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Endocrinology Vol. 141, No. 11 4050-4055
Copyright © 2000 by The Endocrine Society

ARTICLES

A Novel Missense Mutation (G2320R) in Thyroglobulin Causes Hypothyroidism in rdw Rats1

Akira Hishinuma, Sen-Ichi Furudate, Masamichi Oh-Ishi, Narumi Nagakubo, Takashi Namatame and Tamio Ieiri

Department of Clinical Laboratory Medicine (A.H., T.I.), and Institute for Medical Sciences (T.M.), Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan; Department of Laboratory Animal Science (S.F.), School of Medicine, and Department of Physics (M.O.), School of Science, Katasato University, Sagamihara, Kanagawa 228-8555, Japan; and Sumikin Bio-Science (N.N.), Sagamihara, Kanagawa 229-1124, Japan

Address all correspondence and requests for reprints to: Akira Hishinuma, M.D., Ph.D., Department of Clinical Laboratory Medicine, Dokkyo University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi, 321-0293, Japan. E-mail: a-hishi{at}dokkyomed.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The rdw rat is a hereditary hypothyroid variant initially derived from the Wistar-Imamichi strain. Proteome analysis by two-dimensional gelelectrophoresis showed that molecular chaperones accumulated in the thyroid glands, suggesting retention of abnormal proteins in the endoplasmic reticulum (ER). Anatomical studies indicated that thyroglobulin (Tg) was not secreted into the follicular lumina, but retained in the dilated ER. Sequencing of the entire Tg complementary DNA from the rdw rat revealed a missense mutation (G2320R) in the acetylcholinesterase-like domain at the 2320th amino acid residue. Carbohydrate residues of the G2320R Tg mutant were of the high-mannose ER type, as shown by sensitivity to the treatment with endoglycosidase H. Molecular chaperones, GRP94, GRP78, and calreticulin, were all accumulated in the rdw rat thyroid glands. Computer analysis of protein secondary structure predicted that the mutation would cause extension of the helix where ß-sheet and turns were formed in the normal Tg. Altered folding of Tg might account for the impaired intracellular transport of Tg and activated premature degradation by the same mechanism as in ER storage diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE rdw rat was initially isolated as a hereditary dwarf strain from a closed colony of Csk: Wistar-Imamichi rats (1). Previous studies showed that serum GH concentrations and pituitary GH messenger RNA (mRNA) levels were decreased (2, 3). Marked hypothyroidism was subsequently noted in the rdw rat and treatment with thyroid hormones raised serum GH concentrations and reversed clinical symptoms and other laboratory findings (4), suggesting that hypothyroidism is the primary defect in the rdw rat. Proteome analysis by two-dimensional gel electrophoresis revealed increased expression of molecular chaperones, GRP94, GRP78, and hsp70, as well as decreased tissue content of thyroglobulin (Tg) (5). In the thyroid glands of the rdw rat secretory granules were missing in the follicular epithelial cells. The immunohistochemical analysis in the rdw rat showed that Tg was detected at very low levels in the follicular lumina, while a substantial quantity was noted in the dilated endoplasmic reticulum (ER) (6), suggesting that Tg was not transported to the Golgi.

Hereditary hypothyroidism is caused by mutations of the Tg gene in many animal species, as well as in humans. Nonsense mutations in the Tg gene cause congenital hypothyroidism in the Afrikander cattle (7) and Dutch goats (8), whereas a missense mutation is responsible in the cog/cog mouse (9). In humans, Tg gene abnormalities were identified as a 3' splice site mutation (10), nonsense mutations (11, 12), and missense mutations (13, 14). Among these mutations, the missense mutations of the mouse (9) and the humans (13, 14) proved to cause impaired intracellular transport of Tg.

Intracellular transport of Tg requires several steps before secretion into the follicular lumina. Immediately after translation, Tg forms high molecular weight aggregates with molecular chaperones in the ER, from which monomers dissociate and subsequently form homodimers in the Golgi (15, 16, 17). The first steps in N-glycosylation occur in the ER, whereas subsequent transformation of carbohydrate residues to complex-type units takes place in the Golgi (18). Susceptibility to endoglycosidase H is dependent on the exposure of the inner core, which makes Golgi-type complex carbohydrates resistant to the enzyme.

