The type-1 ribosome-inactivating protein OsRIP1 triggers caspase-independent apoptotic-like death in HeLa cells.

Ribosome-inactivating proteins (RIPs) are capable of removing a specific adenine from 28S ribosomal RNA, thus inhibiting protein biosynthesis in an irreversible manner. In this study, recombinant OsRIP1, a type 1 RIP from rice (Oryza sativa L.), was investigated for its anti-proliferative properties. Human cervical cancer HeLa cells were incubated in the presence of OsRIP1 for 24-72 h. OsRIP1 treatment yielded an anti-proliferation response of the HeLa cells and resulted in apoptotic-like blebbing of the plasma membrane without causing DNA fragmentation. OsRIP1 labeled with FITC accumulated at the cell surface. Pull-down assays identified ASPP1 (Apoptosis-Stimulating Protein of p53 1) and IFITM3 (interferon-induced transmembrane protein 3) as potential interaction partners for OsRIP1. Transcript levels for several critical genes related to different signaling pathways were quantified by RT-qPCR. OsRIP1 provoked HeLa cells to undergo caspase-independent cell death, associated with a significant transcriptional upregulation of the apoptotic gene PUMA, interferon regulatory factor 1 (IRF1) and the autophagy-related marker LC3. No changes in caspase activities were observed. Together, these data suggest that apoptotic-like events were involved in OsRIP1-driven caspase-independent cell death that might trigger the IRF1 signaling pathway and LC3-mediated autophagy.

A review of Vicieae lectins studies: End of the book or a story in the writing?

Vicieae tribe, Leguminosae family (Fabaceae), has been extensively studied. In particular, the study of lectins. The purification, physicochemical and structural characterizations of the various purified lectins and the analysis of their relevant biological activities are ongoing. In this review, several works already published about Vicieae lectins are addressed. Initially, we presented the purification protocols and the physicochemical aspects, such as specificity for carbohydrates, optimal activity in the face of variations in temperature and pH, as well metals-dependence. Following, structural characterization studies are highlighted and, finally, various biological activities already reported are summarized. Studies on lectins in almost all genera (Lathyrus, Lens, Pisum and Vicia) are considered, with the exception of Vavilovia which studies of lectins have not yet been reported. Like other leguminous lectins, Vicieae lectins present heterogeneous profiles of agglutination profiles for erythrocytes and other cells of the immune system, and glycoproteins. Most Vicieae lectins consist of two subunits, α and ß, products of a single precursor protein derived from a single gene. The differences between the isoforms result from varying degrees of proteolytic processing. Along with the identification of these molecules and their characteristics, biological activities become very relevant and robust for both basic and applied research.

Comprehensive review on Caelsalpinioideae lectins: From purification to biological activities.

Lectins are a class of proteins with specific and reversible carbohydrate binding properties. Plant lectins constitute the group of these proteins most studied, placing emphasis on the legume family. The Caesalpinioideae subfamily is part of Leguminosae and second only to Papilionoideae with more published works on lectins. Classically, Caesalpinioideae is formed by 171 genera and 2250 species. It presents 13 genera with reports of lectins, featuring the Bauhinia genus with the greatest number of species having purified and characterized lectins. Comparing genera, the lectins in this subfamily do not have similar physicochemical or structural properties. Collectively, however, antibacterial, antiviral, and anticancer activities have been reported, as well as applications as biosensors and biomarkers. This review aims to summarize the available data on purified lectins from species of the Caesalpinioideae subfamily, demonstrating the characteristics of these molecules and the potential for their application in future studies of new lectins, as well as of application in several areas.

Reviewing Mimosoideae lectins: A group of under explored legume lectins.

