We have examined the interaction of certain forms of mouse (m) tumor and bovine (b) pituitary TSH with standard bTSH on the activation of adenylate cyclase in human thyroid membranes. Tumor extract, serum from tumor-bearing mice, culture medium from dispersed cell incubations, and two preparations of purified bTSH (Sigma and Pierce) were fractionated on Sephadex G-100 (1.2 X 200 cm). For each fraction, TSH bioactivity was measured by stimulation of adenylate cyclase activity in human thyroid membranes, and immunoactivity was determined by RIA. On G-100, Pierce bTSH had multiple immunoactive components with partition coefficients (Kav) of 0.28-0.32 and ratios of biological over immunological activity (B/I) of 0.59-1.42. Sigma bTSH, mouse tumor, serum, and medium were even more heterogeneous (Kav = 0.23-0.32), with a lower range of B/I (0.04-1.0). When single doses (125-2000 ng) of those fractions with the highest Kav (0.30-0.32) and lowest B/I (0.04-0.51) were mixed with multiple doses (200-10,000 microU) of Armour TSH standard (B/I = 1), there was 30-56% inhibition of adenylate cyclase activity stimulation. Double reciprocal plots showed competitive inhibition for the low B/I forms from all sources, except for a medium form which showed mixed inhibition. The medium form had the highest inhibitory activity. There were no inhibitors in G-100 fractions from the Kav regions devoid of TSH immunoactivity or from the same Kav regions of normal mouse serum. To determine the chemical differences between different forms, affinity chromatography on Concanavalin A, wheat germ agglutinin, and soybean agglutinin was employed. Compared with the apparent higher molecular weight form with higher B/I, the apparent lower molecular weight form with lower B/I contained decreased amounts or availability of alpha-mannose and increased amounts or availability of beta-N-acetyl-D-galactosamine and/or beta-galactose; both forms appear to contain similar beta-N-acetyl-D-glucosamine residues, presumably in the inner core.
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.
A common genetic variant (V) of luteinizing hormone (LH), with two mutations (Trp(8)Arg and Ile(15)Thr) and an extra glycosylation consensus site (Asn(13)-Ala-Thr), is associated with abnormalities of reproductive function. To address the molecular basis of the functional differences between V- and wild-type (WT)-LH, recombinant (rec) forms of WT- and V-LH were synthesized in human embryonic kidney (HEK 293) cells. The rec hormones synthesized were rigorously purified employing affinity, immunoaffinity and ion exchange chromatographies (final purity approximately 12 000 IU/mg, 180-fold purification, 28% recovery). Functional properties of the hormone preparations were compared in vitro and in vivo. The molecular size of both rec LHs was 31 kDa, as determined by SDS-PAGE. Although the mutations in V-LHbeta did not significantly affect the affinity of LH receptor (LHR) binding (Kd approximately 0.4 nmol/L), V-LH had higher in vitro biopotency than WT-LH, in terms of mLTC-1 mouse Leydig tumor cell cAMP and progesterone (P) production, and steroidogenic acute regulatory protein (StAR) expression. In addition, in HEK 293 cells expressing the human LHR, V-LH demonstrated 1.8-fold higher response of inositol trisphosphate (IP(3)) production than WT-LH. Furthermore, HEK 293 cells expressing the ElK1 trans-reporting plasmids displayed 2.7-fold greater luciferase response to V-LH than WT-LH, documenting stimulation of the mitogen-activated protein kinase (MAPK) pathway. The in vivo half-life of V-LH was clearly faster (5-9 min) than that of WT-LH (12-22 min) and human chorionic gonadotropin (hCG; 50-70 min), when injected into rat circulation. It is worth noting that analysis by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) demonstrated clear differences in structures of carbohydrate side chains attached to the two forms of rec LHs, including incomplete processing of high mannose glycans (Man(5,8,9)) in V-LH, suggesting different pathways in its intracellular trafficking. Collectively, the present findings provide the molecular basis for the qualitative and quantitative differences in LH action that are observed in carriers of the V-LHbeta allele.
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