Published: March 22, 2011 r2011 American Chemical Society 2440 dx.doi.org/10.1021/pr101248e | J. Proteome Res. 2011, 10, 2440–2464 ARTICLE pubs.acs.org/jpr Pseudechis australis Venomics: Adaptation for a Defense against Microbial Pathogens and Recruitment of Body Transferrin Dessislava Georgieva, †,‡ Jana Seifert, †,§ Michaela € Ohler, § Martin von Bergen, § Patrick Spencer, || Raghuvir K. Arni, ^ Nicolay Genov, # and Christian Betzel* ,‡ ‡ Institute of Biochemistry and Molecular Biology, University of Hamburg, Laboratory of Structural Biology of Infection and Inflammation, c/o DESY, Notkestrasse 85, Build. 22a, 22603 Hamburg, Germany § Department of Proteomics, Helmholtz Centre for Environmental Research-UFZ, Permoser Strasse 15, 04318 Leipzig, Germany ) Centro de Biotecnologia, Instituto de Pesquisas Energeticas e Nucleares, Av. Lineeu Prestes 2242, 05508-000 S ~ ao Paulo, Brazil ^ Department of Physics, IBILCE/UNESP, Cristov ~ ao Colombo 2265, CEP 15054-000, S ~ ao Jos e do Rio Preto, SP Brazil # Institute of Organic Chemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria ’ INTRODUCTION Human envenoming by poisonous snakes is of public health significance and a serious medical problem. Snake venoms are very rich and incompletely explored sources of pharmacologi- cally important compounds. The best example for the develop- ment of pharmaceutical products based on investigations on venom compounds is captopril, an inhibitor of the angiotensin I-converting enzyme (ACE inhibitor), used for treatment of hypertension. 1 The knowledge of the venom composition is necessary for the improvement of antivenoms used for the neutralization of snakebite consequences, for quality control of venom preparations 2 and for structure-based design of novel drugs, especially for the blood pressure regulation and the treatment of coagulopathy. Snake venom components have a significant potential for clinical applications as diagnostic agents. 3 The venomics of a large number of viperid snakes have been investigated with respect to their pharmacological and medical application. 2,415 During the past decade, investigations were focused on the molecular origin and evolution of the snake venom proteome. 1623 Methods for the venom proteome anal- ysis with special attention to the structure, function and role of metalloproteinases in the viperid snakebite pathogenesis were developed. 2428 Proteomic and transcriptomic approaches were successfully combined in investigations on the venom composi- tions of South American snakes. 29,30 Australian elapid snakes are among the most venomous in the world. 31 Their bites cause morbidity and in some cases mortality. 32 However, in comparison to viperid snakes, consider- ably less information about the elapid venom proteome is available. Venom compositions of Naja naja atra, 33 Pseudonaja textilis, 34 Micrurus surinamensis (fish eating coral snake) 35 and Received: December 16, 2010 ABSTRACT: The venom composition of Pseudechis australis,a widely distributed in Australia reptile, was analyzed by 2-DE and mass spectrometric analysis. In total, 102 protein spots were identified as venom toxins. The gel is dominated by horizontal trains of spots with identical or very similar molecular masses but differing in the pI values. This suggests possible post- translational modifications of toxins, changing their electrostatic charge. The results demonstrate a highly specialized biosynth- esis of toxins destroying the hemostasis (PIII metallopro- teases, SVMPs), antimicrobial proteins (L-amino acid oxidases, LAAOs, and transferrin-like proteins, TFLPs), and myotoxins (phospholipase A 2 s, PLA 2 s). The three transferrin isoforms of the Australian P. australis (Elapidae snake) venom are highly homologous to the body transferrin of the African Lamprophis fuliginosus (Colubridae), an indication for the recruitment of body transferrin. The venomic composition suggests an adaptation for a defense against microbial pathogens from the prey. Transferrins have not previously been reported as components of elapid or other snake venoms. Ecto-5 0 -nucleotidases (5 0 -NTDs), nerve growth factors (VNGFs), and a serine proteinase inhibitor (SPI) were also identified. The venom composition and enzymatic activities explain the clinical manifestation of the king brown snakebite. The results can be used for medical, scientific, and biotechnological purposes. KEYWORDS: snake venomics, Pseudechis australis, 2-D electrophoresis, electrospray mass spectrometry, venom transferrin, enzyme activity
