Increasing diversity of human thyroperoxidase generated by alternative splicing - Characterization by molecular cloning of new transcripts with single- and multispliced mRNAs
Increasing diversity of human thyroperoxidase generated by alternative splicing - Characterization by molecular cloning of new transcripts with single- and multispliced mRNAs
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DOI：
10.1074/jbc.m209513200
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发表时间：
2003-02-07
影响因子：
4.8
通讯作者：
Franc, JL
中科院分区：
文献类型：
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作者：
Ferrand, M;Le Fourn, V;Franc, JL
人甲状腺过氧化物酶（hTPO）基因由17个外显子组成。迄今为止确定的最长的完整cDNA序列包含编码933个氨基酸的多肽的全长hTPO（TPO 1）。在正常甲状腺组织中存在几种编码hTPO亚型的mRNA，包括外显子10缺失的TPO 2和外显子16缺失的TPO zanelli。在本研究中，我们建立了两个新的单剪接转录，TPO 4和TPO 5，缺乏外显子14和8，分别存在。在将TPO 4 cDNA转染到中国仓鼠卵巢细胞中后，观察到TPO 4能够到达细胞表面，具有酶活性，并且能够被一组12种针对hTPO的单克隆抗体识别，而TPO 5不能正确折叠并且不能到达细胞表面。在正常组织中，采用定量逆转录PCR检测TPO 4 mRNA的表达。这种缺失的TPO mRNA占总TPO mRNA的32 +/-11%。在相同的组织中，TPO 2、TPO zanelli和TPO 5分别为35 +/-12%、36 +/-14%和接近10%。这四种物种（不包括TPO 1）的总和超过100%，可能是由于存在多剪接的mRNA。这种可能性进行了测试，并确定了三个新的变体：TPO 2/3，缺乏外显子10和16，TPO 2/4，缺乏外显子10和14，和一个意想不到的变体，TPO 6，对应于外显子10，12，13，14和16的缺失。总之，这些结果表明存在五个新的成绩单。其中之一，TPO 4，编码一种酶活性蛋白，而TPO 5不能正确折叠。其他新剪接的mRNA变体的功能意义仍有待阐明，但这些结果可能有助于解释从甲状腺中纯化的hTPO的异质性。
The human thyroperoxidase (hTPO) gene is composed of 17 exons. The longest complete cDNA sequence determined so far contains a full-length hTPO (TPO1) encoding a 933-amino acid polypeptide. Several mRNA species encoding for hTPO isoforms are present in normal thyroid tissues, including TPO2 with exon 10 deleted and TPOzanelli with exon 16 deleted. In the present study, we established the existence of two new single-spliced transcripts, TPO4 and TPO5, lacking exons 14 and 8, respectively. Upon transfecting the TPO4 cDNA into Chinese hamster ovary cells, it was observed that TPO4 is able to reach the cell surface, is enzymatically active, and is able to be recognized by a panel of 12 monoclonal antibodies directed against hTPO, whereas TPO5 does not fold correctly and is unable to reach the cell surface. In normal tissues, the expression of TPO4 mRNA was examined by performing quantitative reverse transcription PCR. This deleted TPO mRNA amounted to 32 +/- 11% of the total TPO mRNAs. In the same tissues, the TPO2, TPOzanelli, and TPO5 amounted to 35 +/- 12%, 36 +/- 14%, and similar to10%, respectively. The sum of these four species (not including TPO1) was more than 100%, possibly due to the presence of multispliced mRNAs. This possibility was tested, and three new variants were identified: TPO2/3, lacking exons 10 and 16, TPO2/4, lacking exons 10 and 14, and an unexpected variant, TPO6, corresponding to the deletion of exons 10, 12, 13, 14, and 16. In conclusion, these results indicate the existence of five new transcripts. One of them, TPO4, codes for an enzymatically active protein, whereas TPO5 is unable to fold correctly. The functional significance of the other newly spliced mRNA variants still remains to be elucidated, but these results might help to explain the heterogeneity of the hTPO purified from the thyroid gland.