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

Ana Marisa Chudzinski-Tavassi, Miryam Paola Alvarez-Flores, Linda Christian Carrijo-Carvalho and Maria Esther Ricci-Silva

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53697

**1. Introduction**

Few species of butterflies and moths (order Lepidoptera) are involved in human envenoming [1]. Caterpillars are the larval forms of moths and butterflies. Toxins are usually found in the caterpillar's hairs and spines with defense purposes. The majority of medically important encounters with lepidopterans occur with exposure to the caterpillar's urticating hairs or spines, but hemolymph can also have toxic properties [1, 2]. A variety of clinical effects have been described, which depend on the family and species involved, ranging from local to systemic reactions [3, 4].

In most occasions, the adverse effects caused by caterpillars are self-limited and can be treated with topical antipruritics [4]. However, for the envenoming by the South American *Lonomia obliqua* caterpillars (Figure 1), named lonomism, the antilonomic serum produced at the Butantan Institute in Brazil is the only effective treatment to reestablish the coagulation parameters in poisoned patients and to avoid the complications seen in severe cases such as intracerebral hemorrhage and acute renal failure [5-10].

In 1989, an outbreak of accidents with this species became a serious public health threat in southern Brazil, with high fatality rates [5, 11-15]. Since then, many studies have been carried out to understand the pathophysiological mechanisms of envenoming [14] and to identify the toxins responsible for adverse reactions.

© 2013 Chudzinski-Tavassi et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Chudzinski-Tavassi et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Figure 1.** A) *Lonomia obliqua* caterpillar (5th instar) and B) pupa.

*L. obliqua* is the caterpillar that has the most studied venom, which main components have been isolated and characterized [14, 16, 17]. Table 1 lists the biological activities and toxins isolated and characterized from the bristle extract or hemolymph of *L. obliqua*. *In vivo* studies reported an antithrombotic effect caused by the bristle extract, while most *in vitro* studies reported procoagulant activities [14, 16-23]. It is well known, for a wide range of animal venoms, that procoagulant toxins can cause *in vivo* activation of the coagulation system. The hemostatic disturbances observed in the envenoming by *L. obliqua* caterpillars, result in a consumption coagulopathy (resembling a disseminated intravascular coagulation) and secondary fibrinolysis, which can lead to the hemorrhagic syndrome [6].

The principal components in the caterpillar's venom have been initially identified by isolating toxins through classical purification methods and following the main activities observed in the whole bristle extract (Figure 2). However, this approach provides knowledge restricted only to the most abundant toxins, and usually reveals that activities which are directly associated to the main symptoms and effects observed in the envenoming outcomes. Experi‐ mental assays were specifically developed to test the hemostatic and enzymatic activities of *Lonomia* toxins and their actions on the coagulation cascade. This knowledge has been valuable for description and management of the envenoming syndrome, but with the classical ap‐ proach, low abundant components and unexpected effects are usually overlooked. Possible interaction of venom components, cross-reactions and secondary effects, useful to provide a systemic view of the pathways involved in the toxin's effects are often unnoticed.

In the last years, methods applied in genomic, transcriptomic and proteomic analyses have been applied with the aims of cataloging and classifying the toxins based on their structure and activity (Figure 3). Thus, it was possible to analyze the envenoming processes at the molecular level. For example, significant advance was achieved through two independent transcriptome studies, which generated a list of putative toxic proteins from *L. obliqua* bristles and hemolymph [20, 43]. In addition, significant advance was achieved as a result of micro‐ array study [44]. Moreover, by coupling proteomics and immunochemical approaches, some immunogenic components were identifying in the bristle extract, especially those related to hemostasis [9]. These components were detected by the antilonomic hyperimmune serum

**Source MW (kDa) Characteristics and observed effects Reference**

produced in bacteria and yeast.

sequence similar to Lopap.

produced in bacteria.

