Structural and functional characterization of a P-III metalloproteinase, leucurolysin-B, from Bothrops leucurus venom

Available online at www.sciencedirect.com

ABB Archives of Biochemistry and Biophysics 468 (2007) 193–204 www.elsevier.com/locate/yabbi

Structural and functional characterization of a P-III metalloproteinase, leucurolysin-B, from Bothrops leucurus venom Eladio F. Sanchez a,d,*, Lucilene M. Gabriel a, Sileia Gontijo a, Luiza H. Gremski b,e, Silvio S. Veiga b, Karla S. Evangelista c, Johannes A. Eble c, Michael Richardson a a

Research and Development Center, Ezequiel Dias Foundation, 30510-010 Belo Horizonte, Brazil b Department of Cell Biology, Federal University of Parana, 81531-990 Curitiba, Brazil c Institute for Physiological Chemistry, Mu¨nster University, Mu¨nster, Germany d Post-Graduate Program, Hospital Santa Casa, Belo Horizonte, Brazil e Department of Medicine, UNIFESP, Sa˜o Paulo, Brazil Received 11 July 2007, and in revised form 14 September 2007 Available online 12 October 2007

Abstract Leucurolysin-B (leuc-B) is an hemorrhagic metalloproteinase found in the venom of Bothrops leucurus (white-tailed-jararaca) snake. By means of liquid chromatography consisting of gel filtration on Sephracryl S-200, S-300 and ion-exchange on DEAE Sepharose, leuc-B was purified to homogeneity. The proteinase has an apparent molecular mass of 55 kDa as revealed by the reduced SDS–PAGE, and represents approximately 1.2% of the total protein in B. leucurus venom. The partial amino acid sequence of leuc-B was determined by automated Edman sequencing of peptides derived from digests of the S-reduced and alkylated protein with trypsin. Leuc-B exhibits the characteristic motif of metalloproteinases, HEXXHXXGXXH and a methionine-containing turn of similar conformation (‘‘Metturn’’), which forms a hydrophobic basis for the zinc ions and the three histidine residues involved as ligands. Leuc-B has been characterized as a P-III metalloproteinase and possesses a multidomain structure including a metalloproteinase, a disintegrin-like (ECD sequence instead of the typical RGD motif) and a cysteine-rich C-terminal domain. Leuc-B contains three potential sites of N-glycosylation. The enzyme only cleaves the Ala14–Leu15 peptide bond of the oxidized insulin B-chain and preferentially hydrolyzes the Aa-chain of fibrinogen and the a-chain of fibrin. Its proteolytic activity was completely inhibited by metal chelating agents but not by other typical proteinase inhibitors. In addition, its enzymatic activity was stimulated by the divalent cations Ca2+ and Mg2+ but inhibited by Zn2+ and Cu2+. The catalytic activity of leuc-B on extracellular matrix proteins could readily lead to loss of capillary integrity resulting in hemorrhage occurring at those sites (MHD = 30 ng in rabbit), with alterations in platelet function. In summary, here we report the isolation and the structure–function relationship of a P-III snake venom metalloproteinase.  2007 Elsevier Inc. All rights reserved. Keywords: Leucurolysin-B; Metalloproteinase; Disintegrin-like protein; Snake venom; Bothrops leucurus

Venoms produced by poisonous snakes are cocktails of biologically active proteins and peptides which kill or weaken their preys. Venoms of the viperidae family (vipers and pit vipers), are particularly rich sources of metalloproteases (SVMPs)1 and serine proteases (SVSPs). These *

Corresponding author. Fax: +55 31 3371 1753. E-mail address: [email protected] (E.F. Sanchez). 1 Abbreviations used: Leuc-B, leucurolysin-B; Leuc-a, leucurolysin-a; DMC, dimethylcasein; FUNED, Ezequiel Dias Foundation; EDTA, ethylenediaminetetraacetic acid; FN, fibronectin; EN, enactin; vWF, von Willebrand factor; PMSF, phenylmethanesulfonil fluoride; PRP, platelet-rich plasma; 0003-9861/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2007.10.002

enzymes affect various physiological processes including the coagulation cascade, fibrinolysis, platelet function and blood pressure [1,2]. SVMPs constitute the ‘‘reprolysin’’ group of zinc-dependent endopeptidases which together with matrix metalloproteases (MMPs), astacins and SBTI, soybean trypsin inhibitor; SVMPs, snake venom metalloproteinases; ECM, extra cellular matrix; MDCs, metalloproteinase and disintegrin-like cysteine-rich proteins; ADAMs, a disintegrin-like and metalloproteinase proteins; TFA, trifluoroacetic acid; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; MHD, minimum hemorrhagic dose.

194

E.F. Sanchez et al. / Archives of Biochemistry and Biophysics 468 (2007) 193–204

serralysins belong to the ‘‘metzincin’’ superfamily [1–4]. They are secreted as zymogen precursor proteins followed by processing to mature active forms in the venom gland [5–7]. Based on their domain structures they are classified into four major groups P-I (protein class I) to P-IV [5–8], with different group members often being found in the same venom. P-I SVMPs refers to the short metalloproteases consisting of the protease domain only. They can have either weak or no hemorrhagic activities. However, the exact mechanism of venom-induced hemorrhage is not fully explained. P-II proteases are synthesized with metalloprotease and disintegrin domains, and it has been proposed that most of them are readily hydrolyzed to yield a disintegrin as well as a P-I class SVMP [9]. High molecular mass (Mr) hemorrhagic metalloproteases (class P-III) contain a disintegrin-like and a cysteine-rich domain following the proteinase domain. The disintegrin-like domain contains a disulfide-bonded XECD sequence instead of the RGD/KGD motif that is the characteristic integrin-binding motif of disintegrins. P-III SVMPs are usually strong hemorrhagins and show restricted target specificity. The class P-IV SVMPs have an additional disulfide-linked C-type lectin domain. Similar domain structures are found in various transmembrane glycoproteins called ADAMs or MDCs [10], which function in physiological processes such as neurogenesis, fertilization, blood coagulation and cytokine shedding [11]. It has been documented that the proteinase domain of SVMPs primarily functions by degrading capillary basement membranes as well as the surrounding stroma to allow the escape of blood from the capillary [12]. These enzymes can also digest some coagulation factors including fibrinogen, vWF, fibrin and FN, which increases the hemorrhagic effect [13]. Furthermore, it is well established that disintegrins and P-III SVMPs are potent inhibitors of platelet aggregation, thus, further potentiating the hemorrhagic effect [14,15]. Bothrops (lancehead) snakes are medically relevant species found in Central and South America. The venom of these snakes contains metalloproteinases which cause local bleeding and/or severe systemic injury in envenomed human beings and mammals [8,16]. In Brazil, Bothrops snakes are the major cause of severe envenoming being responsible for approximately 90% of all recorded snakebites [17]. Among a great number of Bothrops species, B. leucurus has a limited range of distribution and is found in the Atlantic forest areas of the northeast of Brazil, including the States of Ceara and Bahia to Espirito Santo in the southeast. In these regions, which include the metropolitan area of Salvador City, B. leucurus is the principal species causing venomous snakebite in humans [18]. Unlike other Bothrops snakes (e.g. B. jararaca), the venom of the ‘‘white-tailed-jararaca’’ has not been studied in as much detail. Moreover, commercial bothropic antivenom has proved to be ineffective in neutralizing the lethal effects of B. leucurus venom compared with the other Bothrops (B. jararaca, B. alternatus, B. moojeni, B.

