Bruno Di Jeso
Thyroglobulin (Tg) is the most highly expressed protein in the thyroid gland. Its vastly higher abundance than other proteins in thyroid extracts tends to yield an exaggerated estimate of its expression relative to that of other thyroid proteins, because most Tg in the thyroid gland is in fact extracellular, having been secreted into the lumen of thyroid follicles, where it is stored as colloid. Nevertheless, even when extracellular storage is not considered, Tg is still the predominant thyroid protein, and is the most important one, because its encoded structure provides for the coupling reaction that underlies the biosynthesis of the active thyroid hormones thyroxine (T4) and triiodothyronine (T3) (see section on thyroid hormone synthesis in Chapter 4).
Tg has a number of remarkable features. First, it falls within the top 1% of all proteins in terms of the molecular mass of its monomeric structure (~330 kDa). Approximately 10% of this mass (i.e., ~30 kDa) consists of covalently bound carbohydrates, the result of posttranslational modifications as Tg passes along the intracellular secretory pathway of thyroid follicular cells (thyrocytes). Indeed, Tg has long served as a model protein for analysis of the carbohydrate side-chain structures that are formed during N-linked glycoprotein processing (1). In addition, the polypeptide backbone of Tg, amounting to ~90% of its total molecular mass (i.e., ~300 kDa), consists of a signal peptide sequence plus ~2750 residues (± 5 residues, depending upon the species) (2). The secreted polypeptide contains at least 66 tyrosine residues (in human Tg, with additional tyrosines in other species). The percentage of tyrosine residues in Tg that become iodinated normally varies with the dietary iodine supply, but it is not unusual for 20% of them to contain iodine, with three quarters of these as residues of monoiodotyrosine (MIT) and diiodotyrosine (DIT) that are not used for the formation of T4 and T3 (3). Thus, Tg protein serves as the primary internal reservoir of recycling iodine in the body, upon which future biosynthesis of thyroid hormones is based.
PHYLOGENY AND ONTOGENY OF THYROGLOBULIN
The appearance of Tg in evolution is associated with thyroid hormone biosynthesis. A colloid-filled follicular lumen surrounded by thyrocytes (presumably a response to decreasing availability of iodide as animals migrated away from the sea to a terrestial environment) and the Tg protein are present in all vertebrates (4). During mammalian embryonic thyroid folliculogenesis (5), as well as folliculogenesis in reconstituted mammalian thyrocyte cultures and in vivo models, intracellular lumens (membrane-bound internal cavities) form within thyrocytes. The cells then associate with one another, after which the intracellular lumens are externalized to form a central, apical extracellular space, which is a hallmark of Tg storage (6). The intracellular lumens have a close physical relationship with the Golgi complex through which newly synthesized Tg traverses (7), and iodination of Tg can occur within them (8), resulting in thyroid hormone formation [nevertheless, their quantitative contribution to overall iodination or hormonogenesis is probably insignificant, because the intracellular availability of hydrogen peroxide in vertebrates is limited (9)]. Although not well studied, some sea-dwelling prevertebrates have specialized pharyngeal epithelial cells that do not aggregate to form obvious follicles (perhaps such cells bear weak evolutionary relationships to salivary epithelial cells that exhibit basolateral iodide uptake and do not enclose an apical lumen), but do form Tg-like iodoproteins (10). Any such means to produce and secrete iodoproteins would not efficiently conserve iodide. Nevertheless, even in prevertebrates, formation of T4 and T3 is likely to be linked to a Tg evolutionary precursor (11), and within hagfish or lamprey eels, Tg-like protein is already present (12) along with demonstrable follicles. These findings suggest evolutionarily conserved roles for Tg protein in iodine storage (particularly in land-dwelling and freshwater organisms) and iodothyronine formation. Cloning and sequencing of Tg-like complementary DNA (cDNA) derived from prevertebrates could provide valuable new insights into the evolutionary origins of T4-forming sites and the domain structure of Tg molecules.
THE THYROGLOBULIN GENE AND ITS MESSENGER RNA
The functionality of the Tg messenger RNA (mRNA) was initially established when a 33S thyroidal mRNA fraction injected into Xenopus oocytes resulted in production of immunoreactive Tg protein in the eggs (13), while a similar 33S thyroidal mRNA fraction obtained from a strain of Dutch goats with congenital goiter did not result in production of detectable Tg protein (14). The complete Tg mRNA sequence was eventually deduced by sequencing of fragments of bovine Tg cDNA (2) and those of other species (Fig. 5.1), which allowed for many structural insights (15) and confirmed that there is only one Tg gene in mammals. The human Tg gene spans approximately 300 kB on the long arm of chromosome 8, of which only ~3% represents the exons [48 of them (16)], separated in many instances by large intronic sequences. Mutations within introns as well as imprecision in the mRNA splicing mechanism have led to the possibility of production of Tg splice variants with no change in Tg exon sequence (2,17,18,19). Alternative splicing is also promoted by increasing thyrotropin (TSH) stimulation (20). This might serve as an evolutionary adaptation to the high frequency of variations in the Tg coding sequence in the population (21), some of which might be less competent for hormonogenesis (22). Nevertheless, TSH action on its receptor is not an absolute requirement for Tg production (23), although there are requirements for the transcription factors TTF-1, TTF-2 (24), and Pax-8 (25), in conjunction with other factors (26). There are also reports of patients with congenital goiter whose thyrocytes may not maintain a sufficient steady-state level of Tg mRNA (27,28). While this could represent a class of transcriptional defects (29), active translation (polyribosome formation) is one of the critical features promoting mRNA stability. Therefore, poorly translated Tg mRNAs (from splice forms, or from exon sequence changes bearing frame-shifts and/or premature termination codons) are likely to correlate with low mRNA abundance of those forms.
FIGURE 5.1. The nucleotide and encoded 2746 amino-acid sequence of murine thyroglobulin (Tg) (described in single-letter code), derived from Genbank accession number AF076186. In addition, the location of selected point mutations in the coding sequence of Tg associated with thyroid pathology in several different species has been identified on this map by the normal amino acid presented in enlarged, bold type. In order, from amino to carboxyl terminus, this includes: a premature stop codon at position 277 in human Tg (260), a premature stop codon at position 296 in goat Tg (261), a premature stop codon at position 697 in bovine Tg (106), a Glu to His change in exon 10 in human (126,127), a Cys to Arg substitution at position 1263 in human Tg (124), a stop codon at position 1510 in human Tg (28), an Arg to His substitution at position 2223 in human Tg (123), a Leu to Pro substitution at position 2263 in murine Tg (112), and a Gly to Arg substitution at position 2320 in rat Tg (120,121).
