Proteomic analysis of digestive tract peptidases and lipases from the invasive gastropod Pomacea canaliculata.

BACKGROUND: The invasive gastropod Pomacea canaliculata has received great attention in the last decades as a result of its negative impact on crops agriculture, yet knowledge of their digestive physiology remains incomplete, particularly the enzymatic breakdown of macromolecules such as proteins and lipids. RESULTS: Discovery proteomics revealed aspartic peptidases, cysteine peptidases, serine peptidases, metallopeptidases and threonine peptidases, as well as acid and neutral lipases and phospholipases along the digestive tract of P. canaliculata. Peptides specific to peptidases (139) and lipases (14) were quantified by targeted mass spectrometry. Digestion begins in the mouth via diverse salivary peptidases (nine serine peptidases; seven cysteine peptidases, one aspartic peptidase and 22 metallopeptidases) and then continues in the oesophagus (crop) via three luminal metallopeptidases (Family M12) and six serine peptidases (Family S1). Downstream, the digestive gland provides a battery of enzymes composed of aspartic peptidase (one), cysteine peptidases (nine), serine peptidases (12) and metallopeptidases (24), including aminopeptidases, carboxypeptidases and dipeptidases). The coiled gut has M1 metallopeptidases that complete the digestion of small peptides. Lipid extracellular digestion is completed by triglyceride lipases. CONCLUSION: From an integrative physiological and anatomical perspective, P. canaliculata shows an unexpected abundance and diversity of peptidases, which participate mainly in extracellular digestion. Moreover, the previously unknown occurrence of luminal lipases from the digestive gland is reported for the first time. Salivary and digestive glands were the main tissues involved in the synthesis and secretion of these enzymes, but plausibly the few luminally exclusive peptidases are secreted by ventrolateral pouches or epithelial unicellular glands. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Integrative Proteomic Analysis of Digestive Tract Glycosidases from the Invasive Golden Apple Snail, Pomacea canaliculata.

The freshwater snail Pomacea canaliculata, an invasive species of global significance, possesses a well-developed digestive system and diverse feeding mechanisms enabling the intake of a wide variety of food. The identification of glycosidases in adult snails would increase the understanding of their digestive physiology and potentially generate new opportunities to eradicate and/or control this invasive species. In this study, liquid chromatography coupled to tandem mass spectrometry was applied to define the occurrence, diversity, and origin of glycoside hydrolases along the digestive tract of P. canaliculata. A range of cellulases, hemicellulases, amylases, maltases, fucosidases, and galactosidases were identified across the digestive tract. The digestive gland and the contents of the crop and style sac yield a higher diversity of glycosidase-derived peptides. Subsequently, peptides derived from 81 glycosidases (46 proteins from the public database and 35 uniquely from the transcriptome database) that were distributed among 13 glycoside hydrolase families were selected and quantified using multiple reaction monitoring mass spectrometry. This study showed a high glycosidase abundance and diversity in the gut contents of P. canaliculata which participate in extracellular digestion of complex dietary carbohydrates. Salivary and digestive glands were the main tissues involved in their synthesis and secretion.

Characterization of a Bioactive Acyclotide from Palicourea rigida.

The extraction and purification of parigidin-br3, a cyclotide analogue belonging to the "bracelet" subfamily, from Palicourea rigida leaves is discussed. Unlike conventional cyclotides, parigidin-br3 has free N- and C-termini, as identified by MALDI-TOF/TOF analysis and confirmed by gene structure elucidation, and is one of a small number of acyclotides discovered during recent years. Parigidin-br3 showed cytotoxic activity against MCF-7 (breast cancer) and CACO2 (colorectal adenocarcinoma) cells, with IC50 values of ∼2.5 µM and less than 10% hemolytic activity. Overall, parigidin-br3 is a promising new molecule with cytotoxic properties against tumor cell lines and, unlike many synthetic acyclic analogues, demonstrates that cytotoxic activity is not limited to conventional (i.e., cyclic) cyclotides.

Identification and structural characterization of novel cyclotide with activity against an insect pest of sugar cane.

Cyclotides are a family of plant-derived cyclic peptides comprising six conserved cysteine residues connected by three intermolecular disulfide bonds that form a knotted structure known as a cyclic cystine knot (CCK). This structural motif is responsible for the pronounced stability of cyclotides against chemical, thermal, or proteolytic degradation and has sparked growing interest in this family of peptides. Here, we isolated and characterized a novel cyclotide from Palicourea rigida (Rubiaceae), which was named parigidin-br1. The sequence indicated that this peptide is a member of the bracelet subfamily of cyclotides. Parigidin-br1 showed potent insecticidal activity against neonate larvae of Lepidoptera (Diatraea saccharalis), causing 60% mortality at a concentration of 1 µm but had no detectable antibacterial effects. A decrease in the in vitro viability of the insect cell line from Spodoptera frugiperda (SF-9) was observed in the presence of parigidin-br1, consistent with in vivo insecticidal activity. Transmission electron microscopy and fluorescence microscopy of SF-9 cells after incubation with parigidin-br1 or parigidin-br1-fluorescein isothiocyanate, respectively, revealed extensive cell lysis and swelling of cells, consistent with an insecticidal mechanism involving membrane disruption. This hypothesis was supported by in silico analyses, which suggested that parigidin-br1 is able to complex with cell lipids. Overall, the results suggest promise for the development of parigidin-br1 as a novel biopesticide.