The Cholinesterase-like Domain is Required for Folding and Secretion of Thyroglobulin.
ABSTRACT Thyroid hormone synthesis requires secretion of thyroglobulin, a precursor protein comprising Cys-rich regions I, II, and III (referred to collectively as regions I-II-III) followed by a cholinesterase-like (ChEL) domain. Secretion of mature thyroglobulin requires extensive folding and oligomerization in the ER. Multiple reports have linked mutations in the ChEL domain to thyroid diseases in humans and other animals; these mutations block thyroglobulin from exiting the ER and induce ER stress. The principal hypothesis in this thesis is that thyroglobulin requires the ChEL domain to form its native tertiary and quaternary structure. We report that, in a cell culture system, thyroglobulin with misfolding mutations in the ChEL domain also impairs folding in other domains. A truncated thyroglobulin (regions I-II-III) devoid of the ChEL domain is incompetent for folding and cellular export. However, co-expression of a secretory ChEL domain (which is efficiently secreted) with the truncated thyroglobulin rescued secretion of regions I-II-III by promoting their oxidative maturation and folding. The data indicate that the ChEL domain functions as an intramolecular chaperone. Moreover, a functional ChEL domain engineered to be retained intracellularly co-retains truncated thyroglobulin even as it facilitates the oxidative maturation of regions I-II-III. This indicates that the ChEL domain is also employed as a molecular escort for thyroglobulin. Thyroglobulin becomes a homodimer within the endoplasmic reticulum. The ChEL domain sequence contains predicted helical structures that are homologous to the dimerization helices of acetylcholinesterase. I have found that the ChEL domain can dimerize with itself, just as acetylcholinesterase does, and this is likely to drive the dimerization of wild-type thyroglobulin. Insertion of an N-linked glycan into one of the putative dimerization helices blocks detectable homodimerization of the isolated ChEL domain. Interestingly, co-expression of upstream regions of thyroglobulin, I-II-III, either in cis or in trans, overrides the dimerization defect of such a mutant. These data suggest that intermonomer interactions of the ChEL domain of thyroglobulin are enhanced by additional interactions with upstream regions of thyroglobulin. I conclude that the ChEL domain is required to form a native structure of Tg which is essential for export from the endoplasmic reticulum, and for thyroid hormone. Ph.D. Cellular & Molecular Biology University of Michigan, Horace H. Rackham School of Graduate Studies http://deepblue.lib.umich.edu/bitstream/2027.42/61755/1/jaeminl_1.pdf
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ABSTRACT: Secretion of thyroglobulin (Tg, a large homodimeric glycoprotein) is essential to deliver Tg to its site of iodination for thyroxine biosynthesis. An L2263P missense mutation in Tg has been proposed as the molecular defect causing congenital goitrous hypothyroidism in cog/cog mice due to perturbed Tg homodimerization, resulting in its retention within the endoplasmic reticulum. The mutation falls within a carboxyl-terminal region of Tg with high structural similarity to the entirety of acetylcholinesterase (AChE), a secretory protein that also forms homodimers. We provide new evidence that authentic AChE and the cholinesterase-like domain of Tg share a common tertiary structure. Moreover, we find that a Tg truncation, deleted of the cholinesterase-like region (but not a comparably sized deletion of internal Tg regions), blocks Tg export. Appending to this truncation a cDNA encoding authentic AChE results in translation of a chimeric protein in which AChE is present in a native, enzymatically active (albeit latent) conformation, and this fully rescues Tg secretion. Introduction of the cog mutation inhibits AChE enzyme activity, and established denaturing mutations of AChE block secretion of the Tg. Additional studies show that the native structure of the AChE region functions as a "dimerization domain," facilitating intracellular transport of Tg to the site of thyroid hormonogenesis.Journal of Biological Chemistry 05/2004; 279(17):17085-9. · 4.65 Impact Factor
