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• W2015156954 abstract "Thyroglobulin (Tg, precursor for thyroid hormone synthesis) is a large secreted glycoprotein composed of upstream regions I-II-III, followed by the ∼570 residue cholinesterase-like (ChEL) domain. ChEL has two identified functions: 1) homodimerization, and 2) binding to I-II-III that facilitates I-II-III oxidative maturation required for intracellular protein transport. Like its homologs in the acetylcholinesterase (AChE) family, ChEL possesses two carboxyl-terminal α-helices. We find that a Tg-AChE chimera (swapping AChE in place of ChEL) allows for dimerization with monomeric AChE, proving exposure of the carboxyl-terminal helices within the larger context of Tg. Further, we establish that perturbing trans-helical interaction blocks homodimerization of the Tg ChEL domain. Additionally, ChEL can associate with neuroligins (a related family of cholinesterase-like proteins), demonstrating potential for Tg cross-dimerization between non-identical partners. Indeed, when mutant rdw-Tg (Tg-G2298R, defective for protein secretion) is co-expressed with wild-type Tg, the two proteins cross-dimerize and secretion of rdw-Tg is partially restored. Moreover, we find that AChE and soluble neuroligins also can bind to the upstream Tg regions I-II-III; however, they cannot rescue secretion, because they cannot facilitate oxidative maturation of I-II-III. These data suggest that specific properties of distinct Tg ChEL mutants may result in distinct patterns of Tg monomer folding, cross-dimerization with wild-type Tg, and variable secretion behavior in heterozygous patients. Thyroglobulin (Tg, precursor for thyroid hormone synthesis) is a large secreted glycoprotein composed of upstream regions I-II-III, followed by the ∼570 residue cholinesterase-like (ChEL) domain. ChEL has two identified functions: 1) homodimerization, and 2) binding to I-II-III that facilitates I-II-III oxidative maturation required for intracellular protein transport. Like its homologs in the acetylcholinesterase (AChE) family, ChEL possesses two carboxyl-terminal α-helices. We find that a Tg-AChE chimera (swapping AChE in place of ChEL) allows for dimerization with monomeric AChE, proving exposure of the carboxyl-terminal helices within the larger context of Tg. Further, we establish that perturbing trans-helical interaction blocks homodimerization of the Tg ChEL domain. Additionally, ChEL can associate with neuroligins (a related family of cholinesterase-like proteins), demonstrating potential for Tg cross-dimerization between non-identical partners. Indeed, when mutant rdw-Tg (Tg-G2298R, defective for protein secretion) is co-expressed with wild-type Tg, the two proteins cross-dimerize and secretion of rdw-Tg is partially restored. Moreover, we find that AChE and soluble neuroligins also can bind to the upstream Tg regions I-II-III; however, they cannot rescue secretion, because they cannot facilitate oxidative maturation of I-II-III. These data suggest that specific properties of distinct Tg ChEL mutants may result in distinct patterns of Tg monomer folding, cross-dimerization with wild-type Tg, and variable secretion behavior in heterozygous patients. Thyroid hormone synthesis requires secretion of thyroglobulin (Tg) 2The abbreviations used are: TgthyroglobulinChELcholinesterase-likeERendoplasmic reticulumDMEMDulbecco's modified Eagle's mediumGFPgreen fluorescent proteinNLneuroliginendo Hendoglycosidase HAChEacetylcholinesterase. to the apical luminal cavity of thyroid follicles in the thyroid gland (1Di Jeso B. Arvan P. Braverman L.E. Utiger R. The Thyroid. 9th Ed. Lippincott Williams & Wilkins, Philadelphia2004: 77-95Google Scholar). After secretion, Tg iodination (by thyroid peroxidase (2Taurog A. Biochimie. 