Holzer G et al. (2016), Thyroglobulin Represents a Novel Molecular Arch...

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J Biol Chem 2016 Aug 05;29132:16553-66. doi: 10.1074/jbc.M116.719047.

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Thyroglobulin Represents a Novel Molecular Architecture of Vertebrates.

Holzer G , Morishita Y , Fini JB , Lorin T , Gillet B , Hughes S , Tohmé M , Deléage G , Demeneix B , Arvan P , Laudet V .

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Thyroid hormones modulate not only multiple functions in vertebrates (energy metabolism, central nervous system function, seasonal changes in physiology, and behavior) but also in some non-vertebrates where they control critical post-embryonic developmental transitions such as metamorphosis. Despite their obvious biological importance, the thyroid hormone precursor protein, thyroglobulin (Tg), has been experimentally investigated only in mammals. This may bias our view of how thyroid hormones are produced in other organisms. In this study we searched genomic databases and found Tg orthologs in all vertebrates including the sea lamprey (Petromyzon marinus). We cloned a full-size Tg coding sequence from western clawed frog (Xenopus tropicalis) and zebrafish (Danio rerio). Comparisons between the representative mammal, amphibian, teleost fish, and basal vertebrate indicate that all of the different domains of Tg, as well as Tg regional structure, are conserved throughout the vertebrates. Indeed, in Xenopus, zebrafish, and lamprey Tgs, key residues, including the hormonogenic tyrosines and the disulfide bond-forming cysteines critical for Tg function, are well conserved despite overall divergence of amino acid sequences. We uncovered upstream sequences that include start codons of zebrafish and Xenopus Tgs and experimentally proved that these are full-length secreted proteins, which are specifically recognized by antibodies against rat Tg. By contrast, we have not been able to find any orthologs of Tg among non-vertebrate species. Thus, Tg appears to be a novel protein elaborated as a single event at the base of vertebrates and virtually unchanged thereafter.

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Species referenced: Xenopus tropicalis
Genes referenced: myc tgm2

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	FIGURE 1.. Schematic organization of human, Xenopus, zebrafish, and lamprey Tgs. A, Tg1 repeats are shown by blue boxes, Tg2 repeats by red boxes, Tg3 repeats by green boxes, and ChEL domain by gray boxes. The linker and the hinge are represented by a light green and dark green line, respectively. The main hormonogenic tyrosines are represented with their positions in each sequence. B, alignment of human, Xenopus, zebrafish, and lamprey Tg. Tg1 repeats are highlighted in light blue, Tg2 repeats are in red, Tg3 repeats are in green, and ChEL domains are in gray. The linker region is indicated by a light green line, and the hinge region is indicated by a dark green line. The donor tyrosines are highlighted in blue, and the acceptor tyrosines are in red. Conserved cysteines are highlighted in yellow with presumptive disulfide bonds of Tg1 repeats shown as black brackets at the top of each line. The 5′-end of Xenopus retrieved after mass sequencing is underlined in orange. The RNA probes for in situ hybridization assays are underlined in purple.
	FIGURE 2.. Expression of Tg in zebrafish and Xenopus revealed by ISH. A, for zebrafish embryo experiments, two probes were used, in the 5′ part of the Tg (upper panel) and in the middle (lower panel). ISH were performed at 24, 48, 120, and 144 hpf as indicated. In situ labeling appears in purple. B, for Xenopus tadpole sections, probes were used at stage NF57 and NF59 according to the Nieuwkoop and Faber's classification (73). In situ labeling appears in blue. Negative controls with sense probe show no staining (supplemental Fig. S1).
	FIGURE 3.. Expression and secretion of zebrafish and Xenopus Tg. A, recombinant Tgs of the indicated species were expressed in 293T cells (C) and secreted into the medium (M). Mouse Tg was used as a positive control (lanes 3 and 4). After a 1-day collection of media, both cell lysates and media were analyzed by SDS-PAGE and Western blotting with a polyclonal anti-rodent Tg antibody. Note that secreted Tg has a slower (higher) mobility because of carbohydrate processing that occurs as Tg migrates through the Golgi complex. Incomplete Xenopus Tg lacks a signal peptide, and the protein is poorly expressed (lanes 7 and 8). Adding a mouse signal peptide (SP) rescues its expression and secretion of the incomplete Xenopus Tg (lanes 9 and 10). Full-length Xenopus Tg is expressed and secreted (lanes 11 and 12). Initiating Tg translation with the second methionine of the Xenopus signal peptide is sufficient for expression and secretion (lanes 13 and 14). B, glycosylation analysis of mouse Tg-myc and the incomplete Xenopus Tg-myc by endoglycosidase H digestion proves that intracellularly, mouse Tg begins within the endoplasmic reticulum (endoglycosidase H-sensitive, lanes 1 and 2) and traverses the Golgi complex where the protein becomes endoglycosidase H-resistant lanes 3 and 4). In contrast, the incomplete Xenopus Tg is unglycosylated (lanes 5 and 6), indicating failure to enter the endoplasmic reticulum. Both cell lysates and media were analyzed by SDS-PAGE and Western blotting with a polyclonal anti-Myc antibody; 10 times more protein from the cells expressing the incomplete Xenopus Tg was loaded to detect these bands.
	FIGURE 4.. Role of the Tg1–8 repeat in the secretion of Tg. Cysteines forming the second disulfide bond of Tg repeat 1–8 were mutated individually or in a pair within mouse Tg. Both cell lysates (C) and media (M) were analyzed by SDS-PAGE and Western blotting with a polyclonal anti rodent Tg antibody. Neither the single mutations (lanes 5 and 6 and lanes 7 and 8) nor the double mutation (lanes 9 and 10) block expression or the secretion of the Tg protein.
	FIGURE 5.. Tg phylogeny. Phylogenetic tree built by maximum likelihood using experimentally determined Tg amino acid sequences as well as predicted sequences. Statistical approximate likelihood ratio test (aLRT) branch supports are indicated when >0.7. Agnathes are highlighted in purple, teleosts are in light blue, percomorphs are in dark blue, amphibians are in green, sauropsidas are in orange, and mammals are in red. The bold line supports the percomorphs.
	FIGURE 6.. Prototypic vertebrate thyroglobulin. Vertebrate common features are shown: Tg1 repeats are shown in blue boxes, Tg2 repeats are in red boxes, Tg3 repeat is green boxes, and ChEL domain is by gray boxes. The linker and the hinge are represented by a light green and dark green line, respectively. The main hormonogenic tyrosines found in the investigated vertebrate species are represented with their positions in human numbering.
	Holzer, Morishita et al. Figure S1
	Holzer, Morishita et al. Figure S2
	Holzer, Morishita et al. Figure S3A
	Holzer, Morishita et al. Figure S5
	Holzer, Morishita et al. Figure S6

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