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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-2261
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 8 3215-3218
Copyright © 2006 by The Endocrine Society

BRIEF REPORT

C-Terminal Amino Acid Alteration rather than Late Termination Causes Complete Deficiency of Thyroxine-Binding Globulin CD-NeuIsenburg

Lars C. Moeller, Anja Fingerhut, Harald Lahner, Helmut Grasberger, Bernd Weimer, Joachim Happ, Klaus Mann and Onno E. Janssen

Division of Endocrinology (L.C.M., A.F., H.L., K.M., O.E.J.), Department of Medicine, University Hospital of Essen Medical School, 45122 Essen, Germany; Department of Medicine (H.G.), The University of Chicago, Chicago, Illinois 60637; and Endokrinologische Gemeinschaftspraxis (B.W., J.H.), 60329 Frankfurt am Main, Germany

Address all correspondence and requests for reprints to: Onno E. Janssen, M.D., Division of Endocrinology, Department of Medicine, University Hospital of Essen Medical School, Hufelandstr. 55, 45122 Essen, Germany. E-mail: onno.janssen{at}uni-essen.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: T4-binding globulin (TBG) is the main transport protein for T4 in blood and a member of the superfamily of serine proteinase inhibitors. So far, 14 mutations leading to familial complete TBG deficiency have been reported. Eleven of these are caused by mutations leading to truncation of the molecule, and three are caused by single amino acid substitutions.

Objective: We report and study the complete deficiency TBG variant found in a patient from NeuIsenburg, Germany (TBG-CDNI).

Methods: Direct DNA sequencing was used to identify the TBG-CDNI mutation in the propositus, which was confirmed by allele-specific amplification. Site-directed mutagenesis and expression in Xenopus oocytes was used to study the secretion defect of TBG-CDNI and several variants by Western blot and T4-binding assay.

Results: The deletion of two nucleotides in codon 384 (1211_1212delTC) causes a frameshift altering the last 11 residues, introduces a new glycosylation site, and elongates the molecule by seven new amino acids. In contrast to normal TBG, TBG-CDNI was not secreted by Xenopus oocytes. Elongation of normal TBG by seven alanines did not affect its secretion or binding properties. On the other hand, neither disruption of its new glycosylation site nor termination of TBG-CDNI at the normal length repaired its secretion defect.

Conclusions: In this first late termination variant of complete TBG deficiency, alteration of ß-strand 5B, located in the core of the molecule, rather than elongation of the molecule or introduction of a new glycosylation site, suffices to disrupt secretion of TBG-CDNI.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T4-BINDING GLOBULIN (TBG) is the main transport protein for T4 and T3 in blood and a member of the serine protease inhibitor (serpin) superfamily. The 54-kDa protein is synthesized in the liver and consists of one single polypeptide chain of 395 amino acids. TBG is encoded by a single gene, located on the long arm of the human X-chromosome (Xq22.2) (1). Thus, familial TBG variants follow an X-linked pattern (2).

By concentration, TBG variants can be classified as excess and partial or complete deficiency, the latter defined by serum levels of TBG in RIA or T4-binding analysis below the current limit of detection of 0.005 mg/liter (normal range 10–25 mg/liter) (3). So far, 14 mutations have been described to cause complete TBG deficiency (4, 5), leading to truncated molecules in 11 complete TBG deficiency variants (TBG-CD). Single amino acid substitutions with possible aberrant posttranslational processing have been described in TBG-CD5 (3, 6), TBG-CD-Kumamoto (7), and TBG-CDT1 (5).

We describe a new complete deficiency variant, TBG-CD-NeuIsenburg (TBG-CDNI), in which a frameshift mutation in codon 384 results in 11 amino acid substitutions, late termination of translation with elongation by another seven amino acids, and introduction of a new canonical glycosylation site. In vitro expression of TBG-CDNI and appropriate C-terminal variants was used to determine which of these alterations causes the secretion defect.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Thyroid function tests

Automated chemiluminescence immunoassay systems were used for the determination of TSH, free T4, free T3, total T4, and total T3 as well as thyroglobulin and thyroperoxidase antibodies (ADVIA Centaur; Bayer Vital, Fernwald, Germany) and TBG (IMMULITE 2000, DPC Biermann, Bad Nauheim, Germany). TSH receptor antibodies were determined by a commercial immunometric assay (Brahms, Berlin, Germany).