neuwiedi and B. jararacussu) species venom included in the antigenic pool [19]. For this reason, there is an urgent need to identify an effective alternative for treating Bothrops accidents in these regions. In view of the importance of understanding the toxicology and the pathophysiology of envenomenation by the white-tailed-jararaca, our laboratory works on the purification of individual components to elucidate their structure–function relationships which could lead to potentially useful clinical or research agents. A number of these components are implicated in a variety of local toxic effects as well as in profound disturbances in the hemostatic events of the victims. Like other viperidae snakes exhibiting fibrinolytic activity, B. leucurus venom contains two separate classes of fibrinolytic SVMPs with Mrs of approximately 23 and 55 kDa. In a recent work, we isolated and characterized a fibrinolytic P-I SVMP termed leuc-a. Leuc-a is composed of 200 amino acid residues with no hemorrhagic effect when injected (up to 100 lg) subcutaneously into mice and possessed an inhibitory effect on platelet aggregation induced by ADP [20]. The venom also contains a 35 kDa thrombin-like enzyme (leucurobin) [21] as well as other procoagulant components e.g. pro-thrombin activator (Sanchez et al., unpublished) that may provoke disseminated intravascular coagulation. In addition, two phospholipases A2 have been isolated and some of their structural and biological properties characterized. Based on their N-terminal sequences, the enzymes were termed D-PLA2 and K-PLA2, according to the amino acid residue (Asp or Lys) at position 48 [22]. In the present communication, we report the purification of a novel hemorrhagic 55 kDa SVMP from the venom of the white-tailed-jararaca. The isolated proteinase, has been called leucurolysin-B (leuc-B). We have determined the partial amino acid sequence of leuc-B and elucidated that it has a P-III class domain structure similar to other SVMPs. The results of experimental studies on its biochemical and functional properties are also presented. Material and methods Venom of B. leucurus was obtained from snakes captured in the south of Bahia State, and raised at the serpentarium of the Ezequiel Dias Foundation, Belo Horizonte, Brazil. SBTI; PMSF; oxidized insulin Bchain; bovine and human fibrinogen essentially plasminogen free; human placental type IV collagen and gelatin were obtained from Sigma Chemical (St. Louis, MO, USA). Collagen; ADP; human plasminogen were purchased from Calbiochem (San Diego, CA, USA). Fibronectin (FN) was purified from fresh human plasma (obtained from Hemepar, Curitiba, PR, Brazil) by gelatin–Sepharose affinity chromatography (Sigma) as described [23]. Vitronectin was purified from fresh human plasma by heparin–Sepharose affinity chromatography (Sigma) as described [24]. Laminin and laminin–entactin complex were purified from EHS tumor as described [25,26]. Type I collagen was purified from rat tail tendon according to [27]. N-glycosidase F (PNGase F) was from New England Biolabs (Beverly, MA, USA). All other chemicals were of analytical reagent grade.

E.F. Sanchez et al. / Archives of Biochemistry and Biophysics 468 (2007) 193–204

195

Venom fractionation

SDS–PAGE

Isolation procedures were performed at 4 C. Test tube fractions were collected and their protein concentrations determined by their absorbance at 280 nm. After each purification step, fractions were tested for hemorrhagic and proteolytic activity using dimethylcasein (DMC) and fibrin as substrates as described below. For the first step of purification, crude B. leucurus venom (2 g) was dissolved in 12 ml of 50 mM ammonium acetate buffer (pH 7.3) containing 0.3 M NaCl and centrifuged at 6000g to remove the insoluble material. The solution (1680 mg protein) was loaded onto two (2.5 · 100 cm each) columns in series packed with Sephacryl S-200, equilibrated and eluted with the same buffer at a flow rate of 7 ml/h and fractions of 7 ml/tube were collected. Proteinase activity was assayed on DMC and fibrin as substrates, and SDS–PAGE was performed on selected fractions. Active fractions were pooled and concentrated using a Centricon 10 microcentrifugal concentrator (Amicon, Inc., Danvers, MA). For the second step, the material (29.7 mg) from the first gel filtration was applied to a Sephacryl S-300 column (1.5 · 100 cm), equilibrated and eluted with 25 mM Hepes buffer, pH 7.5 containing 2 mM CaCl2 at a flow rate of 7 ml/h. The major fraction showing hemorrhagic and proteolytic activities was collected, dialyzed against distilled water and lyophilized. This material (26 mg) was dissolved in 2 ml of 50 mM Hepes buffer, containing 2 mM CaCl2 and applied to a DEAE Sepharose CL 6B column (1.6 · 20 cm), equilibrated with the same buffer. Bound proteins were eluted by using a linear salt gradient from 0 to 0.3 M NaCl at a flow rate of 12 ml/h. Purified leuc-B was analyzed by SDS–PAGE (10% or 12% gels) followed by staining with Coomassie blue.

SDS–PAGE was performed using 10% or 12% separating gels and a 4% upper gel according to the method of Laemmli [29].

Quantitation of protein Protein concentrations were estimated with the BCA reagent (Pierce Chemical Co.), using serum albumin (BSA) as a standard.

Reduction and S-pyridylethylation Purified leuc-B (2 mg) was reduced and alkylated with 4-vinyl pyridine essentially as described [28]. The material was dissolved in 1 ml of 0.1 M Tris–HCl, pH 8.6, containing 6 M guanidine–HCl. To this solution 30 ll of b-mercaptoethanol was added under nitrogen, and the sample incubated at 50 oC for 4 h. Then, 40 ll of vinyl pyridine was added and the solution was incubated at 37 C for further 2 h. The reduced and alkylated protein was desalted on a Vydac C4 (214TP54) column (4.6 mm · 25 cm), using a gradient of 0–70% acetonitrile in 0.1% aqueous TFA over 70 min at a flow rate of 1 ml/min. The collected protein was lyophilized. The S-pyridyl-ethylated protein (PE) (1 mg) was dissolved in 100 ll of 8 M urea and then made up to 1 ml with 0.1 M ammonium bicarbonate pH 7.9 and digested at 37 C with trypsin for 4 h, using 2% (w/w) enzyme:substrate. After lyophilization the peptides produced were separated by reverse phase HPLC on a Vydac C18 column (4.6 mm · 25 cm) (small pore, 201SP54) using an extended gradient of 0–50% acetonitrile in 0.1% aqueous TFA for 150 min at a flow rate of 1 ml/min. The amino acid sequences of the purified peptides were determined by Edman degradation using an automatic protein sequencing system PPSQ-21A (Shimadzu).

N-glycosidase digestion of leuc-B Leuc-B (200 lg) was dissolved in 90 ll of denaturing buffer (0.5% SDS, 1% b-mercaptoethanol). The protein solution was denatured by boiling for 10 min. After addition of 10 ll of reaction buffer (0.05 M phosphate, pH 7.5), 10 ll of 10% NP-40 and 2 U of recombinant PNGase F, the sample was incubated at 37 C for 22 h. The reaction was terminated by boiling for 5 min and 5 ll PAGE loading buffer was added to an aliquot (5 ll) of the reaction mixture. The samples were subsequently subjected to SDS–PAGE (10% or 12% gels) and stained with Coomasie blue.

Proteolytic specificity Substrate specificity was determined using oxidized insulin B-chain as substrate according to [30]. One milligram of the substrate was incubated with 0.3 lg leuc-B at an enzyme to substrate (w/w) ratio of 1:58 in 1 ml of 10 mM Tris–HCl buffer, pH 8.1 at 37 C. At specific time intervals (5, 10, 20, 30 and 60 min), 75 ll aliquots were withdrawn from the reaction mixture, and the reaction was stopped by adding 10 ll glacial acetic acid and kept frozen. The peptides resulting from the digestion of the insulin B-chain by leuc-B were separated on a column of Vydac C18 small pore (Vydac 201SP54). Elution was performed with a gradient of acetonitrile (0–60%, v/ v) in aqueous 0.1% TFA (v/v) at a flow rate of 1 ml/min for 60 min. Peptides were detected by their absorbance at 214 nm and collected manually. The amino acid sequences of the eluted peptides were determined. The position cleaved by the proteinase was deduced by comparing the amino acid sequences with the known amino acid alignment of the insulin B-chain.

Enzymatic activity assays of leuc-B To determine enzymatic activity of the proteinase during purification and to assess activity of the purified leuc-B, a DMC assay was used as described [31]. Fibrinolytic activity. Fibrin plate lysis by leuc-B was measured as previously described [32]. One unit was defined as lyzed zone (mm) per 1 lg. In these assays, three NIH U/ml of human thrombin (Sigma) was added to a bovine fibrinogen solution (2.5 mg/ml) in Tris–HCl buffered saline, pH 7.4, containing 0.15 M NaCl. The mixture was kept standing until clotting was complete (2 h at room temperature). Different amounts of leuc-B in a 10-ll volume were then added and incubated for 15 h at 37 C. To measure areas of lysis accurately, at the end of the incubation period the plate was flooded with 10% TCA solution. The haloes of lysis were measured against a dark background and compared with those of a control in which the proteinase was replaced by the buffer solution. Fibrin hydrolysis was also demonstrated by SDS–PAGE (12% gel). Digestion of fibrinogen. Human fibrinogen was dissolved at a final concentration of 2.5 mg/ml in 25 mM Tris–HCl buffer (pH 7.4) containing 0.154 M NaCl. The purified proteinase (3 lg) was added to 0.5 ml of fibrinogen solution at a molar ratio of enzyme to substrate of 1:150 (w/w) and the enzyme reaction was carried out at 37 C. At intervals (15, 30 and 60 min), 50-ll aliquots were withdrawn and mixed with an equal volume of denaturing solution (10 M urea, 4% b-mercaptoethanol, 4% SDS). The samples were reduced and denatured overnight at room temperature before being analyzed by SDS–PAGE (12% gel).

