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.
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.
Joshi
L
L
Molecular and Cellular Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Murata
Y
Y
Wondisford
F E
FE
Szkudlinski
M W
MW
Desai
R
R
Weintraub
B D
BD
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002241:Carbohydrates / Q000502:physiology*; D002522:Chlorocebus aethiops; D006063:Chorionic Gonadotropin / Q000032:analysis* / Q000737:chemistry; D018997:Chorionic Gonadotropin, beta Subunit, Human; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D017931:DNA Primers / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D006207:Half-Life; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D008297:Male; D008715:Methionine / Q000032:analysis / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics; D012794:Sialic Acids / Q000502:physiology; D013431:Sulfates / Q000032:analysis / Q000378:metabolism; D013936:Thymidine / Q000032:analysis / Q000378:metabolism; D013972:Thyrotropin / Q000737:chemistry* / Q000235:genetics / Q000493:pharmacokinetics*; D014316:Tritium
D002241:Carbohydrates; D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D017931:DNA Primers; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012794:Sialic Acids; D013431:Sulfates; D014316:Tritium; D013972:Thyrotropin; D008715:Methionine; D000242:Cyclic AMP; D019158:N-Acetylneuraminic Acid; D013936:Thymidine