The cholinesterase-like domain of thyroglobulin functions as an intramolecular chaperone

Abstract

Thyroid hormonogenesis requires secretion of thyroglobulin, a protein comprising Cys-rich regions I, II, and III (referred to collectively as region I-II-III) followed by a cholinesterase-like (ChEL) domain. Secretion of mature thyroglobulin requires extensive folding and glycosylation in the ER. Multiple reports have linked mutations in the ChEL domain to congenital hypothyroidism in humans and rodents; these mutations block thyroglobulin from exiting the ER and induce ER stress. We report that, in a cell-based system, mutations in the ChEL domain impaired folding of thyroglobulin region I-II-III. Truncated thyroglobulin devoid of the ChEL domain was incompetent for cellular export; however, a recombinant ChEL protein ("secretory ChEL") was secreted efficiently. Coexpression of secretory ChEL with truncated thyroglobulin increased intracellular folding, promoted oxidative maturation, and facilitated secretion of region I-II-III, indicating that the ChEL domain may function as an intramolecular chaperone. Additionally, we found that the I-II-III peptide was cosecreted and physically associated with secretory ChEL. A functional ChEL domain engineered to be retained intracellularly triggered oxidative maturation of I-II-III but coretained I-II-III, indicating that the ChEL domain may also function as a molecular escort. These insights into the role of the ChEL domain may represent potential therapeutic targets in the treatment of congenital hypothyroidism.

Figure 3. Oxidation state of secreted Tg.

At each chase time, cells were lysed in buffer including 20 mM N-ethylmaleimide, and the lysates and chase media immunoprecipitated with anti-Tg and analyzed by nonreducing 4% SDS-PAGE and fluorography. (A) PC Cl3 cells were pulse labeled for 10 minutes with ³⁵S-labeled amino acids and then chased for the times indicated. Immunoprecipitates were either undigested or digested with PNGase F as indicated. Folding intermediates A, B, and C, which have been characterized in previous studies (28), are shown. Also identifed are 2 closely spaced Tg disulfide isomer bands labeled D and E, respectively. Because of glycosylation differences, the mature E isoform does not comigrate with Tg secreted to the medium at 1 hour of chasing. After PNGase F digestion to remove N-glycans, all Tg forms exhibit a faster (shifted-down) mobility. Under these conditions, it is now apparent that secreted Tg comigrates with the intracellular E isomer, identifying the most oxidized band as the most mature folded form of Tg. (B) Results of an experiment identical to the one represented in A, except using recombinant Tg expressed in 293 cells, without PNGase F digestion. (C) Results of a repeat experiment of that shown in panel B, but including PNGase F digestion. The position of a 181-kDa prestained molecular weight standard is shown at left.

Figure 1. Free Cys thiols in Tg.

293 cells were transiently transfected with an expression vector encoding wild-type Tg, the rdw Tg mutant (G2300R), or empty vector. At 48 hours after transfection, cells were pulse labeled for 30 minutes with ³⁵S-labeled amino acids and chased for 4 hours in the absence or presence of BFA (5 μg/ml) where indicated. At this time, chase media (lanes labeled “Tg secreted”) and cell lysates (all other lanes) were immunoprecipitated with anti-Tg. The immunoprecipitates were denatured in 2× SDS gel sample buffer lacking reducing agents and mock incubated or incubated with AMS (5 mM, 30°C for 1 hour). At the end of the incubation, samples were boiled in the presence of 20 mM DTT and analyzed by 4% SDS-PAGE and fluorography. A slowed mobility (shift up) of the Tg band after AMS is indicative of free reactive thiols in the Tg molecule that are not apparent in secreted, wild-type Tg.

Figure 2. Arrested disulfide maturation of Tg bearing a mutation in the ChEL domain.

(A) 293 cells were transiently transfected with vector DNA encoding wild-type Tg (bearing a C-terminal myc-6xHis epitope tag that does not block Tg secretion; ref. 37) or empty vector (control [Con]). Transfected cells were then pulse labeled for 30 minutes with ³⁵S-labeled amino acids and chased at 37°C for up to 4 hours in the absence or presence of BFA (5 μg/ml) where indicated. At each chase time, cells were lysed, immunoprecipitated with anti-Tg, and analyzed by nonreducing 4% SDS-PAGE and fluorography. A region of the gel enriched in disulfide-linked Tg adducts A, B, and C (28) is indicated. Two additional forms of Tg monomer — folding intermediate D and the E band representing fully oxidized mature Tg — are shown. (B) Results of an experiment identical to the one represented in A, except that the cells were chased at either 25°C (left) or 20°C (right). At each time, chase medium was also collected (as indicated), but at these temperatures the media contained no labeled Tg. (C) Results of an experiment identical to the one represented in A, except that cells were transfected with plasmid DNA encoding cog (L2263P) or rdw (G2300R) mutations within the Tg ChEL domain, and the cell lysis buffer included 20 mM N-ethylmaleimide. No detectable Tg was secreted, so analysis of the supernatant is not shown.