In the present study, we analyzed the Tg complementary DNA (cDNA) from the rdw rat and identified a missense mutation (G2320R). Since the full-length cDNA for the rat Tg has not been reported, we first cloned the cDNA by PCR with primers, the sequences of which are based on the reported 5' and 3' cDNA sequences (19, 20). We also show that the G2320R Tg is not transported properly, but retained in the ER, and that the molecular chaperones, GRP94, GRP78, and calreticulin accumulate in the thyroid glands of the rdw rat.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Because of the autosomal recessive inheritance of the rdw trait and infertility of the mutants, we maintained the rdw rat by mating heterozygous animals that bear offspring with homozygous (rdw/rdw), heterozygous (rdw/+), and normal (+/+) traits. Wistar-Imamichi rats (Imamichi Institute, Ibaraki, Japan), from which the rdw rat was derived, and closely related F344 rats (Japan SLC, Hamamatsu, Japan) were included in this study as controls.

Cloning of the rat Tg cDNA
The sequence in between the reported 5' and 3' sequences of the rat Tg cDNA (GenBank accession no. M35965 and no. X02318) was determined by a PCR-based method. Total RNA was extracted from the thyroid glands of normal animals (+/+) by the RNeasy Mini kit (QIAGEN, Hilden, Germany). RT-PCR was performed as previously reported (14). Briefly, total RNA was reverse transcribed with random hexamers by MuLV reverse transcriptase (Perkin-Elmer Corp., Norwalk, CT). The cDNA was used for nested PCR by the Expand High-Fidelity PCR system (Roche Molecular Biochemicals, Mannheim, Germany) for 45 cycles, which consisted of denaturation at 98 C for 4 sec, primer annealing at 55 C for 30 sec, and primer extension at 72 C for 5 min in a GeneAmp 9600 Thermal Cycler (Perkin-Elmer Corp.). For the first PCR the forward primer 5'-GTGTCCAAGGAGCTGTGAGATAAG-3' and the reverse primer 5'-CAGGCCGAGACCCTATGTCAG-3' were derived from the reported 5' and 3' rat Tg cDNA sequences, respectively. Similarly, for the second PCR, the sequences for the forward and reverse primers were 5'-CGATGCAGATGGGGAGTTTATG-3' and 5' TCAGAATCTTTCCAGAGGTAGACC-3', respectively. The 5-kb RT-PCR product was purified by the GeneClean III (BIO 101, Carlsbad, CA) and ligated to the pCR2.1 vector (Original TA Cloning kit, Invitrogen, San Diego, CA). Deletion mutants were produced by the Kilo-Sequence Deletion kit (Takara, Tokyo, Japan) and sequenced by the Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer Corp.).

Analysis of the Tg cDNA from the rdw rat
Total RNA was isolated from the thyroid glands pooled from three animals with the rdw/rdw, rdw/+, and +/+ traits, as well as the Wistar-Imamichi and F344 rats. Total RNA was reverse transcribed, and RT-PCR of the entire rat Tg cDNA was performed in five segments with the primers listed in Table 1Go. The RT-PCR products were directly sequenced with the forward and reverse primers used in the RT-PCR and additional primers listed in Table 1Go by the Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer Corp.). Total RNA was also isolated from one lobe of the thyroid glands of individual littermates, leaving the other lobe for the proteome analysis.


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  		Table 1. Primers used for PCR and sequencing of Tg cDNA

 
Genomic DNA analysis by PCR-RFLP
Genomic DNA was isolated from the thyroid glands by the QIAamp Tissue kit (QIAGEN). The PCR reaction was performed by the Expand High-Fidelity PCR system (Roche Molecualr Biochemicals) with the forward primer 5'-GGCAAGAGAACTAGTGTGGATTCAGA-3' and the reverse primer 5'-GACCCCCAGTCTGTAGTTAGCAGT-3'. The PCR products were purified with the QIA PCR Purification kit (QIAGEN) and treated with the restriction enzyme MboI (Roche Molecular Biochemicals) at 37 C for 1 h.

Treatment with endoglycosidase H
Thyroid tissue extracts were prepared by homogenizing one lobe of the thyroid glands from single animals in 100 µl Tris buffer (10 mM; pH 8.0) that contained a cocktail of protease inhibitors (Complete Protease Inhibitor Cocktail Set, Roche Molecular Biochemicals). The tissue homogenate was centrifuged at 18,000 x g twice for 30 min each time. Protein concentrations in the supernatant was determined by the Bradford method using the Bio-Rad Laboratories, Inc. Protein Assay Kit (Bio-Rad Laboratories, Inc., Richmond, CA). An aliquot containing 6 µg protein in 4 µl was digested with 0.3 mU/liter endoglycosidase H (Roche Molecular Biochemicals) in a buffer that contained 250 mM sodium citrate (pH 5.3), 2.5% SDS, 50 mM ethylenediamine tetraacetic acid, and 5% 2-mercaptoethanol (2-ME) for 15 min at room temperature. An equal volume of an electrophoresis buffer (0.24 M Tris-HCl; pH 8.7, 15% glycerol, 2.5% SDS, and 5% 2-ME) was subsequently added to the samples. Electrophoresis was carried out on a 4–15% gradient polyacrylamide gel using the Phast System (Amersham Pharmacia Biotech, Uppsala, Sweden).

