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*(L)-HISTIDINE
*L-TYROSINE
The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 5 2477-2483
Copyright © 2004 by The Endocrine Society

Partial Deficiency of Thyroxine-Binding Globulin-Allentown Is Due to a Mutation in the Signal Peptide

Anja Fingerhut, Sirimon Reutrakul, Sebastian D. Knuedeler, Lars C. Moeller, Carol Greenlee, Samuel Refetoff and Onno E. Janssen

Division of Endocrinology, Department of Medicine (A.F., S.D.K., O.E.J.), University of Essen, 45122 Essen, Germany; Departments of Medicine (Si.R., L.C.M., Sa.R.) and Pediatrics (Sa.R.), and Committees on Genetics and Molecular Medicine (Sa.R.), The University of Chicago, Chicago, Illinois 60637; and Endocrinology & Metabolism, Atlanta Diabetes Associates (C.G.), Atlanta, Georgia 30309

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We present an unusual variant of T4-binding globulin (TBG) found in a family from Allentown, Pennsylvania (TBG-AT). The heterozygous proposita presented serum total T4 and TBG levels ranging from low to normal. TBG gene sequencing revealed a C-to-T substitution in codon –2 (CAC to TAC) leading to the substitution of the normal histidine by a tyrosine within the signal peptide. No mutation within the mature peptide was found. Allele-specific PCR confirmed the H(–2)Y mutation in the propositas mother and son. T4-binding analysis of TBG in serum from the proposita and son showed normal affinity but reduced capacity when compared with the unaffected father. Heat stability and isoelectric focusing of TBG-AT were normal. In vitro expression of a recombinant TBG-AT in Xenopus oocytes revealed a diminished secretory efficiency and confirmed the normal binding affinity and heat stability of the small amount of secreted TBG-AT. This study has defined impaired cotranslational processing as a hitherto unrecognized cause of hereditary TBG deficiency.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROXINE-BINDING GLOBULIN (TBG), a 54-kDa glycoprotein synthesized by the liver, is the main transport protein for thyroid hormones in blood (1, 2, 3). By virtue of sequence homology, TBG belongs to the superfamily of serine proteinase inhibitors (serpins) (4), which consists of a variety of heterogeneous proteins including {alpha}1-antitrypsin (also known as proteinase inhibitor, PI), {alpha}1-antichymotrypsin, antithrombin III (AT3), and cortisol-binding globulin (CBG) (5). TBG and CBG are the only serpins that transport small lipophilic molecules having lost the serpin-characteristic function of proteinase inhibition (6). The human TBG gene is located on the X chromosome (Xq 22.2) (7) and encodes a single polypeptide chain of 415 amino acids (8). After cleavage of a 20-amino-acid signal peptide, the mature TBG of 395 amino acids is secreted into the bloodstream (9, 10).

Due to the presence of a single TBG gene on the X chromosome (7), most familial TBG defects follow a X-linked inheritance pattern (11). Inherited TBG abnormalities are classified under four types depending on their serum concentration: complete deficiency, partial deficiency, TBG excess, and normal concentration but altered properties. While TBG excess is caused by gene replication (12), deficiency or altered TBG is caused by point mutations resulting in amino acid substitutions in the mature protein or in truncations caused by stop codons (1, 2, 13, 14, 15, 16, 17, 18, 19, 20). More rarely, TBG defects are caused by aberrant mRNA processing due to mutations in the acceptor splice site (21) or by exon skipping (22) and a probable defect in TBG-specific transcription factors (23). Complete TBG deficiency is defined as the absence of detectable TBG in the serum of affected hemizygous males based on the current ability to detect TBG levels as low as 0.03% of the average normal levels in adults (24, 25).

We now present the characterization, sequence analysis, and in vitro expression of an unusual partial TBG abnormality found in a family from Allentown, Pennsylvania (TBG-AT). A single amino acid substitution (His->Tyr) at position –2 within the signal peptide was found and verified by in vitro expression to cause a variable diminution of secretion. No mutation was found in the mature protein, explaining the normal binding affinity and heat stability of TBG-AT. This is the first report of a partial deficiency of TBG caused by a mutation in the signal peptide.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Restriction endonucleases, DNA molecular size markers, and Taq polymerase were purchased from New England Biolabs (Frankfurt, Germany, and Beverly, MA), Amersham Biosciences (Freiburg, Germany) and Promega (Mannheim, Germany, and Madison, WI), respectively, and were used according to the manufacturers’ suggestions. Rabbit polyclonal antihuman TBG antisera had been raised and characterized previously (24, 25). An alkaline phosphatase-conjugated goat antirabbit secondary antibody was obtained from Stratagene (Amsterdam, The Netherlands). 125I T4 was purchased from PerkinElmer (Rodgau-Jügesheim, Germany). All other reagents were of analytical grade from Merck (Darmstadt, Germany) or Sigma-Aldrich (Taufkirchen, Germany).

