Erythrina velutina Willd. alkaloids : piecing biosynthesis together from transcriptome analysis and metabolite profiling of seeds and leaves

Introduction: Natural products of pharmaceutical interest often do not reach the drug market due to the associated low yields and difficult extraction. Knowledge of biosynthetic pathways is a key element in the development of biotechnological strategies for plant specialized metabolite production. Erythrina species are mainly used as central nervous system depressants in folk medicine and are important sources of bioactive tetracyclic benzylisoquinoline alkaloids (BIAs), which can act on several pathology-related biological targets. Objectives: In this sense, in an unprecedented approach used with a non-model Fabaceae species grown in its unique arid natural habitat, a combined transcriptome and metabolome analyses (seeds and leaves) is presented. Methods: The Next Generation Sequencing-based transcriptome (de novo RNA sequencing) was carried out in a NextSeq 500 platform. Regarding metabolite profiling, the High-resolution Liquid Chromatography was coupled to DAD and a micrOTOF-QII mass spectrometer by using electrospray ionization (ESI) and Time of Flight (TOF) analyzer. The tandem MS/MS data were processed and analyzed through Molecular Networking approach. Results: This detailed macro and micromolecular approach applied to seeds and leaves of E. velutina revealed 42 alkaloids, several of them unique. Based on the combined evidence, 24 gene candidates were put together in a putative pathway leading to the singular alkaloid diversity of this species. Conclusion: Overall, these results could contribute by indicating potential biotechnological targets formodulation of erythrina alkaloids biosynthesis as well as improve molecular databases with omic data from a non-model medicinal plant, and reveal an interesting chemical diversity of Erythrina BIA harvested in Caatinga

Erythrina velutina Willd. alkaloids: Piecing biosynthesis together from transcriptome analysis and metabolite profiling of seeds and leaves

Introduction: Natural products of pharmaceutical interest often do not reach the drug market due to the associated low yields and difficult extraction. Knowledge of biosynthetic pathways is a key element in the development of biotechnological strategies for plant specialized metabolite production. The scarce studies regarding non-model plants impair advances in this field. Erythrina spp. are mainly used as central nervous system depressants in folk medicine and are important sources of bioactive tetracyclic benzylisoquinoline alkaloids, which can act on several pathology-related biological targets. Objective: Herein the purpose is to employ combined transcriptome and metabolome analyses (seeds and leaves) of a non-model medicinal Fabaceae species grown in its unique arid natural habitat. The study tries to propose a putative biosynthetic pathway for the bioactive alkaloids by using an omic integrated approach. Methods: The Next Generation Sequencing-based transcriptome (de novo RNA sequencing) was carried out in a Illumina NextSeq 500 platform. Regarding the targeted metabolite profiling, Nuclear Magnetic Resonance and the High-Performance Liquid Chromatography coupled to a micrOTOF-QII, High Resolution Mass Spectrometer, were used. Results: This detailed macro and micromolecular approach applied to seeds and leaves of E. velutina revealed 42 alkaloids by metabolome tools. Based on the combined evidence, 24 gene candidates were put together in a putative pathway leading to the singular alkaloid diversity of this species. Conclusion: These results contribute by indicating potential biotechnological targets Erythrina alkaloids biosynthesis as well as to improve molecular databases with omic data from a non-model medicinal plant. Furthermore, they reveal an interesting chemical diversity in Erythrina velutina harvested in Caatinga. Last, but not least, this data may also contribute to tap Brazilian biodiversity in a rational and sustainable fashion, promoting adequate public policies for preservation and protection of sensitive areas within the Caatinga

In depth analysis on the carbohydrate-binding properties of a vasorelaxant lectin from Dioclea lasiophylla Mart Ex. Benth seeds

Lectins are a class of proteins or glycoproteins capable of recognizing and interacting with carbohydrates in a specific and reversible manner. Owing to this property, these proteins can interact with glycoconjugates present on the cell surface, making it possible to decipher the glycocode, as well as elicit biological effects, such as inflammation and vasorelaxation. Here, we report a structural and biological study of the mannose/glucose-specific lectin from Dioclea lasiophylla seeds, DlyL. The study aimed to evaluate in detail the interaction of DlyL with Xman and high-mannose N-glycans (MAN3, MAN5 and MAN9) by molecular dynamics (MD) and the resultant in vitro effect on vasorelaxation using rat aortic rings. In silico analysis of molecular docking was performed to obtain the initial coordinates of the DlyL complexes with the carbohydrates to apply as inputs in MD simulations. The MD trajectories demonstrated the stability of DlyL over time as well as different profiles of interaction with Xman and N-glycans. Furthermore, aortic rings assays demonstrated that the lectin could relax pre-contracted aortic rings with the participation of the carbohydrate recognition domain (CRD) and nitric oxide (NO) when endothelial tissue is preserved. These results confirm the ability of DlyL to interact with high-mannose N-glycans with its expanded CRD, supporting the hypothesis that DlyL vasorelaxant activity occurs primarily through its interaction with cell surface glycosylated receptors. Communicated by Ramaswamy H. Sarm

Vatairea guianensis lectin stimulates changes in gene expression and release of TNF‐α from rat peritoneal macrophages via glycoconjugate binding

Using a rat model of peritonitis, we herein report the inflammatory effect induced by the lectin isolated from Vatairea guianensis (VGL) seeds in the context of interactions between VGL and both toll-like receptor 4 (TLR4) and tumor necrosis factor receptor 1 (TNFR1). Peritoneal macrophages were stimulated with VGL for dose-dependent gene expression and release of TNF-alpha. In vivo results showed that VGL (1 mg/kg; intraperitoneal) induced peritonitis in female Wistar rats. Leukocyte migration, macrophage activation, and protein leakage were measured 3 and 6 hours after induction. In vitro, peritoneal macrophages were stimulated with VGL for gene expression and TNF-alpha dosage (mean +/- SEM (n = 6), analysis of variance, and Bonferroni's test (P < .05)). In silico, VGL structure was applied in molecular docking with representative glycans. It was found that (a) VGL increases vascular permeability and stimulates leukocyte migration, both rolling and adhesion; (b) lectin-induced neutrophil migration occurs via macrophage stimulation, both in vitro and in vivo; (c) lectin interacts with TLR4 and TNFR1; and (d) stimulates TNF-alpha gene expression (RT-PCR) and release from peritoneal macrophages. Thus, upon lectin-glycan binding on the cell surface, our results suggest that VGL induces an acute inflammatory response, in turn activating the release of peritoneal macrophages via TNF-alpha and TLR and/or TNFR receptor pathways