Immunoblot analysis
An aliquot from each thyroid tissue extract was electrophoresed either in a native sample buffer (0.24 M Tris-HCl; pH 8.7, and 15% glycerol) or in a denaturing sample buffer (the native buffer plus 2.5% SDS and 5% 2-ME) on a 4–15% gradient polyacrylamide gel using the Phast System. Some of the gels were stained with Coomassie brilliant blue; the rest of the gels were transferred onto nitrocellulose membranes using the Phast System semidry blotting method. The membranes were incubated in 3% BSA in Tris saline (150 mM NaCl and 10 mM Tris, pH 7.5) for 2 h. Each membrane was then placed in Tris saline containing rabbit anti-actin antibody (Biomedical Technologies, Stoughton, MA), rat anti-GRP94 antibody (StressGen, Victoria, Canada), mouse anti-GRP78 antibody (StressGen, Victoria, Canada), and rabbit anti-calreticulin antibody (Affinity BioReagents, Inc. Golden, CO). The membranes were then reacted with peroxidase-conjugated secondary antibody, and stained with 4-chloro-1-naphthol (Sigma, St. Louis, MO) in the presence of H2O2.

Protein secondary structure prediction
The deduced C-terminal 541 amino acids in the acetylcholinesterase-like domain of the rat Tg was submitted for computer analysis (nn Predict-UCSF) of the protein secondary structure prediction via the Internet (www.cmpharm.ucsf.edu/nomi/nnpredict.html).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Using the reported 5'- and 3'-sequences of the rat Tg cDNA, we successfully determined the entire nucleotide sequence of the rat Tg cDNA (GenBank accession no. AB035201) by the PCR method. The complete rat Tg cDNA with 8461 nucleotides contained an ORF of 8307 nucleotides, which consists of a 20-residue signal peptide and a 2748-residue mature protein (Fig. 1Go). The homology of the rat Tg with the mouse (9), bovine (21), and human (14, 22) Tg is 90%, 76%, and 78%, respectively, at the nucleotide level; and 90%, 71%, and 74%, respectively, at the amino acid level. The most conserved amino acid is cysteine, whose number and location are almost invariable among the rat, mouse, bovine, and human Tg. Among 123 cysteine residues in the rat Tg, only one residue at position 2454 is missing in the mouse Tg. Based on the location of cysteine residues, two amino acids, glycine and alanine, at position 1847 and 1848, are missing in the mouse Tg compared with the rat Tg. The tyrosine residues, which are iodinated for the synthesis of thyroid hormones, are also well conserved in the Tg. Among 75 tyrosine residues, 5 locations are different between the rat and mouse Tg. Tyrosine residues at position 977 and 2644 are missing and those at position 1288, 1681, and 1722 are added to the mouse Tg. Among 5 substitutions, 4 tyrosine residues are replaced by aromatic amino acids, histidine and phenylalanine. The possible hormonogenic tyrosine residues at position 24, 2572, 2765, 1309, 2586, and 704 in the human Tg (23) are conserved in the rat Tg at position 25, 2574, 2766, 1310, 2588, and 704, respectively.



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  		Figure 1. The rat Tg cDNA sequence and deduced amino acid sequence. G at nucleotide 6958 was substituted with C in the rdw rat and the corresponding amino acid substitution is G to R. The hormonogenic tyrosine residues at position 25, 2574, 2766, 1310, 2588, and 704 are in italics.

 
Direct sequencing of the Tg cDNA from the rdw rat showed that guanine at nucleotide 6958 was substituted with cytosine (6958G->C); the corresponding amino acid substitution was glycine with arginine (G2320R) (Fig. 1Go). The nucleotide at position 6958 was guanine in both the Wistar-Imamichi and F344 rats. Genomic DNA analysis also showed the nucleotide substitution 6958G->C in the rdw rat by the PCR-RFLP method using the restriction enzyme MboI, which digests only the mutant allele, but not the normal allele (Fig. 2Go). MboI completely digested the PCR product in the rdw/rdw rat, whereas it partially digested the PCR product in the rdw/+ rat. The PCR products in the +/+ rat and the Wistar-Imamichi rat were resistant to MboI digestion.