Proteolytic activity upon the extracellular matrix proteins To further investigate the proteolytic activity of leuc-B, purified ECM proteins were used as substrates for identification. The proteolytic cleavage of ECM proteins were carried out by incubating the purified proteins with different rates of leuc-B in vitro. Distinct enzyme:substrate ratios were used according the susceptibility of matrix molecules to leuc-B. For analysis of FN, EN and vitronectin digestions we used a rate of 1:100 (enzyme:substrate). Concerning laminin and collagens (types I and IV) a rate of 1:50 (enzyme:substrate) was used. Digestion reactions were performed for the indicated quantity ratios and time intervals at 37 C, except for type IV collagen, which was incubated at 25 C. Profiles of digestions were analyzed on SDS–PAGE under reducing conditions [29].

Platelet aggregation assay Platelet aggregation assay was performed with fresh human plateletrich-plasma (PRP) as described in previous report [31]. A PACKS-4

196

E.F. Sanchez et al. / Archives of Biochemistry and Biophysics 468 (2007) 193–204

platelet aggregation chromogenic kinetic system (Helena Laboratories, Beamont, TX) was used to monitor platelet aggregation at 37 C. Platelet aggregation was initiated by the addition of collagen (2 lg/ml) or ADP (10 lM). Inhibition of collagen induced platelet aggregation was conducted by adding leuc-B (100–1000 nM final concentration) 3 min prior to the addition of the agonist. The extent of the inhibition of platelet aggregation was estimated by comparison with the maximum aggregation induced by the control dose of collagen and then expressed as a percentage. Replicate experiments were conducted with PRP from different donors, since the results showed some variability in the intensity of inhibition.

Binding assays of a1b1 and a2b1 integrins to immobilized leuc-B Recombinant soluble a1b1 and a2b1 integrins were produced as described previously [33,35]. The binding of the a1b1 and a2b1 to immobilized leuc-B were measured by ELISA. Microtiter plates were coated with leuc-B (40 lg/ml) in TBS (50 mM Tris/HCl, pH 7.4, 150 mM NaCl), containing 2 mM MgCl2 and 1 mM CaCl2. They were also coated with jararhagin (10 lg/ml) in the same buffer. In parallel, the collagen IV fragment CB3 and collagen I, both coated at a concentration of 5 lg/ml, were used as positive controls to test the binding activities of the integrins a1b1 and a2b1, respectively. After washing and blocking the wells with 1% BSA solution in TBS, pH 7.4, containing 2 mM MgCl2, a1b1 and a2b1 integrins were added in the same buffer either in the presence of 1 mM MnCl2 or 1 mM MnCl2 plus the integrin-activating antibody 9EG7 or 10 mM EDTA. Bound integrin was detected by ELISA with antiserum directed against the integrin b1 subunit and an anti-rabbit-antibody conjugated to alkaline phosphatase (Sigma) as described previously [33–35].

tion II (tubes 24–29) which possessed hemorrhagic and proteolytic activities was collected, desalted and lyophilized. Analysis of fraction II by SDS–PAGE (12% gel) showed other protein bands. Therefore, the lyophilized fraction II (26 mg) was further purified by anion-exchange chromatography on a column of DEAE Sepharose CL 6B, at pH 7.5 using a gradient of NaCl as eluent. Chromatography of fraction II on a DEAE column resulted in four protein fractions (a–d) (Fig. 1, 3rd step). Proteolytic activity on DMC was found in fractions a, b and d, however, the hemorrhagic effect was concentrated in fraction d. Active fraction d which eluted at a concentration of 0.1– 0.2 M NaCl, corresponds to leuc-B. Based on the consideration of original source (B. leucurus venom) and because it is the second metalloproteinase to be isolated, we named the purified protein leucurolysin-B (leuc-B). The final step resulted in an overall yield of 24.5%. The specific activity of the purified leuc-B was 33 U/mg, corresponding to a purification factor of 20.6 with respect to hemorrhagic activity. Leuc-B represented 1.2% (w/w) of the total venom protein. Table 1 summarizes the purification procedure of leuc-B. Primary structure of leuc-B

Purification of leuc-B

Our attempts to determine the N-terminal sequence of leuc-B by automated Edman degradation of the intact protein were unsuccessful, probably due to the presence of a blocked amino acid (possible pyrrolidone carboxylic acid) at the terminus. This type of blocked N-terminus has been reported in several of the other high molecular mass SVMPs [7,36]. We were also unable to isolate any of the putative peptides which should have been produced by the tryptic digestion of the N-terminal sequence of residues 1–85 which is present in many of the larger SVMPs. However, the tryptic peptides resulting from the hydrolysis of the sequence 86–422 were obtained in high yield and con-

Using three chromatographic steps, we isolated a novel SVMP from the venom of white-tailed-jararaca (B. leucurus). First, the crude venom (2 g, 1680 mg protein) of B. leucurus was separated into seven fractions (1–7) by gel filtration on a Sephracryl S-200 column (Fig. 1, 1st step, not shown). Proteolytic activity on DMC and fibrin was found in fractions 1 and 4. Fraction 1 (tubes 133–137) which contained the high molecular mass proteins showing hemorrhagic (55%) and proteolytic (22%) activities was concentrated for further purification. Fraction 4 (tubes 185–191) contained proteins with Mrs of approx. 24 kDa exhibiting proteolytic (29%) and hemorrhagic (5%) activities. A 23 kDa metalloproteinase termed leuc-a was recently purified from this fraction [20]. Leuc-a was characterized as a P-I SVMP showing direct fibrinolytic effect without hemorrhagic activity. For the second step of purification, a concentrated fraction 1 (29.7 mg) was then subjected to gel filtration chromatography on a Sephacryl S300 column (Fig. 1 2nd step not shown). The major frac-

Fig. 1. Purification of leuc-B from B. leucurus venom (3rd step). Fraction II from the 2nd step was applied to a DEAE ion-exchange column (20 · 1.6 cm). The column was pre-equilibrated with 50 mM Hepes buffer, pH 7.5 containing 2 mM CaCl2. Elution was performed at a flow rate of 12 ml/h with a buffer gradient as indicated. The active fractions (tubes 30– 34) containing leuc-B were pooled.

Other methods Clotting activity was tested with bovine fibrinogen and citrated human plasma as described in [19]. Amidolytic activity was tested with the peptide p-nitroanilides Tos-Gly-Pro-Lys-pNA, Bz-Pro-Phe-Arg-pNA and DLBAPNA [31]. Plasminogen activation assays were measured with human plasminogen and the plasmin substrate Tos-Gly-Pro-Lys-pNA as described [31].

Results

E.F. Sanchez et al. / Archives of Biochemistry and Biophysics 468 (2007) 193–204

197

Table 1 Purification of leucurolysin-B (leuc-B) from Bothrops leucurus snake venom Step

Crude venon Sephacryl S-200 Sephacryl S-300 DEAE SepharoseCL-6B a b c

Protein

Hemorrhagic activity

Proteolytic activity c

mg

%

MDH (lg)

Sp. activity U/mga (103)

Total (U)

Yield (%)

PF

Sp. activity U/mgb (103)

Total (U)

Yield (%)

PFc

1680 29.7 26.0 20.0

100 1.8 1.6 1.2

0.24 0.90 0.10 0.03

1.6 4.5 10 33

2688 134 260 660

100 4.9 9.7 24.5

1 3 6 20.6

8.5 35.4 25.2 34.7

14,280 1051 655 694

100 7.4 4.6 4.8

1 4 3 4

One unit of hemorrhagic activity is one minimum hemorrhagic dose (MHD) per mg. One unit of DMC-hydrolyzing activity was defined as DA340/min. The enzyme activity was expressed as units/mg. PF, purification factor.

