RevIewS

The role of thyroglobulin in thyroid

hormonogenesis
Cintia E. Citterio   1,2, Héctor M. Targovnik1,2 and Peter Arvan   3*
Abstract | In humans, the thyroid hormones T3 and T4 are synthesized in the thyroid gland in
a process that crucially involves the iodoglycoprotein thyroglobulin. The overall structure of
thyroglobulin is conserved in all vertebrates. Upon thyroglobulin delivery from thyrocytes to the
follicular lumen of the thyroid gland via the secretory pathway , multiple tyrosine residues can
become iodinated to form mono-iodotyrosine (MIT) and/or di-iodotyrosine (DIT); however,
selective tyrosine residues lead to preferential formation of T4 and T3 at distinct sites.
T4 formation involves oxidative coupling between two DIT side chains, and de novo T3 formation
involves coupling between an MIT donor and a DIT acceptor. Thyroid hormone synthesis is
stimulated by TSH activating its receptor (TSHR), which upregulates the activity of many thyroid
gene products involved in hormonogenesis. Additionally , TSH regulates post-translational
changes in thyroglobulin that selectively enhance its capacity for T3 formation — this process is
important in iodide deficiency and in Graves disease. 167 different mutations, many of which are
newly discovered, are now known to exist in TG (encoding human thyroglobulin) that can lead to
defective thyroid hormone synthesis, resulting in congenital hypothyroidism.

Metamorphosis
An abrupt developmental
change in the shape or form
of an animal.
1
Universidad de Buenos Aires,
Facultad de Farmacia y
Bioquímica, Departamento de
Microbiología, Inmunología y
Biotecnología/Cátedra de
Genética, Buenos Aires,
Argentina.
2
CONICET-Universidad de
Buenos Aires, Instituto de
Inmunología, Genética y
Metabolismo (INIGEM),
Buenos Aires, Argentina.
3
Division of Metabolism,
Endocrinology & Diabetes,
University of Michigan
Medical School, Ann Arbor,
MI, USA.
*e-mail: parvan@umich.edu
https://doi.org/10.1038/
s41574-019-0184-8
Many biological processes are regulated by thyroid
hormones in vertebrates and some invertebrates 1,2
including metamorphosis (in echinoderms3–5 and in
some chordates, including teleosts, and many amphib-
ians6) and control of seasonality7, as well as growth,
development, heart rate and thermogenesis in mam-
mals8. In many of these processes, thyroid hormones
primarily regulate the gene expression controlling oxi-
dative metabolism9. In vertebrates, thyroid hormones
are members of the family of peptide hormones, and
in this case, a protein precursor generates an amino
acid-derived bioactive product of ~0.8 kDa. In humans,
once secreted into the bloodstream, T4 is long lived
(t1/2 = ~7 days), whereas T3, which exhibits far more
potent hormone action, is short lived (t1/2 ≤ 12 hours)10.
Through the activation of thyroid hormone recep-
tors, T3 modulates gene expression to regulate devel-
opment and many of the phenotypes (growth, heart
rate and thermogenesis) described above 11,12. In a
negative-feedback loop, thyroid hormone levels
undergo continuous surveillance by central hypotha-
lamic control over pituitary secretion of TSH, which
activates thyroidal TSH receptors (TSHRs) to stimulate
thyroid hormone synthesis13,14.
In this Review, we highlight the role of thyroglob-
ulin in hormonogenesis within the thyroid gland and
consider the topic from evolutionary, biochemical,
­molecular, cellular and physiological aspects.
Origins of thyroid hormonogenesis
In humans, the primary, original source of thy-
roid hormones is the iodoglycoprotein thyroglobu-
lin (Supplementary Fig. 1), which is the most highly
expressed protein in the thyroid gland. Interestingly,
thyroid-hormone-receptor-mediated bioactivity has
been traced back in evolution to aquatic life forms that
precede the first appearance of the thyroglobulin (TG)
gene15. Specifically, in both sea urchin16,17 and amphi-
oxus18, many orthologues of genes involved in thyroid
hormone synthesis are already present19–22, although
these species lack a gene encoding a vertebrate-like
thyroglobulin. Amphioxus, which does not have
a thyroid follicular structure, accumulates iodide
and synthesizes thyroid hormone in the pharyngeal
endostyle 18; this organ has functional equivalence to
the vertebrate thyroid gland23, including the expres-
sion of transcription termination factor 1 (TTF1),
homeobox protein NKX2.1 and paired box protein
PAX8, which are required for thyroid specification24.
Biosynthesis of a thyroid-hormone-containing pro-
tein with a sedimentation coefficient of 17S–19S has
been reported in the amphioxus endostyle25; however,
genome database searches reveal no clear TG homo-
logue. Nevertheless, amphioxus genes encoding pro-
teins with thyroglobulin type 1 modules and other
proteins bearing a putative thyroglobulin-cholinesteras
e-like (ChEL) domain (thyroglobulin domains are

