华夏医学 2000 0 13 1
关键词： 期刊 huaxiayx 0 1-7 国外专家论坛 fur -->
Runhua Wang
( Bioscience Centre,Department of Biological Sciences1 , )
Thiang Max C.M.Chung
( Department ofBiochemistry2 , )
Karen S. C. Max C.M.Chung
( BioprocessingTechnology Centre, National University of Singapore3 , Singapore)
Abstract ： By means of Superdex 75gel filtration and Mono Q ion exchange chromatography, we have isolated a novel plateletaggregation inducer, termed rhodoaggregin, from the crude venom of Calloselasma rhodostoma(Malayan pit viper). The native molecular mass of rhodoaggregin (as estimated by gelfiltration chromatography) was 66 kDa while its isoelectric point (pI) was determined tobe 3.45. Under reducing conditions of SDS-PAGE, rhodoaggregin exhibited two distinctivebands with molecular masses of 18 kDa ( α subunit) and 15 kDa ( β subunit). In the absence of reducing agents, however, twobands were also observed, but with apparent molecular masses of 28 (major) and 52 (minor) kDa, respectively. Furthermore, mass spectrometric analysis also showed that rhodoaggreginhad a molecular mass of 30155.39±3.25. These molecular weight data suggest thatrhodoaggregin probably exists as a tetrameric structure consisting of two disulfide-linked heterodimers. N-terminal amino acid sequence analysis showed that the two subunits ofrhodoaggregin exhibit a high degree of homology with each other and with those of theC-type lectin related proteins (CLPs) from other snake venoms. Functional platelet assaysshowed that rhodoaggregin induced platelet aggregation in rabbit and human whole blood,platelet-rich-plasma (PRP) and washed platelets with a lag period and in an all-or-nonemanner. The concentration of rhodoaggregin that induced maximal aggregation is estimatedat about 0.04 μ g/ml(or 0.7 nM).
Key words ： a novel platelet aggregation inducer;the venom of calloselasma rhodostoma(Malayanpit viper);Molecular analyses;gel filtration chromatogrophy,SDS-PAGE and RP-HPLC ▲
INTRODUCTION
C-typelectin related proteins (CLPs) is a group of snake venom proteins that are structurallyhomologous to the carbohydrate-recognition domain (CRD) of animal C-type lectins [ 1 ] . Most of these proteins exist as heterodimers linked by asingle inter-chain disulfide bond, with molecular masses of ～ 30 kDa. Despite their striking structural similarity, thisgroup of proteins has different effects on blood coagulation and platelet aggregation.Some of these proteins exhibit anticoagulant activities by binding to the coagulantfactors X and/or IX [ 2 ～ 4 ] ; whereas other CLPs inducevaried effects on platelet functions by modulating the interactions between von Willebrandfactor (vWF) and platelet glycoprotein Ib (GPIb) [ 5 ～ 9 ] . For example, botrocetin from Bothrops jararaca venombinds to vWF and forms an activated complex that induces platelet agglutination [ 5 ] . On the other hand, alboaggregins from Trimeresurusalbolabris venom [ 6 ～ 7 ] , echicetin from Echis carinatus venom [ 8 ] and agkicetin from Agkistrodon acutus venom [ 9 ] all bind to platelet GPIb and function as receptorblockers for vWF binding. However, alboaggregins induce direct platelet agglutinationwhereas echicetin and agkicetin inhibit platelet agglutination.
Recently, severalhigher molecular weight multimers of CLPs with different effects on platelet aggregationhave also been reported [ 7,10,22,26 ] . For example, convulxin from the South American rattlesnake Crotalus durissusterificus is a 72-kDa protein that consists of three heterodimers forming an α 3 β 3 complex [ 12 ] . Furthermore, in marked contrast to other CLPs that acton platelets by modulating the interactions between vWF and GPIb, convulxin was reportedto induce platelet aggregation via GPVI collagen receptor [ 11,13 ] . Similarly, the 50 kDa-alboaggregin was found to potentlyinduce platelet aggregation of platelet-rich-plasma [ 22 ] . On the other hand, flavocetin-A and -B, with nativemolecular masses of 149 and 139 kDa, respectively, inhibit platelet aggregation at highshear stress [ 26 ] .
