PARENT SESSION
PITUITARY AND GONADOTROPINS

Tuesday, August 3, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(357) DESIGN OF TETHERED BOVINE LUTROPIN (LH) ANALOGS.

Ben-Menahem, David ¹, Nakav, Sigal¹, Dantes, Ada², Pen, Svetlana³, Braw-Tal, Ruth³, Amsterdam, Abraham², ¹ Dept Clin Pharmacol, Beer Sheva, Israel² Dept Mol Cell Biol, Rehovot, Israel³ Inst of Animal Science, Bet Dagan, Israel

ABSTRACT- The objective was to genetically engineer single chain (SC) analogs of bovine LH and examine their bioactivity. A key consideration in the design is the spacing between the fused subunit domains, to preserve a functional conformation. In SC human (h) gonadotropins, the carboxyl terminal peptide (CTP) of the choriogonadotropin (CG) subunit serves as an effective spacer. Although a "linker less" arrangement or alternative serine/glycine linkers have also been used in tethered gonadotropins, the CTP-spaced design appears to be optimal for human gonadotropins because this sequence originated from the human genome and has determinants that enhance secretion and circulatory survival. However, gonadotropin beta subunits of non-primate, non-equid species lack a CTP, precluding the use of a homologous CTP in SC analogs in these species. Whether the CTP domain of the hCG subunit (denoted as huCTP) is a feasible linker for bovine SC gonadotropins or inter-species restrictions would interfere with biological properties of the CTP when applied as a spacer has not been previously determined. To address these issues, we tethered the bovine LH subunits using various linkers and examined the impact of the huCTP and a factitious CTP on the intracellular properties and bioactivity of the analogs. The non-natural, factitious CTP (designated boCTP) was generated using frame-shift mutations which extend the bovine LH reading frame, combined with nucleotide mutation, to create a stop codon. We constructed four tethered bovine LH variants including three with a truncated LH subunit and two with the and subunit domains linked via the heterologous huCTP or the homologous boCTP. The variants were expressed in CHO cells and secretion into the media was examined by SDS-PAGE. The presence of either the boCTP or huCTP linkers enhanced secretion 2-3 fold. All the tethered analogs were bioactive as estimated by induction of steroid production in immortalized granulosa cells expressing the rat LH receptor. Furthermore, they were about equally potent, as judged by their EC50s (0.7-0.9 ng/ml). The study demonstrates that a CTP-linker is advantageous for the biosynthesis of the SC bovine gonadotropins and illustrates successful strategies for analog design for potential application in assisted reproduction protocols in domestic animals.

KEY WORDS: Single Chain Analog, LH, Recombinant