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Key points
• The first definitive evidence of a complete TG gene appears with the development
of the vertebrates, and once appearing in evolution, the entire structure of
thyroglobulin, as well as its ability to be secreted, has been retained thereafter.
• The synthesis of T3 and T4 within thyroglobulin involves oxidative coupling between
iodinated tyrosine residues on thyroglobulin.
• The main T3-forming site within thyroglobulin couples a mono-iodotyrosine donor at
the antepenultimate residue of one monomer with a di-iodotyrosine acceptor in the
same residue of the apposed monomer within a dimer.
• Post-translational modifications of thyroglobulin include phosphorylation for which
the secretory pathway kinase FAM20C has been implicated.
• TSH stimulation of thyrocytes promotes post-translational modifications that can
alter thyroglobulin structure in a way that favours T3 formation upon iodination,
whereas defects in TSH-mediated stimulation result in thyroglobulin with diminished
capacity to form T3.
• 167 TG mutations exist that can cause congenital hypothyroidism; although the
disease is usually inherited as an autosomal recessive trait, patients with congenital
hypothyroidism bearing monoallelic mutations of TG have recently been reported.
described in detail below) have been reported 19. A
best guess of the ancestral amphioxus TG-related
gene (Bf_123169) predicts an encoded protein of
~2,400 amino acids; it lacks the ChEL domain (needed
for thyroglobulin function), but an independently
expressed ChEL domain could potentially function in a
thyroglobulin-like heteromeric complex.
The complete TG gene probably first appeared
through intragenic duplication and gene fusion
events26. The first definitive evidence of a complete
TG gene appears with the development of the verte-
brates27 (Fig. 1a,b), and once appearing, the entire struc-
ture encoded by TG seems to have been conserved
throughout all vertebrates thereafter27,28. The earliest
vertebrates bearing TG that have been studied to date
are lamprey larvae, which show an exocrine secretion
of iodoprotein-containing thyroid hormones29 from
the pharyngeal endostyle before metamorphic transi-
tion into adult lamprey, which have a true endocrine
thyroid gland29,30. When comparing the thyroglobulin
protein from lamprey to zebrafish to Xenopus to human,
although there are variations, the overall modular struc-
tures including the most critical disulfide-bond-forming
cysteine residues, as well as the regional structure of
­thyroglobulin, are all retained27,31 (Fig. 1b).
Thyroglobulin in hormonogenesis
The role of iodide. Iodide is initially absorbed in the
gastrointestinal tract and enters the systemic circulation;
upon reaching the thyroid gland, iodide traverses the
basolateral plasma membrane and enters the cytoplasm
of thyroid follicular epithelial cells (hereafter referred to
as thyrocytes) via the activity of the sodium–iodide sym-
porter (NIS; encoded by SLC5A5)32. Thyroidal uptake
of iodide is increased by iodide efflux across the apical
Endostyle membrane into the follicular lumen, which is mediated
A longitudinal, ciliated, by the activities of pendrin33, anoctamin 1 (ref.34) and
grooved organ located on the ClC5 (refs35,36) (Fig. 2). The effect of NIS enables thyro-
ventral wall of the pharynx of cytes to concentrate iodide roughly 30–60-fold within
chordates that has functional
equivalence to the vertebrate
the cytosol of thyrocytes37,38, and the activity of apical
thyroid gland, among other transporters and iodination machinery further increases
functions. overall iodide sequestration in the thyroid gland.
The availability of H2O2 at the apical membrane of
thyrocytes enables thyroid peroxidase (TPO) to catalyse
the oxidation of iodide39, which is necessary for protein
iodination in the follicular lumen. Most of the pro-
tein substrate for iodination is provided by secreted
thyroglobulin, the concentration of which is 100–400 mg
per mL in the follicular lumen40. The H2O2-generating
system at the apical surface involves the activity of
dual-function oxidase 2 (DUOX2) and DUOX1, which
both belong to the NADPH oxidase family. DUOX2 is
more efficient in the production of H2O2 and more highly
expressed than DUOX1 (ref.41). The DUOX maturation
factors, DUOXA2 and DUOXA1, facilitate the intra-
cellular delivery of DUOX2 and DUOX1 to the plasma
membrane and regulate the activity of these enzymes42.
Reactive iodide in the follicular lumen preferen-
tially reacts with tyrosine residues that are closest to
the apical plasma membrane; this is an area enriched in
newly secreted thyroglobulin protein that is also highly
accessible for endocytic internalization. The process of
synthesis and release of thyroid hormone from newly
synthesized thyroglobulin has been termed ‘first come,
first served’43,44. Both singly iodinated tyrosine residues
(mono-iodotyrosine (MIT)) and doubly iodinated tyro­
sine residues (di-iodotyrosine (DIT)) are formed during
iodination of thyroglobulin. In addition, some thyroglob-
ulin dimers (17S; with each monomer ~330 kDa) can
undergo a crosslinking side reaction to generate cova-
lent dimers (19S; ~660 kDa) as well as tetramers (27S)
and even higher-order covalent complexes that together
can constitute up to 30% of the total thyroglobulin in the
follicular lumen45,46. These intermolecular covalent link-
ages include disulfide bonds, 3-3ʹ-dityrosine bridges and
γ-glutamyl-lysine bridges40,45,47,48; however, non-iodinated
thyroglobulin dimers have no i­ ntermolecular crosslinks
between the monomer partners47.
Thyroglobulin iodination is favoured at specific
sites on the protein, which are thought to be based
largely on tyrosine residue exposure at the surface of
the thyroglobulin tertiary and/or quaternary structure
and might additionally be affected by the sequence of
immediately flanking amino acids. Although the ability
of thyroglobulin to be iodinated is not unique, the effi-
ciency of thyroid hormone synthesis in thyroglobulin,
even under low-iodide conditions, is remarkable. At the
time of iodination, selective DIT and MIT residues of
thyroglobulin undergo a coupling reaction for de novo
formation of thyroid hormones within the thyroglob-
ulin polypeptide backbone. Classic studies report that
TPO shows no marked specificity over lactoperoxidase
or myeloperoxidase (all three enzymes are able to cat-
alyse iodination) in driving hormonogenic coupling
within thyroglobulin49. Rather, T3 and T4 formation is
based upon the native conformation of the thyroglobu-
lin substrate, which has been evolutionarily selected for
efficient thyroid hormone synthesis50 as well as iodide
storage. Under conditions of a normal iodide supply,
thyroidal thyroglobulin contains, on average, 2.5 res-
idues of T4 and 0.7 residues of T3 per thyroglobulin
dimer51, but thyroid hormone-rich and hormone-poor
thyroglobulin molecules coexist, in variable proportions,
within thyroid follicles.
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a Region I Region II Region III

NH2 Linker Hinge ChEL domain COOH

T4
T3
b
Human

Y5 Y130 Y1291 Y2554 Y2747

Mouse

Y5 Y130 Y1290 Y2552 Y2744

Domestic cows

Y5 Y130 Y1291 Y2555 Y2748

Frog

Y5 Y130 Y1287 Y2559 Y2750

Zebrafish

Y5 Y125 Y2505 Y2707

Lamprey

Y5 Y131 Y1339 Y2604

Fig. 1 | regional structure and primary hormonogenic sites found within thyroglobulin. a | In vertebrates, the regions of
monomeric thyroglobulin are composed of cysteine-rich repeats within the regions I, II, III and the carboxy-terminal
cholinesterase-like (ChEL) domain. Thyroglobulin contains ~70 tyrosine residues; however, only a few tyrosine residues are
capable of synthesizing thyroid hormones. T4 is primarily formed at a conserved site near the amino terminus (red), and T3 is
mainly synthesized at conserved sites in the carboxy terminus (blue). A typical thyroid hormone distribution for a thyroglobulin
dimer is 2.5 residues of T4 and 0.7 residues of T3. b | The localizations of hormonogenic tyrosine residues are shown in human
(Homo sapiens), mouse (Mus musculus), domestic cows (Bos taurus), frog (Xenopus tropicalis), zebrafish (Danio rerio) and
lamprey (Petromyzon marinus) thyroglobulins. Acceptor T4 and acceptor T3 hormonogenic tyrosine residues are indicated
with light red squares and light blue squares, respectively. Dashed lines indicate missing thyroglobulin sequence in lamprey.

Thyroglobulin trafficking. Follicular thyroglobulin is
re-internalized into thyrocytes via endocytosis44, which
promotes thyroglobulin proteolysis that liberates thy-
roid hormones from the polypeptide backbone, leading
to thyroid hormone secretion via the basolateral plasma
membrane into the bloodstream (Fig. 2). TSH stimulates
endocytic uptake and lysosomal degradation of thy-
roglobulin52,53, and the rate of thyroid hormone produc-
tion correlates with endocytic transfer to lysosomes54,55.
During TSH-stimulated endocytosis, some digestive
proteolytic enzymes are apically regurgitated from thy-
rocytes, which enables the predigestion of thyroglobu-
lin before completion of endocytic internalization and
lysosomal delivery40,52,56–59. In addition, some intact thy-
roglobulin might directly enter the bloodstream by misdi-
rected secretion, by leakage from disrupted follicles or by
transcytosis from the follicular lumen to the basolateral
surface of thyrocytes43,44,60,61. Furthermore, some endo-
cytically internalized thyroglobulin can potentially be
apically recycled back to the thyroid follicular lumen62–66.
A large cohort of both cathepsin proteases and plasma
glutamate carboxypeptidase57–59,67,68 is thought to contrib-
ute to the ultimate lysosomal degradation of thyroglobulin,
which leads to the liberation of thyroid hormones26,31,69,70.
The monocarboxylate transporter 8 (MCT8), which is
expressed in multiple cell types, can facilitate the efflux
of thyroid hormones from the thyroid gland as well as
their influx into various cells and tissues. In mice, MCT10
shows an overlapping expression with MCT8 in organs
such as the liver, kidney and thyroid and is particularly
important in facilitating thyroid hormone transport when
MCT8 is missing71. In addition, l-type amino acid trans-
porters LAT1 and/or LAT2 can function as secondary thy-
roid hormone transporters72–74. Upregulation of lysosomal
thyroglobulin processing and MCT8-mediated thyroid
hormone transport are physiologically coordinated for the
release and delivery of thyroid hormones to the body71,75.
The lysosomal proteolysis of thyroglobulin also yields a
large supply of uncoupled iodotyrosines. MIT and DIT
can be released into the bloodstream but are substantially

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Follicular lumen Oxidation of I–

I+ Iodination
I
–
of tyrosines Coupling
DUOX
Anoctamin 1 system
T4
PDS ClC5 H2O2 Thyroglobulin T3
dimer Apical membrane
TPO

