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

6.Thyroid Hormone Transport Proteins and the Physiology of Hormone Binding

Salvatore Benvenga

This chapter includes part of the data published in the eighth edition of the text by Robbins (1). Because the sterol hormone–binding proteins (SHBP) also bind thyroid hormone (TH), they do contribute to TH binding in serum. Since TH and sterol-derived hormones are hydrophobic, they must be associated with proteins for transport in blood and delivery to distant sites of action. Multiple plasma proteins are involved in the transport of these hormones. For example, cortisol has cortisol-binding globulin (CBG) as a major carrier, and albumin and sex hormone–binding globulin (SHBG) as minor carriers (2). The opposite is true for dihydrotestosterone, whereas estradiol (E2) has both albumin and SHBG as a major and CBG as a minor carrier (2). Of the total circulating 1,25(OH)2D3 and less potent 25(OH)D, 85% and 90% are bound to the α2-globulin vitamin D–binding protein (VDBP) and the remainder to albumin, a VDBP homologue (3). However, about half of the intestinal absorbed vitamin D is carried by lipoproteins (4).

There are three conventional major carriers for TH (Fig. 6.1) [thyroxine-binding globulin (TBG), transthyretin (TTR), and albumin (Table 6.1)], plus a number of minor carriers (5)(Table 6.2), some of which are also SHBP. TBG and CBG belong to the family of the serine protease inhibitors (SERPINs). In human plasma, the free fraction of TH [~0.03% of total thyroxine (T4) and ~0.3% of the biologically more potent triiodothyronine (T3)] is lower than free steroid hormones (> 1%), similar to the free fraction of 25(OH)D (0.03%) and the more potent 1,25(OH)2D3 (0.4%). To add to the parallelism between TH and vitamin D, VDBP in the Emydidae family of turtles is a high-affinity binder of both vitamin D and T4 (6). Finally, considering that one TBG homologue, lipoproteins, and TTR are carriers of retinoids (or the retinoid precursor retinol), then it is of interest that such diverse hormones, which share the ancestors of the corresponding nuclear receptors, also share a number of plasma carriers. Indeed, nine hydrophobic repeats in domain 3 of α-fetoprotein (AFP)—a homologue of albumin and VDBP that binds TH, steroid hormones, and retinoids (7,8)—has structural homology with the heptad dimerization repeats of the nuclear receptor superfamily (9).

FIGURE 6.1. Iodine 125 thyroxine (T4) binding to serum proteins as assessed by zone electrophoresis on agarose gel in a euthyroid adult. Three aliquots from the same incubation mixture (serum plus radiolabeled T4) were run: one aliquot for counting radioactivity, one for protein staining (Coomassie blue), and one for lipid staining (Oil-red-o). Note the inter-position of the highest peak of radioactivity (i.e., the TBG peak) and that the α1 tail of the thyroxine-binding globulin peak overlaps the left part of the α-lipoprotein area (i.e., high-density lipoprotein). The most anodal and cathodal radioactive peaks are trans thyretin and immunoglobulin, respectively. (From Benvenga S, Lapa D, Trimarchi F. Thyroxine binding to members and nonmembers of the serine protease inhibitor family. J Endocrinol Invest 2002;25:32–38, with permission.)

TABLE 6.1. MAIN CHARACTERISTICS OF THE MAJOR PROTEIN TRANSPORTERS OF THYROID HORMONES IN HUMANS


 

TBG

TTR

Albumin


Gene

  Family

SERPINS

Transthyretin

Alb/αFP/VDBP

  Chromosome

Xq22.2

18q12.1

4q11-q13

  Coding exons

5

4

15

Protein

  Site of synthesis

L

L, P islets, CP, R, K

L

  Molecular mass (kd)

54

54 (tetramer)

65

  Residues (n)a

395

127

595

  Electrophoretic mobilityb

α-globulin

Prealbumin

Albumin

  Serum concentration

~12–30 mg/L

~150–400 mg/L

~3.5–5.0 g/dL

 ~0.22–0.56 µM

~2.8–7.4 µM

~540–770 µM

  Half-life (days)

5

2

15

Binding sites

   Ka for T4 (M-1)

Primary

No. 1, ~1.0 × 1010

No. 1, ~7.0 × 107

No. 1–5, ~7.0 × 105

      Secondary

 No. 1, ~7.0 × 105

No. 1, ~5.0 × 104

  Ka for T3 (M-1)

      Primary

No. 1, ~5.0 × 108

No. 1, ~1.0 × 107

No. 1, ~1.0 × 105

      Secondary

 No. 1, ~5.0 × 105

No. 1–5, ~7.0 × 103

  % Serum T4 carriedc

Electrophoresis

64d(68)

13 (11)

9 (20)

      Immunoprecipitation

49d

12

7

  % Serum T3 carried (electrophoresis)

80

9

11

Other ligands

 Retinol-binding protein

Steroids, vitamin D, ions, lipids, drugs, etc.


