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

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 rho-dostoma ). 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 -a-mino 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)(2)(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 PROCEDURES
Materials-Lyophilized crude venom from the western diamondback rattlesnake was purchased from Sigma. Benzyloxycarbonyl-Val-Ala-Asp-CH 2 OC(O)-2,6-dichlorobenzene (Z-VAD) and benzyloxycarbonyl-Asp-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 with the same buffer at the rate of 0.4 ml/min and collected (4 ml/fraction). The fractions containing apoptosis-inducing activity (fractions 24 -32) were pooled and dialyzed against 2 liters of distilled water for 2 days. The active fractions were applied to isoelectric focusing by Rotofor (Bio-Rad) with a constant voltage of 12 watts for 6 h using ampholyte 3/10. The fractions containing apoptosis-inducing activity were pooled (pI, 6.0 -6.5), subsequently loaded on a Superdex 200 HR 10/30 column (Pharmacia) connected to a TSK 3000 PWXL (Toso, Tokyo, Japan) equipped with a fast protein liquid chromatography system (Pharmacia), and eluted with 50 mM sodium phosphate buffer containing 0.15 M sodium chloride (pH 7.0) at the rate of 0.2 ml/min. The elution profile was monitored at 280 nm. SDS-polyacrylamide gel electrophoresis analysis was carried out as described previously (16), and proteins were stained using a two-dimensional silver stain kit (Daiichi, Tokyo, Japan).
Cell Lines and Cell Culture-Human umbilical vein endothelial cells obtained from InvitroCYTE (Seattle, WA) were cultured and maintained in endothelial cell growth medium with low serum growth supplement according to the manufacturer's instructions. The cells were cultured in collagen-coated dishes (Iwaki Grass, Tokyo, Japan). Human promyelocytic leukemia HL-60, human ovarian carcinoma A2780, and mouse endothelial KN-3 cells were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan) and maintained in RPMI 1640 medium (Nissui) supplemented with 10% heat-inactivated fetal bovine serum and 100 mg/ml kanamycin in a humidified atmosphere of 5% CO 2 and 95% air. All experiments were performed using cells during the exponential growth phase.
Nuclear Morphological Changes-Cells (0.5 ϫ 10 6 ) were treated with 10 g/ml purified apoxin I for 18 h, except for HL-60 cells, which were treated for 4 h, in the growth medium and then harvested. After cells were washed in phosphate-buffered saline and fixed in methanol/acetate (3:1) for 10 min, cells were stained with 20 l of 1 g/ml 4,6diamidino-2-phenylindole (Sigma); one drop of fixed cells was placed on a glass slide. Nuclear morphology was observed by a Nikon UFX-IIA fluorescent microscope as described previously (17).
DNA Fragmentation Assay-After treatment with apoxin I, HL-60 cells (0.5 ϫ 10 6 ) were suspended in 20 l of 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 0.5 mg/ml proteinase K (Sigma). After incubation at 50°C for 1 h, 10 l of 0.5 g/ml RNase A solution was added, and the suspension was incubated for an additional hour. The sample was * This work was supported by a special grant for advanced research on cancer and grants-in-aid for Cancer Research from the Ministry of Education, Science and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Amino Acid Sequence-The purified apoxin I was blotted onto a polyvinylidene difluoride membrane (ProBlott, Applied Biosystems) and applied to Applied Biosystems 477A and Beckman LF3400D peptide sequencers. Data were analyzed by the sequencers automatically, and we confirmed the data by checking the chromatograms.
Measurement of LAO Activity-The reaction mixtures (1 ml) containing the 1 g/ml or 2 g/ml purified apoxin I, 50 g/ml horseradish peroxidase (5 milliunits/ml), 10 M O-dianisidine, and 10 M L-leucine or D-leucine in 100 mM Tris-HCl (pH 8.5) were incubated at 25°C, and the initial rate was measured as the increase in absorbance at 436 nm (19) with a Beckman DU 640 spectrophotometer.
Subcellular Distribution of Apoxin I-Purified apoxin I was labeled with fluorescein 5Ј-isothiocyanate as described previously (20). HL-60 cells were treated with the fluorescein 5Ј-isothiocyanate-labeled apoxin I (5 g/ml) for 4 h at 37°C, and observed by fluorescent microscopy.

