**3.** *Lonomia obliqua* **toxins**

The *L. obliqua* caterpillar venom contain non-protein and protein components [21]. The procoagulant proteins present in the venom cause hemostatic disturbances mainly mediated by thrombin formation, the key enzyme of blood coagulation. Physiologically, thrombin generation from prothrombin occurs by assembling of the prothrombinase complex, which consists of factor Xa (catalytic factor), factor Va (non-enzymatic cofactor), calcium and a phospholipid membrane surface. Despite figured as a cascade of subsequent activation of coagulation factors, blood coagulation is currently conceived in a cell based-model [69]. In Figure 7 a simplified cascade model of hemostasis is illustrated, showing the known interac‐ tions of the *L. obliqua* venom and toxins.

Several molecules and activities were reported in bristles or hemolymph (Table 1). Some of them are related to the pathophysiology of the envenoming others to the development process of the animal such as regulation of the cell cycle [16]. Donato and colleagues [70] identified in the bristle extract a direct factor X activator which is calcium-independent, and a prothrombin activator. The prothrombin and factor X activators were later isolated and named Lopap and Losac, respectively [22, 24]. Interestingly, both molecules are no longer similar with any wellknown procoagulant molecule from human or any other species.

The recombinant protein (rLopap) was obtained in enzymatically active form as monomer of 21 kDa with a polyhistidine tag after purification by immobilized metal-chelate affinity chromatography. Partial amino acid sequences of native Lopap lead to identification of its respective clone from the cDNA library of *L. obliqua* bristles [20], encoding for a signal peptide (16 aa residues) and the mature protein (185 aa residues). cDNA of mature protein, consisting in a transcript with 603 bp open reading frame, was subcloned into the pAE vector and expressed in the bacteria E. coli BL21(DE3) with a fusion tag (His6). Protein was recovered in inclusion bodies after cell lysis and subjected to refolding and purification after solubilization

Stuart factor activator. Lopap = *L. obliqua* prothrombin activator protease.

**Figure 7.** Schematic overview of hemostasis. Dark double-bars indicate where inhibitors act. HMWK = high molecularweight-kininogen. PK = Prekallikrein. KK = Kallikrein. CI-INH = CI-inhibitor. TFPI = tissue factor pathway inhibitor. PL = phospholipids. Ca = calcium ions. AT = antithrombin. FDA = Fibrinopeptide A. FDB = Fibrinopeptide B. TAFI = Throm‐ bin-activatable fibrinolysis inhibitor. FnDP = Fibrin degradation products. FgDP = Fibrinogen degradation products. α2- AP = α2-antiplasmin. Known interactions of the *L. obliqua* venom are indicated in the black boxes. Losac = *L. obliqua*

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Interestingly, the deduced amino acid sequence of Lopap showed no similarity with other prothrombin activators or serine proteases, but was similar to lipocalin family members, either from insects or mammals [71]. Lopap sequence alignment with other lipocalins is shown in Figure 8. Members of lipocalin family usually share only about 30% of similarity in amino acid

in urea [71].

### **3.1. Lopap: Functional characterization, recombinant production and bioinformatics analysis**

Lopap (*Lonomia obliqua* Prothrombin Activator Protease) was purified from the bristle extract as a 69-kDa protein through gel filtration followed by reverse-phase chromatography. The purified protein was subjected to trypsin hydrolysis and partial amino acid sequences of Nterminal and internal fragments were obtained through Edman degradation [24].

Different metals may be used (iron, zirconium, gallium, etc) and peptides eluted by acidic or basic conditions, releasing mono-phosphorylated and multi-phosphorylated peptides,

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

The simultaneous screening of thousands of proteins from complex samples in a fast and sensitive manner can be performed using protein arrays. Amongst the different protein microarray applications are biomarker discovery, protein interaction studies, enzymesubstrate profiling, immunological profiling and vaccine development. As our interest is in the immune response, an antibody microarray can be used for identification of antigens that react specifically with the antibodies spotted on a solid support, with the complex formed then

A large number of not yet identified proteins are considered as unknowns, but higher probabilities of identifications are reached when different methodologies are applied for analysis of complex samples. The combination of several proteomic techniques described here could improve the detection of immunogenic compounds and create new perspectives for

The *L. obliqua* caterpillar venom contain non-protein and protein components [21]. The procoagulant proteins present in the venom cause hemostatic disturbances mainly mediated by thrombin formation, the key enzyme of blood coagulation. Physiologically, thrombin generation from prothrombin occurs by assembling of the prothrombinase complex, which consists of factor Xa (catalytic factor), factor Va (non-enzymatic cofactor), calcium and a phospholipid membrane surface. Despite figured as a cascade of subsequent activation of coagulation factors, blood coagulation is currently conceived in a cell based-model [69]. In Figure 7 a simplified cascade model of hemostasis is illustrated, showing the known interac‐