In the presentstudy, we report the isolation and partial characterization of rhodoaggregin, a potentplatelet aggregation inducer from the venom of Calloselasma rhodostoma (Malayan pitviper). N-terminal amino acid sequence analysis showed that rhodoaggregin belonged to theCLP superfamily and it probably exists as a α 2 β 2 tetrameric complex in the native state.
MATERIALS AND METHODS
Materials
Crude venom ofCalloselasma rhodostoma was purchased from Sigma Chemical Co. (St. Louis, MO). FPLC andHPLC columns were from Amersham-Pharmacia Biotech and Vydac, respectively, and peptidesequencing chemicals/reagents were from PE-ABD (Foster City, CA). All buffer salts andorganic solvents were from standard commercial sources and of the highest qualityavailable.
Purification of rhodoaggregin
Calloselasmarhodostoma crude venom (100 mg) was dissolved in 3.0 ml of 0.1 M ammonium hydrogencarbonate (pH 8.0) and centrifuged at 12 000 rpm for 10 min at 4 ℃ to remove particulate material. The supernatant was thenfractionated by a HiLoad Superdex 75 column (2.6cm×60cm) equilibrated with the samebuffer using a FPLC system. The fractions from peak I (containing proteins of interestbased on platelet assays) were pooled and loaded directly onto a Mono Q HR 5/5 columnpre-equilibrated with 20 mM Tris-HCl, pH 8.2. Elution was performed with a linear gradientof 0 - 0.5 M NaCl in 20 mM Tris-HCl buffer, pH 8.2 over 30 min.
Separation of subunits
Purifiedrhodoaggregin was reduced and s-pyridylethylated (s-PE) based on the method of Polgar etal. [ 14 ] . The subunits weresubsequently separated by RP-HPLC using a pH stable C8 Vydac column (4.1mm×250mm) .
Molecular weight determination
Molecular weightsof the native protein and its subunits were determined by gel filtration, SDS-PAGE andelectrospray mass spectrometry.
(A) Gel filtration. The native molecular weight of rhodoaggregin was estimated bygel filtration chromatography on a Superose 12 HR 10/30 column using a FPLC system. Thecolumn was equilibrated and eluted with 0.1 M ammonium hydrogen carbonate at a flow rateof 0.3 ml/min. Molecular weight standard proteins used included glutamate dehydrogenase(290,000), lactate dehydrogenase (142,000), enolase (67,000), adenylate kinase (32,000)and cytochrome C (12,400) (Oriental Yeast Company, Japan).
(B) SDS-PAGE. Nonreducing and reducing (5 % 2-mercaptoethanol) SDS-PAGE wereperformed as described previously [ 15 ] .
(C) Electrospray ionization mass spectrometry. Mass spectrometry was carried outusing a Perkin-Elmer Sciex API 300 LC/MS/MS system, a triple-stage quadruple instrumentequipped with an ionspray interface. The ionspray voltage was set to 4000 V, orificevoltage at 75 V, and the interface temperature at 60 ℃ . Nitrogen was used as a curtain gas with a flow rate of0.6 litres/min, and as a nebulizer gas at 30 psi. A Shimadzu LC-10AD series pump systemwas used for solvent delivery.
Determination of the isoelectric point
The isoelectricpoint of rhodoaggregin was determined using agarose IEF in a pH range of 3-10 (Pharmalyte)based on the manufacturer's protocol.
Determination of N-terminal amino acid sequence
The N-terminalamino acid sequences of the subunits of rhodoaggregin were determined by automated Edmandegradation using an Applied Biosystems 477A pulsed liquid-phased sequencer equipped withan on-line PTH amino acid analyzer (120A).
Platelet aggregation
Blood drawn fromthe central arteries of rabbit ears was anticoagulated with 0.11 M trisodium citrate (1 ∶ 9, v/v). Platelet-rich-plasma(PRP) was obtained by centrifugation of the blood for 20 min at 375 g and 20 (C. Washedplatelets were prepared as described previously [ 16 ] . Platelet aggregation in whole blood was measured by theimpedance method [ 17 ] , using a Chrono-log Model500-CA whole-blood aggregometer (Chronolog, Havertown, PA, USA), under continuous stirringat 1000 rpm. Platelet aggregation in PRP and washed platelets, on the other hand, weremonitored by light transmission [ 18 ] .