1999; 81: 557-562Crossref PubMed Scopus (70) Google Scholar)) leads to a coupling reaction between di-iodotyrosyl residues 5 and 130 to form thyroxine within the amino-terminal portion of the Tg polypeptide (3Marriq C. Lejeune P.J. Venot N. Vinet L. Mol. Cell. Endocrinol. 1991; 81: 155-164Crossref PubMed Scopus (35) Google Scholar, 4Dunn A.D. Corsi C.M. Myers H.E. Dunn J.T. J. Biol. Chem. 1998; 273: 25223-25229Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Hormonogenic iodination depends not upon the specificity of the peroxidase (5Lamas L. Taurog A. Endocrinology. 1977; 100: 1129-1136Crossref PubMed Scopus (39) Google Scholar) but upon the sequence and structure of Tg (6Turakulov I. Saatov T. Babaev T.A. Rasuleva G. Makhmudov V. Biokhimiia. 1976; 41: 1004-1007PubMed Google Scholar, 7Xiao S. Dorris M.L. Rawitch A.B. Taurog A. Arch. Biochem. Biophys. 1996; 334: 284-294Crossref PubMed Scopus (22) Google Scholar). No other thyroidal proteins are known to effectively substitute for Tg in this role. The Tg primary sequence (∼2,750 amino acids after signal peptide cleavage) encodes three disulfide-rich regions comprising 80% of the overall Tg polypeptide, called I-II-III (8Veneziani B.M. Giallauria F. Gentile F. Biochimie. 1999; 81: 517-525Crossref PubMed Scopus (19) Google Scholar, 9van de Graaf S.A. Ris-Stalpers C. Pauws E. Mendive F.M. Targovnik H.M. de Vijlder J.J. J. Endocrinol. 2001; 170: 307-321Crossref PubMed Scopus (119) Google Scholar), followed by ∼570 residues of cholinesterase-like (ChEL) domain (10Lee J. Di Jeso B. Arvan P. J. Clin. Invest. 2008; 118: 2950-2958Crossref PubMed Scopus (55) Google Scholar) with homology to acetylcholinesterase (AChE) (see Fig. 1A) (11Schumacher M. Camp S. Maulet Y. Newton M. MacPhee-Quigley K. Taylor S.S. Friedmann T. Taylor P. Nature. 1986; 319: 407-409Crossref PubMed Scopus (311) Google Scholar, 12Swillens S. Ludgate M. Mercken L. Dumont J.E. Vassart G. Biochem. Biophys. Res. Commun. 1986; 137: 142-148Crossref PubMed Scopus (73) Google Scholar, 13Mori N. Itoh N. Salvaterra P.M. Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 2813-2817Crossref PubMed Scopus (25) Google Scholar). Although I-II-III contains the conserved amino-terminal hormonogenic site, I-II-III by itself is incompetent for hormonogenesis, because its export from the endoplasmic reticulum (ER), leading to secretion, requires ChEL (10Lee J. Di Jeso B. Arvan P. J. Clin. Invest. 2008; 118: 2950-2958Crossref PubMed Scopus (55) Google Scholar, 14Park Y.N. Arvan P. J. Biol. Chem. 2004; 279: 17085-17089Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). thyroglobulin cholinesterase-like endoplasmic reticulum Dulbecco's modified Eagle's medium green fluorescent protein neuroligin endoglycosidase H acetylcholinesterase. The ChEL domain is a commonly affected site of mutation in human congenital hypothyroidism with deficient Tg, including the recently described A2215D (15Caputo M. Rivolta C.M. Esperante S.A. Gruneiro-Papendieck L. Chiesa A. Pellizas C.G. Gonzalez-Sarmiento R. Targovnik H.M. Clin. Endocrinol. 2007; 67: 351-357Crossref PubMed Scopus (40) Google Scholar, 16Pardo V. Rubio I.G. Knobel M. Aguiar-Oliveira M.H. Santos M.M. Gomes S.A. Oliveira C.R. Targovnik H.M. Medeiros-Neto G. Thyroid. 2008; 18: 783-786Crossref PubMed Scopus (26) Google Scholar), R2223H (17Caron P. Moya C.M. Malet D. Gutnisky V.J. Chabardes B. Rivolta C.M. Targovnik H.M. J. Clin. Endocrinol. Metab. 2003; 88: 3546-3553Crossref PubMed Scopus (61) Google Scholar), G2300D, R2317Q (18Kitanaka S. Takeda A. Sato U. Miki Y. Hishinuma A. Ieiri T. Igarashi T. J. Hum. Genet. 