Hemolymph 35 αβ fibrinogenase activity; enable to affect fibrin cross-linked.

extracellular matrix.

baculovirus/insect.

by prostanoids and histamine.

culture.

Bristle extract NI Nociception facilitated by prostaglandin

Bristle extract NI Kinin release from low molecular weight

pressure.

Bristle extract NI Direct platelet aggregation and ATP secretion;

substrate, Ca2+-independent; N-terminal

Cell survival in HUVEC. Recombinant form

blood cells *in vitro*, Ca2+-independent; intravascular hemolysis *in vivo*.

β-endohexosaminidase activity; degradation of

polio viruses. Recombinant form produced in

production; edematogenic response facilitated

kininogen; edema formation and fall in arterial

activity inhibited by *p*-bromophenacyl bromide, a specific PLA2 inhibitor.

consumption coagulopathy *in vivo;* cell survival in endothelial cell culture. Recombinant form

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

[18, 19, 21, 24-26]

177

[20-22, 28]

[29-31]

[32-34]

[35]

[36]

[39]

[40]

[41, 42]

[37, 38]

[27]

http://dx.doi.org/10.5772/53697

Bristle extract 21 Serine protease, activity increased by Ca2+;

Bristle extract 45 Serine protease, Ca2+-independent;

FXa-like Bristle extract 21 Hydrolytic activity on S-2222 chromogenic

Phospholipase A2-like Bristle extract 15 Indirect hemolytic activity in human and rat red

Bristle extract 49

NI: None isolated, studies carried out using the whole venom

**Table 1.** Toxins and activities described in *L. obliqua* venom.

53

Antiapoptotic Hemolymph 51 Activity on *Spodoptera frugiperda* (Sf-9) cell

Antiviral Hemolymph 20 Antiviral activity against measles, influenza and

produced at the Butantan Institute, and abundant proteins were identified.

**Activity (toxin)**

Prothrombin activation (Lopap)

Factor X activation

Fibrinogenolytic (Lonofibrase)

Hyaluronidase (Lonoglyases)

Nociceptive and Edematogenic

Kallikrein-kinin system activation

aggregation

Platelet adhesion and

(Losac)


**Table 1.** Toxins and activities described in *L. obliqua* venom.

**Figure 1.** A) *Lonomia obliqua* caterpillar (5th instar) and B) pupa.

Applications

176

*L. obliqua* is the caterpillar that has the most studied venom, which main components have been isolated and characterized [14, 16, 17]. Table 1 lists the biological activities and toxins isolated and characterized from the bristle extract or hemolymph of *L. obliqua*. *In vivo* studies reported an antithrombotic effect caused by the bristle extract, while most *in vitro* studies reported procoagulant activities [14, 16-23]. It is well known, for a wide range of animal venoms, that procoagulant toxins can cause *in vivo* activation of the coagulation system. The hemostatic disturbances observed in the envenoming by *L. obliqua* caterpillars, result in a consumption coagulopathy (resembling a disseminated intravascular coagulation) and

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

The principal components in the caterpillar's venom have been initially identified by isolating toxins through classical purification methods and following the main activities observed in the whole bristle extract (Figure 2). However, this approach provides knowledge restricted only to the most abundant toxins, and usually reveals that activities which are directly associated to the main symptoms and effects observed in the envenoming outcomes. Experi‐ mental assays were specifically developed to test the hemostatic and enzymatic activities of *Lonomia* toxins and their actions on the coagulation cascade. This knowledge has been valuable for description and management of the envenoming syndrome, but with the classical ap‐ proach, low abundant components and unexpected effects are usually overlooked. Possible interaction of venom components, cross-reactions and secondary effects, useful to provide a

systemic view of the pathways involved in the toxin's effects are often unnoticed.

