Protein L-Isoaspartyl Methyltransferase Catalyzes in Vivo Racemization of Aspartate-25 in Mammalian Histone H2B

Abstract

Protein L-isoaspartyl methyltransferase (PIMT) has been implicated in the repair or metabolism of proteins containing atypical L-isoaspartyl peptide bonds. The repair hypothesis is supported by previous studies demonstrating in vitro repair of isoaspartyl peptides via formation of a succinimide intermediate. Utilization of this mechanism in vivo predicts that PIMT modification sites should exhibit significant racemization as a side reaction to the main repair pathway. We therefore studied the D/L ratio of aspartic acid at specific sites in histone H2B, a known target of PIMT in vivo. Using H2B from canine brain, we found that Asp 25 (the major PIMT target site in H2B) was significantly racemized, exhibiting D/L ratios as high as 0.12, whereas Asp 51 , a comparison site, exhibited negligible racemization (D/L < 0.01). Racemization of Asp 25 was independent of animal age over the range of 2-15 years. Using H2B from 2-3-week mouse brain, we found a similar D/L ratio (0.14) at Asp 25 in wild type mice, but substantially less racemization (D/L ‫؍‬ 0.035) at Asp 25 in PIMT-deficient mice. These findings suggest that PIMT functions in the repair, rather than the metabolic turnover, of isoaspartyl proteins in vivo. Because PIMT has numerous substrates in cells, these findings also suggest that D-aspartate may be more common in cellular proteins than hitherto imagined and that its occurrence, in some proteins at least, is independent of animal age. Protein L-isoaspartyl methyltransferase (PIMT)1 selectively methylates atypical L-isoaspartyl sites in proteins (1-4). The formation of isoaspartate (see Fig. 1) is a common occurrence at certain Asn-Xaa and Asp-Xaa sites in proteins, especially when such sites fall in a flexible domain (5-7). This mechanism explains the propensity and sequence dependence of spontaneous protein deamidation at asparagine residues under mild conditions (8). The formation of isoaspartyl sites at aspartyl residues is also quite common (5, 7, 9) but has not been well appreciated because isomerization of Asp-Xaa linkages alters neither the mass nor the charge (at neutral pH) of a polypeptide.The formation of isoaspartate in proteins is generally viewed as a deleterious event associated with protein aging (10). It has been suggested that PIMT-dependent methylation of isoaspartyl sites serves to either repair the damaged sites or to tag the damaged proteins for degradation (11,12). A repair function is supported by in vitro studies using PIMT and defined polypeptide substrates (13-15). These studies indicate the pathway shown in Fig. 2 whereby methylation serves to activate the atypical ␣-carboxyl of the isoaspartyl site. This activation promotes rapid, spontaneous demethylation via formation of the same succinimide intermediate that occurs during isoaspartate formation. Each cycle of methylation/demethylation converts ϳ15-30% of the isoaspartyl linkage to a normal aspartyl linkage; however, after multiple cycles of methylation/demethylation, a majority of isoaspartyl sites are converted to normal asp...

Changes in leukocyte gene expression profiles induced by antineoplastic chemotherapy

“…In rat nucleosomes, the histone H2B was found to accumulate isoAsp in the Asp25-Gly26 bond in the N-terminal tail. In chicken nucleosomes, isoAsp accumulated mainly in histone H2A and, to a lesser extent, in histone H2B (20)(21)(22).…”

Comparison of the in vitro and in vivo stability of a succinimide intermediate observed on a therapeutic IgG1 molecule

“…28,29 The succinimide intermediate can also undergo racemization resulting in D-Asp residues being incorporated into the molecule. 14,30 Thus, pharmacokinetic (PK) information on the therapeutic antibody and an estimate of the kinetics of deamidation for different asparagine residues on the molecule are important for assessing the exposure and criticality of the PTM.…”

Collapse of Homochirality of Amino Acids in Proteins from Various Tissues during Aging

“…The earlier studies simply reported the presence of d-Asp in whole tissues, and the specific sites at which Asp residues racemize to form d-Asp were not known. Recently, the specific sites of d-Asp were identified in proteins from lens [4] [5], the b-amyloid protein in brain [9], histone of canine brain [10], and type-I collagen tellopeptide in urine [14]. We have also studied the mechanism of formation of d-Asp in a specific lens protein [21].…”