RESULTS
Purification ofrhodoaggregin
Thecrude venom of Calloselasma rhodostoma was separated into five main fractions by Superdex75 gel filtration chromatography (Fig.1A). Fraction I, which was found to induce plateletaggregation potently in rabbit and human whole blood, PRP and washed platelets, was thussubjected to further fractionation on a Mono Q column. This purification step resulted inthree fractions (Fig. 1B) of which only fraction 3 exhibited marked aggregatory activitytowards platelets. This fraction was designated as rhodoaggregin. It is an acidic proteinwith an isoelectric point (pI) of 3.45.
FIGURE 1: Purificationof rhodocetin. (A) Gel filtration chromatography of C. rhodostoma crude venom on a HiLoad26/60 Superdex 75 column. Elution was performed at 2 ml/min. (B) Anion exchangechromatography on a Mono Q HR 5/5 column. Fractions from the peak I of gel filtration weredirectly applied to a Mono Q HR 5/5 column that had been equilibrated with 20 mM Tris-HClbuffer, pH 8.2. The protein was eluted at a flow rate of 1 ml/min with a linear gradientof 0 ～ 0.5 M NaCl in 20mM Tris-HCl buffer, pH8.2 over 30 min Determination of themolecular weight of rhodoaggregin
The nativemolecular weight of rhodoaggregin was determined to be 66 kDa by gel filtrationchromatography on a Superose 12 column (Fig. 2). On reducing SDS-PAGE, rhodoaggreginexhibited two bands with molecular masses of 18 and 15 KDa (designated as α and β subunits, respectively) (Fig.3). Under nonreducingconditions, however, two distinctive bands with apparent molecular masses of 28 and 52KDa, respectively, were also observed (Fig. 3).
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FIGURE 2: Determinationof the native MW of rhodoaggregin. Gel filtration chromatography of standard proteins andpurified rhodoaggregin was carried out as described in Materials and Methods. The standardcurve was constructed using the molecular weights of standard proteins versus their Ve/Vovalues |
FIGURE 3: SDS-PAGEof rhodoaggregin under nonreducing (NR) and reducing (R) conditions. SDS-PAGE wasperformed using 12.5% separating gel. Molecular masses of the markers are indicated on theright. Thesubunits of rhodoaggregin could be reduced and separated by RP-HPLC (Fig. 4). Massspectrometric analysis showed that the molecular masses for the α and β subunits were 16535.62±1.98 and 15209.89±1.61 Da (Fig.5), respectively. Purified rhodoaggregin, which eluted as a single peak in RP-HPLC, gave amolecular mass of 30155.39 ( 3.25 Da when analyzed by electrospray ionization (ESI) massspectrometry (Fig.5). |
FIGURE 4: Separationof the reduced and s-pyridylethylated subunits of rhodoaggregin by RP-HPLC. The reducedand s-pyridylethylated rhodoaggregin was subjected to RP-HPLC on a C8 Vydac column (4.6 ～ 250 mm). Solvent A, 0.1 % (v/v)trifluoroacetic acid; B, 0.085 % (v/v) trifluoroacetic acid in 70 % (v/v) acetronitrile.Elution was performed at a flow rate of 1ml/min using a linear gradient from 5 to 65%solvent B in 45 min. The protein was detected by absorbance at 214 nm.
FIGURE 5: Electrosprarymass spectrometry spectra of rhodoaggregin and its reduced and s-pyridylethylatedsubunits.
N-terminal amino acidsequence analysis
The N-terminalamino acid sequences of the two subunits of rhodoaggregin were determined by sequencingthe individual reduced and s-pyridylethylated subunits separated by RP-HPLC (Fig. 4). Ahigh degree of sequence identity was observed between the two subunits. When aligned withthose of other known members of CLP superfamily from snake venoms (Fig. 6), the α and β subunits of rhodoaggregin also showed a high degree ofsequence identity with rhodocetin [ 28 ] , convulxin [ 10 ] , botrocetin [ 19 ] , alboaggregin-B [ 20 ] , echicetin [ 14 ] , ECLV IX/X-bp [ 2 ] , habu IX/X-bp [ 3 ] , habu IX-bp [ 4 ] and jararaca GPIb-bp [ 21 ] .