Subjects and characterization of serum samples

All members of the Allentown family gave informed consent for this study, which was approved by the Institutional Review Board. Blood was obtained from all members of the Allentown family for genetic and binding studies. Thyroid function tests were performed by commercial laboratories, including Quest Nichols Institute (San Juan Capistrano, CA) and Health Network Laboratories (Allentown, PA).

Measurement of T4 binding and heat denaturation

T4 binding was measured by a method previously described in detail (25). Briefly, TBG preparations were incubated with 125I T4 in the presence of increasing amounts of unlabelled T4. TBG-bound T4 was separated from free T4 with anion exchange resin beads (Sigma-Aldrich), and the protein-bound 125I activity was determined. The affinity constant Ka and TBG concentration were determined by the method of Scatchard (26). Heat denaturation was performed as previously described (27).

Sequencing of the TBG gene

Genomic DNA was obtained from all members of the Allentown family by extraction from peripheral blood mononuclear cells. The coding regions (exons 1–4) and noncoding exon 0, as well as adjacent exon-intron junctions of the TBG gene, were amplified by PCR using the oligonucleotide primer and PCR conditions as previously described (28). The PCR products were isolated and used for automatic sequencing on an ABI Prism 377 DNA sequencer (PerkinElmer, Foster City, CA).

Allele-specific amplification

Allele-specific amplification of normal and mutant TBG genes was performed on genomic DNA isolated from all members of the Allentown family. The common antisense primer was 5'-GATGGTGTTTGGCTTGAGGTCTTG-3', the sense primer specific for the normal allele was 5'-CTTGGGCTTCATGCTACAATCC-3', and the sense primer specific for the mutant allele was 5'-CTTGGGCTTCATGCTACAATCT-3' (the underlined T indicates the TBG-AT mutation). The PCR was run with an initial denaturation at 96 C for 3 min, followed by 25 cycles consisting of 96 C for 30 sec, 60 C for 45 sec, 72 C for 1 min, and a final extension at 72 C for 3 min. A total of 130 random alleles from Caucasian individuals were also screened for TBG-AT by the same method.

Construction of vectors and preparation of synthetic mRNA

A vector containing the full-length cDNA of normal TBG (TBG-N) (29) was used to construct a TBG-AT vector by site-directed mutagenesis using the Altered Sites II in Vitro Mutagenesis System (Promega) as recommended by the supplier. The coding region of the TBG-AT mutant was verified by automated sequencing. Synthetic mRNA was prepared with the mMessage mMachine Transcription Kit (Ambion, Austin, TX) and T7 RNA polymerase according to the suppliers’ recommendations. Expression of the recombinant TBG in microinjected oocytes from Xenopus laevis has been described in detail previously (27).

Western blot analysis of recombinant TBG

Medium from cultured oocytes was used without further purification (27). To obtain cytosolic fractions, microinjected oocytes were washed twice with buffer, transferred to a 1.5-ml Eppendorf tube, and disrupted with a loose-fitting pestle. After freezing and thawing three times in liquid nitrogen, the oocyte extract was centrifuged at 13,000 rpm for 15 min, and the supernatant was transferred to a new tube. Aliquots of the centrifuged oocyte extracts and culture medium were subjected to SDS-PAGE and transferred onto nitrocellulose. TBG-N and TBG-AT were detected with a rabbit polyclonal anti-TBG antiserum and visualized with an alkaline phosphatase-conjugated secondary antibody followed by nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate detection.

