Islam M R, Shah G N, Sly W S
E. A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Health Science Center, Missouri 63104, USA.
Arch Biochem Biophys. 1996 Sep 15;333(2):385-93. doi: 10.1006/abbi.1996.0405.
When cDNAs for human and rodent beta-glucuronidases were expressed in COS-7 cells using several different promoters, rodent beta-glucuronidases were produced three times more than human beta-glucuronidase, although their transcriptional levels were similar. Similar observations were also recorded in LMTK- cells using SV40 early or chicken beta-actin promoters. In hopes of enhancing yields of recombinant human beta-glucuronidase for enzyme replacement therapy, we sought to determine the region within the linear sequences responsible for the higher levels of expression of the rodent cDNAs. To do so, we made various rat-human chimeric cDNAs utilizing conserved restriction enzyme sites. The levels of products expressed from these chimeric cDNAs in COS cells were assessed by activity assay and by metabolic labeling of the proteins followed by immunoprecipitation and SDS-PAGE. From the results of these expression studies, we identified a 155-bp ClaI (643)-AflII (797) fragment in the rat open reading frame responsible for the increased rate of translation of the rat beta-glucuronidase (RBG) cDNA. Replacement of the homologous ClaI (683)-AflII (838) fragment in human beta-glucuronidase (HBG) with this 155-bp fragment from RBG increased the translation level of the resulting chimeric HRaH. Conversely, substitution of the 155-bp human fragment for that of rat in RBG cDNA reduced the total synthesis of the resulting chimeric HHaR. Placement of the 155-bp segment between the initiation ATG and the promoter has only negative effects on the expression of either cDNA. A more stable secondary structure of the human cDNA in this region might explain a reduced rate of translation. However, secondary structure analysis of mRNAs from the 155-bp fragment of rat and human cDNAs predicted that, while both can form stem-loops, the rat fragment (delta Gzero = -42.1 kcal/ mol) is actually more stable than the human fragment (delta Gzero = -32.1 kcal/mol). In fact, the free energy of stability of the first 50 bp within this ClaI-AflII fragment from rat (-10.3 kcal/mol) indicates that the secondary structure is considerably more stable than the corresponding 50 bp from human (-2.1 kcal/mol). This segment of the rat sequence also contains a tar-like sequence in a stem-loop. Although tar-like sequences can enhance rates of translation, altering this sequence by mutagenesis had no effect on the rate of synthesis of rat beta-glucuronidase. Thus, although the region conferring enhanced rate of synthesis from rat cDNA has been identified, the mechanism by which it does so is not yet clear.
当使用几种不同的启动子在COS - 7细胞中表达人和啮齿动物β - 葡萄糖醛酸酶的cDNA时，尽管它们的转录水平相似，但啮齿动物β - 葡萄糖醛酸酶的产量是人β - 葡萄糖醛酸酶的三倍。在使用SV40早期或鸡β - 肌动蛋白启动子的LMTK - 细胞中也记录到了类似的观察结果。为了提高用于酶替代疗法的重组人β - 葡萄糖醛酸酶的产量，我们试图确定线性序列中负责啮齿动物cDNA更高表达水平的区域。为此，我们利用保守的限制性酶切位点制备了各种大鼠 - 人嵌合cDNA。通过活性测定以及对蛋白质进行代谢标记，随后进行免疫沉淀和SDS - PAGE，评估了这些嵌合cDNA在COS细胞中表达的产物水平。从这些表达研究的结果中，我们在大鼠开放阅读框中鉴定出一个155 bp的ClaI（643） - AflII（797）片段，该片段负责大鼠β - 葡萄糖醛酸酶（RBG）cDNA翻译速率的提高。用来自RBG的这个155 bp片段替换人β - 葡萄糖醛酸酶（HBG）中的同源ClaI（683） - AflII（838）片段，提高了所得嵌合HRaH的翻译水平。相反，在RBG cDNA中用155 bp的人片段替换大鼠片段，降低了所得嵌合HHaR的总合成量。将155 bp片段置于起始ATG和启动子之间对任何一种cDNA的表达都只有负面影响。该区域中人cDNA更稳定的二级结构可能解释了翻译速率的降低。然而，对大鼠和人cDNA的155 bp片段的mRNA进行二级结构分析预测，虽然两者都能形成茎环结构，但大鼠片段（ΔGzero = -42.1 kcal/mol）实际上比人片段（ΔGzero = -32.1 kcal/mol）更稳定。事实上，来自大鼠的这个ClaI - AflII片段内前50 bp的稳定性自由能（-10.3 kcal/mol）表明其二级结构比来自人的相应50 bp（-2.1 kcal/mol）稳定得多。大鼠序列的这一片段在一个茎环中还包含一个类似tar的序列。尽管类似tar的序列可以提高翻译速率，但通过诱变改变该序列对大鼠β - 葡萄糖醛酸酶的合成速率没有影响。因此，尽管已经鉴定出赋予大鼠cDNA合成速率提高的区域，但其作用机制尚不清楚。
