Wetsel W C, Mellon P L, Weiner R I, Negro-Vilar A
Reproductive Neuroendocrinology Section, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Endocrinology. 1991 Sep;129(3):1584-95. doi: 10.1210/endo-129-3-1584.
An immortalized hypothalamic neuronal cell line was recently developed by genetically targeting the expression of the simian virus-40 large T-antigen in LHRH neurons. These GT1 cells were subcloned to GT1-1, GT1-3, and GT1-7 cells, and they have been shown to express the mRNA for pro-LHRH and secrete LHRH-like immunoreactive (IR) materials into the media. The purpose of our study was to biochemically and immunologically characterize the IR materials within and secreted from these cells. Both LHRH- and GnRH-associated peptide (GAP)-like IR materials were present and were secreted from these four cell lines. Up to 3% of the total cellular protein was composed of LHRH and GAP materials. When materials from the cell lysate and media were separated according to mol wt (Mr), at least three different pro-LHRH species were detected. These precursors contained both LHRH- and GAP-like IR determinants, and they eluted in the void volume and at approximately 10,000-12,000 and 8,400-8,500 Mr. A material that contained GAP-like IR eluted at approximately 6,500-6,800 Mr. This species is probably mouse GAP-(1-56) because it eluted on a reverse phase column in the approximate position of rat GAP-(1-56). Cell lysates contained a single LHRH-like IR form which coeluted on a size-exclusion column with synthetic LHRH. This material stimulated secretion of LH from anterior pituitary cells in a dose-response manner. By comparison, two different molecular forms of LHRH were detected in media at approximately 1,500 and 540 Mr. HPLC analyses revealed these peaks to be heterogeneous and to contain at least (Gln1)LHRH-(Gly11,Lys12,Arg13), (Gln1)LHRH-(Gly1,Lys12), LHRH-(Gly11), and LHRH. These experiments demonstrate that the cells contain and secrete multiple molecular forms of the pro-LHRH and that processing of the prohormone must involve 1) cleavage by an endopeptidase to give GAP-(1-56) and a C-terminally extended LHRH, 2) removal of C-terminal basic amino acids by a carboxypeptidase, 3) amidation of LHRH-(Gly11) to LHRH, and 4) cyclization of glutamine to pyroglutamate at the N-terminal of LHRH. These results provide the first evidence for intermediates in the metabolic pathway of pro-LHRH to LHRH.
最近，通过基因靶向在促性腺激素释放激素（LHRH）神经元中表达猿猴病毒40大T抗原，建立了一种永生化下丘脑神经元细胞系。这些GT1细胞被亚克隆为GT1-1、GT1-3和GT1-7细胞，并且已证明它们表达促LHRH的信使核糖核酸（mRNA），并将LHRH样免疫反应性（IR）物质分泌到培养基中。我们研究的目的是对这些细胞内以及分泌的IR物质进行生物化学和免疫学特性分析。LHRH和促性腺激素释放激素相关肽（GAP）样IR物质均存在于这四种细胞系中并从这些细胞系中分泌出来。细胞总蛋白中高达3%由LHRH和GAP物质组成。当根据分子量（Mr）对细胞裂解物和培养基中的物质进行分离时，检测到至少三种不同的促LHRH种类。这些前体同时含有LHRH和GAP样IR决定簇，它们在空体积以及约10000 - 12000和8400 - 8500 Mr处洗脱。一种含有GAP样IR的物质在约6500 - 6800 Mr处洗脱。该种类可能是小鼠GAP-(1 - 56)，因为它在反相柱上的洗脱位置与大鼠GAP-(1 - 56)大致相同。细胞裂解物中含有一种单一的LHRH样IR形式，它在尺寸排阻柱上与合成LHRH共洗脱。这种物质以剂量反应方式刺激垂体前叶细胞分泌促黄体生成素（LH）。相比之下，在培养基中检测到两种不同分子形式的LHRH，分子量约为1500和540 Mr。高效液相色谱（HPLC）分析表明，这些峰是异质的，并且至少含有（谷氨酰胺1）LHRH -（甘氨酸11、赖氨酸12、精氨酸13）、（谷氨酰胺1）LHRH -（甘氨酸1、赖氨酸12）、LHRH -（甘氨酸11）和LHRH。这些实验表明，这些细胞含有并分泌多种分子形式的促LHRH，并且激素原的加工过程必定涉及：1）由一种内肽酶切割产生GAP-(1 - 56)和C末端延长的LHRH；2）由一种羧肽酶去除C末端碱性氨基酸；3）将LHRH-(甘氨酸11)酰胺化为LHRH；4）在LHRH的N末端将谷氨酰胺环化为焦谷氨酸。这些结果为促LHRH向LHRH代谢途径中的中间体提供了首个证据。
