Enzymic methylation of arginyl residues in -Gly-Arg-Gly- peptides

NG-Methylation of arginine residues in many nucleic-acid-binding proteins a re formed post-translationally, catalysed by S-adenosylmethionine: protein- arginine N-methyltransferase in their glycine-rich and arginine-rich motifs . The amino acid sequences of the stimulator of HIV-I TAR (Tat-responsive e lement) RNA-binding protein (SRB) and fibronectin also show the presence of the internal -Gly-Arg-Gly- (-GRG-) sequence, which is potentially methylat able by the methyltransferase. To investigate the sequence requirement for methylation of these proteins, several synthetic oligopeptides with differe nt chain lengths and sequences similar to the -GRG- regions of SRB and fibr onectin were synthesized. Whereas the heptapeptide AGGRGKG (residues 16-22 in SRB) served as the methyl acceptor for the methyltransferase with a K-m of 50 mu M, the 19-mer peptide (residues 10-28 in SRB) was methylated with a K, of 8.3 mu M, indicating that a greater peptide chain length yields a b etter methyl acceptor. Product analysis of the methylated [methyl-C-14]SRB- peptide by HPLC indicated the formation of N-G-monomethylarginine and N-G,N -G-dimethyl(asymmetric)-arginine. Synthetic peptides containing the cell at tachment sequence [Arg-Gly-Asp ('RGD')] in fibronectin, GRGDSPK, GGRGDSI'K and GGGRGDSPK, were also studied; whereas GRGDSPK was a poor methyl accepto r, the longer peptides were better methyl accepters. To provide an understa nding of the effect of methylation on fibronectin peptide, arginine-unmethy lated and methylated GGRGDSPK were compared for their effect on the mitogen esis induced by beta-hexosaminidase A and an agonistic antibody (mAb(15)) i n bovine tracheal smooth-muscle cells;whereas the former inhibited 35-67 % of mitogenesis at a concentration of 5-10 mu M, the latter did not block mi togenesis. This lack of inhibition by the insertion of a methyl group on th e arginyl residue of the cell attachment sequence might be due to the hindr ance of the binding of fibronectin peptide to integrins.