Analysis of signal peptide mutations with SignalP

All 26 known human serpins were analyzed with the SignalP World Wide Web prediction server (Version 1.1, http://www.cbs.dtu.dk/ services/SignalP/index.html) (30). SignalP uses artificial neuronal networks to recognize the cleavage site (C-score) and to distinguish between signal peptide and nonsignal peptide sequences (S-score). The C-score networks are trained to be high at position +1, immediately after the cleavage site. The S-score networks are trained to be high at all positions before the cleavage site but low at the 30 positions after the cleavage site, generating a steep slope between positions –1 and +1. Prediction of cleavage site localization is further optimized by averaging the height of the C-score and the slope of the S-score in a combined cleavage site score (Y-score). SignalP has a correlation of 0.97 in signal peptide discrimination and correctly predicts the cleavage site localization in more than 70% of eukaryotic test sequences (i.e. data that were not used to train the networks). Due to redundancy in signal peptides, prediction accuracy on sequences with some degree of homology to the sequences in the datasets of the neuronal networks, which include most of the known serpins, will in general be higher. All amino acid sequences were obtained from the Entrez-PubMed Protein Databank (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi). Maximum performance of SignalP is obtained with peptide lengths of 50–70 amino acids; thus, the first 60 residues of the 17 known human-secreted serpins, including their eight known signal peptide variants (TBG, THBG_human no. P05543; angiotensinogen, ANGT_human no. P01019; antithrombin III, ANT3_human no. P01008; {alpha}2-antiplasmin, A2AP_human no. P08697; {alpha}1-antitrypsin [proteinase inhibitor (PI)], A1AT_human no. P01009; {alpha}1-antichymotrypsin, AACT_human no. P01011; plasminogen activator inhibitor 1, PAI1_human no. P05121; corticosteroid-binding globulin, CBG_human no. P08185; C1 inhibitor, IC1_human no. P05155; kallistatin, KAIN_human no. P29622; heparin cofactor II, HEP2_human no. P05546; glia-derived nexin, GDN_human no. P07093; protein C inhibitor, IPSP_human no. P05154; collagen-binding protein 2, CBP2_human no. P50454; heat shock protein 47, HS47_human no. P29043; neuroserpin, NEUS_human no. P99574; pigment epithelium-derived factor, PEDF_human no. P36955) and of the nine known human cytosolic (nonsecreted) serpins (plasminogen activator inhibitor 2, PAI2_human no. P05120; leukocyte elastase inhibitor, ILEU_human no. P30740; placental thrombin inhibitor, PTI6_human no. P35237; cytoplasmic antiproteinase 3, SPB9_human no. P50453; squamous cell carcinoma antigen 1, SCC1_human no. P29508; squamous cell carcinoma antigen 2, SCC2_human no. P48594; bomapin, SB10_human no. P48595; megsin, SPB7_human no. O75635; hurpin, HSPI_human no. CAA04937) were used for analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Clinical history and thyroid function tests of the proposita

The proposita was 18 yr old when, in 1999, she was found to have a low serum TBG concentration. She presented 18 months earlier to her family physician with the complaint of abdominal pain. Thyroid function tests were obtained as routine evaluation. Total T4 (TT4) was 44 nmol/liter (normal range, 64–161) and TSH was 2.7 mU/liter (normal range, 0.4–5.5). Treatment with 125 µg levothyroxine (L-T4) daily was begun. Although the abdominal pain resolved spontaneously, serum TT4 was 67 nmol/liter, but TSH was below the limit of detection of 0.1 mU/liter. Two months later, while still on L-T4, her TT4 had risen to 135 nmol/liter, whereas TSH was still low at 0.05 mU/liter (normal range, 0.4–3.8) and TBG was 17 ng/liter (normal range, 17–36). L-T4 was discontinued. When thyroid function tests were repeated 7 months later, she was in the last trimester of pregnancy. Her TT4 was 100 nmol/liter, TT3 was 1.92 nmol/liter (normal range, 1.38–2.84), free T4 by dialysis was 14 pmol/liter (normal range, 10–26), TSH was 3.2 mU/liter, and TBG was 32 mg/liter. Both TT4 and TT3, although in the normal range, were below the expected values for the third trimester of pregnancy. From 3–18 months after the delivery of a healthy male infant, three blood samples were obtained from the proposita. Over this interval of 15 months, TT4 levels ranged from 26–136 nmol/liter and TBG concentrations ranged from 9–19 mg/liter (normal range, 14–26 nmol/liter). The infant had normal TSH on neonatal screening. The family was of Caucasian background. At the time of the investigation, all family members were clinically euthyroid and had normal serum TSH and free T4 levels. They declined serial blood sampling to determine TBG levels and their changes with time, as observed in the proposita.

Characterization of serum TBG-AT

Scatchard analysis of T4 binding revealed reduced TBG binding capacity and a normal affinity for the T4 ligand (Ka) in sera from the proposita and her son compatible with partial TBG deficiency. Her father had normal TBG (Fig. 1AGo). TBG capacities were 278 nM (15.0 mg/liter) in the proposita, 227 nM (12.2 mg/liter) in the son, and 328 nM (17.7 mg/liter) in the unaffected father. Heat denaturation at 60 C revealed a normal half-life of the variant TBG-AT (Fig. 1BGo). Isoelectric focusing of TBG-AT showed a pattern identical to normal TBG (TBG-N) (data not shown).