The development of Tg cDNA expression vectors for mammalian cells (30) has allowed the study of full-length recombinant versions leading to Tg secretion from heterologous cells (31). Such an approach overlooks the complexity of Tg mRNA splicing, although it increases focus on the exon sequences. It is important to note that the Tg protein has never been studied by x-ray crystallography, because crystals of the pure protein have not been obtained, perhaps due to the numerous posttranslational modifications of Tg (see later in the chapter). Thus, insights into the internal structure of the Tg protein have been limited largely to primary sequence analysis—although negative-staining electron microscopy has demonstrated that a major form of Tg isolated from thyroid gland is dimeric (32).
REGIONAL STRUCTURE OF THYROGLOBULIN DOMAINS DEDUCED FROM THE PRIMARY SEQUENCE
The (12S) Tg monomer in all vertebrates is comprised mainly of four regions (Fig. 5.2), the first three containing distinct cysteine-rich repeats (2). Region I, found within the first ~1200 residues, contains 10 of the 11 so-called “Tg type 1 repeats” of roughly 60 amino acids each. Each repeat surrounds a central WCV (Trp-Cys-Val) sequence plus at least three additional cysteines (one expanding the motif to CWCV, in which each of the two Cys residues are partnered into different disulfide bonds), thereby shaping and stabilizing the domain. Type 1A repeats have a total of six cysteine residues in the domain (three disulfide bonds), which are also found in several other proteins, including equistatin and the thyropin family of cysteine protease inhibitors (33,34), entactin/nidogen (35), insulin-like growth factor-binding protein-6 (IGFBP-6) (36), and the carcinoma-associated antigen GA733–2 (37). Type 1B repeats have only four cysteine residues (38). The disulfide bond pattern Cys1-Cys2, Cys3-Cys4, Cys5-Cys6 prevails in all Tg type 1A repeats (37) and the sequence homology between different proteins bearing the repeat (Fig. 5.3) suggests that this domain has evolved with a common fold designed to be protease resistant and to function as a protease inhibitor/regulator (39). Region II (Fig. 5.2) contains the 11th Tg type 1 repeat, while region III contains the so-called type 3 repeat units that have little or no homology with other proteins in existing databases (2). Because cysteine side chains are internally located within the various Tg repeats, they form a series of domains internally “stapled together” (via disulfide bridges) while being tethered end-to-end with intervening peptide sequences. Consistent with such a view, limited digestion using a variety of proteases tends to cleave between the repeats, liberating them intact from the parent Tg molecule (40).
FIGURE 5.2. Sketch of the regional structure (below), including repeat domains (above), in the primary sequence of Tg. Note that the region and domain alignments are not drawn precisely to scale. As shown in the sketch (below), the amino-acid sequence of Tg begins with a cleavable signal peptide (open box), the conserved thyroxine hormonogenic site A (see text) (hatched box), the ten type 1 (A+B) repeat domain units contained in region I, the Cys-rich region II which comprises the 11th type 1 repeat, the region III with repeat units that do not closely match known sequences in the database, and the acetylcholinesterase-homology region followed by the hormonogenic site C (see text) (hatched box).
FIGURE 5.3. An alignment of polypeptide sequences containing thyroglobulin (Tg) type 1 repeats, showing the relative positioning of the conserved Cys residues. The last sequence shown is derived from human Tg.
The fourth and most carboxyl-terminal region, the acetylcholinesterase (AChE) region, (Fig. 5.2) Tg has 31% identity and 47% similarity to the entire length of acetylcholinesterase (41,42). All six cysteine residues involved in cholinesterase intrachain disulfide bonding are conserved in Tg (43), and it is likely that these two proteins share a common folding pattern in their respective tertiary structures.
Altogether, human (and bovine) Tg has 122 cytseines (mouse Tg has 121 Cys residues). When studied by nonreducing SDS-PAGE, the mobility of monomeric Tg increases dramatically, indicating that its molecular radius decreases progressively as a function of time after Tg biosynthesis (44), in comparison with the mobility of the same Tg molecules that have been fully “opened” (i.e., linearized) by reducing all disulfide bonds with dithiothreitol (Fig. 5.4). These results indicate that the vast majority of cysteine thiols are involved in intradomain disulfide pairing within the four major regions of Tg (45). That few unpaired Cys residues remain (46) may explain why Tg homodimers are not covalently associated during transit through the intracellular secretory pathway of thyrocytes (47,48). Nevertheless, extracellular Tg may begin to form intermolecular disulfide bridges when it reaches the oxidizing/iodinating environment of the follicular lumen, resulting in cross-linked dimers (19S), tetramers (27S), and higher order assemblies (49).
FIGURE 5.4. Kinetic resolution of the maturation of intrachain disulfide bonds of thyroglobulin (Tg). As disulfide bonds are formed within newly synthesized Tg and the structure of the various repeat domains is reorganized, the molecular radius of Tg decreases. When subjected to acrylamide gel electrophoresis under nonreducing conditions, this Tg penetrates further into the gel (lower band). When the Tg protein is intentionally denatured and all disulfide bonds are disrupted, and the protein subjected to electrophoresis under reducing conditions, the penetration of the newly synthesized Tg is unchanged at all times shown. (From Di Jeso B, Ulianich L, Pacifico F, et al. The folding of thyroglobulin in the calnexin/calreticulin pathway and its alteration by a loss of Ca2+ from the endoplasmic reticulum. Biochem J 2003;370:449, with permission.)
THYROGLOBULIN TRANSLATION AND INITIAL POLYPEPTIDE FOLDING
The initial stages of Tg biosynthesis are similar to those of other secretory glycoproteins. During translation, nascent Tg polypeptide chains, while still emerging from the ribosome-studded polyribosomes, are directed to the cytosolic side of the endoplasmic reticulum by virtue of the Tg signal peptide sequence that promotes association with the signal recognition particle and its receptor in the membranes of the endoplasmic reticulum (50). From there, the nascent Tg polypeptide is translocated into the lumen of the endoplasmic reticulum via the intramembrane Sec61 protein complex, with assistance from molecular chaperones on the lumenal side (51). After translocation of ~100 amino acids, the Tg signal peptide is excised by a specific protease (52), while ongoing translation is coupled to the translocation of the large remaining nascent polypeptide.
