Structural and functional features of a class VI chitinase from cashew (Anacardium occidentale L.) with antifungal properties.

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, ß-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.

Structural characterization of a Vatairea macrocarpa lectin in complex with a tumor-associated antigen: A new tool for cancer research.

Legume lectins are the most thoroughly studied group of lectins and have been widely linked to many pathological processes. Their use as immunohistochemistry markers for cell profiling and cancer diagnosis have made these molecules important tools for immunological studies and have stimulated the prospection and characterization of new lectins. The crystal structures of a recombinant seed lectin from Vatairea macrocarpa (rVML) and its complexes with GalNAcα1-O-Ser, GalNAc and α-lactose, have been determined at 1.90, 1.97, 2.70 and 1.83Å resolution, respectively. Small angle X-ray scattering and calorimetry assays have confirmed the same pH stable oligomerization pattern and binding profiles proposed for its wild-type counterpart. In silico analyzes have explored the potential of this recombinant lectin as new tool for cancer research through a comparative profile with other legume lectins widely used for cancer diagnosis and prognosis. The results suggest the recognition of specific epitopes exhibited on different cancer cells as a process that relies on the disposition of hydrophobic clusters and charged regions around the lectin carbohydrate-binding site, favouring the anchorage of different groups in the antigen boundaries, highlighting the different potential of each analyzed lectin. In conclusion, the experimental results and comparative analysis show that rVML is as a promising tool for cancer research, able to bind with high affinity specific tumor-associated antigens, highly stable and easily produced.

ConBr, a lectin from Canavalia brasiliensis seeds, modulates signaling pathways and increases BDNF expression probably via a glycosylated target.

In the central nervous system, many receptors, ion channels and neurotransmitter transporters are glycoproteins, where the glycan chains are modulator elements. Lectins are proteins, which recognize and bind carbohydrate complexes. We have previously shown that ConBr, a lectin purified from Canavalia brasiliensis seeds, produced antidepressant-like effect and blocked hippocampal neurotoxicity induced by quinolinic acid and glutamate. Noteworthy, all these effects occurred in a dependence of its carbohydrate recognition domain. Therefore, the present study was undertaken in order to elucidate intracellular signaling pathways regulated by ConBr that may be potentially associated with the antidepressant and neuroprotective effects previously reported to be dependent on carbohydrate interaction. ConBr (10 µg/site) was injected into the ventricle (i.c.v.) of mice, and the hippocampi were removed 0.5, 1, 3, 6, 8, 12, 18, and 24 h after treatment. Our results showed that in the period of 0.5-3 h, ConBr induced activation of the protein kinases Akt, ERK1, and PKA. Furthermore, the phosphorylation of CREB-Ser133 was stimulated by ConBr (1-6 h), while brain-derived neurotrophic factor (BDNF) mRNA was increased at 12 h and BDNF protein at 18-24 h. Our data suggest that an early activation of protein kinases may trigger CREB-dependent BDNF transcription, resulting in a subsequent increase of BDNF protein in response to ConBr. Later, increment of Akt phosphorylation was observed 24 h after ConBr administration, possibly due to BDNF/TrkB-dependent activation of Akt. Our findings indicate that ConBr is a multifunctional molecule capable to activate signaling pathways involved in neuroplasticity and neuroprotection.

ConBr, a lectin from Canavalia brasiliensis seeds, protects against quinolinic acid-induced seizures in mice.

Lectins are proteins capable of reversible binding to carbohydrates or glycoconjugates. In the central nervous system of mammals, lectins with affinity for mannose/glucose or galactose can modulate cellular communication. ConBr, a lectin isolated from the seeds of Canavalia brasiliensis, previously showed antidepressant effect in the forced swimming test in mice, with involvement of the monoaminergic system. In this study, we investigated the neuroprotective effects of ConBr against quinolinic acid (QA), a well-known NMDA agonist that produces severe neurotoxicity when administered in vivo. ConBr (10 µg/site) administered via intracerebroventricular (i.c.v.) showed a neuroprotective activity against seizures induced by QA (36.8 nmol/site; i.c.v.) when administered 15 min prior to QA, with a percentage of protection around 50%. ConBr was also able to significantly decrease the severity of the seizures but without changes in the latency of the first convulsion or the duration of the seizures. This effect was dependent on the structural integrity of the ConBr protein and its binding capacity to oligosaccharides residues. ConA, a lectin with high similarity to ConBr, did not reverse the QA-induced seizures. Moreover, ConBr was able to protect against hippocampal cell death caused by QA, which was measured by propidium iodide incorporation. QA caused activation of JNK2 and improved the phosphorylation of Ser831 and 845 on the AMPA receptor GluR1 subunit, and both of these effects were counteracted by ConBr. Our data suggest that the lectin ConBr may exert a modulatory action on NMDA receptors, which inhibits its activity in response to QA.