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Published: March 22, 2011
r 2011 American Chemical Society 2440 dx.doi.org/10.1021/pr101248e | J. Proteome Res. 2011, 10, 2440–2464
ARTICLE
pubs.acs.org/jpr
Pseudechis australis Venomics: Adaptation for a Defense againstMicrobial Pathogens and Recruitment of Body TransferrinDessislava Georgieva,†,‡ Jana Seifert,†,§ Michaela €Ohler,§ Martin von Bergen,§ Patrick Spencer,||
Raghuvir K. Arni,^ Nicolay Genov,# and Christian Betzel*,‡
‡Institute of Biochemistry and Molecular Biology, University of Hamburg, Laboratory of Structural Biology of Infection andInflammation, c/o DESY, Notkestrasse 85, Build. 22a, 22603 Hamburg, Germany§Department of Proteomics, Helmholtz Centre for Environmental Research-UFZ, Permoser Strasse 15, 04318 Leipzig, Germany
)Centro de Biotecnologia, Instituto de Pesquisas Energ�eticas e Nucleares, Av. Lineeu Prestes 2242, 05508-000 S~ao Paulo, Brazil^Department of Physics, IBILCE/UNESP, Crist�ov~ao Colombo 2265, CEP 15054-000, S~ao Jos�e do Rio Preto, SP Brazil#Institute of Organic Chemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
’ INTRODUCTION
Human envenoming by poisonous snakes is of public healthsignificance and a serious medical problem. Snake venoms arevery rich and incompletely explored sources of pharmacologi-cally important compounds. The best example for the develop-ment of pharmaceutical products based on investigations onvenom compounds is captopril, an inhibitor of the angiotensinI-converting enzyme (ACE inhibitor), used for treatment ofhypertension.1 The knowledge of the venom composition isnecessary for the improvement of antivenoms used for theneutralization of snakebite consequences, for quality control ofvenom preparations2 and for structure-based design of noveldrugs, especially for the blood pressure regulation and thetreatment of coagulopathy. Snake venom components have asignificant potential for clinical applications as diagnostic agents.3
The venomics of a large number of viperid snakes have beeninvestigated with respect to their pharmacological and medical
application.2,4�15 During the past decade, investigations werefocused on the molecular origin and evolution of the snakevenom proteome.16�23 Methods for the venom proteome anal-ysis with special attention to the structure, function and role ofmetalloproteinases in the viperid snakebite pathogenesis weredeveloped.24�28 Proteomic and transcriptomic approaches weresuccessfully combined in investigations on the venom composi-tions of South American snakes.29,30
Australian elapid snakes are among the most venomous in theworld.31 Their bites cause morbidity and in some casesmortality.32 However, in comparison to viperid snakes, consider-ably less information about the elapid venom proteome isavailable. Venom compositions of Naja naja atra,33 Pseudonajatextilis,34 Micrurus surinamensis (fish eating coral snake)35 and
Received: December 16, 2010
ABSTRACT: The venom composition of Pseudechis australis, awidely distributed in Australia reptile, was analyzed by 2-DE andmass spectrometric analysis. In total, 102 protein spots wereidentified as venom toxins. The gel is dominated by horizontaltrains of spots with identical or very similar molecular massesbut differing in the pI values. This suggests possible post-translational modifications of toxins, changing their electrostaticcharge. The results demonstrate a highly specialized biosynth-esis of toxins destroying the hemostasis (P�III metallopro-teases, SVMPs), antimicrobial proteins (L-amino acid oxidases,LAAOs, and transferrin-like proteins, TFLPs), and myotoxins(phospholipase A2s, PLA2s). The three transferrin isoforms ofthe Australian P. australis (Elapidae snake) venom are highlyhomologous to the body transferrin of the African Lamprophisfuliginosus (Colubridae), an indication for the recruitment ofbody transferrin. The venomic composition suggests an adaptation for a defense against microbial pathogens from the prey.Transferrins have not previously been reported as components of elapid or other snake venoms. Ecto-50-nucleotidases (50-NTDs),nerve growth factors (VNGFs), and a serine proteinase inhibitor (SPI) were also identified. The venom composition and enzymaticactivities explain the clinical manifestation of the king brown snakebite. The results can be used for medical, scientific, andbiotechnological purposes.
