 735 amino acids of TPO have 42% sequence identity with MPO (141), and the homology between TPO and MPO in the vicinity of the heme group is as high as 74% (140). Based on sequence analyses, which have revealed domains homologous to epidermal growth factor, complement C4b, and mitochondrial cytochrome C oxidase I, the TPO gene may be a mosaic gene (141). The structure of MPO has been solved at 3 Å resolution (159), and more recently at 1.8 Å resolution (160,161). Theoretical three-dimensional structures have been proposed for other mammalian peroxidases using the MPO model as a scaffold (151), but the three-dimensional structure of TPO has not been modeled (2). TPO, analogous to MPO (159), may form a disulfide-linked dimer in its native form, based on immunoblot experiments under nonreducing conditions (162,163), a model that has been questioned (145).

Proper folding and membrane insertion are essential for enzyme activity. Immunohistochemical studies localize TPO at the apical membrane (164), and abundant immunopositivity is also found in the cytoplasm (165). Stimulation with TSH acutely increases TPO immunoreactivity at the apical membrane and increases enzymatic activity (166,167). Thus, TPO is probably brought to the apical through the secretory pathway. The sorting and trafficking of TPO are cell type-dependent (168). In stably transfected heterologous cell systems, TPO is often largely retained in intracellular compartments (168,169,170,171). In contrast, it reaches the plasma membrane efficiently in stably transfected rat PCCl3 thyroid cells (168). These findings suggest that continuous membrane insertion of TPO requires the presence of thyroid-specific factors.

Functional Characterization of Thyroid Peroxidase

Before the cloning of the TPO cDNA, catalytically active TPO was purified by solubilizing membrane fractions of thyroid tissue with detergent and limited proteolytic digestion (146,147), eventually combined with immunoaffinity chromatography (172,173). The catalytic activity of these preparations was determined using several assay procedures, including guaiacol oxidation, iodide oxidation, iodination of albumin, and coupling of DIT to form T4 within thyroglobulin (146). After the molecular cloning of TPO cDNAs, recombinant TPO has been expressed in mammalian cells, insect cells, and yeast [for review, see reference 136]. Recombinant TPO preparations have been particularly useful for immunologic studies, although many have diminished catalytic activity (2,174). Eukaryotic cells may be more suitable as an expression system for obtaining a functional and soluble recombinant TPO, presumably because of their ability to glycosylate the protein fully (174).

Expression and Regulation of Thyroid Peroxidase

The activity of TPO is increased by TSH in vivo (175,176), and this stimulatory effect is the consequence of increased synthesis of TPO (177). The mechanisms leading to stimulation of TPO synthesis vary in different model systems. TSH and other stimulators of the cyclic AMP signaling pathway increase TPO mRNA abundance in cultured thyroid cells (178,179,180,181,182). In FRTL-5 cells, the increase in TPO mRNA levels is due to an increase in mRNA stability (180,181), whereas transcriptional stimulation accounts for the increase in canine thyroid cells (179,183).

TSH-induced stimulation of TPO gene transcription does not require the presence of insulin or insulin-like growth factor 1 (179,180), whereas both stimulators are required for transcription of the Tg gene (180). Phorbol esters, interferon-γ, and interleukin-lα and -1β inhibit TSH-induced TPO mRNA expression in cultured human thyroid cells (184,185,186). In contrast to TSH, follicular thyroglobulin decreases the expression of TTF-1, TTF-2, and Pax-8, actions that decrease the expression of the genes for TPO as well as for NIS, thyroglobulin, and the TSH receptor (40,41).

TSH and forskolin rapidly increase TPO promoter activity without the requirement for protein synthesis (187). This stimulation correlates with an increase in TTF-2 binding to the TPO promoter (188) but does not involve TTF-1 or a cyclic AMP response element (28,189). The structure of the promoter region of the human TPO gene resembles that of the Tg gene promoter, and it contains a TATA box, three binding sites for TTF-1, and a binding site for Pax-8 and TTF-2 in the region between -170 to +1 base pairs relative to the transcriptional start site (Fig. 4B.2) (28).

