Cratylia Mollis Seeds Research Articles

Fluorescent nanoprobes based on quantum dots conjugated to Cramoll to assess surface carbohydrates of Aeromonas spp.

The association of quantum dots (QDs) to carbohydrate-binding proteins – lectins – has revealed novel biotechnological strategies for glycobiology studies. Herein, carboxyl-coated QDs were conjugated by adsorption to Cramoll, a glucose/mannose lectin obtained from Cratylia mollis seeds. Then, the conjugates were optically characterized and used to evaluate the surface carbohydrate profiles of four Aeromonas species isolated from the tambaqui fish (Colossoma macropomum). All the Aeromonas cells were labeled by the conjugate. Inhibition assays with methyl-α-D-mannopyranoside and mannan were performed to confirm the labeling specificity. Cramoll-QDs conjugates presented high brightness and showed similar absorption and emission profiles compared to bare QDs. According to the labeling pattern of Aeromonas spp. by the conjugate, results suggested that A. jandaei and A. dhakensis strains may harbor a higher content of more complex glucose/mannose surface glycans, with more available sites for Cramoll-QDs interaction, than A. hydrophila and A. caviae. Noteworthy, the Cramoll-QDs conjugates demonstrated to be potential tools for bacterial characterization based on superficial carbohydrate detection.

Purification of a lectin from Cratylia mollis crude extract seed by a single step PEG/phosphate aqueous two-phase system

The partitioning and purification of lectins from the crude extract of Cratylia mollis seeds (Cramoll 1,4) was investigated in aqueous two-phase systems (ATPS). A factorial design model (24) was used to evaluate the influence of polyethylene glycol (PEG) molar mass (1500–8000 g/mol), PEG concentration (12.5–17.5% w/w), phosphate (10–15% w/w) concentration, and pH (6–8) on the differential partitioning, purification factor, and yield of the lectin. Polymer and salt concentration were the most important variables affecting partition of lectin and used to find optimum purification factor by experimental Box–Behnken design together with the response surface methodology (RSM). ATPS showed best conditions composed by 13.9% PEG1500, 15.3% phosphate buffer at pH 6, which ensured purification factor of 4.70. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis showed a single band of protein with 26.1 kDa. Furthermore, results demonstrated a thermostable lectin presenting activity until 60 °C and lost hemagglutinating activity at 80 °C. According to the obtained data it can be inferred that the ATPS optimization using RSM approach can be applied for recovery and purification of lectins.

Titanium dioxide nanotubes functionalized with Cratylia mollis seed lectin, Cramoll, enhanced osteoblast-like cells adhesion and proliferation

An alternative to accelerate the osseointegration on titanium dioxide nanotubes (TNTs) used in osseointegrated implants is through the functionalization of these nanostructured surfaces with biomolecules. In this work, we immobilized a lectin with recognized mitogenic activity, the Cramoll lectin, extracted from Cratylia mollis seeds, on surfaces modified by TNTs. For the immobilization of Cramoll on TNTs surfaces, we used the Layer-by-Layer technique (LbL) by growing five alternate layers of poly(allylamine) hydrochloride (PAH) and poly(acrylic) acid (PAA); lastly we incubated the lectin, at different concentrations, with the TNTs-LbL. Before and after the immobilization procedures, the substrate surfaces were characterized by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), and, electrochemical impedance spectroscopy (EIS). We also evaluated the Cramoll activity after immobilization on TNTs by using the lectin interaction with ovalbumin. The lectin did not lose its biological activity, even after immobilization onto nanotubular arrays. In addition, we observed an increase osteoblast-like cell adhesion on the TNTs-LbL-Cramoll system when compared to the bare TNTs surfaces. Moreover, a significative cell proliferation was identified on the substrates when Cramoll was immobilized at concentrations of 80, 160 and 320 μg/mL after 48 h of incubation by using the resazurin assay. Our results suggest that Cramoll was efficiently immobilized on a nanotubular array and this new platform presents a great potential to be tested in implantology.

Treatment with pCramoll Alone and in Combination with Fluconazole Provides Therapeutic Benefits in C. gattii Infected Mice.

