The cholinesterase-like domain, essential in thyroglobulin trafficking for thyroid hormone synthesis, is required for protein dimerization

Abstract

The carboxyl-terminal cholinesterase-like (ChEL) domain of thyroglobulin (Tg) has been identified as critically important in Tg export from the endoplasmic reticulum. In a number of human kindreds suffering from congenital hypothyroidism, and in the cog congenital goiter mouse and rdw rat dwarf models, thyroid hormone synthesis is inhibited because of mutations in the ChEL domain that block protein export from the endoplasmic reticulum. We hypothesize that Tg forms homodimers through noncovalent interactions involving two predicted alpha-helices in each ChEL domain that are homologous to the dimerization helices of acetylcholinesterase. This has been explored through selective epitope tagging of dimerization partners and by inserting an extra, unpaired Cys residue to create an opportunity for intermolecular disulfide pairing. We show that the ChEL domain is necessary and sufficient for Tg dimerization; specifically, the isolated ChEL domain can dimerize with full-length Tg or with itself. Insertion of an N-linked glycan into the putative upstream dimerization helix inhibits homodimerization of the isolated ChEL domain. However, interestingly, co-expression of upstream Tg domains, either in cis or in trans, overrides the dimerization defect of such a mutant. Thus, although the ChEL domain provides a nidus for Tg dimerization, interactions of upstream Tg regions with the ChEL domain actively stabilizes the Tg dimer complex for intracellular transport.

FIGURE 1.

Association of epitope-tagged thyroglobulins. A, 293 cells untransfected (also called Control) or transfected with the constructs shown were pulse-labeled for 30 min with ³⁵S-labeled amino acids and chased for 5 h. Both cells (C) and media (M) were immunoprecipitated with anti-Tg, divided in two equal portions, and either mock-digested (–) or digested (+) with endo H. Secreted Tg and Tg-GFP have acquired endo H resistance, although some glycans on each Tg or Tg-GFP molecule remain sensitive to endo H. B, 293 cells untransfected or transfected with the constructs shown were pulse-labeled for 30 min with ³⁵S-labeled amino acids and chased for 4 h. Both cells and media were immunoprecipitated with anti-Tg. C, 293 cells were transfected with the constructs shown, and media were collected for 24 h. Each sample was divided in two portions and either immunoprecipitated with anti-Myc antibody (IP, left half of panel) or analyzed without immunoprecipitation (right half of panel). Success of co-precipitation was established by SDS-PAGE, electrotransfer, and immunoblotting with anti-GFP. D, cells and media, collected as in C, were divided in two portions and either immunoprecipitated with anti-GFP (IP, left half of gel) or analyzed without immunoprecipitation (right half of gel). Success of co-precipitation was established by SDS-PAGE, electrotransfer, and immunoblotting with anti-Myc. E, 293 cells were transfected with I-II-III-Myc, I-II-III-HA, both, or neither, and the cells were lysed at 48 h in a nondenaturing buffer containing 1% Nonidet P-40. Each sample was divided and either immunoprecipitated with anti-Myc (IP, right panels) or analyzed without immunoprecipitation (No IP, left panels). Lack of co-precipitation was established by SDS-PAGE, electrotransfer, and immunoblotting with anti-HA (upper panels) and anti-Myc (lower panels).

FIGURE 2.

Relationship of the ChEL domain to the full-length Tg protein. A, a summary of the constructs used in this study, including incorporation at the carboxyl terminus of various epitope tags. SP, signal peptide. B, the 581-residue domain extends from residues 2,166 (including a SalI restriction site creating the I_2,167R replacement shown in this figure) to the carboxyl-terminal residue 2,746. Based on secondary structural prediction, two α-helical segments (highlighted) are predicted to match what is referred to as the α-7/8 and α-10 helices of acetylcholinesterase (33, 35). Residues that are bolded and underlined indicate sites that have been mutagenized. Within the α-7/8 helical homology sequence, replacement of A-A-V by N-A-T introduces an N-linked glycosylation acceptor site that could potentially disrupt the four-helix bundle. The site of the D2708C,G2709stop mutation (introduced just downstream of the α-10 helical homology sequence) is also highlighted; introduction of the extra Cys residue at this site is used to test the possible formation of an intersubunit disulfide bond.

