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background: Protease inhibitors (PIs) have attracted attention due to their important roles in plant defense. Objective:: The objective of this work was to characterize and evaluate the antimicrobial activity of the peptides of a family of serine PIs from Capsicum chinense Jacq. seeds. Method: Initially, PIs were extracted from the seeds and subjected to purification by chromatography, resulting in three different peptide enriched fractions (PEFs) termed PEF1, PEF2 and PEF3. Subsequently, the PEF3 was subjected to trypsin inhibition assays, α-amylase activity assays, antimicrobial activity assays on phytopathogenic fungi, and assays to determine the likely mechanisms of action. Result: The PEF3 was composed of three protein bands with molecular masses ranging between 6 and 14 kDa. The amino acid residues of the ~6 kDa band showed high similarity with serine PIs. PEF3 inhibited the activity of the enzymes trypsin, human salivary α-amylase, and Tenebrio molitor larval α-amylase and inhibited the growth of phytopathogenic fungi, showing 83.7% loss of viability in Fusarium oxysporum. PEF3 induced reactive oxygen species in Colletotrichum lindemuthianum and F. oxysporum to dissipate their mitochondrial membrane potential and activated caspases in C. lindemuthianum. Conclusion: Our results reinforce the importance of PIs in plant defense mechanisms against phytopathogenic fungi as well as in their biotechnological applications for the control of plant pathogens.

Fibrinogen-related proteins (FREPs) have been identified in several animals. They are involved in the body's defense, acting as mediators of phagocytosis. Ficolins and intelectins are some of the most studied Fibrinogen-related Domain (FReD)-containing lectins. In this work, we have isolated a singular FReD-containing lectin, which cannot be classified as ficolin or intelectin. ELL (Echinometra lucunter lectin) was isolated from coelomic plasma by affinity chromatography on xanthan gum. Primary structure was determined by tandem mass spectrometry. Moreover, antimicrobial activity of ELL was evaluated against planktonic cells and biofilm of Escherichia coli, Staphylococcus aureus and S. epidermidis. ELL showed hemagglutinating activity in Ca

Inflammation and oxidative stress are processes associated with different human diseases. They are treated using drugs that have several side effects. Seaweed are sources of potentially relevant natural compounds for use as treatment of these disorders. Lectins are able to reversibly interact with complex carbohydrates and modulate cell membrane glycosylated receptors through this interaction. This study aimed to determine the antinociceptive and anti-inflammatory potential of CiL-1 in adult zebrafish by modulation of TRPA1 through lectin-glycan binding. Possible neuromodulation by TRPA1 channel was also evaluated by camphor pretreatment. CiL-1 was efficacious at all tested doses, revealing anti-nociceptive and anti-inflammatory effects in adult zebrafish. This galactose-binding lectin was also able to reduce the content of ROS in brain and liver. In silico analyses showed CiL-1 interactions with both ligands tested. LacNac2 presents the most favorable binding energy with the protein. The interaction occurs at 4 subsites as an extended conformation at the site. LacNac2-Sia had a less favorable curved-shape interaction energy. Based on the predictions made for the oligosaccharides, a tetra-antenate putative glycan was schematically constructed, illustrating an interaction between TRPA1 N-glycan and CiL-1. This binding seems to be related to CiL-1 anti-inflammatory activity as result of receptor modulation.

Plant fungal diseases cause major problems for the global economy. Antimicrobial peptides have aroused great interest in the control of phytopathogens, as they are natural molecules and have a broad spectrum of inhibitory activity. Herein, we have tried to identify and characterize antimicrobial peptides present in fruits of Capsicum chinense and to evaluate their enzymatic and antifungal activities. The retained fraction obtained in the anion exchange chromatography with strong antifungal activity was subjected to molecular exclusion chromatography and obtained four fractions named G1, G2, G3, and G4. The 6.0-kDa protein band of G2 showed similarity with protease inhibitors type II, and it was able to inhibit 100% of trypsin and α-amylase activities. The protein band with approximately 6.5 kDa of G3 showed similarity with sequences of protease inhibitors from genus Capsicum and showed growth inhibition of 48% for Colletotrichum lindemuthianum, 49% for Fusarium lateritium, and 51% for F. solani and F. oxysporum. Additionally, G3 causes morphological changes, membrane permeabilization, and ROS increase in F. oxysporum cells. The 9-kDa protein band of G4 fraction was similar to a nsLTP type 1, and a protein band of 6.5 kDa was similar to a nsLTP type 2. The G4 fraction was able to inhibit 100% of the activities of glycosidases tested and showed growth inhibition of 35 and 50% of F. oxysporum and C. lindemuthianum, respectively. C. chinense fruits have peptides with antifungal activity and enzyme inhibition with biotechnological potential.

H2O2 priming reprograms essential proteins’ expression to help plants survive, promoting responsive and unresponsive proteins adjustment to salt stress. Priming is a powerful strategy to enhance abiotic stress tolerance in plants. Despite this, there is scarce information about the mechanisms induced by H2O2 priming for salt stress tolerance, particularly on proteome modulation. Improving maize cultivation in areas subjected to salinity is imperative for the local economy and food security. Thereby, this study aimed to investigate physiological changes linked with post-translational protein events induced by foliar H2O2 priming of Zea mays plants under salt stress. As expected, salt treatment promoted a considerable accumulation of Na+ ions, a 12-fold increase. It drastically affected growth parameters and relative water content, as well as promoted adverse alteration in the proteome profile, when compared to the absence of salt conditions. Conversely, H2O2 priming was beneficial via specific proteome reprogramming, which promoted better response to salinity by 16% reduction in Na+ content and shoots growth improvement, increasing 61% in dry mass. The identified proteins were associated with photosynthesis and redox homeostasis, critical metabolic pathways for helping plants survive in saline stress by the protection of chloroplasts organization and carbon fixation, as well as state redox. This research provides new proteomic data to improve understanding and forward identifying biotechnological strategies to promote salt stress tolerance.

Diseases caused by phytopathogenic microorganisms are difficult to control and can affect plants at different stages of their development. Several resistance genes and antimicrobial peptides (AMPs) have been identified and related to the resistance process of Capsicum. In recent years, studies have shown that peppers, especially the accession UENF1381, present resistance against phytopathogenic microorganisms. This work aimed identify and characterize AMPs of the leaf and root from Capsicum annuum L. UENF1381 and to analyze the inhibitory activity of the AMPs on different enzyme families and valuated the inhibitory activity on Colletotrichum scovillei. Initially, self-fertilized pepper seeds were cultured for 45 days in glass flasks containing ½-MS medium. Then, leaves were inoculated with inoculum of Xanthomonas euvesicatoria (108 CFU mL−1) or water (control). Leaf and root samples were collected at 12, 24 and 48 h after inoculation to extraction. The activity of extracts on different enzyme families was analyzed by incubating the enzymes (trypsin, α-amylase and β-1,3-glucanase) with 30 μg mL−1 of the obtained extracts. The effect of extracts on fungi was also tested. It was identified that protein of leaf and root extracts from C. annuum L. UENF1381 presented a majority of bands with a low molecular mass (6–14 kDa). We observed that all leaf and root extracts significantly inhibited trypsin and α-amylase activity and able to significantly inhibit C. scovillei growth. With this work we hope to contribute to the use of peptides as potential molecules in microbial control and in the development of new Capsicum cultivars resistant to microorganisms.

