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

Construction of Human Corneal Endothelial cDNA Library and Identification of Novel Active Genes

purpose. To describe genes expressed in human corneal endothelial cells and identify novel genes. methods. Sixteen human donor corneas that had no history of corneal disease, infection, or intraocular surgery were used within 7 days of death. Total RNA was extracted from corneal endothelial cells with attached Descemet membranes. A 3′-directed cDNA library was constructed from mRNA by using a pUC19-based primer. These sequences were compared with each other to determine their frequency and were searched against GenBank for identification. To identify novel specific and abundant transcript genes in corneal endothelial cells, the novel genes were compared with an expressed sequence tag database, the expected sequence extended, and 5′ rapid amplification of cDNA ends–polymerase chain reaction cloning performed. results. The human corneal endothelial cDNA library showed that the most abundant transcript was prostaglandin D2 synthase. The remaining transcript genes that were present in abundance consisted of lactate dehydrogenase-A, gene signature (GS) 3582, which is a novel gene without a known function, and matrix Gla protein. The full-length sequence of GS3582 showed similarity to genes obtained in ovary and testis. conclusions. A human corneal endothelial cDNA library was constructed. An expression profile of corneal endothelium provides probes to monitor physiologic and pathologic conditions of this tissue in terms of gene expression

Profile of miRNAs in small extracellular vesicles released from glioblastoma cells treated by boron neutron capture therapy

Purpose: Boron neutron capture therapy (BNCT) is a tumor cell-selective particle-radiation therapy. In BNCT, administered p-boronophenylalanine (BPA) is selectively taken up by tumor cells, and the tumor is irradiated with thermal neutrons. High-LET α-particles and recoil ⁷Li, which have a path length of 5–9 μm, are generated by the capture reaction between ¹⁰B and thermal neutrons and selectively kill tumor cells that have uptaken ¹⁰B. Although BNCT has prolonged the survival time of malignant glioma patients, recurrences are still to be resolved. miRNAs, that are encapsulated in small extracellular vesicles (sEVs) in body fluids and exist stably may serve critical role in recurrence. In this study, we comprehensively investigated microRNAs (miRNAs) in sEVs released from post-BNCT glioblastoma cells. Method: Glioblastoma U87 MG cells were treated with 25 ppm of BPA in the culture media and irradiated with thermal neutrons. After irradiation, they were plated into dishes and cultured for 3 days in the 5% CO₂ incubator. Then, sEVs released into the medium were collected by column chromatography, and miRNAs in sEVs were comprehensively investigated using microarrays. Result: An increase in 20 individual miRNAs (ratio > 2) and a decrease in 2 individual miRNAs (ratio < 0.5) were detected in BNCT cells compared with non-irradiated cells. Among detected miRNAs, 20 miRNAs were associated with worse prognosis of glioma in Kaplan Meier Survival Analysis of overall survival in TCGA. Conclusion: These miRNA after BNCT may proceed tumors, modulate radiation resistance, or inhibit invasion and affect the prognosis of glioma

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

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

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

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

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

Localization of D-β-Aspartyl Residue-Containing Proteins in Various Tissues

Prior to the emergence of life, it is believed that only L-amino acids were selected for formation of protein and that D-amino acids were eliminated on the primitive Earth. Whilst homochirality is essential for life, the occurrence of proteins containing D-β-aspartyl (Asp) residues in various tissues from elderly subjects has been reported recently. Here, we demonstrate the presence of a D-β-Asp-containing protein in the cardiac muscle of heart, blood vessels of the lung, chief cells of the stomach, longitudinal and circular muscle of the stomach, small intestine and large intestine. Since the D-β-Asp residue occurs through a succinimide intermediate, this isomer may potentially be generated in proteins more easily than initially thought. Formation of the D-β-Asp residue in proteins may be related to stress