2006; 51: 379-382Crossref PubMed Scopus (25) Google Scholar), G2355V, and G2356R and the skipping of exon 45 (which normally encodes 36 amino acids), as well as the Q2638stop mutant (19Rivolta C.M. Targovnik H.M. Clin. Chim. Acta. 2006; 374: 8-24Crossref PubMed Scopus (48) Google Scholar). Additionally, the homozygous Tg-G2300R mutation in the ChEL domain (equivalent to residue 2298 of mature mouse Tg) is responsible for congenital hypothyroidism in rdw/rdw rats (20Kim P.S. Ding M. Menon S. Jung C.G. Cheng J.M. Miyamoto T. Li B. Furudate S. Agui T. Mol. Endocrinol. 2000; 14: 1944-1953Crossref PubMed Scopus (63) Google Scholar, 21Hishinuma A. Furudate S. Oh-Ishi M. Nagakubo N. Namatame T. Ieiri T. Endocrinology. 2000; 141: 4050-4055Crossref PubMed Google Scholar), whereas the Tg-L2263P point mutation is responsible for congenital hypothyroidism in the cog/cog mouse (22Kim P.S. Hossain S.A. Park Y.N. Lee I. Yoo S.E. Arvan P. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 9909-9913Crossref PubMed Scopus (107) Google Scholar). Such mutations, when transplanted into AChE, inhibit enzyme activity (14Park Y.N. Arvan P. J. Biol. Chem. 2004; 279: 17085-17089Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), strongly suggesting that they structurally perturb local folding within this domain. The present state of knowledge highlights the role of the ChEL domain in two critical activities for Tg: 1) homodimerization of Tg within the ER (23Kim P.S. Arvan P. J. Biol. Chem. 1991; 266: 12412-12418Abstract Full Text PDF PubMed Google Scholar, 24Di Jeso B. Pereira R. Consiglio E. Formisano S. Satrustegui J. Sandoval I.V. Eur. J. Biochem. 1998; 252: 583-590Crossref PubMed Scopus (29) Google Scholar) requires ChEL (25Lee J. Wang X. Di Jeso B. Arvan P. J. Biol. Chem. 2009; 284: 12752-12761Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) and 2) ChEL is linked to a rate-limiting process of Tg-oxidative folding within the ER (26Di Jeso B. Ulianich L. Pacifico F. Leonardi A. Vito P. Consiglio E. Formisano S. Arvan P. Biochem. J. 2003; 370: 449-458Crossref PubMed Google Scholar, 27Arvan P. Kim P.S. Kuliawat R. Prabakaran D. Muresan Z. Yoo S.E. Abu Hossain S. Thyroid. 1997; 7: 89-105Crossref PubMed Scopus (38) Google Scholar). Specifically, although upstream regions I-II-III possess 116 of the 122 Tg cysteine residues (10Lee J. Di Jeso B. Arvan P. J. Clin. Invest. 2008; 118: 2950-2958Crossref PubMed Scopus (55) Google Scholar), ChEL binds to I-II-III, functioning as a molecular chaperone to facilitate I-II-III oxidative maturation (10Lee J. Di Jeso B. Arvan P. J. Clin. Invest. 2008; 118: 2950-2958Crossref PubMed Scopus (55) Google Scholar). Indeed, ChEL is not only contiguous with I-II-III in Tg, but the ChEL domain remains associated with I-II-III throughout the secretory pathway and after secretion (10Lee J. Di Jeso B. Arvan P. J. Clin. Invest. 2008; 118: 2950-2958Crossref PubMed Scopus (55) Google Scholar). As best as is currently known, all Tg mutants causing congenital hypothyroidism are defective for intracellular transport (28Vono-Toniolo J. Rivolta C.M. Targovnik H.M. Medeiros-Neto G. Kopp P. Thyroid. 2005; 15: 1021-1033Crossref PubMed Scopus (39) Google Scholar). However, until now, no studies have explored the severity of impairment of mutant Tg protein co-expressed in the presence of wild-type Tg, which is the situation found in simple heterozygous patients who are vastly more common than newborns with congenital hypothyroidism from homozygous or compound heterozygous Tg mutations (29Targovnik H.M. Esperante S.A. Rivolta C.M. Mol. Cell. Endocrinol. 2010; (in press)PubMed Google Scholar). The potential for interaction of non-identical Tg partners in trans is a complex problem; for ChEL mutants this requires investigation of both dimerization and chaperone/escort functions. Notably, ChEL is part of a large family of cholinesterase-like proteins such as the neuroligins, which subserve cell adhesion and neuronal interaction functions (30Gilbert M.M. Auld V.J. Front. Biosci. 