We used a novel approach to study the role of the Asn-linked oligosaccharides for human thyrotropin (hTSH) activity. Mutagenesis of Asn(N) within individual glycosylation recognition sequences to Gln (Q) was combined with expression of wild type and mutant hTSH in cell lines with different glycosylation patterns. The in vitro activity of hTSH lacking the Asn oligosaccharide (Q52/TSH) expressed in CHO-K1 cells (sialylated oligosaccharides) was increased 6-fold compared with wild type, whereas the activities of Q78/TSH and /TSHQ23 were increased 2-3-fold. Deletion of the Asn oligosaccharide also increased the thyrotropic activity of human chorionic gonadotropin, in contrast to previous findings at its native receptor. The in vitro activity of wild type hTSH expressed in CHO-LEC2 cells (sialic acid-deficient oligosaccharides), CHO-LEC1 cells (ManGlcNAc intermediates), and 293 cells (sulfated oligosaccharides) was 5-8-fold higher than of wild type from CHO-K1 cells. In contrast to CHO-K1 cells, there was no difference in the activity between wild type and selectively deglycosylated mutants expressed in these cell lines. Thus, in hTSH, the oligosaccharide at Asn and, specifically, its terminal sialic acid residues attenuate in vitro activity, in contrast to the previously reported stimulatory role of this chain for human chorionic gonadotropin and human follitropin activity. The increased thyrotropic activity of Q52/CG suggests that receptor-related mechanisms may be responsible for these differences among the glycoprotein hormones. Despite their increased in vitro activity, Q52/TSH, and Q78/TSH from CHO-K1 cells had a faster serum disappearance rate and decreased effect on T production in mice. These findings highlight the importance of individual oligosaccharides in maintaining circulatory half-life and hence in vivo activity of hTSH.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				R Nunez Miguel, J Sanders, D Y Chirgadze, J Furmaniak, and B Rees Smith Thyroid stimulating autoantibody M22 mimics TSH binding to the TSH receptor leucine rich domain: a comparative structural study of protein-protein interactions J. Mol. Endocrinol., May 1, 2009; 42(5): 381 - 395. [Abstract] [Full Text] [PDF]

				R Nunez Miguel, J Sanders, D Y Chirgadze, T L Blundell, J Furmaniak, and B Rees Smith FSH and TSH binding to their respective receptors: similarities, differences and implication for glycoprotein hormone specificity J. Mol. Endocrinol., September 1, 2008; 41(3): 145 - 164. [Abstract] [Full Text] [PDF]

				A. D. Nguyen, S. Itoh, V. Jeney, H. Yanagisawa, M. Fujimoto, M. Ushio-Fukai, and T. Fukai Fibulin-5 Is a Novel Binding Protein for Extracellular Superoxide Dismutase Circ. Res., November 26, 2004; 95(11): 1067 - 1074. [Abstract] [Full Text] [PDF]

				V. I. Otto, T. Schurpf, G. Folkers, and R. D. Cummings Sialylated Complex-type N-Glycans Enhance the Signaling Activity of Soluble Intercellular Adhesion Molecule-1 in Mouse Astrocytes J. Biol. Chem., August 20, 2004; 279(34): 35201 - 35209. [Abstract] [Full Text] [PDF]

				C. A. Strott Sulfonation and Molecular Action Endocr. Rev., October 1, 2002; 23(5): 703 - 732. [Abstract] [Full Text] [PDF]

				M. W. Szkudlinski, V. Fremont, C. Ronin, and B. D. Weintraub Thyroid-Stimulating Hormone and Thyroid-Stimulating Hormone Receptor Structure-Function Relationships Physiol Rev, April 1, 2002; 82(2): 473 - 502. [Abstract] [Full Text] [PDF]

				P. R. Manna, L. Joshi, V. N. Reinhold, M. L. Aubert, N. Suganuma, K. Pettersson, and I. T. Huhtaniemi Synthesis, purification and structural and functional characterization of recombinant form of a common genetic variant of human luteinizing hormone Hum. Mol. Genet., February 1, 2002; 11(3): 301 - 315. [Abstract] [Full Text] [PDF]

				Y. Nagayama, H. Namba, N. Yokoyama, S. Yamashita, and M. Niwa Role of Asparagine-linked Oligosaccharides in Protein Folding, Membrane Targeting, and Thyrotropin and Autoantibody Binding of the Human Thyrotropin Receptor J. Biol. Chem., December 11, 1998; 273(50): 33423 - 33428. [Abstract] [Full Text] [PDF]

				M. Grossmann, R. Wong, M. W. Szkudlinski, and B. D. Weintraub Human Thyroid-stimulating Hormone (hTSH) Subunit Gene Fusion Produces hTSH with Increased Stability and Serum Half-life and Compensates for Mutagenesis-induced Defects in Subunit Association J. Biol. Chem., August 22, 1997; 272(34): 21312 - 21316. [Abstract] [Full Text] [PDF]

				M. Grossmann, B. D. Weintraub, and M. W. Szkudlinski Novel Insights into the Molecular Mechanisms of Human Thyrotropin Action: Structural, Physiological, and Therapeutic Implications for the Glycoprotein Hormone Family Endocr. Rev., August 1, 1997; 18(4): 476 - 501. [Abstract] [Full Text] [PDF]

				M. Grossmann, M. W. Szkudlinski, R. Wong, J. A. Dias, T. H. Ji, and B. D. Weintraub Substitution of the Seat-belt Region of the Thyroid-stimulating Hormone (TSH) beta -Subunit with the Corresponding Regions of Choriogonadotropin or Follitropin Confers Luteotropic but Not Follitropic Activity to Chimeric TSH J. Biol. Chem., June 13, 1997; 272(24): 15532 - 15540. [Abstract] [Full Text] [PDF]

				D. Fiete and J. U. Baenziger Isolation of the SO4-4-GalNAcbeta 1,4GlcNAcbeta 1,2Manalpha -specific Receptor from Rat Liver J. Biol. Chem., June 6, 1997; 272(23): 14629 - 14637. [Abstract] [Full Text] [PDF]

				M. Grossmann, R. Wong, N. G. Teh, J. E. Tropea, J. East-Palmer, B. D. Weintraub, and M. W. Szkudlinski Expression of Biologically Active Human Thyrotropin (hTSH) in a Baculovirus System: Effect of Insect Cell Glycosylation on hTSH Activity in Vitro and in Vivo Endocrinology, January 1, 1997; 138(1): 92 - 100. [Abstract] [Full Text] [PDF]

				H. Leitolf, K. P. T. Tong, M. Grossmann, B. D. Weintraub, and M. W. Szkudlinski Bioengineering of Human Thyrotropin Superactive Analogs by Site-directed "Lysine-scanning" Mutagenesis. COOPERATIVE EFFECTS BETWEEN PERIPHERAL LOOPS J. Biol. Chem., August 25, 2000; 275(35): 27457 - 27465. [Abstract] [Full Text] [PDF]