Isoaspartate Formation Research Articles

In vitro aging of calmodulin generates isoaspartate at multiple Asn–Gly and Asp–Gly sites in calcium‐binding domains II, III, and IV

We have determined the major sites responsible for isoaspartate formation during in vitro aging of bovine brain calmodulin under mild conditions. Protein L-isoaspartyl methyltransferase (EC 2.1.1.77) was used to quantify isoaspartate by the transfer of methyl-3H from S-adenosyl-L-[methyl-3H]methionine to the isoaspartyl (alpha-carboxyl) side chain. More than 1.2 mol of methyl-acceptor sites per mol of calmodulin accumulated during a 2-week incubation without calcium at pH 7.4, 37 degrees C. Analysis of proteolytic peptides of aged calmodulin revealed that > 95% of the methylation capacity is restricted to residues in the four calcium-binding domains, which are predicted to be highly flexible in the absence of calcium. We estimate that domains III, IV, and II accumulated 0.72, 0.60, and 0.13 mol of isoaspartate per mol of calmodulin, respectively. The Asn-97-Gly-98 sequence (domain III) is the greatest contributor to isoaspartate formation. Other major sites of isoaspartate formation are Asp-131-Gly-132 and Asp-133-Gly-134 in domain IV, and Asn-60-Gly-61 in domain II. Significant isoaspartate formation was also localized to Asp-20, Asp-22, and/or Asp-24 in domain I, to Asp-56 and/or Asp-58 in domain II, and to Asp-93 and/or Asp-95 in domain III. All of these residues are calcium ligands in the highly conserved EF-hand calcium-binding motif. Thus, other EF-hand proteins may also be subject to isoaspartate formation at these ligands. The results support the idea that isoaspartate formation in structured proteins is strongly influenced by both the C-flanking residue and by local flexibility.

Formation of Isoaspartate at Two Distinct Sites during in vitro Aging of Human Growth Hormone

In vitro aging at pH 7.4, 37 degrees C causes natural sequence recombinant human growth hormone (rhGH), methionyl rhGH, and human pituitary growth hormone to become substrates for bovine brain protein carboxyl methyltransferase, an enzyme that modifies the "side chain" alpha-carboxyl group present at atypical isoaspartyl linkages. The substrate capacity of rhGH increased at a rate of 1.8 methyl-accepting sites/day/100 molecules of hormone. Reversed-phase high performance liquid chromatography (HPLC) of trypsin digests of aged rhGH revealed two altered peptides not present in digests of control rhGH. These two fragments, which had the amino acid compositions of residues 128-134 (Leu-Glu-Asp-Gly-Ser-Pro-Arg) and 146-158 (Phe-Asp-Thr-Asn-Ser-His-Asn-Asp-Asp-Ala-Leu-Leu-Lys), contained the majority of the induced methylation sites, 22 and 58%, respectively. Isoaspartate can result from deamidation of asparagine or isomerization of aspartate. Isomerization of Asp-130, the only candidate site in 128-134, was corroborated by coelution of the altered fragment with the synthetic isoaspartyl peptide upon reversed-phase HPLC. Evidence is presented that the altered 146-158 fragment is a mixture of two peptides resulting from deamidation of Asn-149 to form 70-80% isoaspartate and 20-30% aspartate at this position. The position of isoaspartate in the altered 146-158 fragment was deduced from mass spectrometry, which indicated a single deamidated asparagine; from methylation stoichiometry, which indicated only one methylation site; and from automated Edman degradation, which showed an absence of asparagine and a low yield of aspartate at position 149. These results show that isoaspartate formation from both aspartate and asparagine is a significant, and possibly the major, source of spontaneous covalent alteration of rhGH and that enzymatic carboxyl methylation provides a powerful tool for assessing this type of modification.

Methylation of calmodulin at carboxylic acid residues in erythrocytes. A non-regulatory covalent modification?

The physiological role of protein carboxy-group methylation reactions in human erythrocytes was studied with calmodulin as an endogenous methyl-group acceptor. The steady-state degree of calmodulin carboxy-group methylation is substoichiometric both in intact cells and in a lysed-cell system (about 0.0003 mol of methyl groups/mol of polypeptide). Purified erythrocyte calmodulin is a substrate for a partially purified erythrocyte carboxy-group methyltransferase and can be methylated to the extent of about 0.0007-0.001 mol of methyl groups/mol of polypeptide. This erythrocyte protein methyltransferase displays an apparent specificity for atypical racemized and/or isomerized D-aspartate and L-isoaspartate residues [McFadden & Clarke (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2460-2464; Murray & Clarke (1984) J. Biol. Chem. 259, 10722-10732]. Exposure of calmodulin to elevated temperatures before methylation results in racemization of aspartate and/or asparagine residues, and may result in isoaspartate formation as well. The methylatability of these samples also increases as a function of time of heating, independent of the pH (over the range pH 5-9) or Ca2+ concentration; the most significant increase occurs during the initial 60 min, when calmodulin retains a fraction of its biological activity. These results are consistent with the hypothesis that methylation of calmodulin may occur at these uncommon aspartate residues, but are not consistent with a regulatory role for the methylation reaction.