Immediately upon entry into the endoplasmic reticulum, the linear Tg polypeptide begins to be converted into a native three-dimensional structure. This process is more extensive than coupled translation-translocation, in the sense that while a small peptide hormone precursor such as proinsulin achieves its native state after no more than a few minutes within the endoplasmic reticulum (53), for Tg this takes about an hour. During this process (54), many of the repeat domains are likely to be buried interiorly within the tertiary structure of Tg, based on the fact that it requires progressively increasing concentrations of reducing agents in vitro to gain access to these domains in the native structure (55). Few of the details of the Tg folding process are known, but the basic steps are likely to be similar to those of other secretory glycoproteins. For example, at an estimated translation rate of 6.5 amino acids per second, one can deduce that it should take ~7 minutes for the entire nascent Tg polypeptide to be delivered into the endoplasmic reticulum. In this case, many of the repeating units of region I of Tg will have been engaged in folding for several minutes before some of the downstream domains of Tg arrive within the lumen of the endoplasmic reticulum. Thus, initial folding of the type 1 repeats is likely to be independent of later downstream domains encoded in the Tg sequence. Along the same lines, when the domain of Tg homologous to AChE reaches the endoplasmic reticulum, its initial folding is likely to be independent from that of the N-terminal domains of Tg that are already beyond their earliest stages of folding. Quite possibly, regional folding may even be sequential (56).
The slowness with which nascent Tg buries its many cysteine side-chains while forming a compact structure (Fig. 5.4) suggests that there is ample opportunity for mispairing of Tg disulfides, and indeed there is evidence that some nascent Tg complexes have mispaired intermolecular disulfide bonds (54,57,58). These disulfide-linked complexes are apparently “on-pathway” in normal Tg folding, because their disappearance correlates with the appearance of Tg monomers (and later, dimers). Resolution of disulfide mispairings is likely to require the involvement of protein disulfide isomerase (PDI) or other members of the superfamily of endoplasmic reticulum oxidoreductases (59,60,61,62).
Additional molecular chaperones of the endoplasmic reticulum also play distinct roles in facilitating the early folding of nascent Tg. One member of the heat-shock protein-70 family, known as BiP (63), has been identified repeatedly in protein interactions with Tg both in vitro and within the endoplasmic reticulum (57,64,65,66,67,68,69). BiP is a glucose-regulated protein (GRP), i.e., it is transcriptionally and translationally up-regulated when the extracellular glucose concentration is low (70), when glycosylation is compromised by energy depletion (71) [such as in states of compromised blood flow (68)], or when misfolded Tg or other secretory proteins begin to accumulate in the endoplasmic reticulum for any other reason (such as alterations in redox environment or lumenal calcium concentrations) (72,73). GRP94, a member of the heat-shock protein-90 family that is located in the endoplasmic reticulum and regulated like BiP, also associates with nascent Tg (67). Neither BiP nor GRP94 actively participate in the process of folding Tg (31,74); rather they reversibly associate with newly synthesized Tg molecules, with preferential binding to short hydrophobic sequences, to prevent other inappropriate hydrophobic interactions. Merely by limiting the opportunity for “off-pathway” folding events, the association of the endoplasmic reticulum chaperones promotes “on-pathway” folding of Tg, thus improving the overall yield of native Tg. As this is crucial to the success of secretory protein production in all cells, several (but not all) endoplasmic-reticulum chaperone proteins are essential for life among eukaryotes (75), and the control of their expression is tightly regulated (76,77). Additional chaperones that interact with Tg during its maturation in the endoplasmic reticulum are still being discovered, such as GRP170 (69) and ERp29 (78).
ASPARAGINE-LINKED GLYCOSYLATION AND ITS ROLE IN THYROGLOBULIN FOLDING
Potential asparagine (N)-linked carbohydrate acceptor sites are encoded by the sequence Asn-Xxx-Ser/Thr (Xxx is any amino acid), each of which may accept transfer of a pre-assembled “core oligosaccharide” containing 14 monosaccharide units attached to the asparagine side chain. There are 20 such potential acceptor sites in the sequence of human Tg (and mouse Tg); on average, 16 of these sites are actually utilized by oligosaccharyl transferase (79). This varies between species and amongst the population of Tg molecules within a single species (80), e.g., bovine Tg has a lesser degree of N-glycosylation (81).
One critical link between N-glycosylation and Tg stability stems from the discovery that a family of endoplasmic-reticulum chaperones known as calreticulin and calnexin function as lectins, that is, they recognize sugars (82). Calreticulin is abundantly expressed in the lumen of the endoplasmic reticulum, while calnexin is an abundant integral membrane protein whose lumenal domain is highly homologous to calreticulin (83). The initially added N-linked core glycan has a (GlcNAc)2(Man)9(Glc)3 structure in which the nine mannose (Man) residues form a three-pronged branching structure, and all three glucose (Glc) residues are stacked at the terminus of one branch (Fig. 5.5). This is followed by stepwise removal of all three terminal glucose residues via the sequential actions of the endoplasmic-reticulum glucosidases I and II. During the short period when the core oligosaccharide structure has a single terminal glucose, it is a substrate for binding to calreticulin or calnexin. Both chaperone lectins have the same oligosaccharide-binding specificity, although only one lectin may be bound to the carbohydrate at any one time. Ultimately, removal of the final glucose residue destroys the calreticulin/calnexin binding site. However, if the domain in Tg (or other substrate glycoprotein) has not folded, it can be recognized by UDP-Glc glycoprotein glucosyl transferase (UGT) (Fig. 5.5), a critically important enzyme that adds a single glucose residue back to the same site on the core oligosaccharide (84,85). This in turn re-forms the calreticulin/calnexin binding site, so that chaperone association may proceed again, in a cyclical manner, until the domain has folded to the point that it is no longer a substrate for re-glucosylation by UGT (because UGT is active only on unfolded Tg and not on properly folded Tg) (86,87). Meanwhile, mannosidase activities of the endoplasmic reticulum initiate trimming of Man residues from the other oligosaccharide branches (1), an event that usually occurs in preparation for further modifications that will take place when Tg reaches the Golgi complex (see later in the chapter). The binding of Tg to calnexin under normal conditions is relatively modest (65), because some calnexin dissociation occurs cotranslationally (65,88) and also because calreticulin may be a more important lectin-chaperone for Tg (44).