As in the Tg gene promoter, the Pax-8 and the TTF-1 binding sites overlap, and in vitro the binding of the two transcription factors to DNA is mutually exclusive (190). However, Pax-8 and TTF-1 combine with each other, and the combination synergistically activates the Tg gene promoter (Fig. 4B.2) (191). This particular mechanism may not apply to the TPO promoter (191), although the two transcription factors have synergistic actions on TPO transcription (192). CTF/NF-1, a member of the CCAAT-binding transcription-factor family, which is inducible by cyclic AMP and insulin, cooperates with TTF-2 in increasing the activity of the promoter region of the TPO gene (193). Moreover, the carboxy terminus of Pax-8 associates with the nuclear coactivator p300 to increase TPO gene transcription (194). This suggests that the stimulatory effect of TSH on TPO mRNA expression may be mediated through p300 or CBP (CREB-binding protein), because the TPO promoter does not contain a cyclic AMP response element (28,189,194). A thyroid-specific enhancer region has been identified 5.5 kb upstream of the transcriptional start site in the human TPO gene (28,195). This region has overlapping binding sites for TTF-1 and Pax-8 that may be mutually exclusive (196,197), suggesting that the ratio of TTF-1 to Pax-8 may be important in the regulation of TPO gene expression (197).

Mutations in Thyroid Peroxidase

Recessive TPO defects are among the most frequent causes of inborn abnormalities of thyroid hormone synthesis (see Chapters 48) (198,199,200). The decrease or absence of TPO activity results in a partial or total iodide organification defect, so that affected patients have a substantial discharge of radioiodine after the administration of perchlorate. A survey from the Netherlands confirmed that TPO gene defects are the most common cause of severe defects in iodine organification (201). Of the patients available for molecular studies, 37% were homozygous and 46% were compound heterozygous for mutations affecting the exons or intron/ exon boundaries of the TPO gene. In a small percentage, only one abnormal allele could be identified, and only one of the families did not have a TPO mutation. One patient had a partial maternal isodisomy of chromosome 2p resulting in homozygosity for an inactivating TPO gene mutation (201).

THE HYDROGEN PEROXIDE–GENERATING SYSTEM

H2O2 is an essential and limiting factor in the oxidation of iodide, its organification, and the coupling reaction (Fig. 4B.1) (202). The H2O2-generating system is localized at the apical membrane (203,204), and its generation involves the oxidation of NADPH by an NADPH oxidase (205,206,207,208). This enzyme system contains a membrane-bound flavoprotein using FAD as a cofactor (209,210), and it requires micromolar concentrations of calcium (204,206,211,212,213). A functional NADPH oxidase that generates H2O2 in a Ca2+-dependent manner has been solubilized from plasma membranes of porcine thyroid tissue (210); sequence information derived from this protein led to cloning of a partial cDNA (214).

Cloning of the Thyroid Oxidase Genes

Two cDNAs encoding thyroidal NADPH oxidases have been cloned (214,215). The two oxidases are commonly referred to as THOX1 and THOX2, for “thyroid” oxidase; their official designations are DUOX1 and DUOX2, an eponym derived from “dual” oxidase. Human and porcine cDNA sequences encoding the carboxy-terminal region of an NADPH oxidase (initially called p138Tox) were isolated by reverse transcription-polymerase chain reaction (RT-PCR) using degenerate primers derived from peptide microsequences obtained from purification of a flavoprotein-containing oxidase (210,214). Two full-length cDNAs encoding thyroid NADPH oxidases were cloned using a strategy assuming a homology between the NADPH oxidase systems in thyrocytes and granulocytes (215). The probes used to screen thyroid cDNA libraries were based on gp91Phox (NOX2), a heme-binding oxidase that is part of the enzyme system responsible for the production of superoxide (O2-) in granulocytes (216,217). THOX2 was also detected as an abundant transcript in thyroid cells using serial analysis of gene expression (218).

The two THOX genes are closely linked and located on chromosome 15q15 (214,215). They both consist of 33 exons; the THOX1 gene spans about 36 kb, and the THOX2 gene spans about 22 kb (214,215). Human THOX1 has an open reading frame of 1,551 amino acids, THOX2 of 1,548 residues (215). The protein p138Tox corresponds to THOX2 but lacks the first 338 amino acids (214).