Structure-function study of plant lectins has received attention for elucidating possible mechanisms of lectins in cell models. Lectins have the ability to interact with target glycans and trigger responses able to inhibit the development of various pathogens. In this work, we report amino acid sequencing, prediction of the three-dimensional structure of P. pendula lectin (PpeL) and the effect of this lectin on inhibiting the development of Leishmania infantum promastigote. PpeL was purified on affinity chromatography and has haemagglutinating activity after exposure to temperatures up to 50 °C and at a pH corresponding to the range of 5.0 and 7.0. PpeL consists of 447 amino acids; the theoretical average molecular mass was 47,410 Da. The three-dimensional model of PpeL revealed that it consists of three β-prism domains tandemly arranged with each one presenting a different carbohydrate recognition domain (CRD). PpeL inhibited growth of L. infantum promastigotes (45.6 ± 1.92 %) within 48 h, an effect that occurred via the CRD, suggesting an interaction between PpeL and glycans from L. infantum. Our results confirm the leishmanicidal potential of PpeL, suggesting that this lectin may be a new strategy for the development of antileishmanial drugs.

A chitosanase (CvCsn46) from Chromobacterium violaceum ATCC 12472 was produced in Escherichia coli, purified, and partially characterized. When subjected to denaturing polyacrylamide gel electrophoresis, the enzyme migrated as two protein bands (38 and 36 kDa apparent molecular masses), which were both identified as CvCsn46 by mass spectrometry. The enzyme hydrolyzed colloidal chitosan, with optimum catalytic activity at 50 °C, and two optimum pH values (at pH 6.0 and pH 11.0). The chitosanolytic activity of CvCsn46 was enhanced by some ions (Ca2+, Co2+, Cu2+, Sr2+, Mn2+) and DTT, whereas Fe2+, SDS and β-mercaptoethanol completely inhibited its activity. CvCsn46 showed a non-Michaelis-Menten kinetics, characterized by a sigmoidal velocity curve (R2 = 0.9927) and a Hill coefficient of 3.95. ESI-MS analysis revealed that the hydrolytic action of CvCsn46 on colloidal chitosan generated a mixture of low molecular mass chitooligosaccharides, containing from 2 to 7 hexose residues, as well as D-glucosamine. The chitosan oligomers generated by CvCsn46 inhibited in vitro the mycelial growth of Lasiodiplodia theobromae, significantly reducing mycelium extension and inducing hyphal morphological alterations, as observed by scanning electron microscopy. CvCsn46 was characterized as a versatile biocatalyst that produces well-defined chitooligosaccharides, which have potential to control fungi that cause important crop diseases.

Two new lectins from the green alga Codium isthmocladum (CiL-1 and CiL-2) were isolated. Both lectins could agglutinate human and rabbit erythrocytes. Galactosides and fetuin showed inhibitory effect on CiL-1. CiL-2 was inhibited by GalNAc and porcine stomach mucin. CiL-1 was a monomeric protein of 12 kDa, whereas CiL-2 showed 12 kDa in SDS-PAGE and an oligomeric state in gel filtration. MALDI-ToF-MS of CiL-1 revealed a molecular mass of 12.027 ± 5 Da, while CiL-2 showed molecular mass of 12.264 ± 5 Da. Ninety-eight percent of CiL-1’s primary structure was determined consisting of 112 residues placed in two repeated domains with approximately 60% of similarity. CiL-1 showed similarity with hypothetical proteins from aquatic pathogenic fungi. The N-terminal of CiL-2 showed no similarity to CiL-1 or to any known protein. The three-dimensional model of CiL-1 consists of four two-strand β-sheets disposed in a barrel-like arrangement, connected by loops of variable sizes, with a well-structured hydrophobic core. Binding site prediction suggests the existence of two independent monosaccharide binding sites in CiL-1. The lectins showed no antibacterial activity on Gram-positive and Gram-negative bacteria, but they were able to significantly inhibit the biofilm formation from Staphylococcus aureus and Staphylococcus epidermidis.

The modern pig industry relies on extensive use of artificial insemination with cooled semen. It is important that semen doses maintain their quality during processing, transport and storage before insemination to guarantee maximum fertility rates. However, ejaculates may respond differently to liquid preservation at 17 °C, despite the optimal quality assessed before cooling. Thus, the aim of this study was to identify differences in seminal plasma proteome of ejaculates with a higher or lower seminal resistance to storage at 17 °C. A total of 148 ejaculates from 65 sexually mature healthy boars were classified as: High Resistance to cooling (HR, total motility 60% at 144h) and Low resistance to cooling (LR, total motility60 at 72h). To identify differentially expressed seminal plasma proteins between HR and LR ejaculates, ten ejaculates of each group were analyzed by 2D SDS-PAGE and ESI-Q-TOF mass spectrometry. The proteins associated with HR ejaculates were cathepsin B (spot 2803 and 6601, p 0.01); spermadhesin PSP-I (spots 3101 and 3103, p 0.05); epididymal secretory protein E1 precursor (spot 2101, p 0.05) and IgGFc binding protein (spot 1603, p 0.01). The protein associated with LR group was the Major seminal plasma PSPI (spot 9103, p 0.01). To our knowledge, this is the first report of the association of boar seminal plasma proteins to semen resistance to cold storage at 17 °C. These results suggest the use of these proteins as biomarkers for semen resistance to preservation at 17 °C.

Background Solving primary structure of lectins leads to an understanding of the physiological roles within an organism and its biotechnological potential. Only eight sponge lectins have had their primary structure fully determined. Methods The primary structure of CCL, Chondrilla caribensis lectin, was determined by tandem mass spectrometry. The three-dimensional structure was predicted and the protein-carbohydrate interaction analysed by molecular docking. Furthermore, the anti-leishmanial activity was observed by assays with Leishmania infantum. Results The amino acid sequence consists of 142 amino acids with a calculated molecular mass of 15,443 Da. The lectin has a galectin-like domain architecture. As observed in other sponge galectins, the signature sequence of a highly conserved domain was also identified in CCL with some modifications. CCL exhibits a typical galectin structure consisting of a β-sandwich. Molecular docking showed that the amino acids interacting with CCL ligands at the monosaccharide binding site are mostly the same as those conserved in this family of lectins. Through its interaction with L. infantum glycans, CCL was able to inhibit the development of this parasite. CCL also induced apoptosis after eliciting ROS production and altering the membrane integrity of Leishmania infantum promastigote. Conclusions CCL joins the restricted group of sponge lectins with determined primary structure and very high biotechnological potential owing to its promising results against pathogens that cause Leishmaniasis. General significance As the determination of primary structure is important for biological studies, now CCL can become a sponge galectin with an exciting future in the field of human health.

