Recombinant thyrotropin containing a beta-subunit chimera with the human chorionic gonadotropin-beta carboxy-terminus is biologically active, with a prolonged plasma half-life: role of carbohydrate in bioactivity and metabolic clearance.

Abstract

Recombinant TSH is now successfully being used in clinical studies of thyroid cancer. Because of its therapeutic potential, we have constructed a longer acting analog of TSH by fusing the carboxy-terminal extension peptide (CTEP) of hCG beta onto TSH beta. When coexpressed either with alpha-subunit complementary DNA or alpha minigene in African green monkey (COS-7) and human embryonic kidney (293) cells, the chimera was fully bioactive in vitro and exhibited enhanced in vivo potency associated with a prolonged plasma half-life. The addition of 25 amino acids with 4 O-linked oligosaccharide chains did not affect the assembly and secretion of chimeric TSH. Wild-type (WT) and chimeric TSH secreted by COS-7 and 293 cells displayed wide differences in their plasma half-lives, presumably due to the presence of terminal sialic acid and SO4 on their oligosaccharide chains, respectively. Chimeric and WT TSH secreted by both cell lines demonstrated similar bioactivity in cAMP production, with some differences in [3H]thymidine incorporation. Chimeric TSH appears to be more effective in COS-7 cells than in 293 cells, as judged by growth assay. COS-7-produced chimeric TSH showed the maximum increase in half-life, indicating the importance of sialic acid in prolonging half-life and in vivo potency. Sulfation of both subunits, predominantly beta and to a lesser extent alpha, appears to be responsible at least in part for the increased metabolic clearance of WT and chimeric TSH secreted by 293 cells. Apart from its therapeutic potential, chimeric TSH produced in various cell lines can be used as a tool to delineate the roles of sulfate and sialic acid in the in vivo clearance and, thereby, the in vivo bioactivity.

Effect of acute high dose dobutamine administration on serum thyrotrophin (TSH)

“…Dobutamine may increase TSH clearance or it may decrease production and/ or secretion of either TRH, TSH or both. The rapidity of the observed changes are consistent with the known short T1/2 of TSH (ranging from 10-30 minutes) (Bakke et al, 1961;Joshi et al, 1995). Since dobutamine has a half-life of only 2 minutes (Weber & Tuttle, 1977), the continued effect of dobutamine treatment that was observed 15 minutes after its discontinuation may reflect the longer half-life of TSH compared to dobutamine.…”

Single-chain human gonadotropin analogs induce follicle development in sheep

“…Indeed, the half-lives of both FSH (LaPolt et al 1992) and TSH (Joshi et al 1995, Grossmann et al 1997 are dramatically extended by addition of the CTP segment to the b-subunit. In addition, the oligosaccharide chains of recombinant proteins expressed in CHO cells contain terminal sialic acid residues (Smith et al 1990).…”

“…The linker sequence used in the constructs discussed here is the exon encoding the CTP region of CGb. The CTP segment and the associated O-linked oligosaccharide chains markedly increase the in vivo activity of the gonadotropins by reducing the rate of clearance (LaPolt et al 1992, Joshi et al 1995. The single-chain technology has been extended to include the union of genes encoding two or more types of b-subunits.…”

Expression Profiling, Downstream Signaling and Subunit Interactions of GPA2/GPB5 in the Adult MosquitoAedes aegypti

“…Natural and synthetic linkers function as spacers that connect multidomain proteins, and are commonly used to study unstable or weak protein-protein interactions 34 . The incorporation of a linker sequence between glycoprotein hormone subunits has been performed previously, and does not affect the assembly, secretion or bioactivity of human FSH 35 , TSH 36 and CG 37 . The conversion of two independent glycoprotein hormone subunits into a single polypeptide chain using a glycine-serine repeat linker sequence has also been performed recently with lamprey GPA2/GPB5 38 , which was shown to induce a cAMP response.…”