FIGURE 5.5. Early N-linked carbohydrate-processing events on newly synthesized thyroglobulin (Tg). As the nascent Tg polypeptide enters the lumen of the endoplasmic reticulum, a high fraction of its encoded potential Asn-linked glycosylation acceptor sites bind covalently to a 14-unit oligosaccharide from a lipid-linked donor. Three terminal glucose residues (G) on one branch of the oligosaccharide structure are the site of attack of endoplasmic-reticulum glucosidases I and II. A monoglucosylated oligosaccharide is the site of binding of the endoplasmic reticulum lectin chaperones calreticulin or calnexin. These lectin chaperones are also associated with the thiol reductase ERp57, which assists in the formation/isomerization of Tg disulfide bonds, further facilitating Tg folding (see text). If a glycosylated Tg domain remains unfolded after the sole remaining glucose residue has been removed, this domain serves as a potential substrate for UDP-glycoprotein glucosyl transferase (UGT). UGT re-adds a single glucose residue to the carbohydrate structure, restoring its capacity to bind calreticulin/calnexin and associated ERp57. Ultimately, for Tg that is properly folded, mannose (M) trimming by mannosidases begins in the endoplasmic reticulum and continues in the Golgi complex. For Tg that is misfolded, mannose trimming by endoplasmic-reticulum mannosidase I precedes a process of endoplasmic reticulum-associated degradation that occurs via the ubiquitin-proteasome system (see text).
An additional special feature of calreticulin or calnexin binding is that it brings the folding of nascent Tg into proximity with ERp57, a thiol reductase member of the PDI superfamily that catalyzes formation/isomerization of disulfide bonds (89). Thus, ERp57 disulfide-isomerase function is also dependent upon the same early steps in N-linked carbohydrate processing. Because formation/isomerization of mispaired disulfide bonds is of great importance in the folding of newly synthesized Tg, the activity of ERp57 and the proper processing of the N-glycans of Tg (90) must be viewed as key steps ensuring an adequate rate and efficiency of this process. At the conclusion of the extended period of intrachain disulfide bond formation, which may be rate limiting in achieving proper regional domain structures, the tertiary structure of nascent Tg monomers is now suitable for homodimerization, and this proceeds before exit from the endoplasmic reticulum (54,57,64,91).
These considerations provide a molecular explanation for the established relationship between N-linked glycosylation and the stability of Tg, which in turn affects the efficiency of Tg iodination (92,93). In relation to this, perturbations of divalent cation concentrations in the secretory pathway, particularly those of Ca2+ (and perhaps Mn2+) may destabilize Tg structure (94). Some of this effect may be indirect, because calcium depletion from the endoplasmic reticulum alters carbohydrate processing (95,96,97), reduces Tg binding to calreticulin and calnexin (44), and greatly inhibits export of Tg from this compartment (91). This may be caused by a diminished rate of isomerization/formation of the Tg disulfide bonds needed for folding; under improper glycosylation conditions, increased residence time of newly synthesized Tg in the endoplasmic reticulum correlates with its prolonged association with BiP and GRP94 (44,57,69).
CONGENITAL GOITROUS HYPOTHYROIDISM DUE TO THYROGLOBULIN MISFOLDING
Even without global effects on the environment of the endoplasmic reticulum, mutations altering the amino acid sequence of Tg could yield phenotypes similar to those described earlier. Patients and animals with congenital goiter and hypothyroidism who have abnormal Tg molecules have been described (98,99). In some instances, the endoplasmic reticulum in the thyrocytes was dilated or swollen (100,101), with little or no stainable Tg in the colloid (102), but knowledge of the specific molecular defects at the genetic level was lacking (103). The finding of nonfunctional Tg mRNA was a first step in this direction (14). These disorders are inherited as autosomal recessive traits in virtually all humans and animals. The mating, without inbreeding, of phenotypically unaffected heterozygotes yielding affected compound heterozygous progeny is such a rare event that it may initially be confused with autosomal dominant inheritance (104) (see Chapters 20 and 48). Patients with these disorders may have normal or low levels of Tg, depending on how well their Tg mRNA can be translated (105,106).
Unlike the Tg splicing and frame-shift mutants that have been described (21), there are relatively few descriptions of single missense mutations (amino acid substitutions). Thus, what have been called “Tg synthesis defects” are not typically explained by mutations in the coding region of the Tg gene (22). Only a few particular examples of these defects are described here.
The homozygous cog mutation in mice results in the murine version of cretinism with microcephaly and hypomyelination (107). In addition, the animals are runts, with little or no detectable 19S Tg in their thyroid gland (108). Instead, a peak at 12S extends to above the 27S position, indicating defective dimerization and formation of abnormal Tg complexes, and very little Tg is iodinated (108,109,110). Serum TSH concentrations are very high, and there is massive thyrocyte hyperplasia and hypertrophy, with a swollen endoplasmic reticulum and increased expression of multiple molecular chaperones (111). The folding and dimerization defect, which is to some extent ameliorated at lower temperature (111), is the result of a single L2263P (leucine→proline) substitution in the AChE-homology domain of Tg (112). The misfolded mutant protein is primarily retained within the endoplasmic reticulum until the carbohydrate is trimmed by the endoplasmic-reticulum mannosidase I (112A), after which it is translocated back to the cytosol (113) for degradation by the ubiquitin-proteasome system.
A similar situation seems to prevail in dwarf (rdw/rdw) rats. These rats were initially thought to have growth hormone deficiency (114,115), but in fact their growth failure is secondary to congenital hypothyroidism (116,117). Although their serum TSH concentrations are not as high as those of cog/cog mice, the thyroid glands of rdw/rdw rats also have massive expansion of the endoplasmic reticulum (118) (Fig. 5.6), which contains high levels of multiple molecular chaperones (119). There is likely to be a similar defect in Tg folding and dimerization as in cog/cog mice, as the mutation in rat Tg proved to be due to a single G2320R (glycine→arginine) substitution (120), less than 60 residues away from the site of the cog mutation within the AChE-homology domain that blocks Tg secretion (121). The rdw/rdw rats have little thyroid gland enlargement, suggesting that accumulation of the mutant Tg protein may limit thyroid growth (122).