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  		FIG. 1. Properties of serum TBG in the Allentown family. A, Scatchard analysis. The T4-binding affinity of serum TBG was similar in all family members. The unaffected father ({blacktriangleup}) had a Ka of 1.29 x 1010 mol/liter–1, the heterozygous proposita (•) had a Ka of 1.22 x 1010 mol/liter–1, and the hemizygous affected son ({diamondsuit}) had a Ka of 1.25 x 1010 mol/liter–1. Corresponding maximal binding capacities were 328 nM (17.7 mg/liter), 278 nM (15.0 mg/liter), and 227 nM (12.2 mg/liter). B, Heat denaturation. The rate of heat denaturation was determined as the residual T4-binding of diluted serum samples after incubation at 60 C for increasing time periods. No differences were found in the half-life of denaturation of serum TBG from the normal father, the heterozygous proposita, and the hemizygous affected son.

 
Sequence analysis of the TBG-AT gene

Sequencing of the entire coding region and adjacent exon-intron junctions of the TBG gene from the proposita revealed a C-to-T transition in codon –2 (CAC to TAC), resulting in the substitution of the normal histidine with tyrosine [H(–2)Y] at the C-terminal end of the signal peptide. No mutations were found in the TBG-AT gene sequence coding for the mature protein. Allele-specific amplification confirmed heterozygosity for TBG-AT of the proposita and her mother, and hemizygosity of the son. The nucleotide substitution was not found in DNA from the father and sister (Fig. 2Go). Screening of 130 alleles from unrelated Caucasians failed to identify the nucleotide substitution identified in TBG-AT.



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  		FIG. 2. Genotyping of the Allentown family by allele-specific amplification. PCRs of genomic DNA were performed with oligonucleotides specific for either the normal sequence (n) or the TBG-AT mutation sequence (m). Note that the proposita and her mother are heterozygous for TBG-AT, whereas her son expresses only TBG-AT.

 
Expression of normal and mutant TBG synthetic mRNAs in Xenopus oocytes

To test whether the H(–2)Y mutation is responsible for the partial deficiency of TBG-AT, an appropriate TBG-AT vector was constructed, and mRNAs of TBG-N and TBG-AT were synthesized in vitro and expressed in Xenopus oocytes. Scatchard analysis of T4 binding of the recombinant normal and mutant TBG showed a decreased concentration of secreted TBG-AT, but a normal binding affinity (Fig. 3AGo). Furthermore, heat denaturation at 60 C revealed a normal heat stability of recombinant TBG-AT (Fig. 3BGo). Western blot analysis of culture media from injected oocytes showed a greatly reduced concentration of mature TBG-AT (apparent molecular mass, 60 kDa) compared with TBG-N (Fig. 4Go). Reduced synthesis of TBG-AT as the cause of decreased secretion could be excluded, because analysis of oocyte extracts revealed that TBG-AT was retained in the oocytes, compatible with an intracellular accumulation of TBG-AT (Fig. 4Go).



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  		FIG. 3. Properties of in vitro-synthesized recombinant TBG-N and TBG-AT. A, Scatchard analysis. TBG-N ({blacktriangleup}) and TBG-AT (experiment 1: {diamondsuit}; experiment 2: {diamond}) were expressed in Xenopus oocytes. Scatchard analysis of the secreted proteins revealed reduced expression levels but similar T4-binding affinity of TBG-AT. Serum TBG-N ({triangleup}) was used as a control. B, Heat stability. TBG-variants synthesized by Xenopus oocytes were heated at 60 ± 0.1 C for increasing time periods. Analysis of the residual T4-binding activity revealed similar half-lives of denaturation of recombinant TBG-N ({blacktriangleup}) and TBG-AT ({diamondsuit}).