Figure 6. Secreted I-II-III protein is physically associated with secretory ChEL protein.

(A) 293 cells were transiently transfected with 0.5 μg plasmid DNA encoding I-II-III and cotransfected with 2.5 μg of plasmid DNA encoding the secretory ChEL domain either lacking or containing a myc epitope tag, as indicated. The cotransfected cells or untransfected controls were pulse labeled for 30 minutes and chased in complete media, and the secretion after 6 hours was analyzed by immunoprecipitation with anti-Tg or anti-myc. Immunoprecipitates and coprecipitates were analyzed by reducing 5.5% SDS-PAGE and fluorography. Addition of the myc tag slightly retards the SDS-PAGE mobility of the ChEL domain. Note that anti-myc precipitation of ChEL-myc coprecipitates I-II-III. (B) Cells untransfected (control) or cotransfected and pulse labeled as in A (I-II-III + ChEL) were chased in complete media for the time intervals shown, in the presence or absence of cycloheximide (CHX). The media were immunoprecipitated with anti-Tg and analyzed by reducing SDS-PAGE and fluorography. These lanes were run contiguously; a black line has been added for clarity to separate the samples. Note that prelabeled ChEL secretion proceeded rapidly in the presence of CHX, but prelabeled I-II-III secretion was blocked. The positions of molecular mass markers are shown at left.

Figure 5. ER exit of Tg region I-II-III in the absence and presence of ChEL domain.

(A) 293 cells were either untransfected (control) or transiently transfected with 2 μg plasmid DNA encoding either full-length wild-type mouse Tg or Tg region I-II-III. Cells were pulse labeled and chased and samples prepared and immunoprecipitated with anti-Tg as in Figure 4, with analysis by reducing 4% SDS-PAGE and fluorography. Arrows highlight secretion (or lack thereof) from cells to media. (B) The first lane represents untransfected control cells. The remaining cells transfected to express Tg region I-II-III were pulse labeled as in A and then lysed without or with 4 hours of chasing. Tg immunoprecipitates from cell lysates and media were either mock digested or digested with endoglycosidase H for 1 hour at 37°C, before reducing 4% SDS-PAGE and fluorography. Lanes were run contiguously but have been separated for clarity. The positions of molecular mass standards are shown at left. (C) Lower panels: 293 cells were transiently transfected with 0.5 μg plasmid DNA encoding I-II-III cotransfected with 2.5 μg of plasmid DNA encoding the secretory ChEL domain or untransfected controls. Cells were pulse labeled for 30 minutes with ³⁵S-labeled amino acids and chased for the times indicated. At each chase time, the cells were lysed and both lysates and media immunoprecipitated with polyclonal anti-Tg to recover both I-II-III and secretory ChEL proteins, as revealed by reducing 5.5% SDS-PAGE and fluorography. The upper panel represents a negative control with identical transfection of I-II-III but without cotransfection of the secretory ChEL plasmid.

Figure 4. Efficient exit of the isolated Tg ChEL domain from the ER.

293 cells were transiently transfected with a plasmid encoding the wild-type mouse Tg ChEL domain preceded by the prolactin signal peptide (Secretory ChEL) or were untransfected (293 control). Cells were pulse labeled for 30 minutes with ³⁵S-labeled amino acids and chased for 0 or 4 hours as indicated, at which time the cells were lysed and both lysates and media immunoprecipitated with a rabbit polyclonal anti-Tg. Immunoprecipitates from transfected cells were divided in 2 equal portions and either mock digested or digested with endoglycosidase H (Endo H). Finally, all samples were analyzed by reducing 5.5% SDS-PAGE and fluorography. The band shift observed after digestion of secretory ChEL from the 0 chase time (shift down, lane 2) is indicative of endoglycosidase H sensitivity and defines ChEL that has not yet reached the Golgi complex; in contrast, none of the secreted ChEL shows the same endoglycosidase H sensitivity, indicating intracellular transport via the Golgi complex. The lanes shown were all run on the same gel, although they are presented noncontiguously. The position of the 76-kDa molecular mass standard is shown at left.

Figure 8. ChEL functions as a molecular chaperone for I-II-III.