FIGURE 5.6. Electron photomicrograph a thyrocyte of a rdw/rdw rat, showing enlarged endoplasmic reticulum–derived vacuoles that overcrowd the remaining cytoplasm (arrow). Thyrocytes from some humans with congenital goiter due to defective Tg synthesis have a similar appearance (5,600× magnification). (From Sakai Y, Yamashina S, Furudate SI. Missing secretory granules, dilated endoplasmic reticulum, and nuclear dislocation in the thyroid gland of rdw rats with hereditary dwarfism. Anat Rec 2000;259:60, with permission.)
In humans, single Tg amino-acid substitutions involved in congenital hypothyroid goiter have been reported only rarely. One mutation was in the AChE-homology domain (123); others have involved replacement of a conserved cysteine residue. The homozygous C1263R mutant resulted in only mild hypothyroidism, indicating that the mutant Tg protein can be iodinated and serve as a substrate for hormonogenesis (124). Given the importance of intrachain disulfide bonding within Tg (Fig. 5.4), folding defects associated with replacement of single Cys residues are easy to rationalize.
Simple goiter without hypothyroidism has also been associated with production of defective Tg (125), such as the missense mutation resulting in a glutamine to histidine substitution within exon 10 of the Tg gene (126,127). A variant type of adenomatous goiter, without hypothyroidism, has been attributed to the C1995S (cysteine→serine) mutation (128). By analogy to the cog/cog mice, which as adults have normal serum T4 concentrations (108), humans in whom the Tg is only slightly defective may, by increasing the total number of thyrocytes contributing to hormonogenesis, compensate for the reduced efficiency of hormonogenesis within individual thyrocytes, so that euthyroidism is achieved at the expense of goiter.
POSTTRANSLATIONAL MODIFICATIONS OF THYROGLOBULIN IN LATER STEPS OF THE SECRETORY PATHWAY
As Tg migrates to the Golgi complex, roughly two-thirds of the N-linked core glycans become modified to a “complex glycan” structure (129). This includes additional trimming of mannose residues from the core glycan branches by alpha-mannosidase-II and endomannosidase (130), followed by addition to these branches of other carbohydrates (131), including N-acetylglucosamine, galactose, fucose, sialic acid (132,133), or sulfate (134,135) or phosphate (136,137). [Independently of this, sulfation and phosphorylation may also occur directly on the Tg polypeptide backbone (136,138,139)].
In addition, human Tg has been reported to undergo “O-linked” glycosylation; in this case, the carbohydrate is attached to the side chain of serine or threonine (1). Such O-linked carbohydrate has various structures, including typical glycosaminoglycan chains that may also be sulfated (140,141).
Knowledge of these additional post-translational modifications of Tg has helped to identify normal and abnormal Tg maturation at defined points along the secretory pathway. As an example, both protein and carbohydrate sulfation are thought to occur primarily within the trans-Golgi network (142). Therefore, perturbed secretion of Tg that had been radiolabeled with 35S-methionine might represent delayed export from the endoplasmic reticulum, whereas perturbed secretion of Tg that had been radiolabeled with 35S-sulfate might represent a delay in a late Golgi or post-Golgi step of the Tg secretory pathway. A similar investigative tool involves in vitro digestion of Tg with endoglycosidase H (endo H), which will cleave N-linked oligosaccharides representative of the initial core carbohydrate structure within the endoplasmic reticulum. As Tg contains carbohydrates amounting to ~30kDa of its molecular mass, endo H digestion of Tg that has been confined to the endoplasmic reticulum compartment results in a substantial decrease in molecular mass, observed as an increased mobility of the Tg band by SDS-PAGE. By contrast, when the mannose-trimmed core oligosaccharide becomes modified in the Golgi complex by addition of N-acetylglucosamine (the first of the “complex sugars” to be added), that oligosaccharide can no longer be digested by endo H. Consequently, on endo H digestion, Tg that has migrated to the Golgi compartment loses much less molecular mass, corresponding to only the ~one-third of N-linked core glycans that did not become modified to a “complex glycan” structure. The ratio of this endo H-resistant Tg to endo H-sensitive Tg in the steady state, as well as the rate of acquisition of endo H-resistance in kinetic studies, are both useful indicators of the efficiency of Tg export from the endoplasmic reticulum. Typically, in congenital goitrous hypothyroidism due to a mutation in Tg that affects its folding, the Tg protein never acquires endo H-resistance (Fig. 5.7).
FIGURE 5.7. Thyroglobulin (Tg) glycans normally acquire resistance to digestion with endoglycosidase H upon exit from the endoplasmic reticulum, but defective Tg that does not migrate to the Golgi complex remains persistently endo H-sensitive. A: Newly synthesized Tg in normal (Nl) murine thyroid tissue comprises both post-Golgi, endo H-resistant Tg (R) and pre-Golgi, endo H-sensitive Tg (S). In thyrocytes from cog/cog mice the newly synthesized Tg does not acquire endo H-resistance, being comprised entirely of endo H-sensitive molecules. B: Consistent with its inability to exit the endoplasmic reticulum, newly-synthesized Tg from cog/cog thyrocytes in primary culture disappears from the cells over a one-day period (by a process of endoplasmic reticulum-associated degradation) without being detectably secreted into the extracellular space. (From Kim PS, Hossain SA, Park YN, et al. A single amino acid change in the acetylcholinesterase-like domain of thyroglobulin causes congenital goiter with hypothyroidism in the cog/cog mouse: a model of human ER storage diseases. Proc Natl Acad Sci U S A 1998;95:9909, with permission).
The biological functions of the aforementioned post-translational modifications of Tg glycans (or polypeptide) in the Golgi complex are not clear. One hypothesis is that carbohydrate modifications may control the intracellular targeting of Tg in later stages of the intracellular secretory or endocytotic pathways. For example, Tg that is secreted to the apical side of thyrocytes (i.e., the lumenal side) may have in some of its carbohydrate side chains the lysosomal recognition marker mannose-6-phosphate (143). If so, endocytotic degradation of Tg, which is essential for hormonogenesis, might be directed initially by the well-known mannose 6-phosphate receptors (144); however, this hypothesis has not been confirmed. A second hypothesis is that biosynthetic sulfation (or phosphorylation) of tyrosyl residues could alter the capacity of Tg for subsequent iodination and hormonogenesis (145,146). Such a view is not easily reconciled with the very low degree of tyrosine sulfation or phosphorylation in Tg (134), although, theoretically, these ions could be displaced from Tg at the time of iodination or coupling (147). There are other hypotheses (some described below), but the general view of these Golgi-type post-translational modifications is that they have further structural effects on Tg, such as facilitating multimerization (148), and surface charge effects that increase the solubility of Tg (48).