Several molecules and activities were reported in bristles or hemolymph (Table 1). Some of them are related to the pathophysiology of the envenoming others to the development process of the animal such as regulation of the cell cycle [16]. Donato and colleagues [70] identified in the bristle extract a direct factor X activator which is calcium-independent, and a prothrombin activator. The prothrombin and factor X activators were later isolated and named Lopap and Losac, respectively [22, 24]. Interestingly, both molecules are no longer similar with any well-

**3.1. Lopap: Functional characterization, recombinant production and bioinformatics**

terminal and internal fragments were obtained through Edman degradation [24].

Lopap (*Lonomia obliqua* Prothrombin Activator Protease) was purified from the bristle extract as a 69-kDa protein through gel filtration followed by reverse-phase chromatography. The purified protein was subjected to trypsin hydrolysis and partial amino acid sequences of N-

respectively [67].

Applications

186

detected by fluorescence [59].

effective immunotherapies.

**3.** *Lonomia obliqua* **toxins**

tions of the *L. obliqua* venom and toxins.

**analysis**

known procoagulant molecule from human or any other species.

**Figure 7.** Schematic overview of hemostasis. Dark double-bars indicate where inhibitors act. HMWK = high molecularweight-kininogen. PK = Prekallikrein. KK = Kallikrein. CI-INH = CI-inhibitor. TFPI = tissue factor pathway inhibitor. PL = phospholipids. Ca = calcium ions. AT = antithrombin. FDA = Fibrinopeptide A. FDB = Fibrinopeptide B. TAFI = Throm‐ bin-activatable fibrinolysis inhibitor. FnDP = Fibrin degradation products. FgDP = Fibrinogen degradation products. α2- AP = α2-antiplasmin. Known interactions of the *L. obliqua* venom are indicated in the black boxes. Losac = *L. obliqua* Stuart factor activator. Lopap = *L. obliqua* prothrombin activator protease.

The recombinant protein (rLopap) was obtained in enzymatically active form as monomer of 21 kDa with a polyhistidine tag after purification by immobilized metal-chelate affinity chromatography. Partial amino acid sequences of native Lopap lead to identification of its respective clone from the cDNA library of *L. obliqua* bristles [20], encoding for a signal peptide (16 aa residues) and the mature protein (185 aa residues). cDNA of mature protein, consisting in a transcript with 603 bp open reading frame, was subcloned into the pAE vector and expressed in the bacteria E. coli BL21(DE3) with a fusion tag (His6). Protein was recovered in inclusion bodies after cell lysis and subjected to refolding and purification after solubilization in urea [71].

Interestingly, the deduced amino acid sequence of Lopap showed no similarity with other prothrombin activators or serine proteases, but was similar to lipocalin family members, either from insects or mammals [71]. Lopap sequence alignment with other lipocalins is shown in Figure 8. Members of lipocalin family usually share only about 30% of similarity in amino acid sequence, despite showing conserved secondary and tertiary structures. Furthermore, these proteins have in primary structure three characteristic conserved motifs [72].

[19]. Lopap is the first lipocalin described that displays proteolytic activity. On the other hand, through a peptide mapping approach based on lipocalin conserved motifs found in the Lopap's primary structure, a synthetic peptide was obtained (Figure 10), which has been proposed as a sequence signature among lipocalins, sharing a common role in cell protection and development process [73]. Other lipocalins that have been described with antiapoptotic activity share similar sequences, which have similar conformations in their tridimensional

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**Figure 10.** Lipocalin sequence signature highlighted among lipocalin conserved motifs identified in Lopap sequence [19] and in the model of tridimensional structure. Residues predicted in the catalytic triad are shown in green [25].

structures [73].

**Figure 9.** Model of the tridimensional structure of Lopap [81].


**Figure 8.** Amino acid sequence alignment of Lopap and other lipocalins. Sequences were accessed from protein data bank at NCBI and aligned using BioEdit [80].

Lopap's tridimensional structure obtained by molecular modeling has the characteristic fold of lipocalins, consisting in an eight stranded antiparallel β-barrel (Figure 9) with a hydrophobic pocket for binding of hydrophobic ligands. A serine protease catalytic triad was also predicted [19]. Lopap is the first lipocalin described that displays proteolytic activity. On the other hand, through a peptide mapping approach based on lipocalin conserved motifs found in the Lopap's primary structure, a synthetic peptide was obtained (Figure 10), which has been proposed as a sequence signature among lipocalins, sharing a common role in cell protection and development process [73]. Other lipocalins that have been described with antiapoptotic activity share similar sequences, which have similar conformations in their tridimensional structures [73].

