Enzymic methylation of arginyl residues in -gly-arg-gly- peptides.
N(G)-Methylation of arginine residues in many nucleic-acid-binding proteins are 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-1 TAR (Tat-responsive element) RNA-binding protein (SRB) and fibronectin also show the presence of the internal -Gly-Arg-Gly- (-GRG-) sequence, which is potentially methylatable by the methyltransferase. To investigate the sequence requirement for methylation of these proteins, several synthetic oligopeptides with different chain lengths and sequences similar to the -GRG- regions of SRB and fibronectin 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 microM, the 19-mer peptide (residues 10-28 in SRB) was methylated with a K(m) of 8.3 microM, indicating that a greater peptide chain length yields a better methyl acceptor. Product analysis of the methylated [methyl-(14)C]SRB-peptide by HPLC indicated the formation of N(G)-monomethylarginine and N(G),N(G)-dimethyl(asymmetric)arginine. Synthetic peptides containing the cell attachment sequence [Arg-Gly-Asp ('RGD')] in fibronectin, GRGDSPK, GGRGDSPK and GGGRGDSPK, were also studied; whereas GRGDSPK was a poor methyl acceptor, the longer peptides were better methyl acceptors. To provide an understanding of the effect of methylation on fibronectin peptide, arginine-unmethylated and methylated GGRGDSPK were compared for their effect on the mitogenesis induced by beta-hexosaminidase A and an agonistic antibody (mAb(15)) in bovine tracheal smooth-muscle cells; whereas the former inhibited 35-67% of mitogenesis at a concentration of 5-10 microM, the latter did not block mitogenesis. This lack of inhibition by the insertion of a methyl group on the arginyl residue of the cell attachment sequence might be due to the hindrance of the binding of fibronectin peptide to integrins.
Hyun
Y L
YL
Graduate School of Biotechnology, Korea University, 136, 5-ka Anam-dong, Sungbuk-ku, Seoul, 136-701 Korea.
Lew
D B
DB
Park
S H
SH
Kim
C W
CW
Paik
W K
WK
Kim
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004261:DNA Replication; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008745:Methylation; D009842:Oligopeptides / Q000378:metabolism*; D011484:Protein-Arginine N-Methyltransferases / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats
D005353:Fibronectins; D009842:Oligopeptides; D001120:Arginine; D011484:Protein-Arginine N-Methyltransferases