The Protein Structure of Thyroid Oxidases

THOX genes encode two closely related proteins that have 83% homology. They are related not only to gp91Phox (NOX2) found in granulocytes (216), but also to MOX1, a superoxide-generating oxidase present in nonphago cytic cells (219), and a Caenorhabditis elegans protein (215). Analysis of the secondary structure, and comparison with the model proposed for gp91Phox (NOX2) (216), predicts seven putative transmembrane domains, two everted finger motifs in the first intracellular loop, four NADPH-binding sites, and one FAD-binding site (Fig. 4B.6)(214,215). The intracellular location of the everted finger domains, which are calcium-binding sites, is consistent with the activation of the H2O2 generation system by cytosolic Ca2+in thyroid membranes (208,211), intact follicles (204), and thyroid slices (202,220).

FIGURE 4B.6. Proposed structure of THOX proteins with seven transmembrane domains, two putative Ca2+-binding EF-finger motifs in the first intracellular loop, and cytosolic FAD- and NADPH-binding sites. (From De Deken X, Wang D, Many MC, et al. Cloning of two human thyroid cDNAs encoding new members of the NADPH oxidase family. J Biol Chem 2000;275:23227, with permission.)

As predicted from analysis of the primary structure, which suggests the presence of five possible glycosylation sites (215), the THOX proteins are glycosylated (221,222). Western blots reveal two proteins with a molecular weight of ~180 kDa to 190 kDa. Only the 190-kDa form is resistant to endoglycosidase H digestion, suggesting that it is the completely processed form. After complete deglycosylation, the size of the protein is reduced to ~160 kDa (221).

The amino termini of both THOX proteins have a homology of ~43% with TPO. Remarkably, this domain of THOX1 and its C. elegans homologue have peroxidase activity (223). The finding of a peroxidase activity domain and an NADPH oxidase domain in the same protein led to their official designation as dual oxidases (223). The carboxy-terminal part of THOX1 and THOX2 are highly homologous to the mammalian oxidases gp91Phox (NOX2) and p65Mox (NOX1), and contain the putative intracellular FAD- and NADPH-binding sites. The four histidine residues and arginine residue thought to be involved in heme binding in gp91Phox (NOX2) are conserved (216).

Expression and Regulation of Thyroid Oxidases

The mRNAs for THOX1 and THOX are almost exclusively detected in thyroid tissue (214,215,224). A very small amount of THOX2 mRNA has been found in the stomach (215) and possibly the trachea (225), and an expressed sequence tag for THOX2 has been found in a pancreas library (218). THOX2 transcripts were also detected in the intestinal tract (duodenum, small intestine, colon) of adult rats by RT-PCR analysis (224).

The levels of THOX1 and THOX2 mRNA increase in response to stimulation of the cyclic AMP pathway in human, porcine, canine and rat thyroid cells in vitro (214,215,224). The stimulation is greater in dog than in human thyroid tissue (215). In rats treated with methimazole (MMI), the levels of THOX2 mRNA are reduced (224), a finding that contrasts with the change in cultured cells (214,215,224). Treatment of thyroid cells and follicles with iodide inhibits H2O2generation and NADPH activity in vitro (213,226). While treatment of porcine thyroid follicles with iodide down- regulates the levels of NIS and TPO mRNA, it does not affect the level of THOX2 mRNA (222). However, it does antagonize the cyclic AMP-induced glycosylation of THOX2 to its mature form, possibly explaining the decrease in H2O2 generation (222). This phenomenon may contribute to the mechanisms underlying the antithyroid effect of high iodine concentrations (Wolff-Chaikoff effect) (222).

Immunohistochemical analyses demonstrate THOX protein at the apical membrane of thyrocytes and colocalization with TPO (215,225). The fraction of THOX proteins reaching the apical membrane is, however, modest (221), similar to TPO (227). The low content of THOX proteins at the apical membrane may serve to limit the generation of oxidative agents (221). The THOX proteins and TPO are barely inserted into the membrane of nonthyroidal cells (168,170,171); in nonthyroidal cells transfected with THOX1 and THOX2 cDNAs, the expressed proteins remain restricted to intracellular compartments (221). In addition, a finding that contrasts with the observations in thyrocytes, they are present only as a 180-kDa form (221).