A new mucin-binding lectin (AFL) was isolated from the marine sponge Aplysina fulva. AFL was purified by affinity chromatography on Sepharose™ matrix. Its hemagglutinating activity was independent of divalent ions, and it was weakly inhibited by simple sugars. However, porcine stomach mucin was a powerful inhibitor. In SDS PAGE, piridylethylated AFL showed one band of approximately 16 kDa, whereas in the non-reducing conditions, AFL showed at least two bands of 30 and 70 kDa. Mass spectrometry MALDI-ToF analysis showed one major ion of 31,652 ± 5 Da, which corresponded to a dimer formed by subunits linked by disulfide bonds. The first fifteen amino acids of AFL were determined, and no sequence similarity was observed with any known protein. Internal sequences were obtained by mass spectrometry analysis of tryptic digestion of AFL spots. These peptides showed similarity with a lectin from marine sponge Aplysina lactuca. Secondary structure of AFL was predominantly formed by β-conformations, which were stable at variations of pH and temperature. AFL did not inhibit planktonic growth of Gram-positive and Gram-negative bacteria tested. However, the lectin did significantly reduce the biomass biofilm of the bacteria Staphylococcus aureus, S. epidermidis, and Escherichia coli.

Brazil has one of the largest biodiversities in the world. The search for new natural products extracted from the Brazilian flora may lead to the discovery of novel drugs with potential to treat infectious and other diseases. Here, we have investigated 9 lectins extracted and purified from the Northeastern Brazilian flora, from both leguminous species: Canavalia brasiliensis (ConBr), C. maritima (ConM), Dioclea lasiocarpa (DLasiL) and D. sclerocarpa (DSclerL), and algae Amansia multifida (AML), Bryothamniom seaforthii (BSL), Hypnea musciformis (HML), Meristiella echinocarpa (MEL) and Solieria filiformis (SfL). They were exposed to a panel of 18 different viruses, including HIV and influenza viruses. Several lectins showed highly potent antiviral activity, often within the low nanomolar range. DSclerL and DLasiL exhibited EC50 values (effective concentration of lectin required to inhibit virus-induced cytopathicity by 50%) of 9 nM to 46 nM for HIV-1 and respiratory syncytial virus (RSV), respectively, DLasiL also inhibited feline corona virus at an EC50 of 5 nM, and DSclerL, ConBr and ConM showed remarkably low EC50 values ranging from 0.4 to 6 nM against influenza A virus strain H3N2 and influenza B virus. For HIV, evidence pointed to the blockage of entry of the virus into its target cells as the underlying mechanism of antiviral action of these lectins. Overall, the most promising lectins based on their EC50 values were DLasiL, DSclerL, ConBr, ConM, SfL and HML. These novel findings indicate that lectins from the Brazilian flora may provide novel antiviral compounds with therapeutic potential. ispartof: MEDCHEMCOMM vol:10 issue:3 pages:390-398 ispartof: location:England status: published

Iguana iguana is a lizard found in South America, and its metabolism is directly related to ambient heat. However, the physiology underlying the effect of temperature change on the metabolism of these reptiles has not been fully elucidated. I. iguana hemoglobin (iHb) was isolated and purified by exclusion chromatography. Mass spectrometry, circular dichroism, SAXS and spectroscopic characterization were used to evaluate the primary and secondary structures and oligomeric state of iHb. The secondary structure of intact iHb, which mainly consisted of α-helices, was not stable if heated at 60 °C. Autoxidation rates of iHb were measured at 30 and 40 °C, revealing a suggestive bi-exponential behavior. Furthermore, by lowering the pH from 7.4 to 6.0, an increase in autoxidation rate was observed, indicating a behavior responsive to metabolic changes. Compared to ATP, ADP showed an allosteric effect on iHb oxygenation by lowering its oxygen binding. Alignment of the amino acid sequences revealed that iHb exhibits conserved substitution of a residue at the allosteric site, suggesting that some unknown molecular mechanism accounts for the allosteric effect caused by ADP. The ability of iHB to oxygenate is directly related to metabolic changes, ensuring the survival of Iguana iguana in different environments.

Antimicrobial peptides (AMPs) have been reported to be promising alternatives to chemical preservatives. Thus, this study aimed to characterise AMPs generated from the hydrolysis of wheat gluten proteins using latex peptidases of Calotropis procera, Cryptostegia grandiflora, and Carica papaya. The three hydrolysates (obtained after 16 h at 37 °C, using a 1: 25 enzyme: substrate ratio) inhibited the growth of Aspergillus niger, A. chevalieri, Trichoderma reesei, Pythium oligandrum, Penicillium sp., and Lasiodiplodia sp. by 60-90%, and delayed fungal growth on bread by 3 days when used at 0.3 g/kg. Moreover, the specific volume and expansion factor of bread were not affected by the hydrolysates. Of 28 peptides identified, four were synthesised and exhibited activity against Penicillium sp. Fluorescence and scanning electron microscopy suggested that the peptides damaged the fungal plasma membrane. Bioinformatics analysis showed that no peptide was toxic and that the antigenic ones had cleavage sites for trypsin or pepsin.

HPriming is a powerful strategy to enhance abiotic stress tolerance in plants. Despite this, there is scarce information about the mechanisms induced by H

Made available in DSpace on 2021-06-25T11:23:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-04-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (FUNCAP) The aim of this study was to characterize the protein profile of ovarian follicular fluid (FF) of brown brocket deer (Mazama gouazoubira). Five adult females received an ovarian stimulation treatment and the FF was collected by laparoscopy from small/medium (3.5 mm) follicles. Concentrations of soluble proteins in FF samples were measured and proteins were analyzed by 1-D SDS-PAGE followed by tryptic digestion and tandem mass spectrometry. Data from protein list defined after a Mascot database search were analyzed using the STRAP software tool. For the protein concentration, no significant difference (P> 0.05) was observed between small/medium and large follicles: 49.2 +/- 22.8 and 56.7 +/- 27.4 mu g/mu l, respectively. Mass spectrometry analysis identified 13 major proteins, but with no significant difference (P> 0.05) between follicle size class. This study provides insight into elucidating folliculogenesis in brown brocket deer. Univ Estadual Ceara, Fac Vet, Av Dr Silas Munguba 1700, BR-60714903 Fortaleza, Ceara, Brazil Univ Fed Ceara, Dept Anim Sci, Fortaleza, Ceara, Brazil Univ Estadual Ceara, Hlth Sci Ctr, Fortaleza, Ceara, Brazil Univ Fed Ceara, Lab Mass Spectrometry, Fortaleza, Ceara, Brazil State Univ Sao Paulo, Dept Anim Sci, Jaboticabal, Brazil Univ Ctr Fametro, Mol Genet Res Unit, Fortaleza, Ceara, Brazil State Univ Sao Paulo, Dept Anim Sci, Jaboticabal, Brazil CNPq: 461330/2014-8 (FUNCAP): CI3-0093-0005580100/14