Golgi Iodide recycled

MIT
apparatus
Thyroglobulin
post-translational
modifications

IYD1
ER
DIT
Lysosome

Nucleus

TG gene

NIS Na+/K+-ATPase
Gαq Gαs
PLC AC
Basolateral membrane TSH TSHR
MCT8
Blood vessel

Fig. 2 | Overview of de novo thyroid hormone biosynthesis. Thyroid follicles, which are the functional unit for thyroid
hormone biosynthesis, compose a monolayer of polarized thyrocytes (otherwise known as thyroid follicular epithelial
cells) with the apical surface contacting the follicular lumen and the basolateral membrane facing the bloodstream.
Thyroglobulin is the predominant protein expressed by the thyrocytes and is the primary original source of thyroid
hormones. Hormonogenesis is stimulated by interaction of TSH with its receptor (TSHR), and the central steps involve
the coordination of iodide (I−), H2O2, thyroid peroxidase (TPO) and thyroglobulin. I− traverses the basolateral plasma
membrane of thyrocytes via the activity of the sodium–iodide symporter (NIS) and effluxes across the apical membrane
into the follicular lumen as mediated by the activities of pendrin (PDS), anoctamin 1 and ClC5. A source of H2O2 is
provided by the dual-function oxidase (DUOX) system. In the presence of I− and H2O2, TPO catalyses the iodination of
thyroglobulin tyrosine residues, beginning with the molecules that are closest to the apical plasma membrane. At the
time of iodination, selective di-iodotyrosine (DIT) and mono-iodotyrosine (MIT) residues of thyroglobulin can undergo
the coupling reaction to initiate formation of T4 and T3. Follicular thyroglobulin is re-internalized into the thyrocytes via
endocytosis, which promotes the thyroglobulin proteolysis that liberates thyroid hormones from the polypeptide
backbone, leading to thyroid hormone secretion into the bloodstream via transporters such as MCT8 at the basolateral
plasma membrane. The I− contained within uncoupled MIT and DIT is recycled by iodotyrosine dehalogenase 1 (IYD1).
AC, adenylyl cyclase; ER , endoplasmic reticulum; PLC, phospholipase C.

Thyroid hormonogenic
coupling reaction
A post-translational
modification resulting in
the physical transfer of a
mono-iodotyrosine
or di-iodotyrosine donor to a
di-iodotyrosine acceptor within
a protein.
deiodinated by iodotyrosine dehalogenase 1 (also known
as IYD1) before they escape from the thyroid gland76–78;
this process allows for intrathyroidal iodide recycling,
which is essential to the maintenance of thyroid hormone
synthesis under conditions of low dietary iodide77. The net
result of thyroglobulin iodination, coupling, endocytosis,
proteolysis and thyroid hormone transport is that 100%
of the body’s supply of T4 derives from de novo thyroid
hormone biosynthesis (Fig. 2).
Biochemistry of T4 and T3 synthesis
As mentioned above, H2O2 enables TPO to catalyse
the oxidation of iodide, and reactive iodide can react
with tyrosine residues on thyroglobulin to generate
DIT and MIT (Fig. 3a,b). The next step in the process,
the thyroid hormonogenic coupling reaction, is one of the
least-understood aspects of the biochemistry of thyroid
hormone synthesis. Evidence suggests that coupling
might proceed either through a free radical interme-
diate or through ionic oxidation79. Regardless of the
mechanism, hormonogenic coupling always involves
two iodotyrosine residues, one of which (referred to as
the donor residue) loses its iodophenolic ring to become
dehydroalanine. Either DIT or MIT could be a donor
residue, but to generate bioactive thyroid hormone, the
acceptor residue must be DIT (Fig. 3b). After coupling,
the acceptor residue thereby ultimately bears a two-
ring amino acid side chain with the outer ring derived
either from a DIT donor (destined to become T4) or a
MIT donor (­ destined to become T3), and this two-ring

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 Reviews

a b Thyroglobulin-T3 Thyroglobulin-T4
H2N H2N
NH NH
COOH COOH
NH
O NH O NH
Thyroglobulin-Tyr DIT MIT DIT DIT
I O I O
O
TPO– Fe O I OH I OH I
I–
I OH I OH
H2O OH
I–
H COOH H COOH
TPO– Fe O H2N N H2N N
O NH O NH
H2O2
I I
O O
OH–
O O
TPO– Fe I I I

I OH I OH
H2N H2N
NH NH
COOH COOH
O O

I I

I O I O

I I I
OH OH

c T3 NH2
DIO1, DIO2 ORD DIO1, DIO3 IRD
OH
I I O

HO O I
NH2
T4 NH2 T2 OH
OH I I O
I I O
HO O
HO O I
rT3 NH2
I
OH
DIO1, DIO3 IRD I I O DIO1, DIO2 ORD

HO O

Fig. 3 | Physiological enzymatic reactions involved in thyroid hormone synthesis. a | Thyroid peroxidase (TPO)
catalyses the iodination that leads to the coupling of mono-iodotyrosine (MIT) and di-iodotyrosine (DIT) within
thyroglobulin. b | Coupling during de novo thyroid hormone synthesis entails the transfer of an iodophenoxyl ring from a
donor MIT or DIT residue to an acceptor DIT residue within thyroglobulin, yielding T3 or T4, respectively. c | Additionally ,
type 1 and type 2 iodothyronine deiodinases (DIO1 and DIO2) provide outer ring deiodinase (ORD) activity on the T4
molecule to produce T3. Thyroid hormone inactivation occurs by inner ring deiodination (IRD) catalysed primarily by DIO3
(and secondarily by DIO1). rT3, 3, 3′, 5′-tri-iodothyronine; T2, 3, 3′-tri-iodothyronine.

product is still embedded within the thyroglobulin
­polypeptide chain (Fig. 3b).
In healthy rats, 55% of circulating T3 appears to
be derived directly from the thyroid gland80, whereas
in healthy humans, only ~21% of daily T3 produc-
tion is ordinarily derived from thyroidal secretion81.
Importantly, T3 can be generated from T4 in the cytosol
of cells in selected peripheral tissues (such as kidney and
liver), as well as in thyrocytes, by the actions of type 1
iodothyronine deiodinase (DIO1) and type 2 iodothyro-
nine deiodinase (DIO2)82–85 (Fig. 3c). Importantly, DIO1
contributes primarily to circulating plasma levels of T3
(ref.84), whereas DIO2 contributes to plasma T3 levels and
also might contribute importantly to local T3 generation
in tissues including the brain, pituitary gland, thyroid
gland and brown adipose tissue86. Both DIO1 and DIO2
catalyse deiodination of the outer phenolic ring of the
T4 molecule, which results in an outer ring that emu-
lates the one donated by MIT in the coupling reaction
described above, to produce T3. By contrast, thyroid hor-
mone inactivation — which is also of great physiological
significance — occurs by deiodination of the iodothyro-
nine inner ring, which is catalysed primarily by DIO3,
the third member of the deiodinase group85.
Thyroidal T3 formed de novo within the thyroglobu-
lin protein (Fig. 3b) is released from the thyroid gland in a