firmed the sequence. The amino acid sequences of the tryptic peptides were determined; corresponding to the amino acid residues placed from number 86–422 (Fig. 2). As shown in Fig. 2, the sequence of the amino terminal half (residues 86–205) of leuc-B contains a metalloproteinase structure similar to other SVMPs, including the P-I class, for instance leuc-a isolated from the same B. leucurus venom [20]. This domain contains the conserved zinc-binding motif HEXXHXXGXXH from residues 145 to 155 as well as the methionine-turn, CIM (residues 167–169), that is also involved in zinc-binding of the metzincin superfamily of metalloproteinases. A sequence comparison in Fig. 2, showed that leuc-B has high homology with other P-III SVMPs such as jararhagin and bothropasin from B. jararaca and HR1B from T. flavoviridis. The disintegrin-like domain of leuc-B contains a putative integrin-interacting motif ECD (residues 278–280, or in some cases DCD), that is characteristic for many P-III SVMPs as well as for ADAM metalloproteinases [37]. As expected, the purified enzyme was capable of inhibiting collagen-stimulated platelet aggregation in a concentration-dependent manner (data not shown). An interesting feature found in the structure of leuc-B is the presence of three Asn-linked consensus sequences (Fig. 2). One linked to Asn183 in the metalloproteinase domain, which is also found in the corresponding regions of the three other P-III SVMPs (jararhagin, bothropasin and HR1B), but not in the P-I class leuc-a. The other two N-linked glycosylation motifs in leuc-B are located at residues 385 and 400, respectively. To examine the Asn-linked sugar chains, leuc-B was deglycosylated by digestion with PNGase F which specifically cleaves off Asn-linked sugar chains from glycoproteins. When the covalently attached carbohydrate structures were removed from the native protein (55 kDa) by PNGase F treatment, the deglycosylated leuc-B migrated as a smaller molecular size corresponding to approx. 40 kDa (Fig 3, lane D, deglycosylated; lane N, native). These results suggest that glycosylation at one, two or three of these sites gives rise to the difference in the calculated mass. Other P-III SVMPs like acurhagin from Formosan Agkistrodon acutus and AAVI from A. acutus are glycoproteins with one N-glycosylation site.

Native leuc-B migrated with a mobility of approx. 55 kDa under both non-reduced (not shown) and reduced conditions as examined by SDS–PAGE (Fig. 3, lane N), indicating that it exists as a single polypeptide chain protein. The broad band with several apparent isoforms suggested that the protein was glycosylated. In addition, the enzyme exhibited a gelatinolytic activity, one cleared band (55 kDa) was observed using gelatin zymography (not shown). As observed in Fig. 2, the metalloproteinase domain of leuc-B like HR1B and other toxins of the P-III SVMPs share six conserved cysteinyl residues at positions 120, 160, 162, 167, 184, 200 (leuc-B numbering). In addition to these six cysteinyl residues, other members of the P-III class including jararhagin, bothropasin, acurhagin and catrocolastatin have a seventh cysteinyl residue conserved at position 189 [38]. Sequence comparison of the disintegrinlike region of leuc-B with other disintegrin-like proteins indicated that the toxin from B. leucurus is most similar to HR1B, with 80% amino acid identity, followed by jararhagin and bothropasin, with 77% and 72%, amino acid identity, respectively. Similarly, the cysteine-rich domain of leuc-B, exhibits high homology with the corresponding region of jararhagin (76%), bothropasin (74%) and with HR1B (72%). These P-III hemorrhagic toxins share 12 cysteinyl residues located at conserved positions in their cysrich region. Moreover, almost all the cysteine residues (32 residues) are conserved in the P-III class SVMPs. Proteolytic specificity of leuc-B It is important to characterize the peptide bonds which are hydrolyzed by leuc-B and the manner in which the enzyme acts on potential ECM and plasma proteins substrates such as laminin, type IV collagen, enactin, fibrinogen and fibronectin. To determine the bond specificity, leuc-B was incubated with oxidized insulin B-chain as a model substrate. The site of cleavage of the insulin B-chain by the proteinase was determined from the amino acid sequences of the purified peptides (A and B), which were isolated by reverse phase HPLC of the incubation mixtures after specific incubation periods (5–60 min). Experimental data indicated that leuc-B cleaved only the Ala14–Leu15

198

E.F. Sanchez et al. / Archives of Biochemistry and Biophysics 468 (2007) 193–204

1 10 20 30 40 50 Leuc-B ----------------------------------------------------Jarar EQQRYDPYKYIEFFVVVDQGTVTKNNGDLDKIKARMYELANIVNEIFRYLYMH Bothr EQQKYNPFRYVELFIVVDQGMVTKNNGDLDKIKARMYELANIVNEILRYLYMH HR1B EQQRF-PRRYIKLAIVVDHGIVTKHHGNLKKIRKWIYQLVNTINNIYRSLNIL Leuc-a ------SPRYIELVVVADHGMFKKYNSNLNTIRKWVHEMLNTVNGFYRSMNVD

Leuc-B Jarar Bothr HR1B Leuc-a

Leuc-B Jarar Bothr HR1B Leuc-a

Leuc-B Jarar Bothr HR1B Leuc-a

Leuc-B Jarar Bothr HR1B

60 70 80 90 100 --------------------------------DTVLLNRISHDNAQLLTAIVF VALVGLEIWSNGDKITVKPDVDYTLNSFAEWRKTDLLTRKKHDNAQLLTAIDF AALVGLEIWSNGDKITVKPDVDYTLNSFAEWRKTDLLTRKKHDNAQLLTAIDF VALVYLEIWSKQNKITVQSASNVTLDLFGDWRESVLLKQRSHDCAQLLTTIDF ASLVNLEVWSKKDLIKVEKDSSKTLTSFGEWRERDLLPIRSHDHAQLLTVIFL : ∗∗ ∗∗ ∗ ∗ ∗∗∗ ∗ : 110 120 130 140 150 NENVIGKAYTGGMCDPRYSVGVVMDHSPINRLVADTMAHEMGHNLGIHHDTGS NGPTIGYAYIGSMCHPKRSVGIVQDYSPINLVVAVIMAHEMGHNLGIHHDTGS NGPTIGYAYIGSMCHPKRSVAIVEDYSPINLVVAVIMAHEMGHNLGIHHDTDF DGPTIGKAYTASMCDPKRSVGIVQDYSPINLVVAVIMTHEMGHNLGIPHDGNS DEETIGIAYTAGMCDLSQMVAWGQDY------VAVSMAHELGHNLGMRHDGNQ : ∗∗ ∗∗ : ∗∗ :∗ ∗ : ∗∗∗∗ : ∗∗ ∗ ∗∗ ∗∗∗∗ ∗ ∗∗ 160 170 180 190 200 210 CSCGGHSCIMSRVISHQPLQYFSNCSYIEYWDFITKLNPQCILNEPLRTDIVS CSCGDYPCIMGPTISNEPSKFFSNCSYIQCWDFIMNHNPECIINEPLGTDIIS CSCGDYPCIMGPTISNEPSKFFSNCSYIQCWDFIMKENPQCILNEPLGTDIVS CTCGGFPCIMSPMISDPPSELFSNCSKAYYQTFLTDHKPQCILNAPSKTDIVS CHCNAPSCIMADTLSKGLSFEFSDCSQNQYQTYLTKHNPQCILNKP : : ∗ : ∗∗ : ∗ ∗ ∗∗∗ : ∗ ∗ ∗ ∗∗∗ :∗ ∗∗ : ∗∗ : 220 230 240 250 260 PPVCGNELLEMGEECDCGSPRNCRDLCCDAATCKLHSWVECESGECCDQCRFI PPVCGNELLEVGEECDCGTPENCQNECCDAATCKLKSGSQCGHGDCCEQCKFS PPVCGNELLEVGEECDCGTPENCQNECCDAATCKLKSGSQCGHGDCCEQCKFS PPVCGNELLEAGEECDCGSPENCQYQCCDAASCKLHSWVKCESGECCDQCRFR ∗∗∗ ∗∗ : ∗∗∗ : ∗ ∗ ∗ : ∗∗ : ∗∗ : ∗ ∗∗∗∗∗∗∗∗∗∗∗ ∗ ∗ ∗ ∗∗∗ ∗ : ∗ ∗∗ : 270 280 290 300 310 KAGNVCRPPRKECDVAEACTGQSAQCPTDDFKRNGQPCLNNYAYCYQGNCPIM KSGTECRASMSECDPAEHCTGQSSECPADVFHKNGQPCLDNYGYCYNGNCPIM KSGTECRASMSECDPAEHCTGQSSECPADVFHKNGQPCLDNYGYCYNGNCPIM TAGTECRAAESECDIPESCTGQSADCPTDRFHRNGQFCLYNHGYCYNGKCPIM ∗∗∗ ∗ ∗∗∗∗ ∗ : ∗∗ ∗ ∗: ∗∗∗ ∗∗ ∗ : ∗∗∗ : ∗ : ∗ ∗ ∗∗ ∗ ∗∗ 320 330 340 350 360 370 YHQCYALFGSDATMAQDSCFQVNKKGNEYFYCRLENGINIPCAQEDVKCGRLF YHQCYALFGADVYEAEDSCFKDNQKGNYYGYCRKENGKKIPCAPEDVKCGRLY YHQCYALFGADVYEAEDSCFKDNQKGNYYGYCRKENGKKIPCAPEDVKCGRLY FYQCYFLFGSNATVAEDDCFNNNKKGDKYFYCRKENEKYIPCAQEDVKCGRLF : ∗∗∗ ∗∗ ∗ : ∗ : ∗ ∗∗ : ∗: ∗ ∗: ∗ ∗∗∗ ∗∗ ∗∗∗ ∗ ∗∗∗ ∗ ∗ ∗ ∗∗ 380 390 400 410 420 CHNMKYEQD--CNYSDR------GMVDNGTKCAEGKVCNSNR-----QAYQR CKDNSPGQNNPCKMFYSNDDEHKGMVLPGTKCADGKVCSNGHCVDVATAY CKDNSPGQNNPCKMFYSNDDEHKGMVLPGTKCADGKVCSNGHCVDVATAY CDNK----KYPCHYNYSEDLDF-GMVDHGTKCADGKVCSNRQCVDVNEAYKS ∗ : ∗: ∗ ∗∗∗ ∗∗ ∗∗∗ : ∗∗∗∗ ∗∗ : ,