Measurement of T4 binding to TBG

T4 binding was measured as previously described in detail (8). Briefly, TBG preparations were incubated with 125I T4 in the presence of increasing amounts of unlabeled T4. TBG-bound T4 was separated from free T4 with anion exchange resin beads (Sigma-Aldrich, St. Louis, MO), and the protein-bound 125I activity was determined.

Sequencing of the TBG gene

Genomic DNA isolated from peripheral blood leukocytes served as a template to amplify the coding regions (exons 1–4) and adjacent exon-intron junctions of the TBG gene by PCR as described previously (9). The PCR products were used for automated sequencing with fluorescent dye terminators (ABI PRISM System 377; Applied Biosystems, Foster City, CA).

Construction of vectors

A vector containing TBG cDNA (TBG-N) was available (10) and used to construct a TBG-CDNI vector by cassette mutagenesis. The part of the patient’s genomic DNA harboring the mutation and the extended 3' part of the coding region, including the new stop codon, was amplified by PCR with the reverse primer introducing a new BglII restriction site, cut with XbaI and BglII and ligated into the TBG-N vector. Three more recombinant TBG vectors were created by site-directed mutagenesis (altered sites II in vitro mutagenesis system; Promega, Mannheim, Germany). TBG-N-7Ala has seven alanines added to the C terminus of normal TBG, and in TBG-CDNI-Glydef, the new glycosylation site at position 391 of TBG-CDNI is disrupted. TBG-CDNI-stop has a stop codon at position 396, terminating the molecule at normal length, eliminating TBG-CDNI’s seven additional amino acids. The mutagenesis primers were purchased from Amersham Pharmacia Biotech (Freiburg, Germany), and their sequences are available upon request.

Expression of recombinant TBGs in Xenopus oocytes

Synthetic mRNA (sRNA) was prepared with the mMessage mMachine T7 transcription kit (Ambion, Austin, TX) according to the supplier’s recommendations. Expression of the recombinant TBG in microinjected oocytes from Xenopus leavis has been described in detail previously (6).

Western blot analysis of recombinant TBG

Medium from cultured oocytes was used without further purification (11). To obtain cytosolic fractions, microinjected oocytes were disrupted and the oocyte extract was centrifuged at 13,000 rpm for 15 min. Aliquots of the supernatant and culture medium were subjected to SDS-PAGE and transferred onto nitrocellulose. TBG was detected with a rabbit polyclonal anti-TBG antiserum (12) and visualized with an alkaline phosphatase-conjugated secondary antibody followed by nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate detection.

Structure analysis of TBG-CDNI

The crystal structure of TBG is not available but assumed to be very similar to {alpha}1-proteinase inhibitor (PI) (13). Thus, TBG structure models were obtained by submitting a homology structure of intact {alpha}1-proteinase inhibitor with the TBG sequence to the automated protein modeling server SWISS-MODEL (GlaxoWellcome Experimental Research, Geneva, Switzerland) (14). The model was modified and refined using the Swiss-PDB Viewer (GlaxoWellcome Experimental Research) and POV-Ray programs (POV-Ray Team, Indianapolis, IN).


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Case report

The propositus, a 33-yr-old male of Turkish origin, initially presented with diffuse goiter. Clinically, he was euthyroid. Thyroid function tests revealed normal TSH (0.8 mU/liter; normal 0.3–3.0 mU/liter), free T4 (25.5 pmol/liter; normal 10.3–25.8 pmol/liter), and free T3 (4.65 pmol/liter; normal 4.62–9.24 pmol/liter). However, total T4 and T3 were low at 26 nmol/liter (normal 60–150 nmol/liter) and 0.6 nmol/liter (1.16–3.08 nmol/liter), respectively. Serum TBG was undetectable by both RIA and T4 binding by a resin-uptake method with a lower limit of detection of 1 and 0.05% of mean normal TBG values, respectively. Tg, TPO, and TSH receptor antibodies were negative. On follow-up, his goiter had responded well to iodine supplementation and he was still euthyroid.