αPF, α-fetoprotein; CP, choroid plexus; K, kidney; Ka, affinity constant; L, liver; P, pancreas; R, retina; T3, triiodothyronine; T4, thyroxine; TBG, thyroxine-binding globulin; TTR, transthyretin; VDBP, vitamin D–binding protein.

aMature protein. The corresponding precursors are 415, 147, and 609 residues long.

bTBG peaks between α1 and α2 (inter-α globulin zone). The coexistence of the normal and variant (alloalbumin) protein causes the appearance of a bifid albumin peak (bisalbuminemia) in heterozygotes but no perturbation of thyroid homeostasis

cPercentages for T3 and those in parentheses for T4 are from the 8th edition of this chapter (1). The other percentages for T4 at electrophoresis and immunoprecipation are from reference 5. For comparative purposes with some steroids, albumin binds 6.3%, 21%, and 60.8% of circulating cortisol, dihydrotestosterone, and estradiol, respectively (2)

dThe large discrepancy between zone electrophoresis (ZE) and radioimmunoprecipitation (RIP) is seen because other α-migrating proteins bind T4 (see reference 5 and Table 6.2). Assuming that the two α-globulins not tested by RIP in reference 5 bind ~0.7% of T4 each, the average proportion of circulating T4 carried by α1-AT, α1-ACT, AT-III, cortisol-binding globulin, and high-density lipoprotein would add up to 6.3%. Hence, the 64% carried by TBG at ZE (5) is overestimated by at least 6%. If the 49% proportion of serum T4 bound by TBG at RIP is correct, then there still is 9% of T4 (58% – 49% = 9%), which is carried by other alpha-globulins to be indentified

 

TABLE 6.2. MINOR THYROID HORMONE PLASMA CARRIERS


 

Lipoproteina

 

 

VLDL

LDL

HDL

Ig

SERPINb

Othersc


% T4, GFC

0.04

0.3

2.9

ND

ND

ND

ZE

0.15

1.2

8.6

2

ND

ND

RIP

1.7 (VLDL + LDL)

 

3.5

4 (2 IgG, 2 IgM)

2.8

~0.6

% T3, GFC

0.02

0.05

6.0

ND

ND

ND

ZE

ND

ND

ND

2

ND

ND

RIP

ND

ND

ND

4 (2 IgG, 2 IgM)

ND

ND

Ka for T4(M-1)d

106

4 × 106

106

106–1010

~2 × 106

ND

Ka for T3 (M-1)

≤105

≤105

≤105

105–109

Ka for T4

ND

Synthesise

L

From VLDL

L, I, P

B-lymphocytes

L

L


GFC, gel filtration chromatography; HDL, high-density lipoprotein; I, intestine; Ig, Immunoglobulin; Ka, affinity constant; L, liver; LDL, low-density lipoprotein; ND, not determined; P, plasma; RIP, radioimmunoprecipitation; T3, triiodothyronine; T4, thyroxine; VLDL, very low-density lipoprotein; ZE, zone electrophoresis.

aChylomicrons, which are synthesized in the intestine, are not listed because normally they are present only in postprandial plasma. Their structural apo is apoB-48, which consists of the first 2152 residues of the full-length apoB-100 (4536 residues). Thus, apoB-48 contains the first two of the three thyroid hormone–binding sites of apoB-100 [amino acids 380–392, 1310–1338 and 4223–4510 (data from reference 32)]. Chylomicrons also contain apoA-I, A-IV, and, by transfer from the HDL, apoE and the apoCs

bThe following SERPINs were tested and demonstrated to be T4 binders in reference 5: α1-antitrypsin (α1-AT), α1-antichymotrypsin (α1-ACT), antithrombin III (AT-III) and corticosteroid-binding globulin (CBG). The first two were also tested by RIP, with 125I-T4 binding averaging 1.0% and 0.4% of total activity added to human serum, and Scatchard analysis (Ka = 1.0 and 1.0 × 106 M-1 and number of binding sites 0.9 and 1.0 respectively). The listed value of 2.8% by RIP assumes that AT-III and CBG bind 0.7% each. Binding to α1-AT, α1-ACT, and the non-SERPIN α1-acid glycoprotein, which are all positive acute-phase reactants, should be much greater than listed here during stressful conditions and nonthyroid illnesses, because serum levels of the three proteins increase considerably. Under the same conditions, serum levels of TTR and albumin, and sometimes TBG, decrease. For comparison with corticosteroids, CBG binds 89.7% of circulating cortisol (2), 4% of testosterone, 2% of progesterone, and < 0.1% of estradiol (5)

cThese other non-SERPIN proteins were tested and demonstrated to be T4 binders in reference 5: α1-acid glycoprotein (orosomucoid) and the β-globulin sex hormone–binding globulin (SHBG). The former only was tested by RIP, and an average of 0.30% of 125I-T4 was bound. The listed value of 0.6% assumes a similar value for SHBG. However, as explained in the text and Table 6.1, other α-globulins that are minor thyroid hormone plasma carriers should exist. For comparison with some steroids, SHBG binds 0.2%, 37.3%, and 78.4% of circulating cortisol, estradiol, and dihydrotestosterone, respectively (2)

dConcerning lipid-free apos, only Ka for T4 of human apoA-1 and apoE has been experimentally determined by Scatchard analysis (34). Depending on the apo preparations used, Ka for T4 was 4.4 to 7.5 × 107 M- (apoA-1), and 2.4 to 4.0 × 107 M-1 (apoE). Ka of bovine apoA-1 or rabbit apoE matched the corresponding human apo (34).

eTissue synthesis of apos is always in the liver, except apoA-IV and B-48 (intestine). ApoA-I is also synthesized in the intestine and brain, and apoE is in a variety of tissues (brain, in particular)