Cryptococcus gattii is one of the main causative agents of cryptococcosis in immunocompetent individuals. Treatment of the infection is based on the use of antimycotics, however, the toxicity of these drugs and the increase of drug-resistant strains have driven the search for more effective and less toxic therapies for cryptococcosis. pCramoll are isolectins purified from seeds of Cratylia mollis, a native forage plant from Brazil, which has become a versatile tool for biomedical application. We evaluated the effect of pCramoll alone and in combination with fluconazole for the treatment of mice infected with C. gatti. pCramoll alone or in combination with fluconazole increased the survival, reduced the morbidity and improved mice behavior i.e., neuropsychiatric state, motor behavior, autonomic function, muscle tone and strength and reflex/sensory function. These results were associated with (i) decreased pulmonary and cerebral fungal burden and (ii) increased inflammatory infiltrate and modulatory of IFNγ, IL-6, IL-10, and IL-17A cytokines in mice treated with pCramoll. Indeed, bone marrow-derived macrophages pulsed with pCramoll had increased ability to engulf C. gattii, with an enhanced production of reactive oxygen species and decrease of intracellular fungal proliferation. These findings point toward the use of pCramoll in combination with fluconazole as a viable, alternative therapy for cryptococcosis management.

Encapsulation into Stealth Liposomes Enhances the Antitumor Action of Recombinant Cratylia mollis Lectin Expressed in Escherichia coli.

This study evaluated the in vivo antitumor potential of the recombinant lectin from seeds of Cratylia mollis (rCramoll) expressed in Escherichia coli, free or encapsulated in stealth liposomes, using mice transplanted with sarcoma 180. rCramoll-loaded stealth liposomes (rCramoll-lipo) were formulated by hydration of the lipid film followed by cycles of freezing and thawing, and about 60% of rCramoll was encapsulated. This novel preparation showed particle size, polydispersity index, and pH suitable for the evaluation of antitumor activity in vivo. Tumor growth inhibition rates were 59% for rCramoll and 75% for rCramoll-lipo. Histopathological analysis of the experimental groups showed that both free and encapsulated lectin caused no changes in the kidneys of animals. Hematological analysis revealed that treatment with rCramoll-lipo significantly increased leukocyte concentration when compared with the untreated and rCramoll group. In conclusion, the encapsulation of rCramoll in stealth liposomes improves its antitumor activity without substantial toxicity; this approach was more successful than the previous results reported for pCramoll loaded into conventional liposomes. At this point, a crucial difference between the antitumor action of free and encapsulated rCramoll was found along with their effects on immune cells. Further investigations are required to elucidate the mechanism(s) of the antitumor effect induced by rCramoll.

Immunomodulatory effects of pCramoll and rCramoll on peritoneal exudate cells (PECs) infected and non-infected with Staphylococcus aureus

Peritoneal exudate cells (PECs) play important roles in host defense against Staphylococcus aureus and other pathogens. In this study we evaluated the potentials of native (pCramoll or Cramoll 1,4) and recombinant (rCramoll) lectins from seeds of Cratylia mollis as immunomodulatory tools on mice PECs infected and non-infected with S. aureus. Both lectins significantly enhanced nitric oxide, superoxide and cytokines (IL-1β, IL-6, IFN-γ and TNF-α). pCramoll and rCramoll downregulated the induction of TNF-α and IL-6 and upregulated the expression of IL-1β, IFN-γ in S. aureus infected PECs. Phagocytic activity of S. aureus was also enhanced in 27.1% and 22.47% by pCramoll and rCramoll, respectively. Our results showed that pCramoll induced stronger effects than rCramoll, which could be explained by the different hemagglutinating activities of C. mollis isolectins and nature fragmentation, although the biologic meaning should be studied in detail using in vivo models. Future works will be focused on the molecular mechanisms involved in these actions, using in vitro and in vivo models, to support the use of these lectins as biotechnological tool in immunological studies.

Aqueous two-phase systems: new strategies for separation and purification of lectin from crude extract of Cratylia mollis seeds

Plant lectins are reversible carbohydrate-binding proteins (or glycoproteins) of non-immune origin that can agglutinate cells or precipitate polysaccharides and glycoconjugates. A polymer/salt aqueous two-phase system (ATPS) based on polyethylene glycol (PEG)/sodium citrate was used for the extraction of lectins from a crude extract (CE) of Cratylia mollis seeds in 24 and 22 previously experimental designs. The effects of pH (4, 5, 6 and 7), PEG molar mass (1500, 4000 and 8000g/mol), PEG concentration (15, 18.5 and 22% w/w) and citrate concentration (10, 15, 20 and 22% w/w) were analysed to establish the optimal extraction conditions. The optimal performance system consisted of 20% (w/w) citrate and 22% (w/w) PEG 8000g/mol at pH 5.0 using 20% (w/w) crude extract, which allowed for a 125% activity yield of lectins in the bottom phase, with a purification factor of 13.28. A direct comparison of chromatography and ATPS processes for the recovery of lectin Cramoll 1,4 from the CE revealed a reduction in the number of unit operations (from 10 to 4). An electrophoretic study revealed three bands with molar masses of 30, 16 and 14kDa, consistent with what has been previously observed for Cramoll 1,4. After purification using ATPS, the lectin was stable until 60°C, with a decrease in hemagglutinating activity (512–128) at 80°C. These results indicate that liquid–liquid extraction is an adequate tool for separating and purifying lectins from the CE of C. mollis seeds while maintaining their biological activity, which may have great potential for biotechnological applications.