Sequence analysis of the TBG-CDNI gene

To test for a genetic defect as the cause of absent TBG in the propositus’ serum, the entire coding region and adjacent exon-intron junctions of his TBG gene were sequenced. This revealed a two-nucleotide deletion in codon 384 (1211_1212delTC). The mutation was verified by allele-specific amplification (data not shown).

Expression of TBG-N and TBG-CDNI sRNAs in Xenopus oocytes

To examine whether the deletion in codon 384 is responsible for the complete deficiency of TBG-CDNI, sRNAs of TBG-N (normal TBG) and TBG-CDNI were synthesized in vitro and injected in Xenopus oocytes. T4-binding analysis of the recombinant normal and mutant TBGs failed to detect TBG-CDNI in oocyte culture media, with a lower detection limit of approximately 1% of mean TBG-N levels. Western blot analysis detected TBG-N in both cytosol and culture media of injected oocytes, whereas TBG-CDNI was detectable only in the cytosol and completely absent in the medium (Fig. 1AGo).


Figure 1
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  		FIG. 1. Western blot of TBG variants expressed in vitro. A, Normal TBG and TBG-CDNI were expressed in Xenopus oocytes. Oocyte extracts and culture medium were run on SDS-PAGE, blotted, probed with a polyclonal anti-TBG-antibody, incubated with an alkaline phosphatase (AP)-conjugated secondary antibody, and visualized with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. Both TBG-N and TBG-CDNI were expressed within the oocytes [cytosolic TBG (cTBG)], whereas no TBG was found in noninjected controls. However, only TBG-N was secreted into the medium [mature TBG (mTBG)]. B, Western blot of C-terminal TBG variants expressed in vitro. Whereas the extension of normal TBG (N) with seven alanines (N-7Ala) did not impair its secretion, neither disruption of TBG-CDNI’s new glycosylation site (TBG-CDNI-GlyDef) nor its termination at the length of normal TBG (TBG-CDNI-stop) abolished the secretion defect.

 
Structure analysis of TBG-CDNI

The structure of TBG-CDNI was modeled on the serpin homolog {alpha}1-antitrypsin as described previously (13). The two-nucleotide deletion in codon 384 causes three distinct changes in the molecule (Fig. 2Go). First, whereas codon 384 still encodes leucine, the next 11 amino acids are changed due to the frameshift. Second, this also disrupts the usual stop codon, leading to the addition of seven amino acids to the molecule. Third, the fifth glycosylation site of TBG-N (Asn391Pro392Thr393) is disrupted and a new one created (Asn392Gly393Ser394). Whereas the normal fifth site is unlikely to be used as it contains a proline and is located within five amino acids from the C terminus, the new site probably is because it does not have these limiting factors (15). To study these three distinct features of TBG-CDNI, appropriate vectors were constructed that extend normal TBG by seven amino acids (TBG-N-7Ala), disrupt the new glycosylation site (TBG-CDNI-GlyDef), and terminate TBG-CDNI at normal length (TBG-CDNI-stop).


Figure 2
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  		FIG. 2. Structure of TBG-N and TBG-CDNI modeled on intact {alpha}1-antitrypsin. A, TBG-N. {alpha}-Helices are shown in red, ß-strands in yellow, and glycosylation sites in green. B, TBG-CDNI. The 2-bp deletion in codon 384, shown in light blue spheres, is located at the beginning of ß-strand s5B. The resulting frameshift leads to the substitution of the next 11 amino acids, shown as dark blue spheres; introduction of a new glycosylation site, shown as green spheres; disruption of the stop codon; and addition of another seven amino acids, shown as pink spheres.