Leuc-B Jarar Bothr HR1B

Leuc-B Jarar Bothr HR1B

Leuc-B Jarar Bothr HR1B

Fig. 2. Comparison of the amino acid sequence of leucurolysin-B with the sequences of other SVMPs. The amino acid sequence of leuc-B (this work) was aligned with those of other P-III SVMPs, jararhagin (Jarar) from B. jararaca (GenBank Accession No. P30431), bothropasin (Bothr) from B. jararaca (AAC61986), HR1B (trimerelysin I) from T. flavoviridis [36] and leuc-a, a P-I SVMP from the same B. leucurus [20]. Zinc binding motif (HEMGHNLGIH), and the methionine 169 of the basement (Met-turn) are invariant and are expressed in boldface. Disintegrin-like (ECD) sequences are expressed in boldface. Gaps were inserted to obtain maximum degrees of similarity. Putative N-linked glycosylation sites are in grey box. PyroGlu residue is conventionally assigned as residue 1 [55]. Numbers on the top indicate the residue number of leuc-B. (*) positions fully conserved; (:) strongly similar residues.

bond after 20 min (not shown). The cleavage site was identified by the sequences of the purified peptides A and B after 30 and 60 min incubation (Fig. 4). Other multidomain P-III hemorrhagic toxins including mutalysin I from bushmaster venom [30], hemorrhagic protease IV from timber rattlesnake venom [39], and hemorrhagic factor b from the Chinese habu snake [40] were also characterized by

their restricted substrate specificity. If the specificity of leuc-B is compared with some non-hemorrhagic P-I class SVMPs, e.g. leuc-a [20] and neuwiedase [56] from the venoms of B. leucurus and B. neuwiedi, the latter enzymes also cleaved the peptide bond Tyr16–Leu17 in addition to the first cleavage of the Ala14–Leu15 bond in the oxidized insulin B-chain.

E.F. Sanchez et al. / Archives of Biochemistry and Biophysics 468 (2007) 193–204

199

Table 2 Effect of several reagents on the proteolytic activity of leucurolysin-B Compound added

Concentration (mM)

Residual activity (%)

Enzyme EDTA PMSF SBTI DDT Ca2+ Zn2+ Mg2+ Zn2+ + Ca2+ Zn2+ + Mg2+ Ca2+ + EDTA Mg2+ + EDTA

– 3 3 200 lg/ml 2 2 2 2 2 (each) 2 (each) 2 (each) 2 (each)

100 3.3 ± 1.2 85 ± 4.5 65 ± 2.9 0 114 ± 4.3 0 125 ± 3.2 0 0 0 0

The purified leuc-B (1 lg) was treated with each reagent at the indicated concentrations in 50 mM Hepes buffer, pH 8.0 (1 ml final volume) at 37 C for 30 min. Proteolytic activity was measured with DMC as substrate. These values represent the means ± SD (n = 4).

Fig. 3. Characterization and deglycosylation of leuc-B. SDS–PAGE (12%, w/v, reduced conditions) analysis of purified leuc-B (5 lg) (lane N) and after incubation with N-glycosidase F (2 U) at 37 C for 22 h (lane D). The gel was stained with Coomassie blue. Molecular mass markers are shown on the left.

Enzymatic activity The general proteolytic activity of leuc-B was tested by the sensitive DMC assay. Rate of proteolysis was dependent on pH and temperature. It was optimal at pH 7.5– 9.3 and 30 C (not shown). Table 2 summarizes the effect

of several inhibitors and cations on DMC hydrolyzing activity. DMC degradation by leuc-B was strongly inhibited by pre-incubation with a chelating agent EDTA (3 mM), however, PMSF (3 mM), a reagent for active-site serine residues of serine proteinases had no effect. These results were in agreement with the hemorrhagic effect of the enzyme in mouse and rabbit models (data not shown). Furthermore, the internal disulfide bonds were also found to be critical for structural integrity or enzymatic activity of leuc-B, since pre-incubation with 2 mM DTT completely abolished the DMC hydrolysis. The effect of several cations on DMC hydrolyzing activity was examined. The relative

Fig. 4. Cleavage site in oxidized insulin B-chain by leuc-B. (Upper panel) HPLC profiles of the products of digestion of oxidized insulin B-chain by leuc-B. Chromatography of an incubate containing 1 mg of insulin B-chain with 0.3 lg enzyme was performed at 0, 30 and 60 min (under conditions as described in the text). The solvent systems used were: A, 0.1% TFA in H2O (v/v); and B, acetonitrile containing 0.1% TFA. The column was eluted with a linear gradient from 0% to 60% of solvent B in 60 min at a flow rate of 1 ml/min. (Lower panel) The arrow indicate the cleavage site of oxidized insulin B-chain. The proteolytic specificity of leuc-B (this work) was determined by automated Edman degradation of the HPLC-purified peptides (A and B) and comparing their amino acid sequences to the known sequence. The arrowhead indicates the cleavage site of oxidized insulin B-chain.

200

E.F. Sanchez et al. / Archives of Biochemistry and Biophysics 468 (2007) 193–204

activity of the enzyme was slightly enhanced in the presence of 2 mM Ca2+ (114%) as well as Mg2+ (125%). However, its proteolytic activity was strongly inactivated by 2 mM Zn2+ and Cu2+ (not shown). To examine whether leuc-B recovers its enzymatic activity by reconstituting it with calcium or magnesium, the inactivated enzyme was incubated with DMC in the presence of Ca2+ and Mg2+. As can be seen in Table 2, leuc-B treated with EDTA or Zn2+ could not be reversed by the addition of Ca2+ or Mg2+. Therefore, the effect of metal chelation on its enzymatic activity and its primary structure indicate the metalloproteinase nature of leuc-B.

Fibrin and fibrinogen degradation by leuc-B SVMPs have fibrin(ogen)olytic activities. They preferentially degrade either the Aa- or Bb-chains of fibrin(ogen). Thus, the proteolytic enzymes in snake venoms have been defined as a- or b-fibrinogenases [1,41]. The fibrin(ogenol)ytic activity of leuc-B was investigated by incubating with human fibrin(ogen) that is also required for platelet aggregation. Leuc-B digests the a-chains of fibrin and fibrinogen dose-dependently in vitro (not shown). The purified leuc-B was incubated with fibrinogen or fibrin for different periods of time (0–60 or 0–120 min, respectively). When the reaction products were analyzed by SDS–PAGE (reduced conditions), it was evident that the enzyme is an a-fibrinogenase with the Aa-chains of fibrinogen (not shown) and a-chains of fibrin (Fig. 5) being degraded within 15 min. Under the experimental conditions the intensities of the Bb- and c-chains bands of fibrinogen (not shown) and b-chains bands of fibrin do not change over time, indicating in both cases that these chains do not undergo lysis. In agreement with this concept, a P-I

Fig. 5. Digestion of fibrin by leuc-B. Human fibrin was incubated with leuc-B for the indicated time intervals and aliquots (5 lg) were subjected to SDS–PAGE (12%) under reducing conditions. Fibrin control (no leuc-B) after 120 min incubation is shown (lane C). Molecular mass markers are shown at the right. Arrow on the right side of the gel indicates the main degradation products.