The levels of THOX mRNA are variable in benign and malignant human thyroid tissues (225,228). In thyroid tissue from patients with Graves' disease, only a few cells are stained with anti-THOX antibodies (225). In multinodular goiters, the average number of positive cells is similar to that of normal thyroid tissue, but in hypofunctioning tissue THOX is more abundant (225). In general, as determined by immunostaining, the thyroid tissue content of NIS and THOX is inversely related (225).

Cotransfection of THOX proteins with TPO and p22Phox, one of the proteins required for NADPH oxidase activity of gp91Phox (NOX2), into CHO cells does not result in insertion of THOX or TPO into the cell membrane, and does not result in H2O2 generation (221). Similarly, transfection of THOX cDNAs into PLB-XCGD cells, a human myeloid cell line devoid of NADPH oxidase activity, does not result in H2O2 generation (221). These findings indicate that additional components are required to achieve membrane targeting and full enzymatic activity of THOX (221).

Mechanism of Hydrogen Peroxide Generation

The biochemical mechanisms resulting in H2O2 generation are controversial (2). The H2O2-generating system involves a Ca2+-dependent NADPH oxidase—most likely THOX2—but it is unclear whether H2O2 is formed directly or through a process that includes the formation of O2- as an intermediate step. In one proposed model, O2 is first oxidized to O2- and then converted to H2O2 by a superoxide dismutase (Fig. 4B.7)(206,229,230). In this model, both steps are thought to occur on the cytosolic side of the membrane, and the H2O2 then traverses to the luminal side of the membrane, where it reacts with TPO. In a second model, O2 is directly converted to H2O2 by a complex Ca2+-dependent NADPH oxidase system containing a flavoprotein (Fig. 4B.7) (209,231,232,233,234). The secondary structure of the THOX proteins is consistent with the functional domains predicted by this model (214,215). Further functional characterization of the THOX proteins, together with the isolation of additional components of this enzyme system, will provide a more thorough understanding of the mechanisms underlying H2O2 generation.

FIGURE 4B.7. Proposed mechanisms of hydrogen peroxide (H2O2) formation in thyrocytes. Two substantially different mechanisms are shown. A: NADPH oxidase in the apical membrane interacts with NADPH, Ca2+, and O2 to generate superoxide anions (O2-) on the cytosolic side of the membrane. Through the action of superoxide dismutase (SOD), the O2- is converted to H2O2, which traverses the apical membrane to react with TPO. (From Nakamura Y, Makino R, Tanaka T, Ishimura Y, Ohtaki S. 1991 Mechanism of H2O2 production in porcine thyroid cells: evidence for intermediary formation of superoxide anion by NADPH-dependent H2O2-generating machinery. Biochemistry 1991;30:4880, with permission.) B: NADPH oxidase in the apical membrane is a complex flavoprotein, which is activated by Ca2+. Activation occurs when Ca2+ binds to an inhibitory protein or to an autoinhibitory domain. The activated flavoprotein transfers electrons from NADPH to O2 in some as yet undisclosed manner to form H2O2 directly on the luminal side of the apical membrane. (From Dème D, Doussiere J, De Sandro V, et al. The Ca2+/NADPH-dependent H2O2 generator in thyroid plasma membrane: inhibition by diphenyleneiodonium. Biochem J 1994;301: 75, with permission.)

Regulation of Hydrogen Peroxide Activity

The generation of H2O2 is Ca2+-dependent, and it is rapidly activated by agents stimulating the phosphatidyl inositol pathway (204,212,226,235,236). In addition, TSH stimulates the production of H2O2 through the cyclic AMP pathway. Chronic TSH stimulation was initially reported to decrease H2O2 generation in porcine and FRTL-5 cells (236), but in more recent studies performed in canine and porcine cells, TSH and other stimulators of the cyclic AMP pathway clearly increased H2O2 generation (Fig. 4B.8)(213,233). This effect requires protein syn thesis, since co-incubation with cycloheximide partially inhibits the TSH-mediated stimulation (233). In particulate fractions obtained from human and porcine thyroid tissue, the Ca2+- and NADPH-dependent H2O2-generating activity is inducible by TSH and down-regulated by transforming growth factor-β (208). These tissues also have a small amount of Ca2+-independent, NADH-dependent H2O2-generating activity that is not stimulated by TSH or forskolin, but whether this weak constitutive activity is exerted by THOX1 or another unidentified enzyme is not known (208).

