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Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 19�26 |
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doi:10.1111/j.1745-7270.2008.00372.x |
L-amino acid oxidase from Naja atra
venom activates and binds to human platelets
Rui Li1, Shaowen Zhu1, Jianbo
Wu1,
Wanyu Wang1,
Qiumin Lu1*,
and Kenneth J. Clemetson2
1 Kunming Institute of Zoology, Chinese Academy of
Sciences, Kunming 650223, China
2 Theodor Kocher Institute, University of Berne,
Berne CH-3012, Switzerland
Received: August
15, 2007�������
Accepted: September
30, 2007
This work was
supported by the grants from the Natural Science Foundation� of China
(30770431), the Natural Science Foundation of Yunnan Science and Technology
Committee, Yunnan Province (2007C102M) and the Western Light Project of Chinese
Academy of Sciences
Abbreviations:
BCIP, 5-bromo-4-chloro-3-indolyl phosphate p-toluidine� salt;
biotin-NHS, biotinamidocaproate N-hydroxysuccinimide ester; BSA, bovine serum
albumin; Fcg, Fc receptor g chain; FITC, fluorescein-isothiocyanate; GPVI,
glycoprotein VI; LAAO, L-amino acid oxidase; LAT, T lymphocyte adapter protein;
NA-LAAO, L-amino acid oxidase from Naja atra venom; NBT, p-nitro blue
tetrazolium chloride; PBS, phosphate-buffered saline; PLC, phospholipase C;
PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecyl
sulfate-polyacrylamide gel electrophoresis; Src, Src kinase; Syk, spleen
tyrosine kinase; TBS, Tris-buffered saline.
*Corresponding author: Tel/ Fax, 86-871-5192476; E-mail,
[email protected]
An L-amino
acid oxidase (LAAO), NA-LAAO, was purified from the venom of Naja atra.
Its N-terminal sequence shows great similarity with LAAOs from other snake
venoms. NA-LAAO dose-dependently induced aggregation of washed human�
platelets. However, it had no activity on platelets in platelet-rich plasma. A
low concentration of NA-LAAO greatly promoted the effect of hydrogen peroxide,
whereas hydrogen peroxide itself had little activation effect on platelets.
NA-LAAO induced tyrosine phosphorylation of a number of platelet� proteins�
including Src kinase, spleen tyrosine kinase, and phospho�lipase C g2. Unlike
convulxin, Fc receptor g chain and T lymphocyte adapter protein are not
phosphorylated in NA-LAAO-activated platelets, suggesting an activation
mechanism different� from the glycoprotein VI pathway. Catalase� inhibited� the
platelet aggregation and platelet protein� phosphorylation induced by NA-LAAO.
NA-LAAO bound to fixed platelets as well as to platelet lysates of Western�
blots. Furthermore, affinity chromatography of platelet proteins� on an
NA-LAAO-Sepharose 4B column isolated a few platelet membrane proteins,
suggesting that binding of NA-LAAO to the platelet� membrane might play a role
in its action on platelets.
Keywords������� L-amino acid oxidase; Naja
atra; platelet; hydrogen peroxide
Venom L-amino acid oxidases (LAAOs) are homodimeric flavoenzymes that catalyze� the oxidative deamination of an L-amino acid substrate� to an a-keto acid along with the production of ammonia and hydrogen peroxide. They are widely distributed� in venomous snake families of Viperidae, Crotalidae� and Elapidae [1]. Each subunit contains� three domains: an FAD-binding domain; a substrate�-binding domain; and a helical domain [2]. Although� the mechanisms� are uncertain, venom LAAOs are reported to have various biological acti�vities including induction of apoptosis, induction� of oedema and haemolysis, antibacterial function, and platelet activation or inhibition. All the effects are thought to be at least partly related to H2O2 production because catalase, an H2O2 scavenger, inhibits the actions of venom LAAOs [1].
The reported effects of LAAOs on platelets are quite controversial. LAAO from Echis colorata inhibits ADP-induced platelet aggregation. LAAOs from Agkistrodon halys blomhoffii, Naja naja kaouthia, and king cobra inhibit� agonist-induced or shear stress-induced platelet aggregation� [3-5]. These reports suggested that the interaction� between activated platelet integrin aIIbb3 and fibrinogen was inhibited by the continuous generation of H2O2. LAAOs from some other snakes have been reported to have the totally opposite effect on platelets. LAAOs from Eristocophis macmahoni, Bothrops alternatus, and Trimeresurus jerdonii induce human platelet aggregation through formation of H2O2 [6-8]. It is still not clear how H2O2 functions in LAAO-induced platelet aggregation.
