Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 1926
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,
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 phospholipase 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 activities 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, 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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