 


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  		FIG. 4. Western blot of TBG-N and TBG-AT expressed in Xenopus oocytes. Normal (N) and mutant (AT) TBG 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-conjugated secondary antibody, and visualized with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. The endogenous alkaline phosphatase (ap) served as a control for the amount of protein loaded onto the gel. Both TBG-N and TBG-AT were expressed within the oocytes (cTBG, cytosolic TBG), whereas no TBG was found in noninjected (ni) controls. However, TBG-AT was secreted into the culture medium in much lower amounts than TBG-N (mTBG, mature TBG).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous publications have reported amino acid substitutions, frame shift mutations, and defects in mRNA processing as causes of TBG deficiency. In the latter, severe defects of mRNA prevent synthesis of the protein and thus causes complete TBG deficiency (21, 22). All frame shift mutations also cause complete deficiency, because the C terminus of TBG is part of the protein core (31) and, thus, even small deletions affect folding of the molecule (18), causing its degradation in the endoplasmic reticulum (ER) (32). Most amino acid substitutions cause variable degrees of partial TBG deficiency, often affecting T4-binding affinity, heat stability, and/or isoelectric-focusing pattern (2). A common polymorphism (TBG-Poly, L283F) which is associated with several other TBG mutations, does not interfere with its synthesis and secretion and does not affect the normal properties of TBG when present alone (2). The only report of a partial TBG deficiency with apparently normal function and properties had autosomal dominant inheritance in contrast to the X-linked inheritance of all other TBG defects. It is most likely caused by a defective transcription factor (23).

The Allentown variant of TBG reported in this study is the first partial TBG deficiency caused by a mutation in the signal peptide. The affected proposita had TBG levels that fluctuated from low to normal as indicated by her TT4 and TBG concentrations. TBG had normal T4-binding affinity, heat stability, and isoelectric focusing pattern. Expression of recombinant TBG-AT confirmed the presence of a secretion defect and the normal properties of the mature protein. These data suggest that the H(–2)Y mutation interferes with the efficiency of cotranslational processing but does not change the cleavage site of the TBG signal peptide nor its further processing in the ER. This concept is further supported by the retention and accumulation of TBG-AT in the Xenopus oocytes (Fig. 4Go). This contrasts with other TBG deficiency variants that are also retained in the cells but are rapidly degraded in the ER [TBG-Gary (33), TBG-CD5 (27), TBG-CD Japan (32)]. The reason for the wide variation of serum TBG in the proposita remains unclear. It is possible that it was caused by intermittent secretion of the variant TBG-AT. Unfortunately, we were unable to obtain blood samples from other members of the family to assess if indeed TBG-AT is variably secreted in all affected individuals.

The signal peptide plays an important role in the translation by ribosomes. The nascent protein is bound via the signal peptide to the signal recognition particle (SRP), which guides the complex to the ER. Translocation of the protein into the ER is followed by posttranslational processing, which includes signal peptide cleavage, disulfide bond formation, glycosylation, and folding. The sequences of signal peptides are extremely heterogeneous, but three conserved features have been recognized and shown to be essential for protein export. The N-terminal region of five to eight amino acids is hydrophilic due to the presence of positively charged basic residues, and in prokaryotes, the charge affects the rate of protein translocation (34). A hydrophobic core of seven to 15 amino acids is vital for cotranslational processing of the protein (35). The polar C- terminal region of approximately six amino acids contains the signal peptide cleavage site. In this region positions –1 and –3 are usually occupied by small neutral residues (36), which are thought to fit into the active site on the cleavage enzyme (37).

In the superfamily of serpins, signal peptides are also highly heterogeneous. Of the 26 known human serpins, nine are cytosolic, nonsecreted proteins. The signal peptides of the 17 secreted human serpins range in length from 15–33 amino acids (Table 1Go). Apart from the common features outlined above and including a prevalence for small residues (Ala, Gly, Ser, Cys) and Val and Leu in positions –1 and –3, no further homology is found.


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  		TABLE 1. Serpin signal peptides and known variants

 
Eight serpin signal mutations had been reported previously (Table 1Go). In two individuals from Dublin, Ireland, with a family history of recurrent thrombosis (38), a V(–3)E mutation in the signal peptide of AT3 (AT3-Dublin) produced a shift of the cleavage site and partial AT3 deficiency (39). Venous thrombosis in an individual from Dunedin, New Zealand, was associated with a L(–10)P mutation in the AT3 gene (AT3-New Zealand), found to prevent cotranslational processing by disrupting cleavage of the signal peptide (40). A family with {alpha}1-antitrypsin (PI) deficiency from Wrexham, United Kingdom, was compound heterozygous for the mutations E342L (PI-Z) (41) and S(–19)L in the signal peptide (PI-Wrexham) (42). It remained unclear whether the PI-Wrexham mutation contributes to the defect, because the Z mutation by itself causes complete PI deficiency (43).