**Figure 9.** Model of the tridimensional structure of Lopap [81].

sequence, despite showing conserved secondary and tertiary structures. Furthermore, these

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

**Figure 8.** Amino acid sequence alignment of Lopap and other lipocalins. Sequences were accessed from protein data

Lopap's tridimensional structure obtained by molecular modeling has the characteristic fold of lipocalins, consisting in an eight stranded antiparallel β-barrel (Figure 9) with a hydrophobic pocket for binding of hydrophobic ligands. A serine protease catalytic triad was also predicted

bank at NCBI and aligned using BioEdit [80].

proteins have in primary structure three characteristic conserved motifs [72].

Applications

188

**Figure 10.** Lipocalin sequence signature highlighted among lipocalin conserved motifs identified in Lopap sequence [19] and in the model of tridimensional structure. Residues predicted in the catalytic triad are shown in green [25].

Lopap shows specific proteolytic activity towards prothrombin. It displays serine proteaselike activity and activates human prothrombin through hydrolysis of Arg284-Thr285 and Arg320- Ile321 peptide bounds, generating active thrombin, without formation of the intermediate meizothrombin [24]. This mechanism is similar to prothrombin activation by FXa in absence of the prothrombinase complex (Figure 11), previously described [74]. This is the unique prothrombin activation mechanism described for an exogenous serine protease, which is independent of prothrombinase complex components. All other exogenous prothrombin activators (metalloproteases and serine proteases) currently described from snake venoms fit into four groups, sharing similar mechanisms of action [75].

have no effect on modulation of coagulation and fibrinolysis through endothelial cell response, since it does not modify von Willebrand factor (vWF) and tissue plasminogen activator (t-PA)

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The Lopap-derived peptide obtained through chemical synthesis (Survicalin) reproduces the Lopap's modulation on endothelial cells and neutrophils cell, triggering antiapoptotic activity [73]. Survicalin also induces fibroblast responses, decreasing caspase-3 and increasing Bcl-2, Ki-67, IL-1β and the receptors for IL-8 and IL-6. Enhanced production of extracellular matrix proteins, such as collagen, fibronectin, tenascin and laminin is also induced by Survicalin in

Losac is the first factor X activator purified from a lepidopter secretion. It was obtained from caterpillar's bristle extract as a single polypeptide chain protein of about 45 kDa [22]. Some years later, from a cDNA library of *L. obliqua* bristle transcripts [19], the specific clone encoding for Losac was identified and the recombinant protein produced in bacteria system (for details, see section 3.3). Studies using the native or recombinant form of Losac (rLosac) revealed specificity toward factor X [20, 22, 28]. Moreover, Losac had no effect on fibrin or fibrinogen, indicating its specificity for blood coagulation activation, and it was recognized by the antilonomic serum produced in Butantan Institute. Thus, it is plausible that this protein

Biochemical characterization of Losac has shown that, although its sequence did not show an equivalent among other factor X activators, Losac possess a similar mechanism of action than RVV-X, a factor X activator purified from Russell's viper venom *Daboia russelli* [28, 82, 83]. Like RVV-X, factor X activation by Losac can be accelerated in the presence of calcium and phos‐ pholipids, two important cofactor in the assembling of blood coagulation complexes [69]. In spite of this, Losac can activate factor X independently of these cofactors. Moreover, both activators require a stable conformation of factor X and the presence of the Gla-domain of factor X for an appropriated activity. Interestingly, the cleavage fragments of factor X gener‐ ated by both activators were quite similar. Although there are strong functional similarities, the major difference is in the structure of both activators. Apparently, Losac activates factor X through a serine protease-like activity, while RVV-X has a typical metalloproteinase structure

A model proposed by Morita [83] and crystallographic studies of RVV-X [85] support the hypothesis that it primarily recognizes the calcium-bound conformation of Gla-domain in factor X through an exosite formed by the light chains, followed by the catalytic conversion of factor X to factor Xa. Despite the structural differences between Losac and RVV-X, it remains possible that they share a similar mechanisms for recognition of factor X involving calcium ions, phospholipids and the Gla-domain of factor X followed by its proteolytic conversion to

Besides its role in coagulation [22, 28], Losac is also capable of inducing proliferation and inhibiting endothelial cell death while stimulating the release of NO, a known molecule with

release or tissue factor procoagulant activity on endothelial cell surface [45, 77].

participates in the consumption coagulopathy observed in patients.

fibroblast culture [79].

[20, 28, 84].

active factor X.

