Isomeric Replacement of a Single Aspartic Acid Induces a Marked Change in Protein Function: The Example of Ribonuclease A

lα-Aspartic acid (Asp) residues in proteins are nonenzymatically isomerized to abnormal lβ-, dα-, and dβ-Asp isomers under physiological conditions. Such an isomerization of Asp residues is considered to be a trigger of protein denaturation because it either elongates the main chain or induces a different orientation of the side chain within the protein structure or both. However, previous studies have found no direct evidence of the effects of Asp isomers on protein function. Therefore, the production of Asp-isomer-containing proteins is required to verify the effects of Asp isomerization. Here, we describe the production of an Asp-isomer-containing protein using the expressed protein ligation. As a model protein, bovine pancreatic ribonuclease A (RNase A, EC 3.1.27.5), which catalyzes the cleavage of phosphodiester bonds in RNA, was used. In this study, lα-Asp at position 121 in RNase A was replaced by lβ-, dα-, and dβ-Asp. The objective aspartic acid at position 121 is located near the active site and related to RNA cleavage. The RNase A with lα-Asp at position 121 showed a normal activity. By contrast, the catalytic activity of lβ-, dα-, and dβ-Asp-containing RNase A was markedly decreased. This study represents the first synthesis and analysis of a protein containing four different Asp isomers

Identification of ᴅ-amino acid-containing peptides in human serum

<div><p>Biologically uncommon d-aspartate (d-Asp) residues have been shown to accumulate in proteins associated with age-related human disorders, such as cataract and Alzheimer disease. Such d-Asp-containing proteins are unlikely to be broken down completely because metabolic enzymes recognize only proteins or peptides composed exclusively of l-amino acids. Therefore, undigested d-Asp-containing peptides may exist in blood and, if detectable, may be a useful biomarker for associated diseases. In this study, we investigated d-amino acid-containing peptides in adult human serum by a qualitative d-amino acid analysis based on a diastereomer method and LC-MS/MS method. As a result, two d-Asp-containing peptides were detected in serum, both derived from the fibrinogen β-chain, a glycoprotein that helps in the formation of blood clots. One of the peptides was fibrinopeptide B, which prevents fibrinogen from forming polymers of fibrin, and the other was same peptide with C-terminal Arginine missing. To our knowledge, this is the first report of the presence of d-amino acid-containing peptides in serum and the approach described will provide a new direction on the serum proteome and fragmentome.</p></div

Isolation of peptides from human serum.

<p>RP-HPLC chromatogram of the serum peptides from the donor aged ~60 years. The detailed RP-HPLC conditions are described in Materials and Methods. Arrows indicate d-Asp-containing peptides.</p

Potential mechanism of isomerization of Asp/Asn in proteins and peptides.

<p>Potential mechanism of isomerization of Asp/Asn in proteins and peptides.</p

Determination of the d/l ratio of Asp in human serum-derived peptides.

<p>Elution profile of the enantiomeric separation of Asp derivatives using Boc-l-Cys-OPA. Aspartate residues from the hydrolysates of peptide peaks 16 (<b>a</b>) and 18 (<b>b</b>) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189972#pone.0189972.g001" target="_blank">Fig 1</a> (serum sample from the donor aged ~60 years) are shown. The d/l ratio of Asp was estimated as 0.08 in both peaks by calculating the peak areas of the chromatograms.</p

Identification of d-Asp-containing peptides by LC-MS/MS analysis.

<p>(<b>a</b>) LC-MS chromatogram of peak 16 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189972#pone.0189972.g001" target="_blank">Fig 1</a>. This peptide was separated into two peaks with the same mass ([M+2H]²⁺ = 777.5), indicating two isomers of the same peptide. (<b>b-1</b>) Tandem mass spectrum of the large peak in (<b>a</b>). (<b>b-2</b>) Tandem mass spectrum of the small peak in (<b>a</b>). (<b>c</b>) LC-MS chromatogram of peak 18 ([M+2H]²⁺ = 698.9) in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189972#pone.0189972.g001" target="_blank">Fig 1</a>. (<b>d</b>) Tandem mass spectrum of the peak in (<b>c</b>). Both peptides were identified as fibrinogen β-chain-specific peptides (peak 16, ¹QGVNDNEEGFFSAR¹⁴; and peak 18, ¹QGVNDNEEGFFSA¹³). The N-terminal Gln residue was converted to pyro-Glu in both peptides.</p