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ABSTRACT: Acetylcholinesterase (AChE) exists as AChE(H) and AChE(T) subunits, which differ by their C-terminal H or T peptides, generating glycophosphatidylinositol-anchored dimers and various oligomers, respectively. We introduced mutations in the four-helix bundle interface of glycophosphatidylinositol-anchored dimers, and analyzed their effect on the production and oligomerization of AChE(H), of AChE(T), and of truncated subunits, AChE(C) (without H or T peptide). Dimerization was reduced for all types of subunits, showing that they interact through the same contact zone; the formation of amphiphilic tetramers (Torpedo AChE(T)) and 13.5 S oligomers (rat AChE(T)) was also suppressed. Oligomerization appeared totally blocked by introduction of an N-linked glycan on the surface of helix alpha(7,8). Other point mutations did not affect the synthesis or the catalytic properties of AChE but reduced or blocked the secretion of AChE(T) subunits. Secretion of AChE(T) was partially restored by co-expression with Q(N), a secretable protein containing a proline-rich attachment domain (PRAD); Q(N) organized PRAD-linked tetramers, except for the N-glycosylated mutants. Thus, the simultaneous presence of an abnormal four-helix bundle zone and an exposed T peptide targeted the enzyme toward degradation, indicating a cross-talk between the catalytic and tetramerization domains.Journal of Biological Chemistry 11/2001; 276(40):37379-89. · 4.65 Impact Factor
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ABSTRACT: The three-dimensional structure of acetylcholinesterase from Torpedo californica electric organ has been determined by x-ray analysis to 2.8 angstrom resolution. The form crystallized is the glycolipid-anchored homodimer that was purified subsequent to solubilization with a bacterial phosphatidylinositol-specific phospholipase C. The enzyme monomer is an alpha/beta protein that contains 537 amino acids. It consists of a 12-stranded mixed beta sheet surrounded by 14 alpha helices and bears a striking resemblance to several hydrolase structures including dienelactone hydrolase, serine carboxypeptidase-II, three neutral lipases, and haloalkane dehalogenase. The active site is unusual because it contains Glu, not Asp, in the Ser-His-acid catalytic triad and because the relation of the triad to the rest of the protein approximates a mirror image of that seen in the serine proteases. Furthermore, the active site lies near the bottom of a deep and narrow gorge that reaches halfway into the protein. Modeling of acetylcholine binding to the enzyme suggests that the quaternary ammonium ion is bound not to a negatively charged "anionic" site, but rather to some of the 14 aromatic residues that line the gorge.Science 09/1991; 253(5022):872-9. · 31.03 Impact Factor
THE CHOLINESTERASE-LIKE DOMAIN IS REQUIRED FOR FOLDING AND
SECRETION OF THYROGLOBULIN
A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
(Cellular and Molecular Biology)
in The University of Michigan
Professor Peter Arvan, Chair
Professor Ronald W. Holz
Associate Professor Kathleen L. Collins
Associate Professor Ursula H. Jakob
Associate Professor Billy Tsai
© Jaemin Lee
To my mother and father for their love and support
and to my beloved brother’s family
First, I would like to thank Dr. Peter Arvan, my thesis mentor for all his support
and guidance which allowed me to finish my thesis work. I have had difficulty in
progress of the research. I could not overcome it and finish my thesis without his
encouragement and patience. I am glad to learn a lot from him to be an independent
research scientist. I also enjoyed scientific discussions and other conversations with him.
Thank you, Peter.
I also want to express my appreciation to my committee members, Drs. Kathleen
Collins, Ronald Holz, Ursula Jakob, and Billy Tsai for their invaluable guidance and
suggestions during my thesis period. Thanks to their advices, I could bring my work here.
I thank our collaborators, Drs. Paul Kim and Bruno Di Jeso for sharing scientific
ideas and conducting research together. I also want to thank Dr. Zhaohui Xu for helpful
discussion about the structure of cholinesterase.
I would like to thank Dr. Amy Chang and people in the Chang lab for getting
together in the lab meeting and journal club. I enjoyed and learned a lot during the
My thanks also go to people in the Arvan lab, both past and present. I want to
share my gladness to finish my thesis with them: Xiang Zhao, Jose Ramos-Castaneda
(Pepe), Young-nam Park, Saeyoull Cho, Yukihiro Yamaguchi, Regina Kuliawat, Yi
Zhang, Xiaofan Wang, Gautam Rajpal, Ming Liu, Roberto Lara-Lemus, Dennis Larkin,
Israel Hodish, Leena Haataja, Aaron Kellogg, and Elizabeth Cole. I will never forget
those days with them. I specially want to say thank to Xiang as a friend and former
roommate. I also would like to say my sincere thank to Xiaofan for her help and support
during last years of my thesis.
Most of all, I must say thanks to my parents and brother’s family for their
understanding, care, support, and their love to me. Thank you very much, Jaephil, Mom,
and Dad. I love you!