FIGURE 4B.8. Hydrogen peroxide generation and basal (125I)-iodide uptake in canine thyrocytes cultured for 5 days in me dium containing varying concentrations of thyrotropin (TSH). The Ca2+-phosphatidylinositol cascade was stimulated with carbachol. (From Raspé E, Dumont JE. Tonic modulation of dog thyrocyte H2O2 generation and I- uptake by thyrotropin through the cyclic adenosine 3′,5′-monophosphate cascade. Endocrinology 1995;136:965.)

H2O2 generation is inhibited by high concentrations of iodide (220,226), and iodohexadecanal, an intermediary that may be involved in autoregulatory processes in the thyroid (237,238). In contrast, low concentrations of iodide mildly stimulate H2O2 generation in thyroid tissue from several species (239). This effect is more pronounced in thyroid tissue previously deprived of iodide. The concentration-dependent control of H2O2 generation by iodide permits efficient hormone synthesis when iodide is scarce, while avoiding excessive hormone synthesis when iodide is abundant (239).

Defects in Hydrogen Peroxide Generation

Confirmation for the physiological role of THOX2 in H2O2 generation comes from patients with congenital hypothyroidism who have THOX2 gene mutations (see Chapters 48) (240). One patient with severe permanent hypothyroidism and a total iodide organification defect had a homozygous nonsense mutation in the THOX2 gene. Three other patients had transient congenital hypothyroidism and a partial iodide organification defect; they had monoallelic nonsense mutations in the THOX2 gene. All four mutations resulted in severe truncations of THOX2, eliminating the putative H2O2-generating domain. No mutations were found in the THOX1gene. The finding of biallelic mutations in the patient with a total iodide organification defect provides strong evidence that THOX2 is essential for normal H2O2 and thyroid hormone synthesis. The fact that THOX1 is unable to compensate for the deficiency in THOX2 suggests that the enzymes have different functional roles. The milder, transient hypothyroidism in the patients with monoallelic THOX2 mutations indicates that two functioning alleles are needed to meet the large requirements for thyroid hormone synthesis early in life (240). Whether these patients are at risk for subclinical or overt hypothyroidism during other periods when thyroid secretion increases, for example during pregnancy, is unknown.

Deficient H2O2 generation has been proposed as the explanation for euthyroid goiter and decreased iodide organification in a few sporadic patients, because in vitro addition of an H2O2-generating system to thyroid homogenates or slices from these patients restored normal organification (241,242). In two siblings with hypothyroidism, goiter, and decreased iodide organification, very low NADPH oxidase activity was detected in thyroid tissue, suggesting impaired H2O2generation, but the molecular defect is not known (243).

IODINATION AND THYROID HORMONE SYNTHESIS

The synthesis of thyroid hormone occurs at the apical membrane. Iodide is oxidized by TPO in the presence of H2O2 (Fig. 4B.1). The iodination of selected tyrosyl resi dues of thyroglobulin, a reaction referred to as organification, then leads to the formation of the monoiodotyrosine (MIT) and diiodotyrosine (DIT). Lastly, two iodotyrosyls couple to form the thyronines T4 and T3. Iodination and coupling are both catalyzed by TPO, and, although presented as sequential steps, they occur simultaneously.

Oxidation of Thyroid Peroxidase to Compound I and Compound II

The heme in native peroxidases is in the ferric form (FeIII) (Fig. 4B.9). The reaction product of TPO or other peroxidases with H2O2, a two-electron reaction resulting in the reduction of H2O2 to H2O2 and oxidation of the enzyme, is referred to as compound I (TPO-O) (244). One elec tron is removed from the iron, giving rise to an oxyferryl (FeIV=O) intermediate, and the second electron is removed from the porphyrin ring, a state referred to as π-cation radical. A second, unstable form of compound I, which probably does not occur in the thyroid, is a protein radical in which the second electron is withdrawn from an aromatic amino acid of the apoprotein (245). The one-electron reduction of compound I generates compound II, and a further one-electron reduction brings the enzyme back to its native state (Fig. 4B.9).