The aim of the study was to assess the antihyperglycemic, antilipidemic, and antioxidant effects of a lectin isolated from Bryothamnion seaforthii (BSL), on rats with streptozotocin (STZ)-induced diabetes. The disease model was induced by low-dose injections of STZ. Diabetic rats were treated with NaCl 150 mM, metformin, and BSL at different concentrations. Blood collection was carried out at 0, 30, 60, 90, and 120 days after hyperglycemia confirmation via the assessment of seric glucose, total cholesterol, and triglycerides, assessment of the enzymatic levels of glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD), and the determination of insulin resistance by a homeostasis model of assessment-insulin resistance (HOMA-IR) as well as a homeostasis model of assessment of β-cells resistance (HOMA-β). The BSL-treated animals at all three concentrations showed a significant reduction in levels of glucose, cholesterol, total cholesterol, and triglycerides. Moreover, BSL increased the enzymatic activity of GPx and SOD. Index assessments of HOMA-IR and HOMA-β confirmed that BSL treatment significantly decreased insulin resistance and β-cell hypersecretion, respectively. In conclusion, BSL treatment might exert hypoglycemic and hypolipidemic effects, diminish insulin resistance, and ameliorate pancreatic β-cell function along with enzymatic activities toward oxidative stress caused by diabetes mellitus type 2 (T2DM).

A type II lectin, designated as DlyL2, was purified from Dioclea lasiophylla Mart. ex Benth seeds and some of its physicochemical properties determined. The lectin demonstrated specificity for α-lactose and N-acetyl- d -galactosamine and was able to interact with porcine stomach mucin. DlyL2 has 0.78 % carbohydrates in its composition, therefore can be considered a glycoprotein. In addition, its hemagglutinating activity remained stable at a temperature of 90 °C and in a pH range from 5 to 10. Metal chelation treatment did not affect DlyL2 activity suggesting it's not a metalloprotein. Dlyl2 showed an apparent mass of 31 kDa and average molecular mass of 26.371 kDa. Primary structure data could be generated from the partial amino acid sequence of DlyL2, with about 192 residues sequenced by a combination of Edman degradation and tandem mass spectrometry. The lectin is similar to other type II lectins from Diocleinae subtribe, as well as lectins derived from species of more ancient tribes of the Fabaceae family. In addition, DlyL2 exhibited no toxicity to Artemia sp. nauplii. The present study expands the knowledge about the specificity, structural and physicochemical properties of Diocleinae type II lectins.

Background: An ideal strategy for cancer treatment is the specific induction of tumor cell death, sparing normal cells. Marine sponges are rich biological reservoirs of biomolecules, especially lectins, which have attracted considerable attention due to potential biological effect on human cells. Lectins are proteins that bind specific carbohydrate signatures and some gained further interest for their capacity to bind tumor associated carbohydrates antigens and induce tumor cell apoptosis. Objective: This study aimed to evaluate the antitumor potential of H3, a lectin, recently reported from marine sponge Haliclona caerulea on the human breast cancer cell line MCF7. Results: H3 reduced MCF7 cell viability with an IC50 of 100 µg/ml, without a significant effect on normal cells. At 24 h, H3 induced a significant arrest in the G1 cell cycle phase. Consistently, almost 50% of the cells were in early apoptosis and showed remarkable increased expression of caspase-9 (CASP 9). H3 impaired dramatically the adhesiveness of MCF7 cells in culture. Assays conducted with Lysotracker Red probe showed increased organelle acidity, suggesting autophagic cell death, which was further supported by increased expression of microtubuleassociated protein light chain 3 (LC3) and observable conversion of LC3-I in LC3-II by western blot. Conclusion: The apoptotic effect of H3 may be related to a balance between apoptotic and autophagic cell death, mediated by increased expression of CASP 9 and LC3-II. To the best of our knowledge this is the first report about a sponge lectin triggering both apoptosis and autophagy in MCF7 cell.

A new lectin from the marine red alga Meristiella echinocarpa (MEL) was isolated and biochemically characterized. MEL is a monomeric protein of 28 kDa with specificity for yeast mannan. Hemagglutination activity of MEL was stable between pH 5 and 10, temperatures up to 50 °C, and neither EDTA nor divalent ions affected it. The complete amino acid sequence of MEL was determined through a combination of tandem mass spectrometry and DNA cloning. As a new member of the OAAH-lectin family, the primary structure of MEL consists of 267 amino acid residues distributed in four tandem repeat domains, sharing at least 48% of identity. Theoretical secondary structure of MEL was composed of 3% α-helix, 40% β-sheet, 19% β-turn, and 38% coil. Melting temperatures of the lectin in the absence and presence of mannan were 54 and 61 °C, respectively. Furthermore, MEL was able to recognize and agglutinate pathogenic bacterial strains, such as multidrug-resistant Salmonella and Vibrio alginolyticus.

This study was conducted to assess the effect of lipid supplementation on the proteomic profile of milk fat globule membrane (MFGM) and mammary gland tissue (MGT) in lactating goats. Thirty-eight crossbred goats received a total mixed ration (TMR) based on chopped elephant grass and concentrate during 35 days post-partum. They were divided into three diet groups two weeks after kidding, namely, Control group (CG; n = 12), fed baseline TMR; Flaxseed group (FG; n = 13), fed ground flaxseed added to the TMR for 21 days (12 % total dry matter); and microalgae Chlorella group (MCG; n = 13), fed microalgae Chlorella added to the TMR for 21 days (0.05 % live weight). Blood plasma concentrations of total protein, albumin, and glutathione peroxidase were determined. Weekly milk samples were used to estimate milk yield and protein and fat levels. MGT samples were biopsied on the 35th day of the experiment. The protein profile of MFGM and MGT was assessed using 1D SDS–PAGE and mass spectrometry. The MCG group showed lower dry matter intake and higher milk fat than the other diets. The milk protein level was not affected by the diet group. The 22 protein bands identified in MFGM showed no difference between diets. The multivariate model selected eight protein bands (VIP > 1) to segregate the diet groups. Nine out of 44 protein bands identified in MGT showed lower expression in the FG group, which had a lipid level of the diet of 8 %. The multivariate model involved 11 band proteins (VIP > 1) in the diet group segregation. Thus, the increased lipid level in the diet or milk of lactating goats had no marked effects on the MFGM proteome. High lipid diet interfered with the expression of proteins in the mammary glands, which points to the greater sensitivity of this organ to the effort of lipotoxicity.