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Thyroglobulin short unique
tail sequence
Refers to the final ~32
residues of the thyroglobulin
sequence containing the
antepenultimate tyrosine
residue that can form T3 and is
conserved throughout all
vertebrates.
manner similar to the release of T4 described above. This
process does not involve the deiodination of T4 within
thyroglobulin84,87. Furthermore, hormone-containing
thyroglobulin is localized within the thyroid follicular
lumen as well as in membrane-bound endocytic and
lyso­somal vesicles88, which are sites inaccessible to DIO1
and DIO2 — the catalytic sites of these enzymes topo­
logically face the cytosol85. Therefore, thyroglobulin
itself is not a substrate for deiodination89, and the extent
of thyroidal release of T3 derived from thyroglobulin is
largely dependent upon the pre-existing T3 content of
thyroglobulin, which is formed during its iodination.
Notably, animals with whole-body double knockout of
DIO1 and DIO2 deiodinases exhibit completely normal
circulating T3 levels, which derive entirely from thyroidal
secretion84. Moreover, such animals possess as much or
more T3-containing thyroglobulin — the synthesis of
which is regulated exclusively by the hypothalamus–
pituitary–thyroid axis — as that found in the thyroid
glands of control animals87.
TG gene expression
Specific transcription factors such as the paired-domain
protein PAX8, the homeodomain protein NKX2.1, the
forkhead-domain protein E1 (FOXE1), homeobox-
containing transcription factor NKX2.5, the haemato-
poietically expressed homeobox HEX (also known as
PRH) and transcriptional co-regulators are all involved
in the differentiation programme for human thyroid
follicular cells90,91, including transcription of the TG
gene26,91. Among these transcription factors, PAX8
and NKX2.1 are stimulated by the transcriptional
co-activator with PDZ-binding motif (TAZ; also known
as WWTR1), resulting in their biochemical association
and synergistic mode of action, which has a prominent
role in thyroid cell differentiation and thyroid gene tran-
scription91. By contrast, HEX acts as a repressor of the
TG promoter92,93. TSH action on its receptor enables pos-
itive induction of thyroid-specific genes including TG91,
largely through the modulation of intracellular cAMP
levels13,94, which can stimulate TG enhancer function95.
The human TG gene (a ~270 kb single-copy gene
localized on chromosome 8q24.22) is expressed at very
high levels in thyrocytes26, which is a distinguishing fea-
ture of the thyroid gland. The TG gene is organized into
48 exons with sizes in the range of 63–1,101 nucleotides
and containing introns that range in size, including one
of 64 kb (refs31,96) that encodes the human SRC-like
adaptor protein (SLAP)97. TG (human thyroglobulin,
NCBI Reference Sequence: NG_015832.1) encodes
an mRNA containing a 41-nucleotide 5ʹ-untranslated
region followed by a single open reading frame of
8,307 bases and 105 bp of a 3ʹ-untranslated sequence.
In human thyroid tissues, thyroglobulin mRNA is
very heterogeneous with 12 predicted transcript vari-
ants (NCBI Reference Sequences: XM_017013797.1,
XM_005251042.4, XM_017013796.1, XM_017013800.1,
XM_017013799.1, XM_005251040.4, XM_017013798.1,
XM_017013795.1, XM_005251038.4, XM_017013793.1,
XM_017013794.1 and XM_006716622.3) in addition to
the full-length thyroglobulin mRNA (NCBI Reference
Sequence: NM_003235.4).
Thyroglobulin protein structure
The newly synthesized thyroglobulin precursor protein
contains an ~19-residue signal peptide (Supplementary
Fig. 1), which is immediately removed upon successful
delivery of thyroglobulin into the endoplasmic reticu-
lum (ER)98. Thyroglobulin is synthesized as a mono­
meric protein with a 12S sedimentation coefficient
(~330 kDa). The multidomain monomeric protein has
distinct modules: four thyroglobulin type 1 repeats, a
linker segment, six additional type 1 repeats, a hinge
segment, three thyroglobulin type 2 repeats, a final
type 1 repeat, five thyroglobulin type 3 repeats and a
carboxy-terminal ChEL domain of ~570 residues, fol-
lowed by a thyroglobulin short unique tail sequence of ~32
residues27,31,70 (Supplementary Fig. 1). Thyroglobulin
modules are cysteine-rich repeat domains bearing
intradomain disulfide bonds99. Type 1 modules contain
the central CWCV sequence and are subdivided into
type 1A (containing six cysteine residues) and type 1B
(containing four cysteine residues, in the 9th and 11th
repeats)100. Type 2 modules have two cysteine residues
within a short span of 14–17 residues99. Type 3 mod-
ules are subdivided into type 3A (with eight cysteine
­residues) and type 3B (with six cysteine residues)99.
Overall, the monomeric structure of thyroglobulin
is divided into four regions (Supplementary Fig. 1):
region I contains the first 10 of the 11 thyroglobulin
type 1 modules, including both the linker and hinge seg-
ments (Fig. 1a); region II contains the type 2 repeats and
the embedded 11th type 1 repeat; region III contains the
type 3 repeats; and region IV contains the ChEL domain
plus the short unique tail sequence26 (Supplementary
Fig. 1). The carboxy-terminal ChEL portion of thy-
roglobulin is a member of the classic α-hydrolase and
β-hydrolase fold family, which is best represented by
acetylcholines­terase (with a central β-sheet flanked on
both sides by α-helices)101, to which the thyroglobulin
ChEL domain exhibits 47% residue similarity102,103.
Although the 3D structure of thyroglobulin has not
been elucidated, strong circumstantial evidence exists
for interactions between different regions. The ChEL
domain is implicated in the conformational matura-
tion and export of newly synthesized thyroglobulin
through the thyroid secretory pathway to the follicular
lumen98,104,105. Interestingly, when engineered to bear a
signal peptide for delivery into the ER, regions II and III
(expressed together) and ChEL both behave as fully
folded proteins, which pass the conformational require-
ments for ER quality control to function as efficient and
independently successful secretory proteins106. However,
contiguous thyroglobulin regions I, II and III are not
competent for intracellular transport and require a sep-
arately expressed secretory ChEL to enable complete
folding and export through the secretory pathway106.
After a period of monomer folding, thyroglobulin
self-associates noncovalently into a homodimer in the
ER (~660 kDa)107. Moreover, thyroglobulin can fur-
ther multimerize in the follicular lumen, as described
above45,46,108. Although the intermonomer contact sur-
face might not be limited to ChEL, this domain has been