TSH clearly regulates the carbohydrate processing of Tg, affecting both the number of N-linked glycosylation sites that are initially utilized and the subsequent modifications of those glycans to a complex structure (135,149,150). This may include an increase in the number of oligosaccharides bearing galactose and sialic acid, as well as alterations of sulfation. Perhaps the most interesting of these changes is in respect to the domain structure of Tg. Specifically, in region I (the first ~1200 amino acids) of bovine Tg there are 6 sequential N-linked glycans, and these bear complex or hybrid-type oligosaccharides (at positions 91, 464, 476, 835, 928, and 1121) (81). The situation appears essentially identical in human Tg (79). Thus, a TSH-stimulated increase in the number of complex oligosaccharide units is likely to have a more profound structural effect on the C-terminal half of the Tg molecule. Future studies should focus on the last two or three N-glycosylation sites of Tg [i.e., positions 2104 and 2277 of bovine Tg and positions 2275 and 2562 of human Tg, which are ordinarily high mannose units (79,81)], because changes in glycosylation near the C-terminus of Tg might create structural alterations that would increase the exposure of the C-terminal iodination site and thereby help to account for the increase in production of T3 that results from increased TSH secretion or the action of TSH receptor-stimulating antibodies in patients with Graves' thyrotoxicosis.
THYROGLOBULIN TRAFFICKING IN THE EXOCYTOTIC PATHWAY
Thyrocytes are highly polarized epithelial cells with distinct apical and basolateral regions (151). The secretion of newly synthesized proteins by these cells is directed to both regions (152,153), but Tg is transported almost completely through the apical region into the follicular lumen (154). The apical targeting and storage of Tg is known to go awry in Graves' disease, thyroiditis, and many thyroid cancers. Still, very little is known about the molecular mechanisms of sorting that allow for normal Tg targeting to the apical surface of the cells for iodination, even while other proteins are selectively targeted basolaterally (155,156,157,158,159,160). The apical secretory pathway of polarized epithelial cells may be created in the trans-Golgi network by the creation and merging (in the plane of the membrane) of lipid-protein microdomains known as rafts, which serve as a platform upon which apical secretory proteins can be ferried (161). These rafts are especially enriched in glycosphingolipids. Two key features of the rafts are that they are not solubilized at 4°C in the presence of 1% Triton X-100 (unlike other membranes as well as a large fraction of cellular proteins that are soluble in the presence of this non-ionic detergent), and because they are lipid-rich they rise to the top of a sucrose-flotation gradient. Several newly synthesized apical-plasma-membrane proteins are recovered in the raft fraction (162). The raft fraction has been used as a source of antigen to identify novel proteins that may be involved mechanistically in membrane transport to the apical cell surface. One such protein, VIP17 (163), also known as MAL (164), is a membrane protein whose expression in some epithelial cells is correlated with the efficiency of apical-plasma-membrane protein delivery (165). MAL is expressed in human thyrocytes both at the mRNA and protein level (where it is restricted to the apical zone of thyroid follicles), as well as in some thyroid-cell culture lines such as FRT, in which it is recovered in the raft fraction (166). These features raise the possibility of Tg transport via a raft pathway from the Golgi complex to the thyroid follicular lumen (163).
In PC Cl3 thyrocytes, the steady-state pool size of Tg in the Golgi complex or in post-Golgi compartments is small, as compared with that in the endoplasmic reticulum. Nevertheless, from the endo H-resistant Tg pool (i.e., Tg having passed through the Golgi complex), most is recovered in Triton X-100 insoluble rafts, in spite of the fact that secreted Tg is completely soluble in Triton X-100 (167). The endo H-resistant portion of recombinant Tg expressed in heterologous epithelial cells is also recovered in a raft fraction, and depletion of MAL from these cells inhibits apical secretion of Tg (168). These findings suggest that Tg may be actively selected for apical delivery by association with one or more other raft components. Although the mechanism of this selection is unknown, it may be mediated by one or more novel lectins (169). One such lectin isolated from the raft fraction, known as VIP36, specifically binds N-acetylgalactosamine (170,171), raising again the idea that carbohydrate modifications of Tg occurring in the Golgi could serve as recognition signals for post-Golgi protein trafficking. One potentially serious problem with this model is that treatment of thyrocytes with various inhibitors of carbohydrate processing (e.g., castanospermine) does not prevent the apical secretion of Tg (90,172). Additionally, several basolaterally secreted thyroid proteins also have N-linked glycans (55,160), and it remains to be proven that carbohydrate processing of these proteins in the Golgi is appreciably different from that of apically secreted glycoproteins.
The sheer abundance of Tg is likely to stoichiometrically overwhelm the expression level of any putative Tg sorting receptor for apical secretion. However, if Tg were to undergo higher-order self-assembly while still within the Golgi complex (which presently is merely speculation) (148), such a mechanistic problem might be circumvented. In fact, such a mechanism appears to exist in other peptide hormone-producing endocrine cells that must package high concentrations of a subset of newly synthesized protein products into secretory granules (173). Tg has certain features that suggest it might be incorporated into classical secretory granules, including the presence in thyrocytes of Tg secretory compartments that have been described as apical vesicles or secretory granules (174,175). In addition, Tg exocytosis is to some extent acutely stimulated by TSH both in vivo (176) and in primary thyrocyte cultures (156,157,158,160,177). Moreover, in filter-polarized thyrocyte monolayers, certain heterologous (recombinant) peptide hormones are also secreted apically (153). The recent observations that an increasing number of classical secretory granule proteins also may utilize the raft mechanism for their Golgi-based sorting makes such a possibility plausible for Tg as well (172,173,174,175,176,177,178,179,180,181,182).
