Purification and Biological Activities of Abelmoschus esculentus Seed Lectin

The Abelmoschus esculentus (Malvaceae) plant originated in Africa and has spread across a number of tropic countries, including northeastern Brazil. The plant has been used to treat various disorders, such as cancer, microbial infections, hypoglycemia, constipation, urine retention and inflammation. The lectin of A. esculentus (AEL) was isolated by precipitation with ammonium sulfate at a saturation level of 30/60 and purified by ion exchange chromatography (Sephacel-DEAE). The electrophoresis (SDS-PAGE) profile of the AEL showed two protein bands of apparent molecular mass of approximately 15.0 and 21.0?kDa. The homogenity of the protein was confirmed by electrospray mass spectrometry (ESI-MS), which revealed the presence of a 10.29-kDa monomer and a 20.58-kDa dimer. The AEL exhibits agglutinating activity against rabbit (74.41 UH/mP) and human type ABO erythrocytes (21.00 UH/mP). This activity does not require the presence of divalent cations and is specifically inhibited by lactose, fructose and mannose. The intravenous treatment with 0.01, 0.1 and 1?mg/kg of AEL inhibited the paw edema elicited by carrageenan by approximately 15, 22 and 44?%, respectively, but not that induced by dextran. In addition, treatment with 0.1, 1 and 10?mg/kg of AEL also inhibited the abdominal writhing induced by acetic acid by approximately 52, 57 and 69?%, respectively. In conclusion, AEL is a new lectin with a molecular mass of 20.0?kDa, which is -composed of a 10.291-Da monomer and a 20.582-kDa dimer, that exhibits anti-inflammatory, antinociceptive and hemagglutinating activities. In addition, the lectin hemagglutinating property is both metallo-independent and associated with the lectin domain.     

Crystal structure of Dioclea rostrata lectin: insights into understanding the pH-dependent dimer-tetramer equilibrium and the structural basis for carbohydrate recognition in Diocleinae lectins

The legume lectins from the subtribe Diocleinae, often referred to as concanavalin A-like lectins, are a typical example of highly similar proteins that show distinct biological activities. The pH-dependent oligomerization that some of these lectins undergo and the relative position of amino acids within the carbohydrate-binding site are factors that have been reported to contribute to these differences in the activities of Diocleinae lectins. In the present work, we determined the amino acid sequence and the crystal structure of the lectin of Dioclea rostrata seeds (DRL), with the aim of investigating the structural bases of the different behavior displayed by this lectin in comparison to other Diocleinae lectins and determining the reason for the distinct pH-dependent dimer-tetramer equilibrium. In addition, we discovered a novel multimeric arrangement for this lectin.     

Identification of a new quaternary association for legume lectins

Lotus tetragonolobus lectin (LTA) is a fucose-specific legume lectin. Although several studies report a diverse combination of biological activities for LTA, little is known about the mechanisms involved in l-fucosyl oligosaccharide recognition. The crystal structure of LTA at 2.0A resolution reveals a different legume lectin tetramer. Its structure consists of a homotetramer composed of two back-to-back GS4-like dimers arranged in a new mode, resulting in a novel tetramer. The LTA N-linked carbohydrate at Asn4 and the unusual LTA dimer-dimer interaction are related to its particular mode of tetramerization. In addition, we used small angle X-ray scattering to investigate the quaternary structure of LTA in solution and to compare it to the crystalline structure. Although the crystal structure of LTA has revealed a conserved metal-binding site, its l-fucose-binding site presents some punctual differences. Our investigation of the new tetramer of LTA and its fucose-binding site is essential for further studies related to cross-linking between LTA and complex divalent l-fucosyl carbohydrates.     

A lectin from Platypodium elegans with unusual specificity and affinity for asymmetric complex N-glycans

Lectin activity with specificity for mannose and glucose has been detected in the seed of Platypodium elegans, a legume plant from the Dalbergieae tribe. The gene of Platypodium elegans lectin A has been cloned, and the resulting 261-amino acid protein belongs to the legume lectin family with similarity with Pterocarpus angolensis agglutinin from the same tribe. The recombinant lectin has been expressed in Escherichia coli and refolded from inclusion bodies. Analysis of specificity by glycan array evidenced a very unusual preference for complex type N-glycans with asymmetrical branches. A short branch consisting of one mannose residue is preferred on the 6-arm of the N-glycan, whereas extensions by GlcNAc, Gal, and NeuAc are favorable on the 3-arm. Affinities have been obtained by microcalorimetry using symmetrical and asymmetrical Asn-linked heptasaccharides prepared by the semi-synthetic method. Strong affinity with K_d of 4.5 μm was obtained for both ligands. Crystal structures of Platypodium elegans lectin A complexed with branched trimannose and symmetrical complex-type Asn-linked heptasaccharide have been solved at 2.1 and 1.65 Å resolution, respectively. The lectin adopts the canonical dimeric organization of legume lectins. The trimannose bridges the binding sites of two neighboring dimers, resulting in the formation of infinite chains in the crystal. The Asn-linked heptasaccharide binds with the 6-arm in the primary binding site with extensive additional contacts on both arms. The GlcNAc on the 6-arm is bound in a constrained conformation that may rationalize the higher affinity observed on the glycan array for N-glycans with only a mannose on the 6-arm.     