The three-dimensional structure of Dioclea reflexa seed lectin (DrfL) was studied in detail by a combination of X-ray crystallography, molecular docking and molecular dynamics. DrfL was purified by affinity chromatography using Sephadex G-50 matrix. Its primary structure was obtained by mass spectrometry, and crystals belonging to orthorhombic space group P2(1)2(1)2(1) were grown by the vapor diffusion method at 293 K. The crystal structure was solved at 1.765 A and was very similar to that of other lectins from the same subtribe. The structure presented R-factor and R-free of 21.69% and 24.89%, respectively, with no residues in nonallowed regions of Ramachandran plot. Similar to other Diocleinae lectins, DrfL was capable of relaxing aortic rings via NO induction, with CRD participation, albeit with low intensity (32%). In silica analysis results demonstrated that DrfL could strongly interact with complex N-glycans, components of blood vessel glycoconjugates. Despite the high similarity among Diocleinae lectins, it was also reported that each lectin has unique CRD properties that influence carbohydrate binding, resulting in different biological effects presented by these molecules. (C) 2017 Elsevier B.V. All rights reserved.

A new lectin from Aplysia dactylomela eggs (ADEL) was isolated by affinity chromatography on HCl-activated Sepharose™ media. Hemagglutination caused by ADEL was inhibited by several galactosides, mainly galacturonic acid (Ka = 6.05 × 106 M−1). The primary structure of ADEL consists of 217 residues, including 11 half-cystines involved in five intrachain and one interchain disulfide bond, resulting in a molecular mass of 57,228 ± 2 Da, as determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. ADEL showed high similarity with lectins isolated from Aplysia eggs, but not with other known lectins, indicating that these lectins could be grouped into a new family of animal lectins. Three glycosylation sites were found in its polypeptide backbone. Data from peptide-N-glycosidase F digestion and MS suggest that all oligosaccharides attached to ADEL are high in mannose. The secondary structure of ADEL is predominantly β-sheet, and its tertiary structure is sensitive to the presence of ligands, as observed by CD. A 3D structure model of ADEL was created and shows two domains connected by a short loop. Domain A is composed of a flat three-stranded and a curved five-stranded β-sheet, while domain B presents a flat three-stranded and a curved four-stranded β-sheet. Molecular docking revealed favorable binding energies for interactions between lectin and galacturonic acid, lactose, galactosamine, and galactose. Moreover, ADEL was able to agglutinate and inhibit biofilm formation of Staphylococcus aureus, suggesting that this lectin may be a potential alternative to conventional use of antimicrobial agents in the treatment of infections caused by Staphylococcal biofilms.

Infections caused by Candida tropicalis have increased significantly worldwide in parallel with resistance to antifungal drugs. To overcome resistance novel drugs have to be discovered. The objective of this work was to purify and characterize a cysteine protease inhibitor from the seeds of the Amazon rainforest tree Cassia leiandra and test its inhibitory effect against C. tropicalis growth. The inhibitor, named ClCPI, was purified after ion exchange and affinity chromatography followed by ultrafiltration. ClCPI is composed of a single polypeptide chain and is not a glycoprotein. The molecular mass determined by SDS-PAGE in the absence or presence of β-mercaptoethanol and ESI-MS were 16.63 kDa and 18.362 kDa, respectively. ClCPI was stable in the pH range of 7.0–9.0 and thermostable up to 60 °C for 20 min. ClCPI inhibited cysteine proteases, but not trypsin, chymotrypsin neither alpha-amylase. Inhibition of papain was uncompetitive with a Ki of 4.1 × 10−7 M and IC50 of 8.5 × 10−7 M. ClCPI at 2.6 × 10−6 M reduced 50% C. tropicalis growth. ClCPI induced damages and morphological alterations in C. tropicalis cell surface, which led to death. These results suggest that ClCPI have great potential for the development of an antifungal drug against C. tropicalis.

A new mannose/N-acetyl- d glucosamine-specific lectin, named MaL, was purified from seeds of Machaerium acutifolium by precipitation with ammonium sulfate, followed by affinity and ion-exchange chromatography. MaL haemagglutinates either native rabbit erythrocytes or those treated with proteolytic enzymes. MaL is highly stable by the ability to maintain its haemagglutinating activity after exposure to temperatures up to 50 °C. The lectin haemagglutinating activity was optimum between pH 6.0 and 7.0 and inhibited after incubation with d -mannose and N-acetyl- d -glucosamine and α-methyl- d -mannopyranoside. MaL is a glycoprotein with relative molecular mass of 29 kDa (α-chain), 13 kDa (β-chain) and 8 kDa (γ-chain) with secondary structure composed of 3% α-helix, 44% β-sheet, 21% β-turn, and 32% coil. The orofacial antinociceptive activity of the lectin was also evaluated. MaL (0.03 mg mL−1) reduced orofacial nociception induced by capsaicin, an effect that occurred via carbohydrate recognition domain interaction, suggesting an interaction of MaL with the transient receptor potential cation channel subfamily V member 1 (TRPV1) receptor. Our results confirm the potential pharmacological relevance of MaL as an inhibitor of acute orofacial mediated by TRPV1.

A partial cDNA sequence from Anacardium occidentale CCP 76 was obtained, encoding a GH19 chitinase (AoChi) belonging to class VI. AoChi exhibits distinct structural features in relation to previously characterized plant GH19 chitinases from classes I, II, IV and VII. For example, a conserved Glu residue at the catalytic center of typical GH19 chitinases, which acts as the proton donor during catalysis, is replaced by a Lys residue in AoChi. To verify if AoChi is a genuine chitinase or is a chitinase-like protein that has lost its ability to degrade chitin and inhibit the growth of fungal pathogens, the recombinant protein was expressed in Pichia pastoris, purified and biochemically characterized. Purified AoChi (45 kDa apparent molecular mass) was able to degrade colloidal chitin, with optimum activity at pH 6.0 and at temperatures from 30 °C to 50 °C. AoChi activity was completely lost when the protein was heated at 70 °C for 1 h or incubated at pH values of 2.0 or 10.0. Several cation ions (Al3+, Cd2+, Ca2+, Pb2+, Cu2+, Fe3+, Mn2+, Rb+, Zn2+ and Hg2+), chelating (EDTA) and reducing agents (DTT, β-mercaptoethanol) and the denaturant SDS, drastically reduced AoChi enzymatic activity. AoChi chitinase activity fitted the classical Michaelis-Menten kinetics, although turnover number and catalytic efficiency were much lower in comparison to typical GH19 plant chitinases. Moreover, AoChi inhibited in vitro the mycelial growth of Lasiodiplodia theobromae, causing several alterations in hyphae morphology. Molecular docking of a chito-oligosaccharide in the substrate-binding cleft of AoChi revealed that the Lys residue (theoretical pKa = 6.01) that replaces the catalytic Glu could act as the proton donor during catalysis.

