Apoxin I, a Novel Apoptosis-inducing Factor with L-Amino Acid Oxidase Activity Purified from Western Diamondback Rattlesnake Venom

Abstract

Venom of the western diamondback rattlesnake (Crotalus atrox) induces apoptosis in human umbilical vein endothelial cells, which could result in hemorrhage in tissues bitten by the snake. To identify the hemorrhagic factor, we purified a novel protein, apoxin I, from rattlesnake venom. Apoxin I induced apoptosis in human umbilical vein endothelial, human promyelocytic leukemia HL-60, human ovarian carcinoma A2780, and mouse endothelial KN-3 cells. Amino acid sequence analysis of the apoxin I showed close similarity to L-amino acid oxidase from the Malayan pit viper (Calloselasma rhodostoma). The purified apoxin I oxidized L-leucine but not D-leucine to produce H 2 O 2 . The apoxin I-induced apoptosis was inhibited by catalase, a H 2 O 2 scavenger. These results indicate that the H 2 O 2 produced by L-amino acid oxidation by apoxin I is involved in the apoxin I-induced apoptosis and in hemorrhage caused by rattlesnake venom.Apoptosis is a physiological process by which cells undergo controlled cell death, accompanied by nuclear condensation and fragmentation prior to loss of membrane integrity (1-3). Many kinds of stimuli have been reported to induce apoptosis in a variety of cell systems. These include ligation of Fas and tumor necrosis factor receptors by their ligands (4 -8), deprivation of growth factors (9), and cytotoxic stimuli such as anticancer drugs (10 -12) and ionized radiation (13). In addition to these well characterized stimuli, hemorrhagic snake venoms from the western diamondback rattlesnake (Crotalus atrox), mamushi (Agkistrodon halys blomhottii), puff adder (Bitis arietans), and habu (Trimeresurus flavoviridis) induce apoptosis in vascular endothelial cells (14), although the active component in the venom is not identified. We report here the identification of the apoptosis-inducing factor apoxin I from venom of the western diamondback rattlesnake that has Lamino acid oxidase (LAO, 1 EC 1.4.3.2) activity. EXPERIMENTAL PROCEDURESMaterials-Lyophilized crude venom from the western diamondback rattlesnake was purchased from Sigma. Benzyloxycarbonyl-Val-AlaAsp-CH 2 OC(O)-2,6-dichlorobenzene (Z-VAD) and benzyloxycarbonylAsp-CH 2 OC(O)-2,6-dichlorobenzene (Z-Asp) were synthesized at Kirin Brewery Co. Ltd (Gunma, Japan) as described (15) and dissolved in Me 2 SO at 10 mg/ml. Trolox (Aldrich) was dissolved in 1 M NaHCO 3 at a concentration of 300 mM, and the pH was adjusted to 7.0. For the experiments, the solution was diluted to the desired concentration with medium. Catalase and all other chemicals were purchased from Wako Pure Chemicals (Tokyo, Japan) and Sigma.Purification of Apoxin I-Lyophilized crude venom (100 mg) from the western diamondback rattlesnake (Sigma) was dissolved in 5 ml of distilled water, and the insoluble material was removed by centrifugation (1,000 ϫ g) for 10 min at 4°C. The supernatant was applied to Sephadex G-100 column (Pharmacia Biotech Inc.; 3 ϫ 60 cm) equilibrated with 50 mM sodium phosphate buffer containing 0.15 M sodium chloride (pH 7.0), and the proteins were eluted ...

Purification and Cloning of an Apoptosis-Inducing Protein Derived from Fish Infected with Anisakis simplex, a Causative Nematode of Human Anisakiasis

“…Thus, Fig 1 protein appears functionally unrelated to AIP. While the physiological role of snake venom LAO is unclear, its antibacterial and apoptosis-inducing activities have been demonstrated (6,7). LAO is also not an ER rumen protein and does not induce apoptosis in HL-60 cells at concentrations lower than 2.5 g/ml even by 24-h treatment (6), while treatment with 20 ng/ml of AIP induced apoptosis in HL-60 cells within 2 h (Fig.…”

Characterization and Expression of l-Amino Acid Oxidase of Mouse Milk

“…One cDNA fragment was obtained, and the entire nucleotide sequence from the 5Ј-to 3Ј-end was determined in the present experiments. The nucleotide sequence of this cDNA has high similarity to those for snake venom L-amino acid oxidase (LAO) (8), snake venom Apoxin 1 (9), and Fig1 protein in the mouse B cell (10). LAO catalyzes the oxidative deamination of particular L-amino acids (i.e.…”

Human IL4I1 is a secreted l-phenylalanine oxidase expressed by mature dendritic cells that inhibits T-lymphocyte proliferation

“…Most of the previously described LAAO enzymes present proapoptotic effects that have been related to H 2 O 2 production. 8,26,27 We had no difficulties in developing stable cell lines with constitutive or inducible expression of active IL4I1 in HEK293 cells. While the enzyme is probably functionally active inside the cell, it is highly compartmentalized, restricted to vesicles ( Figure 3B and data not shown) where it is likely to be physically separated from its substrate, and hence metabolically inactive.…”