Glycobiology 2011 doi: 10.1093/glycob/cwr025

Fine specificities of two lectins from Cymbosema roseum seeds: a lectin specific for high-mannose oligosaccharides and a lectin specific for blood group H type II trisaccharide

Tarun K Dam, Benildo S Cavada, Celso S Nagano, Bruno AM Rocha, Raquel G Benevides, Kyria S Nascimento, Luiz AG de Sousa, Stefan Oscarson and C Fred Brewer

The legume species of Cymbosema roseum of Diocleinae subtribe produce at least two different seed lectins. The present study demonstrates that C. roseum lectin I (CRL I) binds with high affinity to the “core” trimannoside of N-linked oligosaccharides. Cymbosema roseum lectin II (CRL II), on the other hand, binds with high affinity to the blood group H trisaccharide (Fucα1,2Galα1-4GlcNAc-). Thermodynamic and hemagglutination inhibition studies reveal the fine binding specificities of the two lectins. Data obtained with a complete set of monodeoxy analogs of the core trimannoside indicate that CRL I recognizes the 3-, 4- and 6-hydroxyl groups of the α(1,6) Man residue, the 3- and 4-hydroxyl group of the α(1,3) Man residue and the 2- and 4-hydroxyl groups of the central Man residue of the trimannoside. CRL I possesses enhanced affinities for the Man5 oligomannose glycan and a biantennary complex glycan as well as glycoproteins containing high-mannose glycans. On the other hand, CRL II distinguishes the blood group H type II epitope from the Lewisx, Lewisy, Lewisa and Lewisb epitopes. CRL II also distinguishes between blood group H type II and type I trisaccharides. CRL I and CRL II, respectively, possess differences in fine specificities when compared with other reported mannose and fucose recognizing lectins. This is the first report of a mannose-specific lectin (CRL I) and a blood group H type II-specific lectin (CRL II) from seeds of a member of the Diocleinae subtribe.
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Plant Cell Environmental 2011 DOI: 10.1111/j.1365-3040.2011.02366.x

Role of peroxidases in the compensation of cytosolic ascorbate peroxidase knockdown in rice plants under abiotic stress

Aurenivia Bonifacio, Marcio o. Martins, Carolina W. Ribeiro, Adilton V. Fontenele, Fabricio E. L. Carvalho, Márcia Margis-Pinheiro, Joaquim A. G. Silveira

Current studies, particularly in Arabidopsis, have demonstrated that mutants deficient in cytosolic ascorbate peroxidases (APXs) are susceptible to the oxidative damage induced by abiotic stress. In contrast, we demonstrate here that rice mutants double silenced for cytosolic APXs (APx1/2s) up-regulated other peroxidases, making the mutants able to cope with abiotic stress, such as salt, heat, high light and methyl viologen, similar to non-transformed (NT) plants. The APx1/2s mutants exhibited an altered redox homeostasis, as indicated by increased levels of H2O2 and ascorbate and glutathione redox states. Both mutant and NT plants exhibited similar photosynthesis (CO2 assimilation and photochemical efficiency) under both normal and stress conditions. Overall, the antioxidative compensatory mechanism displayed by the mutants was associated with increased expression of OsGpx genes, which resulted in higher glutathione peroxidase (GPX) activity in the cytosolic and chloroplastic fractions. The transcript levels of OsCatA and OsCatB and the activities of catalase (CAT) and guaiacol peroxidase (GPOD; type III peroxidases) were also up-regulated. None of the six studied isoforms of OsApx were up-regulated under normal growth conditions. Therefore, the deficiency in cytosolic APXs was effectively compensated for by up-regulation of other peroxidases. We propose that signalling mechanisms triggered in rice mutants could be distinct from those proposed for Arabidopsis.

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Clinical Microbiology Infection DOI: 10.1111/j.1469-0691.2010.03405.x

Mycotic aneurysm caused by Burkholderia pseudomallei: report of a Brazilian strain genetically related to Thai strains

J. J. C. Sidrim, M. F. G. Rocha, T. J. P. G. Bandeira, R. A. Cordeiro, B. M. Carvalho, T. B. Grangeiro, M. A. Holanda, R. A. C. Lima, L. G. A. Valente, A. K. F. Costa, R. S. N. Brilhante.

Melioidosis, a severe infectious disease caused by Burkholderia pseudomallei that is prevalent in Southeast Asia and Northern Australia, has been sporadically reported in Brazil since 2003. We report a case of aortic aneurysm with blood culture positive for B. pseudomallei. The phylogenetic analysis of 16S ribosomal DNA showed this isolate to be evolutionarily grouped with the MSHR346 strains from Thailand.
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Biochimie 2011 doi:10.1016/j.biochi.2010.11.003

Crystal structure of Bn IV in complex with myristic acid: A Lys49 myotoxic phospholipase A2 from Bothrops neuwiedi venom

