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

Young Lan Hyun, D. Betty Lew, Seung Hee Park, Chan Wha Kim, Woon Ki Paik, Sangduk Kim

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

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 μM, the 19 mer peptide (residues 10-28 in SRB) was methylated with a K(m) of 8.3 μM, indicating that a greater peptide chain length yields a better methyl acceptor. Product analysis of the methylated [methyl-14C]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 accepters. 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 β-hexosaminidase A and an agonistic antibody (mAb15) in bovine tracheal smooth-muscle cells; whereas the former inhibited 35-67% of mitogenesis at a concentration of 5-10 μM, 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.

Original languageEnglish
Pages (from-to)573-578
Number of pages6
JournalBiochemical Journal
Volume348
Issue number3
DOIs
Publication statusPublished - 2000 Jun 15

Fingerprint

Methylation
Fibronectins
glycyl- arginyl-glycyl-aspartyl-seryl-prolyl-lysine
Peptides
Arginine
Methyltransferases
Chain length
Protein-Arginine N-Methyltransferases
Hexosaminidase A
omega-N-Methylarginine
S-Adenosylmethionine
Oligopeptides
RNA-Binding Proteins
Integrins
Glycine
Nucleic Acids
Smooth Muscle Myocytes
Muscle
HIV-1
Amino Acid Sequence

Keywords

  • Fibronectin-peptide
  • N(G)-methylarginine
  • Protein methylase I
  • RNA-binding protein
  • S-adenosylmethionine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Enzymic methylation of arginyl residues in -Gly-Arg-Gly- peptides. / Hyun, Young Lan; Lew, D. Betty; Park, Seung Hee; Kim, Chan Wha; Paik, Woon Ki; Kim, Sangduk.

In: Biochemical Journal, Vol. 348, No. 3, 15.06.2000, p. 573-578.

Research output: Contribution to journalArticle

Hyun, Young Lan ; Lew, D. Betty ; Park, Seung Hee ; Kim, Chan Wha ; Paik, Woon Ki ; Kim, Sangduk. / Enzymic methylation of arginyl residues in -Gly-Arg-Gly- peptides. In: Biochemical Journal. 2000 ; Vol. 348, No. 3. pp. 573-578.
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AU - Kim, Sangduk

PY - 2000/6/15

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N2 - 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 μM, the 19 mer peptide (residues 10-28 in SRB) was methylated with a K(m) of 8.3 μM, indicating that a greater peptide chain length yields a better methyl acceptor. Product analysis of the methylated [methyl-14C]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 accepters. 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 β-hexosaminidase A and an agonistic antibody (mAb15) in bovine tracheal smooth-muscle cells; whereas the former inhibited 35-67% of mitogenesis at a concentration of 5-10 μM, 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.

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KW - S-adenosylmethionine

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