IODINATION AND IODOTYROSYL COUPLING WITHIN THYROGLOBULIN
After exocytosis, especially after stimulation with TSH (175,183), Tg that has fully matured within the secretory pathway undergoes iodination in the lumen of the thyroid follicles. It is nearly impossible to study iodination and hormonogenesis in human Tg in vivo. While this can be done in vivo in animals (especially rodents), there are likely to be differences in Tg structure (e.g., glycosylation) and iodination among different species. Normally (as described above), only a subset of the tyrosine residues of Tg becomes iodinated (184), and the fraction that is iodinated is further diminished in iodide-poor environments (see section on iodine deficiency in Chapter 11).
It is important to understand how the iodination sites are selected, because it has long been recognized that iodination of Tg provides a much higher yield of the hormonally active iodothyronines, T4 and T3, than iodination of any other protein, and that most of the specificity of the iodination sites (and subsequent coupling to form iodothyronines) is somehow encoded in the structure of Tg itself (13).
While denatured Tg can be readily iodinated in vitro, it is no longer competent for hormonogenesis (185,186), indicating that there are stringent steric requirements within Tg that allow thyroid peroxidase to catalyze efficiently not only iodination but also formation of T4 and T3 from the iodotyrosyl residues (13). By contrast, T4 and T3 synthesis in vitro can be catalyzed not only by thyroid peroxidase but also by myeloperoxidase (187) and lactoperoxidase (188), although the efficiency of hormonogenesis and maintenance of the integrity of Tg structure varies with the conditions used. Provided certain critical domains of Tg are in the native state, iodination of isolated (iodine-poor) Tg in vitro probably provides a relatively faithful estimation of the iodotyrosyl selectivity that occurs in vivo (189). Using this methodology, efforts have been directed to explore the hypothesis that hormonogenic sites occur at limited and fixed positions in the Tg protein, involving discrete donor and acceptor sites that have relatively precise physical and spatial relationships. For identification of most sites of iodination and hormonogenesis, there has been good agreement between studies of Tg iodinated in vivo and in vitro. Site identification has rested largely on the sequencing of peptides after limited proteolytic digestion of Tg to find iodotyrosines and iodothyronines and their neighboring amino acids, in order to identify the iodopeptide location within the context of the complete Tg sequence. Because the coupling reaction involves iodophenyl transfer from a donor MIT or DIT residue to an acceptor DIT residue, this method is relatively straightforward for locating the acceptor residues, but it will not identify donor iodotyrosine residues. For donor residue identification, it is necessary to identify the dehydroalanine residues that remain within the polypeptide chain after transfer of the iodophenyl moiety to a DIT residue (190,191,192,193,194).
The conclusion of these studies is that, from the many tyrosines in the Tg molecule, three specific tyrosine residues located near the amino and carboxyl ends of the protein are the primary acceptor sites for hormonogenesis, including positions 5 (“site A”), 2554 (“site B”) and 2747 (“site C”) of human Tg, respectively (195). Site A (Tyr5) is the predominant hormonogenic site. It is the most conserved between species, the first to become iodinated in some species, and normally is reserved for formation of T4 (196,197,198,199,200,201). Site B (Tyr2554) is the second most highly utilized site for T4 formation, comprising one-fourth to one-fifth of the T4 content of Tg (3). Site C (position 2747) tends to be favored for T3 formation [with even greater predominance in certain rodent species (197)], although it may account for as much as one-seventh of T4 formation in human Tg (3). In addition to these three sites there are four ancillary hormonogenic sites, termed sites “D” (position 1291), “G” (position 2568), “N” (position 685), and “R” (equivalent to position 632 although not present in human Tg) that may be favored in certain species under certain conditions, but which tend to be more variable than the three primary iodination sites (3).
The variability of specific tyrosine sites for iodination and hormonogenesis is likely to reflect at least three features: tyrosine exposure on the outer surface of Tg; the amino acids flanking the iodination sites, which may provide a biochemical basis favoring iodination and coupling; and the physical proximity of paired “donor” and “acceptor” iodotyrosines, thereby promoting the coupling reaction. It seems plausible that positioning tyrosines near the ends of the protein is likely to increase their surface exposure, although a definitive statement awaits three-dimensional structural analyses of Tg. As regards flanking information, in vitro iodination of human Tg reveals three patterns of neighboring amino acids that favor both early tyrosine iodination as well as T4 formation: Asp/Glu-Tyr; Ser/Thr-Tyr-Ser; and Glu-X-Tyr (195). With respect to proximity of iodotyrosyl pairs, increasing evidence suggests that the donor sites may be closely positioned within the same regions of Tg as their paired acceptor sites (202). Evidence from in vitro iodination of full-length bovine Tg suggests that Tyr130 is an important outer ring donor for T4 formation at Tyr5 (194). Although not completely physiological (203), coupling between Tyr5 and Tyr130 can also be achieved by in vitro iodination of a fragment containing only the first 171 residues of human Tg (192), which is facilitated by N-glycosylation in this domain (204). Mutagenic replacement of 4 of the 5 other tyrosines in the vicinity with phenylalanine (at positions 29, 89, 97, and 192) does not dminish the content of T4 generated in vitro in such a fragment (205). T4 formation has also been demonstrated in a fragment of rat Tg just large enough to encode iodination sites B and C (206). These findings increase the likelihood that the primary donor and acceptor tyrosine sites are near each other within the same monomer of the Tg homodimer.
THYROGLOBULIN ACTIVITY IN COLLOID BEFORE INTERNALIZATION INTO THYROCYTES
The state of the Tg in the colloid may vary, depending upon the levels of TSH and iodination (207,208,209). At the time of exocytosis, Tg is actively iodinated and hormone formation is occurring (210). The proximity of these newly completed Tg molecules to the surface of the apical plasma membrane makes them most readily available for endocytotic internalization. Especially with TSH stimulation (or in Graves' disease), the newly-secreted and iodinated molecules contribute maximally to the pool of iodinated Tg that provides most of the soon-to-be-secreted T4 and T3; this has come to be known as the “last come, first served” hypothesis (211,212).