2005; 10: 2177-2192Crossref PubMed Scopus (40) Google Scholar, 31De Jaco A. Comoletti D. King C.C. Taylor P. Chem. Biol. Interact. 2008; 175: 349-351Crossref PubMed Scopus (13) Google Scholar). In this report we have studied non-identical cholinesterase-like domains, either from structural relatives or from ChEL mutants, to examine interaction in trans with wild-type Tg domains and to examine the extent to which they can substitute for ChEL function in Tg secretion. Such studies shed new light on the folding, dimerization, and export of mutant Tg protein in the presence of co-expressed wild-type Tg. Lipofectamine 2000, Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, penicillin, and streptomycin were from Invitrogen (Carlsbad, CA); Zysorbin was from Zymed Laboratories Inc.; complete protease inhibitor mixture was from Roche Applied Science (Indianapolis, IN); brefeldin A, N-ethylmaleimide, and protein A-agarose were from Sigma; protein G-agarose was from EMD Chemicals (Gibbstown, NJ); dithiobis(succinimidyl propionate) was from ThermoFisher Scientific, Inc. (Waltham, MA); peptide-N-glycosidase F from New England Biolabs (Beverly, MA); and Trans35S-Label was from MP Biomedicals (Irvine, CA). Chicken polyclonal anti-Myc and rabbit polyclonal anti-FLAG were from Immunology Consultants, Inc. (Newberg, OR); rabbit anti-chicken IgY was from Jackson ImmunoResearch (West Grove, PA). Rabbit polyclonal anti-Tg (containing antibodies against epitopes at both N- and C-terminal regions of the protein) has been previously described (32Kim P.S. Kwon O.Y. Arvan P. J. Cell Biol. 1996; 133: 517-527Crossref PubMed Scopus (99) Google Scholar). A cDNA encoding the “T-form” of hAChE (matching the exon 4 to exon 6 splice form (33Massoulié J. Bon S. Perrier N. Falasca C. Chem. Biol. Interact. 2005; 157–158: 3-14Crossref PubMed Scopus (55) Google Scholar)) was employed as template for further mutagenesis. AChE-Myc was constructed using the QuikChange site-directed mutagenesis kit (Stratagene) with the following primer and its complement (5′-CGGTGCTCAGATCTGGAACAGAAGCTCATCAGTGAAGAGGACCTCTAGGCGGCCGCTTCC-3′). The Tg-AChEΔCys mutation was introduced with the same kit using Tg-AChE as template (14Park Y.N. Arvan P. J. Biol. Chem. 2004; 279: 17085-17089Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and the following primer and its complement (5′-CCAGTTCGACCACTACAGCAAGCAGGATCGCAGCTCAGATCTGTGATAGTCGACTCTAG-3′). Tg-GFP, Tg-Myc, Tg-AChE, truncated Tg regions I-II-III and I-II-III-Myc, and secretory ChEL, ChEL-Myc, ChEL-HA, ChEL-CD, and ChELG-CD constructs have been previously described (10Lee J. Di Jeso B. Arvan P. J. Clin. Invest. 2008; 118: 2950-2958Crossref PubMed Scopus (55) Google Scholar, 14Park Y.N. Arvan P. J. Biol. Chem. 2004; 279: 17085-17089Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 25Lee J. Wang X. Di Jeso B. Arvan P. J. Biol. Chem. 2009; 284: 12752-12761Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Each final product was confirmed by direct DNA sequencing. The rdw mutation encoding Tg-G2298R (34Menon S. Lee J. Abplanalp W.A. Yoo S.E. Agui T. Furudate S. Kim P.S. Arvan P. J. Biol. Chem. 2007; 282: 6183-6191Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) was introduced to create rdw-Tg-GFP. The cDNAs encoding epitope-tagged neuroligin 1, 2, 3, and 4 truncation constructs (devoid of splice inserts) have been described previously (35Comoletti D. Flynn R.E. Boucard A.A. Demeler B. Schirf V. Shi J. Jennings L.L. Newlin H.R. Südhof T.C. Taylor P. Biochemistry. 