Lectins are proteins capable of specific and reversible binding to mono- and/or oligosaccharides, and within this group, Legume lectins are the most studied. However, most of these studies focus on the Papilionoideae subfamily, with Caesalpinioideae and Mimosoideae lectins being significantly less explored in the literature. The Mimosoideae subfamily consists of at least 79 genera and 3275 species, but, to date, only about 14 lectins have been purified, a fact which shows the lack of studies for this group. Based on their purification protocols, as well as physicochemical and structural properties, Mimosoideae lectins are very heterogeneous. Despite the few studies, a wide variety of biological activities have been tested, including, for example, inflammatory, anticancer, antibacterial, and antifungal. In this context, the present review aims to summarize the available data regarding the purification, physicochemical and structural properties, as well as biological activities, of lectins extracted from plants of the Mimosoideae subfamily in order to bring more insight to researchers interested in further exploring the potential of these molecules.

Molecular dynamics and binding energy analysis of Vatairea guianensis lectin: a new tool for cancer studies.

The Tn antigen is an epitope containing N-acetyl-D-galactosamine present in the extracellular matrix of some carcinoma cells in humans, and it is often used as a biomarker. Lectins are proteins capable of binding to carbohydrates and can be used as a molecular tool to recognize antigens and to differentiate cancer cells from normal cells. In this context, the present work aimed to characterize the interaction of Vatairea guianensis seed lectin with N-acetyl-D-galactosamine and the Tn antigen by molecular dynamics and molecular mechanics/Poisson-Boltzmann solvent-accessible surface area analysis. This study revealed new interacting residues not previously identified in static analysis of the three-dimensional structures of Vatairea lectins, as well as the configuration taken by the carbohydrate recognition domain, as it interacts with each ligand. During the molecular dynamics simulations, Vatairea guianensis lectin was able to bind stably to Tn antigen, which, as seen previously for other lectins, enables its use in cancer research, diagnosis, and therapy. This work further demonstrates the efficiency of bioinformatics in lectinology.

Purification and partial characterization of a new lectin from Parkia panurensis Benth. ex H.C. Hopkins seeds (Leguminosae family; Mimosoideae subfamily) and evaluation of its biological effects.

Lectins are proteins that have as one of their main characteristics recognizing and reversibly binding to carbohydrates. In this work, it was possible to purify and characterize a lectin from Parkia panurensis (Leguminosae family; Mimosoideae subfamily) seeds by a combination of the techniques: protein precipitation, along with affinity and then ion exchange chromatography using the Sepharose-mannose and diethylaminoethyl matrices, respectively. The pure lectin, called PpaL, has affinity by D-mannose, D-glucose and derivatives. PpaL was stable over a wide range of temperature and pH, and it showed an SDS-PAGE profile of only one protein band with apparent mass of 45 kDa, subsequently confirmed by mass spectrometry, and presented a molecular mass of 50,566 ± 1 Da. PAGE analysis and molecular exclusion chromatography demonstrated that PpaL is presented as a dimer in solution. Partial sequencing of the primary structure resulted in a total of 334 amino acid residues with approximately 97% similarity to Parkia biglobosa and Parkia platycephala seed lectins. PpaL was shown to be toxic against Artemia nauplii and had an LC50 of 20 µg/mL. The effects of biological activities presented by these proteins make them important biotechnological tools, demonstrating the importance of bioprospection of new lectins.

Dalbergieae lectins: A review of lectins from species of a primitive Papilionoideae (leguminous) tribe.

Lectins are (glyco)proteins capable of reversibly binding to specific carbohydrates, thus having various functions and applications. Plant lectins are the best studied, and the Leguminoseae family is highlighted in a number of published works, especially species of the Papilionoideae subfamily. Dalbergieae is one of the tribes in this subfamily comprising 49 genera and over 1300 species. From this tribe, about 26 lectins were studied, among which we can highlight the Arachis hypogaea lectin, widely used in cancer studies. Dalbergieae lectins demonstrate various carbohydrate specificities and biological activities including anti-inflammatory, vasorelaxant, nociceptive, antibacterial, antiviral among others. Structurally, these lectins are quite similar in their three-dimensional folding but present significant differences in oligomerization patterns and in the conservation of carbohydrate-recognition domain. Despite the existence of structural data from some lectins, only sparse literature has reported on this tribe's diversity, not to mention the range of biological effects, determined through specific assays. Therefore, this work will review the most important studies on Dalbergieae lectins and their potential biomedical applications.