FIGURE 3.

Heterodimerization of the ChEL domain with Tg. 293 cells were either untransfected or transiently transfected to express wild-type Tg (wt Tg) or that plus secretory ChEL bearing a carboxyl-terminal Myc epitope tag (ChEL-Myc). Cells were pulse-labeled for 30 min with ³⁵S-labeled amino acids and chased for 5 h, and the media were immunoprecipitated with either anti-Tg (which recovers both proteins, left panel) or anti-Myc (right panel). The samples were analyzed by SDS-PAGE and fluorography (which, after anti-Myc immunoprecipitation, was overexposed ∼2-fold). The positions of prestained molecular mass standards are included (at left).

FIGURE 4.

The ChEL domain and authentic AChE share capabilities in protein dimerization. A, primary sequence alignment at the carboxyl-terminal end of Tg and acetylcholinesterase indicates a nonhomologous peptide extension that bears an extra, unpaired cysteine (upward arrow) immediately following the α-10 helix (denoted Helix) known to be engaged in the homodimerization of AChE. Mouse Tg engineered to terminate with an extra, unpaired cysteine at residue 2,708 (called Tg-CD) is shown with a downward arrow on the primary sequence. B, 293 cells transiently transfected to express human AChE (hAChE) or empty vector were labeled with ³⁵S-labeled amino acids for 2 h and chased further for 3 h in complete medium before immunoprecipitation of AChE from cell lysate (C) and medium (M). The samples were analyzed by nonreducing SDS 8%-PAGE and fluorography. Bands recovered from cells transfected with empty vector (lanes marked vector) represent nonspecific background. The position of monomeric AChE in the medium is indicated with an unlabeled arrow. C, 293 cells were either untransfected controls (C) or transiently transfected to express secretory ChEL (wt) or secretory ChEL-CD (“CD”). Cells were pulse-labeled for 30 min with ³⁵S-labeled amino acids and chased for 4 h, at which time the cell lysates and media immunoprecipitated with a rabbit polyclonal anti-Tg. Immunoprecipitates were analyzed by SDS-PAGE under reducing or nonreducing conditions, as indicated. The position of covalent ChEL-CD dimer is shown. The position of monomeric ChEL-CD in the medium (comprised of monomers and/or noncovalent dimers) is indicated with an unlabeled arrow. The positions of prestained molecular mass standards are included (at left).

FIGURE 5.

Covalent assembly of Tg-CD indicates tail-to-tail engagement of Tg homodimers. A, 293 cells were either mock-transfected or transiently transfected (as indicated at the bottom) with a vector encoding a Tg-CD construct containing D2708C,G2709stop. Left panel, cells were pulse-labeled for 30 min with ³⁵S-labeled amino acids and chased in the presence of BFA (5 μg/ml) for the times indicated. Cell lysates were immunoprecipitated with anti-Tg and analyzed by nonreducing SDS 4%-PAGE and fluorography. A smear of high molecular weight bands termed A, B, and C corresponds to those previously found to be Tg adducts with ER oxidoreductases (43), as well as a newly described Tg oxidative folding intermediate D band and the mature E band (14). Right panel, the same experiment but without BFA, in which both cell lysates and chase media were collected at 1 and 2 h of chase. Immunoprecipitated Tg from each cell lysate was divided into equal portions and either mock-digested or digested with endoglycosidase H before nonreducing SDS-PAGE and fluorography. Intracellular Tg-CD monomers are mostly endo H-sensitive. Intracellular covalent dimers are subdivided into a faster migrating endo H-sensitive population and a slower migrating population of endo H-resistant Tg-CD. In the media, the positions of covalent Tg-CD dimer and noncovalent dimer (indistinguishable from monomer by SDS-PAGE but proven to be dimer in the sucrose gradients of Fig. 6) are shown. B, as a control for the Tg-CD mutation, a D2708S,G2709stop mutant is efficiently secreted, but none of the secreted molecules form a covalent dimer.

