A soluble form of the HLA-G antigen is encoded by a messenger ribonucleic acid containing intron 4.
A soluble form of the HLA-G antigen is encoded by a messenger ribonucleic acid containing intron 4.
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DOI：
10.4049/jimmunol.153.12.5516
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发表时间：
1994-12
影响因子：
4.4
通讯作者：
Tomoyuki Fujii;A. Ishitani;D. Geraghty
中科院分区：
文献类型：
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作者：
Tomoyuki Fujii;A. Ishitani;D. Geraghty
HLA-G初级转录物被选择性剪接以产生编码三种替代膜结合蛋白的mrna。除了这些形式外，还描述了一种可溶性HLA-G蛋白，它不是由三种替代mrna中的任何一种直接编码的。为了解释可能导致可溶性HLA-G Ag表达的过程，我们研究了HLA-G RNA的蛋白水解加工和其他选择性剪接可能发挥的潜在作用。通过产生由逆转录病毒启动子驱动的HLA-G cDNA表达的转染细胞，可以排除膜结合HLA-G的蛋白水解加工作为产生可溶性HLA-G的机制，因此我们将重点放在选择性剪接上作为解释。pcr扩增的cDNA分析显示，在所检测的所有样品中都存在相对丰富的转录物，该转录物由内含子4序列中断的全长HLA-G mRNA序列组成。该mRNA的开放阅读框继续进入内含子4，在α 3结构域后终止21个氨基酸，从而排除了跨膜编码区，产生了具有高电荷羧基端的蛋白质。将含有cDNA的内含子4插入逆转录病毒表达载体，转染LCL。221，然后通过二维等电聚焦/SDS-PAGE分析将I类蛋白与天然可溶性G进行比较，证明该信息编码了可溶性HLA-G蛋白。此外，还发现了一个类似的内含子，内含来自HLA-G2 mRNA的信息，这表明这种替代HLA-G蛋白存在可溶性形式。这些发现与其他可溶性I类分子和可溶性HLA-G Ag的潜在功能有关。
The HLA-G primary transcript is alternatively spliced to yield mRNAs encoding three alternative membrane bound proteins. In addition to these forms, a soluble HLA-G protein has been described which is not encoded directly by any of the three alternative mRNAs. To explain the process which might lead to the expression of a soluble HLA-G Ag, we investigated the potential roles proteolytic processing and additional alternative splicing of HLA-G RNA might play. By generating transfected cells with HLA-G cDNA expression driven by a retroviral promoter, it was possible to rule out proteolytic processing of the membrane-bound HLA-G as a mechanism of generating soluble HLA-G, resulting in our focus on alternative splicing as an explanation. Analysis of PCR-amplified cDNA revealed a relatively abundant transcript present in all samples examined which consisted of the full length HLA-G mRNA sequence interrupted by intron 4 sequence. The open reading frame in this mRNA continues into intron 4 terminating 21 amino acids after the alpha 3 domain, thus excluding the transmembrane encoding region and yielding a protein with a highly charged carboxyl terminus. Transfection of the intron 4 containing cDNA, inserted into a retroviral expression vector, into LCL .221 followed by comparison of the class I protein to native soluble G by two dimensional isoelectric focusing/SDS-PAGE analysis, demonstrated this message encoded the soluble HLA-G protein. In addition, a similar intron containing message derived from the HLA-G2 mRNA was found, suggesting the existence of a soluble form of this alternative HLA-G protein. These findings are discussed in relation to other soluble class I molecules and with regard to potential functions of the soluble HLA-G Ag.