Volatile organic compounds (VOCs) from the red seaweeds Pterocladiella capillacea and Osmundaria obtusiloba were obtained by static headspace extraction and then analyzed qualitatively by gas chromatography mass spectrometry (GC-MS) and quantitatively by gas chromatography (GC) equipped with a flame ionization detector (FID). In total, 31 constituents were identified in the two species of algae. In P. capillacea, among the 21 constituents (100%), the major ones were hexanal (50.4%), 2-pentylfuran (9.2%), and heptadecene (8.8%). In O. obtusiloba, of the 21 constituents (100%), the most representatives were heptadecene (57.3%), hexanal (20.5%), and 1-pentadecene (2.6%). This is the first report on the identification and quantification of VOCs in the Rhodophyta, P. capillacea and O. obtusiloba.

The identification of biomarkers associated with seminal traits could aid in the selection of higher quality ejaculates and benefit the swine industry. The objective of this study was to identify boar seminal plasma proteins associated with sperm motility and morphology. Twenty ejaculates from fifteen adult boars from a commercial boar stud were used for this work. After routine semen collection and analysis, ejaculates were classified into two groups: high-quality semen (HQS) and low-quality semen (LQS), based on sperm motility and morphology. Semen samples were processed for seminal plasma separation and analysis by 2D SDS-PAGE. Total and progressive sperm motility differed between groups (p < 0.001), as well sperm morphology (p < 0.05). The intensity of spots identified as Major seminal plasma PSP-I (PSP-I) and cathepsin B (CTSB) was higher in LQS as compared to HQS samples (p < 0.05). Also, PSP-I was positively associated with major and sperm cauda defects. Sperm motility was negatively correlated with both PSP-I and cathepsin B. We conclude that high concentrations of Major seminal plasma PSP-I and cathepsin B in boar seminal plasma are associated with reduced total and progressive sperm motility and low sperm morphology and might be used as biomarkers for semen quality.

Vicilins are 7S globulins which constitute the major seed storage proteins in leguminous species. Variant vicilins showing differential binding affinities for chitin have been implicated in the resistance and susceptibility of cowpea to the bruchid Callosobruchus maculatus . These proteins are members of the cupin superfamily, which includes a wide variety of enzymes and non-catalytic seed storage proteins. The cupin fold does not share similarity with any known chitin-biding domain. Therefore, it is poorly understood how these storage proteins bind to chitin. In this work, partial cDNA sequences encoding β-vignin, the major component of cowpea vicilins, were obtained from developing seeds. Three-dimensional molecular models of β-vignin showed the characteristic cupin fold and computational simulations revealed that each vicilin trimer contained 3 chitin-binding sites. Interaction models showed that chito-oligosaccharides bound to β-vignin were stabilized mainly by hydrogen bonds, a common structural feature of typical carbohydrate-binding proteins. Furthermore, many of the residues involved in the chitin-binding sites of β-vignin are conserved in other 7S globulins. These results support previous experimental evidences on the ability of vicilin-like proteins from cowpea and other leguminous species to bind in vitro to chitin as well as in vivo to chitinous structures of larval C. maculatus midgut.

A novel lectin present in Dioclea reflexa seeds (DrfL) was discovered and described in this study. DrfL was purified in a single step by affinity chromatography in a Sephadex G-50 column. The lectin strongly agglutinated rabbit erythrocytes and was inhibited by α-methyl-D-mannoside, D-mannose, and D-glucose. The hemagglutinating activity of DrfL is optimum at pH 5.0-7.0, stable up to 50 °C, and dependent on divalent cations. Similar to other lectins of the subtribe Diocleinae, the analysis by mass spectrometry indicated that DrfL has three chains (α, β, and γ) with masses of 25,562, 12,874, and 12,706 Da, respectively, with no disulfide bonds or glycosylation. DrfL showed inflammatory activity in the paw edema model and exhibited low cytotoxicity against Artemia sp.

Phase equilibrium of aqueous two-phase systems (ATPS) containing triblock copolymer, L64, (EO)13(PO)30(EO)13 and sulfate salts were investigated in this work. It was analyzed the effect of the temperature at 278.15 K to 298.15 K and type of salt. The rise of the temperature increased the slope of the tie line (STL). The sodium sulfate showed better capability to induce phase separation.

In this study, the amino acid sequence and anti-inflammatory effect of Bauhinia bauhinioides (BBL) lectin were evaluated. Tandem mass spectrometry revealed that BBL possesses 86 amino acid residues. BBL (1 mg/kg) intravenously injected in rats 30 min prior to inflammatory stimuli inhibited the cellular edema induced by carrageenan in only the second phase (21% – 3 h, 19% – 4 h) and did not alter the osmotic edema induced by dextran. BBL also inhibited carrageenan peritoneal neutrophil migration (51%), leukocyte rolling (58%) and adhesion (68%) and the neutrophil migration induced by TNF-α (64%). These effects were reversed by the association of BBL with galactose, demonstrating that the carbohydrate-binding domain is essential for lectin activity. In addition, BBL reduced myeloperoxidase activity (84%) and TNF-α (68%) and IL1-β (47%) levels. In conclusion, the present investigation demonstrated that BBL contains highly homologous isolectins, resulting in a total of 86 amino acid residues, and exhibits anti-inflammatory activity by inhibiting neutrophil migration by reducing TNF-α and IL1-β levels via the lectin domain. Copyright © 2015 John Wiley & Sons, Ltd.

A new chromophore-containing agglutinin (Haliclona manglaris agglutinin (HMA)) was isolated from the tropical sponge H. manglaris. HMA was purified by a combination of hydrophobic interaction chromatography and ion exchange chromatography. Native HMA is a heterotrimer formed by two β-chains (15kDa) and one α-chain (22kDa). HMA is a glycoprotein and possesses three intrachain disulfide bonds. Hemagglutinating activity of HMA was stable at neutral pH and temperatures up to 60°C. HMA was only inhibited by thyroglobulin. Mass spectrometry sequencing and Edman degradation revealed a unique amino acid sequence of about 30%. Moreover, HMA has an organic chromophore of 581Da, and this characteristic seems to be important to its antioxidant activity. Interestingly, while HMA showed no toxicity against Artemia nauplii and was unable to agglutinate bacterial cells, it did show a high capacity to protect β-carotene against oxidation. Thus, our findings suggest the putative involvement of HMA in the protection of the sponge against oxidation.