2006; 45: 12816-12827Crossref PubMed Scopus (108) Google Scholar); each were truncated before the O-glycosylation attachment site and transmembrane domain rendering them secretable proteins, and each construct is composed of a cleavable pre-trypsin signal peptide followed by the FLAG octapeptide (DYKDDDDK), a linker peptide of 4–8 residues, followed by the neuroligin sequence beginning with the first amino acid of the mature protein. The precise truncation points were made by introducing stop codons at NL1–639, NL2–616, NL3–640, and NL4–620 generating the proteins NL1–638, NL2–615, NL3–639, and NL4–619, respectively. Transiently transfected 293 cells expressing ChEL-CD and ChELG-CD (25Lee J. Wang X. Di Jeso B. Arvan P. J. Biol. Chem. 2009; 284: 12752-12761Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) were pulse-labeled with 35S-amino acids for 30 min and chased in complete media for 5 h at 37 °C. Chase media were collected and treated with a protease inhibitor mixture, and 0.5 ml of the treated media was overlaid atop 11.5-ml 5 to 10% linear gradients of sucrose in 0.3 m NaCl, 0.05 m Tris, pH 7.5. The gradients were spun at 187,000 × g (33,000 rpm) in an SW41 rotor for 18 h at 15 °C. At the conclusion of the velocity gradient centrifugation, 400-μl fractions were collected sequentially by puncture from the bottom of each tube, and each was diluted to 1 ml in 0.3 m NaCl, 0.05 m Tris, pH 7.5. Each fraction was immunoprecipitated with anti-Tg and analyzed by non-reducing SDS-PAGE and fluorography. HEK293 cells (simply called 293 cells) were cultured in DMEM with 10% fetal bovine serum in 6-well plates at 37 °C in a humidified 5% CO2 incubator. Plasmids were transiently transfected using Lipofectamine 2000 transfection reagent, following the manufacturer's instructions. Transfected 293 cells were starved for 30 min in Met/Cys-free DMEM, then pulse-labeled with 180 μCi/ml 35S-amino acids. The cells were then washed with an excess of cold Met/Cys and chased in complete DMEM plus serum. At each time point, cells were lysed in buffer containing 1% Nonidet P-40, 20 mmN-ethylmaleimide, 0.1% SDS, 0.1 m NaCl, 2 mm EDTA, 25 mm Tris, pH 7.4, and a protease inhibitor mixture. For immunoprecipitation, anti-Tg antibody was incubated with cell or media samples overnight at 4 °C, and the immunoprecipitate was recovered with protein A-agarose. For co-immunoprecipitation studies, lysates prepared in the same lysis buffer lacking SDS were incubated overnight at 4 °C with immunoprecipitating antibodies and protein A- or protein G-agarose. Immunoprecipitates (or co-precipitates) were washed three times before boiling in SDS sample buffer with or without reducing agent, resolved by SDS-PAGE, and analyzed by fluorography or phosphorimaging. For measurement of the catalytic activity of recombinant Tg-AChE and Tg-AChEΔCys, 293 cells expressing each construct to be tested were lysed under native conditions (300 μl of 100 mm NaCl, 1% Nonidet P-40, 2 mm EDTA, 25 mm Tris, pH 7.5). One-sixtieth of each cell lysates was then used directly for colorimetric acetylthiocholinesterase activity as described before (36Ellman G.L. Courtney D. Andres Jr., V. Feather-Stone R.M. Biochem. Pharmacol. 1961; 7: 88-95Crossref PubMed Scopus (21516) Google Scholar). Activity measurements for each construct were performed at least three times with mean ± S.D. values calculated. Parallel samples of cell lysate from the identical transfections were used for immunoblotting with anti-Tg to examine recombinant protein expression level normalized to cell protein. The specific activity was calculated as enzymatic activity per unit of immunoreactive protein. Cells were incubated in phosphate-buffered saline containing 2 mm dithiobis(succinimidyl propionate) for 30 min at room temperature. Cells were