Characterization and determination of the biological activities of noncleavable high molecular weight forms of inhibin A and activin A
Characterization and determination of the biological activities of noncleavable high molecular weight forms of inhibin A and activin A
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DOI：
10.1210/me.10.9.1055
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发表时间：
1996-09-01
影响因子：
--
通讯作者：
Sullivan, J
中科院分区：
文献类型：
--
作者：
Mason, AJ;Farnworth, PG;Sullivan, J
在哺乳动物293细胞中重组表达人α-和β A-活化素亚基cDNA导致分泌20- 53 K游离α-亚基衍生产物、30- 105 K α β A-活化素二聚体和24- 110 K β A-活化素二聚体。本研究证实，这些产品的大小的广泛变化是由于蛋白水解加工位点的不完全切割和α C亚基中氨基酸编号302处的N-连接糖基化位点的差异糖基化。通过蛋白水解加工位点和N-连接的糖基化位点的诱变，结合通过免疫沉淀和一维和二维SDS-PAGE（SDS/SDS-β-ME）分析转染产物，确定这些产物中的每一种的身份。在293细胞中瞬时表达α-和β A-亚基的加工位点突变体，以产生微克量的不可切割的55 K和65 K α-二聚体，以及不可切割的110 K激活素A二聚体。纯化55 K和65 K活化素A形式，并发现在大鼠垂体细胞生物测定中具有完全的生物活性。110 K高分子量（HMW）形式的人活化素A在垂体测定中未显示任何FSH释放活性。由于放射性标记的55 K和65 K腺苷二酸A和110 K激活素A在与大鼠垂体细胞孵育72小时后保持完整，因此这些二聚体似乎没有通过在其他位点的蛋白水解裂解转化为较低分子量形式，这些结果首次表明，55 K和65 K双链菌素A具有固有的生物活性，并且不需要裂解成32 K形式，activation.相反，110 K激活素A前体裂解为24 K形式似乎是活性所必需的。
Recombinant expression of human alpha- and beta A-inhibin subunit cDNAs in mammalian 293 cells results in the secretion of 20-53K free alpha-subunit-derived products, 30-105K alpha beta A-inhibin dimers, and 24-110K beta A-activin dimers. The present study verifies that the wide variation in the size of these products is due to incomplete cleavage of the proteolytic processing sites and the differential glycosylation of the N-linked glycosylation site at amino acid number 302 in the alpha C- subunit. The identity of each of these products was established by mutagenesis of proteolytic processing sites and N-linked glycosylation sites, combined with the analysis of transfection products by immunoprecipitation and one- and two-dimensional SDS-PAGE (SDS/SDS-beta-ME). Transient expression of processing site mutants of the alpha- and beta A-subunits in 293 cells was used to generate microgram quantities of noncleavable 55K and 65K inhibin dimers, and noncleavable 110K activin A dimers. The 55K and 65K inhibin A forms were purified and found to be fully biologically active in a rat pituitary cell bioassay, The 110K high molecular weight (HMW) form of human activin A failed to show any FSH-releasing activity in the pituitary assay. Since radioactively labeled 55K and 65K inhibin A and 110K activin A remained intact after incubation with rat pituitary cells for 72 h, there appears to be no conversion of these dimers to lower molecular weight forms by proteolytic cleavage at additional sites, These results show for the first time that 55K and 65K inhibin A are intrinsically biologically active and do not require cleavage to the 32K form for activation. In contrast, cleavage of the 110K activin A precursor to the 24K form would appear to be necessary for activity.