Defective protein folding and intracellular retention of thyroglobulin-R19K mutant as a cause of human congenital goiter

Abstract

It has been suggested that a thyroglobulin (Tg)-R19K missense mutation may be a newly identified cause of human congenital goiter, which is surprising for this seemingly conservative substitution. Here, we have examined the intracellular fate of recombinant mutant Tg expressed in COS-7 cells. Incorporation of the R19K mutation largely blocked Tg secretion, and this mutant was approximately 90% degraded intracellularly over a 24-h period after synthesis. Before its degradation, the Tg-R19K mutant exhibited abnormally increased association with molecular chaperones BiP, calnexin, and protein disulfide isomerase, and was unable to undergo anterograde advance from the endoplasmic reticulum (ER) through the Golgi complex. Inhibitors of proteasomal proteolysis and ER mannosidase-I both prevented ER-associated degradation of the Tg-R19K mutant and increased its association with ER molecular chaperones. ER quality control around Tg residue 19 is not dependent upon charge but upon side-chain packing, because Tg-R19Q was efficiently secreted. Whereas a Tg mutant truncated after residue 174 folds sufficiently well to escape ER quality control, introduction of the R19K point mutation blocked its secretion. The data indicate that the R19K mutation induces local misfolding in the amino-terminal domain of Tg that has global effects on Tg transport and thyroid hormonogenesis.

Figure 1

Transient Expression of Wild-Type and Mutant Tg cDNAs COS-7 cells were transiently transfected with cDNAs encoding full-length myc-his tagged Tg. Forty-eight hours after transfection, the media were changed to serum-free media and collected for a further 24 h. Cell lysates and media were analyzed by reducing SDS-PAGE and Western blotting with anti-myc. A, Empty vector (control), wild-type, R19K, R835Q, and V2452L Tg. B, Empty vector (control), wild-type, Q851H, L2263P, C1244R, and C1976S Tg. These results have been replicated three times.

Figure 2

Intracellular Disposal of Tg-R19K A, Transiently transfected COS-7 cells expressing Tg-R19K were pulse-labeled for 30 min with ³⁵S-amino acids and chased for the times indicated. Cell lysates immunoprecipitated with anti-myc were subjected to reducing SDS 4%-PAGE and autoradiography. B, Quantitation of the kinetics of loss of intracellular Tg and Tg-R19K from the data shown in panel A, as measured by scanning densitometry. The data in this figure are representative of two replicate experiments. WT, Wild type.

Figure 3

Increased Association of BiP, Calnexin, and PDI with Tg-R19K COS-7 cells transfected with either wild-type Tg (A) or Tg-R19K (B) were pulse-labeled for 1 h with ³⁵S-amino acids and chased for the times indicated. Cells lysed under nondenaturing conditions were immunoprecipitated (IP) with the antibodies to Tg, BiP, Calnexin (Clnx), and PDI and analyzed for the recovery of labeled Tg by reducing SDS 4%-PAGE and phosphorimaging. C, For the Tg-R19K mutant at each chase time, the labeled Tg recovered by coprecipitation with anti-BiP, Clnx, or PDI was quantified as a fraction of that recovered by direct immunoprecipitation with anti-Tg. D, Pulse-labeling and 3 h chase of COS-7 cells cotransfected with Tg or Tg-R19K cDNA and a cDNA encoding SEAP. Preimmune serum from a rabbit (before Tg immunization) recovers no radiolabeled myc/6xhis-tagged wild-type (WT) Tg from transfected COS-7 cells, or from the medium bathing these cells, whereas immunoprecipitation with anti-myc specifically recovers Tg and Tg-R19K. Although Tg-R19K is not secreted, coexpressed SEAP is labeled and secreted equally well from these cells as demonstrated by specific immunoprecipitation, SDS-PAGE, and autoradiography.

Figure 4

Tg-R19K Fails to Undergo Intracellular Transport to the Golgi Complex COS-7 cells transiently expressing Tg-R19K were pulse-labeled with ³⁵S-amino acids for 1 h and chased for the times indicated. At each chase time, cell lysates were digested with endo H followed by immunoprecipitation with anti-Tg. The samples were analyzed by SDS 4%-PAGE under reducing conditions; these results have been replicated three times.

Figure 5

Effect of MG132 and KIF on Intracellular Disposal of Tg-R19K COS-7 cells transiently transfected to express either Tg-R19K (R19K) or cog Tg were labeled with ³⁵S-amino acids for 1 h and chased in the presence of 20 μm MG132 for 18 h or 100 μm KIF for 24 h. A, The cells were lysed, immunoprecipitated (IP) with anti-Tg, and analyzed by reducing SDS-PAGE and phosphorimaging. The percentages of labeled Tg recovered under each condition were quantified from triplicate samples. B, The cells expressing Tg-R19K were lysed under nondenaturing conditions, immunoprecipitated with anti-chaperones antibodies, and coprecipitated Tg was analyzed by reducing SDS-PAGE and phosphorimaging. The data in this figure are representative of two replicate experiments. Clnx, Calnexin.

Figure 6

Misfolding Induced by the Tg-R19K Mutation Acts Locally within the Amino-Terminal Region A truncated Tg cDNA was created replacing Cys175 with a stop codon (C175Z). Into this truncated Tg cDNA template, the R19K mutation was introduced (Tg-R19K,C175Z). COS-7 cells were transiently transfected with empty vector (Control), or full-length Tg-R19K, or the truncated Tg cDNAs. At 48 h after transfection, the media were changed to serum-free media and collected for a further 24 h. Lysates of cells (left panel), and secreted media (right panel) were analyzed by reducing SDS 4–15%-PAGE followed by Western blotting using an antibody against the amino terminus of mouse Tg. The data shown here have been replicated twice.

Figure 7

Expression of Mutants Containing R/K or K/R Substitutions at the Amino Terminus of Tg Upper portion, Alignment of amino-terminal Tg sequence between mouse and human, identifying the positions to be mutagenized (arrows). Lower portion, COS-7 cells were transiently transfected with full-length Tg cDNAs encoding the mutants shown, and the cell lysates and media analyzed by reducing SDS-PAGE Western blotting as in Fig. 1. The data in this figure are representative of three experiments. WT, Wild type.