then washed in phosphate-buffered saline containing 20 mmN-ethylmaleimide and then lysed in buffer containing both 20 mmN-ethylmaleimide and 50 mm glycine to quench residual cross-linker. The lysates were then analyzed by immunoprecipitation and SDS-PAGE under reducing conditions. The Tg ChEL domain has 6 Cys residues, which engage in 3 disulfide bonds shown schematically in full-length Tg at the top of Fig. 1A. Related constructs employed or engineered for this study include secretory ChEL (including modifications such as carboxyl-terminal Myc tag, extra unpaired Cys residue, or N-linked glycosylation site), AChE (bearing a carboxyl-terminal Myc tag), FLAG-tagged neuroligins 1, 2, 3, or 4, and a Tg-AChE chimera (bearing or lacking a carboxyl-terminal Cys residue) (Fig. 1A). Amino acid sequence alignment of the first ∼500 residues of the ChEL domains of mouse, rat, and human Tg with rat, mouse, and human AChE and human neuroligins 1–4 reveals areas of identity (Fig. 1B, green) as well as 80 and 60% conservation, respectively (red and yellow). Comparison of the three-dimensional structure of AChE from crystal structure coordinates, that of a representative neuroligin and that predicted for ChEL by ESyPred3D (Fig. 1C) (37Lambert C. Léonard N. De Bolle X. Depiereux E. Bioinformatics. 2002; 18: 1250-1256Crossref PubMed Scopus (525) Google Scholar), supports a shared overall protein architecture between these AChE paralogs. We recently hypothesized that Tg homodimerization may be initiated by noncovalent interactions involving two predicted α-helices at the carboxyl terminus of each ChEL domain, with the potential for Tg to form a 4-helix bundle similar to that reported in the dimerization of AChE (25Lee J. Wang X. Di Jeso B. Arvan P. J. Biol. Chem. 2009; 284: 12752-12761Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). However, upstream regions of Tg, comprising >2000 amino acids, might potentially interfere with accessibility of the α-helices in the ChEL domain. We therefore examined a full-length Tg chimera in which catalytically active AChE replaced ChEL in Tg (14Park Y.N. Arvan P. J. Biol. Chem. 2004; 279: 17085-17089Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), to determine whether, in the context of Tg, these carboxyl-terminal α-helices were available for dimerization with co-transfected monomeric AChE. Formation of an AChE intermolecular disulfide bond engaging an unpaired carboxyl-terminal cysteine, neighboring the α-helices, is a natural reporter of such dimerization (38Kerem A. Kronman C. Bar-Nun S. Shafferman A. Velan B. J. Biol. Chem. 1993; 268: 180-184Abstract Full Text PDF PubMed Google Scholar). Notably, Tg-AChE expressed alone already formed covalent homodimers intracellularly (Fig. 2A). When co-expressed with AChE, more Tg-AChE monomers were consumed (i.e. fewer Tg-AChE monomers were recovered), involving cross-dimerization (i.e. Tg-AChE–AChE) that appeared to compete with Tg-AChE homodimerization (Fig. 2A). The ability to form covalent homodimers or heterodimers was eliminated when expressing the Tg-AChEΔCys chimera that deleted the unpaired cysteine at the carboxyl terminus (Fig. 2B), proving that the carboxyl-terminal segment must be accessible for the apposition of monomer partners even when conjoined to the large upstream Tg regions I-II-III. We introduced an extra, unpaired Cys residue at the tail of the ChEL domain to create a similar opportunity for covalent dimerization of ChEL monomers (called “ChEL-CD”), while also engineering an N-linked glycosylation site into the first of the two dimerization helices (called “ChELG-CD”) that blocks formation of the intermolecular covalent bond (Fig. 1A) (25Lee J. Wang X. Di Jeso B. Arvan P. J. Biol. Chem. 2009; 284: 