FIGURE 6: Comparisonof the N-terminal amino acid sequences of rhodoaggregin with those of CLPs from othersnake venom. The α and β subunits ofN-terminal amino acid sequences of rhodoaggregin are compared with those of CLPs fromother snake venoms, including convulxin (10), botrocetin (19), alboaggregin B (20),echicetin (14), ECLV IX/X-bp (2), habu IX/X-bp (3), habu IX-bp (4) and jararaca GPIb-bp(21). Effect of rhodoaggregin onplatelet aggregation
Rhodoaggreginitself could induce platelet aggregation in rabbit and human whole blood, PRP and washedplatelets with a lag period in an all-or-none manner (Fig. 7). For example, at aconcentration of 0.03 μ g/ml,it did not induce any aggregation, but at 0.04 μ g/ml, it resulted in maximal aggregation (Fig. 7).Interestingly, the initial latent period of aggregation was a direct function of proteinconcentration. |
FIGURE 7: Plateletaggregation induced by various concentrations of rhodoaggregin. Various concentrations ofrhodoaggregin (0.03-5.0 μ g/ml) were added to washed rabbit platelet suspension to induce plateletaggregation. The arrows mark the addition of rhodoaggregin; (T denotes the change in lighttransmission DISCUSSION Recently,several high molecular weight multimeric potent platelet agonists/antagonists isolatedfrom snake venoms have been identified as CLP superfamily members [ 6 ～ 8, 10, 11 ， 22 ] . These proteins usually possess molecular masses inexcess of 50 kDa under nonreducing conditions of SDS-PAGE, but upon reduction gave rise toheterodimeric 12 - 15 kDa subunits with N-terminal amino acid sequences that are highlyhomologous to the CLPs. These proteins are also usually found in venoms that alreadycontained the well-characterized CLPs. For example, in addition to alboaggregin B (MW ～ 23,000) [ 6 ] , Trimeresurus albolabris venom also containedalboaggregins A (MW ～ 52,000)and C (MW ～ 121,000)which showed characteristic of heterodimeric structures with apparent molecular masses of14-18 kDa [ 7 ] . Other examples ofmultimeric CLPs are (i) flavocetin-A (MW ～ 149,000) and flavocetin-B (MW ～ 139,000), which are antagonists of platelet aggregationisolated from Trimeresurus flavoviridis venom [ 26 ] , and (ii) convulxin, a potent platelet aggregationinducer from the venom of the South American rattlesnake, Crotalus durissus terificus,which exists as a 72-kDa hexameric structure α 3 β 3 complex) [ 10 ～ 12 ] . It is worth noting that these multimeric CLPs mustpossess very stable quaternary structures since they do not seem to be dissociated intotheir individual nonreduced heterodimers of molecular masses ～ 30 kDa in the presence of SDS [ 7, 22, 10, 26 ] .
In the presentstudy we report the isolation and characterization of a novel multimeric CLP which is apotent platelet agonist, termed rhodoaggregin, from the venom of Calloselasma rhodostoma.FPLC - gel filtration chromatography of rhodoaggregin on a Superose column yielded amolecular mass of 66 kDa for the native protein (Fig.2), while under nonreducing SDS-PAGE,rhodoaggregin exhibited a 52 kDa band, in addition to a 28 kDa band (Fig. 3). In thisrespect, rhodoaggregin's stability towards SDS may differ from the other multimeric CLPs(see above). We postulate that the 28 kDa band probably represents the nonreducedheterodimer ( αβ )whereas the 52 kDa component is the tetrameric structure ( α 2 β 2 ). This result is corroborated by massspectrometric analysis of the native molecule where it showed a molecular mass of30,155.39 Da (Fig. 5). SDS-PAGE analysis in the presence of reducing agents, revealed thatrhodoaggregin was comprised of α (18 kDa) and β 15(kDa) subunits linked by a disulfide bond, which arecharacteristic features of all CLPs (Fig. 3). N-terminal amino acid sequence analysis alsoshowed that these two subunits exhibited a high degree of sequence identity with eachother and with CLPs from other snake venoms. (Fig 6). In all these respects, rhodoaggregincan be considered as a new member of multimeric CLPs as exemplified by convulxin [ 10 ] , alboaggregin A and C [ 7 ] and flavocetin-A and -B [ 26 ] .
