Isoaspartate in peptides and proteins: formation, significance, and analysis

Aswad, Dana W.; Paranandi, Mallik V.; Schurter, Brandon T.

doi:10.1016/s0731-7085(99)00230-7

Cited by 227 publications

(199 citation statements)

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“…Cyclic imide; Hydroxylamine; Isoaspartate; IsoAsp; Protein L-isoaspartyl-O-methyltransferase; Sadenosyl-L-methionine; Succinimide Formation of isoaspartyl (isoAsp) 2 peptide bonds (also known as β-aspartyl bonds) at AsnXaa or Asp-Xaa linkages constitutes a major source of spontaneous protein damage under physiological conditions [1][2][3]. Formation of isoAsp arises from deamidation of asparaginylXaa bonds or direct isomerization of aspartyl-Xaa bonds.…”

Section: Introductionmentioning

confidence: 99%

Selective cleavage of isoaspartyl peptide bonds by hydroxylamine after methyltransferase priming

Zhu

Aswad

2007

Analytical Biochemistry

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Formation of atypical isoaspartyl (isoAsp) sites in peptides and proteins via the deamidation-linked isomerization of asparaginyl-Xaa bonds or direct isomerization of aspartyl-Xaa bonds is a major contributor to spontaneous protein damage under mild conditions. This nonenzymatic reaction reroutes the Asx-Xaa peptide bond through the β-carbonyl of asparaginyl or aspartyl residues, thereby adding an extra carbon to the polypeptide backbone. Formation of isoAsp has been implicated in protein inactivation, aggregation, degradation, and autoimmunity. Knowing the location of isoAsp sites in proteins is important for understanding mechanisms of protein damage and for characterizing protein pharmaceuticals. Here we present a simple nonradioactive method for direct localization of isoAsp residues in peptides or proteins. Using three model peptides, we demonstrate that isoAsp linkages can be cleaved selectively and in high yield by a two-step process in which (i) the isoAsp linkage is converted into a succinimide on incubation with S-adenosyl-L-methionine and the commercially available enzyme, protein L-isoaspartyl-O-methyltransferase, and (ii) the succinimidyl bond is then cleaved by hydroxylamine under conditions that minimize cleavage of the traditional hydroxylamine-sensitive Asn-Gly and related peptide bonds. Location of the isoAsp linkage is then inferred by identifying the cleavage products by mass spectrometry or N-terminal sequencing. KeywordsCyclic imide; Hydroxylamine; Isoaspartate; IsoAsp; Protein L-isoaspartyl-O-methyltransferase; Sadenosyl-L-methionine; Succinimide Formation of isoaspartyl (isoAsp) 2 peptide bonds (also known as β-aspartyl bonds) at AsnXaa or Asp-Xaa linkages constitutes a major source of spontaneous protein damage under physiological conditions [1][2][3]. Formation of isoAsp arises from deamidation of asparaginylXaa bonds or direct isomerization of aspartyl-Xaa bonds. This process occurs via a succinimide (cyclic imide) intermediate generated when the C-flanking amide nitrogen makes a nucleophilic attack on the side-chain carbonyl group of the Asx residue (Fig. 1). Hydrolysis of the succinimide generates isoAsp and aspartyl linkages in a ratio that typically ranges from 70:30 to 85:15 [4,5]. The propensity for isoAsp formation is determined by local sequence and structural flexibility. Extensive in vitro studies indicate that sequences containing Asn-Gly, Asn-Ser, Asp-Gly, and Asn-His are most susceptible to isoAsp formation, particularly in highly flexible regions [6,7]. Under normal physiological conditions, the aberrant isoAsp linkage is converted back to a normal peptide bond by protein L-isoaspartyl-O-methyltransferase (PIMT, EC 2.1.1.77), a highly conserved and ubiquitous enzyme (Fig. 1) The overall content of isoAsp in a polypeptide can be conveniently determined by its ability to accept methyl groups from S-adenosyl-L-methionine (AdoMet) in a reaction catalyzed by the PIMT enzyme [15,16]. PIMT is available commercially as part of an isoAsp detection kit that can be used in e...

