Pahlich Steffen, Bschir Karim, Chiavi Claudio, Belyanskaya Larisa, Gehring Heinz
Biochemisches Institut, Universität Zürich, Switzerland.
Proteins. 2005 Oct 1;61(1):164-75. doi: 10.1002/prot.20579.
The multifunctional Ewing Sarcoma (EWS) protein, a member of a large family of RNA-binding proteins, is extensively asymmetrically dimethylated at arginine residues within RGG consensus sequences. Using recombinant proteins we examined whether type I protein arginine methyltransferase (PRMT)1 or 3 is responsible for asymmetric dimethylations of the EWS protein. After in vitro methylation of the EWS protein by GST-PRMT1, we identified 27 dimethylated arginine residues out of 30 potential methylation sites by mass spectrometry-based techniques (MALDI-TOF MS and MS/MS). Thus, PRMT1 recognizes most if not all methylation sites of the EWS protein. With GST-PRMT3, however, only nine dimethylated arginines, located mainly in the C-terminal region of EWS protein, could be assigned, indicating that structural determinants prevent complete methylation. In contrary to previous reports this study also revealed that trypsin is able to cleave after methylated arginines. Pull-down experiments showed that endogenous EWS protein binds efficiently to GST-PRMT1 but less to GST-PRMT3, which is in accordance to the in vitro methylation results. Furthermore, methylation of a peptide containing different methylation sites revealed differences in the site selectivity as well as in the kinetic properties of GST-PRMT1 and GST-PRMT3. Kinetic differences due to an inhibition effect of the methylation inhibitor S-adenosyl-L-homocysteine could be excluded by determining the corresponding K(i) values of the two enzymes and the K(d) values for the methyl donor S-adenosyl-L-methionine. The study demonstrates the strength of MS-based methods for a qualitative and quantitative analysis of enzymic arginine methylation, a posttranslational modification that becomes more and more the object of investigations.
多功能尤文肉瘤（EWS）蛋白是RNA结合蛋白大家族的成员之一，在RGG共有序列内的精氨酸残基上广泛发生不对称二甲基化。我们使用重组蛋白研究了I型蛋白精氨酸甲基转移酶（PRMT）1或3是否负责EWS蛋白的不对称二甲基化。通过GST-PRMT1对EWS蛋白进行体外甲基化后，我们使用基于质谱的技术（基质辅助激光解吸电离飞行时间质谱和串联质谱）在30个潜在甲基化位点中鉴定出27个二甲基化精氨酸残基。因此，PRMT1识别EWS蛋白的大部分（如果不是全部）甲基化位点。然而，对于GST-PRMT3，只能确定9个主要位于EWS蛋白C末端区域的二甲基化精氨酸，这表明结构决定因素阻止了完全甲基化。与先前的报道相反，本研究还表明胰蛋白酶能够在甲基化精氨酸后进行切割。下拉实验表明，内源性EWS蛋白与GST-PRMT1有效结合，但与GST-PRMT3的结合较少，这与体外甲基化结果一致。此外，对含有不同甲基化位点的肽进行甲基化显示，GST-PRMT1和GST-PRMT3在位点选择性以及动力学特性方面存在差异。通过测定两种酶的相应抑制常数（Ki）值和甲基供体S-腺苷-L-甲硫氨酸的解离常数（Kd）值，可以排除甲基化抑制剂S-腺苷-L-高半胱氨酸抑制作用导致的动力学差异。该研究证明了基于质谱的方法在酶促精氨酸甲基化定性和定量分析中的优势，酶促精氨酸甲基化是一种越来越成为研究对象的翻译后修饰。