FIGURE 4B.9. Oxidation of iodide, organification, and coupling. The heme in native peroxidases is in the ferric form (FeIII). The reaction product of thyroid peroxidase (TPO) with hydrogen peroxide (H2O2), a two-electron reaction that results in the reduction of H2O2 to H2O and oxidation of the enzyme, is referred to as compound I (TPO-O). One electron is removed from the iron, giving rise to an oxyferryl (FeIV=O) intermediate; the second electron is removed from the porphyrin ring, a state referred to as π-cation radical. Although the nature of the iodinating species (OI-, HOI, or I+) has not been determined with certainty, iodide is then oxidized in a two-electron reaction, and selected tyrosyls are iodinated. In the coupling reaction, successive one-electron oxidations of hormonogenic tyrosyl residues in thyroglobulin (TG), mediated by compound I and compound II of TPO, are proposed to result in the for mation of diiodotyrosine radicals, before two iodotyrosyls are coupled in a nonenzymatic reaction to form thyroxine (or triiodothyronine). DIT, diiodotyrosine; Tyr, tyrosine.

Oxidation of Iodide

In order to become an iodinating agent, iodide must be oxidized. The nature of the iodinating species has not been determined with certainty; several models have been proposed (2). An early model was based on a one-electron oxidation that generated radicals of iodine and tyrosine bound to TPO, which then formed MIT, and in a subsequent reaction DIT (246,247). It is more likely that iodide is oxidized in a two-electron reaction to form either hypoiodite (OI-) (248,249,250), hypoiodous acid (HOI) (251,252), or iodinium (I+) (253,254,255,256).

 

Iodination and Coupling of Tyrosyl Residues

In the iodination reaction, only a subset of the 132 tyrosyl residues of the thyroglobulin dimer are iodinated, giving rise to MIT and DIT (118,257,258,259). During the coupling reaction, a tyrosyl residue donates its iodinated phenyl group to become the outer ring of the iodothyronine amino acid at an acceptor site, leaving dehydroalanine at the donor site (245,260). The location of the hormonogenic iodotyrosyl residues within thyroglobulin creates an optimal spatial alignment, facilitating the coupling reaction, and is highly conserved among various species (see Chapters 5) (4,118,258,259,261). The main hormonogenic acceptor sites in human thyroglobulin are at positions 5, 1291, 2554, 2568, and 2747 (4,118,258,259). Donor sites include tyrosine residues 130, 847, and 1448. The most important T4-forming site is located at tyrosine 5, and tyrosine 130 is the dominant donor site (262).

Iodination and the intramolecular coupling reaction have been studied in vitro using reconstituted systems containing TPO, thyroglobulin, radiolabeled iodide, and an H2O2--generating glucose/glucose-oxidase system (Fig. 4B.10)(2,245,247,256). Peroxidase-catalyzed iodination of selected tyrosine residues within thyroglobulin probably occurs on the enzyme (2,248,249,250,252,263). As in thyroglobulin from normal thyroid glands, MIT and DIT are the most abundant iodinated residues in these in vitro systems (264). Evidence for the catalytic role of TPO in the coupling reaction was obtained by incubating thyroglobulin labeled with iodine-131 with or without TPO (265). In the presence of TPO, the formation of T4 and T3 is significantly increased at the expense of a decrease in DIT and to a lesser extent in MIT (265). Analogous to rats fed an iodide-deficient diet (266), T3 formation is more pronounced at lower iodide concentrations (265). The coupling reaction is stimulated by low concentrations of free DIT (265,267), a phenomenon thought to be caused by oxidation of free DIT, which then facilitates electron transfer from peptide-linked DIT to the prosthetic heme group (268,269). Formation of T4 decreases with increasing iodide concentrations, which may play a role in the autoregulatory effect of iodide on thyroid function (245).

FIGURE 4B.10. Time course of iodination and of formation of diiodotyrosine, mono iodotyrosine, thyroxine (T4), and triiodothyronine (T3) in thyroglobulin iodinated under the conditions indicated. The thyroglobulin contained 0.038% iodine (two atoms iodine per molecule). The value for A410/A280 for thy roid peroxidase was 0.38. The reaction was started by addition of glucose oxidase (100 U/mg). (From Taurog A. Hormone synthesis: thyroid iodine metabolism. In: Braverman L, Utiger R, eds. Werner and Ingbar's The thyroid: a fundamental and clinical text, 8th ed. Philadelphia: Lippincott, Williams & Wilkins, 2000:61, with permission.)