One century of ConA and 40 years of ConBr research: A structural review.

Lectins are proteins that can bind specifically and reversibly to carbohydrates. This capacity gives lectins multiple biological roles and biotechnological applications. Although lectins can be found in all organisms, plant lectins, especially legume lectins, are undoubtedly the most thoroughly studied. Among legume lectins, the lectin from Canavalia ensiformis (ConA) and Canavalia brasiliensis (ConBr), both from Diocleinae subtribe, are two of the most well-known lectins. It has been 100 years since the first report of ConA and 40 years since the first report of ConBr, making 2019 an important year for lectinology. Structural data of these lectins in combination with biological activity tests clearly indicate that even a small shift in amino acid sequence can affect the tertiary and quaternary structures, consequently affecting the biological activity of these proteins. It is in this context that the present paper aims to review the structural data of ConA and ConBr, focusing on the primary structure, crystallography, tertiary and quaternary structures of these lectins, as well as their binding sites. This paper also expands the structural data by employing molecular dynamics to evaluate carbohydrate-binding properties and structural stability. It is anticipated that these data will increase knowledge about the structure-function relationships of these proteins.

Structural analysis, molecular docking and molecular dynamics of an edematogenic lectin from Centrolobium microchaete seeds.

Lectins represent a class of proteins or glycoproteins capable of reversibly binding to carbohydrates. Seed lectins from the Dalbergieae tribe (Leguminosae) have structural variability, carbohydrate specificity, and biological effects, such as inflammation, vasorelaxation and cancer antigen binding. To comprehensively address these factors, the present work aimed to establish and characterize the three-dimensional structure of Centrolobium microchaete lectin (CML) by homology modeling, investigate protein-carbohydrate interactions and evaluate its inflammatory effect on mice. Molecular docking was performed to analyze interactions of the lectin with monosaccharides, disaccharides and N-glycans. Two dimannosides, methyl mannose-1,3-α-D-mannose (MDM) and mannose-1,3-α-D-mannose (M13), were used in molecular dynamics (MD) simulations to study the behavior of the carbohydrate-recognition domain (CRD) over time. Results showed an expanded domain within which hydrophobic interactions with the methyl group in the MDM molecule were established, thus revealing novel interactions for mannose-specific Dalbergieae lectins. To examine its biological activities, CML was purified in a single step by affinity chromatography on Sepharose-mannose matrix. The lectin demonstrated inflammatory response in the paw edema model and stimulated leukocyte migration to the animal peritoneal cavities, an effect elicited by CRD. For the first time, this work reports the molecular dynamics of a lectin from the Dalbergieae tribe.

Canavalia bonariensis lectin: Molecular bases of glycoconjugates interaction and antiglioma potential.

CaBo is a mannose/glucose-specific lectin purified from seeds of Canavalia bonariensis. In the present work, we report the CaBo crystal structure determined to atomic resolution in the presence of X-man, a specific ligand. Similar to the structural characteristics of other legume lectins, CaBo presented the jellyroll motif, a metal binding site occupied by calcium and manganese ions close to the carbohydrate-recognition domain (CRD). In vitro test of CaBo cytotoxicity against glioma cells demonstrated its ability to decrease the cellular viability and migration by induction of autophagy and cell death. Molecular docking simulations corroborate previous data indicating that the lectin's biological activities occur mostly through interactions with glycoproteins since the lectin interacted favorably with several N-glycans, especially those of the high-mannose type. Together, these results suggest that CaBo interacts with glycosylated cell targets and elicits a remarkable antiglioma activity.

Structural studies and nociceptive activity of a native lectin from Platypodium elegans seeds (nPELa).

