The formation of L-Isoaspartate (IsoAsp) residue in proteins and peptides is a spontaneous nonenzymatic post-translational modification (PTM) occurring under physiological conditions. This modification can significantly impact protein and peptide structure, function, stability, and the ability to form fibrills. The enzyme Isoaspartyl O-methyl transferase (PIMT), also known as L-Isoaspartyl/D-aspartyl protein carboxyl methyltransferase (PCMT), catalyzes repair of IsoAsp-containing proteins and peptides by transfer of methyl residue to the carboxyl group side chain consecutively leading to the formation of aspartate. Although IsoAsp accumulation in proteins is recognized as an age-dependent PTM, PIMT-deficient organisms without aging symptoms exhibit early physiological abnormalities, disrupted calcium signaling, and epileptic seizures, ultimately resulting in premature death. Administration of an adenovirus expressing PIMT partially alleviates some symptoms and extends the organism’s lifespan. In a young PIMT-deficient mouse, IsoAsp levels increased ninefold, indicating the important role of PIMT in the adjustment of this PTM concentration. However, PIMT activity declines with age due to reduced co-factor S-adenosyl methionine (SAM) levels, a methyl donor in converting IsoAsp to a methyl ester, and subsequent formation of Asp residue.
The known age-dependent accumulation of IsoAsp residues has traditionally focused research on its occurrence in aging subjects and relatively long-lived proteins. However, the PIMT involvement in developmental processes and the substantial accumulation of IsoAsp in PIMT-deficient organisms demonstrates that IsoAsp formation occurs throughout an organism’s lifetime. Here, we explore IsoAsp formation in a short-lived peptide Gal.
Neuroendocrine peptides serve as intercellular signaling molecules, pivotal in several physiological processes, including development. A notable example is the expression of Gal in the ventrobasal thalamus of mice, which coincides with the formation of the whisker map and influences subcortical circuit wiring. Consequently, alterations to the mature peptide sequence, such as the isomerization to IsoAsp or D-amino acids, can profoundly impact the peptide’s functionality and stability. Despite this, the presence of IsoAsp in short-lived neuroendocrine peptides and its potential physiological and pathological implications remains largely unexplored.
We identified the presence of IsoAsp residues in galanin (Gal) peptides using liquid chromatography trap ion mobility mass spectrometry (LC-TIMS-MS), PIMT, and endoproteinase AspN. Significant IsoAsp residues were characterized in matured Gal peptides extracted from the rat hypothalamus. Our findings show that IsoAsp facilitates amyloid fibril formation in rat Gal. Notably, the action of PIMT significantly reduces or even prevents Gal fibrillization.
Recent studies reveal that Gal and prolactin are stored as amyloid fibrils in the secretory granules of female rats. Given that IsoAsp residues are known to promote amyloid fibril formation, the occurrence of IsoAsp in Gal may contribute to its fibrillization within secretory granules.
The discovery of IsoAsp in Gal expands our understanding of aspartate isomerization as an age-related PTM affecting long-lived proteins to its occurrence in short-lived neuropeptides, potentially influencing their biological function, stability, and accumulation. Our findings help explain the physiological abnormalities experienced by PIMT-knocked-out organisms since neuropeptides play a vital role in development.
Rat Gal (GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-NH 2), isomers were initially identified in hypothalamic extracts by LC-TIMS-MS. Two well-resolved chromatographic peaks formed by ions with identical molecular mass but demonstrating noticeable mobility differences were analyzed by tandem mass spectrometry. The fragment ions for both peaks confirmed that they represent Gal. The sequence of endogenous Gal was validated against the fragment ions of synthetic Gal standard. The elution of identical peptides at different retention times and showing different ion mobility suggests a possible structural difference between them due to a zero-Dalton modification. We hypothesized that one of the two Gal peptides contains an isomerized amino acid resulting from aspartate isomerization. In rat Gal, aspartate residues are located at positions 17 and 24, while asparagine residues are found at positions 5 and 18. These residues can potentially isomerize under harsh extraction conditions; hereafter, an isotopically labeled Gal synthetic standard (molecular mass difference +9 Da) was spiked into a rat hypothalamic tissue homogenate during analyte extraction. A single chromatographic peak was observed for the isotopically labeled synthetic standard at m/z 793.90, z = 4 + . In contrast, the occurrence of Gal peptide at multiple retention times (rt) previously observed in the hypothalamic extract remained, suggesting that the observation of Gal isomeric peaks is not caused by our analyte extraction protocol.
Dehydration and cyclization of aspartate residues lead to the formation of four isomers: L-Asp, L-isoAsp, D-Asp, and D-isoAsp.