SVMP leuc-a found in the same B. leucurus venom has been characterized as an a-fibrinogenase [20]. Hydrolysis of extracellular proteins To further characterize the enzymatic function of leucB, its enzymatic activity was investigated using types I and IV collagens as well as their gelatins, fibronectin (FN), laminin, enactin (EN) and vitronectin. The multidomain proteinase containing the metalloproteinase, disintegrin-like and cysteine rich (MDCs) domains was able to hydrolyze type I collagen and its gelatin. As shown in Fig. 6, a1a1-chains were first degraded followed by the a1a2-chains. In the case of type IV collagen from basement membrane (BM), the molecule contains two different polypeptides, a1-chain (Mr  200 kDa) and a2-chain (Mr  180 kDa). In the experiments presented here, the a1-chain was rapidly digested in 1 h, whereas a2-chain was resistant to hydrolysis. As can be seen in Fig. 6, this pattern does not change substantially during 6 h incubation. In a like manner to type I gelatin, leuc-B is a very effective proteinase on type IV gelatin substrate that was degraded completely in 6 h (not shown). Laminin binds to collagen type IV, heparan sulfate proteoglycan, to enactin/nidogen, and to itself to create an integrated structure within the BM. Laminin from EHS

Fig. 6. Proteolytic digestion of collagens type I and IV by leuc-B. SDS– PAGE analysis of collagen I (panel at the left) and collagen IV (panel at the right) after incubation with leuc-B for 1 and 6 h at 37 C at rate of 1:50 (enzyme:substrate). Aliquots of the incubation mixtures of type I collagen were analyzed by SDS–PAGE (5%). Typical type I collagen chains (a1a1, a1a2, a1 and a2) are shown as indicated. Purified type IV collagen sample was incubated with leuc-B under similar experimental conditions and then subjected to SDS–PAGE (7.5%). Typical type IV collagen chains (a1, a2) are indicated. All experiments were performed under reducing conditions. Position of the molecular mass markers are shown on the left of the gels. Lane C depicts control molecules (without any leuc-B treatment).

E.F. Sanchez et al. / Archives of Biochemistry and Biophysics 468 (2007) 193–204

201

tumor appears as two major bands, a1 and b1, c1 of approx. 400 and 210 kDa, respectively. From SDS–PAGE, laminin appears to be resistant to hydrolysis by leuc-B, no evidence of degradation products were observed even after 24 h incubation (not shown). FNs are a group of extracellular glycoproteins that are composed of structurally similar subunits varying in size between 210 and 250 kDa. One form of FN is found in plasma (plasma FN). Plasma FN appears as a band of approx. 210 kDa. The digestion pattern of FN by leuc-B showed the major degradation product of 80 kDa after 1 h and other fragments which appeared below FN control were observed after 6 h (Fig. 7). Plasma vitronectin is known to be involved in the processes that follow platelet stimulation, specifically in the binding of heparin, in the growth of endothelial cells and in fibrinolysis [42]. Fig. 7 shows two physiologically occurring forms of vitronectin: a one-chain (75 kDa) form, and the nicked two-chain (65 kDa) form. Enzymatic reaction of leuc-B with plasma vitronectin resulted in rapid degradation of the 75 band followed by the 65 kDa band. The major degradation product with apparent Mr of 42 kDa was observed after 1 h incubation, while the 75-chain was digested after 6 h. Finally, EN is a sulfated glycoprotein and when isolated from BM is generally observed as a band of 158 kDa. The

EN substrate was totally cleaved by leuc-B giving rise to two major digestion fragments of 100 and 50 kDa after 1 h (not shown).

Fig. 7. Proteolytic digestion of fibronectin and vitronectin by leuc-B. (Panel at the left) Purified human plasma FN samples were incubated with leuc-B for 1 and 6 h (37 C) at a substrate to enzyme ratio of 1:100 (w/w). Aliquots of the incubation mixture were analyzed by 7.5% SDS–PAGE. (Panel at the right) Purified human plasma vitronectin sample was incubated with leuc-B for 1 and 6 h (37 C) at rate of 1:100 (enzyme:substrate). Aliquots of the incubation mixtures were analyzed by SDS–PAGE (12%). Molecular masses of 75 and 65 kDa (left) indicate the typical profile of vitronectin in SDS–PAGE. Experiments were performed under reducing conditions. Lane C depicts control molecules (without any leuc-B treatment).

Fig. 8. Binding of a1b1 and a2b1 integrins to leuc-B and jararhagin. Leuc-B (40 lg/ml), jararhagin (10 lg/ml), and CB3 (5 lg/ml), all dissolved in TBS, pH 7.4, containing 2 mM MgCl2, as well as collagen I (5 lg/ml) in 0.1 M acetic acid were coated onto a microtiter plate. After blocking with 1% BSA solution in TBS, pH 7.4, 2 mM MgCl2, the wells were incubated with the integrins a1b1 (white bars) and a2b1 (gray bars), each at 50 nM in the same buffer, to which either 1 mM MnCl2 (coarsely hatched) or 1 mM MnCl2 and 200 nM 9EG7 (fine hatched) or 10 mM EDTA (striped) were added. Bound integrins were detected as described under Methods. Collagen IV fragment CB3 had been isolated according to [57]. Means and SD of duplicate measurements of a representative experiment are shown.

Effect of leuc-B on platelet aggregation and binding to a1b1 and a2b1 integrins It has been documented that the majority of SVMPs reported so far have inhibitory effects on platelet aggregation induced by several agonists. Leuc-B inhibited the human collagen-induced aggregation in a dose-dependent manner (data not shown). To study the action of the disintegrin-like domain of leuc-B on platelet aggregation, the enzyme was inactivated by EDTA treatment, since there is the possibility that leuc-B might affect platelet function by its enzymatic activity. The inactive enzyme showed less effect than the native proteinase in inhibiting platelet aggregation (not shown). These results indicate that enzymatic activity of leuc-B is also involved in the inhibitory effect on platelet aggregation. To discriminate the action of leuc-B towards collagenbinding integrins or collagens, we performed the binding assays of leuc-B to the major collagen receptors a1b1 and a2b1 integrin of endothelial cells and platelets, respectively, in an ELISA (Fig. 8). Leuc-B did not interact with either of the integrins ruling out the hypothesis that leuc-B may cause severe bleeding and hemorrhages by blocking the col-

