Abstract
Thyroglobulin (precursor for thyroid hormone synthesis) is a large secreted glycoprotein comprising contiguous region I (multiple type-1 repeating units engaging the first ∼1,191 residues, followed by a ∼245-residue hinge region), regions II-III (multiple type-2 and 3 repeating units, comprising ∼720 residues), and the C-terminal cholinesterase-like (ChEL) domain (∼570 residues). A signal peptide attached to ChEL makes an independent secretory protein that binds to I-II-III, stabilizing it and rescuing the secretion of I-II-III that would otherwise be trapped in the endoplasmic reticulum (ER). In this study, we found that a signal peptide attached to regions II-III also makes for an efficient secretory protein that neither demonstrably interacts nor has its secretion enhanced by the presence of secretory ChEL. By contrast, region I, either with or without the hinge region, cannot be secreted on its own and remains in the ER where it is bound to ER chaperones BiP and GRP94. Whereas ChEL can rescue secretion of I-II-III, it can rescue I-II only very weakly, and region I not at all. Yet, ChEL begins to rescue region I in cells that also co-express secretory II-III. The data suggest that conformational maturation of region I is a limiting step in the thyroglobulin maturation process, and this step is facilitated by the presence of both regions II-III and ChEL. Mutations causing hypothyroidism might induce solely local/regional misfolding or may interfere more globally by impeding interactions between regions that are required for thyroglobulin secretion.
Highlights
The primary sequence of Tg (ϳ2,746 residues in mouse Tg after signal peptide cleavage) comprises disulfide-rich contiguous regions: region I; II-III; and the C-terminal cholinesterase-like (ChEL) domain (ϳ570 residues)
We engineered a construct in which region II-III (HA epitope-tagged) immediately follows a signal peptide sequence to direct the protein into the endoplasmic reticulum (ER)
We found that II-III-HA was efficiently secreted; and second, we found that introduction of the Q2139X mutation blocked this secretion (Fig. 2A), indicating that Q2139X introduces local misfolding into II-III
Summary
Materials—Lipofectamine 2000, Zysorbin, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were from Invitrogen. Zysorbin was from Zymed Laboratories Inc. Complete protease inhibitor mixture was from Roche Applied Bioscience. Brefeldin A and protein A-agarose were from Sigma. Protein G-agarose was from EMD Chemicals (Gibbstown, NJ). Peptide-Nglycosidase F was from New England Biolabs. Trans35S-Label was from MP Biomedicals (Irvine, CA). Rabbit polyclonal antimyc was from Immunology Consultants, Inc. Mouse monoclonal anti-HA epitope was from Covance (Princeton, NJ). Rabbit polyclonal anti-Tg (containing antibodies against epitopes at both N- and C-terminal regions of the protein) has been described previously [17]
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