**3.2. Losac: Functional characterization**

**Figure 11.** Prothrombin activation mechanisms indicating the Lopap hydrolysis sites and its generated products.

When administered *in vivo*, Lopap induces blood clotting into microvessels, resulting in fibrinogen consumption and blood incoagulability [76]. These effects resemble the consump‐ tion coagulopathy triggered by the whole venom, indicating the involvement of this pro‐ thrombin activator as a key toxin in envenoming [26]. In addition, Lopap is able to modulate endothelial cell responses promoting cell survival, IL-8, nitric oxide (NO) and PGI2 release, expression of the cell adhesion molecules ICAM-1 and E-selectin, but not VCAM-1 and PCAM-1 [45, 77, 78]. Lopap also displays cytoprotective activity in neuthrophils and do not modify expressions of L-selectin and β2 integrin. Secretion of the proinflammatory cytokines IL-6 and TNF-α is not changed by Lopap treatment in both cell cultures [78]. Lopap seems to have no effect on modulation of coagulation and fibrinolysis through endothelial cell response, since it does not modify von Willebrand factor (vWF) and tissue plasminogen activator (t-PA) release or tissue factor procoagulant activity on endothelial cell surface [45, 77].

The Lopap-derived peptide obtained through chemical synthesis (Survicalin) reproduces the Lopap's modulation on endothelial cells and neutrophils cell, triggering antiapoptotic activity [73]. Survicalin also induces fibroblast responses, decreasing caspase-3 and increasing Bcl-2, Ki-67, IL-1β and the receptors for IL-8 and IL-6. Enhanced production of extracellular matrix proteins, such as collagen, fibronectin, tenascin and laminin is also induced by Survicalin in fibroblast culture [79].

### **3.2. Losac: Functional characterization**

Lopap shows specific proteolytic activity towards prothrombin. It displays serine proteaselike activity and activates human prothrombin through hydrolysis of Arg284-Thr285 and Arg320- Ile321 peptide bounds, generating active thrombin, without formation of the intermediate meizothrombin [24]. This mechanism is similar to prothrombin activation by FXa in absence of the prothrombinase complex (Figure 11), previously described [74]. This is the unique prothrombin activation mechanism described for an exogenous serine protease, which is independent of prothrombinase complex components. All other exogenous prothrombin activators (metalloproteases and serine proteases) currently described from snake venoms fit

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

**Figure 11.** Prothrombin activation mechanisms indicating the Lopap hydrolysis sites and its generated products.

When administered *in vivo*, Lopap induces blood clotting into microvessels, resulting in fibrinogen consumption and blood incoagulability [76]. These effects resemble the consump‐ tion coagulopathy triggered by the whole venom, indicating the involvement of this pro‐ thrombin activator as a key toxin in envenoming [26]. In addition, Lopap is able to modulate endothelial cell responses promoting cell survival, IL-8, nitric oxide (NO) and PGI2 release, expression of the cell adhesion molecules ICAM-1 and E-selectin, but not VCAM-1 and PCAM-1 [45, 77, 78]. Lopap also displays cytoprotective activity in neuthrophils and do not modify expressions of L-selectin and β2 integrin. Secretion of the proinflammatory cytokines IL-6 and TNF-α is not changed by Lopap treatment in both cell cultures [78]. Lopap seems to

into four groups, sharing similar mechanisms of action [75].

Applications

190

Losac is the first factor X activator purified from a lepidopter secretion. It was obtained from caterpillar's bristle extract as a single polypeptide chain protein of about 45 kDa [22]. Some years later, from a cDNA library of *L. obliqua* bristle transcripts [19], the specific clone encoding for Losac was identified and the recombinant protein produced in bacteria system (for details, see section 3.3). Studies using the native or recombinant form of Losac (rLosac) revealed specificity toward factor X [20, 22, 28]. Moreover, Losac had no effect on fibrin or fibrinogen, indicating its specificity for blood coagulation activation, and it was recognized by the antilonomic serum produced in Butantan Institute. Thus, it is plausible that this protein participates in the consumption coagulopathy observed in patients.

Biochemical characterization of Losac has shown that, although its sequence did not show an equivalent among other factor X activators, Losac possess a similar mechanism of action than RVV-X, a factor X activator purified from Russell's viper venom *Daboia russelli* [28, 82, 83]. Like RVV-X, factor X activation by Losac can be accelerated in the presence of calcium and phos‐ pholipids, two important cofactor in the assembling of blood coagulation complexes [69]. In spite of this, Losac can activate factor X independently of these cofactors. Moreover, both activators require a stable conformation of factor X and the presence of the Gla-domain of factor X for an appropriated activity. Interestingly, the cleavage fragments of factor X gener‐ ated by both activators were quite similar. Although there are strong functional similarities, the major difference is in the structure of both activators. Apparently, Losac activates factor X through a serine protease-like activity, while RVV-X has a typical metalloproteinase structure [20, 28, 84].