Canatoxin-, concanavalin A- and canavalin-cross-reactive materials during maturation of Canavalia brasiliensis (Mart.) seeds

The distribution of three cross-reactive materials (CRMs), a toxic protein analogous to canatoxin, CNTX-CRM, a lectin analogous to concanavalin A, Con A-CRM, and a major storage protein, canavalin-CRM, was investigated during successive stages of maturation of Canavalia brasiliensis Mart. seeds. The data obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunological analyses indicated that these proteins share extensive homology with the analogous proteins found in Canavalia ensiformis seeds. The changes in CNTX-CRM and Con A-CRM levels throughout the maturation process were assayed by rocket immunoelectrophoresis. Synthesis of Con A-CRM was detectable at 30 days post-anthesis (DPA) while its hemagglutinating activity appeared only at 35 DPA. The CNTX-CRM was detected as a biologically active protein from 30 DPA onwards. The behavior of CNTX-CRM during maturation of C. brasiliensis seeds was quite distinct from that of Con A-CRM, pointing to different biological roles of these proteins in the seed.     

Success of maximum likelihood phylogeny inference in the four-taxon case

We used simulated data to investigate a number of properties of maximum- likelihood (ML) phylogenetic tree estimation for the case of four taxa. Simulated data were generated under a broad range of conditions, including wide variation in branch lengths, differences in the ratio of transition and transversion substitutions, and the absence of presence of gamma-distributed site-to-site rate variation. Data were analyzed in the ML framework with two different substitution models, and we compared the ability of the two models to reconstruct the correct topology. Although both models were inconsistent for some branch-length combinations in the presence of site-to-site variation, the models were efficient predictors of topology under most simulation conditions. We also examined the performance of the likelihood ratio (LR) test for significant positive interior branch length. This test was found to be misleading under many simulation conditions, rejecting too often under some simulation conditions. Under the null hypothesis of zero length internal branch, LR statistics are assumed to be asymptotically distributed chi 2(1); with limited data, the distribution of LR statistics under the null hypothesis varies from chi 2(1).      

Establishment of a doxycycline-regulated cell line with inducible,doubly-stable expression of the wild-type p53 gene from p53-deleted hepatocellular carcinoma cells

p53 is important in the development of hepatocellular carcinoma (HCC) and in therapeutic approaches, but the mechanism whereby it inhibits HCC growth is still unclear. The aim of the present study was to establish a HCC cell system in which p53 levels can be regulated. Full-length wild-type p53 cDNA obtained by PCR was cloned into a retroviral response vector controlled by the tetracycline responsive element (RevTRE-p53). The regulatory vectors RevTet-Off and RevTRE-p53 were transfected into a packaging cell line, PT67. Hep3B cells in which the p53 gene was deleted were infected with RevTet-Off viral particles from the PT67. Three G418-resistant cell clones with high luciferase expression and low background were infected with RevTRE-p53. By screening dozens of RevTRE-p53-infected clones with hygromycin we identified the one with the highest expression of p53 and the lowest background after doxycycline treatment. The results showed that p53 expression in this cell clone could be simply turned on or off by removing or adding doxycycline. Furthermore, it was found that the level of p53 protein was negatively and sensitively related to the doxycycline concentration. In conclusion, we have established a HCC cell line in which p53 expression can be switched on or off and regulated in a dose- and time-dependent manner.     

Detecting substitution-rate heterogeneity among regions of a nucleotide sequence

Likelihood-ratio statistics are proposed to test for heterogeneity in nucleotide substitution rate among regions of a DNA sequence. The tests examine three-sequence phylogenies, and two specific tests are proposed: a test to detect rate heterogeneity among genic regions within a sequence, over all evolutionary lineages; and a test to detect rate heterogeneity among regions in a specific evolutionary lineage. Simulations examine the ability of tests to detect a single region that varies in nucleotide substitution rate relative to the remainder of the sequence. A 50-bp region with a fivefold substitution-rate increase can be detected > or = 90% of the time when it is found in all three lineages of the phylogeny, and a 50-bp region of fivefold rate increase can be detected with approximately 70% power when it is found in only one evolutionary lineage. Simulation also examines the effect of transition- and transversion-rate differences. The tests are applied to published DNA sequences. While the tests are powerful, significant results can be difficult to interpret biologically.      

Purification of a thermostable antinociceptive lectin isolated from Andira anthelmia

Andira anthelmia (tribe Dalbergieae), a plant from Brazilian Amazon, possesses a seed lectin that was purified by affinity chromatography in sepharose–mannose. This novel Dalbergieae lectin, named AAL, agglutinated rabbit erythrocytes treated with trypsin. The hemagglutinating activity of AAL was maintained after incubation at a wide range of temperature (40 to 70 °C) and pH, was shown to be dependent on divalent cations, and was inhibited by d ‐mannose and d ‐sucrose. AAL showed an electrophoretic profile in sodium dodecyl sulfate–polyacrylamide gel electrophoresis similar to other lectins of the tribe Dalbergieae, presenting a double band of molecular weight with approximately 20 kDa and other minor bands of 17, 15, and 13 kDa, being the smaller fragment glycosylated. AAL injected by intravenous route in mice showed antinociceptive activity in two behavioral tests (writhing and formalin). In the writhing test induced by acetic acid, AAL showed inhibitory effect at 0.01 mg/kg (68%), 0.1 mg/kg (46%) and 1 mg/kg (74%). In the formalin test, AAL (0.1 mg/kg) inhibited by 48% the licking time in the inflammatory phase, an effect that was recovered by the lectin association with mannose. In conclusion, AAL presents analgesic effect involving the lectin domain via peripheral mechanisms of inflammatory nociception. This activity highlights the importance of lectins as tools to be used for understanding the interaction of protein–carbohydrate in processes associated to inflammatory pain. Copyright © 2015 John Wiley & Sons, Ltd.