THYROXINE-BINDING GLOBULIN

TBG is the major plasma carrier of TH in most of the large mammals. Of 100,000 molecules of circulating TBG, approximately 20,000 are occupied by T4 as compared to approximately 300 of TTR and 3 of albumin. Unlike TTR and albumin, TBG is a glycoprotein. The four carbohydrate chains contain the following oligosaccharide residues per TBG molecule: 10 of sialic acid, 9 to 13 each of galactose and mannose, and about 20 of glucosamine. The carbohydrate moiety contributes, with TH, to its molecular stability and is responsible for TBG microheterogeneity in the pI (isoelectri point) 4.2 to 5.0 range (the greater the sialic acid content, the greater the anodal mobility). TBG crystallographic structure has not yet been determined, unlike that of other homologues [e.g., α1-antitrypsin (α1-AT)]. Based on α1-AT structure, the T4 binding site of TBG is in a β-barrel formed by three strands of sheet C (amino acids 212–223,279–286, and 357–368) and four strands of sheet B (amino acids 220–225, 233–242, 245–252, and 370–379), particularly important being Phe249. Segments 215–291 and 365–395 are the part of human TBG most conserved in mammal TBG (> 95% similarity) (10), and indeed, the acid dissociation constant (Ka) for T4 of mammal and hTBG match (10). For compounds affecting TH binding to TBG and other carriers, see Chapters 11 and Chapters 13.

The large discrepancy between zone electrophoresis (ZE) and radioimmunoprecipitation (RIP) (Table 6.1) is seen because TBG migration overlaps with other α-globulins, whereas TTR and albumin do not comigrate with other proteins. At an average concentration of 2 mg/dL in a serum containing 7.0 g/dL total protein and 0.98 g/dL total α-globulins, TBG accounts for only 0.2% of the total plasma α-globulins. Thus, a number of other α-globulins could bind TH and be confused with TBG in ZE. This is the case of high-density lipoproteins (HDLs), the α-migrating lipoproteins (Fig. 6.1), which bind approximately 3% of circulating T4. Other binders are the TBG homologues α1-AT, α1-chymotrypsin (α1-ACT), antithrombin III (AT-III), and CBG, and the nonhomologous α1-acid glycoprotein (AGP) and SHBG, a β-globulin (5) (Table 6.2). Additional T4 carriers could include AFP (7,8,9) and lipocalins (see later section Additional Proteins). SERPINs have one TH site with relative affinity T4 > T3, but Ka for T4 is much lower than in TBG (5). Conservation of most residues in the β-strand 3B, including TBG Phe249, occurs in these SERPINs and TBG.

Recently the binding of E2, progesterone, testosterone, cortisol, aldosterone, and all-trans-retinoic acid to the inhibitory SERPINs AT-III, heparin cofactor II, plasminogen activator inhibitor I, and protein C inhibitor (PCI) was studied. Only all-trans-retinoic acid bound to PCI (fivefold more than buffer vs. twofold for the other hormones), specifically with a dissociation constant (Kd) of 2.4 µM and one site per mole (11). All trans-retinoic acid is also bound by plasma lipoproteins (12).

TRANSTHYRETIN

TTR circulates as a homotetramer of known crystallographic structure. The four subunits are arranged symmetrically around a cylindrical channel. The central β-barrel where TH binds is formed by eight strands (strands A–H) (Fig. 6.2), corresponding to amino acids 11–19, 29–32, 42–49, 53–55, 67–74, 91–97, 105–112, and 115–121. Strands G and H contribute 6/10 residues of the TH binding surface. The TH sites of the homotetramer are two, but only one is occupied due to the much lower Ka of the second site, as a result of negativity cooperativity. Crystallography studies have shed light on the structural basis for negative cooperativity (13). The primary site has a larger diameter than the second site, and T4 (or other ligand) binding is followed by a slight collapse of the outer and inner parts of this site, and concomitant opening of the second site. Binding of the second ligand is then followed by collapse of the second site. However, this second collapse is more limited, so that the second molecule of ligand is bound less tightly (13). TTR is predominantly a retinol-binding protein (RBP), because RBP is carried by one in three molecules of TTR, and there are four RBP binding sites, but only one is likely to be occupied. The RBP and TH sites are independent. RBP, in turn, binds retinol (vitamin A) in a 1:1 molar ratio. Retinol, retinoids, carotenoids, and 1% to 2% of TTR itself are carried by lipoproteins (12,14,15). Interaction with TH is affected by several compounds, the strongest and most selective inhibitor being the flavonoid EMD 21388. A number of environmental pollutants also interfere with TH binding to TTR and/or TBG and albumin (16,17,18).

FIGURE 6.2. Schematic drawing of the transthyretin tetramer with triiodothyronine occupying one of the two identical sites in the binding channel. The 3′ iodine is in the distal configuration. The side chain carboxylate is shown at the channel entrance interacting with the ε-amino group of lysine 15. (From Jorgensen EC. Thyroid hormones and analogs. II. Structure activity relationships. In: Li CH, ed. Hormonal proteins and peptides. Vol. 6. New York: Academic, 1978:107, with permission. Drawn from the crystallographic data of Blake and colleagues.)