A model proposed by Morita [83] and crystallographic studies of RVV-X [85] support the hypothesis that it primarily recognizes the calcium-bound conformation of Gla-domain in factor X through an exosite formed by the light chains, followed by the catalytic conversion of factor X to factor Xa. Despite the structural differences between Losac and RVV-X, it remains possible that they share a similar mechanisms for recognition of factor X involving calcium ions, phospholipids and the Gla-domain of factor X followed by its proteolytic conversion to active factor X.

Besides its role in coagulation [22, 28], Losac is also capable of inducing proliferation and inhibiting endothelial cell death while stimulating the release of NO, a known molecule with

antiapoptotic activity [86, 87], and t-PA, a component of fibrinolytic-pathway involved in matrix remodeling [88]. The authors suggest that the cell proliferation and cell viability activities elicited by Losac are probably related to the NO liberation [22], since NO was also described as an endothelial survival factor, inhibiting apoptosis [86, 87]. Moreover, it was also observed that the production/expression of some important molecules involved in inflamma‐ tion and coagulation systems such as ICAM-1, PGI2, DAF, IL-8, vWF and tissue factor were not affected by Losac.

It has been show that hemolymph from some insects can increase cell longevity by inhibiting apoptosis [89, 90]. The increase of *Spodoptera frugiperda* Sf-9 cell growth in almost 3-fold was reported after supplementation with *L. obliqua* hemolymph [91]. This effect was attributed to the presence of three factors with different activities: a potential antiapoptotic factor, a growthpromoting factor, and an enzyme that hydrolyzes sucrose. Furthermore, an antiapoptotic protein of 51 kDa was purified from *L. obliqua* hemolymph [36]. This protein was able to prevent apoptosis in Sf-9 cell culture induced by nutrient deprivation and by Actinomycin D. Later reports [37, 38] described in the hemolymph a potent antiviral activity against human virus.

### **3.3. Molecular cloning and heterologous expression of Losac**

The production of Losac in a recombinant form was important due to the disadvantages of purifying Losac from bristle extract: the use of many caterpillars to prepare the bristle extract and the low yield of native Losac (0.3%) [22]. Cloning and production scheme to obtain rLosac is shown in Figure 12 [28].

Nucleotide and deduced amino acid sequences were compared with data banks in order to identify similar genes and their products. The analysis revealed a high similarity with members of the immunoglobulin-like superfamily of cell adhesion molecules (IgCAMs), especially with neural CAMs (NCAMs) [28]. Members of this group have diverse functions but none was associated with proteolytic activities [94]. Multiple comparison of the deduced amino acid sequence revealed different degrees of identity with IgCAMs: 26% of identity with L1-NCAM from humans, 34% with the protein neuroglian from *Drosofila melanogaster*; and 47-76% with hemolins from lepidopters [95-98]. Although no structural data was reported for Losac, a tertiary structure model was built through homology modeling based on crystal structure of *Hyalophora cecropia* hemolin (HcHemolin, Protein Data Bank code 1BIH). Both proteins share 76% of sequence identity [28] and the same multi-domain structure (four Ig-like domains: D1 to D4) and conserved motifs as shown in Figures 13 and 14. Both structures are composed of β-strands, arranged in a globular shape resembling a horseshoe (Figure 14A), akin to hemolin [97], axonin [99], and the four N-terminal Ig domains of neurofascin [100]. Because Losac shares its main sequence features with hemolins, it can be perfectly classified as one of them. It was demonstrated that Losac activate factor X in a similar way than RVV-X [28]. Nevertheless, unlike Losac, no hemolins or cell adhesion molecules were associated with proteolytic activities.

**Figure 12. 1)**After purification from bristle extract [22], Losac was submitted to a tryptic digestion to obtain partial amino acid sequences of internal fragments. Those sequences were obtained by mass spectrometry and used to screen the cDNA library to identify the transcript encoding the protein. The cDNA library was previously constructed with *L. obliqua* bristle mRNAs that were converted to cDNA and cloned into pGEM-11Zf+ plasmid [19, 20]. **2)** From this cDNA library, a transcript corresponding to the clone LOAH12B08 (GenBankTM accession number CX816408.1), matching the tryptic peptide sequences, was identified. However, this transcript was partially sequenced and the com‐ plete sequence was achieved through the *primer walking strategy* using a specific primer designed from an internal sequence of the transcript allowing uncover the complete sequence of Losac gene [92]. The nucleotide sequence has been deposited in GenBankTM (DQ479435), and the deduced protein has been deposited in the NCBI protein se‐ quence database (ABF21073). **3)** The cDNA that encodes mature Losac was amplified by PCR using a sense and an