It is also possible that LAAOs activate platelets in a receptor�-dependent way. Several recent studies showed that H2O2 production might not be the whole story for the biological activities of LAAOs. LAAO from A. halys showed many binding and cytotoxic effects on different cell lines [9]. Hydrogen peroxide generated in the enzymatic� reactions was not sufficient to explain the degree to which bacterial growth was inhibited by a D-amino acid oxidase from hog kidney and an LAAO from the venom of A. halys. A fluorescence labeling assay showed that both of these enzymes bind to the surface of bacteria [10], and a novel LAAO from Trimeresurus stejnegeri showed dose-dependent inhibition on HIV-1 infection and replication. The presence of catalase resulted in an increase in its antiviral� selectivity. However, under the same conditions, no anti-HIV-1 activity was observed by exogenous addition� of H2O2 [11].
Here, we report the purification and characterization of an LAAO from Naja atra venom, named NA-LAAO. We show that it activates washed human platelets but not platelets� in platelet-rich plasma, and binds directly to platelets.
Materials and Methods
Materials
Lyophilized N. atra venom was from Yunnan Province, China. BSA, EDTA, protein A-Sepharose, peroxidase-conjugated� goat anti-mouse and anti-rabbit antibodies, fura-2/AM, FITC, BCIP, streptavidin-alkaline phosphatase, biotin-NHS, NBT, tetramethyl benzidine, horseradish peroxidase, catalase, L-leucine, and Triton X-100 were from Sigma (St. Louis, USA). Hydrogen peroxide was from Merck (Darmstadt, Germany). Sepharose 4B was from Amersham Biosciences (Piscataway, USA). The SuperSignal chemiluminescence detection system was from Pierce (Rockford, USA), and autoradiography (Fuji RX) films were from FujiFilm (Dielsdorf, Switzerland). Antiphosphotyrosine monoclonal antibody 4G10 was from Lucernachem (Luzern, Switzerland). PVDF membranes (PolyScreen) were from Dupont NEN (Boston, USA). Anti-LAT, anti-PLCg2, anti-Src, and anti-Syk antibodies were from Santa Cruz Biotechnology (Santa Cruz, USA). EMD 132338, an aIIbb3 inhibitor, was a kind gift from Merck. Convulxin was purified as described previously [12].
Purification of LAAO
Chromatography of N. atra venom on an SP-Sephadex C-25 column (5 cm�60 cm; Pharmacia, Uppsala, Sweden) was carried out as described previously [13]. Briefly, crude venom of N. atra (5 g) was dissolved in 20 ml of 50 mM sodium acetate buffer (pH 5.8) and applied to the SP-Sephadex C-25 column pre-equilibrated with the same buffer. Unbound protein was washed out with the same buffer and then a gradient of 0-1 M NaCl in the same buffer was applied at a flow rate of 60 ml/h. Fractions containing LAAO were collected, lyophilised, and loaded on a Fractogel EMD BioSEC 650(S) gel filtration column (1.6 cm120 cm; Merck, Whitehouse Station, USA) pre-equilibrated with 20 mM Tris-HCl buffer (pH 7.4) containing� 0.3 M NaCl, and then eluted at 30 ml/h for 15 h collecting 5 ml fractions. Active fractions were pooled and dialysed against 20 mM Tris-HCl buffer (pH 7.4) and applied� to a Bio-Scale Q2 column (Bio-Rad, Hercules, USA) equilibrated with the same buffer. The bound proteins� were eluted with a 0-0.6 M NaCl gradient in the same buffer at a flow rate of 30 ml/h. The active fractions were analyzed by SDS-PAGE silver staining and stored at 4 �C. The N-terminal amino acid sequence was determined by ABI model 476A protein sequencer (Applied Biosystems, Foster City, USA).
LAAO activity assay
A reaction mixture (200 ml) containing 1 mM L-leucine, 10 mM tetramethyl benzidine, and 10 mU/ml horseradish peroxidase in 0.1 M Tris-HCl buffer (pH 8.5) was incubated� at 25 �C. The reaction was started by adding crude fractions� or purified NA-LAAO and monitored at 450 nm over 10 min. One unit of the enzyme was defined as the oxidation of 1 mmol of L-leucine per minute.
SDS-PAGE, silver staining, and
protein determination
SDS-PAGE was carried out according to Laemmli [14] with a 7%-17% acrylamide gradient, and the gel was silver� stained by the method of Morrissey [15]. Protein determination was carried out by bicinchoninic acid protein assay (Pierce) with bovine albumin as standard.
