Enzymic methylation of arginyl residues in -gly-arg-gly- peptides.
Enzymic methylation of arginyl residues in -gly-arg-gly- peptides.
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DOI：
10.1042/0264-6021:3480573
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发表时间：
2000-06
期刊：
影响因子：
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通讯作者：
Y. Hyun;D. Lew;S. Park;Chan-Wha Kim;W. Paik;Sangduk Kim
中科院分区：
文献类型：
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作者：
Y. Hyun;D. Lew;S. Park;Chan-Wha Kim;W. Paik;Sangduk Kim
许多核酸结合蛋白中精氨酸残基的 N(G)-甲基化是在翻译后形成的，由富含甘氨酸和富含精氨酸的基序中的 S-腺苷甲硫氨酸：蛋白质-精氨酸 N-甲基转移酶催化。 HIV-1 TAR（Tat 反应元件）RNA 结合蛋白 (SRB) 和纤连蛋白刺激物的氨基酸序列也显示存在内部 -Gly-Arg-Gly- (-GRG-) 序列，该序列可能被甲基转移酶甲基化。为了研究这些蛋白质甲基化的序列要求，合成了几种具有不同链长和与SRB和纤连蛋白的-GRG-区域相似的序列的合成寡肽。七肽 AGRGKG（SRB 中的残基 16-22）作为甲基转移酶的甲基受体，K(m) 为 50 microM，而 19 肽（SRB 中的残基 10-28）被甲基化，K(m) 为 8.3 microM，表明肽链长度越大，甲基受体越好。通过HPLC对甲基化[甲基-(14)C]SRB-肽的产物分析表明形成了N(G)-单甲基精氨酸和N(G),N(G)-二甲基(不对称)精氨酸。还研究了纤连蛋白、GRGDSPK、GGRGDSPK 和 GGGRGDSPK 中含有细胞附着序列 [Arg-Gly-Asp ('RGD')] 的合成肽；虽然 GRGDSPK 是较差的甲基受体，但较长的肽是更好的甲基受体。为了了解甲基化对纤连蛋白肽的影响，比较了精氨酸未甲基化和甲基化的 GGRGDSPK 对牛气管平滑肌细胞中 β-己糖胺酶 A 和激动性抗体 (mAb(15)) 诱导的有丝分裂的影响；前者在 5-10 µM 浓度下抑制 35-67% 有丝分裂发生，而后者则不会阻止有丝分裂发生。在细胞附着序列的精氨酰残基上插入甲基缺乏抑制可能是由于纤连蛋白肽与整联蛋白的结合受到阻碍。
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.