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193

antiapoptotic activity [86, 87], and t-PA, a component of fibrinolytic-pathway involved in matrix remodeling [88]. The authors suggest that the cell proliferation and cell viability activities elicited by Losac are probably related to the NO liberation [22], since NO was also described as an endothelial survival factor, inhibiting apoptosis [86, 87]. Moreover, it was also observed that the production/expression of some important molecules involved in inflamma‐ tion and coagulation systems such as ICAM-1, PGI2, DAF, IL-8, vWF and tissue factor were

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

It has been show that hemolymph from some insects can increase cell longevity by inhibiting apoptosis [89, 90]. The increase of *Spodoptera frugiperda* Sf-9 cell growth in almost 3-fold was reported after supplementation with *L. obliqua* hemolymph [91]. This effect was attributed to the presence of three factors with different activities: a potential antiapoptotic factor, a growthpromoting factor, and an enzyme that hydrolyzes sucrose. Furthermore, an antiapoptotic protein of 51 kDa was purified from *L. obliqua* hemolymph [36]. This protein was able to prevent apoptosis in Sf-9 cell culture induced by nutrient deprivation and by Actinomycin D. Later reports [37, 38] described in the hemolymph a potent antiviral activity against human virus.

The production of Losac in a recombinant form was important due to the disadvantages of purifying Losac from bristle extract: the use of many caterpillars to prepare the bristle extract and the low yield of native Losac (0.3%) [22]. Cloning and production scheme to obtain rLosac

Nucleotide and deduced amino acid sequences were compared with data banks in order to identify similar genes and their products. The analysis revealed a high similarity with members of the immunoglobulin-like superfamily of cell adhesion molecules (IgCAMs), especially with neural CAMs (NCAMs) [28]. Members of this group have diverse functions but none was associated with proteolytic activities [94]. Multiple comparison of the deduced amino acid sequence revealed different degrees of identity with IgCAMs: 26% of identity with L1-NCAM from humans, 34% with the protein neuroglian from *Drosofila melanogaster*; and 47-76% with hemolins from lepidopters [95-98]. Although no structural data was reported for Losac, a tertiary structure model was built through homology modeling based on crystal structure of *Hyalophora cecropia* hemolin (HcHemolin, Protein Data Bank code 1BIH). Both proteins share 76% of sequence identity [28] and the same multi-domain structure (four Ig-like domains: D1 to D4) and conserved motifs as shown in Figures 13 and 14. Both structures are composed of β-strands, arranged in a globular shape resembling a horseshoe (Figure 14A), akin to hemolin [97], axonin [99], and the four N-terminal Ig domains of neurofascin [100]. Because Losac shares its main sequence features with hemolins, it can be perfectly classified as one of them. It was demonstrated that Losac activate factor X in a similar way than RVV-X [28]. Nevertheless, unlike Losac, no hemolins or cell adhesion molecules were associated with proteolytic

**3.3. Molecular cloning and heterologous expression of Losac**

not affected by Losac.

Applications

192

is shown in Figure 12 [28].

activities.

**Figure 12. 1)**After purification from bristle extract [22], Losac was submitted to a tryptic digestion to obtain partial amino acid sequences of internal fragments. Those sequences were obtained by mass spectrometry and used to screen the cDNA library to identify the transcript encoding the protein. The cDNA library was previously constructed with *L. obliqua* bristle mRNAs that were converted to cDNA and cloned into pGEM-11Zf+ plasmid [19, 20]. **2)** From this cDNA library, a transcript corresponding to the clone LOAH12B08 (GenBankTM accession number CX816408.1), matching the tryptic peptide sequences, was identified. However, this transcript was partially sequenced and the com‐ plete sequence was achieved through the *primer walking strategy* using a specific primer designed from an internal sequence of the transcript allowing uncover the complete sequence of Losac gene [92]. The nucleotide sequence has been deposited in GenBankTM (DQ479435), and the deduced protein has been deposited in the NCBI protein se‐ quence database (ABF21073). **3)** The cDNA that encodes mature Losac was amplified by PCR using a sense and an

antisense primer designed according to the deduced N- and C-terminal sequences of the mature protein carrying *Nco*I or *Eco*RI restriction sites, respectively. **4)** The cDNA corresponding to mature Losac was sub-cloned into the pAE vector [93]. The PCR product and the pAE vector were restricted with *Nco*I and *Eco*RI, purified, and ligated with T4 DNA ligase and used to transform *E. coli* DH5α cells. **5)** The resulting pAE-Losac plasmid was used to transform *E. coli* BL21(DE3) cells. The recombinant protein was expressed in fusion to a minimal N-terminal His6-tag as a 48.6 kDa protein: Protein was recovered in inclusion bodies after cell lysis and subjected to refolding and purification after solubilization in urea.