P. Delatorre, B.A.M. Rocha, T. Santi-Gadelha, C.A.A. Gadelha, M.H. Toyama and B.S. Cavada

The LYS49-PLA2s myotoxins have attracted attention as models for the induction of myonecrosis by a catalytically independent mechanism of action. Structural studies and biological activities have demonstrated that the myotoxic activity of LYS49-PLA2 is independent of the catalytic activity site. The myotoxic effect is conventionally thought to be to due to the C-terminal region 111–121, which plays an effective role in membrane damage. In the present study, Bn IV LYS49-PLA2 was isolated from Bothrops neuwiedi snake venom in complex with myristic acid (CH3(CH2)12COOH) and its overall structure was refined at 2.2 Å resolution. The Bn IV crystals belong to monoclinic space group P21 and contain a dimer in the asymmetric unit. The unit cell parameters are a = 38.8, b = 70.4, c = 44.0 Å. The biological assembly is a “conventional dimer” and the results confirm that dimer formation is not relevant to the myotoxic activity. Electron density map analysis of the Bn IV structure shows clearly the presence of myristic acid in catalytic site. The relevant structural features for myotoxic activity are located in the C-terminal region and the Bn IV C-terminal residues NKKYRY are a probable heparin binding domain. These findings indicate that the mechanism of interaction between Bn IV and muscle cell membranes is through some kind of cell signal transduction mediated by heparin complexes
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Biochimica et Biophysica Acta - Biomembranes 2011 doi:10.1016/j.bbamem.2011.07.014

Osmotin from Calotropis procera latex: New insights into structure and antifungal properties.

Freitas CD, Lopes JL, Beltramini LM, de Oliveira RS, Oliveira JT, Ramos MV.

This study aimed at investigating the structural properties and mechanisms of the antifungal action of CpOsm, a purified osmotin from Calotropis procera latex. Fluorescence and CD assays revealed that the CpOsm structure is highly stable, regardless of pH levels. Accordingly, CpOsm inhibited the spore germination of Fusarium solani in all pH ranges tested. The content of the secondary structure of CpOsm was estimated as follows: α-helix (20%), β-sheet (33%), turned (19%) and unordered (28%), RMSD 1%. CpOsm was stable at up to 75°C, and thermal denaturation (T(m)) was calculated to be 77.8°C. This osmotin interacted with the negatively charged large unilamellar vesicles (LUVs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-1-glycerol (POPG), inducing vesicle permeabilization by the leakage of calcein. CpOsm induced the membrane permeabilization of spores and hyphae from Fusarium solani, allowing for propidium iodide uptake. These results show that CpOsm is a stable protein, and its antifungal activity involves membrane permeabilization, as property reported earlier for other osmotins and thaumatin-like proteins.
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Carbohydrate Plymers 2011

Effects of a sulfated polysaccharide isolated from the red seaweed Solieria filiformis on models of nociception and inflammation

Ianna Wivianne Fernandes de Araújo, Edfranck de Sousa Oliveira Vanderlei, José Ariévilo Gurgel Rodrigues, Chistiane Oliveira Coura, Ana Luíza Gomes Quinderé, Bruno Pedrosa Fontes, Ismael Nilo Lino de Queiroz, Roberta Jeane Bezerra Jorge, Mirna Marques Bezerra, Antonio Alfredo Rodrigues e Silva, Hellíada Vasconcelos Chaves, Helena Serra Azul Monteiro, Regina Célia Monteiro de Paula, Norma Maria Barros Benevides.

This  work  reports  the  effects  of  a  sulfated  polysaccharide  (SP-Sf),  isolated  from  the  seaweed  Solieria filiformis  and  characterized  by  Fourier  transformed  infrared  (FT-IR),  on  nociception  and  inflammation.Male  Swiss  mice  were  pretreated  with  SP-Sf  30  min  before  receiving  an  injection  of  0.8%  acetic  acid, 1%  formalin  or  30  min  prior  to  a  thermal  stimulus.  We  observed  that  SP-Sf  (1,  3  or  9  mg/kg)  significantly reduced  the  number  of  writhes.  SP-Sf  also  reduced  the  second  phase  of  the  formalin  test  and  did  not  cause a  significant  antinociceptive  effect  in  the  hot  plate  test,  suggesting  that  its  antinociceptive  action  occurs through a peripheral mechanism. SP-Sf (1, 3 or 9 mg/kg) did not show a significant anti-inflammatory effect  in  Wistar  rats  when  administrated  by  the  systemic  route  1  h  before  testing  using  carrageenan  ordextran. Finally, SP-Sf (9 mg/kg) did not show significant signs of toxicity when administrated in mice.
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Biochimie 2011 (doi:10.1016/j.biochi.2011.01.006)

Structural basis for both pro- and anti-inflammatory response induced by mannose-specific legume lectin from Cymbosema roseum.

Rocha BA, Delatorre P, Oliveira TM, Benevides RG, Pires AF, Sousa AA, Souza LA, Assreuy AM, Debray H, de Azevedo WF Jr, Sampaio AH, Cavada BS.