Naja kaouthia36 were determined. Selected spots from 2-DPAGE of Australian snake venoms were analyzed and novelproteins identified.37 Transcriptomic approaches were appliedfor analyzing venom gland genes of Oxyuranus scutellatus,38
Micrurus corallinus39 and Bungarus flaviceps.40
Snakes of the genus Pseudechis, also known as “Black Snakes”or “Bongani Sibanda”, are widespread in all Australian statesexcept for Tasmania. Nine species were recognized: P. australis(King brown snake or mulga snake), P. butleri (Spotted mulgasnake), P. collettii (Collett’s snake), P. guttatus (Blue-bellied blacksnake), P. papuanus (Papuan black snake), P. pailsi, P. porphyr-iacus (Red-bellied black snake), P. rossignolii (Papuan dwarf kingbrown) and P. weigeli (Pygmy mulga snake).41,42 P. australis isone of the longest venomous snakes in the world (2.5�3 m inlength) and is encountered in most Australian states except forVictoria and Tasmania.
In the present paper, we report the proteomic profile of thePseudechis australis venom. The venom components were ana-lyzed by 2-D gel electrophoresis and electrospray mass spectro-metry, and classified into protein families.
’MATERIALS AND METHODS
Collection of the VenomCrude venom, pooled from several specimens of Pseudechis
australis, was a kind gift of Dr. P. Mirtschin (Venom Supplies Pyt.Ltd., Australia). Snakes of both genders were milked and thelyophilized venom was stored at 4 �C.
2-D Gel Electrophoresis and Electrospray Mass Spectrom-etry
Two-dimensional electrophoresis was performed as describedpreviously.43 The venom was suspended in deionized water anddesalted using an Amicon-Ultra 0.5 mL filter (Millipore) with a10 kDa cutoff prior to the 2-D electrophoresis. 200 μg of the totalprotein were mixed with 135 μL DeStreak solution (GEHealthcare) and 0.5% IPG (immobilized pH gradient), pH 3 �10, in nonlinear (NL) buffer (v/v) (GE Healthcare, Uppsala,Sweden). The sample was agitated for 15 min at room tempera-ture and centrifuged for 30 min at 13 000 rpm to removeprecipitates. The equilibration was performed with the super-natant loaded on 7 cm Immobiline DryStrip pH 3�10 NL (GEHealthcare, Uppsala, Sweden). In the first dimension, proteinswere separated by an IPGphore electrophoresis unit overnight(GEHealthcare, Uppsala, Sweden). After isoelectric focusing thestrips were equilibrated for 15 min in equilibration buffercontaining 0.05 M Tris/HCL pH 8.8, 30% glycerol (v/v),6 M urea, 4% sodium dodecyl sulfate and 2% dithioerythrithol.In a second equilibration step the strips were incubated with0.05 M Tris/HCL pH 8.8, 30% glycerol (v/v), 6 M urea,4% sodium dodecylsulfate and 2.5% iodoacetamide for 15 min.The strips were stored at �20 �C until used in the seconddimension, performed on a 10% Tris-tricine-polyacrylamide gel(100 � 100 � 1.0 mm3).44
After electrophoresis both gels were stained overnight withcoomassie brilliant blue and destained as described previously.45
Gels were scanned and imported into the software Delta2Dsoftware package (Decodon, Greifswald, Germany).
Protein spots of interest were cut from the polyacrylamide gelsand digested overnight using trypsin (Sigma, Munich, Germany)according to the protocol from Shevchenko, modified in aprevious study.46 Peptides were desalted (ZipTip pipet tips,
Millipore) and reconstituted in 0.1% formic acid. Samples wereinjected by an autosampler and concentrated on a trappingcolumn (nanoAcquity UPLC column, C18, 180 μm � 2 cm,5 μm, Waters) with water containing 0.1% formic acid at flowrates of 15 μL/min. After 2 min the peptides were eluted onto aseparation column (nanoAcquity UPLC column, C18, 75 μm x10 cm, 1.75 μm, Waters). Chromatography was performed with0.1% formic acid in solvent A (100% water) and B (100% ACN).The peptides were eluted over 8 min with 20 � 80% solvent Bgradient using a nano-HPLC system (nanoAcquity, Waters)coupled to an LTQ-Orbitrap mass spectrometer (Thermo FisherScientific). The capillary voltage in MS andMS/MS experimentswas set to 2000 V. The collision gas was helium at a pressure of0.1 MPa, and the collision energy was 40 V. For an unbiasedanalysis, continuous scanning of eluted peptide ions was carriedout between 300 and 2000 m/z, automatically switching toMS/MS CID mode on ions exceeding an intensity of 5000.For MS/MS measurements, a dynamic precursor exclusion of1 min with an isolation width of 4 m/z was used.
