Structural insights and antimicrobial synergy of a proto-galectin from the marine sponge Aiolochroia crassa.

In this study, we isolated a novel lectin from the marine sponge Aiolochroia crassa, named AcrL. The lectin showed a preference for glycans containing sialic acid terminal residues, as indicated by the strongest inhibition with fetuin and bovine submaxillary mucin. Primary structure determination by mass spectrometry revealed that AcrL is a galectin with conserved amino acid residues typically involved in carbohydrate binding. Structural modeling indicated that AcrL adopts a typical galectin ß-sandwich motif, featuring two anti-parallel ß-sheets with five strands each. Docking calculations revealed a carbohydrate-binding site composed of a main site, capable of hosting galactopyranosides, and an extended site, facilitating the binding of complex carbohydrates. AcrL inhibited significant biofilm formation against Staphylococcus aureus, S. epidermidis, and Escherichia coli with concentrations ranging from 500 to 15.6 µg.mL-1 for S. aureus, 7.8 µg.mL-1 for S. epidermidis, and 500 µg.mL-1 for E. coli. Furthermore, when combined with different antibiotics, AcrL potentiated their effect against pathogenic bacteria. The antimicrobial mechanism of AcrL was investigated using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The analysis indicates that AcrL induces damage to the bacterial membrane. These findings underscore the discovery of a novel galectin in a basal organism and the comprehensive biochemical characterization conducted in this research, highlighting the potential of AcrL as a novel antibacterial agent and emphasizing its importance in combating bacterial infections.

Kinetics characterization of a low immunogenic recombinant l-asparaginase from Phaseolus vulgaris with cytotoxic activity against leukemia cells.

l-asparaginases play a crucial role in the treatment of acute lymphoblastic leukemia (ALL), a type of cancer that mostly affects children and teenagers. However, it is common for these molecules to cause adverse reactions during treatment. These downsides ignite the search for novel asparaginases to mitigate these problems. Thus, this work aimed to produce and characterize a recombinant asparaginase from Phaseolus vulgaris (Asp-P). In this study, Asp-P was expressed in Escherichia coli with high yields and optimum activity at 40 °C, pH 9.0. The enzyme Km and Vmax values were 7.05 mM and 1027 U/mg, respectively. Asp-P is specific for l-asparagine, showing no activity against l-glutamine and other amino acids. The enzyme showed a higher cytotoxic effect against Raji than K562 cell lines, but only at high concentrations. In silico analysis indicated that Asp-P has lower immunogenicity than a commercial enzyme. Asp-P induced biofilm formation by Candida sp. due to sublethal dose, showing an underexplored potential of asparaginases. The absence of glutaminase activity, lower immunogenicity and optimal activity similar to physiological temperature conditions are characteristics that indicate Asp-P as a potential new commercial enzyme in the treatment of ALL and its underexplored application in the treatment of other diseases.

Structural characterization of a galectin from the marine sponge Aplysina lactuca (ALL) with synergistic effects when associated with antibiotics against bacteria.

Lectins presents the ability to interact with glycans and trigger varied responses, including the inhibition of the development of various pathogens. Structural studies of these proteins are essential to better understand their functions. In marine sponges, so far only a few lectins have their primary structures completely determined. Thus, the objective of this work was to structurally characterize and evaluate antibacterial potential, in association with different antibiotics, of the lectin isolated from the marine sponge Aplysina lactuta (ALL). ALL is a homotetramer of 60 kDa formed by four 15 kDa-subunits. The lectin showed affinity only for the glycoproteins fetuin, asialofetuin, mucin type III, and bovine submaxillary mucin type I. The complete amino acid sequences of two isoforms of ALL, named ALL-a and ALL-b, were determined by a combination of Edman degradation and overlapped peptides sequenced by tandem mass spectrometry. ALL-a and ALL-b have 144 amino acids with molecular masses of 15,736 Da and 15,985 Da, respectively. Both structures contain conserved residues typical of the galectin family. ALL is a protein with antibacterial potential, when in association with ampicillin and oxacillin the lectin potentiates its antibiotic effect, included Methicillin-resistant Staphylococcus strains. Thus, ALL shows to be a molecule with potential for the development of new antibacterial drugs.

Structural study and antimicrobial and wound healing effects of lectin from Solieria filiformis (Kützing) P.W.Gabrielson.