**Figure 13.** Multiple amino acid sequence alignment of Losac with other hemolin proteins. Sequence identity is shown for all proteins related to Losac. The structures encompass four constant-type immunoglobulin domains (cIg) (D1-D4). The bars above the sequences correspond to domains 1 (D1, blue), 2 (D2, green), 3 (D3, orange) and 4 (D4, red). Se‐ quence identity is shown for all hemolins related to Losac (%ID). The α-helices and β-sheets observed in *Hyalophora cecropia* hemolin (Protein Data Bank code 1BIH) and Losac are shaded in light and dark gray, respectively. Previously it was predicted that hemolin contain conserved regions and motifs (28, 36). Conserved motifs are shown inside boxes according to the domain they belong. The eight cysteines (in yellow) form four intra-chain disulfide bridges. Losac conserves the well-known motifs involved in cell-adhesion mechanism (KDG motif in D1 and D3), as well as the highly conserved *N*-glycosylation site in D3 (Asn265, in black). An additional *N*-glycosylation site (Asn22) is found in *H. cecropia* and *S. c. ricini* hemolins (D1). The LPS-binding site (NRTS motif: Asn265, Arg266, Thr267 and Ser268) in D3 (orange). In D2 are located the KRLS cAMP/cGMP-dependent protein kinase phosphorylation site and the RRIT motif (green boxes). Positions of the residues forming the putative catalytic site are evidenced with an asterisk. Hemolin sequences (with abbreviation and GenBank™ accession numbers in brackets) are from: *Samia cynthia ricini* (Sc, BAE07175), *Antheraea pernyi* (Ap, AAS99343), *Manduca sexta* (Ms, AAC46915), Losac (Lo, ABF21073) and *H. cecropia* (Hc, AAB34817).

located on D1 (Figure 13 and 14A) and could fit to such activity. Theoretical analysis (Figure 13 and 14) and observed results - total inhibition by PMSF as described in [28] - seem to suggest the presence of a serine protease-like active-site in Losac which would be responsible for the proteolytic activation of factor X [20, 28, 84]. Some molecular techniques, such as Site-directed mutagenesis [101], could be applied in an effort to understand the structural requirements for

**Figure 14.** Three-dimensional structure model of Losac. **(A)** Cartoon view of the predicted model of Losac protein built from the structural coordinates of *H. cecropia* hemolin (PDB code 1BIH) using Modeller 9v1 [102]. Each domain (D1-D4) comprises α-helices and 7 strands arranged in 2 antiparallel β-sheets that are linked together by a disulfide bridge (shown in yellow stick). The residues of the putative catalytic triad predicted in D1 by the program *Catalytic Site Atlas* (http://www.ebi.ac.uk/thornton-srv/databases/CSA/) program, are indicated as stick cyan color by the one let‐ ter code followed by the residue number, D40, H72, S90. **(B)** Electrostatic potential surface of Losac showing inside the box the conserved phosphate-binding site at the D2-D3 interface domains, exactly as was demonstrated for HcHemo‐ lin [97]. **(C)** Surface view of Losac model evidencing the conserved adhesive motif KDG (deep blue) and the LPS-bind‐ ing motif RRVT (green) and NRTS (chocolate), which contains the glycosylation site in N265. All the figures were

Toxins from *Lonomia obliqua* — Recombinant Production and Molecular Approach

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Functionally, hemolins were first associated with the insect immune system because of their over-expression after bacterial infection [103]. Due to their adhesion properties, some hemolins have been involved in the cell adhesion mechanisms. In the last two decades independent studies demonstrated that hemolins are multifunctional molecules involved in a diverse range

ligand binding and selectivity and identification of active site residues.

produced in Pymol v1.5 (http://www.pymol.org/).

At this stage we can only speculate about the mechanism of action of Losac. One possibility is to evaluate structural features that might contribute to the Losac-induced factor X activation. Thus, a search for serine protease active-site was undertaken based on the *Catalytic Site Atlas* (CSA) program analysis. Three possible catalytic residues, Asp(D)40, His(H)72 and Ser(S)90, were

antisense primer designed according to the deduced N- and C-terminal sequences of the mature protein carrying *Nco*I or *Eco*RI restriction sites, respectively. **4)** The cDNA corresponding to mature Losac was sub-cloned into the pAE vector [93]. The PCR product and the pAE vector were restricted with *Nco*I and *Eco*RI, purified, and ligated with T4 DNA ligase and used to transform *E. coli* DH5α cells. **5)** The resulting pAE-Losac plasmid was used to transform *E. coli* BL21(DE3) cells. The recombinant protein was expressed in fusion to a minimal N-terminal His6-tag as a 48.6 kDa protein: Protein was recovered in inclusion bodies after cell lysis and subjected to refolding and purification after solubilization in urea.