TTR is the sole member of its family, a gene of much more ancient lineage than TBG. Indeed, TTR is present in marsupials, rodents, insectivora, birds, reptiles, amphibians, and fish, and open-reading frames for TTR gene–like nucleotide sequences were found in bacteria, yeast and the nematode Cunninghamella elegans (19). Of interest, TTR of birds, reptiles, amphibians, and fish binds T3 preferentially (19). The switch to the preferential binding of T4in mammals was due to selective pressure on the N-terminus of TTR, which became shorter and more hydrophilic during evolution (19). Similar to TBG and apolipoproteins (apos), the T4 binding domain is the part best conserved in the phylum. In nonhuman animals, plasma TTR binds more TH than does TBG, but regardless of species, TTR is the major TH transport protein in the cerebrospinal fluid (CSF) (19). The vast majority of TTR in CSF derives from central nervous system synthesis (19). This synthesis (which is restricted to the choroid plexus, except for very little in the meninges) is absent in amphibians and fish, namely in species without a neocortex, but it does exist in species as ancient as reptiles, which are the first species showing traces of a cortex (19). Also expressed in the ependymal cells of the choroid plexus, but primarily in neurons of the adult brain, is the TBG homologue neuroserpin. Variants of this axonal-secreted protein cause an autosomal-dominant form of dementia (20). Neuroserpin is likely to be a low-affinity TH carrier, and impairment of TH binding could favor the intraneuronal polymerization of neuroserpin (21).

Local amino acid sequence homology exists between the tail of TTR (amino acids 90–127) and the glucagon-secretin family of gastrointestinal hormones (22), while two TTR segments (amino acids 18–53 and 72–117) share homology with the amyloid-related proteins (ARP), including the amyloidogenetic A4 segment of the amyloid β-protein precursor (AβPP) associated with Alzheimer's disease (23). The homology with ARP is relevant, because native TTR and several TTR variants are involved in senile amyloidosis and familial amyloid polyneuropathy, respectively. Finally, amino acids 38–57 of the β-subunit of human luteinizing hormone (LH) are homologous with residues 10–30 of TTR (24).

ALBUMIN

This multiligand protein is also of ancient lineage. It is composed of three repeated domains (amino acids 25–205, 212–397, and 404–595 in humans), each containing two subdomains (A and B). Using crystallographic analysis, four T4 sites were identified in subdomains 2A, 3A, and 3B (25). A fifth site is in the cleft between domains 1 and 2, created by the conformational changes on albumin induced by binding of the fatty acids (26). Fatty acids inhibit TH binding to plasma proteins; on albumin they bind in hydrophobic pockets that are distributed asymmetrically (26). Important for the high affinity of the site in subdomain 2A is Arg218. As mentioned above, albumin binds sterol-derived hormones, and its homologue VDBP binds both T4 and vitamin D in turtles of the Emydidae family (7), whereas the homologue AFP binds TH, steroid hormones, and vitamins (7,8,9).

LIPOPROTEINS

Gel filtration chromatography (GFC) of human plasma demonstrated binding of T4 (~3%), T3 (~6%), and reverse T3 (rT3) (~0.2%) to all classes of lipoproteins: chylomicrons, very low-density lipoproteins (VLDLs), low-density lipoproteins (LDLs), and high-density lipoproteins (HDLs) (27,28) (Table 6.2). However, there might be dissociation of TH from lipoproteins during GFC, because ZE and RIP give higher binding values (28). HDL, the major lipoproteins binder, encompasses a broad spectrum of particles between the approximately 60,000 to 450,000 molecular weight range. Distribution of TH between the numerous HDL subfractions is affected by thyroid status (29,30). For example, in euthyroid plasmas, T4 and T3 are associated with HDL subfractions of 111,000 ± 34,000 and 148,000 ± 31,000 (mean ± SD), respectively. However, in hypothyroid plasmas, the corresponding HDL subfractions have molecular weights of 176,000 ± 24,000 and 94,000 ± 30,000 (29). Interestingly, one HDL particle has the same molecular weight as albumin, has very little lipid, and its only protein moiety is apoA-1 (31). The association of TH with lipoprotein lipids has not been quantified. TH, though, binds specifically to apoA-I, apoA-II, apoA-IV, apoB-100, apoC-I, apoC-II, apoC-III, and apoE (27,28,32,33,34). The Ka for TH of the lipoproteins (i.e., the lipid-associated apos) is one to two orders of magnitude lower, because lipids inhibit TH binding to apos. Unlike with TBG (35), several types of lipids inhibit this binding.

The non-apoB-100 apos contain a single TH site of 107 M1 binding affinity (27,34), with relative affinity T4 = rT3 > T3. The TH site is N-terminal, in the exon 3-coded region (exon 2 for apoA-IV), which contains β-sheet structure. There is remarkable local homology in the TH binding domains of apos and TBG, TTR, and albumin (34). The homologous sequences are amino acids 292–348 and 497–534 of albumin, 72–122 of TTR (a segment that contains 6 of the 10 residues that form the TH binding surface, and strands F, G, and H of the binding channel), and 231–273 and 349–395 of TBG, corresponding to β-strands B2, B3, B4, and C2. Apos, including apoB100, share the hydrophobic motif Y,L/I/M/,X,X,V/L/I, the first two residues being conserved in TBG, TTR, and albumin. Particularly, Y matches F249 of TBG. This motif (located, for instance, at position 18–22 in apoA-I and 36–40 in apoE) is well conserved in the phyla, and it is believed to represent the core of the TH binding site (33,35). ApoB-100 contains three TH sites, one in each of three nonoverlapping fragments generated from the natural cleavage by circulating enzymes (32). Experimental data indicated TH sites at amino acids 380–392, 1310–1338, and 4223–4510 (32), in keeping with the sequences 360–406, 1303–1344, and 4281–4341 found by homology search (33).