found to be both necessary and sufficient for dimeriza-
tion. Each ChEL monomer contributes two α-helices to
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De novo T3 formation
Refers to the synthesis of T3
within the thyroglobulin
polypeptide backbone that
occurs as a result of the
coupling of iodotyrosines.
Nature Reviews | Endocrinology
a four-helix bundle that is used for tail-to-tail homo­
dimerization as occurs in the related protein acetylcho-
linesterase and many of its evolutionary homologues109.
In thyroglobulin, homodimerization thereby brings
the contiguous short unique tail sequences from two
­adjoining monomers into close proximity109,110.
Post-translational processing
During its trafficking through the intracellular secre-
tory pathway, thyroglobulin undergoes substantial
post-translational processing as a noncovalent dimer
before its delivery to the follicular lumen for iodination
and hormonogenesis. Several of these post-translational
modifications might have structural consequences
that could impact thyroglobulin hormonogenic
potential47,59,111–117.
Catalysed disulfide bond formation. Each thyroglob-
ulin monomer has the capacity to form up to 60
intramolecular disulfide bonds118. Cysteine residues
present in the repeating thyroglobulin modules are
covalently bound by intradomain disulfide bonds99.
A conserved pattern of Cys1–Cys2, Cys3–Cys4 and
Cys5–Cys6 disulfides are thought to form in type 1A
repeats100, and type 1B, type 2 and type 3 repeats also
include intradomain disulfide bonding. Last, the six
cysteine residues of the thyroglobulin ChEL domain are
thought to form three intrachain disulfide bonds, which
occur at the same positions within the related protein
acetylcholinesterase119.
The formation of disulfide pairs in thyroglobu-
lin is hypothesized to be catalysed by endogenous
ER oxido­reductases120,121, and folding intermediates
of thyroglobulin bearing mixed disulfide bonds with
ER oxidoreductases can occasionally be detected120,121.
The ER oxidoreductases implicated in thyroglobulin
folding include ERp57, protein disulfide-isomerase
(PDI), ERp72 and P5 (Fig. 4a). Thyroglobulin is such a large
protein that the same monomer can be simultaneously
bound by more than one ER oxidoreductase, each pre-
sumably located at its own preferred binding site to facil-
itate thyroglobulin conformational maturation121,122. In
addition, the isomerase function of ER oxidoreductases
such as ERp57, in conjunction with glycan-dependent
calnexin (CNX) and/or calreticulin (CRT) chaperone
binding, can help to reshuffle non-native disulfides and
to form new disulfide bonds121,123 (Fig. 4a).
Glycosylation of thyroglobulin. Approximately 10%
of the molecular mass of thyroglobulin is composed of
carbohydrate124. Thyroglobulin glycosylation is thought
to influence multiple important functions such as pro-
tein folding and trafficking125, thyroglobulin immuno-
reactivity125, iodination and hormone synthesis126,127.
Only a few individual thyroglobulin glycosylation sites
are conserved between human, Xenopus, zebrafish and
lamprey27; however, the presence of multiple N-linked
and/or O-linked glycans added during thyroglobulin
synthesis and intracellular transport is considered to
be an essential structural feature. In vivo and in vitro
studies show that unglycosylated thyroglobulin loses the
ability to synthesize thyroid hormones126, presumably
Reviews
owing to misfolding. The asparagine (N)-linked oli-
gosaccharides on thyroglobulin include both simple
N-acetylglucosamine plus multiple mannose residues,
which are characteristic of ER processing128, as well as
complex biantennary (Fig. 4b) or triantennary oligo-
saccharides with trimmed mannose residues but con-
taining Golgi-added galactose, fucose and sialic acid129.
Thyroglobulin contains multiple N-linked glycosylation
acceptor sites; of the 20 potential sites in human thy-
roglobulin (Supplementary Fig. 1), 16 are actually used26,
whereas other species might have as few as ten N-linked
oligosaccharides per monomer118. TSH is known to reg-
ulate thyroidal glycosylation, including the stimulation
of oligosaccharyl transferase activity112 and the upreg-
ulation of N-acetylglucosaminyltransferase 1 (ref.59), as
well as increased maturation of thyroglobulin N-linked
oligosaccharides from high-mannose to the complex
type111,126 including increased galactose113 and decreased
2,6-bound sialic acid114.
Human thyroglobulin is also known to contain sul-
fated O-linked glycosaminoglycans130. The O-linked
oligosaccharides contain d-galactosamine attached
to serine and threonine via an O-glycosidic linkage or
contain d-glucuronic acid N-acetyl-d-galactosamine
sulfate disaccharide plus chondroitin 6-sulfate, which
are attached to the polypeptide chain through a
d-galactosyl-d-xylosyl-serine linkage124. Interestingly, it
has been reported that the presence of a single chondroi-
tin 6-sulfate on Ser2730 of native human thyroglobulin
enhances its hormone-forming ability127. The mod-
ifications on each glycosylation site show variability
among the population of thyroglobulin molecules, as
identified in porcine thyroglobulin by liquid chroma-
tography and tandem mass spectrometry131. Although
of potentially great physiological importance, the role of
TSH-regulated changes in the patterns and efficiency
of Golgi-based O-glycosylation of thyroglobulin, and
their effects on thyroid hormone synthesis, remains
poorly understood.
Phosphorylation of thyroglobulin. Thyroglobulin
undergoes phosphorylation, some of which is included
within carbohydrate (50%), serine (30%) and tyrosine
(20%)117,132. Approximately 75–85% of the thyroglobu-
lin phosphotyrosine and phosphoserine residues have
been recovered in tryptic or cyanogen-bromide-derived
peptides comprising <10% of the thyroglobulin protein
and nearly devoid of carbohydrate, which suggests
that these residues are not randomly located along the
thyroglobulin molecule117. Phosphorylation is a Golgi
modification that occurs during thyroglobulin trans-
port through the secretory (and possibly endocytic)
pathway133 and is thought to improve the efficiency of T3
formation87. This fact is of potential physiological impor-
tance because increased de novo T3 formation within
thyroglobulin is known to occur when TSHR activity
is stimulated87. FAM20C is a novel secretory pathway
(casein) serine kinase that catalyses the phosphoryla-
tion of secreted proteins bearing S-X-E motifs (where
X is any amino acid134; Fig. 4c). Interestingly, FAM20C
shows a TSH dose-dependent increase in mRNA expres-
sion level in the PCCL3 thyrocyte cell line87. Moreover,
Reviews

