Forty Years of the Description of Brown Spider Venom Phospholipases-D

toxins Review Forty Years of the Description of Brown Spider Venom Phospholipases-D Luiza Helena Gremski 1 , Hanna Câmara da Justa 1 , Thaís Pereira da Silva 1 , Nayanne Louise Costacurta Polli 1 , Bruno César Antunes 1,2 , João Carlos Minozzo 2 , Ana Carolina Martins Wille 3 , Andrea Senff-Ribeiro 1 , Raghuvir Krishnaswamy Arni 4 Silvio Sanches Veiga 1, * 1 2 3 4 * and Departamento de Biologia Celular, Universidade Federal do Paraná (UFPR), Curitiba 81531-980, PR, Brazil; (L.H.G.); (H.C.d.J.); (T.P.d.S.); (N.L.C.P.); (B.C.A.); (A.S.-R.) Centro de Produção e Pesquisa de Imunobiológicos (CPPI), Piraquara 83302-200, PR, Brazil; Departamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta Grossa, Ponta Grossa 84030-900, PR, Brazil; Centro Multiusuário de Inovação Biomolecular, Departamento de Física, Universidade Estadual Paulista (UNESP), São José do Rio Preto 15054-000, SP, Brazil; Correspondence: ; Tel.: +55-(41)-3361-1776



 Received: 12 February 2020; Accepted: 2 March 2020; Published: 6 March 2020 Abstract: Spiders of the genus Loxosceles, popularly known as Brown spiders, are considered a serious public health issue, especially in regions of hot or temperate climates, such as parts of North and South America. Although the venoms of these arachnids are complex in molecular composition, often containing proteins with distinct biochemical characteristics, the literature has primarily described a family of toxins, the Phospholipases-D (PLDs), which are highly conserved in all Loxosceles species. PLDs trigger most of the major clinical symptoms of loxoscelism i.e., dermonecrosis, thrombocytopenia, hemolysis, and acute renal failure. The key role played by PLDs in the symptomatology of loxoscelism was first described 40 years ago, when researches purified a hemolytic toxin that cleaved sphingomyelin and generated choline, and was referred to as a Sphingomyelinase-D, which was subsequently changed to Phospholipase-D when it was demonstrated that the enzyme also cleaved other cellular phospholipids. In this review, we present the information gleaned over the last 40 years about PLDs from Loxosceles venoms especially with regard to the production and characterization of recombinant isoforms. The history of obtaining these toxins is discussed, as well as their molecular organization and mechanisms of interaction with their substrates. We will address cellular biology aspects of these toxins and how they can be used in the development of drugs to address inflammatory processes and loxoscelism. Present and future aspects of loxoscelism diagnosis will be discussed, as well as their biotechnological applications and actions expected for the future in this field. Keywords: brown spider; venom; phospholipases-D; biochemical and biological activities Key Contribution: The primary goal of this review is to celebrate 40 years of research of Brown spider Phospholipases-D by presenting an overview of the extensive research results related to these toxins, outlining the knowledge that has been gathered over the years and the prospects for advances in studies on these enzymes. Toxins 2020, 12, 164; doi:10.3390/toxins12030164 www.mdpi.com/journal/toxins Toxins 2020, 12, 164 2 of 28 1. Molecular Characteristics of Brown Spider Venoms Loxoscelism, the set of clinical manifestations of envenomation by Brown Spiders (Loxosceles genus), can be cutaneous and systemic. At the bite site, the cutaneous symptoms are edema, erythema and dermonecrosis with gravitational spreading of lesion (the characteristic hallmark of envenoming). The systemic condition, which is less common, may lead to death and includes hematological changes such as intravascular hemolysis, disseminated intravascular coagulation and thrombocytopenia, as well as acute renal failure [1–6]. The volume of venom injected at the time of the bite is low, normally just a few microliters and contains between 60 and 100 micrograms of protein [7]. Loxosceles spider venoms are colorless liquids, predominantly composed of proteins and peptides with molecular masses in the 5 to 45 kDa range [2,5]. The toxins encountered in Loxoscelic venoms can be separated into two major groups: (i) insecticidal toxins (Inhibitor Cystine Knot—ICK—peptides), metalloproteases (Astacins) and phospholipases-D (PLDs), and (ii) the toxins expressed in lower quantities such as; hyaluronidases, serine proteases, serine protease inhibitors (serpins), allergenic factors and a translationally controlled tumor protein (TCTP) [5,8]. The total venom composition was determined by transcriptome analyses of the Loxosceles intermedia venom gland [8]. The toxin-encoding transcripts quantitative profile of L. intermedia venom gland was 56% of Knottins, 23% of Astacins, 20% of PLDs and about 1% of the other toxins [8]. The study of L. laeta venom gland transcripts encoding PLDs showed that these toxins correspond to about 16% of all transcripts [9], although 16 clusters annotated as similar to sphingomyelinase D (PLDs) are in fact similar to Loxosceles ICK peptides (GenBank accession numbers EY189720, EY188468, EY188410, EY189491, EY189459, EY189456, EY188675, EY189643, EY189620, EY189608, EY188487, EY189575, EY188624, EY188618, EY188594, EY188592) and one similar to other neurotoxins (EY188603) when compared to GenBank sequences using the blastx algorithm (E values < 1.00 ×10−5 ). A similar study of transcripts encoding PLDs from L. similis venom glands showed that these toxins correspond to about 15% of the total produced transcripts. Transcript analysis revealed 12 main groups and a predominance of s1 and s11 isoforms, which together correspond to about 72% of transcripts for PLDs [10]. The high abundance of these isoforms is not surprising, since L. similis PLDs of s1 and s11 groups are highly similar to L. intermedia PLDs that comprise 69.7% of L. intermedia PLD transcripts. Interestingly, an antimicrobial peptide (1695.75 Da), recently identified in L. gaucho venom, presented a remarkable similarity to specific regions of phospholipases D from Loxosceles genus. This peptide, referred to as U1-SCRTX-Lg1a, possesses an antibacterial effect on gram-negative bacteria [11]. The authors suggested that U1-SCRTX-Lg1a may have originated by limited proteolysis of a venom PLD. Thus, it is possible that PLD rates evaluated by transcriptomic analyses may be overestimated. Despite the complexity of the venom, both native and recombinant PLDs alone can reproduce virtually all the clinical symptoms of Loxoscelism. Although the PLDs are not the most expressed toxins in Loxosceles venoms, they are the best studied and characterized toxins. Figure 1 summarizes the main aspects and activities of Brown spider PLDs by displaying updated assays and analyses performed for this work. The SDS-PAGE profiles of L. intermedia venom and a recombinant PLD are shown in Figure 1B and reveal an enrichment of proteins in the region of 30 (...truncated)