Nakav S, Dantes A, Pen S, Chadna-Mohanty P, Braw-Tal R, Amsterdam A, Grotjan H E, Ben-Menahem D
Department of Clinical Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
Exp Clin Endocrinol Diabetes. 2006 Mar;114(3):95-104. doi: 10.1055/s-2005-865926.
Single chain variants of the heterodimeric gonadotropins were engineered by tethering the genes of the individual subunits into one polypeptide. In tethered human (h) gonadotropins, the carboxyl terminal peptide (CTP) of the choriogonadotropin (CG) beta subunit serves as an effective linker to enhance the secretion of the analogs compared to variants lacking the CTP. The gonadotropin subunits of non-primate, non-equid species lack a CTP domain that precludes the use of a homologous CTP in tethered analogs in many species. Here we used the bovine LH as a model to examine the impact of the CTP domain of the hCGbeta subunit (denoted as huCTP) and of a previously untranslated CTP-like sequence decoded from the bovine LHbeta gene on the secretion and bioactivity of tethered analogs. This cryptic CTP (designated boCTP) was incorporated into the bovine LHbeta reading frame by deletion frame-shift mutations analogous to these that presumably occurred in primates and equids. We genetically engineered single chain variants in which the beta and alpha subunit domains were linked directly or via the heterologous huCTP or the homologous boCTP sequences and expressed them in CHO cells. The data suggest that the tethered analogs were expressed and N-glycosylated, but unlike the huCTP, the boCTP appears as devoid of mucin O-glycans. The incorporation of the boCTP or huCTP linkers enhanced by about 3fold the rate and efficiency of secretion from the transfected cells. The tether variants were bioactive, as estimated by induction of steroid production in immortalized granulosa cells expressing the rat LH receptor. Furthermore, the variants were about equally potent, as judged by their EC50s (0.7-0.9 ng/ml). Thus, the hCGbeta CTP maintains pro-secretory determinants without inhibiting receptor activation when applied as a linker in tethered bovine LH, implying that these CTP features are preserved when the domain is incorporated into non-primate single chain analogs. The study suggests that the boCTP and huCTP domains are advantageous for the secretion of tethered bovine gonadotropins, and also demonstrates strategies for the design of bioactive LH analogs in ruminant species.
通过将异源二聚体促性腺激素的各个亚基基因连接成一条多肽，构建了单链变体。在连接的人（h）促性腺激素中，与缺乏羧基末端肽（CTP）的变体相比，绒毛膜促性腺激素（CG）β亚基的羧基末端肽（CTP）可作为有效的连接子，增强类似物的分泌。非灵长类、非马属物种的促性腺激素亚基缺乏CTP结构域，这使得在许多物种的连接类似物中无法使用同源CTP。在这里，我们以牛促黄体生成素（LH）为模型，研究了人绒毛膜促性腺激素β亚基的CTP结构域（称为huCTP）和从牛LHβ基因解码的先前未翻译的CTP样序列对连接类似物的分泌和生物活性的影响。通过类似于灵长类和马属动物可能发生的缺失移码突变，将这种隐蔽的CTP（称为boCTP）纳入牛LHβ阅读框。我们通过基因工程构建了单链变体，其中β和α亚基结构域直接连接或通过异源huCTP或同源boCTP序列连接，并在CHO细胞中表达。数据表明，连接类似物得到了表达并进行了N-糖基化，但与huCTP不同，boCTP似乎没有粘蛋白O-聚糖。boCTP或huCTP连接子的加入使转染细胞的分泌速率和效率提高了约3倍。通过在表达大鼠LH受体的永生化颗粒细胞中诱导类固醇生成来估计，连接变体具有生物活性。此外，根据它们的半数有效浓度（EC50s，0.7 - 0.9 ng/ml）判断，这些变体的效力大致相同。因此，当作为连接子应用于连接的牛LH中时，hCGβ CTP保持了促分泌决定因素而不抑制受体激活，这意味着当该结构域被纳入非灵长类单链类似物时，这些CTP特征得以保留。该研究表明，boCTP和huCTP结构域有利于连接的牛促性腺激素的分泌，也展示了反刍动物物种中生物活性LH类似物的设计策略。