a
HS ERp57
CNX/CRT
SH

HS
SH

S-S
PDI ERp57
HS CNX/CRT
S-S
SH

S-
S-S
ERp72

S
HS SH

S-
S-

S
S
S-S
S-S

S-S
SH
P5 SH
SH

HS

b c
ER Polypeptide Cell
membrane
Golgi
Asn-X-Ser/Thr

Polypeptide ----S-X-E--
FAM20C FAM20C
Polypeptide

Asn-X-Ser/Thr
ADP ATP
Polypeptide
Mannose trimming
Golgi
Further mannose
trimming
----S-X-E-- ----S-X-E--
Polypeptide P P

Asn-X-Ser/Thr

Polypeptide

Glucose N-acetylglucosamine Galactose

Mannose Sialic acid Fucose

d Region I Region II Region III

5
NH2 Linker Hinge ChEL domain COOH
Y

PAPS
Sulfotransferase
PAP
5

Y SO3H

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◀ Fig. 4 | Thyroglobulin post-translational modifications. a | Early thyroglobulin folding
is required for thyroid hormonogenesis. Disulfide bond formation in thyroglobulin is
catalysed simultaneously by more than one endoplasmic reticulum (ER) oxidoreductase,
including ER resident protein 57 (ERp57), protein disulfide-isomerase (PDI), ERp72 and
P5. Isomerase function of ER oxidoreductase ERp57 in conjunction with glycan-
dependent calnexin (CNX) and calreticulin (CRT) chaperone binding can help to form
new disulfide bonds and to reshuffle non-native disulfides. b | In the ER , asparagine-
linked glycans on thyroglobulin are considered simple N-acetylglucosamine plus
high-mannose oligosaccharides, with terminal glucose residues that are removed,
which is characteristic of ER processing. In the Golgi, thyroglobulin contains complex
oligosaccharides that have trimmed mannose residues but contain Golgi-added
galactose, fucose and sialic acid in biantennary structures like that shown here or in
triantennary glycan structures (not shown). TSH receptor stimulation favours the
maturation of asparagine-linked oligosaccharides from high-mannose type to the
complex type. c | FAM20C catalyses the phosphorylation of secreted proteins within
S-X-E motifs (where X is any amino acid). Human thyroglobulin phosphoserine-2721
(S, in red) is a FAM20C canonical phosphorylation site, confirmed by mass spectrometry ,
and is conserved between human, mouse and rat. A segment of the thyroglobulin
carboxy-terminal cholinesterase-like (ChEL) domain and short unique tail sequence is
shown within the boxed area. Importantly , phosphorylation is thought to improve the
efficiency of T3 formation in thyroglobulin. d | Sulfation and hormonogenesis are
hypothesized to be competitive reactions, given that Tyr5 of porcine thyroglobulin has
been found to be sulfated. 3'-Phosphoadenosine 5'-phosphosulfate (PAPS) is used by
sulfotransferases in the Golgi complex as a sulfate source for protein sulfation.
small interfering RNA (siRNA)-mediated knockdown of
FAM20C significantly decreased the ability of secreted
thyroglobulin to undergo de novo T3 formation87. In
addition, dephosphorylation of secreted thyroglobulin
partially inhibited its de novo T3 formation87.
Of the potential phosphorylation sites that are both
predicted casein kinase and canonical FAM20C targets,
the serine residue at position 2721 of human thyroglob-
ulin has been shown by mass spectrometry to actually
be a phosphoserine residue135 (Fig. 4c). This site is specif-
ically conserved between human, rat and mouse and is
thereby implicated as a potential regulator of de novo T3
formation within thyroglobulin.
Sulfation of thyroglobulin. Sulfation is a late post-
translational modification step that takes place in the
trans-Golgi network and contributes to the negative
charge of the thyroglobulin molecule. Sulfation involves a
complex enzymatic mechanism that is dependent on the
production of 3ʹ-phosphoadenosine 5ʹ-phosphosulfate
(PAPS) in the cytosol and its subsequent transport into
the trans-Golgi compartment, where it is used as a sub-
strate by protein sulfotransferases136. The sulfate residues
of thyroglobulin are largely present in complex carbo-
hydrates, such as chondroitin sulfate chains127,137; the
average sulfate content in purified thyroidal thyroglob-
ulin is ~1.5 mol sulfate per mol complex oligosaccharide
units115,130. Evidence suggests that human thyroglobulin
derived from thyroid nodules has an increased incorpora-
tion of [35S]sulfate into its oligosaccharide units compared
with that derived from healthy thyroid tissue138,139.
Interestingly, it has been reported that TSH generally
downregulates sulfate incorporation into thyroglobulin
Thyroglobulin and specifically downregulates thyroglobulin tyrosine
non-hormonogenic tyrosine sulfation115. TSH could modulate thyroglobulin sulfa-
residues tion by influencing the relative expression levels of sulfo­
Refers to the tyrosines within
thyroglobulin that are not
transferases and glycosyltransferases111 or by altering
directly involved in thyroid the rate of passage of thyroglobulin through the Golgi
hormone synthesis. complex140. In addition, the same consensus sequence
Nature Reviews | Endocrinology
Reviews
for iodination and hormonogenesis of thyroglobulin
(Asp-Tyr or Glu-Tyr) is also favoured for tyrosine sulfa-
tion141, which strongly suggests that these are competing
reactions. Interestingly, in porcine thyroglobulin, some
of the Tyr5 residues (the most prevalent site of T4 forma-
tion) were found to be sulfated115,142 (Fig. 4d). Conceivably,
the sulfate group could be removed before thyroglobulin
iodination and coupling143, offering a number of spec-
ulative alternatives for possible regulation of de novo
hormonogenesis within thyroglobulin.
Iodination of tyrosine residues within thyroglobu-
lin. The iodine content of human thyroglobulin varies
largely with iodide intake, which can be in the region
of 0.05–1.1% (w:w), that is, 2.5–55 atoms of iodine per
mol of thyroglobulin126. Although thyroglobulin has
~70 tyrosine residues, classic studies have shown that,
under physiological conditions, only 16 sites are iodin­
ated in human thyroid glands, and 13–15 sites are
iodinated in thyroglobulin derived from rat thyroid
glands144. By contrast, a 2011 analysis of unpurified
thyroglobulin from mouse thyroid by shotgun mass
spectrometry revealed as many as 37 iodinated tyro­
sine residues145. As only a few positions are involved in
hormonogenesis (maximally approximately five donor–
acceptor iodotyrosine pairs), these findings emphasize
that thyroglobulin non-hormonogenic tyrosine residues
have an underappreciated iodide storage function that
might have played a critical role in thyroid physiology
during the evolution of land-dwelling vertebrates145.
Nevertheless, studies have shown that the initial iodina-
tion of thyroglobulin tends to occur at preferred tyrosine
residues that are involved in hormonogenesis89, driven
by the native 3D structure of thyroglobulin, which offers
tyrosine residue exposure as well as structural proxim-
ity of donor and acceptor iodotyrosines. Residues that
flank the tyrosine residue may also influence iodina-
tion and hormonogenesis. The Asp-Tyr or Glu-Tyr
motif has been described as a ‘consensus sequence’ in
that it appears to be associated with many (but not all)
hormonogenic tyrosine residues145,146. In many species,
Ser-Tyr-Ser or Thr-Tyr-Ser occurs at the T3 synthesis
site within the carboxy-terminal region of thyroglobu-
lin89,145,147, and Glu-X-Tyr is commonly associated with
non-hormonogenic iodotyrosines89,145,147.
Thyroglobulin hormonogenic sites
In human thyroglobulin, three main hormonogenic
acceptor sites have been identified — Tyr5 (site A),
Tyr2554 (site B) and Tyr2747 (site C) plus the minor
site Tyr1291 (site D) (Fig. 1b) — some studies describe
Tyr685 as a fifth site31,70,147,148. At human Tyr972, both
MIT and DIT were recovered147, although this could vary
between species145.
Site A. In all vertebrate species examined, the most
important T4-forming site is at Tyr5 (refs144,149) (site A),
with Tyr130 (human numbering) serving as an impor-
tant outer ring donor for thyroxine formation at this
site 149 (Fig.  1b) . In most species, Tyr5 accounts for
40–50% of all T4 present in thyroglobulin and 25% of T3.
Furthermore, the tyrosines at positions orthologous to
Reviews

a Region I Region II Region III

Y5 Y130

NH2 Linker Hinge ChEL domain COOH

I I
O

I I
Coupling OH

Y2747
b

I I
O
Dimerization OH
I

Coupling

Fig. 5 | Mechanisms of de novo hormonogenesis at conserved sites on thyroglobulin. a | Formation of T4 at the

evolutionarily conserved Tyr5 site within thyroglobulin involves the di-iodotyrosine (DIT) acceptor undergoing
intramonomeric coupling with a DIT donor residue. b | Formation of T3 at the evolutionarily conserved antepenultimate
tyrosine residue of thyroglobulin involves intermolecular coupling of donor mono-iodotyrosine (MIT) and acceptor DIT
from apposed monomers within the thyroglobulin dimer. Type 1 repeats, light blue boxes; type II repeats, yellow boxes;
type III repeats, darker blue boxes; carboxy-terminal cholinesterase-like (ChEL) domain, pale yellow rectangle; zone of
dimerization, pink double trapezoid.

human thyroglobulin Tyr5 and Tyr130 are conserved
in mammals and other vertebrates (Fig.  1b), and in
these species, they are also thought to serve as main
sites of T4 synthesis27,144–146,150–152. A purified human thy­
roglobulin polypeptide fragment from Asn1 to Met171
(which maintains most of its 3D conformation as
demonstrated by antibody epitope mapping153) is itself
capable of forming T4 upon iodination at Tyr5, with
the loss of the Tyr130 phenolic side chain150, which
demonstrates the potential for intramolecular coup­
ling of these residues at the amino-terminal portion of
thyroglobulin — a conclusion that has been independently
supported by several groups and methodologies149,154
(Figs 1b,5). In mouse thyroglobulin, Tyr239 has been
suggested as an alternative potential iodotyrosyl donor
residue to the hormonogenic Tyr5 (ref.145).
Site B. Iodination of human thyroglobulin at Tyr2554
(site B) is conserved between humans, mammals and
other vertebrates 27,145,152. However, different views
exist about the utilization of site B as a hormonogenic
donor152 or acceptor145, which might depend upon the
species and conditions (such as iodide concentration)
or the technology used for analysis.
Site C. The antepenultimate thyroglobulin tyrosine res-
idue has been conserved throughout evolution for 500
million years27,145,152 (Tyr2747 according to human num-
bering; also known as site C) and is the major T3-forming
site26,110 (Fig. 1b), accounting for ~50% of T3 production
within thyroglobulin26. Interestingly, this conserved res-
idue abundantly forms both MIT (making it a possible
donor residue in coupling; Fig. 3a) and DIT (as expected
for an acceptor residue)147,152. Proteolytic cleavage of thy-
roglobulin at Lys2742 excises a carboxy-terminal SYSK
tetrapeptide that is too small to be captured by mass
spectrometry, causing it to escape detection145 and thus
interfering with definitive identification of this puta-
tive T3-containing fragment. A study recently reported
that upon iodination, the side chain from the antepe-
nultimate residue of mouse thyroglobulin (MIT2744)
closely interacts with and donates to the same residue
(DIT2744) of the apposed monomer within the thy-
roglobulin dimer to form T3 (ref.110) (Figs. 1b,5). This crit-
ical site C falls within the short unique tail sequence that
immediately follows the thyroglobulin ChEL domain.
The development of a simple and specific method to
detect T3 formation upon in vitro iodination of recom-
binant thyroglobulin made it possible, for the first time,
to detect the mechanism of de novo T3 hormonogenesis
at site C110. For example, studies demonstrated that the
secretory ChEL region (lacking regions I, II and III),
which is sufficient to form a secreted homodimer109, is
able to form close contact between the antepenultimate
tyrosine side chain and the side chain of the same residue
in an adjacent secretory ChEL (or that of an adjacent
full-length thyroglobulin)110. Furthermore, secretory
ChEL maintains the potential to synthesize T3 at site C110.
Indeed, an artificially engineered mutation that allows
for covalent (disulfide) ‘stapling’ of the apposed unique

