CONSTRUCTION AND EXPRESSION OF AN OVINE SINGLE CHAIN TRIPLE-DOMAIN CHIMERIC GONADOTROPIN: TANDEM LINKAGE OF THE GENES ENCODING THE α-AND FSHβ AND LHβ SUBUNITS

Abstract

Members of the glycoprotein hormone family (LH, FSH, TSH and CG) are heterodimers composed of common α and hormone-specific β subunits that are non-covalently associated. Both subunits are glycosylated, containing asparagine (N)-linked oligosaccharides and, in the case of the CGβ subunit, O-linked carbohydrates are also present in a cluster of amino acids at the carboxy-terminus. The ovine α subunit consists of 120 amino acids and the mature protein sequence is identical with the corresponding bovine sequence. The ovine FSHβ and LHβ genes encode proteins of 129 and 141 amino acids, respectively and they are most closely related to their bovine counterparts (96% and 98%, respectively). To examine the structure-activity relationships of the ovine gonadotropins, and their role in follicle development and ovulation, the availability of purified hormones is essential. The purity and potency of recombinant gonadotropins is greater than tissue-derived gonadotropins. Thus, the recombinant gonadotropins may modulate the gonadal function of sheep in a more precise and controlled manner. In addition, recombinant hormones are likely free of contaminants and disease causing pathogens and, therefore, may improve overall herd health and productivity. Previous studies in transfected Chinese hamster ovary (CHO) cells demonstrated that the assembly of α and the β subunits is inefficient, resulting in low rates of formation of recombinant human, bovine and equine LH. A number of reports have now shown that covalently linking the β and the α subunits in a single chain results in biologically active analogs. The ability to express a single chain bearing genetically linked α and β subunits by-passes the rate-limiting assembly step, often resulting in a greater yield of secreted analog. Recently, we also reported that a covalently fused triple-domain gonadotropin analog containing human FSHβ, LHβ and α subunit was secreted efficiently and its bioactivity was similar to the heterodimer, suggesting productive interactions between the domains in the tether. Here, we generate ovine triple-domain single chain gonadotropin with the following structure: FSHβ–LHβ–α. This chimera was constructed using bovine cDNAs encoding FSHβ, LHβ or α subunits as templates and PCR mutagenesis to generate the corresponding ovine subunits. Stable cell lines expressing the single chain were metabolically labeled with [35S]cysteine. Lysates (intracellular) and media (secreted) were immunoprecipitated with polyclonal human CGβ antiserum (cross reacts with ovine LHβ subunit) and analyzed by SDS– PAGE. The secreted form of the analog was also analyzed on Western blot under non-reducing conditions. Collectively, these data show that the ovine triple-domain analog is synthesized and secreted from CHO cells. The apparent molecular mass of the secreted form is 80 kDa. The ability to construct dually active single-chain analogs could permit more effective control of the half-life and duration of gonadotropin actions. This should aid in the design of analogs that could be used to promote superovulation and/or out-of-season breeding in sheep. (poster)