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H. Edward Grotjan1 *, Prabhjit Chadha-Mohanty1 , David Ben-Menahem2
Animal Science Dept., Univ. Nebraska, Lincoln, NE 1
Dept. Molec. Biol. and Pharm., Washington Univ. School of Medicine, St. Louis, MO 2
Bovine (b) LH, like human LH, is retained inside cells and degraded when produced as a monomer while co-expression with markedly enhances secretion. In contrast, hCG is efficiently secreted as a monomer suggesting that determinants for intracellular stability reside in the CTP region (Muyan & Boime, Molec Endoc 1998; 12:766). The objective was to determine if bLH analogs with CTP extensions exhibit enhanced intracellular stability and secretion when expressed as monomers. A cDNA sequence encoding a bLH analog with a factitious CTP-like domain was generated by PCR using a frame-shift mutation strategy in conjunction with a dinucleotide mutation downstream from the polyadenylation signal to create a stop codon (designated bLHboCTP). For comparison, a cDNA sequence encoding bovine LH (1-110)-equine LH/CG (111-149) designated bLHeqCTP was created by subcloning. Both cDNAs were inserted into a baculovirus expression vector and expressed in High Five insect cells. Expression of either bLH analog resulted in inefficient secretion and intracellular degradation analogous to the expression of monomeric bLH. Also like bLH, co-expression of analogs with markedly enhanced secretion. The heterodimers formed by both CTP-bearing bLH analogs were biologically active in the MA10 cell bioassay but exhibited reduced efficacy compared to native or recombinant bLH. Thus, the secretion of monomeric bLH was not enhanced by addition of either the factitious or equine LH/CG CTP. Hence, determinants outside the CTP domain may be essential for intracellular stability or they are not present in either of the specific CTP sequences expressed.
This abstract is being presented on Sunday, August 1 at 8:00 AM to 10:15 AM at CUB 2nd Floor Ballroom.