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Section: Introductionmentioning

confidence: 99%

Selective cleavage of isoaspartyl peptide bonds by hydroxylamine after methyltransferase priming

Zhu

Aswad

2007

Analytical Biochemistry

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“…One enzyme that participates in repairing such damaged protein is PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT; EC 2.1.1.77). This enzyme catalyzes the transfer of a methyl group from S-adenosyl-methionine (AdoMet) to the free a-carboxyl group of abnormal L-isoAsp residues as well as the b-carboxyl group of D-aspartyl residues (Lowenson and Clarke, 1992;Aswad et al, 2000). Such PIMT-catalyzed methylation can initiate their conversion back to normal Laspartyl residues and eventually reestablish the protein's inherent functions (Johnson et al, 1987;McFadden and Clarke, 1987).…”

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PROTEIN l-ISOASPARTYL METHYLTRANSFERASE2 Is Differentially Expressed in Chickpea and Enhances Seed Vigor and Longevity by Reducing Abnormal Isoaspartyl Accumulation Predominantly in Seed Nuclear Proteins

et al. 2013

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PROTEIN L-ISOASPARTYL METHYLTRANSFERASE (PIMT) is a widely distributed protein-repairing enzyme that catalyzes the conversion of abnormal L-isoaspartyl residues in spontaneously damaged proteins to normal aspartyl residues. This enzyme is encoded by two divergent genes (PIMT1 and PIMT2) in plants, unlike many other organisms. While the biological role of PIMT1 has been elucidated, the role and significance of the PIMT2 gene in plants is not well defined. Here, we isolated the PIMT2 gene (CaPIMT2) from chickpea (Cicer arietinum), which exhibits a significant increase in isoaspartyl residues in seed proteins coupled with reduced germination vigor under artificial aging conditions. The CaPIMT2 gene is found to be highly divergent and encodes two possible isoforms (CaPIMT2 and CaPIMT29) differing by two amino acids in the region I catalytic domain through alternative splicing. Unlike CaPIMT1, both isoforms possess a unique 56-amino acid amino terminus and exhibit similar yet distinct enzymatic properties. Expression analysis revealed that CaPIMT2 is differentially regulated by stresses and abscisic acid. Confocal visualization of stably expressed green fluorescent protein-fused PIMT proteins and cell fractionation-immunoblot analysis revealed that apart from the plasma membrane, both CaPIMT2 isoforms localize predominantly in the nucleus, while CaPIMT1 localizes in the cytosol. Remarkably, CaPIMT2 enhances seed vigor and longevity by repairing abnormal isoaspartyl residues predominantly in nuclear proteins upon seed-specific expression in Arabidopsis (Arabidopsis thaliana), while CaPIMT1 enhances seed vigor and longevity by repairing such abnormal proteins mainly in the cytosolic fraction. Together, our data suggest that CaPIMT2 has most likely evolved through gene duplication, followed by subfunctionalization to specialize in repairing the nuclear proteome.

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“…Amongst these, deamidation of asparaginyl residues and isomerization of aspartyl residues arise most frequently during aging (Aswad et al 2000). These modifications result in altered aspartyl residues such as L-isoaspartate, in which the peptide backbone is transferred from the a-carboxyl group to the side-chain b-carboxyl group, and D-aspartate, in which the natural L-amino acid is converted to the rare D-isomer.…”

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Down‐regulation of protein l‐isoaspartyl methyltransferase in human epileptic hippocampus contributes to generation of damaged tubulin

Lanthier

Bouthillier

Lapointe

et al. 2002

Journal of Neurochemistry

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Protein L-isoaspartyl methyltransferase (PIMT) repairs the damaged proteins which have accumulated abnormal aspartyl residues during cell aging. Gene targeting has elucidated a physiological role for PIMT by showing that mice lacking PIMT died prematurely from fatal epileptic seizures. Here we investigated the role of PIMT in human mesial temporal lobe epilepsy. Using surgical specimens of hippocampus and neocortex from controls and epileptic patients, we showed that PIMT activity and expression were 50% lower in epileptic hippocampus than in controls but were unchanged in neocortex. Although the protein was down-regulated, PIMT mRNA expression was unchanged in epileptic hippocampus, suggesting post-translational regulation of the PIMT level. Moreover, several proteins with abnormal aspartyl residues accumulate in epileptic hippocampus. Microtubules component b-tubulin, one of the major PIMT substrates, had an increased amount (two-fold) of L-isoaspartyl residues in the epileptic hippocampus. These results demonstrate that the down-regulation of PIMT in epileptic hippocampus leads to a significant accumulation of damaged tubulin that could contribute to neuron dysfunction in human mesial temporal lobe epilepsy.

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Isoaspartate in peptides and proteins: formation, significance, and analysis

Cited by 227 publications

References 25 publications

Selective cleavage of isoaspartyl peptide bonds by hydroxylamine after methyltransferase priming

Selective cleavage of isoaspartyl peptide bonds by hydroxylamine after methyltransferase priming

PROTEIN l-ISOASPARTYL METHYLTRANSFERASE2 Is Differentially Expressed in Chickpea and Enhances Seed Vigor and Longevity by Reducing Abnormal Isoaspartyl Accumulation Predominantly in Seed Nuclear Proteins

Down‐regulation of protein l‐isoaspartyl methyltransferase in human epileptic hippocampus contributes to generation of damaged tubulin

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