12752-12761Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). However, such an assay cannot exclude non-covalent dimerization of ChELG-CD. To obtain stronger evidence of the α-helical contribution to homodimerization of the Tg ChEL domain, we examined the migration of ChEL-CD and ChELG-CD by centrifugation on sucrose velocity gradients. Secretion of ChEL-CD was recovered as a combination of dimers and monomers, and upon analysis of gradient fractions by non-reducing SDS-PAGE, there was perfect correspondence between the presence of an intermolecular disulfide bond and recovery in a distinct peak of faster sedimentation (Fig. 3, upper gradient). By contrast, ChELG-CD was not only blocked in formation of the covalent inter-subunit linkage but was also blocked in faster sedimentation in the sucrose-velocity gradient (Fig. 3, lower). These data directly implicate the carboxyl-terminal α-helices in ChEL dimerization, strongly supporting a 4-helix bundle dimerization mechanism similar to that used by AChE and other cholinesterase-like family members (39Morel N. Leroy J. Ayon A. Massoulié J. Bon S. J. Biol. Chem. 2001; 276: 37379-37389Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 40Comoletti D. Grishaev A. Whitten A.E. Taylor P. Trewhella J. Chem. Biol. Interact. 2008; 175: 150-155Crossref PubMed Scopus (8) Google Scholar). Such a dimerization mechanism might render it possible to obtain cross-dimerization of related cholinesterase-like proteins (Fig. 1, B and C). We therefore extended our analysis to include secretory versions of human neuroligins (NLs) 1, 2, 3, and 4. FLAG-tagged NL1, NL2, NL3, and NL4 were each secreted slowly but efficiently, with release from cells primarily between 5 and 24 h after synthesis (Fig. 4A, lower panel). Within cells that co-expressed secretory ChEL-Myc and FLAG-tagged neuroligins, after chemical cross-linking, ChEL could be observed to interact with each of the NL proteins as detected upon co-immunoprecipitation with anti-Myc and immunoblotting with anti-FLAG (Fig. 4B, left panel). A similar interaction was observed when secretory ChEL was co-expressed with AChE-Myc (co-precipitated with anti-Tg and immunoblotted with anti-Myc (Fig. 4B, right panel)). Even without cross-linker, from media, secreted FLAG-tagged neuroligins could be co-precipitated with ChEL-Myc (Fig. 4C, left) or with ChEL-HA (Fig. 4C, right). The data underscore the ability of non-identical cholinesterase-like partners (Fig. 1, B and C) to cross-dimerize. Cross-dimerization of non-identical partners could have important physiological consequences, as demonstrated when NL3 was co-expressed with secretory ChEL-HA, resulting in increased release of NL3 to the media (while the intracellular amount decreased), indicating enhanced secretion (Fig. 4D). Congenital hypothyroidism with defective Tg is an autosomal recessive trait; thus heterozygous individuals in the population go largely undetected. Because mutant Tg ChEL is more similar to wild-type Tg ChEL than is AChE or neuroligins, it seemed plausible that cross-dimerization between mutant and wild-type Tg partners also might take place. To test the potential for mutant rdw-Tg to be rescued by wild-type Tg, the two proteins were co-expressed in 293 cells and incubated for 24 h to examine secretion. To distinguish these proteins unequivocally, rdw-Tg was tagged with GFP at the carboxyl terminus. When expressed along with an empty vector, rdw-Tg-GFP was recovered in cells but was undetectable in the medium. However, when co-expressed with wild-type Tg-Myc, rdw-Tg-GFP began to be recovered in the medium (Fig. 5, left panels). To confirm that this reflected authentic secretion, rdw-Tg-GFP was subjected to endoglycosidase H (endo H) digestion to assess