While iodination may be catalyzed by the π-cation radical of compound I and coupling by the protein radical (270), it is more likely that both reactions are catalyzed by the π-cation radical form of compound I, at least under conditions of normal iodide intake (245). As summarized in Figure 4B.9, successive one-electron oxidations of hormonogenic tyrosyl residues in thyroglobulin, mediated by compound I and compound II of TPO, are proposed to result in the formation of DIT radicals (245,269,271). Lastly, in a nonenzymatic reaction, two iodotyrosyls, positioned in optimal spatial location, form an unstable quinol ether bond, which is rapidly rearranged to form an iodothyronine, leaving dehydroalanine at the position of the donor iodotyrosyl contributing the outer ring (Fig. 4B.11). In the formation of T4, the outer ring comes from a DIT residue, whereas in the formation of T3 it comes from MIT.

FIGURE 4B.11. Proposed scheme for coupling of diiodotyrosyl residues to form thyroxine within a thyroglobulin (Tg) molecule. The major hormonogenic site at tyrosyl residue 5 is indicated. (From Taurog A. Hormone synthesis: thyroid iodine metabolism. In: Braverman L, Utiger R, eds. Werner and Ingbar's The thyroid: a fundamental and clinical text, 8th ed. Philadelphia: Lippincott, Williams & Wilkins, 2000:61, with permission.)

Inhibition of Thyroid Peroxidase by Thionamide Drugs

The mechanisms underlying the inhibitory effects of thionamides on TPO-catalyzed iodination has been comprehensively reviewed by Taurog (2), and their role in the treatment of patients with thyrotoxicosis, which is discussed in Chapters 45, have been reviewed by Cooper (272).

The thionamides PTU, MMI, and carbimazole (CBZ), which is converted to MMI in vivo (273), are concentrated severalfold in the thyroid gland (274,275,276,277). The uptake of these drugs is stimulated by TSH and inhibited by iodide (2,278). Thionamides inhibit iodination and coupling (275,279,280). In vitro, low doses of MMI and PTU inhibit TPO-catalyzed iodination of thyroglobulin transiently and reversibly, whereas the effect of higher doses is irreversible (Fig. 4B.12)(279,280). The inhibi tory effect of thionamides is also dependent on the ratio between drug and iodide concentration. While it is irreversible at lower iodide concentrations, it is only tran sient at high iodide concentrations (280). Under conditions of reversible inhibition, thionamides are rapidly metab olized to higher oxidation products (281,282,283). Iodination resumes once metabolism of the drug is complete. Under these conditions, thionamides are com petitive inhibitors by competing with tyrosyls for oxidized iodide; oxidation of the drug is the favored reac tion. Conversely, thionamides are only partially oxidized at high drug/ iodide ratios (284). In this situation, TPO is inactivated, presumably by covalent binding of an oxidized form of the drug to the prosthetic heme group of the enzyme, and as a result iodination is irreversibly blocked (284). Data obtained from rats fed an iodide-deficient diet are consistent with this in vitro model, in that intrathyroidal metabolism of radiolabeled PTU and MMI is decreased (275).

FIGURE 4B.12. Scheme for mechanism of inhibition of thyroid peroxidase (TPO)-catalyzed iodination by methimazole (MMI). The reactions associated with reversible inhibition of iodination are shown on the left, irreversible inhibition on the right. The relative rates of reactions 2 and 3 determine whether inhibition of iodination is reversible or irreversible. If reaction 2 is predominant, TPO will be inactivated and inhibition of iodination will be irreversible. The inactivation probably involves covalent binding of an oxidized form of MMI to the heme group of compound I (CpdI). If reaction 3 is predominant, this will lead to extensive drug oxidation, permitting iodination to begin after the drug has been metabolized. Reactions 4 and 7 indicate that oxidized iodide (EOI)- acts both to oxidize MMI and to iodinate tyrosyl residues in thyroglobulin. Reversible inhibition depends on competition between MMI and tyrosyl for (EOI)-. Drug oxidation is the preferred reaction, and as long as sufficient drug is present, (EOI)- is diverted from iodination to drug oxidation. (From Taurog A. Hormone synthesis: thyroid iodine metabolism. In: Braverman L, Utiger R, eds. Werner and Ingbar's The thyroid: a fundamental and clinical text, 8th ed. Philadelphia: Lippincott, Williams & Wilkins, 2000:61, with permission.)