Preparation of washed
platelets and platelet aggregation
Human platelets were isolated from human blood obtained from the Central Laboratory of the Swiss Red Cross Blood Transfusion Service (Berne, Switzerland). For 100 ml of human blood, 30 ml of 100 mM citrate, pH 6.5, was added. Platelet-rich plasma and the platelet pellet were isolated by successive centrifugation. Platelets were resuspended with buffer B containing 113 mM NaCl, 4.3 mM K2HPO4, 24.4 mM NaH2PO4, and 5.5 mM glucose, pH 6.5, and centrifuged� at 250 g for 5 min. The platelet-rich supernatant� was centrifuged at 1000 g for 10 min, and the platelets were washed once more with buffer B. Washed platelets were resuspended in buffer C containing 20 mM HEPES, 140 mM NaCl, 4 mM KCl, and 5.5 mM glucose, pH 7.4, and the platelet count was adjusted to 5108 platelets/ml by dilution with buffer C. Samples were kept at room temperature� until used for aggregation studies. Before aggregation� analysis, 2 mM CaCl2 and 2 mM MgCl2 were added and the platelets were incubated at 37 �C for 2 min. Platelet aggregation was measured by light transmission in an aggregometer (LumiTec, Paris, France) with continuous� stirring at 1100 rpm at 37 �C.
Time course of tyrosine
phosphorylation in platelets
Platelets were treated for aggregation. Aliquots (45 ml) were taken at fixed time points and the platelet suspension was lysed by adding the aliquots into 5 ml HEPES containing 10% SDS, 10 mM N-ethylmaleimide, 20 mM sodium orthovanadate, and 20 mM EDTA. After centrifugation, the supernatants were analyzed by a 7%-17% gradient SDS-polyacrylamide gel and electroblotted onto a PVDF membrane. The membrane was incubated with 2% BSA in TBS overnight. Tyrosine phosphorylated proteins were detected by 4G10 monoclonal antibody followed by peroxidase�-coupled rabbit anti-mouse secondary antibodies. Bound antibodies were detected using chemiluminescence.
Platelet biotinylation and
NA-LAAO-Sepharose 4B affinity chromatography
Human platelets were isolated from buffy coats as described� above but in the presence of 10 mM iloprost. Washed platelets were diluted with PBS to 5109 platelets/ml and incubated with 10 mg biotin-NHS for 1 h at room temperature. Free biotin-NHS was removed by washing the platelets three times with PBS, pH 6.8. Biotinylated platelets were solubilized in PBS containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 100 mM leupeptin, 2 mM N-ethylmaleimide, and 2 mM sodium orthovanadate. After centrifugation (12,000 g for 30 min at 4 �C), the supernatant was applied to a column of NA-LAAO-Sepharose 4B and eluted successively with TBS containing 0.2% octanoyl-N-methylglucamide and 0.1% or 0.5% SDS. The eluted fractions were analyzed using SDS-PAGE silver stain and Western blotting detected with phosphatase-labeled streptavidin followed by NBT/BCIP.
Binding of biotin-labeled
NA-LAAO to blots of platelet� lysates
NA-LAAO in 50 mM NaHCO3 was mixed with biotin-NHS dissolved in Me2SO (0.25 mg biotin-NHS/mg protein). The mixture was incubated at room temperature for 2 h. Unlabeled� biotin-NHS was removed by loading the sample to a Sephadex G-25 column eluted with TBS. Washed platelets (5�108 platelets/ml) were lysed in HEPES containing� 1% Triton X-100, 1 mM N-ethylmaleimide, 2 mM sodium orthovanadate, and 2 mM EDTA. After centrifugation, the supernatants were separated on a 7%-17% gradient SDS-PAGE and transferred onto a PVDF membrane. After blocking with 2% BSA overnight, a solution� containing biotin-labeled NA-LAAO was added to the membrane and incubated for 1 h. The membrane was washed four times with TBS containing 0.1% Tween 20. Bound biotin-NA-LAAO was detected with phosphatase-conjugated streptavidin followed by NBT/BCIP.
Immunoprecipitation
Aliquots (500 ml) of resting as well as activated platelets (5�108 platelets/ml) were solubilized in Tris-buffered saline� containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, 2 mM N-ethylmaleimide, 2 mM benzamidine, and 2 mM sodium orthovanadate. After centrifugation, platelet lysates, precleared with protein A-Sepharose, were stirred for 2 h with specific antibodies before adding 20 ml protein A-Sepharose followed by 6-8 h incubation. After washing, proteins were eluted from the protein A-Sepharose by boiling with 40 ml of 100 mM Tris-HCl, pH 7.5, 5% SDS, and 5 mM EDTA.
