Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Effect of amino acids present at the carboxyl end of succinimidyl residue on the rate constants for succinimidyl hydrolysis in small peptides
Graphical abstract
Introduction
Non-enzymatic post-translational modifications are age-related changes observed in amino acids within a given polypeptide sequence. For example, structural alterations of asparaginyl/aspartyl (Asx) residues have been observed in several proteins, where Asx residues are hotspots for non-enzymatic post-translational modification [1]. Structural alterations at Asx residues have been found in several amyloidogenic proteins, such as Alzheimer's β-amyloid protein, α-synuclein, ß2-microglobulin, and copper‑zinc-superoxide dismutase [2]. The proteins bearing structurally altered residues demonstrate varied fibril formation (e.g., certain forms of fibrillization were drastically accelerated). Thus, the spontaneous structural alteration at Asx residues is a pathogenic factor in various age-related diseases associated with altered protein conformation.
As shown in Fig. 1, the reaction mechanism involving the succinimidyl intermediate is widely used for structurally altering Asx residues [3]. Briefly, the carbonyl group of Asx residue's side chain is attacked by the nitrogen atom of the adjacent peptide bond. Consequently, a five-membered l-succinimidyl ring is formed, which is readily hydrolyzed and generates l-aspartyl and l-isoaspartyl residues at a ratio of 1:3 to 1:4. Some l-succinimidyl intermediates undergo reversible stereoinversion, resulting in the generation of d-aspartyl and d-isoaspartyl residues. Therefore, l-isoaspartate is the major product of Asx-structural alteration via the succinimidyl reaction. Moreover, l-isoaspartate accumulates in proteins damaged by ageing. The l-isoaspartyl residue changes the local charge and inserts an additional carbon on the polypeptide chain. Therefore, the accumulation of l-isoaspartyl residues results in a significant loss of protein function [4,5].
The repair system for l-isoaspartyl residues within a polypeptide chain has been identified in various organisms. The l-isoaspartyl residues are repaired by protein l-isoaspartyl methyltransferase (PIMT), which has been identified in plants, bacteria, insects, nematodes, and mammals [[6], [7], [8], [9], [10], [11], [12], [13]]. The enzyme transfers a methyl group from S-adenosyl-l-methionine (SAM) to the carboxyl group of l-isoaspartate, resulting in the formation of l-isoaspartyl α-methyl ester residues (Fig. 1). The methyl ester on the side chain of the l-isoaspartyl residue facilitates the nucleophilic attack of the peptide bond's amide nitrogen; thus, the formation of a succinimidyl ring is accelerated. The succinimidyl rings are further hydrolyzed, thereby generating l-aspartyl and l-isoaspartyl residues. Repetition of this cycle isomerizes the majority of l-isoaspartyl residues into l-aspartyl residues. Evidence has shown that knockout mice lacking PIMT could not repair l-isoaspartyl residues; consequently, damaged proteins containing l-isoaspartyl residues significantly accumulated in the brain cytosol fraction. Finally, the knockout mice succumbed to fatal seizures and did not survive beyond 42 d after birth [14]. In contrast, the life span of PIMT-overexpressing Drosophila melanogaster was prolonged by 32–39% as compared to the control flies [15].
The rate of succinimidyl formation from Asx residues is regulated by the primary amino acid sequence as well as the conformational flexibility near the Asx residues. Studies using artificial peptides, GXNZG, where X and Z represent any of the 18 different amino acids, revealed that the amino acids at the carboxyl side of asparaginyl residue exerted a substantial influence on the asparaginyl deamidation rate. When Z was a glycine (Gly), histidine (His), serine (Ser), or alanine (Ala) residue, deamidation occurred rapidly [16]. Similar results were obtained using artificial peptides VSNXV [17]. Kinetic studies using the peptide VYPDXA, where X represents Gly, Ser, or Ala, also showed that the rate constant of succinimidyl formation in the peptide bearing a Gly residue was 6.5 times faster than that for a peptide bearing an Ala residue [18]. Succinimidyl formation is required for the conformational flexibility near Asx residues [[19], [20], [21], [22]]. Comparison of the β-linkage isomerization of aspartyl residues between the protein and small peptides showed that the isomerization of some aspartyl residues was considerably suppressed, suggesting that the tertiary structure reduces succinimidyl formation [19].
In contrast, studies related to succinimidyl hydrolysis are limited due to the difficulty in handling succinimidyl residues, which are unstable intermediates with a half-life of only a few hours [3]. In this study, we established procedures for preparing peptides bearing succinimidyl residues using the repair enzyme PIMT, which allowed us to prepare the succinimidyl peptide of the desired sequence just before the experiments were performed. The effect of amino acid residues at the carboxyl side of succinimidyl intermediates on succinimidyl hydrolysis was subsequently elucidated.
Section snippets
Preparation of peptides
The synthetic peptides used in this study are listed below. These peptides were prepared using standard Fmoc chemistry. All amino acids, except Gly and aspartate (Asp) residues, were in an l-configuration. The peptides bearing l-isoaspartyl, d-aspartyl, and d-isoaspartyl residues were prepared using building blocks of Fmoc-l-Asp-OtBu, Fmoc-d-Asp(OtBu)-OH, and Fmoc-d-Asp-OtBu (Watanabe Chemical Co. Ltd., Tokyo, Japan), respectively. The synthesized peptides were purified by reversed-phase
Preparation and characterization of rPIMT
rPIMT bearing a His-tag at the N-terminus was expressed in E. coli at 25 °C overnight to reduce protein aggregation. rPIMT was purified using a Ni2+ resin column and concentrated by dialysis operations (2 mg/mL). The purity of rPIMT was found to be around 80%, judging from the densitometric analysis of Coomassie Brilliant Blue staining after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (data not shown). The purified rPIMT was stable at 4 °C but unstable when frozen. To determine
Discussion
Studies of the rate constant for succinimidyl formation (k1′, k1, and k3 in Fig. 1) have been previously reported [3,[16], [17], [18], [19], [20], [21], [22],26,30] because succinimidyl formations can be studied easily using peptides or proteins bearing asparaginyl or aspartyl residues. In contrast, studies on the rate constants (k2 and k4 in Fig. 1) for succinimidyl hydrolysis are quite limited because of the unstable feature of the succinimidyl intermediate, the half-life of which is reported
Conclusions
We used recombinant PIMT as a tool for the preparation of peptides bearing succinimidyl intermediates and determined the rate constants for the formation/hydrolysis of succinimidyl intermediates to investigate the influence of the amino acid sequence on succinimidyl reactions. The formation/hydrolysis of the succinimidyl intermediate is an essential phenomenon for structural alterations of Asx residues. Computational estimation revealed that structural alterations in Asx residues occurred at a
Author contributions
YS: Conceptualization, Funding acquisition, Project administration, Writing - original draft, Writing - review & editing. SS, TD, AT, HT, SM: Investigation.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by JSPS KAKENHI Grant Number JP16K08207 and JP19K07031.
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2020, Biochemical Journal