Presence of Oxidized Protein Hydrolase in Human Cell Lines, Rat Tissues, and Human/Rat Plasma

Abstract

Oxidized protein hydrolase (OPH), an 80 kDa serine protease whose activity is inhibited by diisopropyl fluorophosphate (DFP), has been isolated from human erythrocytes [Fujino, T. et al. (1998) J. Biochem. 124, 1077-1085]. The presence of OPH in various biological samples was examined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting using an anti-OPH antibody raised against OPH purified from human erythrocytes, and by [(3)H]DFP-labeling and successive SDS-PAGE/fluorography. Solubilized samples of human cell lines including K-562 cells, THP-1 cells and Jurkat cells, and rat tissues including brain, heart, liver, kidney, and testis, inhibited the anti-OPH antibody binding to OPH in ELISA. Immunoblotting of lysates of K-562 cells, THP-1 cells and Jurkat cells showed four immunoreactive protein bands including an 80 kDa protein. Immunoprecipitation of the [(3)H]DFP-labeled K-562 cell lysate and successive SDS-PAGE/fluorography showed the presence of only the 80 kDa DFP-reactive protein with OPH antigenic activity. The level of the 80 kDa immunoreactive protein in K-562 cells rose as the cells differentiated toward erythrocytes. Immunoblotting of human and rat plasma showed two immunoreactive protein bands, including the 80 kDa protein, and SDS-PAGE/fluorography of [(3)H]DFP-labeled rat and human plasma showed the presence of only the 80 kDa DFP-reactive protein. The results indicate that OPH is present in a wide variety of biological samples.

A novel class of bifunctional acylpeptide hydrolases – potential role in the antioxidant defense systems of the Antarctic fish Trematomus bernacchii

“…In mammals, APEH has been found in many different types of cells and tissues, such as blood [12][13][14], brain [35,36], liver [23], and kidney [37]. Therefore, RBCs were chosen as the starting sample to optimize a purification procedure, which allowed us to isolate the most abundant of the two APEH isoforms, corresponding to a 75-kDa band detected on a western blot (Fig.…”

“…Mammalian cells have developed a hierarchy of defense processes, in which antioxidants provide the first defensive mechanism, and proteolytic systems act as secondary defenses [11]. Among these, acylpeptide hydrolase (APEH), first identified as oxidized protein hydrolase [12][13][14], was recently hypothesized to participate in the degradation of oxidized and cytotoxic proteins [15][16][17]. For these reasons, APEH may represent a promising therapeutic target for a wide array of human diseases caused by the accumulation of damaged proteins [18,19].…”

Increased Oxidized Protein Hydrolase Activity in Serum and Urine of Diabetic Rat Models

“…8) The purification 7) of OPH from human erythrocytes and several investigations [8][9][10][11][12][13] to elucidate its physiological function have been carried out by Kikugawa and his group. Based on a homology search regarding the amino acid sequence, OPH has subsequently been identified 9) as the same enzyme as acylpeptide hydrolase (ACPH) 14,15) or acylamino acid-releasing enzyme (AARE) (EC 3.4.19.1), which releases N-acylamino acids from N-acylpeptides; both a chymotrypsin-like endopeptidase activity originally demonstrated with OPH and an exopeptidase activity demonstrated with ACPH are shown to be shared by either OPH or ACPH.…”

“…OPH is known to be present in various tissues, 8) but the mechanism of its regulation remains unknown. Shimizu et al 12) previously evaluated the induction of OPH expression by treating COS-7 cells with H 2 O 2 as oxidative stress by immunological staining and Western blotting.…”

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Oxidative stress, caused by an imbalance between antioxidant activity and oxidant production within cells and plasma, is implicated in the pathogenesis of several diseases, including diabetes mellitus.1-3) Several mechanisms appear to be involved in the genesis of oxidative stress in diabetes mellitus, including glucose autoxidation, 4) protein glycation, 5) and the formation of advanced glycation end-products (AGEs).

6)As a consequence of oxidative stress, severe damage to proteins as well as DNA and lipids occurs.On the other hand, oxidized protein hydrolase (OPH) is a serine protease that preferentially degrades oxidized and glycated proteins rather than the corresponding unmodified proteins compared with other representative serine proteases, trypsin and chymotrypsin.7) The presence of OPH has been shown in a wide variety of biological samples, such as many different types of cells, tissues, and plasma.8) The purification 7) of OPH from human erythrocytes and several investigations [8][9][10][11][12][13] to elucidate its physiological function have been carried out by Kikugawa and his group. Based on a homology search regarding the amino acid sequence, OPH has subsequently been identified 9) as the same enzyme as acylpeptide hydrolase (ACPH) 14,15) or acylamino acid-releasing enzyme (AARE) (EC 3.4.19.1), which releases N-acylamino acids from N-acylpeptides; both a chymotrypsin-like endopeptidase activity originally demonstrated with OPH and an exopeptidase activity demonstrated with ACPH are shown to be shared by either OPH or ACPH.

9,10) Even though long known, the physiological significance of ACPH has been insufficiently clarified.

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