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  		Figure 2. Genomic DNA analysis by the PCR-RFLP method. The PCR products with 240 bp from the Wistar-Imamichi, rdw/rdw, rdw/+, and +/+ rats were digested with the MboI restriction enzyme. The mutation G2320R creates an MboI-sensitive site that gives rise to 176-bp and 64-bp restriction fragments. W-I, Wistar-Imamichi.

 
Thyroid tissue extract from the rdw rat was treated with endoglycosidase H, which only digests high mannose ER-type oligosaccharides, and subjected to SDS-PAGE (Fig. 3Go). In the rdw/rdw rat, the Tg band exhibited a large shift upon endoglycosidase H digestion. In the rdw/+, +/+, and Wistar-Imamichi rats, Tg was resistant to endoglycosidase H treatment.



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  		Figure 3. Endoglycosidase H treatment of Tg. Protein extracts from the Wistar-Imamichi, rdw/rdw, rdw/+, and +/+ rats were treated with endoglycosidase H and subjected to SDS-PAGE. Tg was identified in the gels. Polysaccharides of high-mannose ER-type are sensitive to the endoglycosidase H treatment, whereas those of complex Golgi-type are resistant to the treatment. W-I, Wistar-Imamichi.

 
SDS-PAGE of thyroid tissue extract followed by immunoblot analysis showed that proteins at mol wt 99,000, 78,000, and 60,000 reacted with anti-GRP94, anti-GRP78, and anticalreticulin antibodies, respectively (Fig. 4Go). The levels of the detected proteins in rats increased in the following order: +/+, rdw/+, and rdw/rdw (Fig. 4Go). An antiactin antibody was used as a control in the immunoblot experiments. cDNA analysis at nucleotide 6958 was consistent with the proteome analysis in littermates born to the rdw/+ parents (data not shown).



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  		Figure 4. Immunoblot with anti-actin, anti-GRP94, anti-GRP78, and anticalreticulin antibodies (upper panel) and protein analysis by SDS PAGE. Protein extracts from the Wistar-Imamichi, rdw/rdw, rdw/+, and +/+ rats were subjected to SDS PAGE, and some of the gels were stained with Coomassie brilliant blue. The other gels were transferred onto the nitrocellulose membranes and incubated with the antibodies. The membranes were then reacted with peroxidase-conjugated secondary antibody and stained with 4-chloro-1-naphthol.

 
We submitted the amino acid sequence of the acetylcholinesterase-like domain to the nn Predict-UCSF Internet site for protein secondary structure prediction by a two-layer, feed-forward neural network (24) to explore putative effects of the missense mutation, G2320R, on the structure of the rat Tg. Figure 5Go shows that the glycine residue at position 2320 is well conserved among different species. In the normal rat protein, 3 amino acids, from position 2319 to 2321, constitute a turn, whereas in the mutant protein the arginine residue is involved in an extended helix and only one residue at position 2319 constitutes a turn.



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  		Figure 5. Homology and protein secondary structure. The location of the missense mutation (G2320R) is underlined. PSS, Protein secondary structure; E, ß-sheet; H, helix; -, turn.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have identified a missense mutation (G2320R) in the Tg gene of the rdw rat, which was initially isolated as a dwarf variant from the Wistar-Imamichi strain (1). Previous studies focused on pituitary hormones and pituitary-specific transcription factors (2, 3). However, recent studies revealed that the rdw rat is remarkably hypothyroid and that supplementation of thyroid hormones reversed clinical symptoms and laboratory findings in the rdw rat (4). The missense mutation is responsible for the rdw trait for the following reasons: the missense mutation is specific in the rdw rat and is not detected in the Wistar-Imamichi and F344 rats as well as the +/+ rat; genomic DNA analysis by the PCR-RFLP method gives the same results as sequencing of the Tg cDNA; and the genetic and proteome analysis agreed in littermates.

In addition to the mutation, the present study revealed many features of the Tg gene. The rat is the fourth animal in which the Tg cDNA has been completely sequenced. Homology analysis shows that rat Tg is very closely related to different species, especially mice. The most conserved amino acid is cysteine, whose mutations were reported to cause congenital hypothyroidism and a variant type of adenomatous goiter in humans (14). Using the cysteine residues as landmarks, the rat Tg is only two amino acids larger than the mouse Tg at positions 1847 and 1848. Hormonogenic tyrosine residues are invariant in the rat and human proteins.