Raw data were applied to a database search using ThermoProteome Discoverer software (v1.0 build 43) to carry out atandem ion search algorithm from the MASCOT house server(v2.2.1) by database comparison against all chordata entries inthe National Center for Biotechnology Information (NCBInrdatabase 2010) with 10 ppm tolerance for the precursor and0.8 Da for MS2 fragments. Further, trypsin with a maximum oftwo missed cleavages was selected and variable modifications,such as methionine oxidation and carbamidomethylation ofcysteine, were allowed. Peptides were considered to be identifiedby Mascot when a probability <0.05 (probability based ionthreshold scores >40) was achieved. Proteins were consideredto be identified if at least two peptides were identified. In somecases there is a theoretical possibility that non-P. australis venompeptides do not 100% mirror the corresponding fragmentsbecause similar tryptic peptides of the same mass can possessnonidentical sequences due to different sequential order of pairsof residues.
Enzymatic ActivitiesProteolytic activity was determined by the method of Johnson
et al.47 The venom was assayed using 1.2% casein solution inTris-HCl buffer, pH 7.4, at 37 �C. Undigested casein wasprecipitated with 0.5M perchloric acid and centrifuged. Digestedcasein in the supernatant was determined by measuring theabsorbance at 280 nm. Unit definition: One CTA unit liberatesfrom cow casein 0.1 micro equivalents of tyrosine for 1 min at37.5 �C. One CTA unit is equal to 0.096 proteolytic units as usedby SIGMA Chemical Corporation, St Louis, MO.
Phospholipase A2 activity was determined using the CaymanChemical Secretory PLA2 Assay kit (Ann Arbor, MI) containinga bee venom PLA2 as a standard. 1,2 � dithio analog ofdiheptanoyl phosphatidylcholine was used as a substrate. Therelease of free thiols upon the PLA2 catalyzed hydrolysis of thethioester bond at the sn-2 position was detected spectrophoto-metrically using 5,50-dithiobis(2-nitrobenzoic acid).
L-Amino acid oxidase activity was determined by the methodof Wellner et al.48 using L-phenylalanine as substrate. One unit ofactivity is the amount of enzyme required to give an absorbanceof 0.030 at 300 nm.
Alkaline phosphatase activity was measured by the method ofSulkowski et al.49 using p-nitrophenylphosphate as a substrate.
One unit of activity is defined as the amount of enzyme whichliberates 1 μmole of p-nitrophenol per min.
Acid phosphatase activity was determined by the method ofTu and Chua.50 o-Carboxyphenylphosphate (0.0036 M) wasused as a substrate and the initial rate of hydrolysis of thesubstrate at 25 �C was determined from the increase of theabsorbance at 300 nm due to the liberation of salicylic acid.Venom concentration was adjusted so that the increase of theabsorbance was linear for at least 5 min. One unit of acidphosphatase activity is equivalent to 1 μmole of the substratehydrolyzed per min.
’RESULTS
2-D Gel Electrophoresis of the Pseudechis australis VenomThe proteomic composition of the P. australis venom was
investigated by 2-D electrophoresis. The separated protein bandswere subjected to tryptic digestion and the venom componentswere identified by MS/MS and MASCOT search program(Figure 1, Tables 1 and 2). The oxidized methionine residuesare indicated by Mox. The oxidation of methionine is dueprobably to the procedure of harvesting and sample handling.The samples were dried in the presence of ambient air which isknown to cause oxidation of methionine. The gel ensureddetailed information about the components with molecularmasses 9�110 kDa and pI values between 3 and 10. A total of110 spots were detected and identified on the 2-D gel. Theisolated proteins were assigned to the following protein families:metalloproteases, phospholipases A2, L-amino acid oxidases,transferrin-like proteins, ecto-50-nucleotidases, nerve growthfactors and serine protease inhibitors. The major group is thatof the hemostasis-related SVMPs including 53% of the identifiedproteins. The isoforms of LAAOs comprise the second largestprotein family (20% of the identified toxins). In the third positionare PLA2s representing 18.5% of the identified venom compo-nents. The representatives of the other five protein familiescomprise 8.5% of the analyzed toxins. A characteristic featureof the 2-D gel is the presence of several multiple horizontal trains
of spots with identical or very similar molecular masses butdifferent isoelectric points.