Flow cytometry analysis of
FITC-NA-LAAO binding to fixed platelets
Washed platelets were fixed with 1% formaldehyde in TBS at room temperature for 0.5 h. The fixed platelets were washed twice with TBS and then resuspended in TBS at 5107 platelets/ml. Then FITC-NA-LAAO was added to 0.1 ml platelets and shaken for 10 min at room temperature� in the dark. The platelets were washed twice with TBS and then analyzed by flow cytometry. For competent assay, 10-fold excess of unlabeled NA-LAAO was incubated with the platelets for 10 min at room temperature before adding FITC-labeled NA-LAAO.
Results
Purification of NA-LAAO from N.
atra venom
NA-LAAO was purified from N. atra venom by a three-step chromatography protocol including cation ion exchange� on an SP-Sephadex C-25 column, gel filtration on a Fractogel EMD BioSEC 650(S) column [Fig. 1(A)], and anion ion exchange on a Bio-Rad Q2 column [Fig. 1(B)]. The purified NA-LAAO was homogeneous on SDS-PAGE detected by silver staining, which showed it was a pure protein [Fig. 1(B), insert]. The N-terminal sequence of NA-LAAO was determined to be DDRRSPLEEC, which has high similarity to other venom LAAOs and is identical to the LAAO from N. kaouthia venom [Fig. 1(C)] [4]. The enzyme activity of NA-LAAO was 38.4 U/(mg∙min).
NA-LAAO activated platelets
A high dose (15 mg/ml) of NA-LAAO directly activated washed platelets (Fig. 2). However, it had no activation effect on platelets in platelet-rich plasma (data not shown). Catalase (600 U/ml) inhibited the platelet aggregation (Fig. 2). Several platelet proteins were phosphorylated in NA-LAAO-induced platelet aggregation [Fig. 3(A,B)]. Some of them were identified by immunoprecipitation to be Src, PLCg2, and Syk. However, unlike in convulxin-activated platelets [11], Fcg and LAT were not phosphorylated in NA-LAAO-activated platelets [Fig. 3(C)]. The phosphorylation of the proteins was also inhibited by catalase. In contrast, a low dose of NA-LAAO (1.5 mg/ml) did not induce platelet aggregation, but it potentiated the action of H2O2 (40 mM). H2O2 itself did not induce platelet aggregation and had little effect on platelet protein phosphorylation. Incubation of low doses of NA-LAAO or H2O2 did not change the platelet protein phosphorylation profile. Adding� H2O2 (40 mM) after incubation of a low dose of NA-LAAO for 3 min induced rapid platelet aggregation and phosphorylation of platelet proteins (Fig. 3). The activations were inhibited by catalase (Figs. 2 and 3). It is interesting that after frequent freezing and thawing, NA-LAAO retained its enzymatic activity. However, its activity on platelet aggregation was greatly impaired (data not shown).
Full activation of platelets
by NA-LAAO requires Ca2+ and aIIb/b3 activation
As shown in Fig. 4(A), EDTA and EMD 132338 inhibited the platelet aggregation induced by NA-LAAO. The inhibitory effect of EDTA was stronger than that of EMD 132338. Also the two inhibitors greatly inhibited the phosphorylation of platelet proteins [Fig. 4(B)].
NA-LAAO binds to platelets
NA-LAAO was labeled with FITC and incubated with fixed platelets. In flow cytometry assay, FITC-NA-LAAO bound to platelets and the binding was inhibited by 10-fold excess� unlabeled NA-LAAO [Fig. 5(A)]. Furthermore, biotin-labeled� NA-LAAO could bind to platelet lysate immobilized� on a PVDF membrane. A protein band of 66 kDa and a 72 kDa doublet were seen on the membrane incubated with biotin-NA-LAAO and stained with avidin-coagulated alkaline� phosphatase followed by NBT/BCIP [Fig. 5(B)].
Platelet proteins bind to
NA-LAAO
Biotin-labeled platelets lysed by Triton X-100 were loaded onto an NA-LAAO-Sepharose 4B column. The fractions were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated with avidin-coupled alkaline phosphatase and stained with NBT/BCIP (Fig. 6) or with rabbit anti-GPIb or rabbit anti-GPVI antibodies� (data not shown). There were several protein bands on the membrane stained with avidin-coupled phosphatase� and NBT/BCIP under both reduced and non-reduced conditions. These proteins were neither GPVI nor GPIb, as checked by Western blot analysis.