The lectin DLasiL was isolated from seeds of the Dioclea lasiocarpa collected from the northeast coast of Brazil and characterized for the first time by mass spectrometry, DNA sequencing, inductively coupled plasma-mass spectrometry, electron paramagnetic resonance, and fluorescence spectroscopy. The structure of DLasiL lectin obtained by homology modelling suggested strong conservation of the dinuclear Ca/Mn and sugar-binding sites, and dependence of the solvent accessibility of tryptophan-88 on the oligomerisation state of the protein. DLasiL showed highly potent (low nanomolar) antiproliferative activity against several human carcinoma cell lines including A2780 (ovarian), A549 (lung), MCF-7 (breast) and PC3 (prostate), and was as, or more, potent than the lectins ConBr (Canavalia brasiliensis), ConM (Canavalia maritima) and DSclerL (Dioclea sclerocarpa) against A2780 and PC3 cells. Interestingly, DLasiL lectin caused a G2/M arrest in A2780 cells after 24 h exposure, activating caspase 9 and delaying the on-set of apoptosis. Confocal microscopy showed that fluorescently-labelled DLasiL localized around the nuclei of A2780 cells at lectin doses of 0.5–2 × IC50 and gave rise to enlarged nuclei and spreading of the cells at high doses. These data reveal the interesting antiproliferative activity of DLasiL lectin, and suggest that further investigations to explore the potential of DLasiL as a new anticancer agent are warranted.\ud

A new lectin from the marine sponge Chondrilla caribensis (CCL) was isolated by affinity chromatography in Sepharose 6B media. CCL is a homotetrameric protein formed by subunits of 15,445 ±2Da. The lectin showed affinity for disaccharides containing galactose and mucin. Mass spectrometric analysis revealed about 50% of amino acid sequence of CCL, which showed similarity with a lectin isolated from Aplysina lactuca. Secondary structure consisted of 10% α-helix, 74% β-sheet/β-turn and 16% coil, and this profile was unaltered in a broad range of pH and temperatures. CCL agglutinated Staphylococcus aureus, S epidermidis and Escherichia coli, and it was able to reduce biofilm biomass, but showed no inhibition of planktonic growth of these bacteria. CCL activity was inhibited by α-lactose, indicating that Carbohydrate Recognition Domain (CRD) of the lectin was involved in antibiofilm activity.

As described in the literature, Solieria filiformis lectin (SfL) from the marine red alga S. filiformis was found to have antinociceptive and anti-inflammatory effects. In this study, we characterized two SfL variants, SfL-1 and SfL-2, with molecular mass of 27,552Da and 27,985Da, respectively. The primary structures of SfL-1 and SfL-2 consist of four tandem-repeat protein domains with 67 amino acids each. SfL-1 and -2 showed high similarity to OAAH-family lectins. 3D structure prediction revealed that SfL-1 and -2 are composed of two β-barrel-like domains formed by five antiparallel β-strands, which are connected by a short peptide linker. Furthermore, the mixture of isoforms (SfLs) showed anticancer effect against MCF-7 cells. Specifically, SfLs inhibited 50% of viability in MCF-7 cells after treatment at 125μg.mL-1, while the inhibition of Human Dermal Fibroblasts (HDF) was 34% with the same treatment. Finally, 24h after treatment, 25% of MCF-7 cells were in early apoptosis and 35% in late apoptosis. Evaluation of pro- and anti-apoptotic gene expression of MCF-7 cells revealed that SfLs induced caspase-dependent apoptosis within 24h.

This study was conducted to evaluate the effects of different feeding levels on the proteome of oviduct and uterus tissues of hormonally stimulated goats during the periovulatory period. Forty goats were separated into four different diet groups: Diet 1.0 M (n = 11), Diet 1.3 M (n = 10), Diet 1.6 M (n = 9), Diet 1.9 M (n = 10), fed with 1.0, 1.3, 1.6 and 1.9 times live weight maintenance, respectively. After four weeks of treatment, six hormonally stimulated females per treatment group were randomly selected for collection of uterine and the oviduct tissue samples. Samples were collected after animals were slaughtered in a commercial unit. Feeding goats with 1.3 to 1.9 times more nutrients than a control group directly influenced the proteome of the oviduct and uterus, altering the expression of proteins that participate in biological processes such as apoptosis, antioxidant, and immunological activities. These events are crucial for fertilization and early embryonic survival. Expression of oviduct proteins such as Tubulin Beta 2B, Transferrin and Disulphide-isomerase A3 increased in the 1.9 M group in relation to the other feeding levels. Disulphide-isomerase A4 showed higher expression in the 1.0 M group compared to diets with higher energetic levels. As energy intake increased in the diets, there was higher expression of Alpha-1-antitrypsin and downregulation of Profilin-1 in the uterus of the goats. In conclusion, this study showed that specific proteins of the goat oviduct and uterus expressed during the periovulatory period are modified as the result of nutritional balance.

A new lectin was isolated from the marine sponge Aplysina lactuca (ALL) by combining ammonium sulfate precipitation and affinity chromatography on guar gum matrix. ALL showed affinity for the disaccharides α-lactose, β-lactose and lactulose (Ka=12.5, 31.9 and 145.5M-1, respectively), as well as the glycoprotein porcine stomach mucin. Its hemagglutinating activity was stable in neutral acid pH values and temperatures below 60°C. ALL is a dimeric protein formed by two covalently linked polypeptide chains. The average molecular mass, as determined by Electrospray Ionization Mass Spectrometry (ESI-MS), was 31,810±2Da. ESI-MS data also indicated the presence of three cysteines involved in one intrachain and one interchain disulfide bond. The partial amino acid sequence of ALL was determined by tandem mass spectrometry. Eight tryptic peptides presented similarity with lectin I isolated from Axinella polypoides. Its secondary structure is predominantly β-sheet, as indicated by circular dichroism (CD) spectroscopy. ALL agglutinated gram-positive and gram-negative bacterial cells, and it were able to significantly reduce the biomass of the bacterial biofilm tested at dose- dependent effect.

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.