P�III metalloproteasesP�III SVMPs are the most widely represented family of toxins
in the P. australis venom accounting for 53% of the identifiedtoxins (Figure 2). A group of high molecular mass enzymes wasidentified from spots of multiple horizontal trains in the upperleft part of the 2-D gel (Figure 1, Table 1). Spots 1�5 containP�III metalloproteases with molecular masses of 100�105 kDaand pI values between 4.7 and 5.2. A second group of multipleisoforms of P�III SVMPs is shown in the upper right panel of thegel (Figure 1). Again, horizontal trains of spots with identicalmolecular masses, but differing in the pI values were observed.Metalloproteases (60�85 kDa) were identified in spots 7�26,29, 30�35, 42, 44�47, 59�74, 87, and 88. The peptide analysesshowed a high degree of sequence similarity between the P�IIISVMPs from the P. australis venom and their counterparts fromthe other elapid Australian snakes: Austrelaps superbus (theLowland copperhead), Pseudechis porphyriacus (Red-belliedblack snake), Oxyuranus scutellatus (the Coastal taipan) andNotechis scutatus (Tables 1 and 2).
Proteins with molecular masses in the region of 33�43 kDaand pI values between 4.0 and 7.2 were isolated from spots36�41, 75�77 and 80. They form another group of processedP�III SVMPs (Figure 1). The molecular masses are character-istic for the medium size class II proteases, but sequencesimilarities with the P�III group suggest that these proteinsbelong to a group of processed P�III enzymes.
Antimicrobial Proteins: L-Amino Acid Oxidases and Trans-ferrin-Like Proteins
Multiple isoforms of L-amino acid oxidases were found in theprocessed spots of the 2-DE gel (20% of the identified proteins;Figure 1, Tables 1 and 2). Spots 47, 49, 51, 53, 55, 57, and 61 forma horizontal train in the pI range from 6.7 to 7.8. The proteinspossess similar molecular masses from 58 to 62 kDa. A secondtrain in the same pI interval is formed by spots 48, 50, 52, 54, 56,58 and 60, containing proteins with molecular masses of
Figure 1. 2-D gel pattern of the Pseudechis australis venom. Fractionation was performed under the conditions described in the Materials and Methods.
53�58 kDa. LAAOs with pI values in the acidic region, from 3.7to 4.3 and molecular masses of 56�65 kDa were identified inspots 32, 33, and 35 (Figure 1, Table 1). Peptides from proteinswith molecular weight of 80 kDa (spots 42�46) showedsequence similarity to other snake venom LAAOs. However,the molecular weight of these proteins is not characteristic of themonomeric oxidases, since these proteins oligomerize.51 Alldetected isoforms have a sequence similarity to both LAAOsisolated previously from the P. australis venom.52 Peptides fromthe proteins in spots 32 and 60 have a sequence similarity to theLAAOs from the Oxyuranus scutellatus scutellatus and Naja atravenoms.
Isoforms of transferrin-like proteins were identified from spots44, 45, and 46. The three proteins are in a horizontal in the pIrange of 7.5�7.8 (Figure 1). They have the same molecularmasses of 80 kDa and different isoelectric points, which suggestspossible post-translational modifications. All but one sequencesof the isolated tryptic peptides (Table 1) are 100% identical to
the respective segments in the body transferrin of the Africanhouse snake Lamprophis fuliginosus.53 The exception is a se-quence that has partial identity to the respective segment of thebody protein. The snake plasma protein and the three venomTFLPs possess identical molecular weights. This result suggestsrecruitment of body transferrin into the snake venom. Compar-ison of the five P. australis peptides with the sequence of humanplasma transferrin54 showed identity of the sequencesCGLVPXL and LFGSXXT.
Phospholipases A2
The proteomic analysis showed a large diversity of PLA2s inthe venomics of P. australis (Figure.1, Table 1). These enzymesrepresent 18.5% of all identified toxins (Figure 2). Acidic andbasic monomeric PLA2s were isolated from spots 90, 94 � 108and 110. These spots form a long of proteins with molecularweights of approximately 16 kDa in the pI interval from 5 to 9,again due probably to post-translational modifications. Theidentified enzymes likely belong to Group I since elapid snakevenom PLA2s are members of this group. Most probably, 13 ofthe proteins correspond to those isolated from the P. australisvenom PLA2s labeled as Pa-1G, Pa-3, Pa-5, Pa-9c, Pa-10A, Pa-11,Pa-12A, Pa-12c, Pa-13, Pa-16, Pa-17, Pa-18, and Pa-19.55�58 Theothers are isoforms of these enzymes, which have not beencharacterized before, with different pI values and/or molecularmasses. It should be mentioned that PLA2s isolated from spots90�100 are acidic proteins with pI values between 3.6 and 6.6(Figure 1). Usually, the acidic PLA2s are neither catalyticallyactive (or possess very low enzymatic activity) nor neurotoxic.59
However, Pa-1G is an exception to this rule and it is the firstacidic phospholipase A2 with high neurotoxicity (0.13 μgs/gbody wt).55 One peculiarity of the P. australis venomics is thehigh content of acidic PLA2s, while the single chain phospholi-polytic enzymes from other Australian elapids are almost exclu-sively basic.31 Basic PLA2s are highly homologous in their amino
Table 2. Summary of the Protein Families in the Pseudechis australis Venom Identified after 2-DE
spot no. homologous protein homology with protein from protein family
acid sequences and, at the same time, differ considerably in theircatalytic properties and toxicity. Thus, Pa-11 is enzymatically 30times more active than Pa-13 and considerably more toxic thanthe second protein.57 Pa-13 showed no lethal activity at a doseof 7.4 μg/g mouse56 and Pa10A is another lethal PLA2.