Steady-state level of kit ligand mRNA in goat ovaries and the role of kit ligand in preantral follicle survival and growth in vitro.

The aims of this study were to investigate steady-state level of Kit Ligand (KL) mRNA and its effects on in vitro survival and growth of caprine preantral follicles. RT-PCR was used to analyze caprine steady-state level of KL mRNA in primordial, primary, and secondary follicles, and in small (1-3 mm) and large (3-6 mm) antral follicles. Furthermore, ovarian fragments were cultured for 1 or 7 days in Minimal Essential Medium (MEM(+)) supplemented with KL (0, 1, 10, 50, 100, or 200 ng/ml). Noncultured (control) and cultured fragments were processed for histology and transmission electron microscopy (TEM). RT-PCR demonstrated an increase in steady-state level of KL mRNA during the transition from primary to secondary follicles. Small antral follicles had higher steady-state levels of KL mRNA in granulosa and theca cells than large follicles. After 7 days, only 50 ng/ml of KL had maintained the percentage of normal follicles similar to control. After 1 day, all KL concentrations reduced the percentage of primordial follicles and increased the percentage of growing follicles. KL at 10, 50, 100, or 200 ng/ml increased primary follicles, compared to MEM(+) after 7 days. An increase in oocyte and follicular diameter was observed at 50 ng/ml of KL. TEM confirmed ultrastructural integrity of follicles after 7 days at 50 ng/ml of KL. In conclusion, the KL mRNAs were detected in all follicular categories. Furthermore, 50 ng/ml of KL maintained the integrity of caprine preantral follicle cultured for 7 days and stimulated primordial follicle activation and follicle growth.

Quantitative expression analysis of Bodhesin genes in the buck (Capra hircus) reproductive tract by real-time polymerase chain reaction (qRT-PCR).

Knowledge of the different patterns of gene expression along the male reproductive tract can assist in understanding the physiological processes of species-specific reproduction in mammals. In the present work, expression profiles of buck spermadhesin (bodhesin) genes along the reproductive tract by qRT-PCR were investigated. Total RNA from the seminal vesicle, testis, epididymis, bulbourethral gland and ductus deferens were reverse transcribed and the cDNA produced was submitted to qRT-PCR. For each homologous bodhesin gene, namely Bdh-1, Bdh-2 and Bdh-3, sets of specific primers and recombinant plasmids were prepared for gene quantification. In buck seminal vesicles, Bdh-2 is the homologue predominantly expressed, with a copy number on the order of millions of times more than Bdh-1 and thousand times more than Bdh-3. The copy number of Bdh-3 mRNA is only 10-fold greater than that of Bdh-1. Bodhesin transcripts were detected in all tissues examined, except in ductus deferens. The quantitative analysis also demonstrated clearly the differential gene expression of spermadhesin in bulbourethral gland. The striking differences in bodhesin gene expression indicate that each isoform could have a specific biological function in the buck genital tract, which deserves further detailed studies.

Expression and purification of the recombinant ConBr (Canavalia brasiliensis lectin) produced in Escherichia coli cells.

ConBr, a D-glucose/D-mannose-specific lectin from Canavalia brasiliensis seeds, was produced in Escherichia coli from a (c)DNA clone subcloned to pET15b expression vector. The recombinant lectin (rConBr) was purified by one-step immobilized metal-affinity chromatography using an amino-terminal hexahistidine tag. By SDS-PAGE and Western blot, rConBr was highly pure with an apparent molecular mass of 37 kDa. N-terminal sequence analysis revealed a single sequence, confirming the identity of the expressed protein as the pre-pro-ConBr.