In the trout, lipoproteins (HDL > LDL > VLDL) are the major T4 and T3 carriers (36), but unlike human lipo proteins, the Ka values for T4 and T3 are similar (36). This recalls the evolutionary changes in TH affinity of TTR (19). Lipoproteins also bind the lipid-soluble vitamins (4,14,37) and steroids (38) (see also S. Benvenga, unpublished observation).

IMMUNOGLOBULINS

Immunoglobulins (Igs) are minor TH carriers (Table 6.2 and Fig. 6.1). Unlike ZE, RIP permits discrimination between Ig classes (39,40). Above each one's laboratory cut-off point (i.e., the greatest proportion of TH bound to any Ig class in healthy persons), the serum is said to contain TH autoantibodies (THAbs). These can be T3Ab (approximately half of the cases), T4Ab (approximately one fourth of the cases), or T3 plus T4Ab (approximately one fourth of the cases). Traditionally, THAbs are considered to be IgG, though other Ig classes were not always investigated. Cases of IgA, IgM (41), and IgE THAbs were reported (42). In one patient, THAbs also bound steroids (41,43).

ADDITIONAL PROTEINS

Other minor proteins binding TH include AGP (5), which belongs to the lipocalin family and also includes proteins such as α1-microglobulin, tear prealbumin (a cysteine proteinase inhibitor), and apoD. Lipocalins adopt a β-barrel tertiary structure and are carriers of small hydrophobic ligands. The retinol binder, choroid plexus–secreted lipocalin I has functional homology with TTR (44). ApoD, which circulates predominantly associated with the HDL, is homologous with RBP and binds progestational hormones (45).

PATHOPHYSIOLOGY

Changes in Serum Levels of Thyroxine-Binding Globulin, Transthyretin, and Albumin

Mean serum levels are given in Table 6.1. TBG and TTR are age and gender dependent, but in opposite ways (46,47,48,49,50,51). TBG is highest in neonates (~28 mg/L) and progressively declines to approximately 19 mg/L at 10 to 15 years (46), with a significant decrease between Tanner stages 1 and 5 (47). TBG remains substantially stable there after, with values in premenopausal women greater than in men, because of the increased half-life that derives from the estrogen-induced sialylation of TBG (47). Interestingly, in one man with endogenous hyperestrogenemia and hypertestosteronemia, the androgen antagonized the E2 effect on TBG but failed in the E2 effect on PRL and TSH (49). Pregnancy, contraceptive pills, estrogen receptor modulators, 5-fluorouracil, liver diseases, acute intermittent porphyria, hypothyroidism, and inherited TBG excess (TBG-E) increase serum TBG; androgens, corticosteroids, hyperthyroidism, terminal illnesses, and inherited TBG deficiency (TBG-D) decrease or tend to decrease serum TBG.

Serum TTR in newborns averages 110 mg/L and progressively increases to approximately 300 mg/L at 10 to 14 years (50). After 50 years, TTR levels decrease and average 200 mg/L in octogenarians (51); in adulthood men have higher levels than women. In addition to androgen administration, TTR excess has been reported in pancreatic endocrine tumors (52), chronic renal failure (53), and as a compensatory mechanism in analbuminemia (inherited deficiency of albumin). In maintenance dialysis patients, serum TTR of less than 300 mg/L predicts increased risk for morbidity and mortality (53). Serum TTR decreases in severe nonthyroid illnesses, protein-calorie malnutrition, and inherited α1-AT deficiency (54). Persistently or progressively low serum TTR is predictive of death (54,55). Causes of proteinuria decrease serum TBG, TTR, and albumin but increase lipoproteins. Changes in serum levels of albumin and minor carriers are irrelevant for thyroidologists.

Concentrations of TBG, TTR, and albumin in human CSF are 0.0028, 0.42, and 2.3 µmol/L, with a total protein concentration in human CSF that is approximately 0.5% that in human plasma (19). FT4 in human CSF is approximately 70 pmol/L or approximately 1.7% of total T4 in human CSF (19).

Inherited Abnormalities of Protein Concentration or Thyroid Hormone–Binding Affinity

Changes in either plasma concentration or TH affinity of TBG affect plasma levels of T4, and sometimes T3, in the same direction: hypothyroxinemia when the concentration or Ka of TBG decreases, hyperthyroxinemia when concentration increases. Increased affinity of TBG for TH has not been reported yet. Due to the much lower amount of TH transported by either TTR or albumin, compared with TBG, only an increased protein concentration (never described for albumin) or an increased affinity (which can occur for either TTR or albumin) can enhance plasma levels of T4. Reduced protein concentration and even absence of either protein does not cause hypothyroxinemia. Congenital or acquired absence of TTR has not been reported, unlike congenital absence of albumin (analbuminemia). Importantly, all the inherited or acquired variations in transport proteins leave unchanged both FT4 and FT3, in accordance with the clinical euthyroid status (euthyroid hypothyroxinemia or hyperthyroxinemia).