Legume lectins, despite high sequence homology, express diverse biological activities that vary in potency and efficacy. In studies reported here, the mannose-specific lectin from Cymbosema roseum (CRLI), which binds N-glycoproteins, shows both pro-inflammatory effects when administered by local injection and anti-inflammatory effects when by systemic injection. Protein sequencing was obtained by Tandem Mass Spectrometry and the crystal structure was solved by X-ray crystallography using a Synchrotron radiation source. Molecular replacement and refinement were performed using CCP4 and the carbohydrate binding properties were described by affinity assays and computational docking. Biological assays were performed in order to evaluate the lectin edematogenic activity. The crystal structure of CRLI was established to a 1.8Å resolution in order to determine a structural basis for these differing activities. The structure of CRLI is closely homologous to those of other legume lectins at the monomer level and assembles into tetramers as do many of its homologues. The CRLI carbohydrate binding site was predicted by docking with a specific inhibitory trisaccharide. CRLI possesses a hydrophobic pocket for the binding of α-aminobutyric acid and that pocket is occupied in this structure as are the binding sites for calcium and manganese cations characteristic of legume lectins. CRLI route-dependent effects for acute inflammation are related to its carbohydrate binding domain (due to inhibition caused by the presence of α-methyl-mannoside), and are based on comparative analysis with ConA crystal structure. This may be due to carbohydrate binding site design, which differs at Tyr12 and Glu205 position.
Copyright © 2011 Elsevier Masson SAS. All rights reserved. (Biochimie:)

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Planta 2011 (DOI: 10.1007/s00425-011-1392-1)

Laticifer proteins play a defensive role against hemibiotrophic and necrotrophic phytopathogens.

Souza DP, Freitas CD, Pereira DA, Nogueira FC, Silva FD, Salas CE, Ramos MV.

Proteins from latex of Calotropis procera (CpLP), Plumeria rubra (PrLP), Carica candamarcensis (P1G10) and Euphorbia tirucalli (EtLP) were tested for antifungal activity against phytopathogens. CpLP and P1G10 inhibited each fungi analyzed. PrLP and EtLP did not exert inhibition. CpLP and P1G10 exhibited preferential inhibitory activity towards R. solani (IC(50) = 20.7 and 25.3 µg/ml, respectively). The inhibitory activity was lost after heat treatment or proteolysis, providing evidence for the involvement of proteins in the inhibitory effect. Treatment of CpLP or P1G10 with Dithiothreitol improved both, the endogenous proteolytic activity and the antifungal properties. Conversely, pre-treatment of CpLP or P1G10 with iodoacetamide drastically reduced endogenous proteolytic activities and partially abrogated antifungal activity. Similar results were observed when spores were challenged to germinate in the presence of laticifer proteins. The purified cysteine proteinase CMS2MS2 from Carica candamarcensis latex or papain (E.C. 3.4.22.2), a cysteine proteinase from latex of Carica papaya L., but not trypsin (EC 3.4.21.4) or chymotrypsin (EC 3.4.21.1), two serine proteases, replicated the results obtained with CpLP or P1G10, thus restricting the antifungal property to latex plant cysteine proteinases. CpLP, CMS2MS2 and papain induced production of reactive oxygen species in spores of F. solani, suggesting that inhibition could be linked to oxidative stress. Proteome analysis of CpLP by 2-D electrophoresis and MALDI-TOF-TOF confirmed the existence of various pathogenic-related proteins such as chitinases, peroxidases and osmotins. The results support that laticifer proteins are part of plant defense repertoire against phytopathogenic fungi.

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New Phytologist 2011 (DOI: 10.1111/j.1469-8137.2011.03659.x)

Ascorbate peroxidase-related (APx-R) is a new heme-containing protein functionally associated with ascorbate peroxidase but evolutionarily divergent

Fernanda Lazzarotto, Felipe Karam Teixeira, Silvia Barcelos Rosa, Christophe Dunand, Claudia Lemelle Fernandes, Adilton de Vasconcelos Fontenele, Joaquim Albenisio Gomes Silveira, Hugo Verli, Rogério Margis, Marcia Margis-Pinheiro

Peroxidases are involved in several important processes, such as development and responses to environmental cues. In higher plants, most peroxidases are encoded by large, multigenic families that mainly originated from gene and chromosomal duplications.
Using phylogenetic, genomic and functional analyses, we have identified and characterized a new class of putative heme peroxidases, called ascorbate peroxidase-related (APx-R), which arose specifically in the lineage of plants.
The APx-R protein is structurally related to the ascorbate peroxidases, although the active site contains many conserved substitutions. Unlike all other plant peroxidases, which are encoded by gene families, APx-R is encoded by a single-copy gene in virtually all the species analyzed. APx-R proteins are targeted to the chloroplast and can physically interact with chloroplastic APx proteins. APx-R-knockdown rice (Oryza sativa) plants presented delayed development and a disturbed steady state of the antioxidant system compared with wild type. Moreover, the accumulation of APx-R transcripts was modulated by drought, UV irradiation, cold, and aluminum exposure in rice, suggesting the involvement of APx-R in the environmental stress response.
Our results reveal the existence of a new class of heme peroxidase which seems to play a role in the antioxidant system in plants, probably by modulating the activity of chloroplastic APx proteins.

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