Intracellular events determine the fate of antithrombin Utah.-猫眼课题宝

细胞内事件决定了抗凝血酶犹他的命运。

DOI：

10.1182/blood.v86.9.3461.bloodjournal8693461

发表时间：

1995

期刊：

Blood

影响因子：

20.3

通讯作者：

中科院分区：

医学1区

文献类型：

作者：

W. Sheffield;J. E. Castillo;M. Blajchman

我们试图确定细胞内或细胞外事件是否有助于犹他州突变（Pro 407至Leu）受试者中循环抗凝血酶（AT）水平的降低。使用定点诱变在先前表征的兔AT cDNA内重建该突变。无细胞表达的突变的cDNA产生的AT蛋白，未能与凝血酶反应。兔AT-犹他蛋白在瞬时转染的Cos细胞中的表达导致条件培养基中检测到的AT抗原的量减少10倍，与野生型重组AT相比。这种效应不是由转染效率的变化引起的，因为AT水平被归一化为共转染质粒氯霉素乙酰转移酶的产物。此外，在北方印迹分析中，AT mRNA水平在表达兔AT-犹他或野生型重组兔AT的细胞中是相当的。免疫印迹的条件培养基从两个群体的转染细胞显示，重组AT-犹他州蛋白是完整的。使用永久转染的中国仓鼠卵巢（CHO）细胞重现Cos细胞获得的结果。CHO细胞系的脉冲追踪实验表明，与表达野生型AT的细胞相比，脉冲标记后兔AT-犹他的初始水平和追踪期间随后分泌的速率均降低。当在兔AT背景中重建并在Cos细胞中表达时，还观察到AT-Oslo突变（Ala 404至Thr）的AT分泌减少。在这些实验中，与野生型相比，突变体AT的培养基水平降低了50%。这些结果表明，细胞内的事件，而不是加速清除或其他细胞外的原因，有助于在这些链1C AT突变体中看到的AT分泌的缺乏。

We sought to determine whether intracellular or extracellular events contribute to the decrease in circulating antithrombin (AT) levels that is seen in subjects with the Utah mutation (Pro 407 to Leu). Site-directed mutagenesis was used to recreate this mutation within a previously characterized rabbit AT cDNA. Cell-free expression of the mutated cDNA yielded an AT protein that failed to react with thrombin. Expression of the rabbit AT-Utah protein in transiently transfected Cos cells resulted in a 10-fold decrease in the amount of AT antigen detected in the conditioned media, as compared with that seen with the wild-type recombinant AT. This effect was not caused by variations in transfection efficiency, because AT levels were normalized to the product of a cotransfected plasmid, chloramphenicol acetyl transferase. Moreover, on Northern blot analysis, AT mRNA levels were comparable in cells expressing either the rabbit AT-Utah or wild-type recombinant rabbit AT. Immunoblots of conditioned media from the two populations of transfected cells showed that the recombinant AT-Utah protein was intact. The results obtained with Cos cells were reproduced using permanently transfected Chinese hamster ovary (CHO) cells. Pulse-chase experiments with the CHO lines showed that both initial levels of rabbit AT-Utah after the pulse labeling and the rate of subsequent secretion during the chase period were reduced compared with that seen with cells expressing the wild-type AT. The observed reduction in AT secretion was also observed for the AT-Oslo mutation (Ala 404 to Thr) when recreated in the rabbit AT background, and expressed in Cos cells. In these experiments, the media levels of mutant AT were reduced by 50%, compared with wild-type. These results show that intracellular events, as opposed to accelerated clearance or other extracellular causes, contribute to the paucity of AT secretion seen in these strand 1C AT mutants.

Identification of a serpin-enzyme complex receptor on human hepatoma cells and human monocytes.