Braw-Tal Ruth, Pen Svetlana, Grinberg Moran, Nakav Sigal, Ben-Menahem David
Institute of Animal Science, Agricultural Research Organization, The Volcani Center, Beit Dagan 50250, Israel.
Mol Cell Endocrinol. 2006 Jun 27;252(1-2):136-41. doi: 10.1016/j.mce.2006.03.011. Epub 2006 Apr 27.
Single-chain gonadotropin analogs had been constructed for the purpose of structure-function studies and analog design. Incorporation of a spacer derived from the carboxyl terminal peptide (CTP) of the choriogonadotropin (CG) beta subunit between the tethered subunit domains of the human gonadotropins is beneficial for the secretion of the single-chain variants without compromising biocactivity. Although the CGbeta subunit containing the CTP domain is expressed only in primates and equids, a CTP-like sequence exists in the untranslated region of the LHbeta gene of several mammalian species, including the bovine species. The CTP encrypted in the bovine LHbeta DNA (designated as 'boCTP') and the CTP derived from the human CGbeta subunit (denoted as 'huCTP') served as a linker sequence in the design of bovine single-chain luteinizing hormone (LH) analogs. The purpose of the present study was to evaluate steroidogenesis in cultured bovine theca cells following stimulation with these single-chain analogs. The concentration of the LHbetaboCTPalpha and LHbetahuCTPalpha analogs in the conditioned media of the expressing CHO cells was three- to six-fold higher than that of the "linkerless" LHbetaalpha and LHbeta111alpha variants. The four analogs induced androstenedione and progesterone secretion from the primary theca cells in a dose-dependent manner, but differences in the steroidogenic response were observed. The LHbetaboCTPalpha analog (10 ng/ml) effectively induced androstenedione and progesterone secretion over unstimulated levels (4.0- and 4.4-fold increase for androstenedione and progesterone, respectively). The response to the pituitary bovine LH standard (10 ng/ml) was less pronounced for both steroids (two- to three-fold increase over basal levels). The activities of LHbetahuCTPalpha, LHbetaalpha and LHbeta111alpha were comparable and sightly reduced relative to the LHbetaboCTPalpha activity. The data suggested that LHbetaboCTPalpha was ranked as the most potent and this was even more prominent when analogs were used at a lower dose (1 ng/ml). These data suggest that the design, including the huCTP or boCTP linker, is favorable for the production of single-chain bovine LH analogs. Furthermore, spacing of the tethered subunit domains with the cryptic boCTP sequence that originated from the bovine LHbeta gene appears advantageous for the purpose of stimulating steroid production in the species-specific bioassay. Thus, an effective strategy to produce bioactive single-chain LH analogs in non-primate, non-equid species would be the mutatation of the LHbeta genes with the aim of expressing the cryptic CTP sequence as a spacer derived from the DNA of the same organism.
为了进行结构-功能研究和类似物设计，人们构建了单链促性腺激素类似物。在人促性腺激素的连接亚基结构域之间引入源自绒毛膜促性腺激素（CG）β亚基羧基末端肽（CTP）的间隔区，有利于单链变体的分泌，同时不损害其生物活性。尽管含有CTP结构域的CGβ亚基仅在灵长类动物和马科动物中表达，但在包括牛在内的几种哺乳动物的促黄体生成素（LH）β基因的非翻译区存在类似CTP的序列。牛LHβ DNA中编码的CTP（称为“boCTP”）和源自人CGβ亚基的CTP（称为“huCTP”）在设计牛单链促黄体生成素（LH）类似物时用作连接序列。本研究的目的是评估用这些单链类似物刺激后培养的牛卵泡膜细胞中的类固醇生成。表达CHO细胞的条件培养基中LHβboCTPα和LHβhuCTPα类似物的浓度比“无连接区”的LHβα和LHβ111α变体高3至6倍。这四种类似物以剂量依赖性方式诱导原代卵泡膜细胞分泌雄烯二酮和孕酮，但观察到类固醇生成反应存在差异。LHβboCTPα类似物（10 ng/ml）有效诱导雄烯二酮和孕酮分泌，使其高于未刺激水平（雄烯二酮和孕酮分别增加4.0倍和4.4倍）。垂体牛LH标准品（10 ng/ml）对两种类固醇的反应不太明显（比基础水平增加2至3倍）。LHβhuCTPα、LHβα和LHβ111α的活性相当，相对于LHβboCTPα的活性略有降低。数据表明，LHβboCTPα是最有效的，当以较低剂量（1 ng/ml）使用类似物时，这种情况更为明显。这些数据表明，包括huCTP或boCTP连接区的设计有利于生产单链牛LH类似物。此外，用源自牛LHβ基因的隐蔽boCTP序列隔开连接的亚基结构域，对于在物种特异性生物测定中刺激类固醇生成似乎是有利的。因此，在非灵长类、非马科动物物种中生产具有生物活性的单链LH类似物的有效策略是突变LHβ基因，目的是将隐蔽的CTP序列表达为源自同一生物体DNA的间隔区。