All other signal peptide mutations in human serpins [{alpha}2-antiplasmin A(–1)V; {alpha}1-antichymotrypsin A(–15)T; plasminogen activator inhibitor 1 A(–9)T and V(–7)I; and heparin cofactor II A(–13)T] are neutral, not affecting synthesis, secretion or function (5). However, the A(–15)T antichymotrypsin variant has been found to be associated with the susceptibility to allergic asthma (44) and with chronic obstructive pulmonary disease (45).

To further evaluate serpin signal peptides, all 26 known human serpins were submitted to SignalP analysis (30). This artificial neuronal network www-server correctly predicted signal peptide length of normal AT3 and the 2-residue elongation caused by the V(–3)E mutation of AT3-Dublin (39) (Fig. 5Go). In the complete AT deficiency AT3-New Zealand, SignalP predicted a low signal peptide score indicative of a possible disruption of the cleavage site, compatible with the clinical and biochemical characteristics of this variant. The {alpha}1-antitrypsin mutation PI-Wrexham did not significantly change any of the signal peptide scores and would thus not seem to alter processing, as well as the five other serpin signal mutations (Table 1Go) correctly predicted by SignalP. Furthermore, all nine cytoplasmic, nonsecreted human serpins (plasminogen activator inhibitor 2, leukocyte elastase inhibitor, placental thrombin inhibitor, cytoplasmic antiproteinase 3, squamous cell carcinoma antigen 1 and 2, bomapin, megsin, and hurpin) were predicted not to have signal peptides.



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  		FIG. 5. Analysis of AT3 and TBG signal peptide mutations with SignalP. The impact of the V(–3)E mutation of AT3-Dublin and the H(–2)Y mutation of TBG-AT was analyzed with the SignalP prediction server at http://www.cbs.dtu.dk/services/SignalP/index.html (30 ). Signal peptide length of normal AT3 (AT3-N, dotted lines, left diagram) was correctly predicted to be 32 residues, with the maximal cleavage site score (C-score, green), signal peptide score (S-score, blue), and combined cleavage site score (Y-score, red) all at histidine +1 (*). AT3-D (solid lines) was correctly predicted to have a 34-residue signal peptide, as previously confirmed by amino acid sequencing of the purified serum protein (39 ). Although its S-score (solid blue line) was ambiguous, both the C- and Y-score (solid green and red lines) were shifted to indicate cleavage at serine +3 (**). SignalP also correctly predicted the 20 amino acid length of the signal peptide and the localization of the cleavage site of TBG-N (right diagram, dotted lines) at alanine +1 (*). No significant differences were found for the C-, S- and Y-scores of the H(–2)Y mutation (solid lines) suggestive of normal signal peptide length and cleavage of TBG-AT (solid lines).

 
SignalP also correctly predicted the 20-amino-acid signal peptide length and cleavage site localization of TBG-N (Fig. 5Go). However, no significant differences were found for the H(–2)Y mutation, suggesting normal signal peptide length and cleavage of TBG-AT, as expected from the normal properties of the serum and recombinant protein. Possible explanations for the reduced expression of TBG-AT could be: 1) impaired recognition of the signal peptide by the SRP, leading to improper targeting of the ribosome-nascent polypeptide-SRP-complex to the ER (46, 47); 2) a less efficient cleavage of the mutant signal peptide by the ER signal peptidase (48); or 3) an impaired interaction of the signal peptide with a proposed second contact of the translocation machinery in the ER membrane (49, 50). Because the recognition of the signal peptide by the SRP as well as the interaction of the signal peptide with the translocon machinery is still not fully understood, investigation of artificial TBG signal peptide variants may be warranted.


   Acknowledgments
 
We thank J. Klein and M. Stratmann for expert technical assistance.


   Footnotes
 
This work was supported in part by grants from the National Institutes of Health (DK17050 and RR00050 to Sa.R.) and from the Deutsche Forschungsgemeinschaft (DFG Ja 671/1-3 to O.E.J.). Si.R. was supported by a training grant from the National Institutes of Health (DK07011).

Abbreviations: AT3, Antithrombin III; AT3-D, AT3-Dublin; CBG, corticosterone-binding globulin; ER, endoplasmic reticulum; PI, proteinase inhibitor; PI-Z, common Z mutation causing PI deficiency; SRP, signal recognition particle; TBG, T4-binding globulin; TBG-AT, TBG-Allentown; TBG-N, normal TBG; TT3, total T3; TT4, total T4.

Received September 16, 2003.

Accepted January 23, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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