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Cytoplasm
↑ Expression of TG
Nucleus ↑ Thyroglobulin endocytosis and
degradation
↑ T3 formed within thyroglobulin
Gene expression
CREB ELK1
ERK1/ERK2 NF-KB
CREB Ca2+
MEK1/MEK2
PKA RAP1 RAF PKC
cAMP DAG Ins(1,4,5)P3
Gαq
ATP Gαs PtdIns(4,5)P2
PLC
AC
TSHR
Basolateral
membrane
TSH
Fig. 6 | TSH regulation of thyroid hormone biosynthesis in thyroglobulin. TSH binds to
its G protein-coupled receptor (TSHR) on the basolateral membrane of thyrocytes, which
primarily activates guanine nucleotide-binding protein Gαs to catalyse cAMP synthesis
(this is the major TSH-activated signalling pathway) and the Gαq–Gα11-mediated signalling
pathway.  TSHR stimulation induces the expression of critical genes involved in thyroid
hormone formation, including TG, TPO, NIS and TSHR, and promotes thyroglobulin
endocytosis and degradation for hormone release as well as both dual-function oxidase 2
(DUOX2) and type 1 and type 2 iodothyronine deiodinase (DIO1 and DIO2) activities to
further increase thyroid hormone production. AC, adenylyl cyclase; CREB, cAMP response
element binding; DAG, diacylglycerol; ELK1, ETS transcription factor ; Ins(1,4,5)P3,
inositol-1,4,5-triphosphate; NF-κB, nuclear factor-κB; PKA , protein kinase A ; PLC,
phospholipase C; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate.
tail sequences ten residues away from the antepenulti-
mate T3 site enables increased contact between these
tail sequences and significantly enhances T3 formation
at site C110. Thus, the unique tail sequence of thyroglob-
ulin, which contains site C, has essentially become incor-
porated into the conserved ChEL domain during TG
evolution27: ChEL provides homodimerization, which
enhances MIT–DIT coupling at the ­antepenultimate
tyrosine residues110 (Figs 1b,5).
Minor hormonogenic sites. The ‘minor’ hormonogenic
site D has been reported at position Tyr1291 (human
numbering), which is conserved in bovine thyroglob-
ulin as well as in other mammals145,147. Studies utilizing
a fragment of bovine thyroglobulin encompassing resi-
dues 1218–1591 suggested position 1375 to be a donor
for position 1291 (ref.155). Additionally, more minor
hormonogenic sites have been found that are poorly
conserved between human, Xenopus, zebrafish and
lamprey27. Minor sites such as these might be used to
Nature Reviews | Endocrinology
Reviews
optimize hormone production in special situations, such
as in iodide deficiency. However, a more likely hypo­
thesis is that the tyrosine residues that are less well con-
served in their precise location between species might
function primarily as non-hormonogenic sites needed
for iodine storage.
Regulation of thyroid hormonogenesis
TSH action on its receptor stimulates thyroglobulin
endocytosis from the apical membrane of thyrocytes
for hormonogenesis (Fig. 2) as well as the expression and
activities of critical gene products involved in thyroid
hormone formation. In addition, TSH can stimulate
thyroidal DIO1 and DIO2 deiodinase activities, which
can further increase T3 production86,156 (Fig. 3b). TSH
binds to its G protein-coupled receptor TSHR on the
basolateral membrane of thyrocytes and mainly activates
the guanine nucleotide-binding protein Gαs to stimulate
cAMP synthesis157 (Fig. 6). Activation of the cAMP cas-
cade stimulates the expression of thyroglobulin, TPO,
NIS and TSHR158,159. By contrast, there is no significant
induction of the H2O2-generating DUOX system by TSH
or forskolin (a chemical activator of adenylyl cyclase)
in human thyrocytes in primary culture160. However,
human DUOX2 promoter activity can be positively
induced by TSH and forskolin at high doses in PCCL3
(rat) thyrocytes161. Through a protein kinase A (PKA)-
dependent pathway, TSH also regulates the insertion of
pendrin into the plasma membrane of thyrocytes162.
An additional effect of TSHR activation occurs via
the Gαq–Gα11-mediated phospholipase C (PLC)–diacyl­
glycerol–calcium pathway, which increases production
of H2O2 by the DUOX–DUOXA system163 to enhance
TPO activity164. Mice deficient in Gαq–Gα11-mediated sig-
nalling show impairment of thyroid hormone secretion
in response to TSH; however, these mice can still carry
out Gαs-mediated thyroid hormone synthesis, which still
seems to be the major TSH-activated signalling path-
way165. Thyrocyte-specific deletion of Gαs in adult mice
rapidly leads to impaired thyrocyte function and hypo-
thyroidism with a substantial compensatory increase
in serum TSH166. Interestingly, in vitro studies utiliz-
ing a specific TSHR agonist that activates Gαs showed
that thyroglobulin secreted from PCCL3 rat thyrocytes
was capable of increased T3 synthesis upon iodination
under standard conditions87,167. Moreover, incubation
of PCCL3 cells in the presence of dibutyryl cAMP also
resulted in the secretion of thyroglobulin with enhanced
capability for de novo T3 formation. These findings
are consistent with TSH binding to TSHR and acting
through stimulatory G proteins and cAMP production
to induce modifications of thyroglobulin that alter its
structure such as to enhance its intrinsic ability to form
T3 upon iodination87.
Notably, thyroglobulin is an intrinsic negative-
feedback regulator that can limit the effects of
TSH108,168,169, affecting TSHR-mediated cAMP–PKA
and PLC–PKC signalling pathways and NKX2.1 and
PAX8 expression170 and also potentially exerting effects
on thyroid cell proliferation171. Various metabolites have
also been reported to inhibit thyrocyte function172,173,
the most important of which appears to be iodide itself.
Reviews
Haploinsufficiency
A condition that arises when
there is a complete loss of
function of one copy of a gene,
and the remaining functional
copy of the gene is not
adequate to preserve normal
function in a diploid organism.
Iodide negatively influences the TSH response, and high
doses of iodide can be used to block TSH-driven thyroid
hormone synthesis174.
Graves disease and iodide deficiency. As discussed
above, TSHR stimulation influences several important
thyroglobulin post-translational modifications that
could increase de novo T3 formation87. Two common
thyroid conditions — Graves disease (with an annual
incidence of ~30 cases per 100,000 people) and iodide
deficiency (affecting ~2 billion people worldwide) —
cause hyperthyroidism and hypothyroidism, respec-
tively175; however, both conditions share the feature of
hyperstimulation of thyroidal TSHRs. TSHR hyperac-
tivation can also occur as a consequence of activating
mutations in the TSHR, a rare condition that has been
described primarily in patients of European ances-
try176–179. A relative increase in T3 synthesis is observed in
each of these disorders148,176–178,180,181. Interestingly, incu-
bation of PCCL3 cells with TSHR-stimulating immuno-
globulin G (IgG), or unpurified sera from some patients
with Graves disease, induces increased T3 formation
upon in vitro iodination of secreted thyroglobulin87.
Thus far, the molecular contributions to the relative
increase in T3 synthesis remain incompletely defined,
but the data suggest that de novo T3 formation within
thyroglobulin is one of the important contributors87,148.
Thyroglobulin secreted from thyrocytes with genetic
deletion of TSHRs consistently exhibited decreased
de novo T3 formation compared with T3 synthesized in
the thyroglobulin secreted from thyrocytes with stim-
ulated TSHRs87. By contrast, there is no demonstrable
evidence that TSHR stimulation enhances the efficiency
of de novo T4 formation per thyroglobulin molecule87,182,
despite the fact that overall T4 production clearly rises in
patients with Graves disease.
Genetic disorders caused by TG mutation
Hormonogenesis defects caused by TG mutations are
inherited as an autosomal recessive trait, causing con-
genital hypothyroidism, which has an estimated global
incidence of approximately 1 in 100,000 newborn
babies183. Some patients present with thyroid enlarge-
ment shortly after birth, others later in life, and in a few
individuals no goitre develops. Primary hypothyroidism
and thyroid gland enlargement depend on the severity of
the TG defect, the amount of dietary iodide intake and
patient adherence to thyroid hormone replacement ther-
apy. Untreated severe congenital hypothyroidism can
result in irreversible mental delay and short stature175.
The biochemical profile of patients with homozygous
or compound heterozygous TG mutations is charac­
terized by low or absent serum levels of thyroglobulin,
high serum TSH, normal perchlorate discharge test
(indicating a normal iodide organification mechanism),
low serum levels of circulating T4 and variable levels of
circulating T3 (usually resulting in a relatively increased
T3:T4 ratio)183. The first described human mutation
causing a TG defect associated with congenital hypo-
thyroidism was c.275-3 C>G (Supplementary Table 1).
In 2019, 167 human TG gene mutations have been
identified to date: 21 splice site mutations, 36 nonsense
mutations, 89 missense mutations, 5 insertions or dupli-
cations, 15 deletions and 1 imperfect DNA inversion89,183
(Supplementary Table 1). Interestingly, ~30% of these
human TG mutations have been described within the
past 2 years. TG mutations have also been described in
Afrikaner cattle (p.R697*)184, Dutch goats (p.Y296*)185,
cog/cog mice (p.L2263P)186, rdw/rdw rats (p.G2300R)187
and Wistar Hannover GALAS rats (c.749-1 G>T)188.
The animal models provide a heterogeneous preclin-
ical spectrum of congenital hypothyroidism. Because
of the autosomal recessive inheritance, patients who
present clinically with hypothyroidism are generally
either homozygous or compound heterozygous; how-
ever, a few monoallelic variants have been described.
Conceivably, the apparent absence of a second muta-
tion in human patients could be explained by technical
limitations of direct TG sequencing (neither micro­
deletions involving one or several exons nor muta-
tions in distant regulatory or intronic regions of the
TG gene can be excluded). Moreover, in the setting of TG
haploinsufficiency, patients might develop clinical disease
by the undetected presence of a mutation in a second
gene that also disrupts thyroid homeostasis. With this
in mind, several monoallelic TG mutations have been
found in individuals with mild congenital hypothyroid-
ism189. Moreover, a report exists of a patient with severe
hypothyroidism who harbours pathogenic heterozy-
gous variants of both TG and TPO genes, suggesting
that some patients might develop hypothyroidism as a
consequence of oligogenicity190.
The p.C1058R and p.C1977S mutations are the most
frequently identified TG mutations in the Japanese
population, whereas p.R277* is the most frequently
identified TG mutation found in white individuals in
families from Brazil, Argentina, Spain and France26,191
(Supplementary Table 1). The structural consequence
of the p.R277* mutation is the expression of a truncated
protein, which has a complete loss of the central and
carboxy-terminal hormonogenic domains and, conse-
quently, a limited ability to generate thyroid hormone.
Theoretically, however, the p.R277* thyroglobulin poly-
peptide, if iodinated, should retain its ability for T4 and
T3 synthesis because it still harbours both the acceptor
Tyr5 and the donor Tyr130. More important than any
limitation in the ability of thyroglobulin to generate
active thyroid hormones, most mutant thyroglobulin
proteins are misfolded, which causes their retention
in the ER with premature degradation, as observed in
preclinical (animal) models192. Nevertheless, limited
amounts of mutated thyroglobulin molecules might
reach the follicular lumen, enabling iodination with the
potential synthesis of thyroid hormones, as observed in
many patients with congenital hypothyroid goitre and
defective thyroglobulin.
Remarkably, patients have been identified with
compound heterozygous TG mutations encoding
p.R277*/p.C1981W-p.P2183R or p.C1245R/p.G2356R
(Supplementary Table 1), who exhibited an absolute
increase in plasma levels of T3 (refs193,194). Bioinformatic
studies of thyroglobulin p.C1981W-p.P2183R predict
that the protein could be efficiently secreted193, and
functional studies of recombinant thyroglobulin p.C175*
www.nature.com/nrendo
 Reviews