modification of any of the N-linked glycans of Tg by Golgi glycosylation enzymes. Intracellular rdw-Tg-GFP was endo H-sensitive, indicating failure to reach the Golgi complex, whereas all of the rdw-Tg-GFP molecules recovered from the medium had some of their many N-linked glycans acquire endo H resistance (Fig. 5, middle panel), indicating normal intracellular trafficking through the secretory pathway. Moreover, immunoprecipitation of wt-Tg-Myc from the medium co-precipitated rdw-Tg-GFP (Fig. 5, right panel). The data clearly indicate potential for Tg cross-dimerization, as might occur in states of heterozygosity. The Tg coding sequence links each I-II-III directly to its own contiguous ChEL polypeptide. However, because Tg homodimerizes, there is more than one potential stoichiometry and orientation of ChEL interaction with I-II-III. We proceeded to test the stoichiometry when the two are expressed as separate secretory proteins. After co-transfection of I-II-III-Myc and secretory ChEL, 293 cells were radiolabeled continuously at fixed specific radioactivity with [35S]cysteine in complete medium for 2 days to approach steady state. The medium was then collected and immunoprecipitated with anti-Myc to recover I-II-III-Myc and co-precipitated ChEL. The relative band intensities were quantified from scanned SDS-PAGE fluorograms (Fig. 6). An equimolar ChEL:region I-II-III binding stoichiometry binding should be seen as an intensity ratio of 6:116, reflecting precisely the number of Cys residues in each polypeptide. Indeed, from quadruplicate samples, the molar ratio measured was 1.17 ± 0.28, i.e. an equimolar binding stoichiometry (Fig. 6). ChEL and structurally similar cholinesterase-like family members exhibit potential for cross-dimerization, raising the question of whether such family members could also substitute for ChEL in I-II-III binding. Indeed, AChE-Myc was also found able to associate with I-II-III (Fig. 7A), and this was similarly true for FLAG-NL1, -NL2, -NL3, or -NL4 (Fig. 7B). However, the efficiency of I-II-III interaction, as measured by co-precipitation, was weaker, i.e. only approximately half of that observed for ChEL itself (Fig. 7C). The ability of structurally related cholinesterase-like family members (Fig. 1, B and C) to physically interact with regions I-II-III raised the question of whether such interactions could rescue the secretion of region I-II-III lacking their own ChEL domain (10Lee J. Di Jeso B. Arvan P. J. Clin. Invest. 2008; 118: 2950-2958Crossref PubMed Scopus (55) Google Scholar). In side-by-side comparisons, AChE could not promote significantly more I-II-III secretion than was observed with empty vector alone, and indeed, intracellular recovery of I-II-III appeared diminished (Fig. 8, A and B). Likewise, the secretory neuroligins, despite excellent expression, could not promote I-II-III secretion (Fig. 8C). The data suggest that sequence-specific differences among cholinesterase-like family members alter their ability to rescue I-II-III secretion. Why is association of AChE or neuroligins unproductive for Tg? Like full-length Tg, regions I-II-III go through a series of oxidative folding intermediates, including transient mixed disulfides with ER oxidoreductases (termed “A,” “B,” and “C” (41Di Jeso B. Park Y.N. Ulianich L. Treglia A.S. Urbanas M.L. High S. Arvan P. Mol. Cell. Biol. 2005; 25: 9793-9805Crossref PubMed Scopus (62) Google Scholar)) followed by further oxidative folding of immature “D monomers” to the mature “E isofo" @default.
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• W2015156954 title "Cis and Trans Actions of the Cholinesterase-like Domain within the Thyroglobulin Dimer" @default.
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