60
Isoforms with higher molecular masses were isolated fromspots 82�84, 86 (basic proteins with molecular masses of32�34 kDa) and 92 (also basic protein with molecular massof 45�46 kDa). The enzymes from the first group are dimersbecause their molecular masses correspond to that of two-chaincomplexes. The protein in spot 92 could be an isoform of amultichain PLA2. Australian snakes contain such proteins; forexample, the 45.6 kDa taipoxin, the principle toxin of the O. s.scutellatus venom.31 The identified PLA2 isoforms showedsequence homology to phospholipolytic enzymes from thevenoms of Oxyuranus microlepidotus, Notechis scutatus scutatus,Lapemis hardwickii, and Pseudechis porphyriacus.
Other ProteinsA serine protease inhibitor was isolated from spot 109 of the
2-D gel (Figure 1, Tables 1 and 2). This is a basic polypeptide of9 kDa molecular mass and pI value of 7.4. It is homologous tomulgin-2, a 9.2 kDa protein with serine-type endopeptidaseinhibitor activity (GenBank: AAT4540.1). Three isoforms ofecto-50-nucleotidase with pI values between 8.5 and 8.7 wereidentified in the spots 65, 69, and 70. Spots 79, 101, and 107contain isoforms of venom nerve growth factor (Figure 1,Tables 1 and 2).
oxidase, alkaline phosphatase and acidic phosphatase activities ofthe Pseudechis australis venom. The results are presented inTable 3. The enzymatic activities of the Elapidae snake P. australisvenom are compared with the respective activities of Viperidaesnakes. The data are comparable because the activities weredetermined using the same methods and equipment. The venomPLA2 activity of the Elapidae snake is considerably higher thanthat of Bothrops alternatus, Crotalus d. terrificus, Vipera a. ammo-dytes, and Vipera a. meridionalis, but similar (even less) than thephospholipolytic activity of the Daboia russelli siamensis venom(Table 3). The venom proteinase activity is similar to that of theB. alternatus venom, but considerably higher than that of theother Viperidae snakes. Both P. australis and Vipera a. ammodytesvenoms show the highest LAAO activity. The alkaline phospha-tase activity of the mulga venom is similar to the activities of theother snakes. No acidic phosphatase activity was detected.
’DISCUSSION
P�III Metalloproteases, Phospholipases A2 and 50-Nucleo-tidases in Relation to the Pharmacological Activities of theP. australis Venom
Australian elapid snakes are among the most toxic in theworld.31 The major pathological effects of the P. australisenvenomation are severe disruption of hemostasis,31,61 muscledamage and necrosis.62 Mulga is a member of the nonprocoa-gulant group of elapid snakes.31 The coagulopathy should beattributed to the P�III SVMPs which predominate in thevenomics of P. australis (53% of all identified toxins). The classIII metalloproteases are composed of metalloprotease, disinte-grin-like and cysteine-rich domains.28 The metalloprotease do-main is responsible for the degradation of matrix proteins whilethe nonprotease domains exert anticoagulant effects.63 Thecysteine-rich domain inhibits the collagen-stimulated plateletaggregation.63 The P�III-enzymes induce also muscle damageand myonecrosis.64 In this way the metalloproteases contributesignificantly to the pathogenesis of the P. australis inducedenvenomation.
The other feature of the investigated venom composition isthe absence of serine proteases including enzymes with throm-bin-like activity. The severe disruption of hemostasis caused bythe P. australis bites proceeds without fibrinolysis,31 which is inline with the lack of serine proteases/fibrinogenases in the venomproteome.