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**Figure 13.** Multiple amino acid sequence alignment of Losac with other hemolin proteins. Sequence identity is shown for all proteins related to Losac. The structures encompass four constant-type immunoglobulin domains (cIg) (D1-D4). The bars above the sequences correspond to domains 1 (D1, blue), 2 (D2, green), 3 (D3, orange) and 4 (D4, red). Se‐ quence identity is shown for all hemolins related to Losac (%ID). The α-helices and β-sheets observed in *Hyalophora cecropia* hemolin (Protein Data Bank code 1BIH) and Losac are shaded in light and dark gray, respectively. Previously it was predicted that hemolin contain conserved regions and motifs (28, 36). Conserved motifs are shown inside boxes according to the domain they belong. The eight cysteines (in yellow) form four intra-chain disulfide bridges. Losac conserves the well-known motifs involved in cell-adhesion mechanism (KDG motif in D1 and D3), as well as the highly conserved *N*-glycosylation site in D3 (Asn265, in black). An additional *N*-glycosylation site (Asn22) is found in *H. cecropia* and *S. c. ricini* hemolins (D1). The LPS-binding site (NRTS motif: Asn265, Arg266, Thr267 and Ser268) in D3 (orange). In D2 are located the KRLS cAMP/cGMP-dependent protein kinase phosphorylation site and the RRIT motif (green boxes). Positions of the residues forming the putative catalytic site are evidenced with an asterisk. Hemolin sequences (with abbreviation and GenBank™ accession numbers in brackets) are from: *Samia cynthia ricini* (Sc, BAE07175), *Antheraea pernyi* (Ap, AAS99343), *Manduca sexta* (Ms, AAC46915), Losac (Lo, ABF21073) and *H. cecropia* (Hc, AAB34817).

At this stage we can only speculate about the mechanism of action of Losac. One possibility is to evaluate structural features that might contribute to the Losac-induced factor X activation. Thus, a search for serine protease active-site was undertaken based on the *Catalytic Site Atlas* (CSA) program analysis. Three possible catalytic residues, Asp(D)40, His(H)72 and Ser(S)90, were

**Figure 14.** Three-dimensional structure model of Losac. **(A)** Cartoon view of the predicted model of Losac protein built from the structural coordinates of *H. cecropia* hemolin (PDB code 1BIH) using Modeller 9v1 [102]. Each domain (D1-D4) comprises α-helices and 7 strands arranged in 2 antiparallel β-sheets that are linked together by a disulfide bridge (shown in yellow stick). The residues of the putative catalytic triad predicted in D1 by the program *Catalytic Site Atlas* (http://www.ebi.ac.uk/thornton-srv/databases/CSA/) program, are indicated as stick cyan color by the one let‐ ter code followed by the residue number, D40, H72, S90. **(B)** Electrostatic potential surface of Losac showing inside the box the conserved phosphate-binding site at the D2-D3 interface domains, exactly as was demonstrated for HcHemo‐ lin [97]. **(C)** Surface view of Losac model evidencing the conserved adhesive motif KDG (deep blue) and the LPS-bind‐ ing motif RRVT (green) and NRTS (chocolate), which contains the glycosylation site in N265. All the figures were produced in Pymol v1.5 (http://www.pymol.org/).

located on D1 (Figure 13 and 14A) and could fit to such activity. Theoretical analysis (Figure 13 and 14) and observed results - total inhibition by PMSF as described in [28] - seem to suggest the presence of a serine protease-like active-site in Losac which would be responsible for the proteolytic activation of factor X [20, 28, 84]. Some molecular techniques, such as Site-directed mutagenesis [101], could be applied in an effort to understand the structural requirements for ligand binding and selectivity and identification of active site residues.

Functionally, hemolins were first associated with the insect immune system because of their over-expression after bacterial infection [103]. Due to their adhesion properties, some hemolins have been involved in the cell adhesion mechanisms. In the last two decades independent studies demonstrated that hemolins are multifunctional molecules involved in a diverse range

of cell interaction [104-110]. The high identity among Losac and hemolins suggests that Losac could also assume some of these functions in *L. obliqua*. The adhesive properties of Losac probably are relevant to understanding the human umbilical vein endothelial cell responses observed in previous studies.