202

E.F. Sanchez et al. / Archives of Biochemistry and Biophysics 468 (2007) 193–204

lagen receptors on the endothelial cells or platelets. This mechanism is postulated for jararhagin, another P-III SVMP [15]. Whereas a2b1 integrin weakly bound to jararhagin (Fig. 8, and previously published [34]), leuc-B failed to bind to both a2b1 and a1b1 integrin. A very small signal was seen when a1b1 integrin was incubated with leucB in the presence of EDTA. The physiological significance of this observation remains elusive. Nevertheless, this signal was very weak when compared to the binding of the integrins, a1b1 and a2b1, to their physiological ligands, the triple-helical fragment CB3 of collagen IV and collagen I, respectively, (positive controls in Fig. 8). Discussion SVMPs especially of the pit vipers and true vipers are responsible for local and systemic hemorrhage due to proteolysis of the major components of the ECM. Since a number of them are chimeric proteins composed of distinct domains, they also produce complex effects on the hemostatic system. In this study, we have purified to homogeneity an endopeptidase termed Leuc-B, which represents a novel P-III SVMP found in B. leucurus venom. Recently, our group have reported the isolation and structure–function studies of a P-I SVMP, called leuc-a from the same B. leucurus venom. Later data indicated that leuc-a has potential thrombolytic effect, since it was devoid of hemorrhagic effect and with minimal alterations to the hemostatic system [20,43]. However, until now, the mechanism of hemorrhage produced by SVMPs is not entirely clear [44]. In this report, we have determined the partial amino acid sequence of leuc-B. Its primary structure indicates that leuc-B is classified into the high molecular mass metalloproteinase P-III group comprised of an N-terminal metalloproteinase, a disintegrin-like and a C-terminal Cys-rich domains. The multidomain structure is in agreement with the common precursor model of snake venom metalloproteinase/disintegrin [45]. Leuc-B contains the consensus active-site residues for a zinc-dependent metalloproteinase, HEXXHXXGXXH (Fig. 2), and is in accordance with the data that leuc-B loses its enzymatic activity after treatment with EDTA. Although, Ca2+ or Zn2+ are required for catalytic activity, Mg2+ and additionally Ca2+ enhanced leuc-B activity. The presence of calcium ions located on the surface of a number of SVMPs imply that this cation is important for structural stabilization of the protein [46]. In addition, calcium ions are known to stabilize the tertiary structure of MMPS with collagenase activity [47]. However, Zn2+ and Cu2+ were found to be effective inhibitors of leuc-B proteolytic activity (Table 2). Moreover, until now its not clear whether zinc is necessary for any functional activity, but a role of this cation could not be excluded, since zinc is tightly bound to leuc-B. The inhibitory effect on leuc-B by adding zinc was also reported for leuc-a [20] and other SVMPs [46]. Furthermore, the disruption of intramolecular S–S bonds by DTT treatment resulted in completely inactive enzyme,

implying that disulfide bonds were important for maintaining the molecular structure and activity of leuc-B. On the other hand, it is clear that the primary site of cleavage by SVMPs is Ala14–Leu15 when oxidized insulin B-chain is used as a model substrate. For leuc-B, a major hemorrhagic factor (MHD = 30 ng, in rabbit) from B. leucurus venom, this is the only bond cleaved. In general, members of the P-III SVMPs such as mutalysin-I from bushmaster venom [30] and other P-III toxins, have a restricted specificity and potent hemorrhagic activity [39,40]. At present, specific residues or structural motifs responsible for inducing hemorrhage are still not fully elucidated. Additional studies on the substrate specificity of the metalloprotease would be valuable to understand the structure–function correlation of SVMPs. On the other side, degradation and remodeling of the ECM and basement membranes by proteolytic enzymes are essential steps in several physiological (e.g. wound repair, tissue regeneration and embryonal development) as well as in pathological processes including, reumathoid arthritis, osteoarthritis, periodontitis, tumor development and progression [48]. Thus, the presence of exogenous proteinases which degrade ECM can result in pathological conditions. In this context, and to determine if leuc-B was able to interfere with hemostatic system, its proteolytic effect on fibrin(ogen) was examined. Leuc-B, was able to digest preferentially the Aa-chain of fibrinogen and the a-chain of fibrin. Like leuc-a, the enzyme is capable of promoting anticoagulaion by the extensive hydrolysis of the a-chains of fibrin/fibrinogen. It was proposed that the particular degradation of fibrinogen Aa-chain by jararhagin does not affect platelet aggregation, but interferes with fibrin polymerization [49]. Moreover, one of the action mechanism of leuc-B seems to be to degrade fibrin(ogen) as well as other components of the ECM. Therefore, effective degradation of one or more components of the ECM surrounding capillary endothelial cells by the SVMPs appeared to be the principal mechanism for the hemorrhage production associated with snake envenomation. Numerous studies have shown that disruption of the BM is likely to be the result of the proteolytic activity of the SVMPs upon specific proteins that comprise the BM [8,50]. Under our experimental conditions, leuc-B was able to hydrolyze types I and IV collagens and more efficiently their gelatins, whereas laminin from the EHS tumors was resistant to hydrolysis. In addition, the glycoproteins EN and plasma FN and vitronectin appeared to be good substrates for the venom enzyme. The disintegrin-like domain of leuc-B has an ECD motif at the position corresponding to the RGD sequence of disintegrins, which is the binding motif of various cell surface integrins and is highly similar to that of other P-III SVMPs (Fig. 2). The venom proteinase was able to inhibit the collagen-induced platelet aggregation in a concentration dependent manner. As both native and EDTA-treated leuc-B exhibited distinct potency to inhibit platelet aggregation, it can be assumed that the enzymatic activity of

E.F. Sanchez et al. / Archives of Biochemistry and Biophysics 468 (2007) 193–204

leuc-B plays an independent role in anti-platelet aggregation function. In connection with this, integrins a1b1 and a2b1 are important cellular receptors for collagens. Whereas a2b1 integrin is the only collagen-binding integrin on platelets, endothelial cells mediate their attachment to collagens by both integrin receptor, a1b1 and a2b1. Another P-III class (containing ECD motif), jararhagin, from B. jararaca, seems to inhibit adhesion to collagen (platelet aggregation) in an a2b1 integrin-dependent manner [51] but uses a different mechanism. Jararhagin-derivative peptides such as RKKH present in its metalloproteinase domain bind to integrins via A-type domain and block integrin function [52]. Other P-III SVMPs such as crovidisin from Crotalus viridis [53] and AAV1 from A. acutus [54], also bind collagen and inhibit platelet aggregation. In the case of crovidisin its action mechanism was attributed to its binding to collagen fibers. ELISA binding assays showed that there were no remarkable binding signals for leuc-B either with a1b1 or with a2b1 integrin, whereas a weak interaction of a2b1 integrin with jararhagin in a cation-independent manner was evident (Fig. 8), consistent with [34]. As shown in Fig. 2, the sequence RKKH located in the metalloprotease domain is distant from the active site of jararhagin, which has been showed to be involved in blocking collagen binding to the a2 I-domain. This sequence is replaced by the sequence RISH at the corresponding position in leuc-B. It can be hypothesized that the failure of leuc-B to bind to of the a1b1 and a2b1 is caused by this short sequence (RISH). Why the P-III snake venom-derived collagen antagonists have different working mechanisms remains to be elucidated. Further studies on the structure–function correlation of leuc-B would be valuable to understand the mechanism and biological significance of disintegrin-like domains of SVMPs and ADAM proteins. Owing to their multiple functions, the P-III SVMPs with MDC domains found in viperidae and few elapid (e.g. kaouthiagin from Naja kaouthia) venoms may serve as ideal probes for clarifying the mechanisms underlying hemostatic alterations and thrombosis. In summary, we have isolated and partially characterized a novel P-III SVMP, leuc-B from white-tailed-jararaca venom. The proteinase exhibited inhibitory effect of collagen-induced platelet aggregation, however, it does not bind to a1b1 and a2b1 integrins which interact with collagen. Like members of the ADAM protein family, a mature leuc-B has a multidomain structure composed of a metalloproteinase/disintegrin-like/cysteine-rich domain. Its enzymatic activity on ECM and plasma proteins is mainly responsible for its hemorrhagic and hemostatic alterations. This is supported by the fact that the P-III SVMP leuc-B exhibits high hemorrhagic activity, whereas the P-I SVMP leuc-a is devoid of hemorrhagic effect. However, further investigation on the structure–function relationships of leuc-B are necessary to understand how the enzyme interferes with the hemostatic mechanism, as well as cell-adhesion interactions.