<i>In Vitro</i> Evaluation of Gamma Irradiation on a Gel Formulation of Cratylia Mollis: Rheological Proporties and Microbiological Control

Lectin Cramoll-1,4, obtained from Cratylia mollis seeds (beans camaratu) was structurally characterized, biologically and pharmacologically, but its use as a biopharmaceutical is not well documented. The objective of this study is to propose a biopharmaceutical formulation lectin Cramoll-1,4, test their hemagglutinating properties in vitro as well as the use of gamma radiation as a continuous process of decontamination formulation. It was made of the extraction and purification Cramoll-1,4, was developed a gel formulation using Carbopol? as a vehicle, at concentrations of 50, 100, 200, 300 and 600 μg was irradiated with 60Co gamma rays in a dose of 7.549 kGy·h–1. The proposed formulation at a concentration of 300 μg produced an increase in the hemagglutinating units Cramoll-1,4 due to the synergistic effect caused by gamma radiation. Considering the diverse use of lectins, specific molecular and structural factors, as well as changes resulting from its formulation, concentration, irradiation and route of administration is of utmost importance to continue the studies in vitro, for subsequent application in vivo to characterize the physiological and molecular processes involved in the response and cellular effects.

Characterization of the Unfolding Process of the Tetrameric and Dimeric Forms of Cratylia mollis Seed Lectin (CRAMOLL 1): Effects of Natural Fragmentation on Protein Stability

pCRAMOLL 1 is a major, non-glycosylated isolectin found in seeds of Cratylia mollis, which belongs to the Leguminosae family and the Diocleinae subtribe. The lectin (~25 kDa) consists of 236 amino acids, sharing 82% identity and virtually identical topological architecture with concanavalin A. Both lectins also share the same pH-dependent dimer-tetramer equilibrium and the ability to recognize Glc/Man moieties. Intricate post-translational events occurring in Diocleinae seed cotyledons result in a mixture of intact and fragmented monomers within the oligomeric assemblies of pCRAMOLL 1. In an earlier report, we demonstrated the production, purification, and characterization of the bacterially expressed form of CRAMOLL 1 (rCRAMOLL 1). The recombinant lectin retained sugar-binding activity and several other biophysical properties of pCRAMOLL 1, but its tetramers, which are composed of intact monomers only, show little enhancement in stability when probed with acidification, high temperatures, or hydrostatic pressure. Here we examined the urea-induced unfolding of the nonfragmented tetramers and dimers of rCRAMOLL 1 and compared this behavior with that of the mixed plant lectin counterparts. Using fluorescence, circular dichroism, size-exclusion chromatography, and chemical cross-linking experiments, we posited that the absence of fragmentation lent greater firmness to tetramers, but not to dimers. Dimeric and tetrameric pCRAMOLL 1 unfolded via a compact monomeric intermediate. In contrast, dimers of rCRAMOLL 1 behaved similarly to the plant dimer counterpart, but its tetrameric form remarkably showed no evidence of such partially unfolded monomers. By analyzing the crystal structure of pCRAMOLL 1, we were able to dissect the importance of the fragmentation to lectin stability.