The SfL-1 isoform from the marine red algae Solieria filiformis was produced in recombinant form (rSfL-1) and showed hemagglutinating activity and inhibition similar to native SfL. The analysis of circular dichroism revealed the predominance of ß-strands structures with spectra of ßI-proteins for both lectins, which had Melting Temperature (Tm) between 41 °C and 53 °C. The three-dimensional structure of the rSfL-1 was determined by X-ray crystallography, revealing that it is composed of two ß-barrel domains formed by five antiparallel ß chains linked by a short peptide between the ß-barrels. SfL and rSfL-1 were able to agglutinate strains of Escherichia coli and Staphylococcus aureus and did not show antibacterial activity. However, SfL induced a reduction in E. coli biomass at concentrations from 250 to 125 µg mL-1, whereas rSfL-1 induced reduction in all concentrations tested. Additionally, rSfL-1 at concentrations from 250 to 62.5 µg mL-1, showed a statistically significant reduction in the number of colony-forming units, which was not noticed for SfL. Wound healing assay showed that the treatments with SfL and rSfL-1 act in reducing the inflammatory response and in the activation and proliferation of fibroblasts by a larger and fast deposition of collagen.

A fibrinogen-related Lectin from Echinometra lucunter represents a new FReP family in Echinodermata phylum.

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 Ca2+ presence, which was inhibited by glycoprotein mucin and thyroglobulin. Complete amino acid sequence consisted of 229 residues, including a FReD in the N-terminal. Searches for similarity found that ELL was very close to putative proteins from Strongylocentrotus purpuratus. ELL showed moderate similarity with uncharacterized sea stars proteins and protochordate intelectins. ELL was able to inhibit the planktonic growth of the Gram-positive bacteria and significantly reduce the biofilm formation of all bacteria tested. In conclusion, we identified a new type of FReP-containing lectin with some structural and functional conservation towards intelectins.

Codium isthmocladum lectin 1 (CiL-1): Interaction with N-glycans explains antinociceptive and anti-inflammatory activities in adult zebrafish (Danio rerio).

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.

Structural characterization of a galectin isolated from the marine sponge Chondrilla caribensis with leishmanicidal potential.

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 ß-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.

Antihyperglycemic and antioxidant activities of a lectin from the marine red algae, Bryothamnion seaforthii, in rats with streptozotocin-induced diabetes.

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 ß-cells resistance (HOMA-ß). 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-ß confirmed that BSL treatment significantly decreased insulin resistance and ß-cell hypersecretion, respectively. In conclusion, BSL treatment might exert hypoglycemic and hypolipidemic effects, diminish insulin resistance, and ameliorate pancreatic ß-cell function along with enzymatic activities toward oxidative stress caused by diabetes mellitus type 2 (T2DM).

A new mucin-binding lectin from the marine sponge Aplysina fulva (AFL) exhibits antibiofilm effects.

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 ±â€¯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 ß-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.

Structural characterization of two isolectins from the marine red alga Solieria filiformis (Kützing) P.W. Gabrielson and their anticancer effect on MCF-7 breast cancer cells.

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 ß-barrel-like domains formed by five antiparallel ß-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.

Antibacterial activity of a new lectin isolated from the marine sponge Chondrilla caribensis.

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% ß-sheet/ß-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.

Purification, Biochemical Characterization, and Amino Acid Sequence of a Novel Type of Lectin from Aplysia dactylomela Eggs with Antibacterial/Antibiofilm Potential.

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 ß-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 ß-sheet, while domain B presents a flat three-stranded and a curved four-stranded ß-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.

Isolation, biochemical characterization and antibiofilm effect of a lectin from the marine sponge Aplysina lactuca.

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, ß-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 ß-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.

L-Rhamnose-binding lectin from eggs of the Echinometra lucunter: Amino acid sequence and molecular modeling.

An L-rhamnose-binding lectin named ELEL was isolated from eggs of the rock boring sea urchin Echinometra lucunter by affinity chromatography on lactosyl-agarose. ELEL is a homodimer linked by a disulfide bond with subunits of 11 kDa each. The new lectin was inhibited by saccharides possessing the same configuration of hydroxyl groups at C-2 and C-4, such as L-rhamnose, melibiose, galactose and lactose. The amino acid sequence of ELEL was determined by tandem mass spectrometry. The ELEL subunit has 103 amino acids, including nine cysteine residues involved in four conserved intrachain disulfide bonds and one interchain disulfide bond. The full sequence of ELEL presents conserved motifs commonly found in rhamnose-binding lectins, including YGR, DPC and KYL. A three-dimensional model of ELEL was created, and molecular docking revealed favorable binding energies for interactions between ELEL and rhamnose, melibiose and Gb3 (Galα1-4Galß1-4Glcß1-Cer). Furthermore, ELEL was able to agglutinate Gram-positive bacterial cells, suggesting its ability to recognize pathogens.