The severe coagulopathic effect, caused by the P�III metallo-proteases, is strengthened by the high quantities of PLA2s, thethird largest group of toxins in the venomics of P. australis.Anticoagulant phospholipases A2 can bind and block factors ofthe coagulation cascade.31 A hemotoxic PLA2, potent inhibitor ofthe platelet aggregation, was isolated from the venom of anotherAustralian elapid, Austrelaps superbus.65 It is homologous to anenzyme from the P. australis venom (Table 1).
The king brown snake venom caused rhabdomyolysis fol-lowed by myoglobinuria and nephropathy.62 Neurotoxicity canbe supposed due to the presence of PLA2s in the venom.However, myotoxicity is the major pharmacological effect fol-lowing the P. australis bites.66 This can be explained by a strongand direct myotoxic action of a large quantity of PLA2s on themuscles. Myotoxicity is independent of the enzymatic activity.67
Analysis of the structure�function relationships and crystal-lographic investigations on snake venom PLA2s demonstratedthat the C-terminal part of the polypeptide chain, an exposedhydrophobic surface and interfacial surface charge are importantstructural determinants of the myotoxicity.68,69 Investigations ofthe action of five P. australis venom PLA2s on nerves and musclesdemonstrated that the predominant pharmacological effect is
Table 3. Enzymatic Activities of the Pseudechis australis Venom and Comparison of Elapidae and Viperidae Snake Venomactivities
myotoxicity.70 However, neuromuscular effects of some isolatedking brown snake venom phospholipases A2 have beenobserved.60,71 These toxins produce muscle paralysis by reducingacetylcholine release60 or act postsynaptically to depress themuscle contractility.71 The high content of PLA2s is in accor-dance with the myotoxic effects of the P. australis snakebites.66
P�III metalloproteases also contribute significantly to themyotoxicity.
50-Nucleotidases inhibit the platelet aggregation via increasedadenosine signaling72 acting as anticoagulants. In this way thethree isoforms described in Table 1 strengthen additionally thecoagulopathic effects of the P. australis venom.
Adaptation of the P. australis venom for defense againstmicrobial pathogens. Recruitment of body transferrin intothe snake venom
The venomics of mulga snake reveal a high content ofantibacterial proteins, LAAOs and transferrin-like proteins(22.5% of the identified proteins). Potent antibacterial activityof the P. australis venom was demonstrated against Gram-positive and Gram-negative bacteria.52 This snake feeds uponfrogs containing the Aeromonas hydrophila,73 a heterotrophic,Gram-negative bacterium widespread among amphibians andfish. The pathogen is toxic to many organisms and can survive inaerobic and anaerobic environments. The P. australis venomshowed the highest antibacterial activity toward A. hydrophilaamong 21 tested Elapidae snake venoms,52 which correlates withfeatures of the snake diet. The presence of a large diversity ofLAAO isoforms in the venomics of the king brown snake (20% ofthe identified proteins) can account for the bactericidal effects ofthe venom because these enzymes are active against variousbacteria.52,74,75 L-Amino acid oxidases exert their antibacterialeffect through the hydrogen peroxide liberated after the oxidativedeamination of amino acids. Two L-amino acid oxidases, LAO1and LAO2, were isolated from the venom of P. australis.52 Bothenzymes possess subunit molecular masses of 56 kDa and formaggregates of 142 kDa. The correlation of the subunit molecularmasses with those of the proteins from spots 32 and 35 suggests apossible identity with the two LAAOs, described in the papermentioned above. It is known that LAAOs oligomerize inwater.51 Most probably, the aggregates dissociate under theconditions used for the 2-DE. The pathogen A. hydrophila,present in a high concentration in frogs which comprise asignificant part of the P. australis diet, was the most sensitivebacterium tested with venom LAAOs (LAO1 and LAO2) fromthe same snake.52
The presence of three transferrin isoforms in the P. australisvenom demonstrates recruitment of a body protein into thesnake venom. This result supports the theory that the snakevenom toxins evolve from recruitment of body proteins intothe chemical arsenal of the snake.20 The high degree of sequencesimilarity between the body transferrin, found in the liver of theAfrican house snake Lamprophis fuginosus (a colubrid snake) andthe TFLPs in the venom of the Australian P. australis is surprising.Both snakes inhabit different continents. Moreover, the trans-ferrins mentioned above have the same molecular masses as thehuman protein. Transferrin is a blood plasma protein for irondelivery to the tissues,76 associated with the innate immunesystem. It is produced mainly in the liver. A possible explanationof the transferrin physiological role as a venom protein isconnected with the metal binding properties of this protein.The binding of Fe3þ makes the environment unsuitable for the
bacterial survival, that is, transferrin has a bactericidal effect.Transferrins play a major role in iron transport and defenseagainst microbial pathogens.53 One of the reasons for theincorporation of TFLPs into the snake venom could be strength-ening of the antimicrobial effect of the venom.
