The revised 8307 base pair coding sequence of human thyroglobulin transiently expressed in eukaryotic cells

Academic Medical Centre, University of Amsterdam, The Netherlands.

We developed a transient transfection system for human thyroglobulin (TG) cDNA in both human thyroid cells and in COS-1 cells. Four overlapping TG cDNA fragments were amplified by reverse transcription-PCR from RNA of normal thyroid tissue. The most 5' fragment includes the natural translation initiation site and the sequence encoding the signal peptide (SP). After subcloning, the nucleotide sequence was determined and compared with the published human sequence, resulting in the detection of 30 nucleotide variations. For validation purposes, all variations were screened in 6-12 normal human alleles. Twenty-one were present in all screened alleles and have to be revised in the published nucleotide sequence. Since one variation concerns a triplet insertion, the coding sequence of the mature human thyroglobulin is 8307 nucleotides encoding 2750 amino acids. The TG cDNA constructs were transiently transfected in HTori 3 and COS-1 cells and protein expression was detected using a polyclonal anti-human-TG on fixed cells and after SDS-PAGE. In both cell-lines all four TG protein fragments were expressed. The mannose structures detected on the proteins by lectins and localization after expression in the cells suggest that only the N-terminal TG fragment (containing the SP) is directed to the endoplasmatic reticulum but is unable to reach the Golgi complex. The described expression system in human thyrocytes will be a helpful tool in studying the structure-function relationship of human TG in thyroid hormonogenesis.

This article has been cited by other articles:

				M. Caputo, C. M Rivolta, V. J Gutnisky, L. Gruneiro-Papendieck, A. Chiesa, G. Medeiros-Neto, R. Gonzalez-Sarmiento, and H. M Targovnik Recurrence of the p.R277X/p.R1511X compound heterozygous mutation in the thyroglobulin gene in unrelated families with congenital goiter and hypothyroidism: haplotype analysis using intragenic thyroglobulin polymorphisms J. Endocrinol., October 1, 2007; 195(1): 167 - 177. [Abstract] [Full Text] [PDF]

				R. P. M. Biscolla, J. M. Cerutti, and R. M. B. Maciel Detection of Recurrent Thyroid Cancer by Sensitive Nested Reverse Transcription-Polymerase Chain Reaction of Thyroglobulin and Sodium/Iodide Symporter Messenger Ribonucleic Acid Transcripts in Peripheral Blood J. Clin. Endocrinol. Metab., October 1, 2000; 85(10): 3623 - 3627. [Abstract] [Full Text]

				S. A. R. van de Graaf, C. Ris-Stalpers, G. J. M. Veenboer, M. Cammenga, C. Santos, H. M. Targovnik, J. J. M. de Vijlder, and G. Medeiros-Neto A Premature Stopcodon in Thyroglobulin Messenger RNA Results in Familial Goiter and Moderate Hypothyroidism J. Clin. Endocrinol. Metab., July 1, 1999; 84(7): 2537 - 2542. [Abstract] [Full Text]

				A. Hishinuma, J. Takamatsu, Y. Ohyama, T. Yokozawa, Y. Kanno, K. Kuma, S. Yoshida, N. Matsuura, and T. Ieiri Two Novel Cysteine Substitutions (C1263R and C1995S) of Thyroglobulin Cause a Defect in Intracellular Transport of Thyroglobulin in Patients with Congenital Goiter and the Variant Type of Adenomatous Goiter J. Clin. Endocrinol. Metab., April 1, 1999; 84(4): 1438 - 1444. [Abstract] [Full Text]

				P. S. Kim, S. A. Hossain, Y.-N. Park, I. Lee, S.-E. Yoo, and P. Arvan A single amino acid change in the acetylcholinesterase-like domain of thyroglobulin causes congenital goiter with hypothyroidism in the cog/cog mouse: A model of human endoplasmic reticulum storage diseases PNAS, August 18, 1998; 95(17): 9909 - 9913. [Abstract] [Full Text] [PDF]