Molecular and Cellular Biological Studies on Mechanisms for Biosynthesis and Release of Human Coagulation Factor XIII.

我们已经确定了因子XIII缺乏基因A亚基的6个突变，并确定了这些突变如何损害A亚基合成。在两个剪接异常的病例中发现mRNA水平大大降低。分子模型计算出Arg260Cys， Tyr283Cys和Gly562Arg突变以及密码子464处的过早终止改变了A亚基的构象，表明分子的错误折叠和/或不稳定。携带这些突变的重组A亚基在哺乳动物或酵母细胞中表达。结果表明，突变体合成正常，但由于其不稳定性而迅速消失，我们还对已报道的所有5例B亚基缺乏症进行了特征分析，并发现至少有3例不相关的病例由于奠基者效应而具有相同的突变。因此，我们提出了一个新的因子XIII缺乏分类。一个髓富集转录因子（MZF- 1）和两个普遍存在的转录因子（NF- 1和SP- 1）的启动子元件在5'侧区对a亚基的基础表达很重要。在上游区域发现了与骨髓富集因子（GATA-1和Ets-1）结合的DNA序列，并发现GATA-1元件负责增强子活性。这些转录因子在细胞类型特异性表达中起主要作用，这与其他转谷氨酰胺酶不同。最后，我们发现单核细胞和巨核细胞样细胞系内源性表达因子XIII的A亚基，并且在增殖和分化过程中A亚基的表达量发生了显著变化。

We have identified 6 mutations in the A subunit of the gene for Factor XIII deficiency and have determined how these mutations impair A subunit synthesis. The level of mRNA was found to be greatly reduced in two cases with splicing abnormalities. Molecular modeling calculated that the Arg260Cys, Tyr283Cys, and Gly562Arg mutations as well as premature termination at codon 464, changed the conformation of the A subunit, suggesting misfolding and/or destabilization of the molecule. Recombinant A subunits bearing these mutations were expressed in mammalian or yeast cells. Results indicated that the mutants were synthesized normally, but disappeared rapidly because of their instability, We also characterized all five of the cases with B subunit deficiency that have ever been reported, and found that at least three unrelated cases share the same mutation due to a founder's effect. We therefore proposed a new classification for Factor XIII deficiency.Promoter elements for a myeloid-enriched transcription factor (MZF- 1) and two ubiquitous transcription factors (NF- 1 and SP- 1) in the 5'-flanking region were important for basal expression of the A subunit. DNA sequences for binding of the myeloid-enriched factors (GATA-1 and Ets-1) were recognized in the upstream region, and the GATA-1 element was found to be responsible for the enhancer activity. These transcription factors play a major role in the cell-type-specific expression, which differs from other transglutaminases.Finally, we found that monocytoid and megakaryocytoid cell lines endogeneously expressed the A subunit of Factor XIII, and the amount of expressed A subunit significantly changed during proliferation and differentiation.