HGA-2, a novel galactoside-binding lectin from the sea cucumber Holothuria grisea binds to bacterial cells.

A novel lectin, HGA-2, was isolated from the sea cucumber Holothuria grisea. The protein was isolated by a single chromatographic step using a column of Guar Gum as affinity. HGA-2 showed an apparent molecular mass of 17 kDa and 34 kDa under reducing and nonreducing conditions, respectively. The hemagglutinating activity was specific for rabbit erythrocytes, showing no activity for human blood A, B and O. Its hemagglutinating activity was inhibited by carbohydrates containing galactose, with higher affinity for GalNAc and glycoprotein porcine stomach mucin (PSM). HGA-2 was stable at pH 6-10, significantly declining at pH 5 and a temperature of 40°C, with its activity being abolished at 100 °C. The HGA-2 protein was found to be Ca(2+)-dependent; it was highly toxic against Artemia nauplii and able to recognize and agglutinate cells of Escherichia coli. Amino acid sequences of tryptic peptides of HGA-2 strongly suggest that HGA-2 is a member of the C-type lectin family.

Halilectin 1 (H-1) and Halilectin 2 (H-2): two new lectins isolated from the marine sponge Haliclona caerulea.

Two new lectins named Halilectin 1 (H-1) and Halilectin 2 (H-2) were isolated from the marine sponge Haliclona caerulea using a combination of affinity chromatography on stroma fixed onto Sephadex G-25 and cation and anion exchange chromatography. H-1 is a monomeric protein with a molecular mass of 40 kDa estimated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and 15 kDa estimated using a TSK gel. Conversely, H-2 is a homodimeric protein with 15 kDa monomers linked via weak interactions. H-1 more effectively agglutinates trypsinized rabbit erythrocytes, whereas H-2 more effectively agglutinates native rabbit erythrocytes. The hemagglutinating activity of H-1 could be not inhibited by any tested sugars, but H-2 was inhibited by orosomucoid and porcine stomach mucin. Neither lectin was dependent on divalent ions. H-1 was stable at basic pH range and temperatures up to 50 °C, whereas H-2 was stable at acid pH range and temperatures up to 80 °C. The H. caerulea lectins exhibited dose-dependent toxicity against Artemia nauplii. Additionally, 76% of the primary structure of H-2 was determined using tandem mass spectrometry to contain a unique amino acid sequence with no similarity to any members of the animal lectin family.

Toxicity and binding profile of lectins from the Genus canavalia on brine shrimp.

Lectins are sugar-binding proteins widely distributed in nature with many biological functions. Although many lectins have a remarkable biotechnological potential, some of them can be cytotoxic. Thus, the aim of this study was to assess the toxicity of five lectins, purified from seeds of different species of Canavalia genus. In order to determine the toxicity, assays with Artemia nauplii were performed. In addition, a fluorescence assay was carried out to evaluate the binding of lectins to Artemia nauplii. In order to verify the relationship between the structure of lectins and their cytotoxic effect, structural analysis was carried out to evaluate the volume of the carbohydrate recognition domain (CRD) of each lectin. The results showed that all lectins exhibited different toxicities and bound to a similar area in the digestive tract of Artemia nauplii. Concerning the structural analysis, differences in spatial arrangement and volume of CRD may explain the variation of the toxicity exhibited by each lectin. To this date, this is the first study that establishes a link between toxicity and structure of CRD from Diocleinae lectins.

Characterization of isoforms of the lectin isolated from the red algae Bryothamnion seaforthii and its pro-healing effect.

Lectins are a structurally heterogeneous group of proteins that have specific binding sites for carbohydrates and glycoconjugates. Because of their biotechnological potential, lectins are widely used in biomedical research. The present study aimed to evaluate the healing potential of the lectin isolated from the marine red alga Bryothamnion seaforthii (BSL). The lectin was purified using ion exchange chromatography with DEAE cellulose and characterized using tandem mass spectrometry. For healing tests, skin wounds were induced in the dorsal thoracic region of mice. These animals were randomly divided into three groups and subjected to topical treatment for 12 days with BSL, bovine serum albumin and 150 mM NaCl. To evaluate the potential of each treatment, the animals were anesthetized and sacrificed on days 2, 7 and 12, respectively. The parameters evaluated included the wound area, the proportion of wound closure and the histological diagnosis. The wound closure was more effective with BSL (Postoperative Day 7 and 12) than controls. The luminal epithelium was completely restructured; the presence of collagen in the dermis and the strongly active presence of young skin annexes demonstrate the potential of treatment with BSL compared with controls. Our findings suggest that BSL has pro-healing properties and can be a potential medical process in the treatment of acute wounds.