In the last years, methods applied in genomic, transcriptomic and proteomic analyses have been applied with the aims of cataloging and classifying the toxins based on their structure and activity (Figure 3). Thus, it was possible to analyze the envenoming processes at the molecular level. For example, significant advance was achieved through two independent transcriptome studies, which generated a list of putative toxic proteins from *L. obliqua* bristles

secondary fibrinolysis, which can lead to the hemorrhagic syndrome [6].

and hemolymph [20, 43]. In addition, significant advance was achieved as a result of micro‐ array study [44]. Moreover, by coupling proteomics and immunochemical approaches, some immunogenic components were identifying in the bristle extract, especially those related to hemostasis [9]. These components were detected by the antilonomic hyperimmune serum produced at the Butantan Institute, and abundant proteins were identified.

available information about *L. obliqua* venom, and focus on strategies to unveil molecular aspects of toxins and the perspectives for therapeutic and biotechnological applications.

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

http://dx.doi.org/10.5772/53697

179

**Figure 3.** Schematic representation of the strategies to explore the Lonomia obliqua venom and toxins based on cel‐ lular and molecular approaches. Results obtained indicate promising applications for these proteins and derived pep‐

For many years, direct purification of toxins from venoms was the best procedure to charac‐ terize them with regard to their primary structure. Then, the development of molecular approaches to characterize toxin genes represented an expansion in the understanding of the structure and function of toxin, critical for the development of new treatments directed against the venom toxins (antivenoms). Cloning of cDNAs coding for biochemically isolated toxins has improved their characterization. *Transcriptomic* allows the identification of cellular transcripts in a given cell population, while proteomic studies protein's properties and functions (expression level, structure, post-translational modification, etc.) of proteins expressed by the genome of an organism at a certain point of time. The availability of tech‐ nologies for high throughput analysis has led to integrate toxin expression at mRNA and

tides.

**2. Molecular approach**

**Figure 2.** Schematic representation of the classical strategy employed in initial studies of the *Lonomia obliqua* venom. The bristle extract was analyzed through denaturing electrophoresis (SDS-PAGE) which showed the venom is a com‐ plex mixture of proteins. Screening assays were carried out to investigate possible effects on blood coagulation and fibrinolysis. The venom showed procoagulant activity by decreasing blood clotting time. Two procoagulant compo‐ nents (Lopap, a prothrombin activator and Losac, a factor X activator) were identified and isolated from the bristle extract for further characterization. The specific activity of Lopap and Losac were observed on purified coagulation factor zymogens (FII or FX) using chromogenic substrates to detect generation of active forms of clotting factors (FIIa and FXa) by these toxins. This assay was used in the purification process to identify the active fractions containing each toxin. SDS-PAGE profile shows Lopap (1- multimer, 3- monomer) and Losac (2) are abundant components in the ven‐ om. MM: molecular markers.

Production of recombinant forms of *Lonomia* toxins and discovery of new molecules are opening perspectives in the scientific area for basic and applied researches. These molecules can point out novel mechanisms of action, undiscovered molecular interactions and new classes of enzymes and inhibitors. Interesting, some venom toxins have shown multifunctional properties [19, 22, 28]. The best examples are Lopap (a prothrombin activator with high similarity with lipocalins) and Losac (a factor X activator highly similar to hemolins). Besides activation of blood coagulation, Lopap and Losac can modulate cellular functions and promote cell survival [22, 45]. Both molecules were cloned and produced in its recombinant form in yeast and/or bacteria [19, 25, 28].

Additional studies will be conducted to determine the involvement of the venom components in the envenoming syndrome and their biological significance for physiological processes of the animal, such as insect metamorphosis, which is a combination of growth/activation/ differentiation/programmed cell death signals. Thus, this chapter reviews the currently available information about *L. obliqua* venom, and focus on strategies to unveil molecular aspects of toxins and the perspectives for therapeutic and biotechnological applications.

**Figure 3.** Schematic representation of the strategies to explore the Lonomia obliqua venom and toxins based on cel‐ lular and molecular approaches. Results obtained indicate promising applications for these proteins and derived pep‐ tides.