Thionamides have a more pronounced effect on iodothyronine formation than on iodotyrosine formation (285). This does not necessarily imply a specific inhibitory effect on coupling, since the formation of T4 requires preceding DIT formation. However, under selected in vitro conditions, such as low concentrations of iodide, TPO or H2O2, PTU and MMI may have an inhibitory effect on coupling without inhibiting iodination (285).

CELLULAR UPTAKE AND PROTEOLYSIS OF THYROGLOBULIN

Iodinated thyroglobulin is stored as colloid in the follicular lumen. In response to demand for thyroid hormone secretion, further processing of thyroglobulin requires its reen try into thyroid cells through vesicular internalization, i.e., micropinocytosis (Fig. 4B.1) (Chapters 5). Micropinocytosis may be initiated by both nonselective fluid-phase uptake and by receptor-mediated endocytosis (286,287,288,289). Fusion of the thyroglobulin-containing vesicles with lysosomes results in proteolytic breakdown of the thyroglobulin and release of T4, T3, and the iodotyrosines (4).

Digestion of thyroglobulin with lysosomal extracts results in the preferential release of hormone-rich fractions (290). Several endopeptidases have been identified in the thyroid. They include cathepsins D, B, L, and H (291). Inhibition of these enzymes blocks degradation of thyroglobulin, suggesting that they are important in thyroglobulin breakdown (292). After cleavage of thyroglobulin by endopeptidases, it undergoes further degradation by exopeptidases such as dipeptidyl-peptidases I and II, lysosomal dipeptidase I, and N-acetyl-l-phenolalanyl-L-tyrosine hydrolase (292). Digestion of thyroglobulin in vitro with the endopeptidase cathepsin B and the exopeptidase lysosomal dipeptidase I results first in the release of the dipeptide T4-glutamine, corresponding to the amino-terminal hormonogenic site 5 and glutamine at position 6, and subsequent release of T4 (293). The cysteine-rich type I motif of thyroglobulin, which has been identified as a potent inhibitor of cysteine proteases (120), may play an autoregulatory role in the digestion of thyroglobulin (119).

Aside from degradation of thyroglobulin in lysosomes, it can also be recycled back into the follicular lumen (294). The recycling of immature forms of thyroglobulin back to the apical membrane after endocytosis is thought to involve an asialoglycoprotein receptor (295). Thyroglobulin can also be transported from the apical to the basolateral membrane, where it is released into the bloodstream (296,297). This transepithelial transport or transcytosis is mediated by megalin, a receptor located on the apical membrane of the thyrocytes (288,289).

Ultimately, after degradation of thyroglobulin in the lysosomal pathway, T4 and T3 are secreted into the bloodstream at the basolateral membrane. Specific thyroidal channels mediating the transport of thyroid hormone across the basolateral membrane have not been identified.

DEIODINATION OF MONOIODOTYROSINE AND DIIODOTYROSINE

Minimal amounts of iodotyrosines, MIT and DIT, are released into the circulation, even though they are more abundant than T4 and T3 in thyroglobulin. The majority of these iodotyrosines are deiodinated by an intrathyroidal dehalogenase, and the iodide is recycled for hormone synthesis. This dehalogenase is thought to be an NADPH- dependent flavoprotein (298). An mRNA sequence encoding a putative iodotyrosine dehalogenase protein (DEHAL1), isolated from a thyroid mRNA library, has been identified (Genbank AY259176, AY259177) (299); the protein has not yet been characterized.

Dehalogenase Deficiency

Patients with congenital hypothyroidism and goiter caused by a defective iodotyrosine dehalogenase system have been identified (see Chapters 48). In these patients, MIT and DIT are not deiodinated, but rather leak into the circulation and are excreted in the urine. This leads to substantial loss of iodide, and, if the iodine supply is scarce, to goiter and hypothyroidism (300). The diagnosis of a defective dehalogenase can be formally established by administration of radiolabeled DIT. It is normally deiodinated, but in patients with a defective dehalogenase, most is excreted unaltered in the urine. These patients can be treated with large doses of iodide, as well as with T4. The disorder is inherited as an autosomal recessive trait.

ACKNOWLEDGMENT

Supported by 1R01DK63024–01 from NIH/NIDDK.

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