Spatial distribution and biochemical characterization of serine peptidase inhibitors in the venom of the brazilian sea anemone anthopleura cascaia using mass spectrometry imaging

Sea anemones are known to produce a diverse array of toxins with different cysteine-rich peptide scaffolds in their venoms. The serine peptidase inhibitors, specifically Kunitz inhibitors, are an important toxin family that is believed to function as defensive peptides, as well as prevent proteolysis of other secreted anemone toxins. In this study, we isolated three serine peptidase inhibitors named Anthopleura cascaia peptide inhibitors I, II, and III (ACPI-I, ACPI-II, and ACPI-III) from the venom of the endemic Brazilian sea anemone A. cascaia. The venom was fractionated using RP-HPLC, and the inhibitory activity of these fractions against trypsin was determined and found to range from 59% to 93%. The spatial distribution of the anemone peptides throughout A. cascaia was observed using mass spectrometry imaging. The inhibitory peptides were found to be present in the tentacles, pedal disc, and mesenterial filaments. We suggest that the three inhibitors observed during this study belong to the venom Kunitz toxin family on the basis of their similarity to PI-actitoxin-aeq3a-like and the identification of amino acid residues that correspond to a serine peptidase binding site. Our findings expand our understanding of the diversity of toxins present in sea anemone venom and shed light on their potential role in protecting other venom components from proteolysis.

Unraveling and profiling Tityus bahiensis venom: biochemical analyses of the major toxins

Among the scorpions found in Brazil, Tityus bahiensis is one of the species that causes most of the reported human accidents. In spite of this important constatation, the venom composition description is not available in the literature. Thus, this venom remains not properly studied, segregating this particular species into an abandoned, forgotten condition. In the present study, chromatographic separation (RP-HPLC-C18) and proteomic analyses were employed to unravel the diversity, complexity, and proportional distribution of the main peptides and proteins found in the scorpion venom. Moreover, sequence analyses and the presence of new isoforms and toxins are discussed based on a database comparison with other Tityus toxins. Our results show the presence of a wide diversity of potassium and sodium channel toxins and enzymes, such as metallopeptidases and hyaluronidases, as previously described for other species. However, the current work also describes for the first time, at the protein level, phospholipase, angiotensin-converting enzyme, cysteine-rich proteins, serine peptidase inhibitors peptides, and antimicrobial peptides. Finally, thorough data analyses allowed the description of the venom toxins distribution regarding their diversity and relative quantity. Significance: the work presents the first Tityus bahiensis proteome. We have focused on describing the neurotoxin variability in terms of their isoforms/amino acid substitutions. Understanding the natural variations in the toxins' sequences is essential, once the affinity of these peptides to their respective receptors/ionic channels will vary depending on the specific peptide sequences. Moreover, the current study describes some proteins present in the venom, including enzymes being described for the first time in scorpion venoms, such as PLA2 and ACE. Moreover, we describe the individual relative quantity distribution for the different protein classes identified, as well as their variability in the T.bahiensis venom. Finally, this study also reports the development of a simple straightforward chromatographic method for scorpion venom fractionation.

Identificação e caracterização bioquímica de inibidores de serinopeptidases isolados a partir da peçonha das anêmonas-do-mar Anthopleura cascaia e Aulactinia veratra

Phylum Cnidaria comprises more than 10,000 species and around 10% of them are represented by sea anemones. These animals are underexplored sources of molecules, possessing structurally diverse toxins that can act over a diverse range of pharmacological targets, including enzymes. Sea anemones represent almost 96% of the manually annotated toxins from the phylum, but until now only 5% of its species have been studied about their toxin content. In the present work, the venoms of the sea anemones Anthopleura cascaia and Aulactinia veratra were studied and accessed through mass spectrometry analysis for searching serine peptidase inhibitors. The arsenal of toxins from both venoms was elucidated. Additionally, venom’s fractions were screened for inhibitory activity over trypsin, using time-course fluorescence-based kinetic assays or Mass spectrometry-based analysis. Beyond that, the spatial distribution of serine peptidase inhibitors in both sea anemones’ tissues were shown through Mass Spectrometry Imaging by MALDITOF. In the analysis of toxins composition, it was seen that A. cascaia venom presents at least three types of toxins: cytolysins, phospholipases and a toxin similar to natterin. For A. veratra, the classification based on blastp hit similarity and relying on domain architecture of the toxin’s sequences (translated transcripts) was performed. The thorough examination over toxins sequences led to the identification of 59 proteins and peptides belonging to 14 known toxin’s families of sea anemones and to the acknowledge of 20 peptides presenting 18 new cysteine scaffolds. The venom of this sea anemone mainly relies on neurotoxins from ShK-like, β-defensins, SCRiP, ICK, EGF-like types and on serine peptidase inhibitors from Kazal and Kunitz types. Furthermore, serine peptidase inhibitors from both venoms were isolated and present main distribution over tentacles, mesenterial filaments and pedal disc of these sea anemones, suggesting the preferential stock of these toxins. In conclusion, the methodological approaches applied in this study were able of identifying the presence of serine peptidase inhibitors on the venom and tissue of sea anemones through chromatographic techniques followed by enzymatic assays, and MALDI-Imaging.