TBG-D can be partial (e.g., TBG-PD San Diego or TBG-PD Montreal) or complete (e.g., TBG-CD Buffalo). A catalog of the TBG mutations is available online (56). Some mutations, though, are associated with normal or mildly decreased serum TBG levels. These variants are TBG-poly (Leu283Phe), TBG-slow (Asp171Asn), TBG-Chicago (Tyr309Phe), and the not yet characterized TBG-C1 and TBG-F (56). These five TBG variants represent TBG polymorphism because of their enhanced presence in certain populations. For instance, allele frequency ranges from 16% to 31% for TBG-poly in French Canadians, Japanese, and Han Chinese of Taiwan, and from 2% to 16% for TBG-slow in Pacific Islanders and Black populations of African origin (56,57). TBG-D and TBG-E are inherited as X-linked traits, with a single exception (56). Most often, TBG-D are frame shifts or stop codons, resulting in synthesis of a truncated protein, and they occur at several positions. Based on neonatal screening programs for congenital hypothyroidism, TBG-CD is more frequent in Japanese than in whites (1:1200–1:1900 vs. 1:5000 to 1:13,000) (58). A particular form of TBG-D is due to reduced glycosylation of TBG and other glycoproteins as well (58). TBG-E is rarer (1:6000 to 1:40,000), and its molecular basis is TBG gene amplification (duplication, triplication) (56). An unexpected frequency of association between TBG-E and hyperthyroidism, greater than between TBG-D and hyperthyroidism, has been noted (59).

In addition to TBG-E, another cause of familial euthyroid hyperthyroxinemia (FEH) is the increased T4 affinity of either TTR or albumin (familial dysalbuminemic hyperthyroxinemia, FDH) (60), FDH being the most frequent cause of FEH. FDH prevalence is 0.01% to 1.8%, depending on the ethnic group, with the highest prevalence in Hispanics (61). FDH is caused by a change of Arg218 of albumin to either His or Pro, the latter also producing increased serum T3. Increased affinity for either TH arises because Arg218, which contacts the TH bound in sub domain 2A, causes a localized conformational change, relaxing steric restrictions on TH binding at this site (25). Another mutation, Leu66Pro, causes euthyroid hypertriiodothyroninemia because of a 40-fold increase in the Ka for T3 with a negligible increase of the Ka for T4 (56). Similar to FDH, mutations of TTR with increased TH affin ity are autosomal dominant. Such mutations concern Ala109 or Thr119 (56), whereas wild-type TTR can be associ ated with senile amyloidosis. Mutations at numerous co dons other than 109 and 119, the most common being Val30Met, are associated with familial amyloid polyneuropathy (FAP) (62), and mutations at codons (Val30, Leu58, Ser77, Ile84, Val122) decrease TH affinity (56). Prevalence of FAP is 1:100,000 to 1:1,000,000 (62), but FAP due to TTR mutations is the most common association with autosomal-dominant systemic amyloidosis. Interestingly, mutations of apos (apoA-1 and apoSAA) can result in other inherited amyloidoses (63). ApoA-1 Gly26 Arg causes a TTR-like FAP. ApoA-1, apoSAA, and murine apoA-2 (which is involved in amyloidosis-associated accelerated senescence of the mouse) share local homology with the other amyloid proteins (23). Genetic polymorphism of TTR exists in monkeys but not humans (56). In contrast, there are numerous polymorphic forms of albumin, from very slow to very fast.

Significance of Thyroid Hormone Binding to Plasma Proteins

Studies on patients with a defective gene product or equivalent knock-out mice have illuminated the functions of a particular protein. This is not the case for any of the TH plasma carrier proteins, in contrast with other hormone carriers. For instance, in the rare inherited CBG deficiency, there is an unusually high frequency of hypotension, chronic fatigue, and obesity (64,65). In contrast, patients with TBG complete deficiency or analbuminemia are entirely normal. However, analbuminemic patients and rats have a compensatory increase of other proteins, including TTR, Ig, α1-AT, α2-macroglobulin, transferrin, and ceruloplasmin, but most of all lipoproteins (66). This increase in lipoproteins is associated with increased lipogenesis and gene expression of several apos. TTR null mice have normal thyroid and retinoid status (67), including normal TH entry into and distribution in the brain (67), and normal retinal anatomy and function (68). These findings are surprising considering that the choroid plexus has the highest concentration of TTR messenger RNA in the body (4.4 µg/g wet weight tissue, compared with 0.4 µg/g in liver) (69) and that TTR is the most abundant transcript in human retinal pigment epithelium (70). Considering the conservation of the TH-binding domain of a given protein across species, functions presumably exist. In apos, the highly conserved TH-binding motif Y,L/I/M,X,X,V/I/L is spared by natural mutations (34), which contrasts with the occurrence of mutations in the TH-binding domain of the major TH carriers. Based on a number of data (71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110), certain functions can be hypothesized, for some of which details are given (Table 6.3).

TABLE 6.3. SELECTED POSSIBLE FUNCTIONS OF THYROID HORMONE (TH)–BINDING PROTEINS


Function

Comments (with example references)


Prevention of the urinary loss of TH

Albumin, VDBP, and TTR are internalized by megalin (83), a multiligand endocytotic receptor of the LDL-R family that is expressed on the apical surface of several resorptive epithelia, including KPTs. KPTs contain TTR, apos, and a novel apoA-1 binding protein that might be responsible for apoA-1 resorption (83).