to the total toxic effect. For this reason they are an importantcharacteristic of the venom. The relatively high PLA2 activity ofthe king brown snake venom is in agreement with its destructiveeffects on the body tissues. PLA2s hydrolyze membrane phos-pholipids and liberate lysophospholipids and fatty acids, includ-ing arachidonate. In this way they exert pathological effects onthe prey. The damage of biological membranes leads to changesin the permeability to ions and drugs.77 On the other handlysophospholipids are involved in cell lysis77 and arachidonate isa precursor of mediators of inflammation such as thromboxanes,prostaglandins and leukotriens.78 The catalytic activity of PLA2
leads to a serious disturbance of important physiological pro-cesses in the prey. Taking into consideration the relatively highcontent of these enzymes in P. australis, it can be concluded thatphospholipolytic enzymes play an important role in the lifethreatening effects caused by the mulga snakebite.
The high LAAO activity corresponds to the large quantity ofthese enzymes in the investigated venom. The catalytic activity ofthese enzymes results in the formation of the highly cytotoxichydrogen peroxide, which accounts for the strong antimicrobialeffect of the P. australis venom. L-Amino acid oxidases inducenecrotic and apoptotic cell death.75 Probably, this effect is usedby the snake as a defense against pathogens from the prey.Of course, LAAOs contribute to the total toxicity of the venomaimed at the killing of the small animals used as food.
The proteolytic activity of the P. australis venom is higher thanthat of a number of Viperidae snake venoms but, in principle, it isnot as high as it can be expected from the large quantity ofmetalloproteases. Most probably, the low level of this activity isdue to the high specificity of the P�III metalloproteases in thevenom, hydrolyzing a limited number of peptide bonds.
The individual variations in the alkaline phosphatase activityamong the snake venoms, compared in Table 3, are not drasticand hardly can influence the total toxicity.
’CONCLUDING REMARKS
The venom composition of P. australis demonstrates a highlyspecialized biosynthesis of large quantities of antibacterial toxinsand proteins disrupting the hemostasis or exerting myotoxiceffects. The results of the venom proteome analysis point to anadaptation of the venomic system for tissue destruction, bloodcoagulation blockade and a defense against microbial pathogensfrom the prey. The last hypothesis is supported by the obviousrelationship between the presence of potent antimicrobial pro-teins in the venom, its bactericidal effects and the bacterialcontamination of the food used in the snake diet. The antibac-terial activity can also prevent bacterial infections from the buccalcavity into the venom gland. To our knowledge, the bodytransferrin is unknown as a recruited component of the elapidor other snake venoms. A possible role of the venom transferrincould be strengthening of the antimicrobial effect. The highdegree of sequence homology between the body transferrin ofthe Colubridae African house snake Lamprophis fuliginosus andthe transferrin-like proteins from the Australian snake Pseudechis
australis (Elapidae) venom is surprising. Both snakes inhabitdistant regions with a very little likelihood for interbreeding.
The horizontal spot s of proteins belonging to the predomi-nant families of SVMPs, PLA2s and LAAOs are a characteristicfeature of the 2-D gel and suggest post-translational modifica-tions of these enzymes.
Pseudechis australis has a large venom output, up to 150 mg inone bite, and represents a rich source of pharmacologically activecompounds. Knowledge of the venomic composition revealspossibilities for the preparation of a more efficient antivenom, foradequate treatment of the consequences of the snakebite andfor the design of new medicines. Large quantities of proteinsinfluencing the hemostasis or with antibacterial properties can beobtained from this venom for medical, scientific and biotechno-logical purposes. The venomic composition of P. australis isrelevant to the pathologies associated with the snakebites, inparticular to the hemostatic disorders and myotoxicity.
Author Contributions†These authors have contributed equally to this work.
’ACKNOWLEDGMENT
This work was supported by a grant from the DeutscheForschungsgemeinschaft (project BE 1443-18-1 and BE1443) andfrom FAPESP/CNPq/CAPES. We are grateful to Dr. P. Mirtschin(Venom Supplies, Pyt. Ltd., Australia) for providing us with thevenom sample.
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