The molecular pathogenesis of the missense mutation is manifested as defective intracellular transport of Tg. The mutant Tg is associated with high-mannose ER-type carbohydrates, which were sensitive to the treatment with endoglycosidase H. The molecular chaperones GRP94, GRP78, and calreticulin, were also accumulated in the thyroid glands.

The defective intracellular transport of Tg was also reported in humans (13, 14) and mice (9). All the cases were caused by Tg missense mutations. We reported two missense mutations in humans that caused cysteine substitutions with other amino acids (14). The mutant Tg formed disulfide-linked high molecular weight aggregates in the ER. However, native PAGE of rdw rat thyroid tissue extract showed that the G2320R Tg was heterogeneous, with partial formation of monomers (data not shown), suggesting that correct disulfide bonds were, at least, partially formed.

An interesting but unsolved question in the rdw rat is why the rdw thyroid glands were not goitrous in gross inspection. The rdw rat was hypothyroid; serum T4 concentrations in the rdw/rdw rat were 1.17 µg/dl compared with 4.99 µg/dl in the +/+ rat. Serum TSH concentrations in the rdw/rdw rat were elevated to 9.80 ng/ml compared with 1.10 ng/ml in the +/+ rat though the levels were not as high as those of thyroidectomized rat (35.5 ng/ml) (6). The thyroid glands of the rdw rat were almost proportional to body weights. Because the total body weights of the rdw/rdw rat were only 1/3 of the +/+ rat (146.8 g vs. 416.3 g), the thyroids from single animals were indeed smaller in the rdw rat (19.0 mg vs. 45.3 mg). But, when corrected by body weights the weights of the thyroid glands in the rdw/rdw rat were 1.19 times of those in the +/+ rat (6). Because the thyroids of the rdw rat were not extremely large, we believe that the TSH stimulation might be submaximal, not enough to generate a huge goiter or that responsiveness of the thyroid cells to proliferate by TSH or other growth factors might be reduced in rats. Initially, the rdw rat was isolated as a model of dwarfism (1), and production of GH was greatly reduced (2). Because insulin-like growth factor-1 is one of the major stimulator of thyroid cell proliferation in conjunction with TSH (25), reduced serum GH concentrations might play a role in not developing goiter in the rdw rat.

Computer analysis of the protein secondary structure showed that the G2320R mutation caused an extended stretch of helix and reduced amino acid residues consisting of turn. The mouse model of the Tg mutation (cog/cog) was also caused by a missense mutation (L2263P) in the acetylcholinesterase-like domain of Tg. Kim et al. (9) speculated that the mutation might prevent proper formation of nearby disulfide bond because leucine at position 2263 is close to cysteine at position 2280. In the rat Tg, no cysteine residues are found in the proximity of glycine at position 2320. We believe that the G2320R mutation does not affect disulfide bond formation. Rather, the protein structure prediction showed that the mutation caused the extended helix. This subtle structural change might account for less severe protein folding defects that allow partial monomer formation.

In conclusion, we have shown that the hypothyroidism of the rdw rat was caused by the missense mutation G2320R in the Tg gene and that intracellular transport of the mutant protein was impaired. Because defective intracellular transport of Tg was found not only in rats but also in humans and mice, many mutant forms of Tg from different species cause ER storage disease due to changes in protein folding.


   Footnotes
 
1 This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan, and grants from the Japan Private School Promotion Foundation (all to A.H.). The rat thyroglobulin cDNA sequence reported in this paper has been deposited in the DDBJ/GenBank/EMBL databases with the accession no. AB035201. Back

Received April 17, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Two Distinct Compound Heterozygous Constellations (R277X/IVS34-1G>C and R277X/R1511X) in the Thyroglobulin (TG) Gene in Affected Individuals of a Brazilian Kindred with Congenital Goiter and Defective TG Synthesis
J. Clin. Endocrinol. Metab., February 1, 2004; 89(2): 646 - 657.
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P. Caron, C. M. Moya, D. Malet, V. J. Gutnisky, B. Chabardes, C. M. Rivolta, and H. M. Targovnik
Compound Heterozygous Mutations in the Thyroglobulin Gene (1143delC and 6725G->A [R2223H]) Resulting in Fetal Goitrous Hypothyroidism
J. Clin. Endocrinol. Metab., August 1, 2003; 88(8): 3546 - 3553.
[Abstract] [Full Text] [PDF]

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P. Kopp
Perspective: Genetic Defects in the Etiology of Congenital Hypothyroidism
Endocrinology, June 1, 2002; 143(6): 2019 - 2024.
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