O filo Cnidaria é composto por mais de 10.000 espécies e cerca de 10% destas são anêmonas-do-mar. Estes animais são considerados fontes subexploradas de moléculas, possuindo um diverso arsenal de toxinas que podem agir sobre diferentes alvos farmacológicos, incluindo enzimas. Toxinas de anêmonas-do-mar representam cerca de 96% das toxinas anotadas para o filo Cnidaria, embora apenas 5% de suas espécies tenham sido estudadas quanto à composição de toxinas até o momento. Neste trabalho elucidamos por espectrometria de massas a composição da peçonha das anêmonas Anthopleura cascaia e Aulactinia veratra, buscando a identificação de inibidores de serinopeptidases. O arsenal de toxinas para ambas anêmonas foi elucidado. Ainda, descrevemos as etapas de purificação envolvidas na busca de inibidores e a seleção destes candidatos por meio da inibição da atividade da tripsina, avaliada por duas técnicas distintas։ Cinética enzimática e Espectrometria de massas. Adicionalmente, descrevemos a localização de candidatos a inibidores no tecido das anêmonas através do Imageamento por espectrometria de massas. Na análise sobre a composição de toxinas destas anêmonas, vimos que a peçonha da A. cascaia apresentou a existência três tipos de toxinas incluindo citolisinas, fosfolipases e naterinas. Para a espécie A. veratra, a classificação de toxinas baseadas no blastp hit e na arquitetura de domínios das toxinas foi realizada. Esta análise revelou a presença de 59 proteínas e peptídeos pertencentes a 14 famílias de toxinas de anêmonasdo-mar; além do reconhecimento de 20 peptídeos apresentando 18 novos scaffolds de cisteínas. A peçonha desta anêmona é principalmente composto por neurotoxinas do tipo ShK-like, β-defensinas, SCRiP, ICK, EGF-like e inibidores de serinopeptidases. Os dados obtidos mostram que ambas anêmonas são ricas fontes de inibidores de serinopeptidases, especialmente tipo Kunitz e Kazal. Tais inibidores apresentam distribuição na região dos tentáculos, mesentério e disco pedal das anêmonas, o que pode indicar o estoque preferencial destas toxinas. E conclusão, o conjunto de abordagens metodológicas empregadas neste trabalho foi capaz de atender os objetivos propostos: identificar a presença de inibidores de serinopeptidases na peçonha e tecido de anêmonas, tanto por fracionamento cromatográfico seguido de ensaio enzimático, quanto por MALDI-Imaging.

Magnetite-levan nanoparticles for lectin purification: a single-step strategy for protein isolation from the seeds extract of the plant Cratylia mollis

The ability to reversibly bind carbohydrates is an incredible property from lectins. Such characteristic has led these molecules to be employed in several applications involving medical research and biotechnology. Generally, these proteins follow several steps towards purification. Here, the synthesis, physical characterization, and use of levan-coated magnetite nanoparticles (MNPs-levan) for lectin isolation is described. Canavalia ensiformis and Cratylia mollis were used as sources of Concanavalin A and Cramoll, respectively, that were purified by using MNPs-levan. Mass spectrometry, SDS-PAGE, and hemagglutinating activity were employed to assess the efficiency of the process. Moreover, by using mass spectrometry approaches, a novel lectin, similar to Canavalin, was also identified for C. mollis, corroborating the advantages of using nanoparticles over microparticles. MNPs-levan could also be recycled, making this a low-cost, scalable process that can be efficiently employed over crude samples.