 
Expression of TBG-CDNI derivatives in Xenopus oocytes

Western blot analysis of oocyte medium showed that mere extension of normal TBG (TBG-N-7Ala) does not impair secretion, whereas neither disruption of TBG-CDNI’s new glycosylation site (TBG-CDNI-GlyDef) nor terminating it at normal length (TBG-CDNI-stop) repairs the secretion defect (Fig. 1BGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Of the 14 known complete deficiency variants of TBG, 11 lead to early termination resulting in a truncated molecule (4, 5). Their length ranges from 50 (TBG-CD-Yonago, TBG-CD-Negev) to 383 amino acids (TBG-CD-Harwichport). TBG-CDNI differs from these TBG-CD variants because a mutation in the C terminus, in codon 384, leads to a frame shift with disruption of the original stop codon and thus late termination of translation at a predicted length of 402 instead of 395 amino acids.

The C terminus has been focused on other serpin deficiencies, especially in {alpha}1-PI deficiency. The C terminus of {alpha}1-PI contains a nine-amino acid sequence highly conserved within the serpin family (16). Truncations including Proline 391 (according to {alpha}1-PI sequence), the last amino acid of this sequence, or its substitution by various amino acids severely restricted {alpha}1-PI secretion. In mature TBG, this conserved sequence corresponds to amino acids 384 to 392, which, with the exception of leucine 384, are all altered in TBG-CDNI. C-terminal changes, either truncations or substitutions, can dramatically affect serpin secretion. In {alpha}1-PI deficiency, for example, several naturally occurring variants are caused by the formation of stop codons (e.g. {alpha}1-PI NULLbellingham), leading to a truncated molecule (17). Several variants of pigment epithelium-derived factor, another serpin, with substitutions within ß-sheet B were not secreted in measurable amounts (18).

TBG-CDNI is detectable in the cytosol of injected oocytes and, apparently, in higher amount than TBG-N but not in the culture medium (Fig. 1AGo). We therefore speculate that intracellular protein accumulation is more likely than rapid mRNA or protein degradation as the underlying mechanism in this TBG deficiency variant. The substitution of the amino acid sequence in ß-strand s5B, part of ß-sheet B, extending out of the protein core to the molecule’s surface (13) (Fig. 2Go), probably prevents correct folding, which is detected by the quality control system in the endoplasmic reticulum and downstream compartments of the secretory pathway.

The disruption of ß-sheet B was also present in the variant TBG-CDNI-stop, which terminated TBG-CDNI at the length of normal TBG. This variant was not secreted either, demonstrating that disruption of this part of ß-sheet B suffices to prevent secretion. These findings also explain complete deficiency of TBG-CD-Harwichport, which is terminated at codon 384 (19), lacking exactly this part of the protein.

Extension of normal TBG by seven amino acids did not prevent secretion. Mere elongation of the protein seems not to impact its secretion, possibly because this part is, other than ß-sheet B, not in the core of the molecule and thus less crucial for its tertiary structure.

The fifth glycosylation site in exon 4 of TBG is not used due to inclusion of a proline and location within five amino acids from the C terminus (Ref. 15 and our own unpublished data). In TBG-CDNI, a new canonical glycosylation site is created by frame shift. In the partial TBG deficiency variant TBG-Gary, a single nucleotide substitution creates a new site for N-linked glycosylation and leads to severe impairment of TBG secretion (20). We disrupted the new glycosylation site of TBG-CDNI, which did not repair the secretion defect. Additional N-linked glycosylation of TBG-CDNI therefore cannot be the sole explanation for its deficiency.

In conclusion, ß-sheet B alteration rather than extension by seven amino acids or use of a new glycosylation site appears to be responsible for the secretion defect of TBG-CDNI, lending further support for the crucial role of this part of the molecule for efficient secretion of serpins.


   Footnotes
 
This work was supported in part by Grants DFG Mo 1018/1-1 (to L.C.M.) and Ja 671/1-3 (to O.E.J.) from the Deutsche Forschungsgemeinschaft.

Disclosure of potential conflicts of interest: L.C.M., A.F., H.L., H.G., B.W., J.H., K.M., and O.E.J. have nothing to declare.

First Published Online May 30, 2006

Abbreviations: PI, Proteinase inhibitor; sRNA, synthetic mRNA; TBG, T4-binding globulin; TBG-CD, complete TBG deficiency variant; TBG-CDNI, TBG variant found in a patient from NeuIsenburg, Germany; TBG-N, TBG cDNA.

Received October 12, 2005.

Accepted May 19, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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