A native lectin (nPELa), purified from seeds of the species Platypodium elegans, Dalbergieae tribe, was crystallized and structurally characterized by X-ray diffraction crystallography and bioinformatics tools. The obtained crystals diffracted to 1.6Å resolution, and nPELa structure were solved through molecular substitution. In addition, nPELa has a metal binding site and a conserved carbohydrate recognition domain (CRD) similar to other Dalbergieae tribe lectins, such as PAL (Pterocarpus angolensis) and CTL (Centrolobium tomentosum). Molecular docking analysis indicated high affinity of this lectin for different mannosides, mainly trimannosides, formed by α-1,3 or α-1,6 glycosidic bond, as evidenced by the obtained scores. In addition, molecular dynamics simulations were performed to demonstrate the structural behavior of nPELa in aqueous solution. In solution, nPELa was highly stable, and structural modifications in its carbohydrate recognition site allowed interaction between the lectin and the different ligands. Different modifications were observed during simulations for each one of the glycans, which included different hydrogen bonds and hydrophobic interactions through changes in the relevant residues. In addition, nPELa was evaluated for its nociceptive activity in mice and was reported to be the first lectin of the Dalbergieae tribe to show CRD-dependent hypernociceptive activity.

Structural analysis of Dioclea lasiocarpa lectin: A C6 cells apoptosis-inducing protein.

Lectins are multidomain proteins that specifically recognize various carbohydrates. The structural characterization of these molecules is crucial in understanding their function and activity in systems and organisms. Most cancer cells exhibit changes in glycosylation patterns, and lectins may be able to recognize these changes. In this work, Dioclea lasiocarpa seed lectin (DLL) was structurally characterized. The lectin presented a high degree of similarity with other lectins isolated from legumes, presenting a jelly roll motif and a metal-binding site stabilizing the carbohydrate-recognition domain. DLL demonstrated differential interactions with carbohydrates, depending on type of glycosidic linkage present in ligands. As observed by the reduction of cell viability in C6 cells, DLL showed strong antiglioma activity by mechanisms involving activation of caspase 3.

Crystal structure of Pisum arvense seed lectin (PAL) and characterization of its interaction with carbohydrates by molecular docking and dynamics.

The Pisum arvense lectin (PAL), a legume protein belonging to the Vicieae tribe, is capable of specific recognition of mannose, glucose and its derivatives without altering its structure. In this work, the three-dimensional structure of PAL was determined by X-ray crystallography and studied in detail by a combination of molecular docking and molecular dynamics (MD). Crystals belonging to monoclinic space group P21 were grown by the vapor diffusion method at 293 K. The structure was solved at 2.16 Å and was similar to that of other Vicieae lectins. The structure presented Rfactor and Rfree of 17.04% and 22.08%, respectively, with all acceptable geometric parameters. Molecular docking was performed to analyze interactions of the lectin with monosaccharides, disaccharides and high-mannose N-glycans. PAL demonstrated different affinities on carbohydrates, depending on bond orientation and glycosidic linkage present in ligands. Furthermore, the lectin interacted with representative N-glycans in a manner consistent with the biological effects described for Vicieae lectins. Carbohydrate-recognition domain (CRD) in-depth analysis was performed by MD, describing the behavior of CRD residues in complex with ligand, stability, flexibility of the protein over time, CRD volume and topology. This is a first report of its kind for a lectin of the Vicieae tribe.

Partial characterization and immobilization in CNBr-activated Sepharose of a native lectin from Platypodium elegans seeds (PELa) and comparative study of edematogenic effect with the recombinant form.

The lectin from Platypodium elegans seeds (PELa) was purified by affinity chromatography in a mannose-agarose column. The lectin agglutinated rabbit erythrocytes and the agglutinating effect was inhibited by previous incubation with the glycoprotein fetuin, along with N-acetyl-d-glucosamine, D-mannose and its derivatives. The lectin maintained complete activity in temperatures ranging from 40 to 60°C and pH values ranging from 9 to 10. As a glycoprotein, PELa has a carbohydrate content of 2.2%, and its activity requires divalent cations such as Ca2+ and Mn2+. Based on SDS-PAGE, PELa displays a profile similar to that of other Dalbergieae lectins with the main chain of molecular mass around 30kDa and two subunits of 19kDa and 10 kDa each. Two-dimensional (2D) electrophoresis revealed the presence of isoforms with different isoelectric points, and high-performance size exclusion chromatography (HPSEC) was performed to confirm the purity of the sample. The lectin was immobilized in CNBr-activated Sepharose 4B and successfully captured fetuin in solution, demonstrating that this lectin remains active and capable of binding carbohydrates. PELa showed effects different from those of its recombinant form in both pro- and anti-inflammatory tests.

