Methylation at specific altered aspartyl and asparaginyl residues in glucagon by the erythrocyte protein carboxyl methyltransferase.
Protein carboxyl methyltransferases from erythrocytes and brain appear to catalyze the esterification of L-isoaspartyl and/or D-aspartyl residues but not of normal L-aspartyl residues. In order to identify the origin of these unusual residues which occur in subpopulations of a variety of cellular proteins, we studied the in vitro methylation by the erythrocyte enzyme of glucagon, a peptide hormone of 29 amino acids containing 3 aspartyl residues and a single asparagine residue. Methylated glucagon was digested with either trypsin, chymotrypsin, pepsin, or endoproteinase Arg C, and the labeled fragments were separated by high-performance liquid chromatography and identified. In separate experiments, methyl acceptor sites were determined by digesting glucagon first with proteases and then assaying purified glucagon fragments for methyl acceptor activity. Using both approaches, we found that the major site of methylation, accounting for about 62% of the total, was at the position of Asp-9. Chemical analysis of fragments containing this residue indicated that this site represents an L-isoaspartyl residue. A second site of methylation, representing about 23% of the total, was detected at the position of Asn-28 and was also shown to represent an L-isoaspartyl residue. Methyl acceptor sites were not detected at the positions of Asp-15 or Asp-21. Preincubation of glucagon under basic conditions (0.1 M NH4OH, 3 h, 37 degrees C) increased methylation at the Asn-28 site by 4-8-fold while methylation at the Asp-9 site remained unchanged. These results suggest that methylation sites can originate from both aspartyl and asparaginyl residues and that these sites may be distinguished by the effect of base treatment.
D000595:Amino Acid Sequence; D001216:Asparagine; D001224:Aspartic Acid; D001921:Brain / Q000201:enzymology*; D002918:Chymotrypsin; D004912:Erythrocytes / Q000201:enzymology*; D005934:Glucagon / Q000378:metabolism*; D006801:Humans; D008745:Methylation; D010446:Peptide Fragments / Q000032:analysis; D011496:Protein Methyltransferases / Q000378:metabolism*; D011497:Protein O-Methyltransferase / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism; D013379:Substrate Specificity; D014357:Trypsin
D010446:Peptide Fragments; D001224:Aspartic Acid; D001216:Asparagine; D012436:S-Adenosylmethionine; D005934:Glucagon; D011496:Protein Methyltransferases; D011497:Protein O-Methyltransferase; D002918:Chymotrypsin; D014357:Trypsin