203

Acknowledgments This work was financially supported by the Brazilian agencies Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Fundac¸a˜o de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG), processo CBB 359/06 and CAPES. The authors also acknowledge the financial support to J.A.E. by the Deutsche Forschungsgemeinschaft (Grant EB 177/4-2) and PROBAL programme of DAAD (Grant 415-probral/po-05/30352). References [1] F.S. Markland, Toxicon 36 (1998) 1749–1800. [2] T. Matsui, Y. Fujimura, K. Titani, Biochim. Biophys. Acta 1477 (2000) 146–156. [3] W. Stocker, F. Grams, U. Baumann, P. Reinemer, F.-X. GomisRu¨th, D. McKay, W. Bode, Protein Sci. 4 (1995) 823–840. [4] R.K. Andrews, E.E. Gardiner, N. Asazuma, O. Berlanga, D. Tulasne, B. Nieswandt, A.I. Smith, M.C. Berndt, S.P. Watson, J. Biol. Chem. 276 (2001) 28092–28097. [5] M. Kishimoto, T. Takahashi, Toxicon 40 (2002) 1369–1375. [6] W.-K. You, Y.-J. Jang, K.-H. Chung, O.-H. Jeon, D.S. Kim, Biochem. Biophys. Res. Commun. 339 (2006) 964–970. [7] W.-J. Wang, C.-O. Shih, T.-F. Huang, Biochimie 87 (2005) 1065– 1077. [8] J.B. Bjarnason, J.W. Fox, Meth. Enzymol. 248 (1995) 345–368. [9] M.J. Duffy, D.J. Lynn, AT. Lloyd, C.M. O‘Shea, Thromb. Haemost. 89 (2003) 622–631. [10] T.G. Wolfberg, P.D. Strainght, R.L. Gerena, A.P. Huovila, P. Primakoff, D.G. Myles, J.M. White, Dev. Biol. 169 (1995) 378–383. [11] C.P. Blobel, T.G. Wolfdberg, C.W. Turck, D.G. Myles, P. Primakoff, J.M. White, Nature 356 (1992) 248–252. [12] E.N. Baramova, J.D. Shannon, J.B. Bjarnason, J.W. Fox, Arch. Biochem. Biophys. 275 (1989) 63–71. [13] A.S. Kamiguti, J.R. Slupsky, M. Zuzel, C.R. Hay, Thromb. Haemostasis 72 (1994) 244–249. [14] L.G. Jia, X.M. Wang, J.D. Shannon, J.B. Bjarnason, J.W. Fox, J. Biol. Chem. 272 (1997) 13094–13102. [15] A.S. Kamiguti, C.R. Hay, R.D. Theakston, M. Zuzel, Toxicon 34 (1996) 627–642. [16] J.M. Gutierrez, A. Rucavado, Biochimie 82 (2000) 841–850. [17] Ministe´rio da Sau´de do Brasil-Fundac¸a˜o Nacional de Sau´de, Manual de diagno´stico e tratamento de acidentes por animais pec¸onhentos, DF, Brasilia, 1999. [18] L.R.M. Da Silva, B.T. Nunes, Toxicon 31 (1993) 143–144. [19] K.U. Camey, D.T. Velarde, E.F. Sanchez, Toxicon 40 (2002) 501– 509. [20] C.A. Bello, A.L. Hermogenes, A. Magalhaes, S.S. Veiga, L.H. Gremski, M. Richardson, E.F. Sanchez, Biochimie 88 (2006) 189–200. [21] A. Magalhaes, H.B. Magalhaes, M. Richardson, S. Gontijo, R. Ferreira, A.P. Almeida, E.F. Sanchez, Comp. Biochem. Physiol. Part A Mol. Integrative Physiol. 146 (2007) 565–575. [22] D.A. Higuchi, J.R. Chagas, C.M.V. Bincoleto, A. Magalhaes, M. Richardson, E.F. Sanchez, J.B. Pesquero, J.L. Pesquero, Biochimie 89 (2007) 319–328. [23] E. Engvall, E. Ruoslathi, Int. J. Cancer 20 (1977) 1–5. [24] T. Yatohgo, M. Izumi, H. Kashiwagi, M. Hayashi, Cell Struct. Funct. 13 (1988) 281–292. [25] R. Timpl, H. Rohde, P.G. Robey, S.I. Rennard, J.M. Foidartc, G.R. Martin, J. Biol. Chem. 254 (1979) 9933–9937. [26] M. Paulsson, M. Aumaillay, R. Deutzmann, R. Timpl, K. Beck, Eur. J. Biochem. 166 (1987) 11–19. [27] M.B. Guis, R. Slootweg, J.G.M. Tonino, Arch. Oral Biol. 18 (1973) 253–263.

204

E.F. Sanchez et al. / Archives of Biochemistry and Biophysics 468 (2007) 193–204

[28] K.J. Wilson, P.M. Yuan, in: J.B.C. Findlay, M.J. Geisow (Eds.), Protein Sequencing a Practical Approach, I.R.L. Press, Oxford, New York, Tokyo, 1989, pp. 3–41. [29] U.K. Laemmli, Nature 227 (1970) 680–685. [30] E.F. Sanchez, M.N. Cordeiro, E.B. Oliveira, E.B.L. Juliano, E.S. Prado, C.R. Diniz, Toxicon 33 (1995) 1061–1070. [31] E.F. Sanchez, C.I. Santos, A. Magalhaes, C.R. Diniz, S. Figueiredo, M. Richardson, Arch. Biochem. Biophys. 378 (2000) 131–141. [32] M.I. Estevao-Costa, C.R. Diniz, A. Magalhaes, F.S. Markland, E.F. Sanchez, Thromb. Res. 99 (2000) 363–376. [33] A.J. Eble, B. Beermann, H.-J. Hinz, A. Schmidt-Hederich, J. Biol. Chem. 276 (2001) 12274–12284. [34] P. Zigrino, A.S. Kamiguti, J. Eble, C. Drescher, R. Nischt, J. Biol. Chem. 277 (2002) 40526–40535. [35] A.J. Eble, A. Kassner, S. Niland, M. Mo¨rgelin, J. Grifka, S. Gra¨ssel, J. Biol. Chem. 281 (2006) 25745–25756. [36] H. Takeya, K. Oda, K. Miyata, T. Omori-Satoh, S. Iwanaga, J. Biol. Chem. 265 (1990) 16068–16073. [37] D.P. Cerretti, R.F. DuBose, R.A. Black, N. Nelson, Biochem. Biophys. Res. Commun. 263 (1999) 810–815. [38] J.W. Fox, S.M.T. Serrano, Toxicon 45 (2005) 969–985. [39] D.J. Civello, H.L. Duong, C.R. Geren, Biochemistry 22 (1983) 749– 755. [40] T. Nikai, N. Mori, M. Kishida, Y. Kato, Ch. Takenata, T. Murakami, S. Shigezane, H. Sugihara, Biochim. Biophys. Acta 838 (1985) 122–131. [41] S. Swenson, F.S. Markland, Toxicon 45 (2005) 1021–1039.

[42] D. Chain, B. Korc-Grodzicki, T. Kreisman, S. Shaltiel, Biochem. J. 274 (1991) 387–394. [43] L.H. Gremski, O.M. Chaim, K.S. Paludo, Y.B. Sade, M.F. Otuki, M. Richardson, E.F. Sanchez, S.S. Veiga, Toxicon 50 (2007) 120–134. [44] E.F. Sanchez, S. Swenson, Curr. Pharm. Anal. 3 (2007) 147–157. [45] R.M. Kini, H.J. Evans, Toxicon 30 (1992) 265–293. [46] M.E. Peichoto, P. Teibler, S.P. Mackessey, L. Leiva, O. Acosta, L.R.C. Gonc¸alves, A.M. Tanaka, M.L. Santoro, Biochim. Biophys. Acta 1770 (2007) 810–819. [47] B. Lovejoy, A.M. Hassell, M.A. Luther, D. Weigl, S.R. Jordan, Biochemistry 33 (1994) 8207–8217. [48] R. Ala-aho, V.-M. Ka¨ha¨ri, Biochimie 87 (2005) 273–286. [49] A.S. Kamiguti, J.R. Slupski, M. Zuzel, C.R.M. Hay, Thromb. Haemost. 72 (1994) 244–249. [50] O.-H. Jeon, D.-S. Kim, Eur. J. Biochem. 263 (1999) 526–533. [51] A.S. Kamiguti, P. Gallagher, C. Marcionkiewitz, R.D.G. Theakston, M. Zuzel, J.W. Fox, FEBS Lett. 549 (2003) 129–134. [52] Y. Nymalm, J.S. Puranen, T.K. Nyholm, J. Kapyla, H. Kidron, O.T. Pentikainen, T.T. Airenne, J. Heino, J.P. Slotte, M.S. Johonson, T.A. Salminem, J. Biol. Chem. 103 (2004) 2150–5156. [53] C.Z. Liu, T.F. Huang, Arch. Biochem. Biophys. 337 (1997) 291–299. [54] W.-E. Wang, Biochimie 89 (2007) 105–115. [55] F.-X. Gomis-Ruth, L.F. Kress, J. Kellermann, I. Mayr, R. Huber, W. Bode, J. Mol. Biol. 239 (1994) 513–544. [56] V.M. Rodriguez, A.M. Soares, R. Guerra-Sa, V. Rodrigues, M.R.M. Fontes, J.R. Giglio, Arch. Biochem. Biophys. 381 (2000) 213–224. [57] A. Kern, J. Eble, R. Golbik, K. Ku¨hn, Eur. J. Biochem. 225 (1993) 151–159.