Discussion
This study reports the isolation and characterization of NA-LAAO, an LAAO from the venom of N. atra. Its N-terminal sequence is highly similar to those of other known venom LAAOs. Because the reported functions of venom LAAOs on platelets are controversial, we investigated its activities on human platelets.
The activation or inhibition functions of venom LAAOs on platelets are largely ascribed to their ability to produce H2O2 because catalase, an H2O2 scavenger, inhibits these effects. However, H2O2 production alone is insufficient to account for the effects. There are few reports that H2O2 directly induces platelet aggregation. In our experiments, H2O2 (2500 mM) did not induce platelet aggregation (data not shown). However, several lines of evidence showed that the biological actions of LAAOs are only partly dependent� on H2O2 production, suggesting that there are specific targets or receptors on cells [8-10]. NA-LAAO induced aggregation of washed human platelets at high doses (15 mg/ml). Catalase inhibited this platelet aggregation. However, the amount of H2O2 produced by the enzyme is not enough to explain the aggregation as discussed above. Furthermore, a low dose of NA-LAAO (1.5 mg/ml) did not induce platelet aggregation even after long incubation (data not shown). It is likely that the function� of NA-LAAO is concentration-dependent. After frequently freezing and thawing, NA-LAAO retained its enzymatic activity as assayed by H2O2 production. However, its activity in inducing platelet aggregation was greatly reduced (data not shown). It is possible that freezing� and thawing processes affect part of the structure needed to interact with platelets.
Several studies have shown that H2O2, a reactive oxygen� species, is an intracellular messenger involved in a large number of signal transduction mechanisms, especially those mediated by tyrosine kinases [16-18]. Reactive oxygen species stimulate tyrosine phosphorylation by activating several kinases, such as members of the mitogen-activated protein kinase pathway, Janus kinase, and members of the Src family [19,20]. However, the mechanism remains unknown. A large number of physiological agonists stimulate� H2O2 production in several cell types, including human platelets [21,22]. For example, collagen-induced platelet aggregation is associated with a burst of H2O2 that acts as a second messenger by stimulating arachidonic acid metabolism and the phospholipase C pathway [22]. H2O2 added after incubation a low dose of NA-LAAO with platelets� induced platelet aggregation. In addition, this treatment� induced phosphorylation of a number of platelet proteins including Src, Syk, and PLCg2, resembling that produced by a high dose of NA-LAAO. As H2O2 alone has few effects in activating platelets, it is possible that incubation� of NA-LAAO with platelets sensitizes them to H2O2 and thus induces platelet aggregation when H2O2 is added. aIIbb3 inhibition by EMD 132338 greatly reduced the platelet aggregation and phosphorylation of platelet proteins, suggesting that it is activated in NA-LAAO-treated platelets. EDTA, a divalent cation chelator, completely inhibited� platelet aggregation induced by NA-LAAO, indicating� that calcium influx might play an important role in the activation by NA-LAAO.
Recent studies suggested that H2O2 production alone could not explain the biological activities of LAAOs. LAAO from A. halys strongly associated with 1210 cells but not HeLa cells and the cytotoxic effect is variable depending on cell lines [9]. In addition, H2O2 generated in the enzymatic� reactions was not sufficient to explain the degree to which bacterial growth was inhibited by a D-amino acid oxidase from hog kidney and an LAAO from the venom of A. halys. A fluorescence-labeling assay showed that both of these enzymes could bind to the surface of bacteria [10]. Furthermore, a novel LAAO from T. stejnegeri dose-dependently� inhibited HIV-1 infection and replication. The presence of catalase resulted in an increase in its antiviral selectivity. However, under the same conditions, no anti-HIV-1 activity was observed by exogenous addition of H2O2 [11]. Our results support the above observations. First, FITC-labeled NA-LAAO binds to fixed platelets. This binding� is specific because non-labeled enzyme competes for binding with the labeled one. Second, biotin-labeled NA-LAAO binds to platelet lysate immobilized on PVDF membranes. This interaction is also specific and strong because the non-labeled enzyme inhibited the binding and the enzyme recognized the proteins under the stringent conditions of Western blot analysis. Finally, affinity isolation� by an NA-LAAO Sepharose 4B column revealed several platelet membrane proteins, although these proteins were not identified in this study.
In conclusion, NA-LAAO from N. atra venom induces platelet
aggregation. Both H2O2 production and binding
to platelet membrane proteins might be involved in its action. The enzyme binds
to the platelet membrane to enhance the sensitivity of platelets to H2O2. At the same time, H2O2
released by the enzyme activated platelets by an unknown mechanism.
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