Uniform distribution of TH to all cells of a tissue

In rat liver, distribution of T4 is taken up uniformly by all cells of the hepatic lobule when the organ is perfused with a solution of TBG, TTR, and albumin (74). Perfusion with albumin alone suffices for this purpose

Accumulation of free TH at sites of inflammation

SERPINs have an exposed reactive site loop for cleavage by serine proteases such as neutrophil-derived elastase. As a result of cleavage, the hormone affinity decreases, and free hormone is liberated (75,76). T4 released is locally degraded to DIT and other metabolites with bactericidal properties (75). LDL and HDL, which have antiviral and antimicrobial activities (77,78), are attacked by the same elastase (27). Either lipoprotein favors elastase release from neutrophils (27).

Facilitation of TH entry into cells

Facilitation of TH entry reported for TBG in blood mononuclear cells (27,76). TTR uptake reported for several human or animal cells (76,83), and this uptake is increased in the presence of high T4 or T3concentrations (76). LDL–TH complexes are internalized via the LDL-R, and human skin fibroblasts internalize up to 50% more T4 than they would if cells were exposed to the same concentration of T4but without LDL (80). Only two thirds of T4 internalized via LDL/LDL-R have access to the cell nuclei (81). In contrast, HDL facilitates uptake of a lesser extra-quota of T4 (up to +25%), and independently from HDL-R (81). All of this extra-quota of T4 has access to the cell nuclei. There could be an interplay between TTR and lipoprotein, as part of circulating TTR is transported by lipoprotein. Lipoproteins inhibit TTR uptake, and TTR uptake is partly mediated by megalin (a member of the LDL-R family) (83).

Facilitation of TH exit from cells

All TH plasma carriers favor both T4 and T3 exit from cells (88). On a molar basis, the order of potency is TBG > TTR > albumin > LDL > HDL, but at ambient concentration, the effect of lipoprotein (HDL > LDL) is comparable with that of the nonlipoproteins (TBG = TTR = albumin). Lipoproteins are so effective because their lipids permit a better interaction with the lipids of plasma membrane. All proteins act as sink for the extruded TH, thus maintaining the physical gradient from outside to inside the cells that drives the passive efflux of TH.

Enterohepatic circulation of TH

LDL-R and HDL-R are present in the liver and intestine (27). Apos are synthesized in the intestine, liver, and epithelial cells of the bile ducts, and are present in the bile (27). TH stimulates apo expression of both liver and intestine apos (27). HDL transports bilirubin.

TH distribution in the central and peripheral nervous system, mainly for reparative purposes

SERPINs (including TBG, α1-AT, α1-ACT), TTR, albumin (and the albumin homologues AFP and VDBP), apos, and other TH-binding proteins (e.g., α1-AGP, Ig) are present in CSF (19,27,90,91,92,93). TTR, albumin, AFP, apos, α1-AT, 1-ACT, and α1-AGP are synthesized in the brain (19,27,90,91,92,93,94,96); LDL-R are also expressed in brain (27). Apos and LDL-R genes are overexpressed, so that the corresponding protein products accumulate after either cranial or peripheral nerve injury and subsequent nerve repair and regeneration (27,94).

Regulation of hibernation and seasonal rhythms

(a) Regulation of hibernation. In the golden-mantled ground squirrel, four mRNAs vary seasonally, augmenting in the winter (hibernation) compared with summer. These mRNAs are TBG (15-fold), cathepsin H (5-fold), α2-macroglobulin (4.5-fold), and apoA-1 (2.6-fold) (95). mRNA of albumin did not change, while TTR was not tested. (b)Regulation of the annual transition in reproductive function of seasonally breeding animals through regulation of T4 access to specific hypothalamic areas. In Siberian hamsters, expression of TBG, TTR, and albumin genes is associated with reproductive refractoriness to short day lengths (96). Down-regulation of these genes is associated with reduced hypothalamic T4 uptake, which is reversed by long-day photoperiod treatments that restored responsiveness to short days. Circulating T4 did not vary with states of photoresponsiveness

Transport across placenta and/or oocytes of TH

Maternal serum levels of TBG, CBG, SHBG, and lipoprotein increase in pregnancy (27,97). Synthesis in or uptake by placenta, oocytes, or yolk sac of hormone transport proteins has been shown (98,99,100,101,102,103). Expression of the apos precedes that of the major TH transport proteins in the yolk sac (103).

Nongenomic effects

A clear background exists for apos. In addition to the enterohepatic circulation of TH (see above), TH binding to apos may protect plasma lipoprotein from oxidation (107), contribute to vasodilatation, and stimulate enzyme activity (LCAT, LpL) for which apos have activating properties (108). TH exert several nongenomic effects (106), including interference with oxidation (109), promotion of vasodilatation (110) and stimulation of certain apo-associated enzyme activities (LCAT, LpL) (27,108).