FIGURE 6.

Dimerization of secreted Tg as measured by sucrose velocity gradient centrifugation. In each gradient, fractions (collected from the bottom) were immunoprecipitated with anti-Tg antibodies and analyzed by nonreducing SDS-PAGE. First gradient, endogenous Tg secreted by the metabolically labeled PC Cl3 thyrocyte cell line. Second gradient, media from metabolically labeled 293 cells transfected with empty vector. Third gradient, recombinant Tg secreted from metabolically labeled 293 cells that had been transfected to express wild-type mouse Tg (wt Tg). Fourth gradient, media from metabolically labeled 293 cells that had been transfected to express Tg-CD. With or without the intersubunit disulfide bond, endogenous and recombinant Tg and Tg-CD are recovered in the dimer (DIM) peak, with little or no secreted protein recovered in the monomer (MON) peak.

FIGURE 7.

A-A-V to N-A-T mutagenesis to create an N-linked glycosylation site within the α-7/8 helical sequence (Fig. 2B) perturbs subunit contact (35). A, 293 cells were either untransfected or transiently transfected to express either secretory ChEL-Myc or ChEL-Myc bearing the extra glycosylation site (ChELG-Myc), in conjunction with an equal amount of plasmid DNA encoding secretory ChEL-HA. Cells expressing secretory ChEL-HA alone were included as a negative control. Secretion was collected for 24 h. The media were either immunoprecipitated (IP) with anti-Myc before SDS-PAGE (upper two panels) or analyzed directly without immunoprecipitation (lower two panels). Samples underwent Western blotting (WB) with either anti-Myc (to demonstrate recovery of ChEL-Myc or ChELG-Myc) or anti-HA (to examine the extent of co-precipitation of the dimerization partner). Introduction of an N-glycan slowed the electrophoretic mobility of the ChELG-Myc band and decreased co-precipitation of ChEL-HA by 72% (in three such experiments, co-precipitation decreased 52 ± 18%). B, the G mutation was introduced into secretory ChELG-CD. The listed constructs were transiently expressed in 293 cells; secretory ChEL and secretory ChELG (lacking potential for intersubunit covalent bonding) were included as controls. Cells were metabolically labeled and chased for 4 h, and the media were immunoprecipitated with anti-Tg. The samples were analyzed by SDS 5.5%-PAGE under nonreducing (and reducing) conditions as indicated, with no covalent ChELG-CD dimer detected. C, covalent homodimer synthesized in cells expressing ChEL-CD but not in cells expressing cog-ChEL-CD or rdw-ChEL-CD.

FIGURE 8.

Effect of Tg regions I-II-III on the dimer stability of glycosylated ChELG domain. A, 293 cells were either transiently transfected with the secretory ChELG-CD construct alone or co-transfected in the presence of Tg I-II-III. Cells expressing the covalent dimerizing ChEL-CD construct were analyzed in parallel, as a control. Cells were pulse-labeled for 20 min with ³⁵S-labeled amino acids and chased for 4 h; media were collected and immunoprecipitated with anti-Tg followed by nonreducing SDS 5.5%-PAGE and fluorography. B, 293 cells were transiently transfected to express full-length Tg-CD or TgG-CD bearing the extra N-linked glycosylation site in the ChEL domain. Cells were pulse-labeled for 30 min with ³⁵S-labeled amino acids and chased for 6 h before analysis by Tg immunoprecipitation and nonreducing SDS 4%-PAGE and fluorography. Mobility of the TgG-CD band establishes that the extra glycosylation site is actually used (nevertheless, covalent intersubunit interaction proceeds).