Heterologous expression and purification of a biologically active legume lectin from Cratylia mollis seeds (CRAMOLL 1)

CRAMOLL 1 is a mannose/glucose isolectin isolated from Cratylia mollis seeds. This lectin has 82% sequence identity with Con A and essentially the same quaternary structure. As with Con A, CRAMOLL 1 seems to undergo complex post-translational processing which makes it difficult to the use of traditional molecular cloning for heterologous expression. Here we report the expression and purification of functional recombinant CRAMOLL 1 (rCRAMOLL 1) in Escherichia coli. This was accomplished by introducing a chemically synthesized DNA encoding the mature CRAMOLL 1 amino acid sequence into a bacterial expression vector under T7 promoter control. Most of the recombinant lectin was found in insoluble aggregates (inclusion bodies), but we were able to recover reasonable amounts of soluble lectin in the active form by decreasing the protein induction temperature. The recombinant lectin was purified to homogeneity with one-step affinity chromatography. The plant CRAMOLL 1 (pCRAMOLL 1) and rCRAMOLL 1 share several physicochemical properties such as molecular mass, charge density and secondary and tertiary structures. However, pCRAMOLL 1 has a lower thermodynamic stability than rCRAMOLL 1 when probed by acidification, high temperature or high hydrostatic pressure, and this is probably caused by the presence of tetramers composed of fragmented monomers, which are formed in the plant cotyledon but absent from the recombinant protein. rCRAMOLL 1 behaves identically to its plant counterpart with respect to its specificity for monosaccharides, and to its agglutinating activities against rabbit erythrocytes and Trypanosoma cruzi epimastigote cells.

Purification and primary structure determination of two Bowman–Birk type trypsin isoinhibitors from Cratylia mollis seeds

Two Bowman-Birk type trypsin inhibitors (CmTI 1 and CmTI 2) were purified from Cratylia mollis seeds by acetone precipitation, ion exchange, gel filtration and reverse-phase chromatography. CmTI 1 and CmTI 2, with 77 and 78 amino acid residues, respectively, were sequenced in their entirety and show a high structural similarity to Bowman-Birk inhibitors from other Leguminosae. The putative reactive sites of CmTI 1 are a lysine residue at position 22 and a tyrosine residue at position 49. Different reactive sites, as identified by their alignment with related inhibitors, were found for CmTI 2: lysine at position 22 and leucine at position 49. The dissociation constant K i of the complex with trypsin is 1.4 nM. The apparent molecular mass is 17 kDa without DDT and 11 kDa with reducing agent and heating.

Ultrastructural analysis and immunocytochemical localization of isolectins in Cratylia mollis seeds

Cratylia mollis is a native forage from the semi-arid region of Northeast, State of Pernambuco, Brazil, whose seeds have been considered an important lectin source. Multiple molecular forms of lectins, carbohydrate-binding proteins, have been purified from C. mollis seeds (Cra Iso) allowing several applications of these purified proteins. In this work seeds were processed for ultrastructural analysis and immunocytochemical localization of the two most abundant isolectins, Cra Iso 1 and Cra Iso 3, with glucose/mannose and galactose specificities, respectively. The ultrastructural analysis revealed a typical plant cell: organelles, nucleus and cellular wall were visualized. The localization of isolectins occurred mainly in the amorphous matrix of protein bodies, and in the cellular walls of the embryonic axis. The results showed that the isolectins, which differ in relation to carbohydrate specificity and glycosylation are located in the same cellular compartment suggesting different functions inside the same subcellular organelle. Cra Iso 1 and Cra Iso 3 distribution in the C. mollis seeds was consistent with the subcellular localization of several legume lectins.

Amino acid sequence and tertiary structure of Cratylia mollis seed lectin.

Carbohydrate-protein interactions play a key role in many biological processes. Cramoll is a lectin purified from Cratylia mollis seeds that is taxonomically related to concanavalin A (Con A). Although Cramoll and Con A have the same monosaccharide specificity, they have different glycoprotein binding profiles. We report the primary structure of Cramoll, determined by Edman degradation and mass spectrometry and its 1.77 A crystallographic structure and compare it with the three-dimensional structure of Con A in an attempt to understand how differential binding can be achieved by similar or nearly identical structures. We report here that Cramoll consists of 236 residues, with 82% identity with Con A, and that its topological architecture is essentially identical to Con A, because the Calpha positional differences are below 3.5 A. Cramoll and Con A have identical binding sites for MealphaMan, Mn2+, and Ca2+. However, we observed six substitutions in a groove adjacent to the extended binding site and two in the extended binding site that may explain the differences in binding of oligosaccharides and glycoproteins between Cramoll and Con A.

Liquid-liquid extraction of lectin from Cratylia mollis seeds using reversed micelles

Extraction of lectin from seeds ofCratylia mollis, camaratu bean, with reversed micelles of 100 mM sodium di(2-ethylhexyl) sulfosuccinate/isooctane was performed firstly with affinity-purified lectin. The best conditions were extraction of the seed extract at pH 5 and back-extraction at pH 10, giving yields of 38% and 100%, respectively.