α1–ACT, α1–antichymotrypsin; α1-AGP, α1-acid glycoprotein; α1-AT, α1-antitrypsin; AFP, alpha-fetoprotein; apos, apoliproteins; CBG, corticosteroid-binding globulin; CSF, cerebrospinal fluid; DIT, diiodothyrosine; HDL, high-density lipoprotein; HDL-R, HDL receptor; Ig, immunoglobulin; KPT, kidney proximal tubule; LCAT, lecithin-cholesterol-acyltransferase; LDL-R, low-density lipoprotein receptor; LpL, lipoprotein lipase; SERPINs, serine protease inhibitors; SHBG, sex hormone–binding globulin; T3, triiodothyronine; T4, thyroxine; TBG, thyroxine-binding globulin; TH, thyroid hormone; TTR, transthyretin; VDBP, vitamin D–binding protein.

Two functions are apparent: to limit the urinary loss of TH and to act as a buffer counterbalancing sudden fluctuations in thyroid gland secretion. A third function is to constitute an extracellular reservoir that circulates continuously (in the blood and other fluids such as CSF), steadily releasing free TH and making TH available to tissues for replacing the TH used (74). A fourth function is temporary and local, occurring at sites of inflammation (75,76,77,78). Unrelated to inflammation, apoB-100 and apoE are attacked by plasma enzymes so that both intact and fragmented forms of the two apos circulate in plasma (27,32).

Because sterol hormone delivery to target cells has been described for CBG, SHBG, and VDBP through specific sites on the plasma membrane (79), a fifth function might be facilitation of TH transport (entry or exit) across cells (76,80,81) (Fig. 6.3). Albumin, TTR, or TBG inhibits TH entry (80,81,82), although cell uptake of TTR and TBG was described by others (76,83). The superiority of a minor carrier protein (lipoprotein) over the major carrier proteins was also observed with vitamin D, because its uptake by liver cells was greatest when vitamin D3 was presented to the cells on LDL and least when presented on VDBP (84). Delivery to certain tissues of another lipid-soluble vitamin, vitamin E, is also dependent on LDL interaction with the LDL-R (85), whereas HDL is important for vitamin E efflux from cells (86). The permissive effect of albumin on T3 efflux has been known since the mid 1980s (87). Unlike TH entry, TH exit is facilitated by all proteins, and in a nonsaturable fashion (88)(Fig. 6.3). Importantly, if from the extracellular mixture containing all proteins only a single protein at a time is removed, the efflux of either T4 or T3 remains unaffected (88). These data indicate that redundancy of TH plasma carriers compensate for the absence of single proteins.

FIGURE 6.3. Ten-minute fractional efflux (mean ± SD) of radiolabeled thyroxine or triiodothyronine from human hepatocytes (HepG2 cells) or human skin fibroblasts in the absence (buffer) or presence of individual proteins or mixture of proteins. The protein concentrations approximate the ambient concentrations for hepatocytes (plasma) and fibroblasts (interstitial fluid). (From Benvenga S, Robbins J. Thyroid hormone efflux from monolayer cultures of human fibro blasts and hepatocytes. Effect of lipoproteins and other thyroxine transport proteins. 1998;139:4311–4318, with permission.) Note that the effect of high-density lipoprotein (HDL) and HDL plus low-density lipoprotein (LDL) is disproportionately similar to that produced by the three major thyroid hormone–binding proteins when tested either individually or combined. Independently of cell and hormone, the effect given by all proteins (i.e., HDL + LDL + human serum albumin + transthyretin + thyroxine-binding globulin) did not change when each of these five proteins was removed one at a time (not shown).

Particular functions may be attributed to the presence of lipoprotein receptors or apos in certain tissues or fluids, as reviewed previously (27). These functions include enterohepatic circulation of TH (27), distribution of TH in the nervous system mainly for reparative and seasonal biorhythm regulation purposes (27,90,91,92,93,94,95,96), and TH transport across the placenta or oocytes (27,97,98,99,100,101,102,103). In the placenta, SHBG or apos stimulate secretion of human chorionic gonadotropin or human placental lactogen (104,105).

Considering that TH exerts numerous nongenomic effects (106), most of which are best served by T4, and that part of the LDL-facilitated T4 cell entry does not have access to cell nuclei (81), a number of nongenomic effects can be hypothesized (Table 6.3). Overall, the nongenomic actions summarized in Table 6.3 would have an antiatherogenic effect (107), and the apo-TH binding could potentiate individual antiatherogenic properties of the HDL apos and TH (108,109,110).

THAbs are considered the rarest thyroid autoantibodies (39,40), their rate of detection being low even when investigated in isolated IgG (111). Contributing to the rarity is the fact that THAbs can be transient (40,112,113). However, prevalence of THAb at our center has increased steadily, particularly in patients with autoimmune thyroid diseases (114). Surprisingly, prevalence in autoimmune thyroid diseases is lower than in primary Sjögren's syndrome and rheumatoid arthritis (114). THAbs have iodinated thyroglobulin as the autoantigen (40) and can be induced by viruses (113), drugs (115), or other insults (40).

In summary, the major TH transport proteins, while they individually may be absent without endangering health, together contribute importantly to thyroid homeostasis. On the other hand, TBG, TTR, and minor transport proteins such as lipoproteins also have additional physiologic and pathophysiologic functions, both real and potential, that further investigation will elucidate.

ACKNOWLEDGMENTS

I am appreciative of the helpful discussions with professor Francesco Trimarchi (Head, Sezione di Endocrinologia and Director, Dipartimento di Medicina e Farmacologia Clinica, University of Messina School of Medicine) and Dr. Jacob Robbins (Scientist Emeritus, NIH).

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