Tg also undergoes further structural changes upon iodination and storage in colloid (213). As reviewed above, Tg initially forms noncovalently bound homodimers. To the extent that higher order noncovalent Tg complexes are formed (148), they tend to be quantitatively dissociated under the conditions of sample preparation used for the analysis of oligomerization of cellular Tg by sucrose gradient or native gel electrophoresis. Indeed, noncovalently bound Tg homodimers are relatively easily dissociated by heating, changes of ionic strength, or in the presence of increasing concentrations of various detergents. On the other hand, upon TSH stimulation of Tg secretion into colloid (214), and especially in conjunction with iodination, Tg dimers (215) and higher order oligomers (216) are stabilized. The stability of tetrameric Tg (27S by sucrose velocity gradient) is directly correlated with increasing iodine content (3). This could be due to direct structural modification of Tg by iodide, but more likely is due to secondary structural events within the oxidative environment of the colloid; specifically, interchain covalent cross linking primarily via disulfide bonds (49) and secondarily via N-epsilon-amino lysine cross linking (217) and formation of dityrosine bridges (218,219). Such iodide-rich, multimeric Tg is curiously devoid of T4 and T3 (220), possibly because hormone-rich iodopeptides have already been released from the remaining Tg protein by proteolysis (221). In any event, SDS-PAGE analysis of colloidal Tg under reducing conditions reveals the presence of many iodinated Tg fragments, including a variety of overlapping N-terminal fragments that include the primary “site A” for T4formation (189). The generation of these and other Tg fragments within the colloidal space has been attributed both to direct peptide bond cleavage during the oxidative process of iodination (221,222,223), as well as possible cleavage by secreted proteases (224,225,226) with discrete and distinct cleavage sites. Most of these cleavage fragments are likely to remain associated within the parent molecule under nonreduced conditions because of the extensive disulfide bonded structure of Tg.
THYROGLOBULIN BINDING AND INTERNALIZATION
Tg has the potential to interact with a number of additional proteins, not only within the endoplasmic reticulum but also in later compartments of the secretory pathway. Tg interacts with receptor proteins in the apical plasma membrane, followed by endocytotic internalization. In addition, there may be a sufficiently high concentration of soluble, newly iodinated Tg molecules that an ample number should be available for fluid-phase endocytosis, requiring no receptors, after which they are directed to lysosomes for complete proteolytic digestion, including liberation of T4 and T3 (227). In this case, the high-affinity apical receptors for Tg could be used for apical recycling of Tg, presumably for the purpose of increasing the ultimate efficiency of its iodination (228); for transcytosis to the basolateral (bloodstream) side of the cell (229) [a process which is less easily linked to normal thyroid hormonogenesis (230)]; or for selective uptake of a subset of colloidal Tg molecules en route to lysosomes (231). Given the very large size of Tg, it is not surprising that several peptides derived from the Tg molecule have the capability for binding to surface molecules of thyrocytes and other cells (232). The more difficult question is the physiological importance of such binding (233).
N-acetylglucosamine receptors (228,234,235) may be important in endocytotic routing of poorly-iodinated Tg molecules (236), although recently this notion was modified to suggest modulation or recognition of protein determinants on extracellular Tg (237) by the cell-surface protein disulfide isomerase (PDI) (238). Alternatively, cell-surface PDI-like activity might function in conjunction with the oxidizing environment of the follicular lumen to promote disulfide-linked multimerization of Tg (239); or extracellular PDI might (in conjunction with other extracellularly located chaperones) work oppositely, to solubilize multimeric colloidal Tg (240).
Megalin is another endocytotic receptor for Tg. Initially, it was was thought that megalin-mediated Tg endocytosis might be involved in thyroid hormone synthesis (241). Later studies suggested that Tg does not dissociate from megalin at the low pH of endosomes (242), but rather that megalin binding to Tg facilitates apical-to-basal transcytosis of Tg (243). Addition of receptor-associated protein (RAP) inhibits Tg binding to megalin (241), presumably by a direct interaction between RAP and Tg (244). Tg also contains short heparin-binding sequences, and heparin treatment inhibits Tg binding by megalin (245,246), which may signify a role for one or more heparan-sulfate proteoglycans in the internalization process (247). These experiments have been mostly conducted in cultures of rat thyroid FRTL5 cells, but megalin is expressed in normal thyroid tissue. In rats with aminotriazole-induced goiters, the thyroid content of megalin is increased, suggesting TSH-dependent regulation (243).
Tg binding to the surface of FRTL5 cells has been reported to alter the expression of several thyroid differentiation markers (248,249,250). This effect may be mediated by apical binding of Tg (251), specifically, via an apical version of the asialoglycoprotein receptor (252). Nevertheless, as Tg displays to the cell surface many potential binding sites, it is not clear whether this effect of Tg binding is actually restricted to this receptor, restricted to thyrocytes (253), or requires that Tg be present under native conditions (254). Overall, these studies are intriguing, but need to be pursued further to determine whether Tg is a physiological regulator of the expression of markers of thyroid differentiation in vivo.
PROTEOLYTIC DIGESTION OF THYROGLOBULIN IN THE ENDOSOME-LYSOSOME SYSTEM
Ultimately, Tg is exposed to cathepsins within lysosomes to liberate T4 and T3 from the Tg backbone. Early studies of the endocytotic degradation of Tg were done in collagenase-digested porcine thyroid follicles using [125I]Tg (255). Although confounded by the possibility of Tg binding to the basolateral surface (256), Western blotting with anti-Tg of lysosomes isolated from the thyrocytes revealed slightly smaller-than-normal dimeric Tg which dissociated into fragments upon SDS-PAGE under reducing conditions (257), consistent with early limited proteolysis. Indeed, extracts of thyroid lysosomes can themselves generate such fragments from pure Tg (258). It is difficult to establish the fraction of such fragments that are generated before endocytosis (224,225) versus the fraction generated within the endosome-lysosome system, especially because lysosomes themselves may, under stimulation, undergo exocytosis (226).
In vitro analyses have established that cysteine proteinases mediate proteolytic processing of Tg (3). Individual Tg type 1 repeats are themselves substrates for lysosomal cysteine proteases (33), even as they may function as inhibitors of these enzymes (34). Studies in mice with genetic deficiency of various cathepsins (B, K, or L) have revealed impaired proteolysis of Tg in mice deficient for cathepsin B or cathepsin L, but not cathepsin K-deficient mice (259). Of particular interest, cathepsin K(-/-)/L(-/-) double-mutant mice had low serum free T4 concentrations, suggesting that either or both of these enzymes may participate in liberation of T4 from the Tg backbone.
The authors acknowledge funding from NIH DK40344 and from Ministero dellâ Universitê Ricerca Scientifica Grant No. 2002063745. The authors also thank Drs. E. Consiglio, S. Formisano, Y. N. Park, and P. Kim for helpful discussions and support.
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