Monoallelic mutations
Changes in the genetic
sequence in one of two
homologous alleles (paternal
or maternal) of a single gene.
demonstrate its secretion, leaving open the possibility
that thyroglobulin p.R277* might also be secreted191.
Possibly the increase in plasma T3 might be due to
elevated DIO2 activity194 in the thyroid gland; alterna-
tively, certain mutations in TG might provoke structural
changes that trigger the appearance of cryptic sites of
T3 formation in thyroglobulin that become favoured by
TSH hyperstimulation.
Finally, a few patients with TG mutations do not
develop goitre. For example, a goitre has not been
reported in patients homozygous for p.G2300D195, which
occurs in the position equivalent to the p.G2298R muta-
tion responsible for the phenotype in rdw/rdw dwarf rats
that develop a hypoplastic thyroid gland187. Similar to
goitrous congenital hypothyroidism, misfolded thy-
roglobulin mutants responsible for nongoitrous hypo-
thyroidism in rdw/rdw dwarf rats196 are linked to an
increased ER stress response197, but importantly, the
Tgrdw/rdw rats exhibit proteotoxic thyrocyte cell death198,
which appears to explain the lack of goitre.
Conclusions
Recognizing the importance of thyroglobulin in thy-
roid hormone synthesis could provoke new studies
investigating ancestral pre-vertebrates that synthesize
thyroid hormones despite the absence of a complete
vertebrate-style TG gene. The possibility exists that in
the endostyle of pre-vertebrates, thyroglobulin regions
expressed as separate gene products might potentially
account for thyroid hormone synthesis. Alternatively,
unrelated gene products, whose identification remains
to be determined, might serve as substrates for iodina-
tion and thyroid hormone synthesis, although their hor-
monogenic potential would presumably be less efficient
than that of thyroglobulin, which has been evolutionarily
selected for this function.
Additionally, we now recognize that de novo T3 for-
mation within thyroglobulin is an important contributor
to enhanced T3 production in pathological states during
which TSHRs are hyperstimulated87,148. To understand
the molecular mechanisms underlying the increased T3
production, future studies will need to further clarify
the T3 formation sites within thyroglobulin that are
used in Graves disease, as well as to understand how
TSHR-stimulated post-translational processing impacts
thyroglobulin structure to enhance its hormonogenic
capability. For this, new technologies including X-ray
crystallography of purified thyroglobulin regions or
cryo-electron microscopy, combined with other tools
that have the capability of single-molecule resolution,
might advance the field. Using these technologies in
conjunction with large-scale synthesis of recombinant
thyroglobulin domains could enable direct visualiza-
tion of the proximity between donor and acceptor hor-
monogenic iodotyrosine residues. Furthermore, such
technology might conceivably help in the analysis of the
rare compound heterozygous genotypes of patients who
actually exhibit a selective elevation of plasma T3 levels.
Finally, unusual diseases have been identified, such
as cystinosis199 and Fabry disease200, in which hypothy-
roidism is a common yet poorly understood feature and
in which pathogenic intracellular thyroglobulin traf-
ficking has been implicated. As personalized medicine
continues to expand, unexpected new gene products
might come to light that, in some capacity, modulate or
contribute to thyroid hormonogenesis. As such, further
study is needed (utilizing next-generation sequencing
platforms) of the interesting and rare patients with con-
genital hypothyroidism who have monoallelic mutations
of TG. Such patients might ultimately highlight some of
the most physiologically important genetic interactions
regulating the role of TG in thyroid hormone synthe-
sis. As a purely hypothetical example, genes regulating
autophagy might allow thyroglobulin entrapped in the
ER to be brought to an autophagosomal compartment
in which it might conceivably encounter molecules of
the iodination machinery, bypassing hormonogenesis at
the follicular lumen. Of course, 167 different human TG
mutations exist that are linked to hypothyroidism, and
synthetic interactions with other defective genes might
vary for different mutant TG alleles, altering disease
severity. Clearly, there is still much to learn.
Published online xx xx xxxx

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Acknowledgements
The authors acknowledge the support of NIH grant R01
DK40344.
Author contributions
C.E.C., H.M.T. and P.A. researched data for the article and
made a substantial contribution to discussion of content.
C.E.C. and P.A. wrote, reviewed and/or edited the manuscript
before submission.
Competing interests
The authors declare no competing interests.
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Nature Reviews Endocrinology thanks X. DeDeken, J.
Lado-Abeal and other anonymous reviewer(s) for their
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