A growing interest in post-translational modification of proteins by arginine methylation has been sparked by studies linking protein methylation to regulation of signal transduction, transcription, RNA metabolism and protein targeting within cells (Bedford and Richard, 2005
). Several methylarginine-specific antibodies have been utilized to successfully identify hundreds of putative arginine methylated proteins (Boisvert et al., 2003
). In most cases, the antibody reagents were raised against methyl-peptides derived from confirmed methylarginine proteins; SAM68, SmD3 and nucleolin. An antibody raised against a symmetrical dimethylarginine decapeptide has also previously been described (Boisvert et al., 2002
). In the current work, a novel immunological reagent is described based on a 16-mer peptide sequence which harbors asymmetric dimethylarginines in a motif that is widely represented in many methylarginine proteins. The target peptide was designed with two particular characteristics: seven tandem repeats of the minimal arginine methylation consensus motif, GRG, and asymmetric dimethylation at each and every arginine residue. Unlike other antibodies raised against asymmetric dimethylarginine antigens, anti-mRG was generated using the repetitive tri-amino acid consensus motif that is characteristic of many asymmetric dimethylarginine proteins. Previously generated anti-asymmetric dimethylarginine antibodies have instead employed immunizing peptides, the sequences of which, were derived from single, specific methylated proteins. In addition, a peptide identical in every respect save for the asymmetric dimethyl-modification of the arginine residues was produced as well. The latter peptide serves as a control immunogen for the production of antisera that reacts only poorly with methylarginine in peptides and proteins. Both of the antibodies characterized in this work (anti-mRG and anti-RG) add to the tools available in protein methylation research.
The anti-mRG rabbit polyclonal antibody described in this report is expected to be useful for the analysis of asymmetric dimethylarginine proteins such as the heterogeneous nuclear ribonucleoproteins (Liu and Dreyfuss, 1995
). Preliminary experiments with Y12-reactive SmD3 (cf. Boisvert et al., 2002
; Hebert et al., 2002
) indicate that anti-mRG does not react with symmetrically dimethylated arginine proteins (unpublished results). The number of methylarginine residues, the symmetrical or asymmetrical configuration of the dimethylarginine epitope and peptide length of the immunogen may contribute to the protein recognition profile exhibited by the particular antibody preparations. For example, the SYM10 reagent (Boisvert et al., 2002
) raised against a decapeptide containing 4 symmetric dimethylarginines was poorly reactive with the same decapeptide harboring only a single symmetrically dimethylarginine or a peptide derived from myelin basic protein with a single symmetric dimethylarginine. Despite the specificity of antibody preparations for symmetric and asymmetric dimethylarginine, the methylation status of any protein that reacts with a methylarginine-specific antibody must be confirmed by other means.
Anti-mRG may be generally useful in two broad applications. First, with regard to the detection of known methylproteins, the antibody can be used to detect changes in the relative degree of methylation. Second, anti-mRG provides another experimental approach to the identification of undisclosed methylarginine proteins. In the latter case, the use of the anti-RG reagent can serve as a useful control. For example, there are a number of anti-mRG-reactive proteins that are uniquely present in either the procyclic or bloodstream forms of trypanosomes, none of which react with anti-RG (). In the case of PC12 cells undergoing neuronal differentiation, there is a ~30 kilodalton protein band that reacts more strongly with anti-mRG than with anti-RG in protein lysates from NGF-treated cells (). Based on the findings of our experiments, the use of anti-RG, an immunological reagent prepared against the identical primary peptide sequence as anti-mRG but without any methyl-modification, serves as a suitable means of controlling for cross-reactivity to non-methylarginine epitopes.
The potential involvement of methylarginine proteins in human disease is quite broad (Bedford and Richard, 2005
). Several neurodevelopmental diseases arise from gene mutations that implicate methylarginine proteins in the support of normal neuronal functions. Deletion or loss of function mutations of the survival motor neuron gene 1 (SMN1
) result in spinal muscular atrophy (Lefebvre et al., 1995
). SMN protein binds preferentially to symmetric dimethylarginine proteins important for the assembly and processing of specific RNA-protein complexes (Friesen et al., 2001
). Fragile X syndrome results from dynamic mutation, trinucleotide repeat expansion of the fragile X mental retardation gene 1 (FMR1
). Repression of FMR1
due to the expanded CGG repeat leads to a decrease of FMR1
product, the fragile X mental retardation protein (FMRP). Stetler et al. (2006)
have recently demonstrated the presence of asymmetric dimethylarginine in Flag-tagged FMRP that was immunoprecipitated from mammalian cells. The dimerization and recruitment of FMRP into large stress granules is, furthermore, methylation-dependent (Dolzhanskaya et al., 2006
). Various mutations in MECP2
are responsible for Rett syndrome, one of the most common causes of mental retardation in females. Recombinant MeCP2 protein exhibits three GRG methylation consensus sites, is an excellent in vitro
substrate for PRMT1 methylation and the native protein immunoprecipitated from cell lysates is recognized by anti-mRG (unpublished observations, Aletta lab). MeCP2 is a methyl CpG binding protein that acts as a DNA silencer. Thus, all of these neurodevelopmental genes that are associated with methylarginine modification may conceivably produce abnormal neuronal gene expression when disease-causing mutations are present.
Further experiments will be required to define the role of asymmetric dimethylarginine protein modification in developmental events and to understand better the neurotrophin-stimulated effects on protein methylation. Future work to accomplish these goals will be facilitated by the use of anti-mRG and other methylarginine-specific antibodies.