RESULTS AND DISCUSSION
Crude venom from rattlesnake was first subjected to a Sephadex G-100 gel filtration column followed by a Rotofor isoelectric focusing apparatus; major apoptosis-inducing activity was detected in fractions of pI 6.0 -6.5. The active fractions were collected and applied to fast protein liquid chromatography equipped with a Superdex 200 HR 10/30 column directly connected to a TSK 3000 PWXL column. The activity was eluted as a single peak with a molecular mass of approximately 100 kDa (Fig. 1a). The purification yielded 1.48% of the crude venom proteins. According to the LAO activity (described below), the purified protein was concentrated 56-fold from the crude venom, and the recovery of the LAO activity was 83%. The purified protein, named apoxin I, showed a homogenous single band of 55 kDa in SDS-polyacrylamide gel electrophoresis analysis (Fig. 1b), suggesting that apoxin I has a dimeric conformation under natural conditions. Apoxin I at 10 g/ml induced chromatin condensation and segregation in human umbilical vein endothelial, HL-60, A2780, and KN-3 cells (Fig. 2). The nuclear morphological changes typical of apoptosis were also observed in U937 and K562 cells (not shown). To confirm the induction of apoptosis by apoxin I, we examined a DNA fragmentation assay in the apoxin I-treated cells. Apoxin I above 2.5 g/ml caused fragmentation of nuclear DNA into an oligosomal ladder in HL-60 cells (Fig. 3a), and the fragmentation began to occur 2 h after apoxin I treatment (Fig. 3b). Apoxin I at concentrations lower than 2.5 g/ml did not induce apoptosis in HL-60 cells even by 24 h of treatment. We further examined the apoxin I-induced apoptosis by flow cytometric analysis. As shown in Fig. 4, apoptotic cells containing DNA at less than G 1 dramatically increased when HL-60 cells were treated with 10 g/ml apoxin I for 4 h. In addition, inhibitors of the interleukin-1␤-converting enzyme family proteases Z-VAD and Z-Asp (11, 15) completely prevented the appearance of apoptotic populations in apoxin I-treated cells (Fig. 4, c and d). Z-VAD and Z-Asp also inhibited the nuclear condensation caused by 10 g/ml Apoxin I (not shown). These results indicate that apoxin I induced apoptosis in such cell lines.
We next analyzed the N-terminal amino acid sequence of the purified apoxin I. Nineteen of 20 amino acids were identified (Fig. 5). According to a protein sequence data base search, it was revealed that the sequence has a close similarity (75% identity) to LAO from Malayan pit viper (Calloselasma rhodostoma) venom and a moderate similarity (45% identity) to LAO from king cobra (Ophiophagus hannah) venom (19) (Fig. 5). The LAO from the Malayan pit viper is a homodimeric glycoprotein composed of a 66-kDa polypeptide as determined by SDS-polyacrylamide gel electrophoresis (19), resembling apoxin I. Therefore, we measured the LAO activity of the purified apoxin I. Apoxin I oxidized L-leucine in a dose-and time-dependent manner, whereas it did not oxidize D-leucine (Fig. 6), indicating that apoxin I is a LAO.
Treatment of apoxin I with heat (70°C, 10 min) or repeated freezing and thawing abolished both the LAO-and apoptosisinducing activity (not shown), suggesting the involvement of the LAO activity in apoxin I-induced apoptosis. The oxidation by LAO of L-amino acid under oxygenated conditions results in the production of the corresponding ␣-keto acid, NH 3 , and H 2 O 2 (21,22). Therefore, we tested the effects of scavengers of reactive oxygen species on the apoxin I-induced apoptosis. As shown in Fig. 7, catalase completely inhibited apoptosis in-duced by apoxin I and H 2 O 2 , indicating that H 2 O 2 mediates apoxin I-induced apoptosis. We prepared fluorescein 5Ј-isothiocyanate-labeled apoxin I to investigate subcellular localization of apoxin I by fluorescent microscopy. Our preliminary results showed that apoxin I accumulated in the cellular plasma membrane. Further study is now in progress. According to these results, we tested antioxidants for the inhibition of the apoxin I-induced apoptosis. A membrane antioxidant, trolox (23), but not a water-soluble antioxidant, L-ascorbate (24), inhibited the apoxin I-induced apoptosis (Fig. 7). These results suggest that apoptosis is triggered by membrane oxidation by H 2 O 2 produced by apoxin I accumulating in the cellular plasma membrane.
Venoms of several snake species contain large amounts of LAO, but its biological effects are largely unknown. It was reported that LAO from king cobra (O. hannah) venom induced platelet aggregation (22). The LAO-induced platelet aggregation is inhibited by catalase, suggesting that H 2 O 2 also mediates LAO-induced platelet aggregation (22). We do not know whether all the LAOs from different snake species show similar biological effects; however, it is likely they do, because H 2 O 2 mediated the biological effects of LAO. These observations, together with our present study, suggest that treatment with antioxidants might protect tissues affected by snake bite from various types of harmful snake venom.