Natural antioxidants found in marine macroalgae are bioactive compounds known to play an important role in the prevention of diseases associated with aging cells protecting them against the oxidative damage. The purpose of this study was to evaluate the antioxidant and cytotoxic activity of ethanolic extracts of two species of red seaweeds, Amansia multifida and Meristiella echinocarpa. In vitro antioxidant activity was determined by DPPH radical scavenging assay, ferric-reducing antioxidant power (FRAP) assay, ferrous ion chelating (FIC) assay, β-carotene bleaching (BCB) assay and total phenolic content (TPC) quantification. Cytotoxicity was evaluated with the brine shrimp Artemia sp. lethality test. The TPC values observed in the present study indicated that both species A. multifida and M. echinocarpa are rich in phenolic compounds, reaching values of 45.40 and 28.46 mg gallic acid equivalent (GAE) g−1 of ethanolic extract, respectively. DPPH radical scavenging and ferrous ion chelating showed values of 60% and 17%, respectively. Both seaweed extracts inhibited β-carotene oxidation by approximately 40%. None of the algal extracts were potentially cytotoxic. The results have showed that extracts of both species of marine red algae exhibit antioxidant potential and low toxicity. They are sources of natural antioxidant compounds. Antioxidantes naturais encontrados em macroalgas marinhas são substâncias bioativas conhecidas por desempenhar um papel importante na prevenção de doenças associadas com o envelhecimento, protegendo as células de danos oxidativos. O objetivo deste estudo foi avaliar as atividades antioxidante e citotóxica de extratos etanólicos de duas espécies de algas vermelhas, Amansia multifida e Meristiella echinocarpa. A atividade antioxidante in vitro foi determinada pela capacidade de sequestro do radical DPPH, o poder de redução do ferro (FRAP), o poder de quelação do íon ferroso (FIC), o teste de oxidação acoplada β-caroteno/ácido linoleico (BCB), bem como a quantificação do conteúdo fenólico total. A citotoxicidade foi avaliada pelo teste de letalidade contra Artemia sp. Os valores das substâncias fenólicas observados no presente estudo indicaram que as espécies A. multifida e M. echinocarpa são ricas em substâncias fenólicas, atingindo valores de 45,40 e de 28,86 mg de ácido gálico equivalente (AGE) g−1 de extrato etanólico, respectivamente. A capacidade de seqüestro do radical DPPH e o poder de quelação do íon ferroso foram 60% e 17%, respectivamente. Ambos os extratos algáceos inibiram a oxidação do β-caroteno em torno de 40%. Nenhum dos extratos algáceos foi potencialmente citotóxico. Os resultados mostraram que os extratos destas espécies de rodófitas apresentam potencial antioxidante e baixa toxicidade. Elas são fontes de compostos antioxidantes naturais.

A novel mannose/glucose-binding lectin from Canavalia virosa (designated as ConV) has been purified from seeds of C. virosa by affinity chromatography on a mannose-Sepharose 4B column. ConV strongly agglutinates rabbit erythrocytes and was inhibited by monosaccharides (D-mannose, D-glucose, and α-methyl-D-mannoside) and glycoproteins (ovalbumin and fetuin). SDS-PAGE revealed three bands corresponding to three subunits (α, β, and γ) confirmed by ESI mass spectrometry with exact mass of 25,480 ± 2 Da, 12,864 ± 1 Da, and 12,633 ± 1 Da, respectively. The purified lectin was more stable in pH ranging from 7.0 to 9.0, supported up to 80 ºC without any loss in activity and unaffected by EDTA. ConV showed no toxicity against Artemia sp. nauplii and relaxed endothelized rat aorta, with the participation of the lectin domain. In our tests, the lectin immobilized on CNBr-Sepharose was capable of binding 0.8 mg of ovalbumin per chromatography, allowing the use of ConV as a tool for capture and purification of glycoproteins.

Lectins are comprised of a large family of proteins capable of the specific and reversible recognition of carbohydrates. Legume lectins, the most studied plant lectins, show high structural similarity, but with modifications that imply a variation in the intensity of some biological activities. In this work, the primary and tertiary structures of Canavalia grandiflora (ConGF) were determined. ConGF, a lectin isolated from C. grandiflora seeds, is able to induce relaxant activity in rat aortic rings. The complete sequence of ConGF comprises 237 amino acids. This particular protein has primary sequence variations commonly found in lectins from Dioclea and Canavalia genera. The protein structure was solved at 2.3 Å resolution by X-ray crystallography. An X-Man molecule was modeled into the carbohydrate recognition domain. Still, ConGF (30 and 100 μg mL(-1)) elicited 25% of vasorelaxation (IC50=34.48 ± 5.07 μg mL(-1)) in endothelialized aortic rings. A nonselective inhibitor of nitric oxide blocked ConGF relaxant effect, showing mediation by nitric oxide. Key distances between ConGF carbohydrate recognition domain residues were determined in order to explain this effect, in turn revealing some structural aspects that could differentiate lectins from the Canavalia genera with respect to different efficacy in vasorelaxant effect.

Recent studies have shown that lectins are promising tools for use in various biotechnological processes, as well as studies of various pathological mechanisms, isolation, and characterization of glycoconjugates and understanding the mechanisms underlying pathological mechanisms conditions, including the inflammatory response. This study aimed to purify, characterize physicochemically, and predict the biological activity of Canavalia oxyphylla lectin (CoxyL) in vitro and in vivo. CoxyL was purified by a single-step affinity chromatography in Sephadex® G-50 column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the pure lectin consists of a major band of 30 kDa (α-chain) and two minor components (β-chain and γ-chain) of 16 and 13 kDa, respectively. These data were further confirmed by electrospray ionization mass spectrometry, suggesting that CoxyL is a typical ConA-like lectin. In comparison with the average molecular mass of α-chain, the partial amino acid sequence obtained corresponds to approximately 45% of the total CoxyL sequence. CoxyL presented hemagglutinating activity that was specifically inhibited by monosaccharides (D-glucose, D-mannose, and α-methyl-D-mannoside) and glycoproteins (ovalbumin and fetuin). Moreover, CoxyL was shown to be thermostable, exhibiting full hemagglutinating activity up to 60°C, and it was pH-sensitive for 1 h, exhibiting maximal activity at pH 7.0. CoxyL caused toxicity to Artemia nauplii and induced paw edema in rats. This biological activity highlights the importance of lectins as important tools to better understand the mechanisms underlying inflammatory responses.

A new lectin from the marine sponge Haliclona caerulea (H-3) was isolated using a combination of hydrophobic interaction chromatography and ion-exchange chromatography. H-3 is a protein with three distinct bands on SDS-PAGE: 9 kDa, 16 kDa and 18 kDa. Nevertheless, on gel filtration and N-PAGE, H-3 showed a symmetrical peak and a unique band, respectively. Hemagglutinating activity of H-3 was stable at neutral pH and temperatures up to 60 °C. N-Acetylgalactosamine and porcine stomach mucin were the most potent inhibitors of H-3. Primary structure of the lectin was determined using tandem mass spectrometry, and it showed no similarity to any members of the animal lectin families. Top down fragmentation revealed some posttranslational modifications in H-3, including glycosylation. The glycan composition of H-3 was determined, and its structure was predicted. Furthermore, H-3 is a blue protein, binding to a chromophore(-597) by weak interactions, and this is the first time that the interaction between one lectin and a natural chromophore has been shown.