Recently, twoCLPs, aggretin and rhodocytin, with similar functional properties and identical N-terminalamino acid sequences to rhodoaggregin, have been isolated from the same venom [ 23 ， 24 ] . Aggretin is a potent platelet agonist that inducesplatelet aggregation via glycoprotein Ia/IIa [ 24 ] while rhodocytin has been shown to act independently ofglycoprotein Ib [ 23 ] . They also elicitedplatelet aggregation with a lag period and in an all-or-none manner. Rhodoaggregin showssimilar effects on platelet aggregation, and the concentration at which it producesmaximal aggregation (0.04 μ g/ml or ～ 0.7nM) (Fig.7) is also similar to those of aggretin and rhodocytin. However, although theelectrophoretic patterns of aggretin, rhodocytin and rhodoaggregin under reducingconditions are identical, unlike rhodoaggregin, aggretin and rhodocytin did not showevidence of any multimeric band with Mr > 50,000 under nonreducing conditions [ 23 ， 24 ] . This observation thus indicates that aggretin andrhodocytin may exist as single disulfide-linked heterodimers whereas rhodoaggregin alsoconsists of similar heterodimers but manifests itself as a tetrameric complex ( α 2 β 2 ).
The gene encodingfor aggretin has been recently cloned and fully sequenced [ 27 ] . The mature α and β subunits of aggretin contain 136 and 123 amino acidresidues, respectively. All the cysteine residues in each subunit are well conserved. Themolecular masses of the fragments from the endoproteinase Lys-C digestion of the α and β subunits of rhodoaggregin observed by mass spectrometricanalysis were in agreement with calculated molecular masses of those from deduced aminoacid sequence of aggretin (data not shown). It is therefore further demonstrated that thesubunits of rhodoaggregin and aggretin are identical. Based on the known disulfidepairings of CLPs, we can infer that the intra-subunit disulfide bonds of rhodoaggregin mayoccur as follows: α subunit,Cys 5-16, Cys 33 ～ 131and Cys 106 ～ 123; α subunit, Cys 2 ～ 13, Cys 30 ～ 119 and Cys 33 ～ 111 (Fig. 8). The two subunits are linked by aninter-subunit disulfide bond between Cys 83 ( α ) and Cys 75 ( β ), which have always been found to form the onlyinter-subunit disulfide bond in other CLPs. As expected, unlike all other CLP subunits,which contain 7 cysteine residues, the β subunit of rhodoaggregin contains 8 cysteine residues. Itis thus inferred that the extra cysteine residue in a β subunit of a heterodimer forms an inter-subunit disulfidebridge with a similar cysteine residue from another heterodimer (Fig. 8). Therefore,rhodoaggregin exists as a tetrameric structure in which the two heterodimers are linked byan inter-subunit disulfide bridge (Fig. 8) |
FIGURE 8: Schematiclocations of the disulfide bridges in rhodoaggregin. Bold lines indicate peptide chains;fine lines indicate disulfide bridges; Numbers indicate positions of amino acid residuesfrom the amino termini of α and β subunits;and circled numbers indicate positions of half-cysteinyl residues.
It isinteresting to note that the venom of Calloselasma rhodostoma is the source of severalnovel toxins with unique structures. From the fraction III of gel filtrationchromatography of the venom (Fig. 1A), we have recently purified and characterized a novelplatelet aggregation inhibitor, rhodocetin [ 28 ] . Rhodocetin was also identified as a novel CLP in whichthe two subunits are held together only by noncovalent interactions and not by anyinter-subunit disulfide bridge [ 28 ] . Earlier, a major hemorrhagin (rhodostoxin) that containedthe first four intra-disulfide linkages among all known venom metalloproteinases had beenreported [ 25 ] .
ACKNOWNLEDGMENT
We wishto thank Dr. R. Manjunatha Kini in the Bioscience Centre, Department of BiologicalSciences, National University of Singapore, for his assistance in platelet aggregationassay and Madam Chan Siew Lee for her technical assistance.
[ ExecutiveEditor ： Wang Huijin ]
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Received January 28,2000
