Generation of polyclonal antiserum for the detection of methylarginine proteins

This report describes an approach for the study of the biology of methylarginine proteins based on the generation of immunological reagents capable of recognizing the methylarginine status of cellular proteins. Two forms of an immunizing peptide were prepared based upon an amino acid sequence motif found most prevalently among verified dimethylarginine-containing proteins. One form of the peptide was constructed with 7 arginine residues alternating with 8 glycine residues. None of the arginines used in the synthesis were methylated. The alternative form of the peptide was synthesized with the identical repeating GRG sequence, but with asymmetrical dimethylarginine at each arginine residue. A methylarginine-specific antiserum was generated using the latter peptide. ELISA and western blotting of glycine arginine-rich peptides, each synthesized with or without asymmetric dimethylarginine, demonstrate the methyl specificity of the antiserum. The methylarginine-specific antibody co-localizes with the highly methylated native nucleolin protein conspicuously concentrated in the nucleolus. The methylarginine-specific antiserum recognizes a GRG peptide and bacterially expressed RBP16 only after incubation of the peptide or RBP16 with recombinant protein arginine methyltransferase 1, or cell extracts, respectively. Proteins isolated from cells in different developmental states exhibit different patterns of reactivity observed by western blots. Finally, the methylarginine-specific reagent interacts specifically with the methylarginine of cellular hnRNPA1 and human fragile X mental retardation protein expressed in cultured PC12 cells. An immunological reagent capable of detecting the methylarginine status of cellular methylproteins will facilitate the cellular and molecular analysis of protein arginine methylation in a wide variety of research and biomedical applications.