(A) Cells were either untransfected (control) or transfected with a plasmid encoding I-II-III plus either empty vector or secretory ChEL-KDEL, as indicated. The transfected cells were pulse labeled for 30 minutes with ³⁵S-labeled amino acids and chased for the times indicated. At each chase time, cells were immunoprecipitated with anti-Tg and newly synthesized I-II-III analyzed by nonreducing 4% SDS-PAGE and fluorography. Absence of recovery of I-II-III from untransfected control cells is shown at left. The band seen at time 0 and after chasing for 2 hours appears equivalent to that of the D isoform of full-length Tg. In the presence of the ChEL domain in the ER, a faster-migrating band equivalent to that of the mature E isoform of full-length Tg is detected (filled arrow). (B) Results of an experiment using the same cotransfection protocol and analysis as in A, except the second plasmid is either vector alone or secretory ChEL domain containing the cog mutation, the rdw mutation, or the KDEL appendage. Absence of recovery of I-II-III from untransfected 293 cells is shown in lane 1. The position of a 176-kDa molecular mass marker is shown at left.

Figure 9. Oxidation state of secreted I-II-III.

293 cells were transfected either with empty vector (control) or with secretory ChEL bearing or lacking the KDEL appendage. The cells were pulse labeled for 30 min with ³⁵S-labeled amino acids and then chased for the times indicated (control cells were chased for 2 hours). The cells were lysed, and both lysates and chase medium immunoprecipitated with anti-Tg and subjected to PNGase F digestion to remove N-glycans. The samples were then analyzed by nonreducing 5.5% SDS-PAGE. Under these conditions, it is apparent that secreted I-II-III closely migrates with the intracellular E isomer of I-II-III. A black line has been added to distinguish the medium from the cell lysate. The position of a 176-kDa molecular mass marker is shown at left.

Figure 7. ChEL interaction improves recovery as well as secretion of Tg I-II-III.

(A) 293 cells were triply transfected with empty vector plus a plasmid encoding Tg region I-II-III (always 0.1 μg DNA per well) plus a plasmid encoding the secretory ChEL domain (at different DNA levels as shown). DNA in each transfection totaled 3.1 μg per well. Transfected cells were pulse labeled for 30 minutes with ³⁵S-labeled amino acids and chased for 6 hours, at which time the cells were lysed, and both lysates and media were immunoprecipitated with anti-Tg and analyzed by reducing 5.5% SDS-PAGE and fluorography, as shown. The position of a 76-kDa molecular mass marker is shown at left. The figure has been spliced at the position indicated by a black line (between lanes 2 and 3), but all data were derived from a single exposure of the same gel. (B) Cells were either untransfected or transfected with 0.5 μg plasmid DNA encoding I-II-III plus 2.5 μg of vector DNA or that encoding secretory ChEL. As a positive control, 2 μg of plasmid DNA encoding wild-type Tg was transfected in parallel. Cells were pulse labeled for 30 minutes with ³⁵S-labeled amino acids and chased for either 0 or 16 hours, at which time the cells were lysed and both lysates and media immunoprecipitated with anti-Tg and analyzed by reducing 5.5% SDS-PAGE and phosphorimaging, as shown. The intracellular band density at the 0 chase time was defined as 100%; based on this, the recovery of each band at 16 hours is shown. Total recovery of labeled I-II-III alone at 16 hours was approximately 35%, while total recovery of labeled I-II-III (cells plus media) in the presence of secretory ChEL was approximately 68%.

Figure 10. Intracellular association of secretory ChEL with I-II-III.

293 cells were transfected with empty vector or with plasmid DNAs encoding I-II-III (0.5 μg) plus 1.5 μg of either vector alone (I-II-III) or secretory ChEL with an appended myc epitope tag (ChEL-myc). All cells were pulse labeled for 30 minutes with ³⁵S-labeled amino acids and chased for the times shown in the presence of BFA (5 μg/ml). The cells were lysed and immunoprecipitated either anti-Tg (lanes 1–7) or anti-myc (lanes 8–11). Immunoprecipitates were analyzed by nonreducing 5.5% SDS-PAGE and fluorography, with the I-II-III region shown. The I-II-III band seen at time 0 appears equivalent to that of the D isoform, while ChEL-myc favorably coprecipitates a faster-migrating band equivalent to that of the mature E isoform (arrow). The position of a 176-kDa molecular mass marker is shown at left.

Figure 11. The Tg ChEL domain functions as a molecular escort.

(A) 293 cells were untransfected or transiently cotransfected with 0.5 μg plasmid DNA encoding I-II-III plus 2.5 μg of the constructs indicated. The cells were pulse labeled for 30 minutes with ³⁵S-labeled amino acids and chased in complete medium for 4 hours. Cell lysates and media were immunoprecipitated with anti-Tg and analyzed by SDS-PAGE and fluorography. Note that anti-Tg recognizes both I-II-III and the ChEL protein; the ChEL-KDEL construct is selectively retained intracellularly, while secretory ChEL is released to the medium. Intracellular retention of ChEL-KDEL causes a parallel retention of I-II-III. (B) Cells cotransfected and pulse labeled as in A were chased for 5 hours before analysis as above. Note that the secretory ChEL domain bearing the cog or rdw mutation cannot support the secretion of I-II-III. The positions of molecular mass markers are shown at left.