Different methylation characteristics of protein arginine methyltransferase 1 and 3 toward the Ewing Sarcoma protein and a peptide.
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.
Pahlich
Steffen
S
Biochemisches Institut, Universität Zürich, Switzerland.
Bschir
Karim
K
Chiavi
Claudio
C
Belyanskaya
Larisa
L
Gehring
Heinz
H
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011484:Protein-Arginine N-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D034802:RNA-Binding Protein EWS / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010446:Peptide Fragments; D034802:RNA-Binding Protein EWS; D011994:Recombinant Proteins; D011484:Protein-Arginine N-Methyltransferases