Molecular modeling, docking and dynamics simulations of the Dioclea lasiophylla Mart. Ex Benth seed lectin: An edematogenic and hypernociceptive protein.

Lectins are proteins, or glycoproteins, capable of reversibly binding to specific mono- or oligosaccharides via a noncatalytic domain. The Diocleinae subtribe presents lectins with high structural similarity, but different effects based on biological activity assays. This variability results from small structural differences. Therefore, in this context, the present study aimed to perform a structural analysis of the lectin from Dioclea lasiophylla Mart. ex Benth seeds (DlyL) and evaluate its inflammatory effect. To accomplish this, DlyL was purified in a single step by affinity chromatography on Sephadex® G-50 matrix. DlyL primary structure was determined through a combination of tandem mass spectrometry and DNA sequencing. DlyL showed high similarity with other species from the same genus. Its theoretical three-dimensional structure was predicted by homology modelling, and the protein was subjected to ligand screening with monosaccharides, oligosaccharides and complex N-glycans by molecular docking. Stability and binding of the lectin with α-methyl-d-mannoside were assessed by molecular dynamics. DlyL showed acute inflammatory response with hypernociceptive effect in the paw edema model, possibly by interaction with glycans present at the cell surface.

Structural analysis of Centrolobium tomentosum seed lectin with inflammatory activity.

A glycosylated lectin (CTL) with specificity for mannose and glucose has been detected and purified from seeds of Centrolobium tomentosum, a legume plant from Dalbergieae tribe. It was isolated by mannose-sepharose affinity chromatography. The primary structure was determined by tandem mass spectrometry and consists of 245 amino acids, similar to other Dalbergieae lectins. CTL structures were solved from two crystal forms, a monoclinic and a tetragonal, diffracted at 2.25 and 1.9 Å, respectively. The carbohydrate recognition domain (CRD), metal-binding site and glycosylation site were characterized, and the structural basis for mannose/glucose-binding was elucidated. The lectin adopts the canonical dimeric organization of legume lectins. CTL showed acute inflammatory effect in paw edema model. The protein was subjected to ligand screening (dimannosides and trimannoside) by molecular docking, and interactions were compared with similar lectins possessing the same ligand specificity. This is the first crystal structure of mannose/glucose native seed lectin with proinflammatory activity isolated from the Centrolobium genus.

A novel vasorelaxant lectin purified from seeds of Clathrotropis nitida: partial characterization and immobilization in chitosan beads.

A novel lectin from seeds of Clathrotropis nitida (CNA) was purified and characterized. CNA is a glycoprotein containing approximately 3.3% carbohydrates in its structure. CNA promoted intense agglutination of rabbit erythrocytes, which was inhibited by galactosides and porcine stomach mucin (PSM). The lectin maintained its hemagglutinating activity after incubation in a wide range of temperatures (30-60 °C) and pH (6.0-7.0), and its binding activity was dependent on divalent cations (Ca(+2) and Mg(+2)). SDS-PAGE showed an electrophoretic profile consisting of a single band of 28 kDa, as confirmed by electrospray ionization mass spectrometry, which indicated an average molecular mass of 27,406 ± 2 Da and the possible presence of isoforms and glycoforms. In addition, CNA exhibited no toxicity to Artemia sp. nauplii and elicited reversible and dose-dependent vasorelaxation in precontracted aortic rings. CNA was successfully immobilized on chitosan beads and was able to capture PSM in solution. This study demonstrated that CNA is a lectin that has potential as a biotechnological tool in glycomics and glycoproteomics applications.